Magnetic nanoparticles: Applications in gene delivery and gene therapy.

PubMed

Majidi, Sima; Zeinali Sehrig, Fatemeh; Samiei, Mohammad; Milani, Morteza; Abbasi, Elham; Dadashzadeh, Kianoosh; Akbarzadeh, Abolfazl

2016-06-01

Gene therapy is defined as the direct transfer of genetic material to tissues or cells for the treatment of inherited disorders and acquired diseases. For gene delivery, magnetic nanoparticles (MNPs) are typically combined with a delivery platform to encapsulate the gene, and promote cell uptake. Delivery technologies that have been used with MNPs contain polymeric, viral, as well as non-viral platforms. In this review, we focus on targeted gene delivery using MNPs.

Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

NASA Astrophysics Data System (ADS)

Yuan, Chenyan; An, Yanli; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng

2014-08-01

Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression.

Development of therapeutic microbubbles for enhancing ultrasound-mediated gene delivery.

PubMed

Sun, Ryan R; Noble, Misty L; Sun, Samuel S; Song, Shuxian; Miao, Carol H

2014-05-28

Ultrasound (US)-mediated gene delivery has emerged as a promising non-viral method for safe and selective gene delivery. When enhanced by the cavitation of microbubbles (MBs), US exposure can induce sonoporation that transiently increases cell membrane permeability for localized delivery of DNA. The present study explores the effect of generalizable MB customizations on MB facilitation of gene transfer compared to Definity®, a clinically available contrast agent. These modifications are 1) increased MB shell acyl chain length (RN18) for elevated stability and 2) addition of positive charge on MB (RC5K) for greater DNA associability. The MB types were compared in their ability to facilitate transfection of luciferase and GFP reporter plasmid DNA in vitro and in vivo under various conditions of US intensity, MB dosage, and pretreatment MB-DNA incubation. The results indicated that both RN18 and RC5K were more efficient than Definity®, and that the cationic RC5K can induce even greater transgene expression by increasing payload capacity with prior DNA incubation without compromising cell viability. These findings could be applied to enhance MB functions in a wide range of therapeutic US/MB gene and drug delivery approach. With further designs, MB customizations have the potential to advance this technology closer to clinical application. Copyright © 2014 Elsevier B.V. All rights reserved.

Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing.

PubMed

Liu, Jia; Shui, Sai-Lan

2016-12-28

The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing. Copyright © 2016 Elsevier B.V. All rights reserved.

Biodegradable DNA Nanoparticles that Provide Widespread Gene Delivery in the Brain

PubMed Central

Mastorakos, Panagiotis; Song, Eric; Zhang, Clark; Berry, Sneha; Park, Hee Won; Kim, Young Eun; Park, Jong Sung; Lee, Seulki; Suk, Jung Soo; Hanes, Justin

2016-01-01

Successful gene therapy of neurological disorders is predicated on achieving widespread and uniform transgene expression throughout the affected disease area in the brain. However, conventional gene vectors preferentially travel through low-resistance perivascular spaces and/or are confined to the administration site even with the aid of a pressure-driven flow provided by convection-enhanced delivery. Biodegradable DNA nanoparticles offer a safe gene delivery platform devoid of adverse effects associated with virus-based or synthetic non-biodegradable systems. Using a state-of-the-art biodegradable polymer, poly(β-amino ester), we engineered colloidally stable sub-100 nm DNA nanoparticles coated with a non-adhesive polyethylene glycol corona that are able to avoid the adhesive and steric hindrances imposed by the extracellular matrix. Following convection enhanced delivery, these brain-penetrating nanoparticles were able to homogeneously distribute throughout the rodent striatum and mediate widespread and high-level transgene expression. These nanoparticles provide a biodegradable DNA nanoparticle platform enabling uniform transgene expression patterns in vivo and hold promise for the treatment of neurological diseases. PMID:26680637

Recent advances in dendrimer-based nanovectors for tumor-targeted drug and gene delivery

PubMed Central

Kesharwani, Prashant; Iyer, Arun K.

2015-01-01

Advances in the application of nanotechnology in medicine have given rise to multifunctional smart nanocarriers that can be engineered with tunable physicochemical characteristics to deliver one or more therapeutic agent(s) safely and selectively to cancer cells, including intracellular organelle-specific targeting. Dendrimers having properties resembling biomolecules, with well-defined 3D nanopolymeric architectures, are emerging as a highly attractive class of drug and gene delivery vector. The presence of numerous peripheral functional groups on hyperbranched dendrimers affords efficient conjugation of targeting ligands and biomarkers that can recognize and bind to receptors overexpressed on cancer cells for tumor-cell-specific delivery. The present review compiles the recent advances in dendrimer-mediated drug and gene delivery to tumors by passive and active targeting principles with illustrative examples. PMID:25555748

Design of magnetic gene complexes as effective and serum resistant gene delivery systems for mesenchymal stem cells.

PubMed

Zhang, Tian-Yuan; Wu, Jia-He; Xu, Qian-Hao; Wang, Xia-Rong; Lu, Jingxiong; Hu, Ying; Jo, Jun-Ichiro; Yamamoto, Masaya; Ling, Daishun; Tabata, Yasuhiko; Gao, Jian-Qing

2017-03-30

Gene engineered mesenchymal stem cells (MSCs) have been proposed as promising tools for their various applications in biomedicine. Nevertheless, the lack of an effective and safe way to genetically modify these stem cells is still a major obstacle in the current studies. Herein, we designed novel magnetic complexes by assembling cationized pullulan derivatives with magnetic iron oxide nanoparticles for delivering target genes to MSCs. Results showed that this complexes achieved effective gene expression with the assistance of external magnetic field, and resisted the adverse effect induced by serum proteins on the gene delivery. Moreover, neither significant cytotoxicity nor the interference on the osteogenic differentiation to MSCs were observed after magnetofection. Further studies revealed that this effective and serum resistant gene transfection was partly due to the accelerated and enhanced intracellular uptake process driven by external magnetic field. To conclude, the current study presented a novel option for genetic modification of MSCs in an effective, relatively safe and serum compatible way. Copyright © 2017 Elsevier B.V. All rights reserved.

Artificial Virus as Trump-card to Resolve Exigencies in Targeted Gene Delivery.

PubMed

Ajithkumar, K C; Pramod, Kannissery

2018-01-01

Viruses are potent pathogens that can effectively deliver the genetic material to susceptible host cells. This capability is beneficially utilized to successfully deliver the genetic material. However, the use of virus mediated gene delivery is considered divisive, because the potentially replicable genomes recombine or integrate with the cell DNA resulting in immunogenicity, ranging from inflammation to death. Thus, the need for potentially effective non-viral gene delivery vehicles arises. Non-viral vectors, protein only particles and virus like particles (VLP) can be constructed which contain all the necessary functional moieties. These resemble viruses and are called artificial or synthetic virus. The artificial virus eliminates the disadvantages of viral vectors but retain the beneficial effects of the viruses. Need for further functionalization can be avoided by this approach because incorporation of requisite agents such as cell ligands, membrane active peptides, etc. into proteins is possible. The protein- DNA complexes resemble bacterial inclusion bodies. Nucleic acids influence conformation of protein units which subsequently result in cell uptake and finally to the cell nucleus. Such tunable systems mimic the activities of infected viruses and are used for the safe and effective delivery of drugs and genetic material in gene therapy. The versatility, stability and biocompatible nature of artificial virus along with high transfection efficacy have made it favorite for gene delivery purposes, in addition to being useful for various biomedical and drug delivery applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

FUNCTIONAL NANOPARTICLES FOR MOLECULAR IMAGING GUIDED GENE DELIVERY

PubMed Central

Liu, Gang; Swierczewska, Magdalena; Lee, Seulki; Chen, Xiaoyuan

2010-01-01

Gene therapy has great potential to bring tremendous changes in treatment of various diseases and disorders. However, one of the impediments to successful gene therapy is the inefficient delivery of genes to target tissues and the inability to monitor delivery of genes and therapeutic responses at the targeted site. The emergence of molecular imaging strategies has been pivotal in optimizing gene therapy; since it can allow us to evaluate the effectiveness of gene delivery noninvasively and spatiotemporally. Due to the unique physiochemical properties of nanomaterials, numerous functional nanoparticles show promise in accomplishing gene delivery with the necessary feature of visualizing the delivery. In this review, recent developments of nanoparticles for molecular imaging guided gene delivery are summarized. PMID:22473061

Advanced drug and gene delivery systems based on functional biodegradable polycarbonates and copolymers.

PubMed

Chen, Wei; Meng, Fenghua; Cheng, Ru; Deng, Chao; Feijen, Jan; Zhong, Zhiyuan

2014-09-28

Biodegradable polymeric nanocarriers are one of the most promising systems for targeted and controlled drug and gene delivery. They have shown several unique advantages such as excellent biocompatibility, prolonged circulation time, passive tumor targeting via the enhanced permeability and retention (EPR) effect, and degradation in vivo into nontoxic products after completing their tasks. The current biodegradable drug and gene delivery systems exhibit, however, typically low in vivo therapeutic efficacy, due to issues of low loading capacity, inadequate in vivo stability, premature cargo release, poor uptake by target cells, and slow release of therapeutics inside tumor cells. To overcome these problems, a variety of advanced drug and gene delivery systems has recently been designed and developed based on functional biodegradable polycarbonates and copolymers. Notably, polycarbonates and copolymers with diverse functionalities such as hydroxyl, carboxyl, amine, alkene, alkyne, halogen, azido, acryloyl, vinyl sulfone, pyridyldisulfide, and saccharide, could be readily obtained by controlled ring-opening polymerization. In this paper, we give an overview on design concepts and recent developments of functional polycarbonate-based nanocarriers including stimuli-sensitive, photo-crosslinkable, or active targeting polymeric micelles, polymersomes and polyplexes for enhanced drug and gene delivery in vitro and in vivo. These multifunctional biodegradable nanosystems might be eventually developed for safe and efficient cancer chemotherapy and gene therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

Microneedles As a Delivery System for Gene Therapy

PubMed Central

Chen, Wei; Li, Hui; Shi, De; Liu, Zhenguo; Yuan, Weien

2016-01-01

Gene delivery systems can be divided to two major types: vector-based (either viral vector or non-viral vector) and physical delivery technologies. Many physical carriers, such as electroporation, gene gun, ultrasound start to be proved to have the potential to enable gene therapy. A relatively new physical delivery technology for gene delivery consists of microneedles (MNs), which has been studied in many fields and for many molecule types and indications. Microneedles can penetrate the stratum corneum, which is the main barrier for drug delivery through the skin with ease of administration and without significant pain. Many different kinds of MNs, such as metal MNs, coated MNs, dissolving MNs have turned out to be promising in gene delivery. In this review, we discussed the potential as well as the challenges of utilizing MNs to deliver nucleic acids for gene therapy. We also proposed that a combination of MNs and other gene delivery approaches may lead to a better delivery system for gene therapy. PMID:27303298

Hyaluronic Acid Enhances Gene Delivery into the Cochlea

PubMed Central

Shibata, Seiji B.; Cortez, Sarah R.; Wiler, James A.; Swiderski, Donald L.

2012-01-01

Abstract Cochlear gene therapy can be a new avenue for the treatment of severe hearing loss by inducing regeneration or phenotypic rescue. One necessary step to establish this therapy is the development of a safe and feasible inoculation surgery, ideally without drilling the bony cochlear wall. The round window membrane (RWM) is accessible in the middle-ear space, but viral vectors placed on this membrane do not readily cross the membrane to the cochlear tissues. In an attempt to enhance permeability of the RWM, we applied hyaluronic acid (HA), a nontoxic and biodegradable reagent, onto the RWM of guinea pigs, prior to delivering an adenovirus carrying enhanced green fluorescent protein (eGFP) reporter gene (Ad-eGFP) at the same site. We examined distribution of eGFP in the cochlea 1 week after treatment, comparing delivery of the vector via the RWM, with or without HA, to delivery by a cochleostomy into the perilymph. We found that cochlear tissue treated with HA-assisted delivery of Ad-eGFP demonstrated wider expression of transgenes in cochlear cells than did tissue treated by cochleostomy injection. HA-assisted vector delivery facilitated expression in cells lining the scala media, which are less accessible and not transduced after perilymphatic injection. We assessed auditory function by measuring auditory brainstem responses and determined that thresholds were significantly better in the ears treated with HA-assisted Ad-eGFP placement on the RWM as compared with cochleostomy. Together, these data demonstrate that HA-assisted delivery of viral vectors provides an atraumatic and clinically feasible method to introduce transgenes into cochlear cells, thereby enhancing both research methods and future clinical application. PMID:22074321

Hyaluronic acid enhances gene delivery into the cochlea.

PubMed

Shibata, Seiji B; Cortez, Sarah R; Wiler, James A; Swiderski, Donald L; Raphael, Yehoash

2012-03-01

Cochlear gene therapy can be a new avenue for the treatment of severe hearing loss by inducing regeneration or phenotypic rescue. One necessary step to establish this therapy is the development of a safe and feasible inoculation surgery, ideally without drilling the bony cochlear wall. The round window membrane (RWM) is accessible in the middle-ear space, but viral vectors placed on this membrane do not readily cross the membrane to the cochlear tissues. In an attempt to enhance permeability of the RWM, we applied hyaluronic acid (HA), a nontoxic and biodegradable reagent, onto the RWM of guinea pigs, prior to delivering an adenovirus carrying enhanced green fluorescent protein (eGFP) reporter gene (Ad-eGFP) at the same site. We examined distribution of eGFP in the cochlea 1 week after treatment, comparing delivery of the vector via the RWM, with or without HA, to delivery by a cochleostomy into the perilymph. We found that cochlear tissue treated with HA-assisted delivery of Ad-eGFP demonstrated wider expression of transgenes in cochlear cells than did tissue treated by cochleostomy injection. HA-assisted vector delivery facilitated expression in cells lining the scala media, which are less accessible and not transduced after perilymphatic injection. We assessed auditory function by measuring auditory brainstem responses and determined that thresholds were significantly better in the ears treated with HA-assisted Ad-eGFP placement on the RWM as compared with cochleostomy. Together, these data demonstrate that HA-assisted delivery of viral vectors provides an atraumatic and clinically feasible method to introduce transgenes into cochlear cells, thereby enhancing both research methods and future clinical application.

Safe delivery of optical power from space.

PubMed

Smith, M; Fork, R L; Cole, S

2001-05-07

More than a billion gigawatts of sunlight pass through the area extending from Earth out to geostationary orbit. A small fraction of this clean renewable power appears more than adequate to satisfy the projected needs of Earth, and of human exploration and development of space far into the future. Recent studies suggest safe and efficient access to this power can be achieved within 10 to 40 years. Light, enhanced in spatial and temporal coherence, as compared to natural sunlight, offers a means, and probably the only practical means, of usefully transmitting this power to Earth. We describe safety standards for satellite constellations and Earth based sites designed, respectively, to transmit, and receive this power. The spectral properties, number of satellites, and angle subtended at Earth that are required for safe delivery are identified and discussed.

Nanoparticle-mediated gene delivery.

PubMed

Jin, Sha; Leach, John C; Ye, Kaiming

2009-01-01

Nonviral gene delivery has been gaining considerable attention recently. Although the efficacy of DNA transfection, which is a major concern, is low in nonviral vector-mediated gene transfer compared with viral ones, nonviral vectors are relatively easy to prepare, less immunogenic and oncogenic, and have no potential of virus recombination and no limitation on the size of a transferred gene. The ability to incorporate genetic materials such as plasmid DNA, RNA, and siRNA into functionalized nanoparticles with little toxicity demonstrates a new era in pharmacotherapy for delivering genes selectively to tissues and cells. In this chapter, we highlight the basic concepts and applications of nonviral gene delivery using super paramagnetic iron oxide nanoparticles and functionalized silica nanoparticles. The experimental protocols related to these topics are described in the chapter.

Endovascular Gene Delivery from a Stent Platform: Gene- Eluting Stents

PubMed Central

Fishbein, Ilia; Chorny, Michael; Adamo, Richard F; Forbes, Scott P; Corrales, Ricardo A; Alferiev, Ivan S; Levy, Robert J

2015-01-01

A synergistic impact of research in the fields of post-angioplasty restenosis, drug-eluting stents and vascular gene therapy over the past 15 years has shaped the concept of gene-eluting stents. Gene-eluting stents hold promise of overcoming some biological and technical problems inherent to drug-eluting stent technology. As the field of gene-eluting stents matures it becomes evident that all three main design modules of a gene-eluting stent: a therapeutic transgene, a vector and a delivery system are equally important for accomplishing sustained inhibition of neointimal formation in arteries treated with gene delivery stents. This review summarizes prior work on stent-based gene delivery and discusses the main optimization strategies required to move the field of gene-eluting stents to clinical translation. PMID:26225356

Safe delivery and newborn care practices in Sindh, Pakistan: a community-based investigation of mothers and health workers.

PubMed

Hassan, Hamid; Jokhio, Abdul Hakeem; Winter, Heather; Macarthur, Christine

2012-08-01

to determine the prevalence of specific intrapartum practices in Sindh province, Pakistan. a cross-sectional, questionnaire based study. 6 health clinics in Mirpurkhas, Sindh province, rural Pakistan. 225 mothers and 82 health workers. outcome measures were indicators of safe delivery practices and referral following an obstetric complication. Prevalence of unhygienic and unsafe practices in deliveries attended by Traditional Birth Attendants (TBAs) was common. Deliveries by skilled attendants were significantly safer but with some failures in hygienic practices. 29% of women who had experienced an obstetric complication had not received emergency obstetric care. safe delivery practices and newborn care needs to be improved in rural Pakistan. This may be achieved by training health workers and TBAs in safe delivery practices, using safe delivery kits and with an effective referral system. Copyright © 2011 Elsevier Ltd. All rights reserved.

Gadolinium embedded iron oxide nanoclusters as T1-T2 dual-modal MRI-visible vectors for safe and efficient siRNA delivery

NASA Astrophysics Data System (ADS)

Wang, Xiaoyong; Zhou, Zijian; Wang, Zhiyong; Xue, Yunxin; Zeng, Yun; Gao, Jinhao; Zhu, Lei; Zhang, Xianzhong; Liu, Gang; Chen, Xiaoyuan

2013-08-01

This report illustrates a new strategy of designing a T1-T2 dual-modal magnetic resonance imaging (MRI)-visible vector for siRNA delivery and MRI. Hydrophobic gadolinium embedded iron oxide (GdIO) nanocrystals are self-assembled into nanoclusters in the water phase with the help of stearic acid modified low molecular weight polyethylenimine (stPEI). The resulting water-dispersible GdIO-stPEI nanoclusters possess good stability, monodispersity with narrow size distribution and competitive T1-T2 dual-modal MR imaging properties. The nanocomposite system is capable of binding and delivering siRNA for knockdown of a gene of interest while maintaining its magnetic properties and biocompatibility. This new gadolinium embedded iron oxide nanocluster provides an important platform for safe and efficient gene delivery with non-invasive T1-T2 dual-modal MRI monitoring capability.This report illustrates a new strategy of designing a T1-T2 dual-modal magnetic resonance imaging (MRI)-visible vector for siRNA delivery and MRI. Hydrophobic gadolinium embedded iron oxide (GdIO) nanocrystals are self-assembled into nanoclusters in the water phase with the help of stearic acid modified low molecular weight polyethylenimine (stPEI). The resulting water-dispersible GdIO-stPEI nanoclusters possess good stability, monodispersity with narrow size distribution and competitive T1-T2 dual-modal MR imaging properties. The nanocomposite system is capable of binding and delivering siRNA for knockdown of a gene of interest while maintaining its magnetic properties and biocompatibility. This new gadolinium embedded iron oxide nanocluster provides an important platform for safe and efficient gene delivery with non-invasive T1-T2 dual-modal MRI monitoring capability. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr02797j

Biological Gene Delivery Vehicles: Beyond Viral Vectors

PubMed Central

Seow, Yiqi; Wood, Matthew J

2009-01-01

Gene therapy covers a broad spectrum of applications, from gene replacement and knockdown for genetic or acquired diseases such as cancer, to vaccination, each with different requirements for gene delivery. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications today, but both have limitations and risks, including complexity of production, limited packaging capacity, and unfavorable immunological features, which restrict gene therapy applications and hold back the potential for preventive gene therapy. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents which include bacteria, bacteriophage, virus-like particles (VLPs), erythrocyte ghosts, and exosomes. Exploiting the natural properties of these biological entities for specific gene delivery applications will expand the repertoire of gene therapy vectors available for clinical use. Here, we review the prospects for nonviral biological delivery vehicles as gene therapy agents with focus on their unique evolved biological properties and respective limitations and potential applications. The potential of these nonviral biological entities to act as clinical gene therapy delivery vehicles has already been shown in clinical trials using bacteria-mediated gene transfer and with sufficient development, these entities will complement the established delivery techniques for gene therapy applications. PMID:19277019

Biological gene delivery vehicles: beyond viral vectors.

PubMed

Seow, Yiqi; Wood, Matthew J

2009-05-01

Gene therapy covers a broad spectrum of applications, from gene replacement and knockdown for genetic or acquired diseases such as cancer, to vaccination, each with different requirements for gene delivery. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications today, but both have limitations and risks, including complexity of production, limited packaging capacity, and unfavorable immunological features, which restrict gene therapy applications and hold back the potential for preventive gene therapy. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents which include bacteria, bacteriophage, virus-like particles (VLPs), erythrocyte ghosts, and exosomes. Exploiting the natural properties of these biological entities for specific gene delivery applications will expand the repertoire of gene therapy vectors available for clinical use. Here, we review the prospects for nonviral biological delivery vehicles as gene therapy agents with focus on their unique evolved biological properties and respective limitations and potential applications. The potential of these nonviral biological entities to act as clinical gene therapy delivery vehicles has already been shown in clinical trials using bacteria-mediated gene transfer and with sufficient development, these entities will complement the established delivery techniques for gene therapy applications.

A phase1 study of stereotactic gene delivery of AAV2-NGF for Alzheimer's disease.

PubMed

Rafii, Michael S; Baumann, Tiffany L; Bakay, Roy A E; Ostrove, Jeffrey M; Siffert, Joao; Fleisher, Adam S; Herzog, Christopher D; Barba, David; Pay, Mary; Salmon, David P; Chu, Yaping; Kordower, Jeffrey H; Bishop, Kathie; Keator, David; Potkin, Steven; Bartus, Raymond T

2014-09-01

Nerve growth factor (NGF) is an endogenous neurotrophic-factor protein with the potential to restore function and to protect degenerating cholinergic neurons in Alzheimer's disease (AD), but safe and effective delivery has proved unsuccessful. Gene transfer, combined with stereotactic surgery, offers a potential means to solve the long-standing delivery obstacles. An open-label clinical trial evaluated the safety and tolerability, and initial efficacy of three ascending doses of the genetically engineered gene-therapy vector adeno-associated virus serotype 2 delivering NGF (AAV2-NGF [CERE-110]). Ten subjects with AD received bilateral AAV2-NGF stereotactically into the nucleus basalis of Meynert. AAV2-NGF was safe and well-tolerated for 2 years. Positron emission tomographic imaging and neuropsychological testing showed no evidence of accelerated decline. Brain autopsy tissue confirmed long-term, targeted, gene-mediated NGF expression and bioactivity. This trial provides important evidence that bilateral stereotactic administration of AAV2-NGF to the nucleus basalis of Meynert is feasible, well-tolerated, and able to produce long-term, biologically active NGF expression, supporting the initiation of an ongoing multicenter, double-blind, sham-surgery-controlled trial. Copyright © 2014 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

In Vivo Tumor Gene Delivery Using Novel Peptideticles: pH-Responsive and Ligand Targeted Core-Shell Nanoassembly.

PubMed

Alipour, Mohsen; Majidi, Asia; Molaabasi, Fatemeh; Sheikhnejad, Reza; Hosseinkhani, Saman

2018-04-30

Modulating cancer causing genes with nucleic acid based-molecules as cutting-edge approaches need efficient delivery systems to succeed in clinic. Herein, we report design and fabrication of a novel tissue penetrating Peptideticle with charge-structure switching in tumor microenvironment for an effective gene delivery. The comparative in vitro studies indicate that peptideticles identify and bind to tumor endothelial cells and efficiently penetrate into multicellular tumor spheroid. In addition, negatively charged peptideticle at pH 7.4, prevent unwanted interaction while it's sharp charge-structure switching at pH 6.2-6.9 (e.g.in tumor tissue) facilitates malignant cells penetration. More importantly, upon systemic administration into tumor bearing mice, peptideticles effectively localized in tumor tissue and delivered luciferase gene with a 200-fold higher efficiency compared to their non-pH-responsive counterparts. In conclusion, this study presents a robust nanoassembly of safe materials for high efficient tumor gene delivery. This article is protected by copyright. All rights reserved. © 2018 UICC.

Gene delivery for cancer therapy.

PubMed

Zhang, Teng

2014-01-01

Gene therapy has potential in the treatment of human cancers. However, its clinical implication has only achieved little success due to the lack of an efficient gene delivery system. A major hurdle in the current available approaches is in the ability to transduce target tissues at very high efficiencies that ultimately lead to therapeutic levels of transgene expression. This review outlines the characteristics and utilities of several available gene delivery systems, including their advantages and drawbacks in the context of cancer treatment. A perspective of existing challenges and future directions is also included.

Cardiac Gene Therapy: Optimization of Gene Delivery Techniques In Vivo

PubMed Central

Katz, Michael G.; Swain, JaBaris D.; White, Jennifer D.; Low, David; Stedman, Hansell

2010-01-01

Abstract Vector-mediated cardiac gene therapy holds tremendous promise as a translatable platform technology for treating many cardiovascular diseases. The ideal technique is one that is efficient and practical, allowing for global cardiac gene expression, while minimizing collateral expression in other organs. Here we survey the available in vivo vector-mediated cardiac gene delivery methods—including transcutaneous, intravascular, intramuscular, and cardiopulmonary bypass techniques—with consideration of the relative merits and deficiencies of each. Review of available techniques suggests that an optimal method for vector-mediated gene delivery to the large animal myocardium would ideally employ retrograde and/or anterograde transcoronary gene delivery,extended vector residence time in the coronary circulation, an increased myocardial transcapillary gradient using physical methods, increased endothelial permeability with pharmacological agents, minimal collateral gene expression by isolation of the cardiac circulation from the systemic, and have low immunogenicity. PMID:19947886

Composite nanoparticles for gene delivery.

PubMed

Wang, Yuhua; Huang, Leaf

2014-01-01

Nanoparticle-mediated gene and siRNA delivery has been an appealing area to gene therapists when they attempt to treat the diseases by manipulating the genetic information in the target cells. However, the advances in materials science could not keep up with the demand for multifunctional nanomaterials to achieve desired delivery efficiency. Researchers have thus taken an alternative approach to incorporate various materials into single composite nanoparticle using different fabrication methods. This approach allows nanoparticles to possess defined nanostructures as well as multiple functionalities to overcome the critical extracellular and intracellular barriers to successful gene delivery. This chapter will highlight the advances of fabrication methods that have the most potential to translate nanoparticles from bench to bedside. Furthermore, a major class of composite nanoparticle-lipid-based composite nanoparticles will be classified based on the components and reviewed in details.

Association with Amino Acids Does Not Enhance Efficacy of Polymerized Liposomes As a System for Lung Gene Delivery

PubMed Central

Bandeira, Elga; Lopes-Pacheco, Miquéias; Chiaramoni, Nadia; Ferreira, Débora; Fernandez-Ruocco, Maria J.; Prieto, Maria J.; Maron-Gutierrez, Tatiana; Perrotta, Ramiro M.; de Castro-Faria-Neto, Hugo C.; Rocco, Patricia R. M.; Alonso, Silvia del Valle; Morales, Marcelo M.

2016-01-01

Development of improved drug and gene delivery systems directly into the lungs is highly desirable given the important burden of respiratory diseases. We aimed to evaluate the safety and efficacy of liposomes composed of photopolymerized lipids [1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine] associated with amino acids as vectors for gene delivery into the lungs of healthy animals. Lipopolymer vesicles, in particular, are more stable than other types of liposomes. In this study, lipopolymers were associated with l-arginine, l-tryptophan, or l-cysteine. We hypothesized that the addition of these amino acids would enhance the efficacy of gene delivery to the lungs by the lipopolymers. l-Arginine showed the highest association efficiency due to its positive charge and better surface interactions. None of the formulations caused inflammation or altered lung mechanics, suggesting that these lipopolymers can be safely administered as aerosols. All formulations were able to induce eGFP mRNA expression in lung tissue, but the addition of amino acids reduced delivery efficacy when compared with the simple lipopolymer particle. These results indicate that this system could be further explored for gene or drug delivery targeting lung diseases. PMID:27199766

Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice.

PubMed

Quirin, Kayla A; Kwon, Jason J; Alioufi, Arafat; Factora, Tricia; Temm, Constance J; Jacobsen, Max; Sandusky, George E; Shontz, Kim; Chicoine, Louis G; Clark, K Reed; Mendell, Joshua T; Korc, Murray; Kota, Janaiah

2018-03-16

Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 10 12 viral genomes (vg). Intraductal delivery of 1 × 10 11 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 10 11 vg. In a Kras G12D -driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

Factors associated with safe delivery service utilization among women in Sheka zone, southwest Ethiopia.

PubMed

Asres, Abyot; Davey, Gail

2015-04-01

Attempts to predict pregnancy and childbirth complications before they occur have not been successful. Provision of safe delivery service for all births is considered to be a critical intervention for ensuring safe motherhood. Hence the aim of the study was to assess factors associated with safe delivery service utilization among women in Sheka Zone South West Ethiopia. A community based comparative cross sectional survey was conducted among 554 women in Sheka Zone from February to March 2008. Data were collected through structured pre-tested questionnaire and entered into Epinfo version 3.3. Analyses were done with SPSS version 13 computer software with which bivariate and multiple logistic regressions were carried out. Mothers who completed at least secondary school were more likely to give birth at health facility than those uneducated (AOR = 3.26, 95 % CI 1.51-7.06). Women with birth order above four were less likely to give birth in a health facility than those with first order births (AOR = 0.21, 95 %CI 0.10-0.43). Women who had encountered problems in their immediate birth and received prenatal care were more likely to give birth at health facilities AOR = 33.78 95 % CI 16.44-69.39) and (AOR = 2.55, 95 % CI 1.05-6.21) respectively. Factors associated with safe delivery service utilization are related to the women's socioeconomic status and obstetric experiences. Consequently promotion of maternal education, prenatal care utilization, information education and communication on obstetric risks and general health service expansion are needed to ensure safe delivery service.

Gene delivery in tissue engineering and regenerative medicine.

PubMed

Fang, Y L; Chen, X G; W T, Godbey

2015-11-01

As a promising strategy to aid or replace tissue/organ transplantation, gene delivery has been used for regenerative medicine applications to create or restore normal function at the cell and tissue levels. Gene delivery has been successfully performed ex vivo and in vivo in these applications. Excellent proliferation capabilities and differentiation potentials render certain cells as excellent candidates for ex vivo gene delivery for regenerative medicine applications, which is why multipotent and pluripotent cells have been intensely studied in this vein. In this review, gene delivery is discussed in detail, along with its applications to tissue engineering and regenerative medicine. A definition of a stem cell is compared to a definition of a stem property, and both provide the foundation for an in-depth look at gene delivery investigations from a germ lineage angle. © 2014 Wiley Periodicals, Inc.

Predictors of safe delivery service utilization in arsi zone, South-East ethiopia.

PubMed

Abera, Mulumebet; Gebremariam, Abebe; Belachew, Tefera

2011-08-01

Evidence show that lack of access to and use of, essential obstetric care services to be a crucial factor that contributes to the high maternal morbidity and mortality. Skilled attendance during labor, delivery and early post-partum period could reduce deaths due to obstructed labor, hemorrhage, sepsis and eclampsia. There is limited information on the mothers' use of skilled delivery services in the study area. This study assessed the predictors of safe delivery service utilization in Arsi Zone, Southeast Ethiopia. A cross- sectional community based study using quantitative and qualitative methods was conducted from February 15(th) to March 15(th) 2006. A total of 1089 women who had at least one birth one year prior to the study were involved in the study from nine rural and four urban kebeles in three Woredas (Districts) selected using a systematic sampling method from all households in the study area. A pre-tested structured interviewer administered questionnaire was used to collect data. Information on the utilization of safe delivery service and socio-demographic, individual and institutional factors and past obstetric history were collected. Focus Group Discussion guide was used for qualitative data collection. The data were edited, cleaned, and entered into a computer and analyzed using SPSS for windows version 12.0. One thousand seventy four women who had at least one birth were interviewed making a response rate 98.6%. Two hundred seventy one (75.0%) of urban and 373(52.0%) rural women received antenatal care from skilled health professional at least once during their last pregnancy. Thirty-one (4.3%) of rural and 145 (40.4%) of urban women delivered in health institution. In multivariate analysis showed that residential area OR= 8.5, 95%CI; (5.1,13.9), parity OR=0.18, 95%CI; (0.08, 0.42), and ANC service use OR= 4.5, 95%CI; (2.2,8.9), and maternal education OR=4.6, 95%CI; (1.7,12.8), were most significant predictors of safe delivery service use by mothers

In Vivo Fate of Carbon Nanotubes with Different Physicochemical Properties for Gene Delivery Applications.

PubMed

Cifuentes-Rius, Anna; Boase, Nathan R B; Font, Ines; Coronas, Nuria; Ramos-Perez, Victor; Thurecht, Kristofer J; Borrós, Salvador

2017-04-05

Gene therapy has arisen as a pioneering technique to treat diseases by direct employment of nucleic acids as medicine. The major historical problem is to develop efficient and safe systems for the delivery of therapeutic genes into the target cells. Carbon nanotubes (CNTs) have demonstrated considerable promise as delivery vectors due to their (i) high aspect ratio and (ii) capacity to translocate through plasma membranes, known as the nanoneedle effect. To leverage these advantages, close attention needs to be paid to the physicochemical characteristics of the CNTs used. CNTs with different diameters (thinner and thicker) were treated by chemical oxidation to produce shorter fragments. Rigid (thick) and flexible (thin) CNTs, and their shortened versions, were coated with polyallylamine (ppAA) by plasma-enhanced chemical vapor deposition. The ppAA coating leads to a positively charged CNT surface that is able to electrostatically bind the green fluorescent protein plasmid reporter. This study shows how rigidity and length can affect their (i) behavior in biological media, (ii) ability to transfect in vitro, and (iii) biodistribution in vivo. This study also generates a set of basic design rules for the development of more efficient CNT-based gene-delivery vectors.

Gene therapy delivery systems for enhancing viral and nonviral vectors for cardiac diseases: current concepts and future applications.

PubMed

Katz, Michael G; Fargnoli, Anthony S; Williams, Richard D; Bridges, Charles R

2013-11-01

Gene therapy is one of the most promising fields for developing new treatments for the advanced stages of ischemic and monogenetic, particularly autosomal or X-linked recessive, cardiomyopathies. The remarkable ongoing efforts in advancing various targets have largely been inspired by the results that have been achieved in several notable gene therapy trials, such as the hemophilia B and Leber's congenital amaurosis. Rate-limiting problems preventing successful clinical application in the cardiac disease area, however, are primarily attributable to inefficient gene transfer, host responses, and the lack of sustainable therapeutic transgene expression. It is arguable that these problems are directly correlated with the choice of vector, dose level, and associated cardiac delivery approach as a whole treatment system. Essentially, a delicate balance exists in maximizing gene transfer required for efficacy while remaining within safety limits. Therefore, the development of safe, effective, and clinically applicable gene delivery techniques for selected nonviral and viral vectors will certainly be invaluable in obtaining future regulatory approvals. The choice of gene transfer vector, dose level, and the delivery system are likely to be critical determinants of therapeutic efficacy. It is here that the interactions between vector uptake and trafficking, delivery route means, and the host's physical limits must be considered synergistically for a successful treatment course.

Gene Therapy Delivery Systems for Enhancing Viral and Nonviral Vectors for Cardiac Diseases: Current Concepts and Future Applications

PubMed Central

Katz, Michael G.; Fargnoli, Anthony S.; Williams, Richard D.

2013-01-01

Abstract Gene therapy is one of the most promising fields for developing new treatments for the advanced stages of ischemic and monogenetic, particularly autosomal or X-linked recessive, cardiomyopathies. The remarkable ongoing efforts in advancing various targets have largely been inspired by the results that have been achieved in several notable gene therapy trials, such as the hemophilia B and Leber's congenital amaurosis. Rate-limiting problems preventing successful clinical application in the cardiac disease area, however, are primarily attributable to inefficient gene transfer, host responses, and the lack of sustainable therapeutic transgene expression. It is arguable that these problems are directly correlated with the choice of vector, dose level, and associated cardiac delivery approach as a whole treatment system. Essentially, a delicate balance exists in maximizing gene transfer required for efficacy while remaining within safety limits. Therefore, the development of safe, effective, and clinically applicable gene delivery techniques for selected nonviral and viral vectors will certainly be invaluable in obtaining future regulatory approvals. The choice of gene transfer vector, dose level, and the delivery system are likely to be critical determinants of therapeutic efficacy. It is here that the interactions between vector uptake and trafficking, delivery route means, and the host's physical limits must be considered synergistically for a successful treatment course. PMID:24164239

Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice.

PubMed

Iyer, Meera; Berenji, Manijeh; Templeton, Nancy S; Gambhir, Sanjiv S

2002-10-01

Gene therapy involves the safe and effective delivery of one or more genes of interest to target cells in vivo. The advantages of using nonviral delivery systems include ease of preparation, low toxicity, and weak immunogenicity. Nonviral delivery methods, when combined with a noninvasive, clinically applicable imaging assay, will greatly aid in the optimization of gene therapy approaches for cancer. We demonstrate cationic lipid-mediated noninvasive monitoring of reporter gene expression of firefly (Photinus pyralis) luciferase (fl) and a mutant herpes simplex virus type I thymidine kinase (HSV1-sr39tk, tk) in living mice using a cooled charge coupled device (CCD) camera and positron emission tomography (PET), respectively. We observe a high level of fl and tk reporter gene expression predominantly in the lungs after a single injection of the extruded DOTAP:cholesterol DNA liposome complexes by way of the tail vein, seen to be time- and dose-dependent. We observe a good correlation between the in vivo bioluminescent signal and the ex vivo firefly luciferase enzyme (FL) activity in different organs. We further demonstrate the feasibility of noninvasively imaging both optical and PET reporter gene expression in the same animal using the CCD camera and microPET, respectively.

Safe delivery care practices in western Nepal: Does women's autonomy influence the utilization of skilled care at birth?

PubMed

Bhandari, Tulsi Ram; Kutty, V Raman; Sarma, P Sankara; Dangal, Ganesh

2017-01-01

Despite various efforts to increase the utilization of skilled birth attendants (SBA), nearly two-thirds of deliveries take place at home without the assistance of SBAs in Nepal. We hypothesized that the ability of women to take decisions about their own lives-women's autonomy-plays an important part in birth choices. To know this, we conducted a community-based cross-sectional study for assessing women's autonomy and utilization of safe delivery care service in Kapilvastu district of Nepal from June to October 2014. We used multivariate modeling to associate socioeconomic factors and women's autonomy with the utilization of safe delivery care services. Just over one-third of women sought institutional delivery care during the birth of their last child. Out of the total deliveries at health facilities, nearly 58% women visited health facility for self-reported emergency obstructive care. Only 6.2% home deliveries were handled by health workers and 14.7% women used the safe delivery kit for home delivery care. Higher levels of women's education had a strong positive association (odds ratio = 24.11, CI = 9.43-61.64) with institutional delivery care. Stratified analysis showed that when the husband is educated, women's education seems to work partly through their autonomy in decision making. Educational status of women emerged as one of the key predictors of the utilization of delivery care services in Kapilvastu district. Economic status of household and husband's education are other dominant predictors of the utilization of safe delivery care services. Improving the economic and educational status may be the way out for improving the proportion of institutional deliveries. Women's autonomy may be an important mediating factor in this pathway.

Gene delivery with viral vectors for cerebrovascular diseases

PubMed Central

Gan, Yu; Jing, Zheng; Stetler, R. Anne; Cao, Guodong

2017-01-01

Recent achievements in the understanding of molecular events involved in the pathogenesis of central nervous system (CNS) injury have made gene transfer a promising approach for various neurological disorders, including cerebrovascular diseases. However, special obstacles, including the post-mitotic nature of neurons and the blood-brain barrier (BBB), constitute key challenges for gene delivery to the CNS. Despite the various limitations in current gene delivery systems, a spectrum of viral vectors has been successfully used to deliver genes to the CNS. Furthermore, recent advancements in vector engineering have improved the safety and delivery of viral vectors. Numerous viral vector-based clinical trials for neurological disorders have been initiated. This review will summarize the current implementation of viral gene delivery in the context of cerebrovascular diseases including ischemic stroke, hemorrhagic stroke and subarachnoid hemorrhage (SAH). In particular, we will discuss the potentially feasible ways in which viral vectors can be manipulated and exploited for use in neural delivery and therapy. PMID:23276981

Cell-penetrating DNA-binding protein as a safe and efficient naked DNA delivery carrier in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Eun-Sung; Yang, Seung-Woo; Hong, Dong-Ki

Non-viral gene delivery is a safe and suitable alternative to viral vector-mediated delivery to overcome the immunogenicity and tumorigenesis associated with viral vectors. Using the novel, human-origin Hph-1 protein transduction domain that can facilitate the transduction of protein into cells, we developed a new strategy to deliver naked DNA in vitro and in vivo. The new DNA delivery system contains Hph-1-GAL4 DNA-binding domain (DBD) fusion protein and enhanced green fluorescent protein (EGFP) reporter plasmid that includes the five repeats of GAL4 upstream activating sequence (UAS). Hph-1-GAL4-DBD protein formed complex with plasmid DNA through the specific interaction between GAL4-DBD and UAS,more » and delivered into the cells via the Hph-1-PTD. The pEGFP DNA was successfully delivered by the Hph-1-GAL4 system, and the EGFP was effectively expressed in mammalian cells such as HeLa and Jurkat, as well as in Bright Yellow-2 (BY-2) plant cells. When 10 {mu}g of pEGFP DNA was intranasally administered to mice using Hph-1-GAL4 protein, a high level of EGFP expression was detected throughout the lung tissue for 7 days. These results suggest that an Hph-1-PTD-mediated DNA delivery strategy may be an useful non-viral DNA delivery system for gene therapy and DNA vaccines.« less

Intracellular trafficking of hybrid gene delivery vectors.

PubMed

Keswani, Rahul K; Lazebnik, Mihael; Pack, Daniel W

2015-06-10

Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles. Copyright © 2015 Elsevier B.V. All rights reserved.

Non-viral gene delivery strategies for cancer therapy, tissue engineering and regenerative medicine

NASA Astrophysics Data System (ADS)

Bhise, Nupura S.

Gene therapy involves the delivery of deoxyribonucleic acid (DNA) into cells to override or replace a malfunctioning gene for treating debilitating genetic diseases, including cancer and neurodegenerative diseases. In addition to its use as a therapeutic, it can also serve as a technology to enable regenerative medicine strategies. The central challenge of the gene therapy research arena is developing a safe and effective delivery agent. Since viral vectors have critical immunogenic and tumorogenic safety issues that limit their clinical use, recent efforts have focused on developing non-viral biomaterial based delivery vectors. Cationic polymers are an attractive class of gene delivery vectors due to their structural versatility, ease of synthesis, biodegradability, ability to self-complex into nanoparticles with negatively charged DNA, capacity to carry large cargo, cellular uptake and endosomal escape capacity. In this thesis, we hypothesized that developing a biomaterial library of poly(betaamino esters) (PBAE), a newer class of cationic polymers consisting of biodegradable ester groups, would allow investigating vector design parameters and formulating effective non-viral gene delivery strategies for cancer drug delivery, tissue engineering and stem cell engineering. Consequently, a high-throughput transfection assay was developed to screen the PBAE-based nanoparticles in hard to transfect fibroblast cell lines. To gain mechanistic insights into the nanoparticle formulation process, biophysical properties of the vectors were characterized in terms of molecular weight (MW), nanoparticle size, zeta potential and plasmid per particle count. We report a novel assay developed for quantifying the plasmid per nanoparticle count and studying its implications for co-delivery of multiple genes. The MW of the polymers ranged from 10 kDa to 100 kDa, nanoparticle size was about 150 run, zeta potential was about 30 mV in sodium acetate buffer (25 mM, pH 5) and 30 to 100

Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide.

PubMed

Wang, Hong-Xia; Song, Ziyuan; Lao, Yeh-Hsing; Xu, Xin; Gong, Jing; Cheng, Du; Chakraborty, Syandan; Park, Ji Sun; Li, Mingqiang; Huang, Dantong; Yin, Lichen; Cheng, Jianjun; Leong, Kam W

2018-05-08

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications. Copyright © 2018 the Author(s). Published by PNAS.

Dual delivery systems based on polyamine analog BENSpm as prodrug and gene delivery vectors

NASA Astrophysics Data System (ADS)

Zhu, Yu

-SS-BEN) capable of intracellular release of BENSpm using thiolytically sensitive dithiobenzyl carbamate linker. Similar activity on SSAT enzyme induction by Lipo-SS-BEN compared with BENSpm free drug verified the success of this prodrug design. Biodegradability of Lipo-SS-BEN contributed to decreased toxicity compared with nondegradable control LipoBEN. However, decreased enhancement of TRAIL activity was observed for Lipo-SS-BEN when compared with BENSpm, indicating that the lipid-related toxicity diminished the synergism. In addition, compared with LipoBEN and DOTAP, decreased transfection efficiency of Lipo-SS-BEN demonstrated instability of Lipo-SS-BEN in extracellular environment. In order to design a dual delivery vector with reduced vector toxicity and improved linker stability, we employed dendritic polyglycerol (PG) as a safe carrier backbone, onto which BENSpm was conjugated through carbamate linkage (PG-BEN). Polymers with norspermine (PG-Nor) shell and amine-terminated PG (PG-NH2) were synthesized as controls. The BENSpm dual vector PG-BEN demonstrated superior gene delivery function, and showed decreased toxicity compared with the control polymers. However, compared with BENSpm, which depleted all natural polyamines, PG-BEN only down-regulated intracellular putrescine levels. In addition, no free BENSpm was detected in PG-BEN treated cells. These results suggested that in order to take full advantage of BENSpm anticancer activity, alternative linker chemistry needs to be further explored. We then incorporated bis(2-hydroxyethyl) disulfide as a self-immolative linker to synthesize polymer prodrugs of BENSpm (DSS-BEN). The proposed mechanism of BENSpm release from DSS-BEN contains two steps: disulfide bond is first cleaved in the reducing intracellular space, then the intermediate further undergoes slow intramolecular cyclization to release free BENSpm. Cell line-dependent BENSpm release after DSS-BEN treatment was observed using HPLC analysis, demonstrating the

Transferrin-bearing polypropylenimine dendrimer for targeted gene delivery to the brain.

PubMed

Somani, Sukrut; Blatchford, David R; Millington, Owain; Stevenson, M Lynn; Dufès, Christine

2014-08-28

The possibility of using genes as medicines to treat brain diseases is currently limited by the lack of safe and efficacious delivery systems able to cross the blood-brain barrier, thus resulting in a failure to reach the brain after intravenous administration. On the basis that iron can effectively reach the brain by using transferrin receptors for crossing the blood-brain barrier, we propose to investigate if a transferrin-bearing generation 3-polypropylenimine dendrimer would allow the transport of plasmid DNA to the brain after intravenous administration. In vitro, the conjugation of transferrin to the polypropylenimine dendrimer increased the DNA uptake by bEnd.3 murine brain endothelioma cells overexpressing transferrin receptors, by about 1.4-fold and 2.3-fold compared to that observed with the non-targeted dendriplex and naked DNA. This DNA uptake appeared to be optimal following 2h incubation with the treatment. In vivo, the intravenous injection of transferrin-bearing dendriplex more than doubled the gene expression in the brain compared to the unmodified dendriplex, while decreasing the non-specific gene expression in the lung. Gene expression was at least 3-fold higher in the brain than in any tested peripheral organs and was at its highest 24h following the injection of the treatments. These results suggest that transferrin-bearing polypropylenimine dendrimer is a highly promising gene delivery system to the brain. Copyright © 2014 Elsevier B.V. All rights reserved.

How controlled release technology can aid gene delivery.

PubMed

Jo, Jun-Ichiro; Tabata, Yasuhiko

2015-01-01

Many types of gene delivery systems have been developed to enhance the level of gene expression. Controlled release technology is a feasible gene delivery system which enables genes to extend the expression duration by maintaining and releasing them at the injection site in a controlled manner. This technology can reduce the adverse effects by the bolus dose administration and avoid the repeated administration. Biodegradable biomaterials are useful as materials for the controlled release-based gene delivery technology and various biodegradable biomaterials have been developed. Controlled release-based gene delivery plays a critical role in a conventional gene therapy and genetic engineering. In the gene therapy, the therapeutic gene is released from biodegradable biomaterial matrices around the tissue to be treated. On the other hand, the intracellular controlled release of gene from the sub-micro-sized matrices is required for genetic engineering. Genetic engineering is feasible for cell transplantation as well as research of stem cells biology and medicine. DNA hydrogel containing a sequence of therapeutic gene and the exosome including the individual specific nucleic acids may become candidates for controlled release carriers. Technologies to deliver genes to cell aggregates will play an important role in the promotion of regenerative research and therapy.

Electrostatic Surface Modifications to Improve Gene Delivery

PubMed Central

Shmueli, Ron B.; Anderson, Daniel G.

2010-01-01

Importance of the field Gene therapy has the potential to treat a wide variety of diseases including genetic diseases and cancer. Areas covered in this review This review introduces biomaterials used for gene delivery and then focuses on the use of electrostatic surface modifications to improve gene delivery materials. These modifications have been used to stabilize therapeutics in vivo, add cell-specific targeting ligands, and promote controlled release. Coatings of nanoparticles and microparticles as well as non-particulate surface coatings are covered in this review. Electrostatic principles are crucial for the development of multilayer delivery structures fabricated by the layer-by-layer method. What the reader will gain The reader will gain knowledge about the composition of biomaterials used for surface modifications and how these coatings and multilayers can be utilized to improve spatial control and efficiency of delivery. Examples are shown for the delivery of nucleic acids, including DNA and siRNA, to in vitro and in vivo systems. Take home message The versatile and powerful approach of electrostatic coatings and multilayers will lead to the development of enhanced gene therapies. PMID:20201712

Investigation of a thiolated polymer in gene delivery

NASA Astrophysics Data System (ADS)

Bacalocostantis, Irene

Thiol-containing bioreducible polymers show significant potential as delivery vectors in gene therapy, a rapidly growing field which seeks to treat genetic-based disorders by delivering functional synthetic genes to diseased cells. Studies have shown that thiolated polymers exhibit improved biodegradability and prolonged in vivo circulation times over non-thiolated polymers. However, the extent to which thiol concentrations impact the carrier's delivery potential has not been well explored. The aim of this dissertation is to investigate how relative concentrations of free thiols and disulfide crosslinks impact a polymeric carriers delivery performance with respect to DNA packaging, complex stability, cargo protection, gene release, internalization efficiency and cytotoxicity. To accomplish this goal, several fluorescent polymers containing varying concentrations of thiol groups were synthesized by conjugating thiol-pendant chains onto the primary amines of cationic poly(allylamine). In vitro delivery assays and characterization techniques were employed to assess the effect of thiols in gene delivery.

In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles

PubMed Central

Lopes, Cátia DF; Oliveira, Hugo; Estevão, Inês; Pires, Liliana Raquel; Pêgo, Ana Paula

2016-01-01

A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies. PMID:27354797

SYBR safeTM efficiently replaces ethidium bromide in Aspergillus fumigatus gene disruption.

PubMed

Canela, H M S; Takami, L A; Ferreira, M E S

2017-02-08

Invasive aspergillosis is a disease responsible for high mortality rates, caused mainly by Aspergillus fumigatus. The available drugs are limited and this disease continues to occur at an unacceptable frequency. Gene disruption is essential in the search for new drug targets. An efficient protocol for A. fumigatus gene disruption was described but it requires ethidium bromide, a genotoxic agent, for DNA staining. Therefore, the present study tested SYBR safe TM , a non-genotoxic DNA stain, in A. fumigatus gene disruption protocol. The chosen gene was cipC, which has already been disrupted successfully in our laboratory. A deletion cassette was constructed in Saccharomyces cerevisiae and used in A. fumigatus transformation. There was no statistical difference between the tested DNA stains. The success rate of S. cerevisiae transformation was 63.3% for ethidium bromide and 70% for SYBR safe TM . For A. fumigatus gene disruption, the success rate for ethidium bromide was 100 and 97% for SYBR safe TM . In conclusion, SYBR safe TM efficiently replaced ethidium bromide, making this dye a safe and efficient alternative for DNA staining in A. fumigatus gene disruption.

Elastin-like polypeptide matrices for enhancing adeno-associated virus-mediated gene delivery to human neural stem cells.

PubMed

Kim, J-S; Chu, H S; Park, K I; Won, J-I; Jang, J-H

2012-03-01

The successful development of efficient and safe gene delivery vectors continues to be a major obstacle to gene delivery in stem cells. In this study, we have developed an elastin-like polypeptide (ELP)-mediated adeno-associated virus (AAV) delivery system for transducing fibroblasts and human neural stem cells (hNSCs). AAVs have significant promise as therapeutic vectors because of their safety and potential for use in gene targeting in stem cell research. ELP has been recently employed as a biologically inspired 'smart' biomaterial that exhibits an inverse temperature phase transition, thereby demonstrating promise as a novel drug carrier. The ELP that was investigated in this study was composed of a repetitive penta-peptide with [Val-Pro-Gly-Val-Gly]. A novel AAV variant, AAV r3.45, which was previously engineered by directed evolution to enhance transduction in rat NSCs, was nonspecifically immobilized onto ELPs that were adsorbed beforehand on a tissue culture polystyrene surface (TCPS). The presence of different ELP quantities on the TCPS led to variations in surface morphology, roughness and wettability, which were ultimately key factors in the modulation of cellular transduction. Importantly, with substantially reduced viral quantities compared with bolus delivery, ELP-mediated AAV delivery significantly enhanced delivery efficiency in fibroblasts and hNSCs, which have great potential for use in tissue engineering applications and neurodegenerative disorder treatments, respectively. The enhancement of cellular transduction in stem cells, as well as the feasibility of ELPs for utilization in three-dimensional scaffolds, will contribute to the advancement of gene therapy for stem cell research and tissue regenerative medicine.

Safe delivery care practices in western Nepal: Does women’s autonomy influence the utilization of skilled care at birth?

PubMed Central

Kutty, V. Raman; Sarma, P. Sankara; Dangal, Ganesh

2017-01-01

Despite various efforts to increase the utilization of skilled birth attendants (SBA), nearly two-thirds of deliveries take place at home without the assistance of SBAs in Nepal. We hypothesized that the ability of women to take decisions about their own lives—women’s autonomy—plays an important part in birth choices. To know this, we conducted a community-based cross-sectional study for assessing women’s autonomy and utilization of safe delivery care service in Kapilvastu district of Nepal from June to October 2014. We used multivariate modeling to associate socioeconomic factors and women’s autonomy with the utilization of safe delivery care services. Just over one-third of women sought institutional delivery care during the birth of their last child. Out of the total deliveries at health facilities, nearly 58% women visited health facility for self-reported emergency obstructive care. Only 6.2% home deliveries were handled by health workers and 14.7% women used the safe delivery kit for home delivery care. Higher levels of women’s education had a strong positive association (odds ratio = 24.11, CI = 9.43–61.64) with institutional delivery care. Stratified analysis showed that when the husband is educated, women’s education seems to work partly through their autonomy in decision making. Educational status of women emerged as one of the key predictors of the utilization of delivery care services in Kapilvastu district. Economic status of household and husband’s education are other dominant predictors of the utilization of safe delivery care services. Improving the economic and educational status may be the way out for improving the proportion of institutional deliveries. Women’s autonomy may be an important mediating factor in this pathway. PMID:28771579

Gene doping: gene delivery for olympic victory

PubMed Central

Gould, David

2013-01-01

With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. PMID:23082866

Biodegradable nanoparticles for gene therapy technology

NASA Astrophysics Data System (ADS)

Hosseinkhani, Hossein; He, Wen-Jie; Chiang, Chiao-Hsi; Hong, Po-Da; Yu, Dah-Shyong; Domb, Abraham J.; Ou, Keng-Liang

2013-07-01

Rapid propagations in materials technology together with biology have initiated great hopes in the possibility of treating many diseases by gene therapy technology. Viral and non-viral gene carriers are currently applied for gene delivery. Non-viral technology is safe and effective for the delivery of genetic materials to cells and tissues. Non-viral systems are based on plasmid expression containing a gene encoding a therapeutic protein and synthetic biodegradable nanoparticles as a safe carrier of gene. Biodegradable nanoparticles have shown great interest in drug and gene delivery systems as they are easy to be synthesized and have no side effect in cells and tissues. This review provides a critical view of applications of biodegradable nanoparticles on gene therapy technology to enhance the localization of in vitro and in vivo and improve the function of administered genes.

Gene doping: gene delivery for olympic victory.

PubMed

Gould, David

2013-08-01

With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called 'gene doping'. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. © 2012 The Author. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.

Fe₃O₄ Nanoparticles in Targeted Drug/Gene Delivery Systems.

PubMed

Shen, Lazhen; Li, Bei; Qiao, Yongsheng

2018-02-23

Fe₃O₄ nanoparticles (NPs), the most traditional magnetic nanoparticles, have received a great deal of attention in the biomedical field, especially for targeted drug/gene delivery systems, due to their outstanding magnetism, biocompatibility, lower toxicity, biodegradability, and other features. Naked Fe₃O₄ NPs are easy to aggregate and oxidize, and thus are often made with various coatings to realize superior properties for targeted drug/gene delivery. In this review, we first list the three commonly utilized synthesis methods of Fe₃O₄ NPs, and their advantages and disadvantages. In the second part, we describe coating materials that exhibit noticeable features that allow functionalization of Fe₃O₄ NPs and summarize their methods of drug targeting/gene delivery. Then our efforts will be devoted to the research status and progress of several different functionalized Fe₃O₄ NP delivery systems loaded with chemotherapeutic agents, and we present targeted gene transitive carriers in detail. In the following section, we illuminate the most effective treatment systems of the combined drug and gene therapy. Finally, we propose opportunities and challenges of the clinical transformation of Fe₃O₄ NPs targeting drug/gene delivery systems.

Therapeutic gene editing: delivery and regulatory perspectives.

PubMed

Shim, Gayong; Kim, Dongyoon; Park, Gyu Thae; Jin, Hyerim; Suh, Soo-Kyung; Oh, Yu-Kyoung

2017-06-01

Gene-editing technology is an emerging therapeutic modality for manipulating the eukaryotic genome by using target-sequence-specific engineered nucleases. Because of the exceptional advantages that gene-editing technology offers in facilitating the accurate correction of sequences in a genome, gene editing-based therapy is being aggressively developed as a next-generation therapeutic approach to treat a wide range of diseases. However, strategies for precise engineering and delivery of gene-editing nucleases, including zinc finger nucleases, transcription activator-like effector nuclease, and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated nuclease Cas9), present major obstacles to the development of gene-editing therapies, as with other gene-targeting therapeutics. Currently, viral and non-viral vectors are being studied for the delivery of these nucleases into cells in the form of DNA, mRNA, or proteins. Clinical trials are already ongoing, and in vivo studies are actively investigating the applicability of CRISPR/Cas9 techniques. However, the concept of correcting the genome poses major concerns from a regulatory perspective, especially in terms of safety. This review addresses current research trends and delivery strategies for gene editing-based therapeutics in non-clinical and clinical settings and considers the associated regulatory issues.

Therapeutic gene editing: delivery and regulatory perspectives

PubMed Central

Shim, Gayong; Kim, Dongyoon; Park, Gyu Thae; Jin, Hyerim; Suh, Soo-Kyung; Oh, Yu-Kyoung

2017-01-01

Gene-editing technology is an emerging therapeutic modality for manipulating the eukaryotic genome by using target-sequence-specific engineered nucleases. Because of the exceptional advantages that gene-editing technology offers in facilitating the accurate correction of sequences in a genome, gene editing-based therapy is being aggressively developed as a next-generation therapeutic approach to treat a wide range of diseases. However, strategies for precise engineering and delivery of gene-editing nucleases, including zinc finger nucleases, transcription activator-like effector nuclease, and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated nuclease Cas9), present major obstacles to the development of gene-editing therapies, as with other gene-targeting therapeutics. Currently, viral and non-viral vectors are being studied for the delivery of these nucleases into cells in the form of DNA, mRNA, or proteins. Clinical trials are already ongoing, and in vivo studies are actively investigating the applicability of CRISPR/Cas9 techniques. However, the concept of correcting the genome poses major concerns from a regulatory perspective, especially in terms of safety. This review addresses current research trends and delivery strategies for gene editing-based therapeutics in non-clinical and clinical settings and considers the associated regulatory issues. PMID:28392568

Targeted Delivery of CRISPR/Cas9-Mediated Cancer Gene Therapy via Liposome-Templated Hydrogel Nanoparticles.

PubMed

Chen, Zeming; Liu, Fuyao; Chen, Yanke; Liu, Jun; Wang, Xiaoying; Chen, Ann T; Deng, Gang; Zhang, Hongyi; Liu, Jie; Hong, Zhangyong; Zhou, Jiangbing

2017-12-08

Due to its simplicity, versatility, and high efficiency, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has emerged as one of the most promising approaches for treatment of a variety of genetic diseases, including human cancers. However, further translation of CRISPR/Cas9 for cancer gene therapy requires development of safe approaches for efficient, highly specific delivery of both Cas9 and single guide RNA to tumors. Here, novel core-shell nanostructure, liposome-templated hydrogel nanoparticles (LHNPs) that are optimized for efficient codelivery of Cas9 protein and nucleic acids is reported. It is demonstrated that, when coupled with the minicircle DNA technology, LHNPs deliver CRISPR/Cas9 with efficiency greater than commercial agent Lipofectamine 2000 in cell culture and can be engineered for targeted inhibition of genes in tumors, including tumors the brain. When CRISPR/Cas9 targeting a model therapeutic gene, polo-like kinase 1 (PLK1), is delivered, LHNPs effectively inhibit tumor growth and improve tumor-bearing mouse survival. The results suggest LHNPs as versatile CRISPR/Cas9-delivery tool that can be adapted for experimentally studying the biology of cancer as well as for clinically translating cancer gene therapy.

Exploring the role of peptides in polymer-based gene delivery.

PubMed

Sun, Yanping; Yang, Zhen; Wang, Chunxi; Yang, Tianzhi; Cai, Cuifang; Zhao, Xiaoyun; Yang, Li; Ding, Pingtian

2017-09-15

Polymers are widely studied as non-viral gene vectors because of their strong DNA binding ability, capacity to carry large payload, flexibility of chemical modifications, low immunogenicity, and facile processes for manufacturing. However, high cytotoxicity and low transfection efficiency substantially restrict their application in clinical trials. Incorporating functional peptides is a promising approach to address these issues. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we systematically summarize the role of peptides in polymer-based gene delivery, and elaborate how to rationally design polymer-peptide based gene delivery vectors. Polymers are widely studied as non-viral gene vectors, but suffer from high cytotoxicity and low transfection efficiency. Incorporating short, bioactive peptides into polymer-based gene delivery systems can address this issue. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we highlight the peptides' roles in polymer-based gene delivery, and elaborate how to utilize various functional peptides to enhance the transfection efficiency of polymers. The optimized peptide-polymer vectors should be able to alter their structures and functions according to biological microenvironments and utilize inherent intracellular pathways of cells, and consequently overcome the barriers during gene delivery to enhance transfection efficiency. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A Rapid Pathway Toward a Superb Gene Delivery System: Programming Structural and Functional Diversity into a Supramolecular Nanoparticle Library

PubMed Central

Wang, Hao; Liu, Kan; Chen, Kuan-Ju; Lu, Yujie; Wang, Shutao; Lin, Wei-Yu; Guo, Feng; Kamei, Ken-ichiro; Chen, Yi-Chun; Ohashi, Minori; Wang, Mingwei; Garcia, Mitch André; Zhao, Xing-Zhong; Shen, Clifton K.-F.; Tseng, Hsian-Rong

2010-01-01

Nanoparticles are regarded as promising transfection reagents for effective and safe delivery of nucleic acids into specific type of cells or tissues providing an alternative manipulation/therapy strategy to viral gene delivery. However, the current process of searching novel delivery materials is limited due to conventional low-throughput and time-consuming multistep synthetic approaches. Additionally, conventional approaches are frequently accompanied with unpredictability and continual optimization refinements, impeding flexible generation of material diversity creating a major obstacle to achieving high transfection performance. Here we have demonstrated a rapid developmental pathway toward highly efficient gene delivery systems by leveraging the powers of a supramolecular synthetic approach and a custom-designed digital microreactor. Using the digital microreactor, broad structural/functional diversity can be programmed into a library of DNA-encapsulated supramolecular nanoparticles (DNA⊂SNPs) by systematically altering the mixing ratios of molecular building blocks and a DNA plasmid. In vitro transfection studies with DNA⊂SNPs library identified the DNA⊂SNPs with the highest gene transfection efficiency, which can be attributed to cooperative effects of structures and surface chemistry of DNA⊂SNPs. We envision such a rapid developmental pathway can be adopted for generating nanoparticle-based vectors for delivery of a variety of loads. PMID:20925389

Safe Active Scanning for Energy Delivery Systems Final Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Helms, J.; Salazar, B.; Scheibel, P.

The Department of Energy’s Cybersecurity for Energy Delivery Systems Program has funded Safe(r) Active Scanning for Energy Delivery Systems, led by Lawrence Livermore National Laboratory, to investigate and analyze the impacts of active scanning in the operational environment of energy delivery systems. In collaboration with Pacific Northwest National Laboratory and Idaho National Laboratory, active scans across three testbeds including 38 devices were performed. This report gives a summary of the initial literature survey performed on the SASEDS project as well as industry partner interview summaries and main findings from Phase 1 of the project. Additionally, the report goes into themore » details of scanning techniques, methodologies for testing, testbed descriptions, and scanning results, with appendices to elaborate on the specific scans that were performed. As a result of testing, a single device out of 38 exhibited problems when actively scanned, and a reboot was required to fix it. This single failure indicates that active scanning is not likely to have a detrimental effect on the safety and resilience of energy delivery systems. We provide a path forward for future research that could enable wide adoption of active scanning and lead utilities to incorporate active scanning as part of their default network security plans to discover and rectify rogue devices, adversaries, and services that may be on the network. This increased network visibility will allow operational technology cybersecurity practitioners to improve their situational awareness of networks and their vulnerabilities.« less

The transition from linear to highly branched poly(β-amino ester)s: Branching matters for gene delivery

PubMed Central

Zhou, Dezhong; Cutlar, Lara; Gao, Yongsheng; Wang, Wei; O’Keeffe-Ahern, Jonathan; McMahon, Sean; Duarte, Blanca; Larcher, Fernando; Rodriguez, Brian J.; Greiser, Udo; Wang, Wenxin

2016-01-01

Nonviral gene therapy holds great promise but has not delivered treatments for clinical application to date. Lack of safe and efficient gene delivery vectors is the major hurdle. Among nonviral gene delivery vectors, poly(β-amino ester)s are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. However, to date, all research has been focused on vectors with a linear structure. A well-accepted view is that dendritic or branched polymers have greater potential as gene delivery vectors because of their three-dimensional structure and multiple terminal groups. Nevertheless, to date, the synthesis of dendritic or branched polymers has been proven to be a well-known challenge. We report the design and synthesis of highly branched poly(β-amino ester)s (HPAEs) via a one-pot “A2 + B3 + C2”–type Michael addition approach and evaluate their potential as gene delivery vectors. We find that the branched structure can significantly enhance the transfection efficiency of poly(β-amino ester)s: Up to an 8521-fold enhancement in transfection efficiency was observed across 12 cell types ranging from cell lines, primary cells, to stem cells, over their corresponding linear poly(β-amino ester)s (LPAEs) and the commercial transfection reagents polyethyleneimine, SuperFect, and Lipofectamine 2000. Moreover, we further demonstrate that HPAEs can correct genetic defects in vivo using a recessive dystrophic epidermolysis bullosa graft mouse model. Our findings prove that the A2 + B3 + C2 approach is highly generalizable and flexible for the design and synthesis of HPAEs, which cannot be achieved by the conventional polymerization approach; HPAEs are more efficient vectors in gene transfection than the corresponding LPAEs. This provides valuable insight into the development and applications of nonviral gene delivery and demonstrates great prospect for their

A Novel Nonviral Gene Delivery System: Multifunctional Envelope-Type Nano Device

NASA Astrophysics Data System (ADS)

Hatakeyama, Hiroto; Akita, Hidetaka; Kogure, Kentaro; Harashima, Hideyoshi

In this review we introduce a new concept for developing a nonviral gene delivery system which we call "Programmed Packaging." Based on this concept, we succeeded in developing a multifunctional envelope-type nano device (MEND), which exerts high transfection activities equivalent to those of an adenovirus in a dividing cell. The use of MEND has been extended to in vivo applications. PEG/peptide/DOPE ternary conjugate (PPD)-MEND, a new in vivo gene delivery system for the targeting of tumor cells that dissociates surface-modified PEG in tumor tissue by matrix metalloproteinase (MMP) and exerts significant transfection activities, was developed. In parallel with the development of MEND, a quantitative gene delivery system, Confocal Image-assisted 3-dimensionally integrated quantification (CIDIQ), also was developed. This method identified the rate-limiting step of the nonviral gene delivery system by comparing it with adenoviral-mediated gene delivery. The results of this analysis provide a new direction for the development of rational nonviral gene delivery systems.

Regional differences in usage of antenatal care and safe delivery services in Indonesia: findings from a nationally representative survey.

PubMed

Tripathi, Vrijesh; Singh, Rajvir

2017-02-03

Indonesia has shown a nominal increase in antenatal care (ANC) coverage from 93% to 96% in the Indonesia Demographic Health Survey (IDHS)-2012. This is high but for a comprehensive assessment of maternal health coverage in Indonesia, safe delivery services need to be assessed in conjunction with ANC coverage. The study uses survey data from the IDHS-2012 that was conducted among women aged 15-49 years who gave birth during the past 3 years preceding the survey. Socioeconomic and demographic factors affecting ANC coverage and safe delivery services are analysed by segregating the data into 7 regions of Indonesia. Multivariate results show that besides wealth and education differentials, regional differences significantly affect the usage of ANC and safe delivery services across the 7 regions. Univariate analyses show that Sulawesi, Maluku and Western New Guinea islands are at a disadvantage in accessing ANC and safe delivery services. The study recommends that disaggregated regional targets be set in order to further reduce maternal mortality rates in Indonesia. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Non-viral gene delivery strategies for gene therapy: a "ménage à trois" among nucleic acids, materials, and the biological environment. Stimuli-responsive gene delivery vectors

NASA Astrophysics Data System (ADS)

Pezzoli, Daniele; Candiani, Gabriele

2013-03-01

Gene delivery is the science of transferring genetic material into cells by means of a vector to alter cellular function or structure at a molecular level. In this context, a number of nucleic acid-based drugs have been proposed and experimented so far and, as they act on distinct steps along the gene transcription-translation pathway, specific delivery strategies are required to elicit the desired outcome. Cationic lipids and polymers, collectively known as non-viral delivery systems, have thus made their breakthrough in basic and medical research. Albeit they are promising alternatives to viral vectors, their therapeutic application is still rather limited as high transfection efficiencies are normally associated to adverse cytotoxic side effects. In this scenario, drawing inspiration from processes naturally occurring in vivo, major strides forward have been made in the development of more effective materials for gene delivery applications. Specifically, smart vectors sensitive to a variety of physiological stimuli such as cell enzymes, redox status, and pH are substantially changing the landscape of gene delivery by helping to overcome some of the systemic and intracellular barriers that viral vectors naturally evade. Herein, after summarizing the state-of-the-art information regarding the use of nucleic acids as drugs, we review the main bottlenecks still limiting the overall effectiveness of non-viral gene delivery systems. Finally, we provide a critical outline of emerging stimuli-responsive strategies and discuss challenges still existing on the road toward conceiving more efficient and safer multifunctional vectors.

Effective delivery of large genes to the retina by dual AAV vectors

PubMed Central

Trapani, Ivana; Colella, Pasqualina; Sommella, Andrea; Iodice, Carolina; Cesi, Giulia; de Simone, Sonia; Marrocco, Elena; Rossi, Settimio; Giunti, Massimo; Palfi, Arpad; Farrar, Gwyneth J; Polishchuk, Roman; Auricchio, Alberto

2014-01-01

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes. PMID:24150896

Gene therapy progress and prospects: magnetic nanoparticle-based gene delivery.

PubMed

Dobson, J

2006-02-01

The recent emphasis on the development of non-viral transfection agents for gene delivery has led to new physics and chemistry-based techniques, which take advantage of charge interactions and energetic processes. One of these techniques which shows much promise for both in vitro and in vivo transfection involves the use of biocompatible magnetic nanoparticles for gene delivery. In these systems, therapeutic or reporter genes are attached to magnetic nanoparticles, which are then focused to the target site/cells via high-field/high-gradient magnets. The technique promotes rapid transfection and, as more recent work indicates, excellent overall transfection levels as well. The advantages and difficulties associated with magnetic nanoparticle-based transfection will be discussed as will the underlying physical principles, recent studies and potential future applications.

Noninvasive ocular drug delivery: potential transcorneal and other alternative delivery routes for therapeutic molecules in glaucoma.

PubMed

Foldvari, Marianna

2014-01-01

Drug delivery to the eye is made difficult by multiple barriers (such as the tear film, cornea, and vitreous) between the surface of the eye and the treatment site. These barriers are difficult to surmount for the purposes of drug delivery without causing toxicity. Using nanotechnology tools to control, manipulate, and study delivery systems, new approaches to delivering drugs, genes, and antigens that are effective and safe can be developed. Topical administration to the ocular surface would be the safest method for delivery, as it is noninvasive and painless compared with other delivery methods. However, there is only limited success using topical delivery methods, especially for gene therapy. Current thinking on treatments of the future enabled by nanodelivery systems and the identification of target specificity parameters that require deeper understanding to develop successful topical delivery systems for glaucoma is highlighted.

Sonoporation, drug delivery, and gene therapy.

PubMed

Liang, H-D; Tang, J; Halliwell, M

2010-01-01

Ultrasound is a very effective modality for drug delivery and gene therapy because energy that is non-invasively transmitted through the skin can be focused deeply into the human body in a specific location and employed to release drugs at that site. Ultrasound cavitation, enhanced by injected microbubbles, perturbs cell membrane structures to cause sonoporation and increases the permeability to bioactive materials. Cavitation events also increase the rate of drug transport in general by augmenting the slow diffusion process with convective transport processes. Drugs and genes can be incorporated into microbubbles, which in turn can target a specific disease site using ligands such as the antibody. Drugs can be released ultrasonically from microbubbles that are sufficiently robust to circulate in the blood and retain their cargo of drugs until they enter an insonated volume of tissue. Local drug delivery ensures sufficient drug concentration at the diseased region while limiting toxicity for healthy tissues. Ultrasound-mediated gene delivery has been applied to heart, blood vessel, lung, kidney, muscle, brain, and tumour with enhanced gene transfection efficiency, which depends on the ultrasonic parameters such as acoustic pressure, pulse length, duty cycle, repetition rate, and exposure duration, as well as microbubble properties such as size, gas species, shell material, interfacial tension, and surface rigidity. Microbubble-augmented sonothrombolysis can be enhanced further by using targeting microbubbles.

Gene Delivery Strategies to Promote Spinal Cord Repair

PubMed Central

Walthers, Christopher M; Seidlits, Stephanie K

2015-01-01

Gene therapies hold great promise for the treatment of many neurodegenerative disorders and traumatic injuries in the central nervous system. However, development of effective methods to deliver such therapies in a controlled manner to the spinal cord is a necessity for their translation to the clinic. Although essential progress has been made to improve efficiency of transgene delivery and reduce the immunogenicity of genetic vectors, there is still much work to be done to achieve clinical strategies capable of reversing neurodegeneration and mediating tissue regeneration. In particular, strategies to achieve localized, robust expression of therapeutic transgenes by target cell types, at controlled levels over defined time periods, will be necessary to fully regenerate functional spinal cord tissues. This review summarizes the progress over the last decade toward the development of effective gene therapies in the spinal cord, including identification of appropriate target genes, improvements to design of genetic vectors, advances in delivery methods, and strategies for delivery of multiple transgenes with synergistic actions. The potential of biomaterials to mediate gene delivery while simultaneously providing inductive scaffolding to facilitate tissue regeneration is also discussed. PMID:25922572

Synthesis of galactosyl compounds for targeted gene delivery.

PubMed

Ren, T; Zhang, G; Liu, D

2001-11-01

Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.

Gene Delivery to the Airway

PubMed Central

Keiser, Nicholas W.; Engelhardt, John F.

2013-01-01

This unit describes generation of and gene transfer to several commonly used airway models. Isolation and transduction of primary airway epithelial cells are first described. Next, the preparation of polarized airway epithelial monolayers is outlined. Transduction of these polarized cells is also described. Methods are presented for generation of tracheal xenografts as well as both ex vivo and in vivo gene transfer to these xenografts. Finally, a method for in vivo gene delivery to the lungs of rodents is included. Methods for evaluating transgene expression are given in the support protocols. PMID:23853081

Gene delivery systems by the combination of lipid bubbles and ultrasound.

PubMed

Negishi, Yoichi; Endo-Takahashi, Yoko; Maruyama, Kazuo

2016-11-28

Gene therapy is promising for the treatment of many diseases including cancers and genetic diseases. From the viewpoint of safety, ultrasound (US)-mediated gene delivery with nano/ microbubbles was recently developed as a novel non-viral vector system. US-mediated gene delivery using nano/microbubbles are able to produce transient changes in the permeability of the cell membrane after US-induced cavitation while reducing cellular damage and enables the tissue-specific or the site-specific intracellular delivery of gene both in vitro and in vivo. We have recently developed novel lipid nanobubbles (Lipid Bubbles). These nanobubbles can also be used to enhance the efficacy of the US-mediated genes (plasmid DNA, siRNA, and miRNA etc.) delivery. In this review, we describe US-mediated delivery systems combined with nano/microbubbles and discuss their feasibility as non-viral vector systems.

Current and future technological advances in transdermal gene delivery.

PubMed

Chen, Xianfeng

2017-12-19

Transdermal gene delivery holds significant advantages as it is able to minimize the problems of systemic administration such as enzymatic degradation, systemic toxicity, and poor delivery to target tissues. This technology has the potential to transform the treatment and prevention of a range of diseases. However, the skin poses a great barrier for gene delivery because of the "bricks-and-mortar" structure of the stratum corneum and the tight junctions between keratinocytes in the epidermis. This review systematically summarizes the typical physical and chemical approaches to overcome these barriers and facilitate gene delivery via skin for applications in vaccination, wound healing, skin cancers and skin diseases. Next, the advantages and disadvantages of different approaches are discussed and the insights for future development are provided. Copyright © 2017 Elsevier B.V. All rights reserved.

Reducible, Dibromomaleimide-linked Polymers for Gene Delivery

PubMed Central

Tan, James-Kevin Y.; Choi, Jennifer L.; Wei, Hua; Schellinger, Joan G.; Pun, Suzie H.

2014-01-01

Polycations have been successfully used as gene transfer vehicles both in vitro and in vivo; however, their cytotoxicity has been associated with increasing molecular weight. Polymers that can be rapidly degraded after internalization are typically better tolerated by mammalian cells compared to their non-degradable counterparts. Here, we report the use of a dibromomaleimide-alkyne (DBM-alkyne) linking agent to reversibly bridge cationic polymer segments for gene delivery and to provide site-specific functionalization by azidealkyne cycloaddition chemistry. A panel of reducible and non-reducible, statistical copolymers of (2-dimethylamino) ethyl methacrylate (DMAEMA) and oligo(ethylene glycol) methyl ether methacrylate (OEGMA) were synthesized and evaluated. When complexed with plasmid DNA, the reducible and non-reducible polymers had comparable DNA condensation properties, sizes, and transfection efficiencies. When comparing cytotoxicity, the DBM-linked, reducible polymers were significantly less toxic than the non-reducible polymers. To demonstrate polymer functionalization by click chemistry, the DBM-linked polymers were tagged with an azidefluorophore and were used to monitor cellular uptake. Overall, this polymer system introduces the use of a reversible linker, DBM-alkyne, to the area of gene delivery and allows for facile, orthogonal, and site-specific functionalization of gene delivery vehicles. PMID:26214195

Chitosan for gene delivery and orthopedic tissue engineering applications.

PubMed

Raftery, Rosanne; O'Brien, Fergal J; Cryan, Sally-Ann

2013-05-15

Gene therapy involves the introduction of foreign genetic material into cells in order exert a therapeutic effect. The application of gene therapy to the field of orthopaedic tissue engineering is extremely promising as the controlled release of therapeutic proteins such as bone morphogenetic proteins have been shown to stimulate bone repair. However, there are a number of drawbacks associated with viral and synthetic non-viral gene delivery approaches. One natural polymer which has generated interest as a gene delivery vector is chitosan. Chitosan is biodegradable, biocompatible and non-toxic. Much of the appeal of chitosan is due to the presence of primary amine groups in its repeating units which become protonated in acidic conditions. This property makes it a promising candidate for non-viral gene delivery. Chitosan-based vectors have been shown to transfect a number of cell types including human embryonic kidney cells (HEK293) and human cervical cancer cells (HeLa). Aside from its use in gene delivery, chitosan possesses a range of properties that show promise in tissue engineering applications; it is biodegradable, biocompatible, has anti-bacterial activity, and, its cationic nature allows for electrostatic interaction with glycosaminoglycans and other proteoglycans. It can be used to make nano- and microparticles, sponges, gels, membranes and porous scaffolds. Chitosan has also been shown to enhance mineral deposition during osteogenic differentiation of MSCs in vitro. The purpose of this review is to critically discuss the use of chitosan as a gene delivery vector with emphasis on its application in orthopedic tissue engineering.

Selective intraarterial gene delivery into a canine meningioma.

PubMed

Chauvet, A E; Kesava, P P; Goh, C S; Badie, B

1998-05-01

The goal of this study was to evaluate gene delivery to a benign brain tumor. A recombinant adenovirus vector bearing the Escherichia coli beta-galactosidase reporter gene was selectively injected into the vascular supply of a spontaneously occurring canine olfactory groove meningioma. The tumor and a small amount of peritumoral brain tissue were removed 5 days after viral injection and stained with X-Gal to assess gene delivery. The authors noted significant beta-galactosidase gene expression by the tumor, but not by surrounding brain tissue. No obvious viral-related cytotoxicity was noted. The authors found that meningiomas can be successfully transduced by adenovirus vectors by using endovascular techniques.

A chemistry/physics pathway with nanofibrous scaffolds for gene delivery.

PubMed

Wan, Fen; Tang, Zhaohui; He, Weidong; Chu, Benjamin

2010-10-21

This perspective is to introduce a new pathway for non-viral gene delivery by taking advantage of nanofibrous scaffolds as gene storage devices, gene carriers and homing devices. During gene delivery to the target, the DNA has to be protected in order to pass through a set of barriers before reaching the nucleus. The DNA can form a complex with polycations, and numerous publications exist on how to stabilize the DNA fragments by natural and synthetic materials. Electrospun nanofibrous scaffolds can be used to store the DNA, especially in the form of a more stabilized polyplex, and then to deliver the DNA (polyplex) to cells that are attached to the scaffold. While each essential step has been tested experimentally, the overall yet untested process, especially for in vivo experiments, may lead to a promising specific approach for gene/drug storage and delivery. The pathway described herein is based mainly on our understanding of the physics and chemistry of gene storage and delivery processes, in contrast to using pure biological concepts. Novel biodegradable, biocompatible nanofibrous materials with imbedded DNA (e.g., in the polyplex form) can then be designed to fabricate an intelligent scaffold for gene delivery. To achieve the above goal, the first step is to stabilize the DNA so that it can be incorporated into nanofibrous scaffolds. In this respect, we shall discuss the different methods of DNA/gene condensation and complex formation, and then explain the strategy used to incorporate DNA into electrospun nanofibers. Solvent-induced DNA condensation and then encapsulation were achieved. However, the released naked DNA was not sufficiently protected for gene transfection in cells. The objective of the current perspective is to suggest that, instead of the solvent-induced DNA condensation, one can combine the recently developed polyplex formation by using branched polyethyleneimine (bPEI). More importantly, free bPEI can be incorporated into the nanofibers

Examining the Effect of Household Wealth and Migration Status on Safe Delivery Care in Urban India, 1992–2006

PubMed Central

Singh, Prashant Kumar; Rai, Rajesh Kumar; Singh, Lucky

2012-01-01

Background Although the urban health issue has been of long-standing interest to public health researchers, majority of the studies have looked upon the urban poor and migrants as distinct subgroups. Another concern is, whether being poor and at the same time migrant leads to a double disadvantage in the utilization of maternal health services? This study aims to examine the trends and factors that affect safe delivery care utilization among the migrants and the poor in urban India. Methodology/Principal Findings Using data from the National Family Health Survey, 1992–93 and 2005–06, this study grouped the household wealth and migration status into four distinct categories poor-migrant, poor-non migrant, non poor-migrant, non poor-non migrant. Both chi-square test and binary logistic regression were performed to examine the influence of household wealth and migration status on safe delivery care utilization among women who had experienced a birth in the four years preceding the survey. Results suggest a decline in safe delivery care among poor-migrant women during 1992–2006. The present study identifies two distinct groups in terms of safe delivery care utilization in urban India – one for poor-migrant and one for non poor-non migrants. While poor-migrant women were most vulnerable, non poor-non migrant women were the highest users of safe delivery care. Conclusion This study reiterates the inequality that underlies the utilization of maternal healthcare services not only by the urban poor but also by poor-migrant women, who deserve special attention. The ongoing programmatic efforts under the National Urban Health Mission should start focusing on the poorest of the poor groups such as poor-migrant women. Importantly, there should be continuous evaluation to examine the progress among target groups within urban areas. PMID:22970324

Physical non-viral gene delivery methods for tissue engineering.

PubMed

Mellott, Adam J; Forrest, M Laird; Detamore, Michael S

2013-03-01

The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that "fits-all" cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

Physical non-viral gene delivery methods for tissue engineering

PubMed Central

Mellott, Adam J.; Forrest, M. Laird; Detamore, Michael S.

2016-01-01

The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that “fits-all” cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications. PMID:23099792

Part II: Functional delivery of a neurotherapeutic gene to neural stem cells using minicircle DNA and nanoparticles: Translational advantages for regenerative neurology.

PubMed

Fernandes, Alinda R; Chari, Divya M

2016-09-28

Both neurotrophin-based therapy and neural stem cell (NSC)-based strategies have progressed to clinical trials for treatment of neurological diseases and injuries. Brain-derived neurotrophic factor (BDNF) in particular can confer neuroprotective and neuro-regenerative effects in preclinical studies, complementing the cell replacement benefits of NSCs. Therefore, combining both approaches by genetically-engineering NSCs to express BDNF is an attractive approach to achieve combinatorial therapy for complex neural injuries. Current genetic engineering approaches almost exclusively employ viral vectors for gene delivery to NSCs though safety and scalability pose major concerns for clinical translation and applicability. Magnetofection, a non-viral gene transfer approach deploying magnetic nanoparticles and DNA with magnetic fields offers a safe alternative but significant improvements are required to enhance its clinical application for delivery of large sized therapeutic plasmids. Here, we demonstrate for the first time the feasibility of using minicircles with magnetofection technology to safely engineer NSCs to overexpress BDNF. Primary mouse NSCs overexpressing BDNF generated increased daughter neuronal cell numbers post-differentiation, with accelerated maturation over a four-week period. Based on our findings we highlight the clinical potential of minicircle/magnetofection technology for therapeutic delivery of key neurotrophic agents. Copyright © 2016 Elsevier B.V. All rights reserved.

CRISPR-Cas9 gene editing: Delivery aspects and therapeutic potential.

PubMed

Oude Blenke, Erik; Evers, Martijn J W; Mastrobattista, Enrico; van der Oost, John

2016-12-28

The CRISPR-Cas9 gene editing system has taken the biomedical science field by storm, initiating rumors about future Nobel Prizes and heating up a fierce patent war, but also making significant scientific impact. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with CRISPR-associated proteins (Cas) are a part of the prokaryotic adaptive immune system and have successfully been repurposed for genome editing in mammalian cells. The CRISPR-Cas9 system has been used to correct genetic mutations and for replacing entire genes, opening up a world of possibilities for the treatment of genetic diseases. In addition, recently some new CRISPR-Cas systems have been discovered with interesting mechanistic variations. Despite these promising developments, many challenges have to be overcome before the system can be applied therapeutically in human patients and enabling delivery technology is one of the key challenges. Furthermore, the relatively high off-target effect of the system in its current form prevents it from being safely applied directly in the human body. In this review, the transformation of the CRISPR-Cas gene editing systems into a therapeutic modality will be discussed and the currently most realistic in vivo applications will be highlighted. Copyright © 2016 Elsevier B.V. All rights reserved.

Engineering Delivery Vehicles for Genome Editing.

PubMed

Nelson, Christopher E; Gersbach, Charles A

2016-06-07

The field of genome engineering has created new possibilities for gene therapy, including improved animal models of disease, engineered cell therapies, and in vivo gene repair. The most significant challenge for the clinical translation of genome engineering is the development of safe and effective delivery vehicles. A large body of work has applied genome engineering to genetic modification in vitro, and clinical trials have begun using cells modified by genome editing. Now, promising preclinical work is beginning to apply these tools in vivo. This article summarizes the development of genome engineering platforms, including meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas9, and their flexibility for precise genetic modifications. The prospects for the development of safe and effective viral and nonviral delivery vehicles for genome editing are reviewed, and promising advances in particular therapeutic applications are discussed.

Barriers to Liposomal Gene Delivery: from Application Site to the Target.

PubMed

Saffari, Mostafa; Moghimi, Hamid Reza; Dass, Crispin R

2016-01-01

Gene therapy is a therapeutic approach to deliver genetic material into cells to alter their function in entire organism. One promising form of gene delivery system (DDS) is liposomes. The success of liposome-mediated gene delivery is a multifactorial issue and well-designed liposomal systems might lead to optimized gene transfection particularly in vivo. Liposomal gene delivery systems face different barriers from their site of application to their target, which is inside the cells. These barriers include presystemic obstacles (epithelial barriers), systemic barriers in blood circulation and cellular barriers. Epithelial barriers differ depending on the route of administration. Systemic barriers include enzymatic degradation, binding and opsonisation. Both of these barriers can act as limiting hurdles that genetic material and their vector should overcome before reaching the cells. Finally liposomes should overcome cellular barriers that include cell entrance, endosomal escape and nuclear uptake. These barriers and their impact on liposomal gene delivery will be discussed in this review.

Delivery of gene silencing agents for breast cancer therapy

PubMed Central

2013-01-01

The discovery of RNA interference has opened the door for the development of a new class of cancer therapeutics. Small inhibitory RNA oligos are being designed to specifically suppress expression of proteins that are traditionally considered nondruggable, and microRNAs are being evaluated to exert broad control of gene expression for inhibition of tumor growth. Since most naked molecules are not optimized for in vivo applications, the gene silencing agents need to be packaged into delivery vehicles in order to reach the target tissues as their destinations. Thus, the selection of the right delivery vehicles serves as a crucial step in the development of cancer therapeutics. The current review summarizes the status of gene silencing agents in breast cancer and recent development of candidate cancer drugs in clinical trials. Nanotechnology-based delivery vectors for the formulation and packaging of gene silencing agents are also described. PMID:23659575

Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery

PubMed Central

Zhou, Jiangbing; Liu, Jie; Cheng, Christopher J.; Patel, Toral R.; Weller, Caroline E.; Piepmeier, Joseph M.; Jiang, Zhaozhong; Saltzman, W. Mark

2014-01-01

Many synthetic polycationic vectors for non-viral gene delivery show high efficiency in vitro, but their usually excessive charge density makes them toxic for in vivo applications. Here we describe the synthesis of a series of high molecular weight terpolymers with low charge density, and show that they exhibit efficient gene delivery, some surpassing the efficiency of the commercial transfection reagents Polyethylenimine and Lipofectamine 2000. The terpolymers were synthesized via enzyme-catalyzed copolymerization of lactone with dialkyl diester and amino diol, and their hydrophobicity adjusted by varying the lactone content and by selecting a lactone comonomer of specific ring size. Targeted delivery of the pro-apoptotic TRAIL gene to tumour xenografts by one of the terpolymers results in significant inhibition of tumour growth, with minimal toxicity both in vitro and in vivo. Our findings suggest that the gene delivery ability of the terpolymers stems from their high molecular weight and increased hydrophobicity, which compensates for their low charge density. PMID:22138789

Ultrasound-Mediated Local Drug and Gene Delivery Using Nanocarriers

PubMed Central

Zhou, Qiu-Lan; Chen, Zhi-Yi; Yang, Feng

2014-01-01

With the development of nanotechnology, nanocarriers have been increasingly used for curative drug/gene delivery. Various nanocarriers are being introduced and assessed, such as polymer nanoparticles, liposomes, and micelles. As a novel theranostic system, nanocarriers hold great promise for ultrasound molecular imaging, targeted drug/gene delivery, and therapy. Nanocarriers, with the properties of smaller particle size, and long circulation time, would be advantageous in diagnostic and therapeutic applications. Nanocarriers can pass through blood capillary walls and cell membrane walls to deliver drugs. The mechanisms of interaction between ultrasound and nanocarriers are not clearly understood, which may be related to cavitation, mechanical effects, thermal effects, and so forth. These effects may induce transient membrane permeabilization (sonoporation) on a single cell level, cell death, and disruption of tissue structure, ensuring noninvasive, targeted, and efficient drug/gene delivery and therapy. The system has been used in various tissues and organs (in vitro or in vivo), including tumor tissues, kidney, cardiac, skeletal muscle, and vascular smooth muscle. In this review, we explore the research progress and application of ultrasound-mediated local drug/gene delivery with nanocarriers. PMID:25202710

Micelles and Nanoparticles for Ultrasonic Drug and Gene Delivery

PubMed Central

Husseini, Ghaleb A.; Pitt, William G.

2008-01-01

Drug delivery research employing micelles and nanoparticles has expanded in recent years. Of particular interest is the use of these nanovehicles that deliver high concentrations of cytotoxic drugs to diseased tissues selectively, thus reducing the agent’s side effects on the rest of the body. Ultrasound, traditionally used in diagnostic medicine, is finding a place in drug delivery in connection with these nanoparticles. In addition to their non-invasive nature and the fact that they can be focused on targeted tissues, acoustic waves have been credited with releasing pharmacological agents from nanocarriers, as well as rendering cell membranes more permeable. In this article, we summarize new technologies that combine the use of nanoparticles with acoustic power both in drug and gene delivery. Ultrasonic drug delivery from micelles usually employs polyether block copolymers, and has been found effective in vivo for treating tumors. Ultrasound releases drug from micelles, most probably via shear stress and shock waves from collapse of cavitation bubbles. Liquid emulsions and solid nanoparticles are used with ultrasound to deliver genes in vitro and in vivo. The small packaging allows nanoparticles to extravasate into tumor tissues. Ultrasonic drug and gene delivery from nano-carriers has tremendous potential because of the wide variety of drugs and genes that could be delivered to targeted tissues by fairly non-invasive means. PMID:18486269

Synergistic effect of amino acids modified on dendrimer surface in gene delivery.

PubMed

Wang, Fei; Wang, Yitong; Wang, Hui; Shao, Naimin; Chen, Yuanyuan; Cheng, Yiyun

2014-11-01

Design of an efficient gene vector based on dendrimer remains a great challenge due to the presence of multiple barriers in gene delivery. Single-functionalization on dendrimer cannot overcome all the barriers. In this study, we synthesized a list of single-, dual- and triple-functionalized dendrimers with arginine, phenylalanine and histidine for gene delivery using a one-pot approach. The three amino acids play different roles in gene delivery: arginine is essential in formation of stable complexes, phenylalanine improves cellular uptake efficacy, and histidine increases pH-buffering capacity and minimizes cytotoxicity of the cationic dendrimer. A combination of these amino acids on dendrimer generates a synergistic effect in gene delivery. The dual- and triple-functionalized dendrimers show minimal cytotoxicity on the transfected NIH 3T3 cells. Using this combination strategy, we can obtain triple-functionalized dendrimers with comparable transfection efficacy to several commercial transfection reagents. Such a combination strategy should be applicable to the design of efficient and biocompatible gene vectors for gene delivery. Copyright © 2014 Elsevier Ltd. All rights reserved.

Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice.

PubMed

Sato, Masahiro; Inada, Emi; Saitoh, Issei; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

2013-11-01

The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ. © 2013 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptions are made.

PLGA/polymeric liposome for targeted drug and gene co-delivery.

PubMed

Wang, Hanjie; Zhao, Peiqi; Su, Wenya; Wang, Sheng; Liao, Zhenyu; Niu, Ruifang; Chang, Jin

2010-11-01

Chemotherapy is one of the most effective approaches to treat cancers in the clinic, but the problems, such as multidrug resistance (MDR), low bioavailability and toxicity, severely constrain the further application of chemotherapy. Our group recently reported that cationic PLGA/folate coated PEGlated polymeric liposome core-shell nanoparticles (PLGA/FPL NPs). It was self-assembled from a hydrophobic PLGA core and a hydrophilic folate coated PEGlated lipid shell for targeting co-delivery of drug and gene. Hydrophobic drugs can be incorporated into the core and the cationic shell of the drug-loaded nanoparticles can be used to bind DNA. The drug-loaded PLGA/FPL NPs/DNA complexes offer advantages to overcome these problems mentioned above, such as co-delivery of drugs and DNA to improving the chemosensitivity of cancer cells at a gene level, and targeting delivery of drug to the cancer tissue that enhance the bioavailability and reduce the toxicity. The experiment showed that nanoparticles have core-shell structure with nanosize, sustained drug release profile and good DNA-binding ability. Importantly, the core-shell nanoparticles achieve the possibility of co-delivering drugs and genes to the same cells with high gene transfection and drug delivery efficiency. Our data suggest that the PLGA/FPL NPs may be a useful drug and gene co-delivery system. Copyright © 2010 Elsevier Ltd. All rights reserved.

Harnessing hospital purchase power to design safe care delivery.

PubMed

Ebben, Steven F; Gieras, Izabella A; Gosbee, Laura Lin

2008-01-01

Since the Institute of Medicine's well-publicized 1999 report To Err is Human, the healthcare patient safety movement has grown at an exponential pace. However, much more can be done to advance patient safety from a care process design vantage point-improving safety through effective care processes and technology integration. While progress is being made, the chasm between technology developers and caregivers remains a profound void. Why hasn't more been done to expand our view of patient safety to include technology design? Healthcare organizations have not consolidated their purchasing power to expect improved designs. This article will (1) provide an assessment of the present state of healthcare technology management and (2) provide recommendations for collaborative design of safe healthcare delivery systems.

Fe3O4 Nanoparticles in Targeted Drug/Gene Delivery Systems

PubMed Central

Shen, Lazhen; Li, Bei; Qiao, Yongsheng

2018-01-01

Fe3O4 nanoparticles (NPs), the most traditional magnetic nanoparticles, have received a great deal of attention in the biomedical field, especially for targeted drug/gene delivery systems, due to their outstanding magnetism, biocompatibility, lower toxicity, biodegradability, and other features. Naked Fe3O4 NPs are easy to aggregate and oxidize, and thus are often made with various coatings to realize superior properties for targeted drug/gene delivery. In this review, we first list the three commonly utilized synthesis methods of Fe3O4 NPs, and their advantages and disadvantages. In the second part, we describe coating materials that exhibit noticeable features that allow functionalization of Fe3O4 NPs and summarize their methods of drug targeting/gene delivery. Then our efforts will be devoted to the research status and progress of several different functionalized Fe3O4 NP delivery systems loaded with chemotherapeutic agents, and we present targeted gene transitive carriers in detail. In the following section, we illuminate the most effective treatment systems of the combined drug and gene therapy. Finally, we propose opportunities and challenges of the clinical transformation of Fe3O4 NPs targeting drug/gene delivery systems. PMID:29473914

Selective gene silencing by viral delivery of short hairpin RNA

PubMed Central

2010-01-01

RNA interference (RNAi) technology has not only become a powerful tool for functional genomics, but also allows rapid drug target discovery and in vitro validation of these targets in cell culture. Furthermore, RNAi represents a promising novel therapeutic option for treating human diseases, in particular cancer. Selective gene silencing by RNAi can be achieved essentially by two nucleic acid based methods: i) cytoplasmic delivery of short double-stranded (ds) interfering RNA oligonucleotides (siRNA), where the gene silencing effect is only transient in nature, and possibly not suitable for all applications; or ii) nuclear delivery of gene expression cassettes that express short hairpin RNA (shRNA), which are processed like endogenous interfering RNA and lead to stable gene down-regulation. Both processes involve the use of nucleic acid based drugs, which are highly charged and do not cross cell membranes by free diffusion. Therefore, in vivo delivery of RNAi therapeutics must use technology that enables the RNAi therapeutic to traverse biological membrane barriers in vivo. Viruses and the vectors derived from them carry out precisely this task and have become a major delivery system for shRNA. Here, we summarize and compare different currently used viral delivery systems, give examples of in vivo applications, and indicate trends for new developments, such as replicating viruses for shRNA delivery to cancer cells. PMID:20858246

Solid Lipid Nanoparticles as Efficient Drug and Gene Delivery Systems: Recent Breakthroughs

PubMed Central

Ezzati Nazhad Dolatabadi, Jafar; Valizadeh, Hadi; Hamishehkar, Hamed

2015-01-01

In recent years, nanomaterials have been widely applied as advanced drug and gene delivery nanosystems. Among them, solid lipid nanoparticles (SLNs) have attracted great attention as colloidal drug delivery systems for incorporating hydrophilic or lipophilic drugs and various macromolecules as well as proteins and nucleic acids. Therefore, SLNs offer great promise for controlled and site specific drug and gene delivery. This article includes general information about SLN structures and properties, production procedures, characterization. In addition, recent progress on development of drug and gene delivery systems using SLNs was reviewed. PMID:26236652

Progresses towards safe and efficient gene therapy vectors.

PubMed

Chira, Sergiu; Jackson, Carlo S; Oprea, Iulian; Ozturk, Ferhat; Pepper, Michael S; Diaconu, Iulia; Braicu, Cornelia; Raduly, Lajos-Zsolt; Calin, George A; Berindan-Neagoe, Ioana

2015-10-13

The emergence of genetic engineering at the beginning of the 1970's opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors.

Progresses towards safe and efficient gene therapy vectors

PubMed Central

Chira, Sergiu; Jackson, Carlo S.; Oprea, Iulian; Ozturk, Ferhat; Pepper, Michael S.; Diaconu, Iulia; Braicu, Cornelia; Raduly, Lajos-Zsolt; Calin, George A.; Berindan-Neagoe, Ioana

2015-01-01

The emergence of genetic engineering at the beginning of the 1970′s opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors. PMID:26362400

Novel Polymeric Nanoparticles for Pulmonary Gene Delivery

NASA Astrophysics Data System (ADS)

Fields, Rachel Jennifer

The lung is an important target for gene and drug therapy of many diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), tubuerculosis (TB) and lung cancer. In fact, the pulmonary route has been employed as a means of delivering drugs for centuries, dating back 4000 years to India where inhaled vapors were used for medicinal purpose. Currently, pulmonary administration of small, hydrophobic drugs leads to rapid local and systemic absorption. However, delivery of large biomacromolecules, such as therapeutic genes, has not yet been accomplished. Here, I test the hypothesis that a rationally engineered nanoparticle (NP) vector can improve delivery of large biomacromolecules. . In this dissertation I tested this hypothesis using a hybrid NP delivery system consisting of a blend of poly(lactic-co-glycolic acid) (PLGA) and a poly(beta-amino ester) (PBAE), a cationic polymer that is particularly useful for delivery of nucleic acids.. PBAE/PLGA nanoparticles (15% PBAE) loaded with plasmid DNA were surface modified with cell-penetrating peptides (CPPs) via a PEGylated phospholipid linker. This optimized NP formulation was able to induce substantial intracellular uptake and transfect lung epithelial cells in vitro while imparting minimal cellular toxicity. In order to determine the most effective method to deliver these NPs to the lung I used fluorescently labeled particles to study the biodistribution of particles after administration to the lung of mice via various administration routes. I determined that the intranasal route was most effective. I further investigated this route and determined that an average of 37.1 +/- 15.1 % of lung cells had NP association after 4hrs. I also investigated the association of particles with different lung cell types like macrophages and alveolar epithelial cells and determined that our best particle formulations associated with approximately 80% of both of these cell types. To demonstrate the ability of the

Gene delivery to skin using biolistics.

PubMed

Heiser, William C

2006-06-01

INTRODUCTION Biolistics ("biological ballistics") or particle bombardment provides a rapid and simple physical procedure for delivering genes into cells (Klein et al. 1987; Yang et al. 1990). The technique has many advantages-plasmids may be used for delivery, DNA theoretically can be delivered to any cell type, and genes may be delivered to cells in vitro, ex vivo, or in vivo. DNA-coated gold particles are distributed evenly along the length of the tubing, which is subsequently cut into short sections of cartridges to be used in a gene gun. The Helios Gene Gun uses a pulse of helium to launch the DNA-coated particles, spreading them onto the target cells.

A Self-Assembled Coumarin-Anchored Dendrimer for Efficient Gene Delivery and Light-Responsive Drug Delivery.

PubMed

Wang, Hui; Miao, Wujun; Wang, Fei; Cheng, Yiyun

2018-06-11

The assembly of low molecular weight polymers into highly efficient and nontoxic nanostructures has broad applicability in gene delivery. In this study, we reported the assembly of coumarin-anchored low generation dendrimers in aqueous solution via hydrophobic interactions. The synthesized material showed significantly improved DNA binding and gene delivery, and minimal toxicity on the transfected cells. Moreover, the coumarin moieties in the assembled nanostructures endow the materials with light-responsive drug delivery behaviors. The coumarin substitutes in the assembled nanostructures were cross-linked with each other upon irradiation at 365 nm, and the cross-linked assemblies were degraded upon further irradiation at 254 nm. As a result, the drug-loaded nanoparticle showed a light-responsive drug release behavior and light-enhanced anticancer activity. The assembled nanoparticle also exhibited a complementary anticancer activity through the codelivery of 5-fluorouracil and a therapeutic gene encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This study provided a facile strategy to develop light-responsive polymers for the codelivery of therapeutic genes and anticancer drugs.

Gene Delivery in Neuro-Oncology.

PubMed

Dixit, Karan; Kumthekar, Priya

2017-09-02

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults with a dismal prognosis despite aggressive multimodal management thus novel treatments are urgently needed. Gene therapy is a versatile treatment strategy being investigated in multiple cancers including GBM. In gene therapy, a variety of vectors or "carriers" are used to deliver genes designed for different anti-tumoral effects. Gene delivery vehicles and approaches to treatment will be addressed in this review. The most commonly studied vectors are viral based, however, driven by advances in biomedical engineering, mesenchymal and neural stem cells, as well as multiple different types of nanoparticles have been developed to improve tumor tropism and also increase gene transfer into tumor cells. Different genes have been studied including suicide genes, which convert non-toxic prodrug into cytotoxic drug; immunomodulatory genes, which stimulate the immune system; and tumor suppressor genes which repair the defect that allow cells to divide unchecked. Gene therapy may be a promising treatment strategy in neuro-oncology as it is versatile and flexible due to the ability to tailor vectors and genes for specific therapeutic activity. Pre-clinical studies and clinical trials have demonstrated feasibility and safety of gene therapy; however, further studies are required to determine efficacy.

Smart Micro/Nano-robotic Systems for Gene Delivery.

PubMed

Pedram, Alireza; Pishkenari, Hossein Nejat

2017-01-01

Small scale robotics have attracted growing attention for the prospect of targeting and accessing cell-sized sites, necessary for high precision biomedical applications and drug/gene delivery. The loss of controlled gene therapy, inducing systemic side effects and reduced therapeutic efficiency, can be settled utilizing these intelligent carriers. Newly proposed solutions for the main challenges of control, power supplying, gene release and final carrier extraction/degradation have shifted these smart miniature robots to the point of being employed for practical applications of transferring oligonucleotides (pDNA, siRNA, mRNA, etc.) in near future. In this paper, different scenarios and their endeavors to address the vital working demands and steps, in particular, carrier attachment and release, cell internalization, manipulation concerns as well as actuation systems are discussed.This review highlights some promising experimental results showing controlled gene release of robotic systems in comparison with current non-specific gene delivery methods. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Three-layered polyplex as a microRNA targeted delivery system for breast cancer gene therapy

NASA Astrophysics Data System (ADS)

Li, Yan; Dai, Yu; Zhang, Xiaojin; Chen, Jihua

2017-07-01

MicroRNAs (miRNAs), small non-coding RNAs, play an important role in modulating cell proliferation, migration, and differentiation. Since miRNAs can regulate multiple cancer-related genes simultaneously, regulating miRNAs could target a set of related oncogenic genes or pathways. Owing to their reduced immune response and low toxicity, miRNAs with small size and low molecular weight have become increasingly promising therapeutic drugs in cancer therapy. However, one of the major challenges of miRNAs-based cancer therapy is to achieve specific, effective, and safe delivery of therapeutic miRNAs into cancer cells. Here we provide a strategy using three-layered polyplex with folic acid as a targeting group to systemically deliver miR-210 into breast cancer cells, which results in breast cancer growth being inhibited.

Three-layered polyplex as a microRNA targeted delivery system for breast cancer gene therapy.

PubMed

Li, Yan; Dai, Yu; Zhang, Xiaojin; Chen, Jihua

2017-07-14

MicroRNAs (miRNAs), small non-coding RNAs, play an important role in modulating cell proliferation, migration, and differentiation. Since miRNAs can regulate multiple cancer-related genes simultaneously, regulating miRNAs could target a set of related oncogenic genes or pathways. Owing to their reduced immune response and low toxicity, miRNAs with small size and low molecular weight have become increasingly promising therapeutic drugs in cancer therapy. However, one of the major challenges of miRNAs-based cancer therapy is to achieve specific, effective, and safe delivery of therapeutic miRNAs into cancer cells. Here we provide a strategy using three-layered polyplex with folic acid as a targeting group to systemically deliver miR-210 into breast cancer cells, which results in breast cancer growth being inhibited.

A Safe Bacterial Microsyringe for In Vivo Antigen Delivery and Immunotherapy

PubMed Central

Le Gouëllec, Audrey; Chauchet, Xavier; Laurin, David; Aspord, Caroline; Verove, Julien; Wang, Yan; Genestet, Charlotte; Trocme, Candice; Ahmadi, Mitra; Martin, Sandrine; Broisat, Alexis; Cretin, François; Ghezzi, Catherine; Polack, Benoit; Plumas, Joël; Toussaint, Bertrand

2013-01-01

The industrial development of active immunotherapy based on live-attenuated bacterial vectors has matured. We developed a microsyringe for antigen delivery based on the type III secretion system (T3SS) of P. aeruginosa. We applied the “killed but metabolically active” (KBMA) attenuation strategy to make this bacterial vector suitable for human use. We demonstrate that attenuated P. aeruginosa has the potential to deliver antigens to human antigen-presenting cells in vitro via T3SS with considerable attenuated cytotoxicity as compared with the wild-type vector. In a mouse model of cancer, we demonstrate that this KBMA strain, which cannot replicate in its host, efficiently disseminates into lymphoid organs and delivers its heterologous antigen. The attenuated strain effectively induces a cellular immune response to the cancerous cells while lowering the systemic inflammatory response. Hence, a KBMA P. aeruginosa microsyringe is an efficient and safe tool for in vivo antigen delivery. PMID:23531551

Self-assembled pentablock copolymers for selective and sustained gene delivery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Bingqi

2011-05-15

The poly(diethylaminoethyl methacrylate) (PDEAEM) - Pluronic F127 - PDEAEM pentablock copolymer (PB) gene delivery vector system has been found to possess an inherent selectivity in transfecting cancer cells over non-cancer cells in vitro, without attaching any targeting ligands. In order to understand the mechanism of this selective transfection, three possible intracellular barriers to transfection were investigated in both cancer and non-cancer cells. We concluded that escape from the endocytic pathway served as the primary intracellular barrier for PB-mediated transfection. Most likely, PB vectors were entrapped and rendered non-functional in acidic lysosomes of non-cancer cells, but survived in less acidic lysosomesmore » of cancer cells. The work highlights the importance of identifying intracellular barriers for different gene delivery systems and provides a new paradigm for designing targeting vectors based on intracellular differences between cell types, rather than through the use of targeting ligands. The PB vector was further developed to simultaneously deliver anticancer drugs and genes, which showed a synergistic effect demonstrated by significantly enhanced gene expression in vitro. Due to the thermosensitive gelation behavior, the PB vector packaging both drug and gene was also investigated for its in vitro sustained release properties by using polyethylene glycol diacrylate as a barrier gel to mimic the tumor matrix in vivo. Overall, this work resulted in the development of a gene delivery vector for sustained and selective gene delivery to tumor cells for cancer therapy.« less

Nano-scale gene delivery systems; current technology, obstacles, and future directions.

PubMed

Garcia-Guerra, Antonio; Dunwell, Thomas L; Trigueros, Sonia

2018-01-07

Within the different applications of nanomedicine currently being developed, nano-gene delivery is appearing as an exciting new technique with the possibility to overcome recognised hurdles and fulfill several biological and medical needs. The central component of all delivery systems is the requirement for the delivery of genetic material into cells, and for them to eventually reside in the nucleus where their desired function will be exposed. However, genetic material does not passively enter cells; thus, a delivery system is necessary. The emerging field of nano-gene delivery exploits the use of new materials and the properties that arise at the nanometre-scale to produce delivery vectors that can effectively deliver genetic material into a variety of different types of cells. The novel physicochemical properties of the new delivery vectors can be used to address the current challenges existing in nucleic acid delivery in vitro and in vivo. While there is a growing interest in nanostructure-based gene delivery, the field is still in its infancy, and there is yet much to discover about nanostructures and their physicochemical properties in a biological context. We carry out an organized and focused search of bibliographic databases. Our results suggest that despite new breakthroughs in nanostructure synthesis and advanced characterization techniques, we still face many barriers in producing highly efficient and non-toxic delivery systems. In this review, we overview the types of systems currently used for clinical and biomedical research applications along with their advantages and disadvantages, as well as discussing barriers that arise from nano-scale interactions with biological material. In conclusion, we hope that by bringing the far reaching multidisciplinary nature of nano-gene delivery to light, new targeted nanotechnology-bases strategies are developed to overcome the major challenges covered in this review. Copyright© Bentham Science Publishers; For

Dendrimer Nanovectors for SiRNA Delivery.

PubMed

Liu, Xiaoxuan; Peng, Ling

2016-01-01

Small interfering RNA (SiRNA) delivery remains a major challenge in RNAi-based therapy. Dendrimers are emerging as appealing nonviral vectors for SiRNA delivery thanks to their well-defined architecture and their unique cooperativity and multivalency confined within a nanostructure. We have recently demonstrated that structurally flexible poly(amidoamine) (PAMAM) dendrimers are safe and effective nanovectors for SiRNA delivery in various disease models in vitro and in vivo. The present chapter showcases these dendrimers can package different SiRNA molecules into stable and nanosized particles, which protect SiRNA from degradation and promote cellular uptake of SiRNA, resulting in potent gene silencing at both mRNA and protein level in the prostate cancer cell model. Our results demonstrate this set of flexible PAMAM dendrimers are indeed safe and effective nonviral vectors for SiRNA delivery and hold great promise for further applications in functional genomics and RNAi-based therapies.

Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

PubMed Central

Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

2015-01-01

The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier. PMID:26123532

Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

NASA Astrophysics Data System (ADS)

Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

2015-06-01

The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier.

A novel gene delivery composite system based on biodegradable folate-poly (ester amine) polymer and thermosensitive hydrogel for sustained gene release

PubMed Central

Yang, Yi; Zhao, Hang; Jia, YanPeng; Guo, QingFa; Qu, Ying; Su, Jing; Lu, XiaoLing; Zhao, YongXiang; Qian, ZhiYong

2016-01-01

Local anti-oncogene delivery providing high local concentration of gene, increasing antitumor effect and decreasing systemic side effects is currently attracting interest in cancer therapy. In this paper, a novel local sustained anti-oncogene delivery system, PECE thermoresponsive hydrogel containing folate-poly (ester amine) (FA-PEA) polymer/DNA (tumor suppressor) complexes, is demonstrated. First, a tumor-targeted biodegradable folate-poly (ester amine) (FA-PEA) polymer based on low-molecular-weight polyethyleneimine (PEI) was synthesized and characterized, and the application for targeted gene delivery was investigated. The polymer had slight cytotoxicity and high transfection efficiency in vitro compared with PEI 25k, which indicated that FA-PEA was a potential vector for targeted gene delivery. Meanwhile, we successfully prepared a thermoresponsive PECE hydrogel composite containing FA-PEA/DNA complexes which could contain the genes and slowly release the genes into cells. We concluded the folate-poly (ester amine) (FA-PEA) polymer would be useful for targeted gene delivery, and the novel gene delivery composite based on biodegradable folate-poly (ester amine) polymer and thermosensitive PECE hydrogel showed potential for sustained gene release. PMID:26883682

Characterisation of gene delivery using liposomal bubbles and ultrasound

NASA Astrophysics Data System (ADS)

Koshima, Risa; Suzuki, Ryo; Oda, Yusuke; Hirata, Keiichi; Nomura, Tetsuya; Negishi, Yoichi; Utoguchi, Naoki; Kudo, Nobuki; Maruyama, Kazuo

2011-09-01

The combination of nano/microbubbles and ultrasound is a novel technique for a non-viral gene deliver. We have previously developed novel ultrasound sensitive liposomes (Bubble liposomes) which contain the ultrasound imaging gas perfluoropropane. In this study, Bubble liposomes were compared with cationic lipid (CL)-DNA complexes as potential gene delivery carriers into tumors in vivo. The delivery of genes by bubble liposomes depended on the intensity of the applied ultrasound. The transfection efficiency plateaued at 0.7 W/cm2 ultrasound intensity. Bubble liposomes efficiently transferred genes into cultured cells even when the cells were exposed to ultrasound for only 1 s. In addition, bubble liposomes were able to introduce the luciferase gene more effectively than CL-DNA complexes into mouse ascites tumor cells. We conclude that the combination of Bubble liposomes and ultrasound is a good method for gene transfer in vivo.

Micro- and nanobubbles: a versatile non-viral platform for gene delivery.

PubMed

Cavalli, Roberta; Bisazza, Agnese; Lembo, David

2013-11-18

Micro- and nanobubbles provide a promising non-viral strategy for ultrasound mediated gene delivery. Microbubbles are spherical gas-filled structures with a mean diameter of 1-8 μm, characterised by their core-shell composition and their ability to circulate in the bloodstream following intravenous injection. They undergo volumetric oscillations or acoustic cavitation when insonified by ultrasound and, most importantly, they are able to resonate at diagnostic frequencies. It is due to this behaviour that microbubbles are currently being used as ultrasound contrast agents, but their use in therapeutics is still under investigation. For example, microbubbles could play a role in enhancing gene delivery to cells: when combined with clinical ultrasound exposure, microbubbles are able to favour gene entry into cells by cavitation. Two different delivery strategies have been used to date: DNA can be co-administered with the microbubbles (i.e. the contrast agent) or 'loaded' in purposed-built bubble systems - indeed a number of different technological approaches have been proposed to associate genes within microbubble structures. Nanobubbles, bubbles with sizes in the nanometre order of magnitude, have also been developed with the aim of obtaining more efficient gene delivery systems. Their small sizes allow the possibility of extravasation from blood vessels into the surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. In contrast, microbubbles, due to their larger sizes, are unable to extravasate, thus and their targeting capacity is limited to specific antigens present within the vascular lumen. This review provides an overview of the use of microbubbles as gene delivery systems, with a specific focus on recent research into the development of nanosystems. In particular, ultrasound delivery mechanisms, formulation parameters, gene-loading approaches and the advantages of nanometric systems will be described. Copyright © 2013

Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein.

PubMed

Kessler, P D; Podsakoff, G M; Chen, X; McQuiston, S A; Colosi, P C; Matelis, L A; Kurtzman, G J; Byrne, B J

1996-11-26

Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the beta-galactosidase (AAV-lacZ) gene into adult BALB/c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies.

Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein

PubMed Central

Kessler, Paul D.; Podsakoff, Gregory M.; Chen, Xiaojuan; McQuiston, Susan A.; Colosi, Peter C.; Matelis, Laura A.; Kurtzman, Gary J.; Byrne, Barry J.

1996-01-01

Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the β-galactosidase (AAV-lacZ) gene into adult BALB/c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies. PMID:8943064

"Combo" nanomedicine: Co-delivery of multi-modal therapeutics for efficient, targeted, and safe cancer therapy.

PubMed

Kemp, Jessica A; Shim, Min Suk; Heo, Chan Yeong; Kwon, Young Jik

2016-03-01

The dynamic and versatile nature of diseases such as cancer has been a pivotal challenge for developing efficient and safe therapies. Cancer treatments using a single therapeutic agent often result in limited clinical outcomes due to tumor heterogeneity and drug resistance. Combination therapies using multiple therapeutic modalities can synergistically elevate anti-cancer activity while lowering doses of each agent, hence, reducing side effects. Co-administration of multiple therapeutic agents requires a delivery platform that can normalize pharmacokinetics and pharmacodynamics of the agents, prolong circulation, selectively accumulate, specifically bind to the target, and enable controlled release in target site. Nanomaterials, such as polymeric nanoparticles, gold nanoparticles/cages/shells, and carbon nanomaterials, have the desired properties, and they can mediate therapeutic effects different from those generated by small molecule drugs (e.g., gene therapy, photothermal therapy, photodynamic therapy, and radiotherapy). This review aims to provide an overview of developing multi-modal therapies using nanomaterials ("combo" nanomedicine) along with the rationale, up-to-date progress, further considerations, and the crucial roles of interdisciplinary approaches. Copyright © 2015 Elsevier B.V. All rights reserved.

Targeted Gene Addition to a Safe Harbor locus in human CD34+ Hematopoietic Stem Cells for Correction of X-linked Chronic Granulomatous Disease

PubMed Central

De Ravin, Suk See; Reik, Andreas; Liu, Pei-Qi; Li, Linhong; Wu, Xiaolin; Su, Ling; Raley, Castle; Theobald, Narda; Choi, Uimook; Song, Alexander H.; Chan, Andy; Pearl, Jocelynn R.; Paschon, David E.; Lee, Janet; Newcombe, Hannah; Koontz, Sherry; Sweeney, Colin; Shivak, David A.; Zarember, Kol A.; Peshwa, Madhusudan V.; Gregory, Philip D.; Urnov, Fyodor D.; Malech, Harry L.

2016-01-01

Gene therapy with genetically modified human CD34+ hematopoietic stem cells (HSCs) may be safer using targeted integration (TI) of transgenes into a genomic ‘safe harbor’ site than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno associated virus (AAV) 6 delivery of donor constructs in human HSCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus-positive HSCs with 6–16% human cell marking were observed following engraftment into mice. In HSCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 in resulted in ~15% gp91phox expression and increased NADPH oxidase activity in ex vivo–derived neutrophils. In mice transplanted with corrected HSCs, 4–11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases. PMID:26950749

Evolving phage vectors for cell targeted gene delivery.

PubMed

Larocca, David; Burg, Michael A; Jensen-Pergakes, Kristen; Ravey, Edward Prenn; Gonzalez, Ana Maria; Baird, Andrew

2002-03-01

We adapted filamentous phage vectors for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (GFP) into the vector backbone and fusing the pIII coat protein to a cell targeting ligand (i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage gene delivery is concentration, time, and ligand dependent. Importantly, targeted phage particles are specific for the appropriate target cell surface receptor. Phage have distinct advantages over existing gene therapy vectors because they are simple, economical to produce at high titer, have no intrinsic tropism for mammalian cells, and are relatively simple to genetically modify and evolve. Initially transduction by targeted phage particles was low resulting in foreign gene expression in 1-2% of transfected cells. We increased transduction efficiency by modifying both the transfection protocol and vector design. For example, we stabilized the display of the targeting ligand to create multivalent phagemid-based vectors with transduction efficiencies of up to 45% in certain cell lines when combined with genotoxic treatment. Taken together, these studies establish that the efficiency of phage-mediated gene transfer can be significantly improved through genetic modification. We are currently evolving phage vectors with enhanced cell targeting, increased stability, reduced immunogenicity and other properties suitable for gene therapy.

Malaria Prevention by New Technology: Vectored Delivery of Antibody Genes

DTIC Science & Technology

2017-10-01

AWARD NUMBER: W81XWH-15-1-0401 TITLE: Malaria Prevention by New Technology : Vectored Delivery of Antibody Genes PRINCIPAL INVESTIGATOR: Gary...CONTRACT NUMBER Malaria Prevention by New Technology : Vectored Delivery of Antibody Genes 5b. GRANT NUMBER W81XWH-15-1-0401 5c. PROGRAM ELEMENT...whole animals. Using a specific technology originally applied to expression of HIV antibodies, we demonstrated that mice can be protected from

Recent progresses in gene delivery-based bone tissue engineering.

PubMed

Lu, Chia-Hsin; Chang, Yu-Han; Lin, Shih-Yeh; Li, Kuei-Chang; Hu, Yu-Chen

2013-12-01

Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches. © 2013.

Safe Prevention of the Primary Cesarean Delivery: ACOG and SMFM Change the Game

PubMed Central

Lothian, Judith A.

2014-01-01

Safe Prevention of the Primary Cesarean Delivery, a joint statement of the American College of Obstetricians and Gynecologists and the Society for Maternal-Fetal Medicine, changes the rules of maternity care management. The statement reviews the research and uses the findings to make practice recommendations. This article discusses the major recommendations and their importance in decreasing risk and increasing safety for mothers and babies. The articles in the current issue of the journal are also reviewed.

The magnetofection method: using magnetic force to enhance gene delivery.

PubMed

Plank, Christian; Schillinger, Ulrike; Scherer, Franz; Bergemann, Christian; Rémy, Jean-Serge; Krötz, Florian; Anton, Martina; Lausier, Jim; Rosenecker, Joseph

2003-05-01

In order to enhance and target gene delivery we have previously established a novel method, termed magnetofection, which uses magnetic force acting on gene vectors that are associated with magnetic particles. Here we review the benefits, the mechanism and the potential of the method with regard to overcoming physical limitations to gene delivery. Magnetic particle chemistry and physics are discussed, followed by a detailed presentation of vector formulation and optimization work. While magnetofection does not necessarily improve the overall performance of any given standard gene transfer method in vitro, its major potential lies in the extraordinarily rapid and efficient transfection at low vector doses and the possibility of remotely controlled vector targeting in vivo.

Lumbar spine intervertebral disc gene delivery: a pilot study in lewis rats.

PubMed

Damle, Sheela R; Rawlins, Bernard A; Boachie-Adjei, Oheneba; Crystal, Ronald G; Hidaka, Chisa; Cunningham, Matthew E

2013-02-01

Basic research toward understanding and treating disc pathology in the spine has utilized numerous animal models, with delivery of small molecules, purified factors, and genes of interest. To date, gene delivery to the rat lumbar spine has only been described utilizing genetically programmed cells in a matrix which has required partial disc excision, and expected limitation of treatment diffusion into the disc. This study was designed to develop and describe a surgical technique for lumbar spine exposure and disc space preparation, and use of a matrix-free method for gene delivery. Naïve or genetically programmed isogeneic bone marrow stromal cells were surgically delivered to adolescent male Lewis rat lumbar discs, and utilizing quantitative biochemical and qualitative immunohistological assessments, the implanted cells were detected 3 days post-procedure. Statistically significant differences were noted for recovery of the β-galactosidase marker gene comparing delivery of naïve or labeled cells (10(5) cells per disc) from the site of implantation, and between delivery of 10(5) or 10(6) labeled cells per disc at the site of implantation and the adjacent vertebral body. Immunohistology confirmed that the β-galactosidase marker was detected in the adjacent vertebra bone in the zone of surgical implantation. The model requires further testing in larger cohorts and with biologically active genes of interest, but the observations from the pilot experiments are very encouraging that this will be a useful comparative model for basic spine research involving gene or cell delivery, or other locally delivered therapies to the intervertebral disc or adjacent vertebral bodies in rats.

Mutated-leptin gene transfer induces increases in body weight by electroporation and hydrodynamics-based gene delivery in mice.

PubMed

Xiang, Lan; Murai, Atsushi; Muramatsu, Tatsuo

2005-12-01

To investigate whether in vivo gene transfer causes leptin-antagonistic effects on food intake, animal body weight and fat tissue weight, the R128Q mutated-leptin gene, an R to Q substitution at position 128 of mouse leptin, was transferred into mouse liver and leg muscle by electroporation and hydrodynamics-based gene delivery. Mutated-leptin gene transfer by electroporation caused significant increases in body weight at 5 days and after (5.4% increase relative to control; p<0.05). Hydrodynamics-based gene delivery of the mutated-leptin gene also caused an increase in body weight (3.0% increase relative to control; p<0.05). Mutated-leptin gene transfer by electroporation significantly increased the tissue weight of epididymal white fat and neuropeptide Y mRNA expression in the hypothalamus compared with those of the control group 3 weeks after gene transfer (p<0.05). These results suggest that mutated-leptin gene transfer successfully produced leptin-antagonistic effects by modulating the central regulator of energy homeostasis. Also, the extent of leptin-antagonistic effects by electroporation was much higher than hydrodynamics-based gene delivery, with at least single gene transfer.

New serine-derived gemini surfactants as gene delivery systems.

PubMed

Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Silva, Sandra G; do Vale, M Luísa; Marques, Eduardo F; de Lima, Maria C Pedroso; Jurado, Amália S

2015-01-01

Gemini surfactants have been extensively used for in vitro gene delivery. Amino acid-derived gemini surfactants combine the special aggregation properties characteristic of the gemini surfactants with high biocompatibility and biodegradability. In this work, novel serine-derived gemini surfactants, differing in alkyl chain lengths and in the linker group bridging the spacer to the headgroups (amine, amide and ester), were evaluated for their ability to mediate gene delivery either per se or in combination with helper lipids. Gemini surfactant-based DNA complexes were characterized in terms of hydrodynamic diameter, surface charge, stability in aqueous buffer and ability to protect DNA. Efficient formulations, able to transfect up to 50% of the cells without causing toxicity, were found at very low surfactant/DNA charge ratios (1/1-2/1). The most efficient complexes presented sizes suitable for intravenous administration and negative surface charge, a feature known to preclude potentially adverse interactions with serum components. This work brings forward a new family of gemini surfactants with great potential as gene delivery systems. Copyright © 2014 Elsevier B.V. All rights reserved.

Au nanoinjectors for electrotriggered gene delivery into the cell nucleus.

PubMed

Kang, Mijeong; Kim, Bongsoo

2015-01-01

Intracellular delivery of exogenous materials is an essential technique required for many fundamental biological researches and medical treatments. As our understanding of cell structure and function has been improved and diverse therapeutic agents with a subcellular site of action have been continuously developed, there is a demand to enhance the performance of delivering devices. Ideal intracellular delivery devices should convey various kinds of exogenous materials without deteriorating cell viability regardless of cell type and, furthermore, precisely control the location and the timing of delivery as well as the amount of delivered materials for advanced researches.In this chapter the development of a new intracellular delivery device, a nanoinjector made of a Au (gold) nanowire (a Au nanoinjector) is described in which delivery is triggered by external application of an electric pulse. As a model study, a gene was delivered directly into the nucleus of a neuroblastoma cell, and successful delivery without cell damage was confirmed by the expression of the delivered gene. The insertion of a Au nanoinjector directly into a cell can be generally applied to any kind of cell, and a high degree of surface modification of Au allows attachment of diverse materials such as proteins, small molecules, or nanoparticles as well as genes on Au nanoinjectors. This expands their applicability, and it is expected that they will provide important information on the effects of delivered exogenous materials and consequently contribute to the development of related therapeutic or clinical technologies.

Engineering of small interfering RNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles for highly efficient and safe gene silencing: A quality by design-based approach.

PubMed

Thanki, Kaushik; Zeng, Xianghui; Justesen, Sarah; Tejlmann, Sarah; Falkenberg, Emily; Van Driessche, Elize; Mørck Nielsen, Hanne; Franzyk, Henrik; Foged, Camilla

2017-11-01

Safety and efficacy of therapeutics based on RNA interference, e.g., small interfering RNA (siRNA), are dependent on the optimal engineering of the delivery technology, which is used for intracellular delivery of siRNA to the cytosol of target cells. We investigated the hypothesis that commonly used and poorly tolerated cationic lipids might be replaced with more efficacious and safe lipidoids as the lipid component of siRNA-loaded lipid-polymer hybrid nanoparticles (LPNs) for achieving more efficient gene silencing at lower and safer doses. However, formulation design of such a complex formulation is highly challenging due to a strong interplay between several contributing factors. Hence, critical formulation variables, i.e. the lipidoid content and siRNA:lipidoid ratio, were initially identified, followed by a systematic quality-by-design approach to define the optimal operating space (OOS), eventually resulting in the identification of a robust, highly efficacious and safe formulation. A 17-run design of experiment with an I-optimal approach was performed to systematically assess the effect of selected variables on critical quality attributes (CQAs), i.e. physicochemical properties (hydrodynamic size, zeta potential, siRNA encapsulation/loading) and the biological performance (in vitro gene silencing and cell viability). Model fitting of the obtained data to construct predictive models revealed non-linear relationships for all CQAs, which can be readily overlooked in one-factor-at-a-time optimization approaches. The response surface methodology further enabled the identification of an OOS that met the desired quality target product profile. The optimized lipidoid-modified LPNs revealed more than 50-fold higher in vitro gene silencing at well-tolerated doses and approx. a twofold increase in siRNA loading as compared to reference LPNs modified with the commonly used cationic lipid dioleyltrimethylammonium propane (DOTAP). Thus, lipidoid-modified LPNs show highly

Gene delivery by a steroid-peptide nucleic acid conjugate.

PubMed

Rebuffat, Alexandre G; Nawrocki, Andrea R; Nielsen, Peter E; Bernasconi, Alessio G; Bernal-Mendez, Eloy; Frey, Brigitte M; Frey, Felix J

2002-09-01

We previously introduced a method called steroid-mediated gene delivery (SMGD), which uses steroid receptors as shuttles to facilitate the nuclear uptake of transfected DNA. Here, we describe a SMGD strategy with peptide nucleic acids (PNAs) that allowed linkage of a steroid molecule to a defined position in a plasmid without disturbing its gene expression. We synthesized and tested several bifunctional steroid derivatives [patent in process of nationalization] and finally selected the compound named DEX-bisPNA, a molecule consisting of a dexamethasone moiety linked to a PNA clamp (bisPNA) through a 30-atom chemical spacer. Dex-bisPNA binds to the glucocorticoid receptor (GR) as well as to reporter plasmids containing the corresponding PNA binding sites, translocates the GR from the cytoplasm into the nucleus, and increases the delivery of plasmid to the nucleus, resulting in enhanced GR-dependent expression of the reporter gene. The SMGD effect was more pronounced in growth-arrested cells than in proliferating cells. The specificity for the GR was shown by the reversion of the SMGD effect in the presence of dexamethasone as well as an enhanced expression in GR-positive cells but not in GR-negative cells. Thus, SMGD with PNA is a promising strategy for nonviral gene delivery into target tissues expressing specific steroid receptors.

A sight on protein-based nanoparticles as drug/gene delivery systems.

PubMed

Salatin, Sara; Jelvehgari, Mitra; Maleki-Dizaj, Solmaz; Adibkia, Khosro

2015-01-01

Polymeric nanomaterials have extensively been applied for the preparation of targeted and controlled release drug/gene delivery systems. However, problems involved in the formulation of synthetic polymers such as using of the toxic solvents and surfactants have limited their desirable applications. In this regard, natural biomolecules including proteins and polysaccharide are suitable alternatives due to their safety. According to literature, protein-based nanoparticles possess many advantages for drug and gene delivery such as biocompatibility, biodegradability and ability to functionalize with targeting ligands. This review provides a general sight on the application of biodegradable protein-based nanoparticles in drug/gene delivery based on their origins. Their unique physicochemical properties that help them to be formulated as pharmaceutical carriers are also discussed.

Innovations in gene and growth factor delivery systems for diabetic wound healing

PubMed Central

Laiva, Ashang Luwang; O'Brien, Fergal J.

2017-01-01

Abstract The rise in lower extremity amputations due to nonhealing of foot ulcers in diabetic patients calls for rapid improvement in effective treatment regimens. Administration of growth factors (GFs) are thought to offer an off‐the‐shelf treatment; however, the dose‐ and time‐dependent efficacy of the GFs together with the hostile environment of diabetic wound beds impose a major hindrance in the selection of an ideal route for GF delivery. As an alternative, the delivery of therapeutic genes using viral and nonviral vectors, capable of transiently expressing the genes until the recovery of the wounded tissue offers promise. The development of implantable biomaterial dressings capable of modulating the release of either single or combinatorial GFs/genes may offer solutions to this overgrowing problem. This article reviews the state of the art on gene and protein delivery and the strategic optimization of clinically adopted delivery strategies for the healing of diabetic wounds. PMID:28482114

Recent Advances in Skin Penetration Enhancers for Transdermal Gene and Drug Delivery.

PubMed

Amjadi, Morteza; Mostaghaci, Babak; Sitti, Metin

2017-01-01

There is a growing interest in transdermal delivery systems because of their noninvasive, targeted, and on-demand delivery of gene and drugs. However, efficient penetration of therapeutic compounds into the skin is still challenging largely due to the impermeability of the outermost layer of the skin, known as stratum corneum. Recently, there have been major research activities to enhance the skin penetration depth of pharmacological agents. This article reviews recent advances in the development of various strategies for skin penetration enhancement. We show that approaches such as ultrasound waves, laser, and microneedle patches have successfully been employed to physically disrupt the stratum corneum structure for enhanced transdermal delivery. Rather than physical approaches, several non-physical route have also been utilized for efficient transdermal delivery across the skin barrier. Finally, we discuss some clinical applications of transdermal delivery systems for gene and drug delivery. This paper shows that transdermal delivery devices can potentially function for diverse healthcare and medical applications while further investigations are still necessary for more efficient skin penetration of gene and drugs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Enhancing endosomal escape for nanoparticle mediated siRNA delivery

NASA Astrophysics Data System (ADS)

Ma, Da

2014-05-01

Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery, including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. Work has been done to develop different endosomal escape strategies based on nanoparticle types, administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers, great progress has been achieved in nanoparticle mediated siRNA delivery.

Selenium nanoparticles: potential in cancer gene and drug delivery.

PubMed

Maiyo, Fiona; Singh, Moganavelli

2017-05-01

In recent decades, colloidal selenium nanoparticles have emerged as exceptional selenium species with reported chemopreventative and therapeutic properties. This has sparked widespread interest in their use as a carrier of therapeutic agents with results displaying synergistic effects of selenium with its therapeutic cargo and improved anticancer activity. Functionalization remains a critical step in selenium nanoparticles' development for application in gene or drug delivery. In this review, we highlight recent developments in the synthesis and functionalization strategies of selenium nanoparticles used in cancer drug and gene delivery systems. We also provide an update of recent preclinical studies utilizing selenium nanoparticles in cancer therapeutics.

Carboxymethylcellulose (CMC) formed nanogels with branched poly(ethyleneimine) (bPEI) for inhibition of cytotoxicity in human MSCs as a gene delivery vehicles.

PubMed

Yang, Han Na; Park, Ji Sun; Jeon, Su Yeon; Park, Keun-Hong

2015-05-20

Specific vehicles are necessary for safe and efficient gene transfection into cells. Nano-type hydrogels (nanogel) comprising carboxymethylcellulose (CMC) complexed with branched type cationic poly(ethleneimine) (bPEI) were used as gene delivery vehicles. When complexes of CMC and bPEI were used in vitro, CMC showed nano-gel type properties, as shown by the results of a viscosity test, and bPEI showed low cytotoxicity comparing to bPEI alone. Together, these properties are shown to maintain high gene transfection efficiency. In viability experiments using three types of adult stem cells, cell viability varied depending on the branch form of PEI and whether or not it is in a complex with CMC. The gene delivery efficacy showed that the CMC nanogel complexed with bPEI (CMC-bPEI) showed more uptaking and gene transfection ability in hMSCs comparing to bPEI alone. In osteogenesis, the CMC-bPEI complexed with OSX pDNA showed more easy internalization than bPEI alone complexed with OSX pDNA in hMSCs. Specific genes and proteins related in osteogenic differentiation were expressed in hMSCs when the CMC-bPEI complexed with OSX pDNA was used. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ranking of healthcare programmes based on health outcome, health costs and safe delivery of care in hospital pharmacy practice.

PubMed

Brisseau, Lionel; Bussières, Jean-François; Bois, Denis; Vallée, Marc; Racine, Marie-Claude; Bonnici, André

2013-02-01

To establish a consensual and coherent ranking of healthcare programmes that involve the presence of ward-based and clinic-based clinical pharmacists, based on health outcome, health costs and safe delivery of care. This descriptive study was derived from a structured dialogue (Delphi technique) among directors of pharmacy department. We established a quantitative profile of healthcare programmes at five sites that involved the provision of ward-based and clinic-based pharmaceutical care. A summary table of evidence established a unique quality rating per inpatient (clinic-based) or outpatient (ward-based) healthcare programme. Each director rated the perceived impact of pharmaceutical care per inpatient or outpatient healthcare programme on three fields: health outcome, health costs and safe delivery of care. They agreed by consensus on the final ranking of healthcare programmes. A ranking was assigned for each of the 18 healthcare programmes for outpatient care and the 17 healthcare programmes for inpatient care involving the presence of pharmacists, based on health outcome, health costs and safe delivery of care. There was a good correlation between ranking based on data from a 2007-2008 Canadian report on hospital pharmacy practice and the ranking proposed by directors of pharmacy department. Given the often limited human and financial resources, managers should consider the best evidence available on a profession's impact to plan healthcare services within an organization. Data are few on ranking healthcare programmes in order to prioritize which healthcare programme would mostly benefit from the delivery of pharmaceutical care by ward-based and clinic-based pharmacists. © 2012 The Authors. IJPP © 2012 Royal Pharmaceutical Society.

Nanomedicines for Back of the Eye Drug Delivery, Gene Delivery, and Imaging

PubMed Central

Kompella, Uday B.; Amrite, Aniruddha C.; Ravi, Rashmi Pacha; Durazo, Shelley A.

2013-01-01

Treatment and management of diseases of the posterior segment of the eye such as diabetic retinopathy, retinoblastoma, retinitis pigmentosa, and choroidal neovascularization is a challenging task due to the anatomy and physiology of ocular barriers. For instance, traditional routes of drug delivery for therapeutic treatment are hindered by poor intraocular penetration and/or rapid ocular elimination. One possible approach to improve ocular therapy is to employ nanotechnology. Nanomedicines, products of nanotechnology, having at least one dimension in the nanoscale include nanoparticles, micelles, nanotubes, and dendrimers, with and without targeting ligands, are making a significant impact in the fields of ocular drug delivery, gene delivery, and imaging, the focus of this review. Key applications of nanotechnology discussed in this review include a) bioadhesive nanomedicines; b) functionalized nanomedicines that enhance target recognition and/or cell entry; c) nanomedicines capable of controlled release of the payload; d) nanomedicines capable of enhancing gene transfection and duration of transfection; f) nanomedicines responsive to stimuli including light, heat, ultrasound, electrical signals, pH, and oxidative stress; g) diversely sized and colored nanoparticles for imaging, and h) nanowires for retinal prostheses. Additionally, nanofabricated delivery systems including implants, films, microparticles, and nanoparticles are described. Although the above nanomedicines may be administered by various routes including topical, intravitreal, intravenous, transscleral, suprachoroidal, and subretinal routes, each nanomedicine should be tailored for the disease, drug, and site of administration. In addition to the nature of materials used in nanomedicine design, depending on the site of nanomedicine administration, clearance and toxicity are expected to differ. PMID:23603534

Electroporation-mediated Delivery of Genes in Rodent Models of Lung Contusion

PubMed Central

Machado-Aranda, David; Raghavendran, Krishnan

2015-01-01

Several of the biological processes involved in the pathogenesis of acute lung injury and acute respiratory distress syndrome after lung contusion are regulated at a genetic and epigenetic level. Thus, strategies to manipulate gene expression in this context are highly desirable not only to elucidate the mechanisms involved but also to look for potential therapies. In the present chapter, we describe mouse and rat models of inducing blunt thoracic injury followed by electroporation-mediated gene delivery to the lung. Electroporation is a highly efficient and easily reproducible technique that allows circumvention of several of lung gene delivery challenges and safety issues present with other forms of lung gene therapy. PMID:24510825

Synthetic Virology: Engineering Viruses for Gene Delivery

PubMed Central

Guenther, Caitlin M.; Kuypers, Brianna E.; Lam, Michael T.; Robinson, Tawana M.; Zhao, Julia; Suh, Junghae

2014-01-01

The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or “bionic” viruses, feature engineered components, or “parts”, that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies – rational, combinatorial, and pseudo-rational – have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavours will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine. PMID:25195922

Synthetic virology: engineering viruses for gene delivery.

PubMed

Guenther, Caitlin M; Kuypers, Brianna E; Lam, Michael T; Robinson, Tawana M; Zhao, Julia; Suh, Junghae

2014-01-01

The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or 'bionic' viruses, feature engineered components, or 'parts', that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies--rational, combinatorial, and pseudo-rational--have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavors will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine. © 2014 Wiley Periodicals, Inc.

Amphiphilic graft copolymer based on poly(styrene-co-maleic anhydride) with low molecular weight polyethylenimine for efficient gene delivery

PubMed Central

Duan, Xiaopin; Xiao, Jisheng; Yin, Qi; Zhang, Zhiwen; Mao, Shirui; Li, Yaping

2012-01-01

Background and methods A new amphiphilic comb-shaped copolymer (SP) was synthesized by conjugating poly(styrene-co-maleic anhydride) with low molecular weight polyethyleneimine for gene delivery. Fourier transform infrared spectrum, 1H nuclear magnetic resonance, and gel permeation chromatography were used to characterize the graft copolymer. Results The buffering capability of SP was similar to that of polyethyleneimine within the endosomal pH range. The copolymer could condense DNA effectively to form complexes with a positive charge (13–30 mV) and a small particle size (130–200 nm) at N/P ratios between 5 and 20, and protect DNA from degradation by DNase I. In addition, SP showed much lower cytotoxicity than polyethyleneimine 25,000. Importantly, the gene transfection activity and cellular uptake of SP-DNA complexes were all markedly higher than that of complexes of polyethyleneimine 25,000 and DNA in MCF-7 and MCF-7/ADR cell lines. Conclusion This work highlights the promise of SP as a safe and efficient synthetic vector for DNA delivery. PMID:23028224

The evolution of heart gene delivery vectors

PubMed Central

Wasala, Nalinda B.; Shin, Jin-Hong; Duan, Dongsheng

2012-01-01

Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer. PMID:21837689

Degradable polyethylenimine derivate coupled to a bifunctional peptide R13 as a new gene-delivery vector

PubMed Central

Liu, Kehai; Wang, Xiaoyu; Fan, Wei; Zhu, Qing; Yang, Jingya; Gao, Jing; Gao, Shen

2012-01-01

Background To solve the efficiency versus cytotoxicity and tumor-targeting problems of polyethylenimine (PEI) used as a nonviral gene delivery vector, a degradable PEI derivate coupled to a bifunctional peptide R13 was developed. Methods First, we synthesized a degradable PEI derivate by crosslinking low-molecular-weight PEI with pluronic P123, then used tumor-targeting peptide arginine-glycine-aspartate-cysteine (RGDC), in conjunction with the cell-penetrating peptide Tat (49–57), to yield a bifunctional peptide RGDC-Tat (49–57) named R13, which can improve cell selection and increase cellular uptake, and, lastly, adopted R13 to modify the PEI derivates so as to prepare a new polymeric gene vector (P123-PEI-R13). The new gene vector was characterized in terms of its chemical structure and biophysical parameters. We also investigated the specificity, cytotoxicity, and gene transfection efficiency of this vector in αvβ3-positive human cervical carcinoma Hela cells and murine melanoma B16 cells in vitro. Results The vector showed controlled degradation, strong targeting specificity to αvβ3 receptor, and noncytotoxicity in Hela cells and B16 cells at higher doses, in contrast to PEI 25 KDa. The particle size of P123-PEI-R13/DNA complexes was around 100–250 nm, with proper zeta potential. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 6 U DNase I/μg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 600 μg/mL sodium heparin. P123-PEI-R13 also revealed higher transfection efficiency in two cell lines as compared with PEI 25 KDa. Conclusion P123-PEI-R13 is a potential candidate as a safe and efficient gene-delivery carrier for gene therapy. PMID:22412301

Preparation and testing of quaternized chitosan nanoparticles as gene delivery vehicles.

PubMed

Li, Guang-Feng; Wang, Jing-Cheng; Feng, Xin-Min; Liu, Zhen-Dong; Jiang, Chao-Yong; Yang, Jian-Dong

2015-04-01

significant difference in gene delivery efficiency was detected between HACC/pEGFP-GDNF and liposome/pEGFP-GDNF complexes (33.8 vs. 34 %, P = 0.363). In this study, HACC was successfully synthesized, and HACC/DNA complex assembled efficiently. HACC showed strong DNA binding affinity and high protection of DNA and was non-cytotoxic to human cells. The particles had appropriate nanostructure, mean diameter, and DNA release time. The results suggest that HACC nanoparticles are a novel tool for efficient and safe gene delivery.

The evolution of heart gene delivery vectors.

PubMed

Wasala, Nalinda B; Shin, Jin-Hong; Duan, Dongsheng

2011-10-01

Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer. Copyright © 2011 John Wiley & Sons, Ltd.

Specific gene delivery to liver sinusoidal and artery endothelial cells.

PubMed

Abel, Tobias; El Filali, Ebtisam; Waern, Johan; Schneider, Irene C; Yuan, Qinggong; Münch, Robert C; Hick, Meike; Warnecke, Gregor; Madrahimov, Nodir; Kontermann, Roland E; Schüttrumpf, Jörg; Müller, Ulrike C; Seppen, Jurgen; Ott, Michael; Buchholz, Christian J

2013-09-19

Different types of endothelial cells (EC) fulfill distinct tasks depending on their microenvironment. ECs are therefore difficult to genetically manipulate ex vivo for functional studies or gene therapy. We assessed lentiviral vectors (LVs) targeted to the EC surface marker CD105 for in vivo gene delivery. The mouse CD105-specific vector, mCD105-LV, transduced only CD105-positive cells in primary liver cell cultures. Upon systemic injection, strong reporter gene expression was detected in liver where mCD105-LV specifically transduced liver sinusoidal ECs (LSECs) but not Kupffer cells, which were mainly transduced by nontargeted LVs. Tumor ECs were specifically targeted upon intratumoral vector injection. Delivery of the erythropoietin gene with mCD105-LV resulted in substantially increased erythropoietin and hematocrit levels. The human CD105-specific vector (huCD105-LV) transduced exclusively human LSECs in mice transplanted with human liver ECs. Interestingly, when applied at higher dose and in absence of target cells in the liver, huCD105-LV transduced ECs of a human artery transplanted into the descending mouse aorta. The data demonstrate for the first time targeted gene delivery to specialized ECs upon systemic vector administration. This strategy offers novel options to better understand the physiological functions of ECs and to treat genetic diseases such as those affecting blood factors.

Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery.

PubMed

Ueki, Shoko; Magori, Shimpei; Lacroix, Benoît; Citovsky, Vitaly

2013-01-01

Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure.

Polyethyleneimine grafted short halloysite nanotubes for gene delivery.

PubMed

Long, Zheru; Zhang, Jun; Shen, Yan; Zhou, Changren; Liu, Mingxian

2017-12-01

Inorganic nanoparticles have attracted much attentions in gene delivery because of their desirable characteristics including low toxicity, well-controlled characteristics, high gene delivery efficiency, and multi-functionalities. Here, natural occurred halloysite nanotubes (HNTs) were developed as a novel non-viral gene vector. To increase the efficiency of endocytosis, HNTs were firstly shortened into an appropriate size (~200nm). Then polyethyleneimine (PEI) was grafted onto HNTs to bind green fluorescence protein (GFP) labeled pDNA. The structure and physical-chemical properties of PEI grafted HNTs (PEI-g-HNTs) were characterized by various methods. PEI-g-HNTs show lower cytotoxicity than PEI. PEI-g-HNTs are positively charged and can bind DNA tightly at designed N/P ratio from 5:1 to 40:1. PEI-g-HNTs/pDNA complexes show much higher transfection efficiency towards both 293T and HeLa cells compared with PEI/pDNA complexes at the equivalent N/P ratio. The transfection efficiencies of PEI-g-HNTs/pDNA complex towards HeLa cell can reach to 44.4% at N/P ratio of 20. PEI-g-HNTs/pDNA complexes possess a higher GFP protein expression than PEI/pDNA from simple western immunoblots. So, PEI-g-HNTs are potential gene vectors with good biocompatibility and high transfection efficiency, which have promising applications in cancer gene therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel Approaches for Retinal Drug and Gene Delivery

PubMed Central

Kim, Stephen J.

2014-01-01

The ARVO 2014 minisymposium on “Novel Approaches for Retinal Drug and Gene Delivery” was held on May 6, 2014 in Orlando, FL. The main intent of the symposium was to review recent advances in retinal drug and gene delivery with specific emphasis on novel approaches that address current limitations and have the potential to translate into clinical practice. The symposium was sponsored by Translational Vision Science and Technology. PMID:25346872

Non-viral delivery of genome-editing nucleases for gene therapy.

PubMed

Wang, M; Glass, Z A; Xu, Q

2017-03-01

Manipulating the genetic makeup of mammalian cells using programmable nuclease-based genome-editing technology has recently evolved into a powerful avenue that holds great potential for treating genetic disorders. There are four types of genome-editing nucleases, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and clustered, regularly interspaced, short palindromic repeat-associated nucleases such as Cas9. These nucleases have been harnessed to introduce precise and specific changes of the genome sequence at virtually any genome locus of interest. The therapeutic relevance of these genome-editing technologies, however, is challenged by the safe and efficient delivery of nuclease into targeted cells. Herein, we summarize recent advances that have been made on non-viral delivery of genome-editing nucleases. In particular, we focus on non-viral delivery of Cas9/sgRNA ribonucleoproteins for genome editing. In addition, the future direction for developing non-viral delivery of programmable nucleases for genome editing is discussed.

Drug-conjugated polymers as gene carriers for synergistic therapeutic effect.

PubMed

Pofali, P A; Singh, B; Dandekar, P; Jain, R D; Maharjan, S; Choi, Y J; Arote, R B; Cho, C S

2016-05-01

The ability to safely and effectively transfer gene into cells is the fundamental goal of gene delivery. In spite of the best efforts of researchers around the world, gene therapy has limited success. This may be because of several limitations of delivering gene which is one of the greatest technical challenges in the modern medicine. To address these issues, many efforts have been made to bind drugs and genes together by polymers for co-delivery to achieve synergistic effect. Usually, binding interaction of drugs with polymers is either physical or chemical. In case of drug-polymer physical interaction, the efficiency of drugs generally decreases because of separation of drugs from polymers in vivo whenever it comes in contact with charged biofluid/s or cells. While chemical interaction of drug-polymer overcomes the aforementioned obstacle, several problems such as steric hindrance, solubility, and biodegradability hinder it to develop as gene carrier. Considering these benefits and pitfalls, the objective of this review is to discuss the possible extent of drug-conjugated polymers as safe and efficient gene delivery carriers for achieving synergistic effect to combat various genetic disorders. © 2015 Wiley Periodicals, Inc.

In vivo correction of anaemia in β-thalassemic mice by γPNA-mediated gene editing with nanoparticle delivery

PubMed Central

Bahal, Raman; Ali McNeer, Nicole; Quijano, Elias; Liu, Yanfeng; Sulkowski, Parker; Turchick, Audrey; Lu, Yi-Chien; Bhunia, Dinesh C.; Manna, Arunava; Greiner, Dale L.; Brehm, Michael A.; Cheng, Christopher J.; López-Giráldez, Francesc; Ricciardi, Adele; Beloor, Jagadish; Krause, Diane S.; Kumar, Priti; Gallagher, Patrick G.; Braddock, Demetrios T.; Mark Saltzman, W.; Ly, Danith H.; Glazer, Peter M.

2016-01-01

The blood disorder, β-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human β-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% β-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration. PMID:27782131

Adenovirus receptors and their implications in gene delivery

PubMed Central

Sharma, Anurag; Li, Xiaoxin; Bangari, Dinesh S.; Mittal, Suresh K.

2010-01-01

Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed. PMID:19647886

In vivo gene delivery and expression by bacteriophage lambda vectors.

PubMed

Lankes, H A; Zanghi, C N; Santos, K; Capella, C; Duke, C M P; Dewhurst, S

2007-05-01

Bacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo. Mice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo. Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer. Unmodified lambda phage particles are capable of transducing mammalian cells in vivo, and may be taken up -- at least in part -- by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer. These experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo, and suggest new approaches that may enhance the efficiency of this process.

Novel gemini cationic lipids with carbamate groups for gene delivery

PubMed Central

Zhao, Yi-Nan; Qureshi, Farooq; Zhang, Shu-Biao; Cui, Shao-Hui; Wang, Bing; Chen, Hui-Ying; Lv, Hong-Tao; Zhang, Shu-Fen; Huang, Leaf

2014-01-01

To obtain efficient non-viral vectors, a series of Gemini cationic lipids with carbamate linkers between headgroups and hydrophobic tails were synthesized. They have the hydrocarbon chains of 12, 14, 16 and 18 carbon atoms as tails, designated as G12, G14, G16 and G18, respectively. These Gemini cationic lipids were prepared into cationic liposomes for the study of the physicochemical properties and gene delivery. The DNA-bonding ability of these Gemini cationic liposomes was much better than their mono-head counterparts (designated as M12, M14, M16 and M18, respectively). In the same series of liposomes, bonding ability declined with an increase in tail length. They were tested for their gene-transferring capabilities in Hep-2 and A549 cells. They showed higher transfection efficiency than their mono-head counterparts and were comparable or superior in transfection efficiency and cytotoxicity to the commercial liposomes, DOTAP and Lipofectamine 2000. Our results convincingly demonstrate that the gene-transferring capabilities of these cationic lipids depended on hydrocarbon chain length. Gene transfection efficiency was maximal at a chain length of 14, as G14 can silence about 80 % of luciferase in A549 cells. Cell uptake results indicate that Gemini lipid delivery systems could be internalised by cells very efficiently. Thus, the Gemini cationic lipids could be used as synthetic non-viral gene delivery carriers for further study. PMID:25045521

Self-Assembled Peptide-Lanthanide Nanoclusters for Safe Tumor Therapy: Overcoming and Utilizing Biological Barriers to Peptide Drug Delivery.

PubMed

Yan, Jin; He, Wangxiao; Yan, Siqi; Niu, Fan; Liu, Tianya; Ma, Bohan; Shao, Yongping; Yan, Yuwei; Yang, Guang; Lu, Wuyuan; Du, Yaping; Lei, Bo; Ma, Peter X

2018-02-27

Developing a sophisticated nanomedicine platform to deliver therapeutics effectively and safely into tumor/cancer cells remains challenging in the field of nanomedicine. In particular, reliable peptide drug delivery systems capable of overcoming biological barriers are still lacking. Here, we developed a simple, rapid, and robust strategy to manufacture nanoclusters of ∼90 nm in diameter that are self-assembled from lanthanide-doped nanoparticles (5 nm), two anticancer peptides with different targets (BIM and PMI), and one cyclic peptide iNGR targeted to cancer cells. The peptide-lanthanide nanoclusters (LDC-PMI-BIM-iNGR) enhanced the resistance of peptide drugs to proteolysis, disassembled in response to reductive conditions that are present in the tumor microenvironment and inhibited cancer cell growth in vitro and in vivo. Notably, LDC-PMI-BIM-iNGR exhibited extremely low systemic toxicity and side effects in vivo. Thus, the peptide-lanthanide nanocluster may serve as an ideal multifunctional platform for safe, targeted, and efficient peptide drug delivery in cancer therapy.

Baculovirus: an Insect-derived Vector for Diverse Gene Transfer Applications

PubMed Central

Airenne, Kari J; Hu, Yu-Chen; Kost, Thomas A; Smith, Richard H; Kotin, Robert M; Ono, Chikako; Matsuura, Yoshiharu; Wang, Shu; Ylä-Herttuala, Seppo

2013-01-01

Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered. PMID:23439502

Carbon Nanotubes in Drug and Gene Delivery

NASA Astrophysics Data System (ADS)

Karimi, Mahdi; Ghasemi, Amir; Mirkiani, Soroush; Moosavi Basri, Seyed Masoud; Hamblin, Michael R.

2017-10-01

Recent important discoveries and developments in nanotechnology have had a remarkable and ever-increasing impact on many industries, especially materials science, pharmaceuticals, and biotechnology. Within this book, the authors describe different features of carbon nanotubes, survey the properties of both the multi-walled and single-walled varieties, and cover their applications in drug and gene delivery.

Biopolymer-Based Nanoparticles for Drug/Gene Delivery and Tissue Engineering

PubMed Central

Nitta, Sachiko Kaihara; Numata, Keiji

2013-01-01

There has been a great interest in application of nanoparticles as biomaterials for delivery of therapeutic molecules such as drugs and genes, and for tissue engineering. In particular, biopolymers are suitable materials as nanoparticles for clinical application due to their versatile traits, including biocompatibility, biodegradability and low immunogenicity. Biopolymers are polymers that are produced from living organisms, which are classified in three groups: polysaccharides, proteins and nucleic acids. It is important to control particle size, charge, morphology of surface and release rate of loaded molecules to use biopolymer-based nanoparticles as drug/gene delivery carriers. To obtain a nano-carrier for therapeutic purposes, a variety of materials and preparation process has been attempted. This review focuses on fabrication of biocompatible nanoparticles consisting of biopolymers such as protein (silk, collagen, gelatin, β-casein, zein and albumin), protein-mimicked polypeptides and polysaccharides (chitosan, alginate, pullulan, starch and heparin). The effects of the nature of the materials and the fabrication process on the characteristics of the nanoparticles are described. In addition, their application as delivery carriers of therapeutic drugs and genes and biomaterials for tissue engineering are also reviewed. PMID:23344060

Construction of a star-shaped copolymer as a vector for FGF receptor-mediated gene delivery in vitro and in vivo.

PubMed

Li, Da; Ping, Yuan; Xu, Fujian; Yu, Hai; Pan, Hongming; Huang, Hongliang; Wang, Qingqing; Tang, Guping; Li, Jun

2010-09-13

The success of cancer gene therapy highly relies on the gene delivery vector with high transfection activity and low toxicity. In the present study, eight-armed polyethylene glycol (EAP) and low molecular weight (LMW) polyethylenimine (PEI) were used as basic units to construct the architecture of a new star-shaped EAP-PEI copolymer (EAPP). MC11, a peptide capable of selectively binding fibroblast growth factor receptor (FGFR) on tumor cell membranes, was further conjugated to EAPP to produce the vector EAPP-MC11 (EAPPM) to enhance tumor targetability. This tumor-targeting vector EAPPM was observed to retard the plasmids mobility at a nitrogen/phosphorus (N/P) ratio of 3. The vector could efficiently condense plasmids within 300 nm nanoparticles with a positive zeta potential at the N/P ratio of 20 or above. While the cytotoxicity of EAPPM polyplexes was similar to that of LMW PEI, it was significantly lower than that of PEI (25 kDa) in HepG2 and PC3 cell lines. In vitro gene transfection with pDNA mediated by EAPPM showed that the transfection efficiency increased 15 times in HepG2 cells but remained at a similar level in PC3 cells in comparison with that of EAPP. By systemic injection of EAPPM/pDNA complexes into a HepG2-bearing mice model, luciferase expression detected in lung, liver, and tumor tissues demonstrated EAPPM could deliver in a targeted manner a reporter gene into tumor tissues, where the luciferase expression of EAPPM was 4 times higher than that of EAPP and even 23 times higher than that of PEI (25 kDa). Furthermore, it was found that the systemic delivery of EAPPM/pCSK-α-interferon complexes in vivo were much more effective in inhibiting tumor growth than EAPP or PEI (25 kDa). These results clearly show that EAPPM is an efficient and safe vector for FGFR-mediated targeted gene delivery both in vitro and in vivo. With low cytotoxicity and high targetability, EAPPM may have great potential as a delivery vector for future cancer gene therapy

Enzyme-synthesized Poly(amine-co-esters) as Non-viral Vectors for Gene Delivery

PubMed Central

Liu, Jie; Jiang, Zhaozhong; Zhou, Jiangbing; Zhang, Shengmin; Saltzman, W. Mark

2010-01-01

A family of biodegradable poly(amine-co-esters) was synthesized in one step via enzymatic copolymerization of diesters with amino-substituted diols. Diesters of length C4–C12 (i.e., from succinate to dodecanedioate) were successfully copolymerized with diethanolamines with either an alkyl (methyl, ethyl, n-butyl, t-butyl) or an aryl (phenyl) substituent on the nitrogen. Upon protonation at slightly acidic conditions, these poly(amine-co-esters) readily turned to cationic polyelectrolytes, which were capable of condensing with polyanionic DNA to form nanometer-sized polyplexes. In vitro screening with pLucDNA revealed that two of the copolymers, poly(N-methyldiethyleneamine sebacate) (PMSC) and poly(N-ethyldiethyleneamine sebacate) (PESC), possessed comparable or higher transfection efficiencies compared to Lipofectamine 2000. PMSC/pLucDNA and PESC/pLucDNA nanoparticles had desirable particle sizes (40–70 nm) for cellular uptake and were capable of functioning as proton sponges to facilitate endosomal escape after cellular uptake. These polyplex nanoparticles exhibited extremely low cytotoxicity. Furthermore, in vivo gene transfection experiments revealed that PMSC is a substantially more effective gene carrier than PEI in delivering pLucDNAto cells in tumors in mice. All these properties suggest that poly(amine-co-esters) are promising non-viral vectors for safe and efficient DNA delivery in gene therapy. PMID:21171165

2-Aminoimidazole facilitates efficient gene delivery in a low molecular weight poly(amidoamine) dendrimer.

PubMed

Wang, Jing; Hu, Xuefeng; Wang, Dongli; Xie, Cao; Lu, Weiyue; Song, Jie; Wang, Ruifeng; Gao, Chunli; Liu, Min

2018-06-20

Functional groups have shown great potential in gene delivery. However, a number of the reported functional groups can only overcome one certain physiological barrier, resulting in limited transfection efficiencies. Based on the structure-activity relationships of both imidazolyl and guanidyl, we designed a novel multifunctional group, 2-aminoimidazole (AM), for gene delivery. On modifying with the AM group, the transfection efficiency of low molecular weight poly(amidoamine) (G2) was 200 times greater than the parent dendrimer in vitro. In contrast, the transfection efficiency of G2 showed a decreasing trend when it was grafted with imidazole. Assays revealed that the AM group played multiple roles in gene delivery, including condensing DNA into monodisperse nanoparticles of 80-90 nm in diameter, achieving nearly ten times higher cellular-uptake efficacy, and enhancing the abilities of endosome/lysosome escape and nuclear localization. What's more, AM showed low toxicity. These results demonstrate that the AM group could be a promising tool in non-viral gene delivery.

Nonviral siRNA delivery for gene silencing in neurodegenerative diseases.

PubMed

Prakash, Satya; Malhotra, Meenakshi; Rengaswamy, Venkatesh

2010-01-01

Linking genes with the underlying mechanisms of diseases is one of the biggest challenges of genomics-driven drug discovery research. Designing an inhibitor for any neurodegenerative disease that effectively halts the pathogenicity of the disease is yet to be achieved. The challenge lies in crossing the blood-brain barrier (BBB)/blood-cerebrospinal fluid barrier (BCSFB) to reach the catalytic pockets of the enzyme/protein involved in the molecular mechanism of the disease process. Designing siRNA with exquisite specificity may result in selective suppression of the disease-linked gene. Although siRNA is the most promising method, it loses its potency in downregulating the gene due to its inherent instability, off-target effects, and lack of on-target effective delivery systems. Viral as well as nonviral delivery methods have been effectively tested in vivo for silencing of molecular targets and have resulted in significant efficacy in animal models of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anxiety, depression, encephalitis, glioblastoma, Huntington's disease, neuropathic pain, and spinocerebellar ataxia. To realize the full therapeutic potential of siRNA for neurodegenerative diseases, we need to overcome many hurdles and challenges such as selecting suitable tissue-specific delivery vectors, minimizing the off-target effects, and achieving distribution in sufficient concentrations at the target tissue without any side effects. Cationic nanoparticle-mediated targeted siRNA delivery for therapeutic purposes has gained considerable clinical importance as a result of its promising efficacy.

Bacteriophages as vehicles for gene delivery into mammalian cells: prospects and problems.

PubMed

Bakhshinejad, Babak; Sadeghizadeh, Majid

2014-10-01

The identification of more efficient gene delivery vehicles (GDVs) is essential to fulfill the expectations of clinical gene therapy. Bacteriophages, due to their excellent safety profile, extreme stability under a variety of harsh environmental conditions and the capability for being genetically manipulated, have drawn a flurry of interest to be applied as a newly arisen category of gene delivery platforms. The incessant evolutionary interaction of bacteriophages with human cells has turned them into a part of our body's natural ecosystem. However, these carriers represent several barriers to gene transduction of mammalian cells. The lack of evolvement of specialized machinery for targeted cellular internalization, endosomal, lysosomal and proteasomal escape, cytoplasmic entry, nuclear localization and intranuclear transcription poses major challenges to the expression of the phage-carried gene. In this review, we describe pros and cons of bacteriophages as GDVs, provide an insight into numerous barriers that bacteriophages face for entry into and subsequent trafficking inside mammalian cells and elaborate on the strategies used to bypass these barriers. Tremendous genetic flexibility of bacteriophages to undergo numerous surface modifications through phage display technology has proven to be a turning point in the uncompromising efforts to surmount the limitations of phage-mediated gene expression. The revelatory outcomes of the studies undertaken within the recent years have been promising for phage-mediated gene delivery to move from concept to reality.

Convection-enhanced delivery of AAV2 in white matter--a novel method for gene delivery to cerebral cortex.

PubMed

Barua, N U; Woolley, M; Bienemann, A S; Johnson, D; Wyatt, M J; Irving, C; Lewis, O; Castrique, E; Gill, S S

2013-10-30

Convection-enhanced delivery (CED) is currently under investigation for delivering therapeutic agents to subcortical targets in the brain. Direct delivery of therapies to the cerebral cortex, however, remains a significant challenge. We describe a novel method of targeting adeno-associated viral vector (AAV) mediated gene therapies to specific cerebral cortical regions by performing high volume, high flow rate infusions into underlying white matter in a large animal (porcine) model. Infusion volumes of up to 700 μl at flow rates as high as 10 μl/min were successfully performed in white matter without adverse neurological sequelae. Co-infusion of AAV2/5-GFP with 0.2% Gadolinium in artificial CSF confirmed transgene expression in the deep layers of cerebral cortex overlying the infused areas of white matter. AAV-mediated gene therapies have been previously targeted to the cerebral cortex by performing intrathalamic CED and exploiting axonal transport. The novel method described in this study facilitates delivery of gene therapies to specific regions of the cerebral cortex without targeting deep brain structures. AAV-mediated gene therapies can be targeted to specific cortical regions by performing CED into underlying white matter. This technique could be applied to the treatment of neurological disorders characterised by cerebral cortical degeneration. Copyright © 2013 Elsevier B.V. All rights reserved.

Non-viral gene delivery regulated by stiffness of cell adhesion substrates.

PubMed

Kong, Hyun Joon; Liu, Jodi; Riddle, Kathryn; Matsumoto, Takuya; Leach, Kent; Mooney, David J

2005-06-01

Non-viral gene vectors are commonly used for gene therapy owing to safety concerns with viral vectors. However, non-viral vectors are plagued by low levels of gene transfection and cellular expression. Current efforts to improve the efficiency of non-viral gene delivery are focused on manipulations of the delivery vector, whereas the influence of the cellular environment in DNA uptake is often ignored. The mechanical properties (for example, rigidity) of the substrate to which a cell adheres have been found to mediate many aspects of cell function including proliferation, migration and differentiation, and this suggests that the mechanics of the adhesion substrate may regulate a cell's ability to uptake exogeneous signalling molecules. In this report, we present a critical role for the rigidity of the cell adhesion substrate on the level of gene transfer and expression. The mechanism relates to material control over cell proliferation, and was investigated using a fluorescent resonance energy transfer (FRET) technique. This study provides a new material-based control point for non-viral gene therapy.

Myostatin propeptide gene delivery by gene gun ameliorates muscle atrophy in a rat model of botulinum toxin-induced nerve denervation.

PubMed

Tsai, Sen-Wei; Tung, Yu-Tang; Chen, Hsiao-Ling; Yang, Shang-Hsun; Liu, Chia-Yi; Lu, Michelle; Pai, Hui-Jing; Lin, Chi-Chen; Chen, Chuan-Mu

2016-02-01

Muscle atrophy is a common symptom after nerve denervation. Myostatin propeptide, a precursor of myostatin, has been documented to improve muscle growth. However, the mechanism underlying the muscle atrophy attenuation effects of myostatin propeptide in muscles and the changes in gene expression are not well established. We investigated the possible underlying mechanisms associated with myostatin propeptide gene delivery by gene gun in a rat denervation muscle atrophy model, and evaluated gene expression patterns. In a rat botulinum toxin-induced nerve denervation muscle atrophy model, we evaluated the effects of wild-type (MSPP) and mutant-type (MSPPD75A) of myostatin propeptide gene delivery, and observed changes in gene activation associated with the neuromuscular junction, muscle and nerve. Muscle mass and muscle fiber size was moderately increased in myostatin propeptide treated muscles (p<0.05). And enhancement of the gene expression of the muscle regulatory factors, neurite outgrowth factors (IGF-1, GAP43) and acetylcholine receptors was observed. Our results demonstrate that myostatin propeptide gene delivery, especially the mutant-type of MSPPD75A, attenuates muscle atrophy through myogenic regulatory factors and acetylcholine receptor regulation. Our data concluded that myostatin propeptide gene therapy may be a promising treatment for nerve denervation induced muscle atrophy. Copyright © 2016 Elsevier Inc. All rights reserved.

Design and assembly of new non-viral RNAi delivery agents by microwave-assisted quaternization (MAQ) of tertiary amines

PubMed Central

Ghosh, Animesh; Mukherjee, Koushik; Jiang, Xinpeng; Zhou, Ying; McCarroll, Joshua; Qu, James; Swain, Pamela M.; Baigude, Huricha; Rana, Tariq M.

2010-01-01

RNA interference (RNAi), a gene-silencing phenomenon whereby double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNA. RNAi has been quickly and widely applied to discover gene functions and holds great potential to provide a new class of therapeutic agents. However, new chemistry and delivery approaches are greatly needed to silence disease-causing genes without toxic effects. We reasoned that conjugation of the cholesterol moiety to cationic lipids would enhance RNAi efficiencies and lower the toxic effects of lipid-mediated RNAi delivery. Here, we report the first design and synthesis of new cholesterol-conjugated cationic lipids for RNAi delivery using microwave-assisted quaternization (MAQ) of tertiary amines. This strategy can be employed to develop new classes of non-viral gene delivery agents under safe and fast reaction conditions. PMID:20722369

Polyethyleneimine Coating Enhances the Cellular Uptake of Mesoporous Silica Nanoparticles and Allows Safe Delivery of siRNA and DNA Constructs

PubMed Central

Xia, Tian; Kovochich, Michael; Liong, Monty; Meng, Huan; Kabehie, Sanaz; Zink, Jeffrey I.; Nel, Andre E.

2014-01-01

Surface-functionalized mesoporous silica nanoparticles (MSNP) can be used as an efficient and safe carrier for bioactive molecules. In order to make the MSNP a more efficient delivery system, we modified the surface of the particles by a functional group that enhances cellular uptake and allows nucleic acid delivery in addition to traditional drug delivery. Non-covalent attachment of polyethyleneimine (PEI) polymers to the surface not only increases MSNP cellular uptake, but also generates a cationic surface to which DNA and siRNA constructs could be attached. While efficient for intracellular delivery of these nucleic acids, the 25 KD PEI polymer unfortunately changes the safety profile of the MSNP that is otherwise very safe. By experimenting with several different polymer molecular weights, it was possible to retain high cellular uptake and transfection efficiency while reducing or even eliminating cationic MSNP cytotoxicity. The particles coated with the 10 KD PEI polymer was particularly efficient for transducing HEPA-1 cells with a siRNA construct that was capable of knocking down GFP expression. Similarly, transfection of a GFP plasmid induced effective expression of the fluorescent protein in > 70% cells in the population. These outcomes were quantitatively assessed by confocal microscopy and flow cytometry. We also demonstrated that the enhanced cellular uptake of the non-toxic cationic MSNP enhance the delivery of the hydrophobic anticancer drug, paclitaxel, to pancreatic cancer cells. In summary, we demonstrate that by a careful selection of PEI size, it is possible to construct cationic MSNP that are capable of nucleotide and enhanced drug delivery with minimal or no cytotoxicity. This novel use of a cationic MSNP extends its therapeutic use potential. PMID:19739605

Eye drop delivery of nano-polymeric micelle formulated genes with cornea-specific promoters.

PubMed

Tong, Yaw-Chong; Chang, Shwu-Fen; Liu, Chia-Yang; Kao, Winston W-Y; Huang, Chong Heng; Liaw, Jiahorng

2007-11-01

This study evaluates the eye drop delivery of genes with cornea-specific promoters, i.e., keratin 12 (K12) and keratocan (Kera3.2) promoters, by non-ionic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles (PM) to mouse and rabbit eyes, and investigates the underlying mechanisms. Three PM-formulated plasmids (pCMV-Lac Z, pK12-Lac Z and pKera3.2-Lac Z) containing the Lac Z gene for beta-galactosidase (beta-Gal) whose expression was driven by the promoter of either the cytomegalovirus early gene, the keratin 12 gene or the keratocan gene, were characterized by critical micelle concentration (CMC), dynamic light scattering (DLS), and atomic force microscopy (AFM). Transgene expression in ocular tissue after gene delivery was analyzed by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) color staining, 1,2-dioxetane beta-Gal enzymatic activity measurement, and real-time polymerase chain reaction (PCR) analysis. The delivery mechanisms of plasmid-PM on mouse and rabbit corneas were evaluated by EDTA and RGD (arginine-glycine-aspartic acid) peptide. The sizes of the three plasmid-PM complexes were around 150-200 nm with unimodal distribution. Enhanced stability was found for three plasmid-PM formulations after DNase I treatment. After six doses of eye drop delivery of pK12-Lac Z-PM three times a day, beta-Gal activity was significantly increased in both mouse and rabbit corneas. Stroma-specific Lac Z expression was only found in pKera3.2-Lac Z-PM-treated animals with pretreatment by 5 mM EDTA, an opener of junctions. Lac Z gene expression in both pK12-Lac Z-PM and pKera3.2-Lac Z-PM delivery groups was decreased by RGD peptide pretreatment. Cornea epithelium- and stroma-specific gene expression could be achieved using cornea-specific promoters of keratin 12 and keratocan genes, and the gene was delivered with PM formulation through non-invasive, eye drop in mice and rabbits. The transfection mechanism of plasmid-PM may

Targeted Gene Transfer to the Brain via the Delivery of Brain-Penetrating DNA Nanoparticles with Focused Ultrasound

PubMed Central

Mead, Brian P.; Mastorakos, Panagiotis; Suk, Jung Soo; Klibanov, Alexander L.; Hanes, Justin; Price, Richard J.

2016-01-01

Gene therapy holds promise for the treatment of many pathologies of the central nervous system (CNS), including brain tumors and neurodegenerative diseases. However, the delivery of systemically administered gene carriers to the CNS is hindered by both the blood-brain barrier (BBB) and the nanoporous and electrostatically charged brain extracelluar matrix (ECM), which acts as a steric and adhesive barrier. We have previously shown that these physiological barriers may be overcome by, respectively, opening the BBB with MR image-guided focused ultrasound (FUS) and microbubbles and using highly compact “brain penetrating” nanoparticles (BPN) coated with a dense polyethylene glycol corona that prevents adhesion to ECM components. Here, we tested whether this combined approach could be utilized to deliver systemically administered DNA-bearing BPN (DNA-BPN) across the BBB and mediate localized, robust, and sustained transgene expression in the rat brain. Systemically administered DNA-BPN delivered through the BBB with FUS led to dose-dependent transgene expression only in the FUS-treated region that was evident as early as 24 h post administration and lasted for at least 28 days. In the FUS-treated region ~42% of all cells, including neurons and astrocytes, were transfected, while less than 6% were transfected in the contralateral non-FUS treated hemisphere. Importantly, this was achieved without any sign of toxicity or astrocyte activation. We conclude that the image-guided delivery of DNA-BPN with FUS and microbubbles constitutes a safe and non-invasive strategy for targeted gene therapy to the brain. PMID:26732553

Integrating mitosis, toxicity, and transgene expression in a telecommunications packet-switched network model of lipoplex-mediated gene delivery.

PubMed

Martin, Timothy M; Wysocki, Beata J; Beyersdorf, Jared P; Wysocki, Tadeusz A; Pannier, Angela K

2014-08-01

Gene delivery systems transport exogenous genetic information to cells or biological systems with the potential to directly alter endogenous gene expression and behavior with applications in functional genomics, tissue engineering, medical devices, and gene therapy. Nonviral systems offer advantages over viral systems because of their low immunogenicity, inexpensive synthesis, and easy modification but suffer from lower transfection levels. The representation of gene transfer using models offers perspective and interpretation of complex cellular mechanisms,including nonviral gene delivery where exact mechanisms are unknown. Here, we introduce a novel telecommunications model of the nonviral gene delivery process in which the delivery of the gene to a cell is synonymous with delivery of a packet of information to a destination computer within a packet-switched computer network. Such a model uses nodes and layers to simplify the complexity of modeling the transfection process and to overcome several challenges of existing models. These challenges include a limited scope and limited time frame, which often does not incorporate biological effects known to affect transfection. The telecommunication model was constructed in MATLAB to model lipoplex delivery of the gene encoding the green fluorescent protein to HeLa cells. Mitosis and toxicity events were included in the model resulting in simulation outputs of nuclear internalization and transfection efficiency that correlated with experimental data. A priori predictions based on model sensitivity analysis suggest that increasing endosomal escape and decreasing lysosomal degradation, protein degradation, and GFP-induced toxicity can improve transfection efficiency by three-fold. Application of the telecommunications model to nonviral gene delivery offers insight into the development of new gene delivery systems with therapeutically relevant transfection levels.

Rapid and efficient gene delivery into the adult mouse brain via focal electroporation

PubMed Central

Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

2016-01-01

In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

Functional Nanostructures for Effective Delivery of Small Interfering RNA Therapeutics

PubMed Central

Hong, Cheol Am; Nam, Yoon Sung

2014-01-01

Small interfering RNA (siRNA) has proved to be a powerful tool for target-specific gene silencing via RNA interference (RNAi). Its ability to control targeted gene expression gives new hope to gene therapy as a treatment for cancers and genetic diseases. However, siRNA shows poor pharmacological properties, such as low serum stability, off-targeting, and innate immune responses, which present a significant challenge for clinical applications. In addition, siRNA cannot cross the cell membrane for RNAi activity because of its anionic property and stiff structure. Therefore, the development of a safe, stable, and efficient system for the delivery of siRNA therapeutics into the cytoplasm of targeted cells is crucial. Several nanoparticle platforms for siRNA delivery have been developed to overcome the major hurdles facing the therapeutic uses of siRNA. This review covers a broad spectrum of non-viral siRNA delivery systems developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and discusses their characteristics and opportunities for clinical applications of therapeutic siRNA. PMID:25285170

A Promising Combo Gene Delivery System Developed from (3-Aminopropyl)triethoxysilane-Modified Iron Oxide Nanoparticles and Cationic Polymers

NASA Astrophysics Data System (ADS)

Zhang, Zubin; Song, Lina; Dong, Jinlai; Guo, Dawei; Du, Xiaolin; Cao, Biyin; Zhang, Yu; Gu, Ning; Mao, Xinliang

2013-05-01

(3-Aminopropyl)triethoxysilane-modified iron oxide nanoparticles (APTES-IONPs) have been evaluated for various biomedical applications, including medical imaging and drug delivery. Cationic polymers (CPs) such as Lipofectamine and TurboFect are widely used for research in gene delivery, but their toxicity and low in vivo efficiency limited their further application. In the present study, we synthesized water-soluble APTES-IONPs and developed a combo gene delivery system based on APTES-IONPs and CPs. This system significantly increased gene-binding capacity, protected genes from degradation, and improved gene transfection efficiency for DNA and siRNA in both adherent and suspension cells. Because of its great biocompatibility, high gene-carrying ability, and very low cytotoxicity, this combo gene delivery system will be expected for a wide application, and it might provide a new method for gene therapy.

Syngeneic AAV pseudo-particles potentiate gene transduction of AAV vectors

USDA-ARS?s Scientific Manuscript database

Gene delivery vectors based on adeno-associated virus (AAV) have emerged as safe and efficient therapeutic platform for numerous diseases. Excessive empty particles were generated as impurities during AAV vector production, but their effects on clinical outcome of AAV gene therapy are unclear. Here,...

Safe and efficient drug delivery system with liposomes for intrathecal application of an antivasospastic drug, fasudil.

PubMed

Ishida, Tatsuhiro; Takanashi, Yoshihiro; Kiwada, Hiroshi

2006-03-01

Pharmacological treatment for cerebral ischemia and cerebral vasospasm following subarachnoid hemorrhage (SAH) cannot attain sufficiently high concentrations of the drugs in the cerebrospinal fluid (CSF) without precipitating systemic side effects. We recently developed a liposomal drug delivery system for intrathecal application that can maintain effective concentrations of cerebral vasodilator, fasudil, in the CSF. A single intrathecal injection of liposomal fasudil could maintain a therapeutic drug concentration in the CSF over a period time due to their sustained-release property, significantly decreasing infarct size in a rat model of acute ischemia and reducing vasoconstriction of the rat and dog basilar artery in a model of SAH. In this review, we are introducing our new less-invasive intrathecal drug delivery system that provides an alternative and safe method to deliver therapeutic agents.

A novel needleless liquid jet injection methodology for improving direct cardiac gene delivery: An optimization of parameters, AAV mediated therapy and investigation of host responses in ischemic heart failure

NASA Astrophysics Data System (ADS)

Fargnoli, Anthony Samuel

Heart disease remains the leading cause of mortality and morbidity worldwide, with 22 million new patients diagnosed annually. Essentially, all present therapies have significant cost burden to the healthcare system, yet fail to increase survival rates. One key employed strategy is the genetic reprogramming of cells to increase contractility via gene therapy, which has advanced to Phase IIb Clinical Trials for advanced heart failure patients. It has been argued that the most significant barrier preventing FDA approval are resolving problems with safe, efficient myocardial delivery, whereby direct injection in the infarct and remote tissue areas is not clinically feasible. Here, we aim to: (1) Improve direct cardiac gene delivery through the development of a novel liquid jet device approach (2) Compare the new method against traditional IM injection with two different vector constructions and evaluate outcome (3) Evaluate the host response resulting from both modes of direct cardiac injection, then advance a drug/gene combination with controlled release nanoparticle formulations.

Lipid nanoparticles as drug/gene delivery systems to the retina.

PubMed

del Pozo-Rodríguez, Ana; Delgado, Diego; Gascón, Alicia R; Solinís, Maria Ángeles

2013-03-01

This review highlights the application of lipid nanoparticles (Solid Lipid Nanoparticles, Nanostructured Lipid Carriers, or Lipid Drug Conjugates) as effective drug/gene delivery systems for retinal diseases. Most drug products for ocular disease treatment are marketed as eye drop formulations but, due to ocular barriers, the drug concentration in the retina hardly ever turns out to be effective. Up to this date, several delivery systems have been designed to deliver drugs to the retina. Drug delivery strategies may be classified into 3 groups: noninvasive techniques, implants, and colloidal carriers. The best known systems for drug delivery to the posterior eye are intravitreal implants; in fact, some of them are being clinically used. However, their long-term accumulation might impact the patient's vision. On the contrary, colloidal drug delivery systems (microparticles, liposomes, or nanoparticles) can be easily administered in a liquid form. Nanoparticular systems diffuse rapidly and are better internalized in ocular tissues than microparticles. In comparison with liposomes, nanoparticles have a higher loading capacity and are more stable in biological fluids and during storage. In addition, their capacity to adhere to the ocular surface and interact with the endothelium makes these drug delivery systems interesting as new therapeutic tools in ophthalmology. Within the group of nanoparticles, those composed of lipids (Solid Lipid Nanoparticles, Nanostructred Lipid Carriers, and Lipid Drug Conjugates) are more biocompatible, easy to produce at large scale, and they may be autoclaved or sterilized. The present review summarizes scientific results that evidence the potential application of lipid nanoparticles as drug delivery systems for the retina and also as nonviral vectors in gene therapy of retina disorders, although much more effort is still needed before these lipidic systems could be available in the market.

Depletion of autophagy receptor p62/SQSTM1 enhances the efficiency of gene delivery in mammalian cells.

PubMed

Tsuchiya, Megumi; Ogawa, Hidesato; Koujin, Takako; Kobayashi, Shouhei; Mori, Chie; Hiraoka, Yasushi; Haraguchi, Tokuko

2016-08-01

Novel methods that increase the efficiency of gene delivery to cells will have many useful applications. Here, we report a simple approach involving depletion of p62/SQSTM1 to enhance the efficiency of gene delivery. The efficiency of reporter gene delivery was remarkably higher in p62-knockout murine embryonic fibroblast (MEF) cells compared with normal MEF cells. This higher efficiency was partially attenuated by ectopic expression of p62. Furthermore, siRNA-mediated knockdown of p62 clearly increased the efficiency of transfection of murine embryonic stem (mES) cells and human HeLa cells. These data indicate that p62 acts as a key regulator of gene delivery. © 2016 Federation of European Biochemical Societies.

Adenovirus-Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New Era of Personalized Medicine

PubMed Central

Lee, Cody S.; Bishop, Elliot S.; Zhang, Ruyi; Yu, Xinyi; Farina, Evan M.; Yan, Shujuan; Zhao, Chen; Zheng, Zongyue; Shu, Yi; Wu, Xingye; Lei, Jiayan; Li, Yasha; Zhang, Wenwen; Yang, Chao; Wu, Ke; Wu, Ying; Ho, Sherwin; Athiviraham, Aravind; Lee, Michael J.; Wolf, Jennifer Moriatis; Reid, Russell R.; He, Tong-Chuan

2017-01-01

With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology, it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies. Despite numerous setbacks, efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases. It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies. There has been a long-lasting interest in using viral vectors, especially adenoviral vectors, to deliver therapeutic genes for the past two decades. Among all currently available viral vectors, adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types. The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development. In fact, among over 2,000 gene therapy clinical trials approved worldwide since 1989, a significant portion of the trials have utilized adenoviral vectors. This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors, including adenoviral biology, approaches to engineering adenoviral vectors, and their applications in clinical and pre-clinical studies with an emphasis in the areas of cancer treatment, vaccination and regenerative medicine. Current challenges and future directions regarding the use of adenoviral vectors are also discussed. It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine. PMID:28944281

Polyplex micelle installing intracellular self-processing functionalities without free catiomers for safe and efficient systemic gene therapy through tumor vasculature targeting.

PubMed

Chen, Qixian; Osada, Kensuke; Ge, Zhishen; Uchida, Satoshi; Tockary, Theofilus A; Dirisala, Anjaneyulu; Matsui, Akitsugu; Toh, Kazuko; Takeda, Kaori M; Liu, Xueying; Nomoto, Takahiro; Ishii, Tekihiko; Oba, Makoto; Matsumoto, Yu; Kataoka, Kazunori

2017-01-01

Both efficiency and safety profiles are crucial for promotion of gene delivery systems towards practical applications. A promising template system was previously developed based on block catiomer of poly(ethylene glycol) (PEG)-b-poly{N'-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide}-cholesteryl [PEG-PAsp(DET)-cholesteryl] with strategies of ligand conjugation at the α-terminus for specific affinity to the targeted cells and cholesteryl conjugation at the ω-terminus for structural stabilization to obtain systemic retention. Aiming for advocating this formulation towards practical applications, in the current study, the binding profile of this polymer to plasmid DNA (pDNA) was carefully studied to address an issue of toxicity origin. Quantification of free polymer composition confirmed that the toxicity mainly results from unbound polymer and polyplex micelle itself has negligible toxicity. This evaluation allowed for identifying an optimal condition to prepare safe polyplex micelles for systemic application that possess maximal polymer-binding but exclude free polymers. The identified polyplex micelles then faced a drawback of limited transfection efficiency due to the absence of free polymer, which is an acknowledged tendency found in various synthetic gene carriers. Thus, series of functional components was strategically compiled to improve the transfection efficiency such as attachment of cyclic (Arg-Gly-Asp) (cRGD) peptide as a ligand onto the polyplex micelles to facilitate cellular uptake, use of endosome membrane disruptive catiomer of PAsp(DET) for facilitating endosome escape along with use of the conjugated cholesteryl group to amplify the effect of PAsp(DET) on membrane disruption, so as to obtain efficient transfection. The mechanistic investigation respecting the appreciated pH dependent protonation behavior of PAsp(DET) permitted to depict an intriguing scenario how the block catiomers manage to escape from the endosome entrapment in response to the p

Influence of molecular weight upon mannosylated bio-synthetic hybrids for targeted antigen presenting cell gene delivery

PubMed Central

Jones, Charles H.; Gollakota, Akhila; Chen, Mingfu; Chung, Tai-Chun; Ravikrishnan, Anitha; Zhang, Guojian; Pfeifer, Blaine A.

2015-01-01

Given the rise of antibiotic resistant microbes, genetic vaccination is a promising prophylactic strategy that enables rapid design and manufacture. Facilitating this process is the choice of vector, which is often situationally-specific and limited in engineering capacity. Furthermore, these shortcomings are usually tied to an incomplete understanding of the structure-function relationships driving vector-mediated gene delivery. Building upon our initial report of a hybrid bacterial-biomaterial gene delivery vector, a comprehensive structure-function assessment was completed using a class of mannosylated poly(beta-amino esters). Through a top-down screening methodology, an ideal polymer was selected on the basis of gene delivery efficacy and then used for the synthesis of a stratified molecular weight polymer library. By eliminating contributions of polymer chemical background, we were able to complete an in-depth assessment of gene delivery as a function of (1) polymer molecular weight, (2) relative mannose content, (3) polymer-membrane biophysical properties, (4) APC uptake specificity, and (5) serum inhibition. In summary, the flexibility and potential of the hybrid design featured in this work highlights the ability to systematically probe vector-associated properties for the development of translational gene delivery candidates. PMID:25941787

Cyclen-based cationic lipids for highly efficient gene delivery towards tumor cells.

PubMed

Huang, Qing-Dong; Zhong, Guo-Xing; Zhang, Yang; Ren, Jiang; Fu, Yun; Zhang, Ji; Zhu, Wen; Yu, Xiao-Qi

2011-01-01

Gene therapy has tremendous potential for both inherited and acquired diseases. However, delivery problems limited their clinical application, and new gene delivery vehicles with low cytotoxicity and high transfection efficiency are greatly required. In this report, we designed and synthesized three amphiphilic molecules (L1-L3) with the structures involving 1, 4, 7, 10-tetraazacyclododecane (cyclen), imidazolium and a hydrophobic dodecyl chain. Their interactions with plasmid DNA were studied via electrophoretic gel retardation assays, fluorescent quenching experiments, dynamic light scattering and transmission electron microscopy. The in vitro gene transfection assay and cytotoxicity assay were conducted in four cell lines. Results indicated that L1 and L3-formed liposomes could effectively bind to DNA to form well-shaped nanoparticles. Combining with neutral lipid DOPE, L3 was found with high efficiency in gene transfer in three tumor cell lines including A549, HepG2 and H460. The optimized gene transfection efficacy of L3 was nearly 5.5 times more efficient than that of the popular commercially available gene delivery agent Lipofectamine 2000™ in human lung carcinoma cells A549. In addition, since L1 and L3 had nearly no gene transfection performance in normal cells HEK293, these cationic lipids showed tumor cell-targeting property to a certain extent. No significant cytotoxicity was found for the lipoplexes formed by L1-L3, and their cytotoxicities were similar to or slightly lower than the lipoplexes prepared from Lipofectamine 2000™. Novel cyclen-based cationic lipids for effective in vitro gene transfection were founded, and these studies here may extend the application areas of macrocyclic polyamines, especially for cyclen.

Gene delivery to the lungs: pulmonary gene therapy for cystic fibrosis.

PubMed

Villate-Beitia, Ilia; Zarate, Jon; Puras, Gustavo; Pedraz, José Luis

2017-07-01

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder where the defective gene, the cystic fibrosis transmembrane conductance regulator (CFTR), is well identified. Moreover, the respiratory tract can be targeted through noninvasive aerosolized formulations for inhalation. Therefore, gene therapy is considered a plausible strategy to address this disease. Conventional gene therapy strategies rely on the addition of a correct copy of the CFTR gene into affected cells in order to restore the channel activity. In recent years, genome correction strategies have emerged, such as zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases. These gene editing tools aim to repair the mutated gene at its original genomic locus with high specificity. Besides, the success of gene therapy critically depends on the nucleic acids carriers. To date, several clinical studies have been carried out to add corrected copies of the CFTR gene into target cells using viral and non-viral vectors, some of them with encouraging results. Regarding genome editing systems, preliminary in vitro studies have been performed in order to repair the CFTR gene. In this review, after briefly introducing the basis of CF, we discuss the up-to-date gene therapy strategies to address the disease. The review focuses on the main factors to take into consideration when developing gene delivery strategies, such as the design of vectors and plasmid DNA, in vitro/in vivo tests, translation to human use, administration methods, manufacturing conditions and regulatory issues.

An efficient nonviral gene-delivery vector based on hyperbranched cationic glycogen derivatives.

PubMed

Liang, Xuan; Ren, Xianyue; Liu, Zhenzhen; Liu, Yingliang; Wang, Jue; Wang, Jingnan; Zhang, Li-Ming; Deng, David Yb; Quan, Daping; Yang, Liqun

2014-01-01

The purpose of this study was to synthesize and evaluate hyperbranched cationic glycogen derivatives as an efficient nonviral gene-delivery vector. A series of hyperbranched cationic glycogen derivatives conjugated with 3-(dimethylamino)-1-propylamine (DMAPA-Glyp) and 1-(2-aminoethyl) piperazine (AEPZ-Glyp) residues were synthesized and characterized by Fourier-transform infrared and hydrogen-1 nuclear magnetic resonance spectroscopy. Their buffer capacity was assessed by acid-base titration in aqueous NaCl solution. Plasmid deoxyribonucleic acid (pDNA) condensation ability and protection against DNase I degradation of the glycogen derivatives were assessed using agarose gel electrophoresis. The zeta potentials and particle sizes of the glycogen derivative/pDNA complexes were measured, and the images of the complexes were observed using atomic force microscopy. Blood compatibility and cytotoxicity were evaluated by hemolysis assay and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, respectively. pDNA transfection efficiency mediated by the cationic glycogen derivatives was evaluated by flow cytometry and fluorescence microscopy in the 293T (human embryonic kidney) and the CNE2 (human nasopharyngeal carcinoma) cell lines. In vivo delivery of pDNA in model animals (Sprague Dawley rats) was evaluated to identify the safety and transfection efficiency. The hyperbranched cationic glycogen derivatives conjugated with DMAPA and AEPZ residues were synthesized. They exhibited better blood compatibility and lower cytotoxicity when compared to branched polyethyleneimine (bPEI). They were able to bind and condense pDNA to form the complexes of 100-250 nm in size. The transfection efficiency of the DMAPA-Glyp/pDNA complexes was higher than those of the AEPZ-Glyp/pDNA complexes in both the 293T and CNE2 cells, and almost equal to those of bPEI. Furthermore, pDNA could be more safely delivered to the blood vessels in brain tissue of Sprague Dawley rats

Intranasal gene delivery for treating Parkinson's disease: overcoming the blood-brain barrier.

PubMed

Aly, Amirah E-E; Waszczak, Barbara L

2015-01-01

Developing a disease-modifying gene therapy for Parkinson's disease (PD) has been a high priority for over a decade. However, due to the inability of large biomolecules to cross the blood-brain barrier (BBB), the only means of delivery to the brain has been intracerebral infusion. Intranasal administration offers a non-surgical means of bypassing the BBB to deliver neurotrophic factors, and the genes encoding them, directly to the brain. This review summarizes: i) evidence demonstrating intranasal delivery to the brain of a number of biomolecules having therapeutic potential for various CNS disorders; and ii) evidence demonstrating neuroprotective efficacy of a subset of biomolecules specifically for PD. The intersection of these two spheres represents the area of opportunity for development of new intranasal gene therapies for PD. To that end, our laboratory showed that intranasal administration of glial cell line-derived neurotrophic factor (GDNF), or plasmid DNA nanoparticles encoding GDNF, provides neuroprotection in a rat model of PD, and that the cells transfected by the nanoparticle vector are likely to be pericytes. A number of genes encoding neurotrophic factors have therapeutic potential for PD, but few have been tested by the intranasal route and shown to be neuroprotective in a model of PD. Intranasal delivery provides a largely unexplored, promising approach for development of a non-invasive gene therapy for PD.

Design and Fabrication of N-Alkyl-Polyethylenimine-Stabilized Iron Oxide Nanoclusters for Gene Delivery

PubMed Central

Liu, Gang; Wang, Zhiyong; Lee, Seulki; Ai, Hua; Chen, Xiaoyuan

2013-01-01

With the rapid development of nanotechnology, inorganic magnetic nanoparticles, especially iron oxide nanoparticles (IOs), have emerged as great vehicles for biomedical diagnostic and therapeutic applications. In order to rationally design IO-based gene delivery nanovectors, surface modification is essential and determines the loading and release of the gene of interest. Here we highlight the basic concepts and applications of nonviral gene delivery vehicles based on low molecular weight N-alkyl polyethylenimine-stabilized IOs. The experimental protocols related to these topics are described in this chapter. PMID:22568910

Water insoluble and soluble lipids for gene delivery.

PubMed

Mahato, Ram I

2005-04-05

Among various synthetic gene carriers currently in use, liposomes composed of cationic lipids and co-lipids remain the most efficient transfection reagents. Physicochemical properties of lipid/plasmid complexes, such as cationic lipid structure, cationic lipid to co-lipid ratio, charge ratio, particle size and zeta potential have significant influence on gene expression and biodistribution. However, most cationic lipids are toxic and cationic liposomes/plasmid complexes do not disperse well inside the target tissues because of their large particle size. To overcome the problems associated with cationic lipids, we designed water soluble lipopolymers for gene delivery to various cells and tissues. This review provides a critical discussion on how the components of water insoluble and soluble lipids affect their transfection efficiency and biodistribution of lipid/plasmid complexes.

Adenoviral Gene Delivery to Primary Human Cutaneous Cells and Burn Wounds

PubMed Central

Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

2006-01-01

The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2 × 108 pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P > 0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P = 0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting

Adenoviral gene delivery to primary human cutaneous cells and burn wounds.

PubMed

Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

2006-01-01

The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2x10(8) pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P>0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P=0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has

Engineering adeno-associated viruses for clinical gene therapy.

PubMed

Kotterman, Melissa A; Schaffer, David V

2014-07-01

Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

Engineering adeno-associated viruses for clinical gene therapy

PubMed Central

Kotterman, Melissa A.; Schaffer, David V.

2015-01-01

Clinical gene therapy has been increasingly successful, due both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among the latter, delivery vectors based on adeno-associated virus (AAV) have emerged as safe and effective – in one recent case leading to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers. PMID:24840552

Design and fabrication of N-alkyl-polyethylenimine-stabilized iron oxide nanoclusters for gene delivery.

PubMed

Liu, Gang; Wang, Zhiyong; Lee, Seulki; Ai, Hua; Chen, Xiaoyuan

2012-01-01

With the rapid development of nanotechnology, inorganic magnetic nanoparticles, especially iron oxide nanoparticles (IOs), have emerged as great vehicles for biomedical diagnostic and therapeutic applications. In order to rationally design IO-based gene delivery nanovectors, surface modification is essential and determines the loading and release of the gene of interest. Here we highlight the basic concepts and applications of nonviral gene delivery vehicles based on low molecular weight N-alkyl polyethylenimine-stabilized IOs. The experimental protocols related to these topics are described in this chapter. Copyright © 2012 Elsevier Inc. All rights reserved.

Hydrodynamic gene delivery in human skin using a hollow microneedle device.

PubMed

Dul, M; Stefanidou, M; Porta, P; Serve, J; O'Mahony, C; Malissen, B; Henri, S; Levin, Y; Kochba, E; Wong, F S; Dayan, C; Coulman, S A; Birchall, J C

2017-11-10

Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potential of a hollow microneedle device for the delivery and subsequent expression of pDNA in human skin. The regulatory approved MicronJet600® (MicronJet hereafter) device was used to deliver reporter plasmids (pCMVβ and pEGFP-N1) into viable excised human skin. Exogenous gene expression was subsequently detected at multiple locations that were distant from the injection site but within the confines of the bleb created by the intradermal bolus. The observed levels of gene expression in the tissue are at least comparable to that achieved by the most invasive microneedle application methods e.g. lateral application of a microneedle. Gene expression was predominantly located in the epidermis, although also evident in the papillary dermis. Optical coherence tomography permitted real time visualisation of the sub-surface skin architecture and, unlike a conventional intradermal injection, MicronJet administration of a 50μL bolus appears to create multiple superficial microdisruptions in the papillary dermis and epidermis. These were co-localised with expression of the pCMVβ reporter plasmid. We have therefore shown, for the first time, that a hollow microneedle device can facilitate efficient and reproducible gene expression of exogenous naked pDNA in human skin using volumes that are considered to be standard for intradermal administration, and postulate a hydrodynamic effect as the mechanism of gene delivery. Copyright © 2017 Elsevier B.V. All rights reserved.

Hybrid biosynthetic gene therapy vector development and dual engineering capacity.

PubMed

Jones, Charles H; Ravikrishnan, Anitha; Chen, Mingfu; Reddinger, Ryan; Kamal Ahmadi, Mahmoud; Rane, Snehal; Hakansson, Anders P; Pfeifer, Blaine A

2014-08-26

Genetic vaccines offer a treatment opportunity based upon successful gene delivery to specific immune cell modulators. Driving the process is the vector chosen for gene cargo packaging and subsequent delivery to antigen-presenting cells (APCs) capable of triggering an immune cascade. As such, the delivery process must successfully navigate a series of requirements and obstacles associated with the chosen vector and target cell. In this work, we present the development and assessment of a hybrid gene delivery vector containing biological and biomaterial components. Each component was chosen to design and engineer gene delivery separately in a complimentary and fundamentally distinct fashion. A bacterial (Escherichia coli) inner core and a biomaterial [poly(beta-amino ester)]-coated outer surface allowed the simultaneous application of molecular biology and polymer chemistry to address barriers associated with APC gene delivery, which include cellular uptake and internalization, phagosomal escape, and intracellular cargo concentration. The approach combined and synergized normally disparate vector properties and tools, resulting in increased in vitro gene delivery beyond individual vector components or commercially available transfection agents. Furthermore, the hybrid device demonstrated a strong, efficient, and safe in vivo humoral immune response compared with traditional forms of antigen delivery. In summary, the flexibility, diversity, and potential of the hybrid design were developed and featured in this work as a platform for multivariate engineering at the vector and cellular scales for new applications in gene delivery immunotherapy.

Influence of molecular weight upon mannosylated bio-synthetic hybrids for targeted antigen presenting cell gene delivery.

PubMed

Jones, Charles H; Gollakota, Akhila; Chen, Mingfu; Chung, Tai-Chun; Ravikrishnan, Anitha; Zhang, Guojian; Pfeifer, Blaine A

2015-07-01

Given the rise of antibiotic resistant microbes, genetic vaccination is a promising prophylactic strategy that enables rapid design and manufacture. Facilitating this process is the choice of vector, which is often situationally-specific and limited in engineering capacity. Furthermore, these shortcomings are usually tied to an incomplete understanding of the structure-function relationships driving vector-mediated gene delivery. Building upon our initial report of a hybrid bacterial-biomaterial gene delivery vector, a comprehensive structure-function assessment was completed using a class of mannosylated poly(beta-amino esters). Through a top-down screening methodology, an ideal polymer was selected on the basis of gene delivery efficacy and then used for the synthesis of a stratified molecular weight polymer library. By eliminating contributions of polymer chemical background, we were able to complete an in-depth assessment of gene delivery as a function of (1) polymer molecular weight, (2) relative mannose content, (3) polymer-membrane biophysical properties, (4) APC uptake specificity, and (5) serum inhibition. In summary, the flexibility and potential of the hybrid design featured in this work highlights the ability to systematically probe vector-associated properties for the development of translational gene delivery candidates. Copyright © 2015 Elsevier Ltd. All rights reserved.

Functionalized nanoparticles for AMF-induced gene and drug delivery

NASA Astrophysics Data System (ADS)

Biswas, Souvik

The properties and broad applications of nano-magnetic colloids have generated much interest in recent years. Specially, Fe3O4 nanoparticles have attracted a great deal of attention since their magnetic properties can be used for hyperthermia treatment or drug targeting. For example, enhanced levels of intracellular gene delivery can be achieved using Fe3O4 nano-vectors in the presence of an external magnetic field, a process known as 'magnetofection'. The low cytotoxicity, tunable particle size, ease of surface functionalization, and ability to generate thermal energy using an external alternating magnetic field (AMF) are properties have propelled Fe3O4 research to the forefront of nanoparticle research. The strategy of nanoparticle-mediated, AMF-induced heat generation has been used to effect intracellular hyperthermia. One application of this 'magnetic hyperthermia' is heat activated local delivery of a therapeutic effector (e.g.; drug or polynucleotide). This thesis describes the development of a magnetic nano-vector for AMF-induced, heat-activated pDNA and small molecule delivery. The use of heat-inducible vectors, such as heat shock protein ( hsp) genes, is a promising mode of gene therapy that would restrict gene expression to a local region by focusing a heat stimulus only at a target region. We thus aimed to design an Fe3O4 nanoparticle-mediated gene transfer vehicle for AMF-induced localized gene expression. We opted to use 'click' oximation techniques to assemble the magnetic gene transfer vector. Chapter 2 describes the synthesis, characterization, and transfection studies of the oxime ether lipid-based nano-magnetic vectors MLP and dMLP. The synthesis and characterization of a novel series of quaternary ammonium aminooxy reagents (2.1--2.4) is described. These cationic aminooxy compounds were loaded onto nanoparticles for ligation with carbonyl groups and also to impart a net positive charge on the nanoparticle surface. Our studies indicated that the

Learning From Experience: Development of a Cognitive Task List to Perform a Safe and Successful Non-Rotational Forceps Delivery.

PubMed

Simpson, Andrea N; Gurau, David; Secter, Michael; Mocarski, Eva; Pittini, Richard; Snelgrove, John; Hodges, Ryan; Windrim, Rory; Higgins, Mary

2015-07-01

Increased rates of delivery by Caesarean section have resulted in a reduction in rates of instrumental deliveries. This has led to a new educational challenge for teaching and development of skills. In teaching trainees, there are subconscious tasks that the supervising staff may not review because they are automatic. This study aimed to create a new tool to meet this challenge: to identify the core steps required to perform a non-rotational forceps delivery safely and successfully. Labour and delivery nursing staff of three large teaching hospitals were asked to identify clinicians they considered to be particularly skilled in non-rotational forceps deliveries. Obstetricians who were identified consistently in this way were invited to participate in the study. After providing written consent, participants were then filmed performing a non-rotational forceps delivery on a model. Two clinicians reviewed all videos and documented verbal and non-verbal components of the assessment. Thematic analysis combined findings into an integrated summary. The initial summary was then circulated to all participants for their approval. Seventeen clinicians were identified and consented. Themes identified included the need for careful assessment of suitability for operative delivery, the role of the multidisciplinary team, the need for careful and appropriate communication with the parents, the technique of delivery itself, and postpartum care and documentation. In the core steps identified, the clinicians balanced respect for the "elegant technique" of non-rotational forceps deliveries with careful assessment and knowing when to stop if safety criteria were not met.

Bioengineered Nanoparticles for siRNA delivery

PubMed Central

Kozielski, Kristen L.; Tzeng, Stephany Y.; Green, Jordan J.

2014-01-01

Short interfering RNA (siRNA) has been an important laboratory tool in the last two decades and has allowed researchers to better understand the functions of non-protein-coding genes through RNA interference (RNAi). Although RNAi holds great promise for this purpose as well as for treatment of many diseases, efforts at using siRNA have been hampered by the difficulty of safely and effectively introducing it into cells of interest, both in vitro and in vivo. To overcome this challenge, many biomaterials and nanoparticles (NPs) have been developed and optimized for siRNA delivery, often taking cues from the DNA delivery field, although different barriers exist for these two types of molecules. In this review, we discuss general properties of biomaterials and nanoparticles that are necessary for effective nucleic acid delivery. We also discuss specific examples of bioengineered materials, including lipid-based NPs, polymeric NPs, inorganic NPs, and RNA-based NPs, which clearly illustrate the problems and successes in siRNA delivery. PMID:23821336

Viral Vectors for Gene Delivery to the Central Nervous System

PubMed Central

Lentz, Thomas B.; Gray, Steven J.; Samulski, R. Jude

2011-01-01

The potential benefits of gene therapy for neurological diseases such as Parkinson’s, Amyotrophic Lateral Sclerosis (ALS), Epilepsy, and Alzheimer’s are enormous. Even a delay in the onset of severe symptoms would be invaluable to patients suffering from these and other diseases. Significant effort has been placed in developing vectors capable of delivering therapeutic genes to the CNS in order to treat neurological disorders. At the forefront of potential vectors, viral systems have evolved to efficiently deliver their genetic material to a cell. The biology of different viruses offers unique solutions to the challenges of gene therapy, such as cell targeting, transgene expression and vector production. It is important to consider the natural biology of a vector when deciding whether it will be the most effective for a specific therapeutic function. In this review, we outline desired features of the ideal vector for gene delivery to the CNS and discuss how well available viral vectors compare to this model. Adeno-associated virus, retrovirus, adenovirus and herpesvirus vectors are covered. Focus is placed on features of the natural biology that have made these viruses effective tools for gene delivery with emphasis on their application in the CNS. Our goal is to provide insight into features of the optimal vector and which viral vectors can provide these features. PMID:22001604

Pullulan-protamine as efficient haemocompatible gene delivery vector: synthesis and in vitro characterization.

PubMed

Priya, S S; Rekha, M R; Sharma, Chandra P

2014-02-15

Biodegradable non-viral vectors with good transfection efficiency is essential for successful gene delivery. The purpose of this study was to design a non-viral vector by conjugating protamine to pullulan and elucidate the potential use of pullulan protamine conjugate (PPA) as an effective, non toxic and haemocompatible gene delivery system. The particle size and surface charge were measured using Nanosizer. Derivatization was confirmed by NMR, FTIR and DSC analyses. Acid base titration revealed the buffering behaviour of the conjugate. The protection of DNA from nuclease enzyme and interaction of plasma components on the stability of nanoplexes were also analysed. The uptake studies confirmed the plasmid delivery into the nucleus and the inhibitor studies determined the uptake mechanism. Transfection experiments revealed the capability of PPA to cellular uptake in C6 cells and facilitate high gene expression. Thus, PPA proves to be a promising non-viral vector. Copyright © 2013 Elsevier Ltd. All rights reserved.

Delivery of Na/I symporter gene into skeletal muscle using nanobubbles and ultrasound: visualization of gene expression by PET.

PubMed

Watanabe, Yukiko; Horie, Sachiko; Funaki, Yoshihito; Kikuchi, Youhei; Yamazaki, Hiromichi; Ishii, Keizo; Mori, Shiro; Vassaux, Georges; Kodama, Tetsuya

2010-06-01

The development of nonviral gene delivery systems is essential in gene therapy, and the use of a minimally invasive imaging methodology can provide important clinical endpoints. In the current study, we present a new methodology for gene therapy-a delivery system using nanobubbles and ultrasound as a nonviral gene delivery method. We assessed whether the gene transfer allowed by this methodology was detectable by PET and bioluminescence imaging. Two kinds of reported vectors (luciferase and human Na/I symporter [hNIS]) were transfected or cotransfected into the skeletal muscles of normal mice (BALB/c) using the ultrasound-nanobubbles method. The kinetics of luciferase gene expression were analyzed in vivo using bioluminescence imaging. At the peak of gene transfer, PET of hNIS expression was performed using our recently developed PET scanner, after (124)I injection. The imaging data were confirmed using reverse-transcriptase polymerase chain reaction amplification, biodistribution, and a blocking study. The imaging potential of the 2 methodologies was evaluated in 2 mouse models of human pathology (McH/lpr-RA1 mice showing vascular disease and C57BL/10-mdx Jic mice showing muscular dystrophy). Peak luciferase gene activity was observed in the skeletal muscle 4 d after transfection. On day 2 after hNIS and luciferase cotransfection, the expression of these genes was confirmed by reverse-transcriptase polymerase chain reaction on a muscle biopsy. PET of the hNIS gene, biodistribution, the blocking study, and autoradiography were performed on day 4 after transfection, and it was indicated that hNIS expression was restricted to the site of plasmid administration (skeletal muscle). Similar localized PET and (124)I accumulation were successfully obtained in the disease-model mice. The hNIS gene was delivered into the skeletal muscle of healthy and disease-model mice by the ultrasound-nanobubbles method, and gene expression was successfully visualized with PET. The

Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo

PubMed Central

White, JD; Thesier, DM; Swain, JBD; Katz, MG; Tomasulo, C; Henderson, A; Wang, L; Yarnall, C; Fargnoli, A; Sumaroka, M; Isidro, A; Petrov, M; Holt, D; Nolen-Walston, R; Koch, WJ; Stedman, HH; Rabinowitz, J; Bridges, CR

2013-01-01

We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy. PMID:21228882

Multifunctional nanocarrier based on clay nanotubes for efficient intracellular siRNA delivery and gene silencing.

PubMed

Wu, Hui; Shi, Yinfeng; Huang, Chusen; Zhang, Yang; Wu, Jiahui; Shen, Hebai; Jia, Nengqin

2014-04-01

RNA interference-mediated gene silencing relating to disease has recently emerged as a powerful method in gene therapy. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. Halloysites are cheap and naturally available aluminosilicate clay nanotubes with high mechanical strength and biocompatibility. In this study, a novel multifunctional nanocarrier based on functionalized halloysite nanotubes (f-HNTs) has been developed via electrostatic layer-by-layer assembling approach for loading and intracellular delivery of therapeutic antisurvivin siRNA and simultaneously tracking their intracellular transport, in which PEI-modified HNTs are used as gene vector, antisurvivin siRNA as gene therapeutic agent, and mercaptoacetic acid-capped CdSe quantum dots as fluorescent labeling probes. The successful assembly of the f-HNTs-siRNA complexes was systematically characterized by transmission electron microscopy (TEM), UV-visible spectrophotometry, Zeta potential measurement, fluorescence spectrophotometry, and electrochemical impedance spectroscopy. Confocal microscopy, biological TEM, and flow cytometry studies revealed that the complexes enabled the efficient intracellular delivery of siRNA for cell-specific gene silencing. MTT assays exhibited that the complexes can enhance antitumor activity. Furthermore, Western blot analysis showed that f-HNTs-mediated siRNA delivery effectively knocked down gene expression of survivin and thereby decreased the levels of target proteins of PANC-1 cells. Therefore, this study suggested that the synthesized f-HNTs were a new effective drug delivery system for potential application in cancer gene therapy.

Antheraea pernyi silk fibroin for targeted gene delivery of VEGF165-Ang-1 with PEI.

PubMed

Ma, Caili; Lv, Linlin; Liu, Yu; Yu, Yanni; You, Renchuan; Yang, Jicheng; Li, Mingzhong

2014-06-01

Vascularization is a crucial challenge in tissue engineering. One solution for this problem is to implant scaffolds that contain functional genes that promote vascularization by providing angiogenic growth factors via a gene delivery carrier. Poly(ethylenimine) (PEI) is a gene delivery carrier with high transfection efficiency but with cytotoxicity. To solve this problem, we utilized Antheraea pernyi silk fibroin (ASF), which has favorable cytocompatibility and biodegradability, RGD sequences and a negative charge, in conjunction with PEI, as the delivery vector for vascular endothelial growth factor (VEGF) 165-angiopoietin-1 (Ang-1) dual gene simultaneous expression plasmid, creating an ASF/PEI/pDNA complex. The results suggested that the zeta potential of the ASF/PEI/pDNA complex was significantly lower than that of the PEI/pDNA complex. Decreased nitrogen and increased oxygen on the surface of the complex demonstrated that the ASF had successfully combined with the surface of the PEI/pDNA. Furthermore, the complexes resisted digestion by nucleic acid enzymes and degradation by serum. L929 cells were cultured and transfected in vitro and improved cytotoxicity was found when the cells were transfected with ASF/PEI/pDNA compared with PEI/pDNA. In addition, the transfection efficiency and VEGF secretion increased. In general, this study provides a novel method for decreasing the cytotoxicity of PEI gene delivery vectors and increasing transfection efficiency of angiogenesis-related genes.

Liver cell-targeted delivery of therapeutic molecules.

PubMed

Kang, Jeong-Hun; Toita, Riki; Murata, Masaharu

2016-01-01

The liver is the largest internal organ in mammals and is involved in metabolism, detoxification, synthesis of proteins and lipids, secretion of cytokines and growth factors and immune/inflammatory responses. Hepatitis, alcoholic or non-alcoholic liver disease, hepatocellular carcinoma, hepatic veno-occlusive disease, and liver fibrosis and cirrhosis are the most common liver diseases. Safe and efficient delivery of therapeutic molecules (drugs, genes or proteins) into the liver is very important to increase the clinical efficacy of these molecules and to reduce their side effects in other organs. Several liver cell-targeted delivery systems have been developed and tested in vivo or ex vivo/in vitro. In this review, we discuss the literature concerning liver cell-targeted delivery systems, with a particular emphasis on the results of in vivo studies.

The next step in gene delivery: molecular engineering of adeno-associated virus serotypes.

PubMed

Wang, Jinhui; Faust, Susan M; Rabinowitz, Joseph E

2011-05-01

Delivery is at the heart of gene therapy. Viral DNA delivery systems are asked to avoid the immune system, transduce specific target cell types while avoiding other cell types, infect dividing and non-dividing cells, insert their cargo within the host genome without mutagenesis or to remain episomal, and efficiently express transgenes for a substantial portion of a lifespan. These sought-after features cannot be associated with a single delivery system, or can they? The Adeno-associated virus family of gene delivery vehicles has proven to be highly malleable. Pseudotyping, using AAV serotype 2 terminal repeats to generate designer shells capable of transducing selected cell types, enables the packaging of common genomes into multiple serotypes virions to directly compare gene expression and tropism. In this review the ability to manipulate this virus will be examined from the inside out. The influence of host cell factors and organism biology including the immune response on the molecular fate of the viral genome will be discussed as well as differences in cellular trafficking patterns and uncoating properties that influence serotype transduction. Re-engineering the prototype vector AAV2 using epitope insertion, chemical modification, and molecular evolution not only demonstrated the flexibility of the best-studied serotype, but now also expanded the tool kit for molecular modification of all AAV serotypes. Current AAV research has changed its focus from examination of wild-type AAV biology to the feedback of host cell/organism on the design and development of a new generation of recombinant AAV delivery vehicles. This article is part of a Special Section entitled "Special Section: Cardiovascular Gene Therapy". Copyright © 2010 Elsevier Ltd. All rights reserved.

Versatile types of polysaccharide-based supramolecular polycation/pDNA nanoplexes for gene delivery

NASA Astrophysics Data System (ADS)

Hu, Yang; Zhao, Nana; Yu, Bingran; Liu, Fusheng; Xu, Fu-Jian

2014-06-01

Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple β-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations with adamantane-functionalized polysaccharides is an effective strategy for the production of new nanoplex delivery systems.Different polysaccharide-based supramolecular polycations were readily synthesized by assembling multiple β-cyclodextrin-cored star polycations with an adamantane-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star polycation and pDNA, the supramolecular polycation/pDNA nanoplexes exhibited similarly low cytotoxicity, improved cellular internalization and significantly higher gene transfection efficiencies. The incorporation of disulfide linkages imparted the supramolecular polycation/pDNA nanoplexes with the advantage of intracellular bioreducibility, resulting in better gene delivery properties. In addition, the antitumor properties of supramolecular polycation/pDNA nanoplexes were also investigated using a suicide gene therapy system. The present study demonstrates that the proper assembly of cyclodextrin-cored polycations

The construction of a novel kind of non-viral gene delivery vector based on protein as core backbone.

PubMed

Li, D; Kong, Y; Yu, H; Lehtinen, A; Huang, H; Shen, F; Min, L; Zhou, J; Tang, G; Wang, Q

2008-04-01

A novel kind of non-viral gene delivery vector based on transferrin (Tf) as the core component was constructed with high transfection efficiency and low toxicity. The synthesis vector of Tf-PEI600 was confirmed by different physicochemical methods, including (1)H nuclear magnetic resonance, gel permeation chromatography, X-ray and thermogravimetric analysis. The cytotoxicity and gene delivery efficiency of the synthesized vector were verified by in vitro experiments. The agarose gel electrophoresis assay indicated that the novel copolymer Tf-PEI600 could efficiently condense plasmid DNA and the condensed nanoparticles exhibited a spherical shape. As the weight ratio of Tf-PEI600 to DNA reached 15.0, the particle size (about 200 nm) and the zeta potential (about 20 mV) of the nanoparticles became optimal for gene delivery. The methylthiazolyl tetrazolium (MTT) assay showed the cytotoxicity of Tf-PEI600 to be similar to that of PEI600 and much lower than that of PEI25kDa. In gene-delivery experiments with COS-7 cells and HepG2 cells, the Tf-PEI600 showed about a 30- to 53-fold higher efficiency than PEI600 and nearly equal to that of PEI25kDa. These data suggest that Tf-PEI600, with the advantages of low toxicity and high gene-delivery efficiency, might have great prospects in the practice of gene delivery. The core-shell structure of Tf-PEI600 also provided a novel strategy for the construction of non-viral gene delivery vectors.

Poly(ethylenimine) conjugated bioreducible dendrimer for efficient gene delivery.

PubMed

Nam, Kihoon; Jung, Simhyun; Nam, Joung-Pyo; Kim, Sung Wan

2015-12-28

Branched poly(ethylenimine) (PEI) 25 kDa is an efficient gene delivery vector with outstanding gene condensation ability and great endosome escape activity. However, it also induces higher cytotoxicity. Transfection efficiency and toxicity of PEI are highly dependent upon their molecular weight and structure. We developed a bioreducible poly(ethylenimine) (PEI (-s-s-)) derived from low molecular weight PEI (1.8 kDa) for efficient gene delivery. Bioreducible core molecule is expected to increase molecular weight and reduce the cytotoxicity of the copolymer. PEI (-s-s-) polyplexes showed higher transfection efficiency and lower cytotoxicity compared to branched PEI 25 kDa, Lipofectamine® 2000 and, FuGENE® 6. In addition, PEI (-s-s-) derivative (16 kDa) formed stable polyplexes with a zeta-potential value of +34 mV and polyplex size of 61 nm. PEI (-s-s-) derivative (16 kDa) showed excellent transfection efficiency: 3.6 times higher than branched PEI 25 kDa in HeLa cells and 7.4 times higher than Lipofectamine® 2000 in H9C2 cell. The derivatives also showed lower cytotoxicity compared with Lipofectamine® 2000 and PEI 25 kDa in various cell types. In addition, newly synthesized PEI (-s-s-) derivatives have high reproducibility. Copyright © 2015 Elsevier B.V. All rights reserved.

One-Pot Parallel Synthesis of Lipid Library via Thiolactone Ring Opening and Screening for Gene Delivery.

PubMed

Molla, Mijanur R; Böser, Alexander; Rana, Akshita; Schwarz, Karina; Levkin, Pavel A

2018-04-18

Efficient delivery of nucleic acids into cells is of great interest in the field of cell biology and gene therapy. Despite a lot of research, transfection efficiency and structural diversity of gene-delivery vectors are still limited. A better understanding of the structure-function relationship of gene delivery vectors is also essential for the design of novel and intelligent delivery vectors, efficient in "difficult-to-transfect" cells and in vivo clinical applications. Most of the existing strategies for the synthesis of gene-delivery vectors require multiple steps and lengthy procedures. Here, we demonstrate a facile, three-component one-pot synthesis of a combinatorial library of 288 structurally diverse lipid-like molecules termed "lipidoids" via a thiolactone ring opening reaction. This strategy introduces the possibility to synthesize lipidoids with hydrophobic tails containing both unsaturated bonds and reducible disulfide groups. The whole synthesis and purification are convenient, extremely fast, and can be accomplished within a few hours. Screening of the produced lipidoids using HEK293T cells without addition of helper lipids resulted in identification of highly stable liposomes demonstrating ∼95% transfection efficiency with low toxicity.

Modification of nanostructured calcium carbonate for efficient gene delivery.

PubMed

Zhao, Dong; Wang, Chao-Qun; Zhuo, Ren-Xi; Cheng, Si-Xue

2014-06-01

In this study, a facile method to modify nanostructured calcium carbonate (CaCO3) gene delivery systems by adding calcium phosphate (CaP) component was developed. CaCO3/CaP/DNA nanoparticles were prepared by the co-precipitation of Ca(2+) ions with plasmid DNA in the presence of carbonate and phosphate ions. For comparison, CaCO3/DNA nanoparticles and CaP/DNA co-precipitates were also prepared. The effects of carbonate ion/phosphate ion (CO3(2-)/PO4(3-)) ratio on the particle size and gene delivery efficiency were investigated. With an appropriate CO3(2-)/PO4(3-) ratio, the co-existence of carbonate and phosphate ions could control the size of co-precipitates effectively, and CaCO3/CaP/DNA nanoparticles with a decreased size and improved stability could be obtained. The in vitro gene transfections mediated by different nanoparticles in 293T cells and HeLa cells were carried out, using pGL3-Luc as a reporter plasmid. The gene transfection efficiency of CaCO3/CaP/DNA nanoparticles could be significantly improved as compared with CaCO3/DNA nanoparticles and CaP/DNA co-precipitates. The confocal microscopy study indicated that the cellular uptake and nuclear localization of CaCO3/CaP/DNA nanoparticles were significantly enhanced as compared with unmodified CaCO3/DNA nanoparticles. Copyright © 2014 Elsevier B.V. All rights reserved.

PLGA-Chitosan nanoparticle-mediated gene delivery for oral cancer treatment: A brief review

NASA Astrophysics Data System (ADS)

Bakar, L. M.; Abdullah, M. Z.; Doolaanea, A. A.; Ichwan, S. J. A.

2017-08-01

Cancer becomes a serious issue on society with increasing of their growth and proliferation, either in well economic developed countries or not. Recent years, oral cancer is one of the most threatening diseases impairing the quality of life of the patient. Scientists have emphasised on application of gene therapy for oral cancer by using nanoparticle as transportation vectors as a new alternative platform in order to overcome the limitations of conventional approaches. In modern medicine, nanotechnologies’ application, such as nanoparticles-mediated gene delivery, is one of promising tool for therapeutic devices. The objective of this article is to present a brief review summarizes on the current progress of nanotechnology-based gene delivery treatment system targeted for oral cancer.

Guidelines for the Delineation of Roles and Responsibilities for the Safe Delivery of Specialized Health Care in the Educational Setting.

ERIC Educational Resources Information Center

Council for Exceptional Children, Reston, VA.

These guidelines were developed by a joint task force of members and staff of four national associations, to be of assistance to persons concerned with the safe delivery of specialized health care in educational settings. The guidelines delineate the roles and responsibilities of personnel involved in the provision of specialized health care. They…

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications.

PubMed

Liu, Chang; Zhang, Li; Liu, Hao; Cheng, Kun

2017-11-28

The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

The potential of toxin-based drug delivery systems for enhanced nucleic acid therapeutic delivery.

PubMed

Shorter, Susan A; Gollings, Alexander S; Gorringe-Pattrick, Monique A M; Coakley, J Emma; Dyer, Paul D R; Richardson, Simon C W

2017-05-01

The potential of gene replacement therapy has been underscored by the market authorization of alipogene tiparvovec (Glybera) and GSK2696273 (Strimvelis) in the EU and recombinant adenovirus-p53 (Gendicine) in China. Common to these systems is the use of attenuated viruses for 'drug' delivery. Whilst viral delivery systems are being developed for siRNA, their application to antisense delivery remains problematic. Non-viral delivery remains experimental, with some notable successes. However, stability and the 'PEG dilemma', balancing toxicity and limited (often liver-tropic) pharmacokinetics/oharmacodynamics, with the membrane destabilizing activity, necessary for nucleocytosolic access and transfection remain a problem. Areas covered: Here we review the use of attenuated protein toxins as a delivery vehicle for nucleic acids, their relationship to the PEG dilemma, and their biological properties with specific reference to their intracellular trafficking. Expert opinion: The possibility of using attenuated toxins as antisense and siRNA delivery systems has been demonstrated in vitro. Systems based upon attenuated anthrax toxin have been shown to have high activity (equivalent to nucleofection) and low toxicity whilst not requiring cationic 'helpers' or condensing agents, divorcing these systems from the problems associated with the PEG dilemma. It remains to be seen whether these systems can operate safely, efficiently and reproducibly, in vivo or in the clinic.

One-step Conjugation of Glycyrrhetinic Acid to Cationic Polymers for High-performance Gene Delivery to Cultured Liver Cell.

PubMed

Cong, Yue; Shi, Bingyang; Lu, Yiqing; Wen, Shihui; Chung, Roger; Jin, Dayong

2016-02-23

Gene therapies represent a promising therapeutic route for liver cancers, but major challenges remain in the design of safe and efficient gene-targeting delivery systems. For example, cationic polymers show good transfection efficiency as gene carriers, but are hindered by cytotoxicity and non-specific targeting. Here we report a versatile method of one-step conjugation of glycyrrhetinic acid (GA) to reduce cytotoxicity and improve the cultured liver cell -targeting capability of cationic polymers. We have explored a series of cationic polymer derivatives by coupling different ratios of GA to polypropylenimine (PPI) dendrimer. These new gene carriers (GA-PPI dendrimer) were systematically characterized by UV-vis,(1)H NMR titration, electron microscopy, zeta potential, dynamic light-scattering, gel electrophoresis, confocal microscopy and flow cytometry. We demonstrate that GA-PPI dendrimers can efficiently load and protect pDNA, via formation of nanostructured GA-PPI/pDNA polyplexes. With optimal GA substitution degree (6.31%), GA-PPI dendrimers deliver higher liver cell transfection efficiency (43.5% vs 22.3%) and lower cytotoxicity (94.3% vs 62.5%, cell viability) than the commercial bench-mark DNA carrier bPEI (25 kDa) with cultured liver model cells (HepG2). There results suggest that our new GA-PPI dendrimer are a promising candidate gene carrier for targeted liver cancer therapy.

One-step Conjugation of Glycyrrhetinic Acid to Cationic Polymers for High-performance Gene Delivery to Cultured Liver Cell

PubMed Central

Cong, Yue; Shi, Bingyang; Lu, Yiqing; Wen, Shihui; Chung, Roger; Jin, Dayong

2016-01-01

Gene therapies represent a promising therapeutic route for liver cancers, but major challenges remain in the design of safe and efficient gene-targeting delivery systems. For example, cationic polymers show good transfection efficiency as gene carriers, but are hindered by cytotoxicity and non-specific targeting. Here we report a versatile method of one-step conjugation of glycyrrhetinic acid (GA) to reduce cytotoxicity and improve the cultured liver cell -targeting capability of cationic polymers. We have explored a series of cationic polymer derivatives by coupling different ratios of GA to polypropylenimine (PPI) dendrimer. These new gene carriers (GA-PPI dendrimer) were systematically characterized by UV-vis,1H NMR titration, electron microscopy, zeta potential, dynamic light-scattering, gel electrophoresis, confocal microscopy and flow cytometry. We demonstrate that GA-PPI dendrimers can efficiently load and protect pDNA, via formation of nanostructured GA-PPI/pDNA polyplexes. With optimal GA substitution degree (6.31%), GA-PPI dendrimers deliver higher liver cell transfection efficiency (43.5% vs 22.3%) and lower cytotoxicity (94.3% vs 62.5%, cell viability) than the commercial bench-mark DNA carrier bPEI (25kDa) with cultured liver model cells (HepG2). There results suggest that our new GA-PPI dendrimer are a promising candidate gene carrier for targeted liver cancer therapy. PMID:26902258

Biocleavable graphene oxide based-nanohybrids synthesized via ATRP for gene/drug delivery.

PubMed

Yang, Xinchao; Zhao, Nana; Xu, Fu-Jian

2014-06-07

Graphene oxide (GO) has been proven to be promising in many biomedical fields due to its biocompatibility, unique conjugated structure, easily tunable surface functionalization and facile synthesis. In this work, a flexible two-step method was first developed to introduce the atom transfer radical polymerization (ATRP) initiation sites containing disulfide bonds onto GO surfaces. Surface-initiated ATRP of (2-dimethyl amino)ethyl methacrylate (DMAEMA) was then employed to tailor the GO surfaces in a well-controlled manner, producing a series of organic-inorganic hybrids (termed as SS-GPDs) for highly efficient gene delivery. Under reducible conditions, the PDMAEMA side chains can be readily cleavable from the GO backbones, benefiting the resultant gene delivery process. Moreover, due to the conjugated structure of the graphene basal plane, SS-GPD can attach and absorb aromatic, water insoluble drugs, such as 10-hydroxycamptothecin (CPT), producing SS-GPD-CPT. The MTT assay and the simultaneous double-staining procedure revealed that SS-GPD-CPT possessed a high potency of killing cancer cells in vitro. With a high aqueous solubility and coulombic interaction with cell membrane, SS-GPDs may have great potential in gene/drug delivery fields.

Mannosylated polyion complexes for in vivo gene delivery into CD11c(+) dendritic cells.

PubMed

Raviv, Lior; Jaron-Mendelson, Michal; David, Ayelet

2015-02-02

Dendritic cells (DCs) possess unique abilities in initiating primary immune responses and thus represent prime targets for DNA-based vaccinations. Here, we describe the design and synthesis of mannosylated polyion complexes (PICs) composed of cationic polyethylenimine (PEI) and hydrophilic polyethylene glycol (PEG) segments, and bearing mono- and trivalent mannose as a ligand for targeting mannose receptor (MR/CD206)-positive DCs. Amino-terminated mannose (Man)-containing ligands in mono- and trivalent presentations (Man- and Man3-, respectively) were prepared and conjugated to PEG via an N-hydroxysuccinimide (NHS)-activated terminal. Thiolated PEI was conjugated to the mannosylated PEG via the maleimide (MAL)-activated terminal. The resulting positively charged diblock copolymers bearing mannoses (Man-PEG-b-PEI and Man3-PEG-b-PEI) were self-assembled with DNA to form PICs with lower surface charge than did their PEI building block and mean hydrodynamic diameters in the range of 100-450 nm, depending on the N/P ratio. Man3-PEG-b-PEI demonstrated a 3-4-fold greater transfection efficiency in MR-positive dendritic cell lines (THP-1, DC2.4), relative to Man-PEG-b-PEI, exhibited low cytotoxicity when compared with PEI, and showed low transfection efficiency in nondendritic HeLa cells. In preliminary in vivo experiments, Man-PEG-b-PEI/DNA and Man3-PEG-b-PEI/DNA demonstrated 2-3-fold higher gene delivery efficiency into CD11c(+) DCs collected from inguinal lymph nodes of C57/BL6 mice, when compared to PEI/DNA complexes, as shown by GFP expression measurements, 24 h post subcutaneous injection. The results indicate that the mannosylated PICs are a safe and effective gene delivery system, showing in vivo specificity toward CD11c(+) DCs.

Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy.

PubMed

Fernandes, Alinda R; Chari, Divya M

2016-09-28

Genetically engineered neural stem cell (NSC) transplant populations offer key benefits in regenerative neurology, for release of therapeutic biomolecules in ex vivo gene therapy. NSCs are 'hard-to-transfect' but amenable to 'magnetofection'. Despite the high clinical potential of this approach, the low and transient transfection associated with the large size of therapeutic DNA constructs is a critical barrier to translation. We demonstrate for the first time that DNA minicircles (small DNA vectors encoding essential gene expression components but devoid of a bacterial backbone, thereby reducing construct size versus conventional plasmids) deployed with magnetofection achieve the highest, safe non-viral DNA transfection levels (up to 54%) reported so far for primary NSCs. Minicircle-functionalized magnetic nanoparticle (MNP)-mediated gene delivery also resulted in sustained gene expression for up to four weeks. All daughter cell types of engineered NSCs (neurons, astrocytes and oligodendrocytes) were transfected (in contrast to conventional plasmids which usually yield transfected astrocytes only), offering advantages for targeted cell engineering. In addition to enhancing MNP functionality as gene delivery vectors, minicircle technology provides key benefits from safety/scale up perspectives. Therefore, we consider the proof-of-concept of fusion of technologies used here offers high potential as a clinically translatable genetic modification strategy for cell therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Thiolated chitosan/DNA nanocomplexes exhibit enhanced and sustained gene delivery.

PubMed

Lee, Dongwon; Zhang, Weidong; Shirley, Shawna A; Kong, Xiaoyuan; Hellermann, Gary R; Lockey, Richard F; Mohapatra, Shyam S

2007-01-01

Thiolated chitosan appears to possess enhanced mucoadhesiveness and cell penetration properties, however, its potential in gene-drug delivery remains unknown. Herein, we report on a highly effective gene delivery system utilizing a 33-kDa thiol-modified chitosan derivative. Thiolated chitosan was prepared by the reaction with thioglycolic acid. Nanocomplexes of unmodified chitosan or thiolated chitosan with plasmid DNA encoding green fluorescenct protein (GFP) were characterized for their size, zeta potential, their ability to bind and protect plasmid DNA from degradation. The transfection efficiency of thiolated chitosan and sustained gene expression were evaluated in various cell lines in vitro and in Balb/c mice in vivo. Thiolated chitosan-DNA nanocomplexes ranged in size from 75 to 120 nm in diameter and from +2.3 to 19.7 mV in zeta potential, depending on the weight ratio of chitosan to DNA. Thiolated chitosan, CSH360, exhibited effective physical stability and protection against DNase I digestion at a weight ratio>or=2.5:1. CSH360/DNA nanocomplexes induced significantly (P<0.01) higher GFP expression in HEK293, MDCK and Hep-2 cell lines than unmodified chitosan. Nanocomplexes of disulphide-crosslinked CSH360/DNA showed a sustained DNA release and continuous expression in cultured cells lasting up to 60 h post transfection. Also, intranasal administration of crosslinked CSH360/DNA nanocomplexes to mice yielded gene expression that lasted for at least 14 days. Thiolated chitosans condense pDNA to form nanocomplexes, which exhibit a significantly higher gene transfer potential and sustained gene expression upon crosslinking, indicating their great potential for gene therapy and tissue engineering.

Gene delivery in conjunction with gold nanoparticle and tumor treating electric field

NASA Astrophysics Data System (ADS)

Tiwari, Pawan K.; Soo Lee, Yeon

2013-08-01

The advances in electrotherapy to treat the diseased biological cell instigate its extension in gene therapy through the delivery of gene into the nucleus. The objective of this study is to investigate the application of moderate intensity alternating electric field, also known as tumor treating electric field on a carrier system consisting of a charged gene complex conjugated to the surface of a gold nanoparticle. The gene delivery mechanism relies on the magnitude and direction of the induced electric field inside the cytoplasm in presence of carrier system. The induced electric field strength is significant in breaking the gene complex-gold nanoparticle bonding, and exerting an electric force pushing the charged gene into the nucleus. The electric force orientation is dependent on the aspect ratio (AR) of the gold nanoparticle and a relationship between them is studied via Maxwell two-dimensional (2D) finite element simulation analyzer. The development of charge density on the surface of carrier system and the required electric field strength to break the bonding are investigated utilizing the Gouy-Chapman-Grahame-Stern (GCGS) theoretical model. A carrier system having the aspect ratio of the gold nanoparticle in the range 1 < AR ≤ 5 and AR = 1 are substantial delivering cationic and anionic genes into the nucleus, respectively.

Gene delivery through the use of a hyaluronate-associated intracellularly degradable cross-linked polyethyleneimine

PubMed Central

Xu, Peisheng; Quick, Griffin; Yeo, Yoon

2009-01-01

For a non-viral gene delivery system to be clinically effective, it should be non-toxic, compatible with biological components, and highly efficient in gene transfection. With this goal in mind, we investigated the gene delivery efficiency of a ternary complex consisting of DNA, an intracellularly degradable polycation, and sodium hyaluronate (DPH complex). Here, we report that the DPH ternary complex achieved significantly higher transfection efficiency than other polymer systems, especially in the presence of serum. The high transfection efficiency and serum tolerance of DPH are attributed to a unique interplay between CLPEI and HA, which leads to (i) the improved stability of DNA in the extracellular environment and at the early stage of intracellular trafficking and (ii) timely dissociation of the DNA-polymer complex. This study reinforces findings of earlier studies that emphasized each step as a bottleneck for efficient gene delivery; yet, it is the first to show that it is possible to overcome these obstacles simultaneously by taking advantage of two distinctive approaches. PMID:19631979

Hydrogel Design for Supporting Neurite Outgrowth and Promoting Gene Delivery to Maximize Neurite Extension

PubMed Central

Shepard, Jaclyn A.; Stevans, Alyson C.; Holland, Samantha; Wang, Christine E.; Shikanov, Ariella; Shea, Lonnie D.

2012-01-01

Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of inductive factors. This report investigates the design of hydrogels that balance the requirements for supporting neurite growth with those requirements for promoting gene delivery. Enzymatically-degradable PEG hydrogels encapsulating dorsal root ganglia explants, fibroblasts, and lipoplexes encoding nerve growth factor were gelled within channels that can physically guide neurite outgrowth. Transfection of fibroblasts increased with increasing concentration of Arg-Gly-Asp (RGD) cell adhesion sites and decreasing PEG content. The neurite length increased with increasing RGD concentration within 10% PEG hydrogels, yet was maximal within 7.5% PEG hydrogels at intermediate RGD levels. Delivering lipoplexes within the gel produced longer neurites than culture in NGF-supplemented media or co-culture with cells exposed to DNA prior to encapsulation. Hydrogels designed to support neurite outgrowth and deliver gene therapy vectors locally may ultimately be employed to address multiple barriers that limit regeneration. PMID:22038654

PEO-PPO-PEO Tri-Block Copolymers for Gene Delivery Applications in Human Regenerative Medicine—An Overview

PubMed Central

Cucchiarini, Magali

2018-01-01

Lineal (poloxamers or Pluronic®) or X-shaped (poloxamines or Tetronic®) amphiphilic tri-block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO-PEO) have been broadly explored for controlled drug delivery in different regenerative medicine approaches. The ability of these copolymers to self-assemble as micelles and to undergo sol-to-gel transitions upon heating has endowed the denomination of “smart” or “intelligent” systems. The use of PEO-PPO-PEO copolymers as gene delivery systems is a powerful emerging strategy to improve the performance of classical gene transfer vectors. This review summarizes the state of art of the application of PEO-PPO-PEO copolymers in both nonviral and viral gene transfer approaches and their potential as gene delivery systems in different regenerative medicine approaches. PMID:29518011

Regulation the morphology of cationized gold nanoparticles for effective gene delivery.

PubMed

Zhang, Peng; Li, Bangbang; Du, Jianwei; Wang, Youxiang

2017-09-01

Recent research indicated that the morphology of nanoparticles could result in distinct biological behaviors, thus played an important role in designing efficient gene delivery systems. Among them, gold nanoparticles (AuNPs) with various shapes were widely studied due to the good biocompatibility and easy modification ability. Our recent research indicated that polyethyleneimine-g-bovine serum albumin (BSA-PEI) as non-viral gene vector showed good colloid stability and high transfection efficiency. In this work, BSA-PEI was utilized to modify gold nanospheres (AuNSs) and gold nanorods (AuNRs) to investigate the influence of the morphology on gene delivery. Both AuNS@BSA-PEI and AuNR@BSA-PEI nanoparticles condensed DNA effectively at N/P ratio above 5 and maintained spherical or rod-like morphology respectively. Due to the higher surface charge density at the tips, the rod-like gene complexes were prone to use the tips to contact with cell membrane, which facilitated to be uptaked by HepG2 cells. The endocytosis inhibition experiments showed some differences in the endocytic pathway. Gene transfection experiment showed that the rod-like complexes had almost 100-fold higher of transfection level than that of spherical complexes at the N/P ratio of 20. This work provided a potential strategy for further design of gene vectors with improved transfection results by adjusting the morphology of gene vectors. Copyright © 2017. Published by Elsevier B.V.

Setting priorities for safe motherhood interventions in resource-scarce settings.

PubMed

Prata, Ndola; Sreenivas, Amita; Greig, Fiona; Walsh, Julia; Potts, Malcolm

2010-01-01

Guide policy-makers in prioritizing safe motherhood interventions. Three models (LOW, MED, HIGH) were constructed based on 34 sub-Saharan African countries to assess the relative cost-effectiveness of available safe motherhood interventions. Cost and effectiveness data were compiled and inserted into the WHO Mother Baby Package Costing Spreadsheet. For each model we assessed the percentage in maternal mortality reduction after implementing all interventions, and optimal combinations of interventions given restricted budgets of US$ 0.50, US$ 1.00, US$ 1.50 per capital maternal health expenditures respectively for LOW, MED, and HIGH models. The most cost-effective interventions were family planning and safe abortion (fpsa), antenatal care including misoprostol distribution for postpartum hemorrhage prevention at home deliveries (anc-miso), followed by sepsis treatment (sepsis) and facility-based postpartum hemorrhage management (pph). The combination of interventions that avert the greatest number of maternal deaths should be prioritized and expanded to cover the greatest number of women at risk. Those which save the most number of lives in each model are 'fpsa, anc-miso' and 'fpsa, sepsis, safe delivery' for LOW; 'fpsa, anc-miso' and 'fpsa, sepsis, safe delivery' for MED; and 'fpsa, anc-miso, sepsis, eclampsia treatment, safe delivery' for HIGH settings. Safe motherhood interventions save a significant number of newborn lives.

MicroRNA delivery for regenerative medicine.

PubMed

Peng, Bo; Chen, Yongming; Leong, Kam W

2015-07-01

MicroRNA (miRNA) directs post-transcriptional regulation of a network of genes by targeting mRNA. Although relatively recent in development, many miRNAs direct differentiation of various stem cells including induced pluripotent stem cells (iPSCs), a major player in regenerative medicine. An effective and safe delivery of miRNA holds the key to translating miRNA technologies. Both viral and nonviral delivery systems have seen success in miRNA delivery, and each approach possesses advantages and disadvantages. A number of studies have demonstrated success in augmenting osteogenesis, improving cardiogenesis, and reducing fibrosis among many other tissue engineering applications. A scaffold-based approach with the possibility of local and sustained delivery of miRNA is particularly attractive since the physical cues provided by the scaffold may synergize with the biochemical cues induced by miRNA therapy. Herein, we first briefly cover the application of miRNA to direct stem cell fate via replacement and inhibition therapies, followed by the discussion of the promising viral and nonviral delivery systems. Next we present the unique advantages of a scaffold-based delivery in achieving lineage-specific differentiation and tissue development. Copyright © 2015 Elsevier B.V. All rights reserved.

DNA Nanotechnology for Precise Control over Drug Delivery and Gene Therapy.

PubMed

Angell, Chava; Xie, Sibai; Zhang, Liangfang; Chen, Yi

2016-03-02

Nanomedicine has been growing exponentially due to its enhanced drug targeting and reduced drug toxicity. It uses the interactions where nanotechnological components and biological systems communicate with each other to facilitate the delivery performance. At this scale, the physiochemical properties of delivery systems strongly affect their capacities. Among current delivery systems, DNA nanotechnology shows many advantages because of its unprecedented engineering abilities. Through molecular recognition, DNA nanotechnology can be used to construct a variety of nanostructures with precisely controllable size, shape, and surface chemistry, which can be appreciated in the delivery process. In this review, different approaches that are currently used for the construction of DNA nanostructures are reported. Further, the utilization of these DNA nanostructures with the well-defined parameters for the precise control in drug delivery and gene therapy is discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Magnetically enhanced adeno-associated viral vector delivery for human neural stem cell infection.

PubMed

Kim, Eunmi; Oh, Ji-Seon; Ahn, Ik-Sung; Park, Kook In; Jang, Jae-Hyung

2011-11-01

Gene therapy technology is a powerful tool to elucidate the molecular cues that precisely regulate stem cell fates, but developing safe vehicles or mechanisms that are capable of delivering genes to stem cells with high efficiency remains a challenge. In this study, we developed a magnetically guided adeno-associated virus (AAV) delivery system for gene delivery to human neural stem cells (hNSCs). Magnetically guided AAV delivery resulted in rapid accumulation of vectors on target cells followed by forced penetration of the vectors across the plasma membrane, ultimately leading to fast and efficient cellular transduction. To combine AAV vectors with the magnetically guided delivery, AAV was genetically modified to display hexa-histidine (6xHis) on the physically exposed loop of the AAV2 capsid (6xHis AAV), which interacted with nickel ions chelated on NTA-biotin conjugated to streptavidin-coated superparamagnetic iron oxide nanoparticles (NiStNPs). NiStNP-mediated 6xHis AAV delivery under magnetic fields led to significantly enhanced cellular transduction in a non-permissive cell type (i.e., hNSCs). In addition, this delivery method reduced the viral exposure times required to induce a high level of transduction by as much as to 2-10 min of hNSC infection, thus demonstrating the great potential of magnetically guided AAV delivery for numerous gene therapy and stem cell applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

Far-red light-mediated programmable anti-cancer gene delivery in cooperation with photodynamic therapy.

PubMed

Wang, Jinhui; He, Hua; Xu, Xin; Wang, Xiao; Chen, Yongbing; Yin, Lichen

2018-07-01

Effective anti-cancer therapy is hurdled by the complicated extracellular and intracellular barriers, and thus a smart gene vector that can enable programmable gene delivery is highly demanded. Photo-manipulation of gene delivery processes features spatial and temporal precision, while majority of current strategies utilizes short-wavelength UV/visible light with poor tissue penetration or high-power-density near-infrared (NIR) light that would cause undesired heat damage. Herein, an ROS-degradable polycation was designed and co-delivered with a photosensitizer (PS), thus realizing photo-programmable gene delivery using far-red light (661 nm) at low optical power density (down to 5 mW cm -2 ). Thioketal-crosslinked polyethylenimine (TK-PEI) was synthesized to condense p53 gene to form nanocomplexes (NCs), and hyaluronic acid (HA) modified with pheophytin a (Pha) was coated onto NCs to enhance their colloidal stability and enable cancer cell targeting. Short-time (8-min) light irradiation produced non-lethal amount of ROS to disrupt the endosomal membranes and facilitate p53 gene release via degradation of TK-PEI, which collectively enhanced p53 expression levels toward anti-cancer gene therapy. Long-time (30-min) light irradiation at the post-transfection state generated lethal amount of ROS, which cooperatively killed cancer cells to strengthen p53 gene therapy. To the best of our knowledge, this study represents the first example of an "one stone, three birds" approach to realize cooperative anti-cancer gene therapy using low-power-density, long-wavelength visible light as a single stimulus. Copyright © 2018 Elsevier Ltd. All rights reserved.

An intestinal Trojan horse for gene delivery

NASA Astrophysics Data System (ADS)

Peng, Haisheng; Wang, Chao; Xu, Xiaoyang; Yu, Chenxu; Wang, Qun

2015-02-01

The intestinal epithelium forms an essential element of the mucosal barrier and plays a critical role in the pathophysiological response to different enteric disorders and diseases. As a major enteric dysfunction of the intestinal tract, inflammatory bowel disease is a genetic disease which results from the inappropriate and exaggerated mucosal immune response to the normal constituents in the mucosal microbiota environment. An intestine targeted drug delivery system has unique advantages in the treatment of inflammatory bowel disease. As a new concept in drug delivery, the Trojan horse system with the synergy of nanotechnology and host cells can achieve better therapeutic efficacy in specific diseases. Here, we demonstrated the feasibility of encapsulating DNA-functionalized gold nanoparticles into primary isolated intestinal stem cells to form an intestinal Trojan horse for gene regulation therapy of inflammatory bowel disease. This proof-of-concept intestinal Trojan horse will have a wide variety of applications in the diagnosis and therapy of enteric disorders and diseases.

An intestinal Trojan horse for gene delivery.

PubMed

Peng, Haisheng; Wang, Chao; Xu, Xiaoyang; Yu, Chenxu; Wang, Qun

2015-03-14

The intestinal epithelium forms an essential element of the mucosal barrier and plays a critical role in the pathophysiological response to different enteric disorders and diseases. As a major enteric dysfunction of the intestinal tract, inflammatory bowel disease is a genetic disease which results from the inappropriate and exaggerated mucosal immune response to the normal constituents in the mucosal microbiota environment. An intestine targeted drug delivery system has unique advantages in the treatment of inflammatory bowel disease. As a new concept in drug delivery, the Trojan horse system with the synergy of nanotechnology and host cells can achieve better therapeutic efficacy in specific diseases. Here, we demonstrated the feasibility of encapsulating DNA-functionalized gold nanoparticles into primary isolated intestinal stem cells to form an intestinal Trojan horse for gene regulation therapy of inflammatory bowel disease. This proof-of-concept intestinal Trojan horse will have a wide variety of applications in the diagnosis and therapy of enteric disorders and diseases.

Spray-Dried Nanoparticle-in-Microparticle Delivery Systems (NiMDS) for Gene Delivery, Comprising Polyethylenimine (PEI)-Based Nanoparticles in a Poly(Vinyl Alcohol) Matrix.

PubMed

Schulze, Jan; Kuhn, Stephanie; Hendrikx, Stephan; Schulz-Siegmund, Michaela; Polte, Tobias; Aigner, Achim

2018-03-01

Nucleic acid-based therapies rely on efficient formulations for nucleic acid protection and delivery. As nonviral strategies, polymeric and lipid-based nanoparticles have been introduced; however, biological efficacy and biocompatibility as well as poor storage properties due to colloidal instability and their unavailability as ready-to-use systems are still major issues. Polyethylenimine is the most widely explored and promising candidate for gene delivery. Polyethylenimine-based polyplexes and their combination with liposomes, lipopolyplexes, are efficient for DNA or siRNA delivery in vitro and in vivo. In this study, a highly potent spray-dried nanoparticle-in-microparticle delivery system is presented for the encapsulation of polyethylenimine-based polyplexes and lipopolyplexes into poly(vinyl alcohol) microparticles, without requiring additional stabilizing agents. This easy-to-handle gene delivery device allows prolonged nanoparticle storage and protection at ambient temperature. Biological analyses reveal further advantages regarding profoundly reduced cytotoxicity and enhanced transfection efficacies of polyethylenimine-based nanoparticles from the nanoparticle-in-microparticle delivery system over their freshly prepared counterparts, as determined in various cell lines. Importantly, this nanoparticle-in-microparticle delivery system is demonstrated as ready-to-use dry powder to be an efficient device for the inhalative delivery of polyethylenimine-based lipopolyplexes in vivo, as shown by transgene expression in mice after only one administration. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rescue Effects and Underlying Mechanisms of Intragland Shh Gene Delivery on Irradiation-Induced Hyposalivation.

PubMed

Hai, Bo; Zhao, Qingguo; Qin, Lizheng; Rangaraj, Dharanipathy; Gutti, Veera R; Liu, Fei

2016-05-01

Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found in an inducible sonic hedgehog (Shh) transgenic mouse model that transient activation of the Hedgehog pathway after irradiation rescued salivary gland function in males by preserving salivary stem/progenitor cells and parasympathetic innervation. To translate these findings into feasible clinical application, we evaluated the effects of Shh gene transfer to salivary glands of wild-type mice on irradiation-induced hyposalivation. Shh or control GFP gene was delivered by noninvasive retrograde ductal instillation of corresponding adenoviral vectors. In both male and female mice, Shh gene delivery efficiently activated Hedgehog/Gli signaling, and significantly improved stimulated saliva secretion and preserved saliva-producing acinar cells after irradiation. In addition to preserving parasympathetic innervation through induction of neurotrophic factors, Shh gene delivery also alleviated the irradiation damage of the microvasculature, likely via inducing angiogenic factors, but did not expand the progeny of cells responsive to Hedgehog/Gli signaling. These data indicate that transient activation of the Hedgehog pathway by gene delivery is promising to rescue salivary function after irradiation in both sexes, and the Hedgehog/Gli pathway may function mainly in cell nonautonomous manners to achieve the rescue effect.

Creating an arsenal of Adeno-associated virus (AAV) gene delivery stealth vehicles.

PubMed

Smith, J Kennon; Agbandje-McKenna, Mavis

2018-05-01

The Adeno-associated virus (AAV) gene delivery system is ushering in a new and exciting era in the United States; following the first approved gene therapy (Glybera) in Europe, the FDA has approved a second therapy, Luxturna [1]. However, challenges to this system remain. In viral gene therapy, the surface of the capsid is an important determinant of tissue tropism, impacts gene transfer efficiency, and is targeted by the human immune system. Preexisting immunity is a significant challenge to this approach, and the ability to visualize areas of antibody binding ("footprints") can inform efforts to improve the efficacy of viral vectors. Atomic resolution, smaller proteins, and asymmetric structures are the goals to attain in cryo-electron microscopy and image reconstruction (cryo-EM) as of late. The versatility of the technique and the ability to vitrify a wide range of heterogeneous molecules in solution allow structural biologists to characterize a variety of protein-DNA and protein-protein interactions at lower resolution. Cryo-EM has served as an important means to study key surface areas of the AAV gene delivery vehicle-specifically, those involved with binding neutralizing antibodies (NAbs) [2-4]. This method offers a unique opportunity for visualizing antibody binding "hotspots" on the surface of these and other viral vectors. When combined with mutagenesis, one can eliminate these hotspots to create viral vectors with the ability to avoid preexisting host immune recognition during gene delivery and genetic defect correction in disease treatment. Here, we discuss the use of structure-guided site-directed mutagenesis and directed evolution to create "stealth" AAV vectors with modified surface amino acid sequences that allow NAb avoidance while maintaining natural capsid functions or gaining desired novel tropisms.

Creating an arsenal of Adeno-associated virus (AAV) gene delivery stealth vehicles

PubMed Central

Agbandje-McKenna, Mavis

2018-01-01

The Adeno-associated virus (AAV) gene delivery system is ushering in a new and exciting era in the United States; following the first approved gene therapy (Glybera) in Europe, the FDA has approved a second therapy, Luxturna [1]. However, challenges to this system remain. In viral gene therapy, the surface of the capsid is an important determinant of tissue tropism, impacts gene transfer efficiency, and is targeted by the human immune system. Preexisting immunity is a significant challenge to this approach, and the ability to visualize areas of antibody binding (“footprints”) can inform efforts to improve the efficacy of viral vectors. Atomic resolution, smaller proteins, and asymmetric structures are the goals to attain in cryo-electron microscopy and image reconstruction (cryo-EM) as of late. The versatility of the technique and the ability to vitrify a wide range of heterogeneous molecules in solution allow structural biologists to characterize a variety of protein–DNA and protein–protein interactions at lower resolution. Cryo-EM has served as an important means to study key surface areas of the AAV gene delivery vehicle—specifically, those involved with binding neutralizing antibodies (NAbs) [2–4]. This method offers a unique opportunity for visualizing antibody binding “hotspots” on the surface of these and other viral vectors. When combined with mutagenesis, one can eliminate these hotspots to create viral vectors with the ability to avoid preexisting host immune recognition during gene delivery and genetic defect correction in disease treatment. Here, we discuss the use of structure-guided site-directed mutagenesis and directed evolution to create “stealth” AAV vectors with modified surface amino acid sequences that allow NAb avoidance while maintaining natural capsid functions or gaining desired novel tropisms. PMID:29723270

Preliminary studies of particle-mediated gene delivery to the joints of dogs.

PubMed

Campbell, S E; Nasir, L; Gault, E A; Argyle, D J; Bennett, D

2007-04-07

This paper describes a preliminary evaluation of particle-mediated bombardment via the Helios gene gun for the delivery of therapeutic genes to synovial cells in culture. A reporter gene, enhanced green fluorescent protein, was delivered to rabbit synovial fibroblasts (HIG-82) using gold particle (1.0 microm) bombardment to evaluate transfection efficiency at helium pressures of 100 and 150 psi. Transfection of cells occurred at these pressures despite some cell death. The in vitro delivery of gold particles to samples of synovial membrane and articular cartilage from a freshly euthanased dog was also studied to examine depth of penetration of gold particles (1.0 microm) at helium pressures of 250 and 500 psi. Light microscopical examination of histological sections of the synovial membrane showed that particles of gold had penetrated the lining cells of the synovium. However, no gold particles had penetrated the articular cartilage even at 500 psi.

Topical mitomycin-C for recalcitrant esophageal strictures: a novel endoscopic/fluoroscopic technique for safe endoluminal delivery.

PubMed

Heran, Manraj K S; Baird, Robert; Blair, Geoffrey K; Skarsgard, Erik D

2008-05-01

Nonsurgical treatment of recalcitrant pediatric esophageal strictures is challenging. The chemotherapy drug mitomycin-C, which reduces collagen synthesis and scar formation, shows anecdotal promise in the topical treatment of these strictures. Mitomycin-C is cytotoxic, and a safe endoluminal delivery system that avoids inadvertent application to adjacent mucosa has not yet been described. We have treated 2 patients with a combined endoscopic/fluoroscopic technique that ensures protected delivery of a mitomycin-soaked pledget directly to the targeted site. Following pneumatic balloon dilation of the stricture under fluoroscopy, flexible esophagoscopy is performed to the disrupted stricture. Through the gastrostomy tract, a 12F to 16F semirigid sheath is introduced over a guide wire and passed retrograde up the esophagus to the stricture. A grasping forceps introduced through the instrument channel of the esophagoscope is advanced through the sheath and grasps a mitomycin-C-soaked pledget. The pledget is drawn back through the sheath up to the stricture where timed, serial radial applications to the stricture are performed without any contamination of the rest of the esophagus or stomach. We describe a novel technique of endoluminal delivery and focused application of mitomycin-C to an esophageal stricture that avoids inadvertent topical application to adjacent mucosa.

Nano-biomimetic carriers are implicated in mechanistic evaluation of intracellular gene delivery

NASA Astrophysics Data System (ADS)

Alipour, Mohsen; Hosseinkhani, Saman; Sheikhnejad, Reza; Cheraghi, Roya

2017-01-01

Several tissue specific non-viral carriers have been developed for gene delivery purposes. However, the inability to escape endosomes, undermines the efficacy of these carriers. Researchers inspired by HIV and influenza virus, have randomly used Gp41 and H5WYG fusogenic peptides in several gene delivery systems without any rational preference. Here for the first time, we have genetically engineered two Nano-biomimetic carriers composed of either HWYG (HNH) or Gp41 (GNH) that precisely provide identical conditions for the study and evaluation of these fusogenic peptides. The luciferase assay demonstrated a two-fold higher transfection efficiency of HNH compared to GNH. These nanocarriers also displayed equivalent properties in terms of DNA binding ability and DNA protection against serum nucleases and formed similar nanoparticles in terms of surface charge and size. Interestingly, hemolysis and cellular analysis demonstrated both of nanoparticles internalized into cells in similar rate and escaped from endosome with different efficiency. Furthermore, the structural analysis revealed the mechanisms responsible for the superior endosomal escaping capability of H5WYG. In conclusion, this study describes the rationale for using H5WYG peptide to deliver nucleic acids and suggests that using nano-biomimetic carriers to screen different endosomal release peptides, improves gene delivery significantly.

Ultrasound-mediated gene delivery of naked plasmid DNA in skeletal muscles: a case for bolus injections.

PubMed

Sanches, Pedro Gomes; Mühlmeister, Mareike; Seip, Ralf; Kaijzel, Eric; Löwik, Clemens; Böhmer, Marcel; Tiemann, Klaus; Grüll, Holger

2014-12-10

Localized gene delivery has many potential clinical applications. However, the nucleic acids (e.g. pDNA and siRNA) are incapable of passively crossing the endothelium, cell membranes and other biological barriers which must be crossed to reach their intracellular targets. A possible solution is the use of ultrasound to burst circulating microbubbles inducing transient permeabilization of surrounding tissues which mediates nucleic acid extravasation and cellular uptake. In this study we report on an optimization of the ultrasound gene delivery technique. Naked pDNA (200 μg) encoding luciferase and SonoVue® microbubbles were co-injected intravenously in mice. The hindlimb skeletal muscles were exposed to ultrasound from a non-focused transducer (1 MHz, 1.25 MPa, PRI 30s) and injection protocols and total amounts as well as ultrasound parameters were systemically varied. Gene expression was quantified relative to a control using a bioluminescence camera system at day 7 after sonication. Bioluminescence ratios in sonicated/control muscles of up to 101× were obtained. In conclusion, we were able to specifically deliver genetic material to the selected skeletal muscles and overall, the use of bolus injections and high microbubble numbers resulted in increased gene expression reflected by stronger bioluminescence signals. Based on our data, bolus injections seem to be required in order to achieve transient highly concentrated levels of nucleic acids and microbubbles at the tissue of interest which upon ultrasound exposure should lead to increased levels of gene delivery. Thus, ultrasound mediated gene delivery is a promising technique for the clinical translation of localized drug delivery. Copyright © 2014 Elsevier B.V. All rights reserved.

Current Progress in Gene Delivery Technology Based on Chemical Methods and Nano-carriers

PubMed Central

Jin, Lian; Zeng, Xin; Liu, Ming; Deng, Yan; He, Nongyue

2014-01-01

Gene transfer methods are promising in the field of gene therapy. Current methods for gene transfer include three major groups: viral, physical and chemical methods. This review mainly summarizes development of several types of chemical methods for gene transfer in vitro and in vivo by means of nano-carriers like; calcium phosphates, lipids, and cationic polymers including chitosan, polyethylenimine, polyamidoamine dendrimers, and poly(lactide-co-glycolide). This review also briefly introduces applications of these chemical methods for gene delivery. PMID:24505233

Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus.

PubMed

Yue, Yongping; Pan, Xiufang; Hakim, Chady H; Kodippili, Kasun; Zhang, Keqing; Shin, Jin-Hong; Yang, Hsiao T; McDonald, Thomas; Duan, Dongsheng

2015-10-15

The ultimate goal of muscular dystrophy gene therapy is to treat all muscles in the body. Global gene delivery was demonstrated in dystrophic mice more than a decade ago using adeno-associated virus (AAV). However, translation to affected large mammals has been challenging. The only reported attempt was performed in newborn Duchenne muscular dystrophy (DMD) dogs. Unfortunately, AAV injection resulted in growth delay, muscle atrophy and contracture. Here we report safe and bodywide AAV delivery in juvenile DMD dogs. Three ∼2-m-old affected dogs received intravenous injection of a tyrosine-engineered AAV-9 reporter or micro-dystrophin (μDys) vector at the doses of 1.92-6.24 × 10(14) viral genome particles/kg under transient or sustained immune suppression. DMD dogs tolerated injection well and their growth was not altered. Hematology and blood biochemistry were unremarkable. No adverse reactions were observed. Widespread muscle transduction was seen in skeletal muscle, the diaphragm and heart for at least 4 months (the end of the study). Nominal expression was detected in internal organs. Improvement in muscle histology was observed in μDys-treated dogs. In summary, systemic AAV gene transfer is safe and efficient in young adult dystrophic large mammals. This may translate to bodywide gene therapy in pediatric patients in the future. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Enhanced thermogenic program by non-viral delivery of combinatory browning genes to treat diet-induced obesity in mice.

PubMed

Park, Hongsuk; Cho, Sungpil; Janat-Amsbury, Margit M; Bae, You Han

2015-12-01

Thermogenic program (also known as browning) is a promising and attractive anti-obesity approach. Islet amyloid polypeptide (IAPP) and irisin have emerged as potential browning hormones that hold high potential to treat obesity. Here, we have constructed a dual browning gene system containing both IAPP and irisin (derived from fibronectin type III domain containing 5; FNDC5) combined with 2A and furin self-cleavage sites. Intraperitoneal administration of the construct complexed with a linear polyethylenimine into diet-induced obese mice demonstrated the elevation of anti-obesogenic effects characterized as the decreased body weight, adiposity, and levels of glucose and insulin. In addition, the construct delivery increased energy expenditure and the expression of core molecular determinants associated with browning. The additional advantages of the dual browning gene construct delivery compared to both single gene construct delivery and dual peptide delivery can be emphasized on efficacy and practicability. Hence, we have concluded that dual browning gene delivery makes it therapeutically attractive for diet-induced obesity treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery of genetic material via living cells.

PubMed

Ward, Eliot; Chan, Emma; Gustafsson, Kenth; Jayasinghe, Suwan N

2010-05-01

The investigations reported in this article demonstrate the ability of bio-electrosprays and cell electrospinning to deliver a genetic construct in association with living cells. Previous studies on both bio-electrosprays and cell electrospinning demonstrated great promise for tissue engineering and regenerative biology/medicine. The investigations described herein widen the applicability of these biotechniques by combining gene therapy protocols, resulting in a novel drug delivery methodology previously unexplored. In these studies a human cell line was transduced with recombinant self-inactivating lentiviral particles. These particles incorporated a green fluorescent protein fused to an endosomal targeting construct. This construct encodes a peptide, which can subsequently be detected on the surface of cells by specific T-cells. The transduced cell line was subsequently manipulated in association with either bio-electrospraying or cell electrospinning. Hence this demonstrates (i) the ability to safely handle genetically modified living cells and (ii) the ability to directly form pre-determined architectures bearing living therapeutic cells. This merged technology demonstrates a unique approach for directly forming living therapeutic architectures for controlled and targeted release of experimental cells/genes, as well as medical cell/gene therapeutics for a plethora of biological and medical applications. Hence, such developments could be applied to personalised medicine.

Carbon nanotubes part II: a remarkable carrier for drug and gene delivery

PubMed Central

Karimi, Mahdi; Solati, Navid; Ghasemi, Amir; Estiar, Mehrdad Asghari; Hashemkhani, Mahshid; Kiani, Parnian; Mohamed, Elmira; Saeidi, Ahad; Taheri, Mahdiar; Avci, Pinar; Aref, Amir R; Amiri, Mohammad; Baniasadi, Fazel; Hamblin, Michael R

2015-01-01

Introduction Carbon nanotubes (CNT) have recently been studied as novel and versatile drug and gene delivery vehicles. When CNT are suitably functionalized, they can interact with various cell types and are taken up by endocytosis. Areas covered Anti-cancer drugs cisplatin and doxorubicin have been delivered by CNT, as well as methotrexate, taxol and gemcitabine. The delivery of the antifungal compound amphotericin B and the oral administration of erythropoietin have both been assisted using CNT. Frequently, targeting moieties such as folic acid, epidermal growth factor or various antibodies are attached to the CNT-drug nanovehicle. Different kinds of functionalization (e.g., polycations) have been used to allow CNT to act as gene delivery vectors. Plasmid DNA, small interfering RNA and micro-RNA have all been delivered by CNT vehicles. Significant concerns are raised about the nanotoxicology of the CNT and their potentially damaging effects on the environment. Expert opinion CNT-mediated drug delivery has been studied for over a decade, and both in vitro and in vivo studies have been reported. The future success of CNTs as vectors in vivo and in clinical application will depend on achievement of efficacious therapy with minimal adverse effects and avoidance of possible toxic and environmentally damaging effects. PMID:25613837

Backflow-free catheters for efficient and safe convection-enhanced delivery of therapeutics.

PubMed

Lueshen, Eric; Tangen, Kevin; Mehta, Ankit I; Linninger, Andreas

2017-07-01

Convection-enhanced delivery (CED) is an invasive drug delivery technique used to target specific regions of the brain for the treatment of cancer and neurodegenerative diseases while bypassing the blood-brain barrier. In order to prevent the possibility of backflow, low volumetric flow rates are applied which limit the achievable drug distribution volumes from CED. This can render CED treatment ineffective since a small convective flow produces narrow drug distribution inside the treatment region. Novel catheter designs and CED protocols are needed to improve the drug distribution inside the treatment region. This is especially important when administering toxic chemotherapeutics which could adversely affect other organs if backflow occurred and these drugs entered the circulating blood stream. In order to help elucidate the causes of backflow and to design backflow-free catheters, we have studied the impact that microfluid flow has on deformable brain phantom gels experimentally as well as numerically. We found that fluid injections into porous media have considerable effects on local transport properties such as porosity and hydraulic conductivity. These phenomena not only alter the bulk flow velocity distribution of the microfluid flow due to the changing porosity, but significantly modify flow direction and even volumetric flow distribution due to induced local hydraulic conductivity anisotropy. These studies led us to the development of novel backflow-free catheters with safe volumetric flow rates up to 10 µL/min. The catheter designs, numerical simulations and experimental results are described throughout this article. Copyright © 2017 IPEM. Published by Elsevier Ltd. All rights reserved.

Bolstering cholesteryl ester hydrolysis in liver: A hepatocyte-targeting gene delivery strategy for potential alleviation of atherosclerosis

PubMed Central

He, Hongliang; Lancina, Michael G.; Wang, Jing; Korzun, William J.; Yang, Hu; Ghosh, Shobha

2017-01-01

Current atherosclerosis treatment strategies primarily focus on limiting further cholesteryl esters (CE) accumulation by reducing endogenous synthesis of cholesterol in the liver. No therapy is currently available to enhance the removal of CE, a crucial step to reduce the burden of the existing disease. Given a central role of hepatic cholesteryl ester hydrolase (CEH) in intrahepatic hydrolysis of CE and subsequent removal of the resulting free cholesterol, in this work, we applied galactose-functionalized polyamidoamine (PAMAM) dendrimer G5 (Gal-G5) for hepatocyte-specific delivery of CEH expression vector. The data presented herein show increased specific uptake of Gal-G5 by the hepatocytes in vitro and in vivo. Furthermore, the upregulated CEH expression in the hepatocytes significantly enhanced intracellular hydrolysis of HDL-CE and subsequent conversion/secretion of hydrolyzed free cholesterol (FC) as bile acids. Increased CEH expression in the liver significantly increased the flux of HDL-CE to biliary as well as fecal FC and bile acids. Meanwhile, Gal-G5 did not induce hepatic or renal toxicity. It was not immunotoxic. Because of these encouraging pre-clinical testing results, using this safe and highly efficient hepatocyte-specific gene delivery platform to enhance the hepatic processes involved in cholesterol elimination is a promising strategy for alleviation of atherosclerosis. PMID:28349866

Targeted Drug Delivery Based on Gold Nanoparticle Derivatives.

PubMed

Gholipourmalekabadi, Mazaher; Mobaraki, Mohammadmahdi; Ghaffari, Maryam; Zarebkohan, Amir; Omrani, Vahid Fallah; Urbanska, Aleksandra M; Seifalian, Alexander

2017-01-01

Drug delivery systems are effective and attractive methods which allow therapeutic substances to be introduced into the body more effectively and safe by having tunable delivery rate and release target site. Gold nanoparticles (AuNPs) have a myriad of favorable physical, chemical, optical, thermal and biological properties that make them highly suitable candidates as non-toxic carriers for drug and gene delivery. The surface modifications of AuNPs profoundly improve their circulation, minimize aggregation rates, enhance attachment to therapeutic molecules and target agents due to their nano range size which further increases their ability to cross cell membranes and reduce overall cytotoxicity. This comprehensive article reviews the applications of the AuNPs in drug delivery systems along with their corresponding surface modifications. The highlighting results obtained from the preclinical trial are promising and next five years have huge possibility move to the clinical setting. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Electrostatically assembled dendrimer complex with a high-affinity protein binder for targeted gene delivery.

PubMed

Kim, Jong-Won; Lee, Joong-Jae; Choi, Joon Sig; Kim, Hak-Sung

2018-06-10

Although a variety of non-viral gene delivery systems have been developed, they still suffer from low efficiency and specificity. Herein, we present the assembly of a dendrimer complex comprising a DNA cargo and a targeting moiety as a new format for targeted gene delivery. A PAMAM dendrimer modified with histidine and arginine (HR-dendrimer) was used to enhance the endosomal escape and transfection efficiency. An EGFR-specific repebody, composed of leucine-rich repeat (LRR) modules, was employed as a targeting moiety. A polyanionic peptide was genetically fused to the repebody, followed by incubation with an HR-dendrimer and a DNA cargo to assemble the dendrimer complex through an electrostatic interaction. The resulting dendrimer complex was shown to deliver a DNA cargo with high efficiency in a receptor-specific manner. An analysis using a confocal microscope confirmed the internalization of the dendrimer complex and subsequent dissociation of a DNA cargo from the complex. The present approach can be broadly used in a targeted gene delivery in many areas. Copyright © 2018 Elsevier B.V. All rights reserved.

Tumor targeting RGD conjugated bio-reducible polymer for VEGF siRNA expressing plasmid delivery

PubMed Central

Kim, Hyun Ah; Nam, Kihoon; Kim, Sung Wan

2014-01-01

Targeted delivery of therapeutic genes to the tumor site is critical for successful and safe cancer gene therapy. The arginine grafted bio-reducible poly (cystamine bisacrylamide-diaminohexane, CBA-DAH) polymer (ABP) conjugated poly (amido amine) (PAMAM), PAM-ABP (PA) was designed previously as an efficient gene delivery carrier. To achieve high efficacy in cancer selective delivery, we developed the tumor targeting bio-reducible polymer, PA-PEG1k-RGD, by conjugating cyclic RGDfC (RGD) peptides, which bind αvβ3/5 integrins, to the PAM-ABP using polyethylene glycol (PEG,1kDa) as a spacer. Physical characterization showed nanocomplex formation with bio-reducible properties between PA-PEG1k-RGD and plasmid DNA (pDNA). In transfection assays, PA-PEG1k-RGD showed significantly higher transfection efficiency in comparison with PAM-ABP or PA-PEG1k-RGD in αvβ3/5 positive MCF7 breast cancer and PANC-1 pancreatic cancer cells. The targeting ability of PA-PEG1k-RGD was further established using a competition assay. To confirm the therapeutic effect, the VEGF siRNA expressing plasmid was constructed and then delivered into cancer cells using PA-PEG1k-RGD. PA-PEG1k-RGD showed 20-59% higher cellular uptake rate into MCF7 and PANC-1 than that of non-targeted polymers. In addition, MCF7 and PANC-1 cancer cells transfected with PA-PEG1k-RGD/pshVEGF complexes had significantly decreased VEGF gene expression (51-71%) and cancer cell viability (35-43%) compared with control. These results demonstrate that a tumor targeting bio-reducible polymer with an anti-angiogenic therapeutic gene could be used for efficient and safe cancer gene therapy. PMID:24894645

An assessment of the safe delivery incentive program at a tertiary level hospital in Nepal.

PubMed

Baral, G

2012-05-01

Maternity incentive program of Nepal known as Safe Delivery Incentive Program (SDIP) was introduced nationwide in 2005 with the intention of increasing utilization of professional care at childbirth. The program provided both childbirth service as well as 'cash' to women giving birth in a health facility in addition to incentives to health provider for each delivery attended, either at home or the facility. Due to a lack of uniformity in its implementation and administrative delays, the program was reformed and even extended to many not-for-profit health institutions in early 2007, and implemented as a 'Safer Mother Program' popularly known as "Aama-Suraksha-Karyakram" since January 2009. This is a system research with observational and analytical components. Plausibility design is selected to evaluate the performance-based funding (PBF) as a system level intervention of maternity care using two instruments: Pay-For-Performance and Conditional-Cash-Transfer. It uses interrupted time-series to control for the natural trend. Research tools used are interviews, the focus group discussions and literature review. Numerical data are presented in simple graphs. While online random number generator was used partly, the purposive sampling was used for qualitative data. There is a gross discrepancy in non-targeted service delivery at the tertiary level health facility. Overflooding of maternity cases has hampered gynecological admission and surgical management delaying subspecialty care and junior physicians' training. With the same number and quality of physical facility and human resource, the additional program has put more strains to service providers and administrators. There should be adequate planning and preparation at all levels of health facilities; implementing a new program should not adversely affect another existing service delivery system. For the optional implementation, hospital organogram should be revised; and physical facilities and the low-risk birthing

Bacteriophage-Derived Vectors for Targeted Cancer Gene Therapy

PubMed Central

Pranjol, Md Zahidul Islam; Hajitou, Amin

2015-01-01

Cancer gene therapy expanded and reached its pinnacle in research in the last decade. Both viral and non-viral vectors have entered clinical trials, and significant successes have been achieved. However, a systemic administration of a vector, illustrating safe, efficient, and targeted gene delivery to solid tumors has proven to be a major challenge. In this review, we summarize the current progress and challenges in the targeted gene therapy of cancer. Moreover, we highlight the recent developments of bacteriophage-derived vectors and their contributions in targeting cancer with therapeutic genes following systemic administration. PMID:25606974

Bacteriophage-derived vectors for targeted cancer gene therapy.

PubMed

Pranjol, Md Zahidul Islam; Hajitou, Amin

2015-01-19

Cancer gene therapy expanded and reached its pinnacle in research in the last decade. Both viral and non-viral vectors have entered clinical trials, and significant successes have been achieved. However, a systemic administration of a vector, illustrating safe, efficient, and targeted gene delivery to solid tumors has proven to be a major challenge. In this review, we summarize the current progress and challenges in the targeted gene therapy of cancer. Moreover, we highlight the recent developments of bacteriophage-derived vectors and their contributions in targeting cancer with therapeutic genes following systemic administration.

Delivery of drugs, growth factors, genes and stem cells via intrapericardial, epicardial and intramyocardial routes for sustained local targeted therapy of myocardial disease.

PubMed

Maslov, Mikhail; Foianini, Stephan; Lovich, Mark

2017-10-01

Local myocardial delivery (LMD) of therapeutic agents is a promising strategy that aims to treat various myocardial pathologies. It is designed to deliver agents directly to the myocardium and minimize their extracardiac concentrations and side effects. LMD aims to enhance outcomes of existing therapies by broadening their therapeutic window and to utilize new agents that could not be otherwise be implemented systemically. Areas covered: This article provides a historical overview of six decades LMD evolution in terms of the approaches, including intrapericardial, epicardial, and intramyocardial delivery, and the wide array of classes of agents used to treat myocardial pathologies. We examines delivery of pharmaceutical compounds, targeted gene transfection and cell implantation techniques to produce therapeutic effects locally. We outline therapeutic indications, successes and failures as well as technical approaches for LMD. Expert opinion: While LMD is more complicated than conventional oral or intravenous administration, given recent advances in interventional cardiology, it is safe and may provide better therapeutic outcomes. LMD is complex as many factors impact pharmacokinetics and biologic result. The choice between routes of LMD is largely driven not only by the myocardial pathology but also by the nature and physicochemical properties of the therapeutic agents.

Exploring advantages/disadvantages and improvements in overcoming gene delivery barriers of amino acid modified trimethylated chitosan.

PubMed

Zheng, Hao; Tang, Cui; Yin, Chunhua

2015-06-01

Present study aimed at exploring advantages/disadvantages of amino acid modified trimethylated chitosan in conquering multiple gene delivery obstacles and thus providing comprehensive understandings for improved transfection efficiency. Arginine, cysteine, and histidine modified trimethyl chitosan were synthesized and employed to self-assemble with plasmid DNA (pDNA) to form nanocomplexes, namely TRNC, TCNC, and THNC, respectively. They were assessed by structural stability, cellular uptake, endosomal escape, release behavior, nuclear localization, and in vitro and in vivo transfection efficiencies. Besides, sodium tripolyphosphate (TPP) was added into TRNC to compromise certain disadvantageous attributes for pDNA delivery. Optimal endosomal escape ability failed to bring in satisfactory transfection efficiency of THNC due to drawbacks in structural stability, cellular uptake, pDNA liberation, and nuclear distribution. TCNC evoked the most potent gene expression owing to multiple advantages including sufficient stability, preferable uptake, efficient pDNA release, and high nucleic accumulation. Undesirable stability and insufficient pDNA release adversely affected TRNC-mediated gene transfer. However, incorporation of TPP could improve such disadvantages and consequently resulted in enhanced transfection efficiencies. Coordination of multiple contributing effects to conquer all delivery obstacles was necessitated for improved transfection efficiency, which would provide insights into rational design of gene delivery vehicles.

Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.

PubMed

Rincon, Melvin Y; VandenDriessche, Thierry; Chuah, Marinee K

2015-10-01

Gene therapy is a promising modality for the treatment of inherited and acquired cardiovascular diseases. The identification of the molecular pathways involved in the pathophysiology of heart failure and other associated cardiac diseases led to encouraging preclinical gene therapy studies in small and large animal models. However, the initial clinical results yielded only modest or no improvement in clinical endpoints. The presence of neutralizing antibodies and cellular immune responses directed against the viral vector and/or the gene-modified cells, the insufficient gene expression levels, and the limited gene transduction efficiencies accounted for the overall limited clinical improvements. Nevertheless, further improvements of the gene delivery technology and a better understanding of the underlying biology fostered renewed interest in gene therapy for heart failure. In particular, improved vectors based on emerging cardiotropic serotypes of the adeno-associated viral vector (AAV) are particularly well suited to coax expression of therapeutic genes in the heart. This led to new clinical trials based on the delivery of the sarcoplasmic reticulum Ca(2+)-ATPase protein (SERCA2a). Though the first clinical results were encouraging, a recent Phase IIb trial did not confirm the beneficial clinical outcomes that were initially reported. New approaches based on S100A1 and adenylate cyclase 6 are also being considered for clinical applications. Emerging paradigms based on the use of miRNA regulation or CRISPR/Cas9-based genome engineering open new therapeutic perspectives for treating cardiovascular diseases by gene therapy. Nevertheless, the continuous improvement of cardiac gene delivery is needed to allow the use of safer and more effective vector doses, ultimately bringing gene therapy for heart failure one step closer to reality. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Cardiology.

Technique of retinal gene therapy: delivery of viral vector into the subretinal space

PubMed Central

Xue, K; Groppe, M; Salvetti, A P; MacLaren, R E

2017-01-01

Purpose Safe and reproducible delivery of gene therapy vector into the subretinal space is essential for successful targeting of the retinal pigment epithelium (RPE) and photoreceptors. The success of surgery is critical for the clinical efficacy of retinal gene therapy. Iatrogenic detachment of the degenerate (often adherent) retina in patients with hereditary retinal degenerations and small volume (eg, 0.1 ml) subretinal injections pose new surgical challenges. Methods Our subretinal gene therapy technique involved pre-operative planning with optical coherence tomography (OCT) and autofluorescence (AF) imaging, 23 G pars plana vitrectomy, internal limiting membrane staining with Membrane Blue Dual (DORC BV, Zuidland, Netherlands), a two-step subretinal injection using a 41 G Teflon tipped cannula (DORC) first with normal saline to create a parafoveal bleb followed by slow infusion of viral vector via the same self-sealing retinotomy. Surgical precision was further enhanced by intraoperative OCT (Zeiss Rescan 7000, Carl Zeiss Meditec AG, Jena, Germany). Foveal functional and structural recovery was evaluated using best-corrected Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity, microperimetry and OCT. Results Two patients with choroideremia aged 29 (P1) and 27 (P2) years, who had normal and symmetrical levels of best-corrected visual acuity (BCVA) in both eyes, underwent unilateral gene therapy with the fellow eye acting as internal control. The surgeries were uncomplicated in both cases with successful detachment of the macula by subretinal vector injection. Both treated eyes showed recovery of BCVA (P1: 76–77 letters; P2: 84–88 letters) and mean threshold sensitivity of the central macula (P1: 10.7–10.7 dB; P2: 14.2–14.1 dB) to baseline within a month. This was accompanied by normalisation of central retinal thickness on OCT. Conclusions Herein we describe a reliable technique for subretinal gene therapy, which is currently used

Safe biodegradable fluorescent particles

DOEpatents

Martin, Sue I [Berkeley, CA; Fergenson, David P [Alamo, CA; Srivastava, Abneesh [Santa Clara, CA; Bogan, Michael J [Dublin, CA; Riot, Vincent J [Oakland, CA; Frank, Matthias [Oakland, CA

2010-08-24

A human-safe fluorescence particle that can be used for fluorescence detection instruments or act as a safe simulant for mimicking the fluorescence properties of microorganisms. The particle comprises a non-biological carrier and natural fluorophores encapsulated in the non-biological carrier. By doping biodegradable-polymer drug delivery microspheres with natural or synthetic fluorophores, the desired fluorescence can be attained or biological organisms can be simulated without the associated risks and logistical difficulties of live microorganisms.

Improving Small Interfering RNA Delivery In Vivo Through Lipid Conjugation.

PubMed

Osborn, Maire F; Khvorova, Anastasia

2018-05-10

RNA interference (RNAi)-based therapeutics are approaching clinical approval for genetically defined diseases. Current clinical success is a result of significant innovations in the development of chemical architectures that support sustained, multi-month efficacy in vivo following a single administration. Conjugate-mediated delivery has established itself as the most promising platform for safe and targeted small interfering RNA (siRNA) delivery. Lipophilic conjugates represent a major class of modifications that improve siRNA pharmacokinetics and enable efficacy in a broad range of tissues. Here, we review current literature and define key features and limitations of this approach for in vivo modulation of gene expression.

A bio-recognition device developed onto nano-crystals of carbonate apatite for cell-targeted gene delivery.

PubMed

Chowdhury, E H; Akaike, Toshihiro

2005-05-20

The DNA delivery to mammalian cells is an essential tool for analyzing gene structure, regulation, and function. The approach holds great promise for the further development of gene therapy techniques and DNA vaccination strategies to treat and control diseases. Here, we report on the establishment of a cell-specific gene delivery and expression system by physical adsorption of a cell-recognition molecule on the nano-crystal surface of carbonate apatite. As a model, DNA/nano-particles were successfully coated with asialofetuin to facilitate uptake by hepatocyte-derived cell lines through the asialoglycoprotein receptor (ASGPr) and albumin to prevent non-specific interactions of the particles with cell-surface. The resulting composite particles with dual surface properties could accelerate DNA uptake and enhance expression to a notable extent. Nano-particles coated with transferrin in the same manner dramatically enhanced transgene expression in the corresponding receptor-bearing cells and thus our newly developed strategy represents a universal phenomenon for anchoring a bio-recognition macromolecule on the apatite crystal surface for targeted gene delivery, having immediate applications in basic research laboratories and great promise for gene therapy. (c) 2005 Wiley Periodicals, Inc.

Messenger RNA Delivery for Tissue Engineering and Regenerative Medicine Applications.

PubMed

Patel, Siddharth; Athirasala, Avathamsa; Menezes, Paula P; Ashwanikumar, N; Zou, Ting; Sahay, Gaurav; Bertassoni, Luiz E

2018-06-07

The ability to control cellular processes and precisely direct cellular reprogramming has revolutionized regenerative medicine. Recent advances in in vitro transcribed (IVT) mRNA technology with chemical modifications have led to development of methods that control spatiotemporal gene expression. Additionally, there is a current thrust toward the development of safe, integration-free approaches to gene therapy for translational purposes. In this review, we describe strategies of synthetic IVT mRNA modifications and nonviral technologies for intracellular delivery. We provide insights into the current tissue engineering approaches that use a hydrogel scaffold with genetic material. Furthermore, we discuss the transformative potential of novel mRNA formulations that when embedded in hydrogels can trigger controlled genetic manipulation to regenerate tissues and organs in vitro and in vivo. The role of mRNA delivery in vascularization, cytoprotection, and Cas9-mediated xenotransplantation is additionally highlighted. Harmonizing mRNA delivery vehicle interactions with polymeric scaffolds can be used to present genetic cues that lead to precise command over cellular reprogramming, differentiation, and secretome activity of stem cells-an ultimate goal for tissue engineering.

3D Porous Chitosan-Alginate Scaffolds as an In Vitro Model for Evaluating Nanoparticle-Mediated Tumor Targeting and Gene Delivery to Prostate Cancer.

PubMed

Wang, Kui; Kievit, Forrest M; Florczyk, Stephen J; Stephen, Zachary R; Zhang, Miqin

2015-10-12

Cationic nanoparticles (NPs) for targeted gene delivery are conventionally evaluated using 2D in vitro cultures. However, this does not translate well to corresponding in vivo studies because of the marked difference in NP behavior in the presence of the tumor microenvironment. In this study, we investigated whether prostate cancer (PCa) cells cultured in three-dimensional (3D) chitosan-alginate (CA) porous scaffolds could model cationic NP-mediated gene targeted delivery to tumors in vitro. We assessed in vitro tumor cell proliferation, formation of tumor spheroids, and expression of marker genes that promote tumor malignancy in CA scaffolds. The efficacy of NP-targeted gene delivery was evaluated in PCa cells in 2D cultures, PCa tumor spheroids grown in CA scaffolds, and PCa tumors in a mouse TRAMP-C2 flank tumor model. PCa cells cultured in CA scaffolds grew into tumor spheroids and displayed characteristics of higher malignancy as compared to those in 2D cultures. Significantly, targeted gene delivery was only observed in cells cultured in CA scaffolds, whereas cells cultured on 2D plates showed no difference in gene delivery between targeted and nontarget control NPs. In vivo NP evaluation confirmed targeted gene delivery, indicating that only CA scaffolds correctly modeled NP-mediated targeted delivery in vivo. These findings suggest that CA scaffolds serve as a better in vitro platform than 2D cultures for evaluation of NP-mediated targeted gene delivery to PCa.

Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa.

PubMed

Cai, Xue; Conley, Shannon M; Nash, Zack; Fliesler, Steven J; Cooper, Mark J; Naash, Muna I

2010-04-01

The purpose of the present study was to test the therapeutic efficiency and safety of compacted-DNA nanoparticle-mediated gene delivery into the subretinal space of a juvenile mouse model of retinitis pigmentosa. Nanoparticles containing the mouse opsin promoter and wild-type mouse Rds gene were injected subretinally into mice carrying a haploinsufficiency mutation in the retinal degeneration slow (rds(+ or -)) gene at postnatal day (P)5 and 22. Control mice were either injected with saline, injected with uncompacted naked plasmid DNA carrying the Rds gene, or remained untreated. Rds mRNA levels peaked at postinjection day 2 to 7 (PI-2 to PI-7) for P5 injections, stabilized at levels 2-fold higher than in uninjected controls for both P5 and P22 injections, and remained elevated at the latest time point examined (PI-120). Rod function (measured by electroretinography) showed modest but statistically significant improvement compared with controls after both P5 and P22 injections. Cone function in nanoparticle-injected eyes reached wild-type levels for both ages of injections, indicating full prevention of cone degeneration. Ultrastructural examination at PI-120 revealed significant improvement in outer segment structures in P5 nanoparticle-injected eyes, while P22 injection had a modest structural improvement. There was no evidence of macrophage activation or induction of IL-6 or TNF-alpha mRNA in P5 or P22 nanoparticle-dosed eyes at either PI-2 or PI-30. Thus, compacted-DNA nanoparticles can efficiently and safely drive gene expression in both mitotic and postmitotic photoreceptors and retard degeneration in this model. These findings, using a clinically relevant treatment paradigm, illustrate the potential for application of nanoparticle-based gene replacement therapy for treatment of human retinal degenerations.-Cai, X., Conley, S. M., Nash, Z., Fliesler, S. J., Cooper, M. J., Naash, M. I. Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

PubMed

Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G; Corydon, Thomas J; Mikkelsen, Jacob Giehm; Aagaard, Lars

2015-08-01

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin

PubMed Central

Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G.; Corydon, Thomas J.; Mikkelsen, Jacob Giehm; Aagaard, Lars

2015-01-01

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo. PMID:26204415

Organizing delivery care: what works for safe motherhood?

PubMed Central

Koblinsky, M. A.; Campbell, O.; Heichelheim, J.

1999-01-01

The various means of delivering essential obstetric services are described for settings in which the maternal mortality ratio is relatively low. This review yields four basic models of care, which are best described by organizational characteristics relating to where women give birth and who performs deliveries. In Model 1, deliveries are conducted at home by a community member who has received brief training. In Model 2, delivery takes place at home but is performed by a professional. In Model 3, delivery is performed by a professional in a basic essential obstetric care facility, and in Model 4 all women give birth in a comprehensive essential obstetric care facility with the help of professionals. In each of these models it is assumed that providers do not increase the risk to women, either iatrogenically or through traditional practices. Although there have been some successes with Model 1, there is no evidence that it can provide a maternal mortality ratio under 100 per 100,000 live births. If strong referral mechanisms are in place the introduction of a professional attendant can lead to a marked reduction in the maternal mortality ratio. Countries using Models 2-4, involving the use of professional attendants at delivery, have reduced maternal mortality ratios to 50 or less per 100,000. However, Model 4, although arguably the most advanced, does not necessarily reduce the maternal mortality ratio to less than 100 per 100,000. It appears that not all countries are ready to adopt Model 4, and its affordability by many developing countries is doubtful. There are few data making it possible to determine which configuration with professional attendance is the most cost-effective, and what the constraints are with respect to training, skill maintenance, supervision, regulation, acceptability to women, and other criteria. A successful transition to Models 2-4 requires strong links with the community through either traditional providers or popular demand. PMID

Application of pulsed-magnetic field enhances non-viral gene delivery in primary cells from different origins

NASA Astrophysics Data System (ADS)

Kamau Chapman, Sarah W.; Hassa, Paul O.; Koch-Schneidemann, Sabine; von Rechenberg, Brigitte; Hofmann-Amtenbrink, Margarethe; Steitz, Benedikt; Petri-Fink, Alke; Hofmann, Heinrich; Hottiger, Michael O.

Primary cell lines are more difficult to transfect when compared to immortalized/transformed cell lines, and hence new techniques are required to enhance the transfection efficiency in these cells. We isolated and established primary cultures of synoviocytes, chondrocytes, osteoblasts, melanocytes, macrophages, lung fibroblasts, and embryonic fibroblasts. These cells differed in several properties, and hence were a good representative sample of cells that would be targeted for expression and delivery of therapeutic genes in vivo. The efficiency of gene delivery in all these cells was enhanced using polyethylenimine-coated polyMAG magnetic nanoparticles, and the rates (17-84.2%) surpassed those previously achieved using other methods, especially in cells that are difficult to transfect. The application of permanent and pulsating magnetic fields significantly enhanced the transfection efficiencies in synoviocytes, chondrocytes, osteoblasts, melanocytes and lung fibroblasts, within 5 min of exposure to these magnetic fields. This is an added advantage for future in vivo applications, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.

Manipulating the membrane penetration mechanism of helical polypeptides via aromatic modification for efficient gene delivery.

PubMed

Zheng, Nan; Song, Ziyuan; Yang, Jiandong; Liu, Yang; Li, Fangfang; Cheng, Jianjun; Yin, Lichen

2017-08-01

The delivery performance of non-viral gene vectors is greatly related to their intracellular kinetics. Cationic helical polypeptides with potent membrane penetration properties and gene transfection efficiencies have been recently developed by us. However, they suffer from severe drawbacks in terms of their membrane penetration mechanisms that mainly include endocytosis and pore formation. The endocytosis mechanism leads to endosomal entrapment of gene cargos, while the charge- and helicity-induced pore formation causes appreciable cytotoxicity at high concentrations. With the attempt to overcome such critical challenges, we incorporated aromatic motifs into the design of helical polypeptides to enhance their membrane activities and more importantly, to manipulate their membrane penetration mechanisms. The aromatically modified polypeptides exhibited higher cellular internalization level than the unmodified analogue by up to 2.5 folds. Such improvement is possibly because aromatic domains promoted the polypeptides to penetrate cell membranes via direct transduction, a non-endocytosis and non-pore formation mechanism. As such, gene cargos were more efficiently delivered into cells by bypassing endocytosis and subsequently avoiding endosomal entrapment, and the material toxicity associated with excessive pore formation was also reduced. The top-performing aromatic polypeptide containing naphthyl side chains at the incorporated content of 20mol% revealed notably higher transfection efficiencies than commercial reagents in melanoma cells in vitro (by 11.7 folds) and in vivo (by 9.1 folds), and thus found potential utilities toward topical gene delivery for cancer therapy. Cationic helical polypeptides, as efficient gene delivery materials, suffer from severe drawbacks in terms of their membrane penetration mechanisms. The main cell penetration mechanisms involved are endocytosis and pore formation. However, the endocytosis mechanism has the limitation of endosomal

Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

PubMed Central

2012-01-01

Background We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns. Results The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively. Conclusions These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns. PMID:22853641

Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

PubMed Central

Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

2013-01-01

Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. PMID:23799175

The expanding role of aerosols in systemic drug delivery, gene therapy, and vaccination.

PubMed

Laube, Beth L

2005-09-01

Aerosolized medications have been used for centuries to treat respiratory diseases. Until recently, inhalation therapy focused primarily on the treatment of asthma and chronic obstructive pulmonary disease, and the pressurized metered-dose inhaler was the delivery device of choice. However, the role of aerosol therapy is clearly expanding beyond that initial focus. This expansion has been driven by the Montreal protocol and the need to eliminate chlorofluorocarbons (CFCs) from traditional metered-dose inhalers, by the need for delivery devices and formulations that can efficiently and reproducibly target the systemic circulation for the delivery of proteins and peptides, and by developments in medicine that have made it possible to consider curing lung diseases with aerosolized gene therapy and preventing epidemics of influenza and measles with aerosolized vaccines. Each of these drivers has contributed to a decade or more of unprecedented research and innovation that has altered how we think about aerosol delivery and has expanded the role of aerosol therapy into the fields of systemic drug delivery, gene therapy, and vaccination. During this decade of innovation, we have witnessed the coming of age of dry powder inhalers, the development of new soft mist inhalers, and improved pressurized metered-dose inhaler delivery as a result of the replacement of CFC propellants with hydrofluoroalkane. The continued expansion of the role of aerosol therapy will probably depend on demonstration of the safety of this route of administration for drugs that have their targets outside the lung and are administered long term (eg, insulin aerosol), on the development of new drugs and drug carriers that can efficiently target hard-to-reach cell populations within the lungs of patients with disease (eg, patients with cystic fibrosis or lung cancer), and on the development of devices that improve aerosol delivery to infants, so that early intervention in disease processes with aerosol

Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes

PubMed Central

Ramirez-Gordillo, Daniel; Trujillo-Provencio, Casilda; Knight, V. Bleu; Serrano, Elba E.

2014-01-01

The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel β1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology. PMID:21959846

Retroviral packaging cells encapsulated in TheraCyte immunoisolation devices enable long-term in vivo gene delivery.

PubMed

Krupetsky, Anna; Parveen, Zahida; Marusich, Elena; Goodrich, Adrienne; Dornburg, Ralph

2003-05-01

The method of delivering a therapeutic gene into a patient is still one of the major obstacles towards successful human gene therapy. Here we describe a novel gene delivery approach using TheraCyte immunoisolation devices. Retroviral vector producing cells, derived from the avian retrovirus spleen necrosis virus, SNV, were encapsulated in TheraCyte devices and tested for the release of retroviral vectors. In vitro experiments show that such devices release infectious retroviral vectors into the tissue culture medium for up to 4 months. When such devices were implanted subcutaneously in SCID mice, infectious virus was released into the blood stream. There, the vectors were transported to and infected tumors, which had been induced by subcutaneous injection of tissue culture cells. Thus, this novel concept of a continuous, long-term gene delivery may constitute an attractive approach for future in vivo human gene therapy.

Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

PubMed

Tolmachov, Oleg E

2015-01-01

Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo

Non-viral vectors based on magnetoplexes, lipoplexes and polyplexes for VEGF gene delivery into central nervous system cells.

PubMed

Villate-Beitia, Ilia; Puras, Gustavo; Soto-Sánchez, Cristina; Agirre, Mireia; Ojeda, Edilberto; Zarate, Jon; Fernández, Eduardo; Pedraz, José Luis

2017-04-15

Nanotechnology based non-viral vectors hold great promise to deliver therapeutic genes into the central nervous system (CNS) in a safe and controlled way. Vascular endothelial growth factor (VEGF) is a potential therapeutic gene candidate for CNS disorders due to its specific roles in brain angiogenesis and neuroprotection. In this work, we elaborated three different non-viral vectors based on magnetic, cationic lipid and polymeric nanoparticles complexed to the phVEGF165aIRESGFP plasmid, which codifies the VEGF protein -extracellular- and the green fluorescent protein (GFP) -intracellular-. Nanoparticles and corresponding nanoplexes -magnetoplexes, lipoplexes and polyplexes- were characterized in terms of size, zeta potential, polydispersity index, morphology and ability to bind, release and protect DNA. Transfection efficiencies of nanoplexes were measured in terms of percentage of GFP expressing cells, mean fluorescent intensity (MFI) and VEGF (ng/ml) production in HEK293, C6 and primary neuronal culture cells. Magnetoplexes showed the highest transfection efficiencies in C6, followed by lipoplexes, and in primary neuronal culture cells, followed by polyplexes. Lipoplexes were the most efficient in HEK293 cells, followed by magnetoplexes. The biological activity of VEGF was confirmed by its proliferative effect in HUVEC cells. Overall, these results provide new insights for VEGF gene delivery into CNS cells using non-viral vectors. Copyright © 2017. Published by Elsevier B.V.

Particle tracking in drug and gene delivery research: State-of-the-art applications and methods.

PubMed

Schuster, Benjamin S; Ensign, Laura M; Allan, Daniel B; Suk, Jung Soo; Hanes, Justin

2015-08-30

Particle tracking is a powerful microscopy technique to quantify the motion of individual particles at high spatial and temporal resolution in complex fluids and biological specimens. Particle tracking's applications and impact in drug and gene delivery research have greatly increased during the last decade. Thanks to advances in hardware and software, this technique is now more accessible than ever, and can be reliably automated to enable rapid processing of large data sets, thereby further enhancing the role that particle tracking will play in drug and gene delivery studies in the future. We begin this review by discussing particle tracking-based advances in characterizing extracellular and cellular barriers to therapeutic nanoparticles and in characterizing nanoparticle size and stability. To facilitate wider adoption of the technique, we then present a user-friendly review of state-of-the-art automated particle tracking algorithms and methods of analysis. We conclude by reviewing technological developments for next-generation particle tracking methods, and we survey future research directions in drug and gene delivery where particle tracking may be useful. Copyright © 2015 Elsevier B.V. All rights reserved.

RNAi-based therapeutic nanostrategy: IL-8 gene silencing in pancreatic cancer cells using gold nanorods delivery vehicles

NASA Astrophysics Data System (ADS)

Panwar, Nishtha; Yang, Chengbin; Yin, Feng; Yoon, Ho Sup; Swee Chuan, Tjin; Yong, Ken-Tye

2015-09-01

RNA interference (RNAi)-based gene silencing possesses great ability for therapeutic intervention in pancreatic cancer. Among various oncogene mutations, Interleukin-8 (IL-8) gene mutations are found to be overexpressed in many pancreatic cell lines. In this work, we demonstrate IL-8 gene silencing by employing an RNAi-based gene therapy approach and this is achieved by using gold nanorods (AuNRs) for efficient delivery of IL-8 small interfering RNA (siRNA) to the pancreatic cell lines of MiaPaCa-2 and Panc-1. Upon comparing to Panc-1 cells, we found that the dominant expression of the IL-8 gene in MiaPaCa-2 cells resulted in an aggressive behavior towards the processes of cell invasion and metastasis. We have hence investigated the suitability of using AuNRs as novel non-viral nanocarriers for the efficient uptake and delivery of IL-8 siRNA in realizing gene knockdown of both MiaPaCa-2 and Panc-1 cells. Flow cytometry and fluorescence imaging techniques have been applied to confirm transfection and release of IL-8 siRNA. The ratio of AuNRs and siRNA has been optimized and transfection efficiencies as high as 88.40 ± 2.14% have been achieved. Upon successful delivery of IL-8 siRNA into cancer cells, the effects of IL-8 gene knockdown are quantified in terms of gene expression, cell invasion, cell migration and cell apoptosis assays. Statistical comparative studies for both MiaPaCa-2 and Panc-1 cells are presented in this work. IL-8 gene silencing has been demonstrated with knockdown efficiencies of 81.02 ± 10.14% and 75.73 ± 6.41% in MiaPaCa-2 and Panc-1 cells, respectively. Our results are then compared with a commercial transfection reagent, Oligofectamine, serving as positive control. The gene knockdown results illustrate the potential role of AuNRs as non-viral gene delivery vehicles for RNAi-based targeted cancer therapy applications.

Selective Gene Delivery for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide-DNA Complexes.

PubMed

Yoshizumi, Takeshi; Oikawa, Kazusato; Chuah, Jo-Ann; Kodama, Yutaka; Numata, Keiji

2018-05-14

Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants, but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals.

Generation of Envelope-Modified Baculoviruses for Gene Delivery into Mammalian Cells.

PubMed

Hofmann, Christian

2016-01-01

Genetically modified baculoviruses can efficiently deliver and express genes in mammalian cells. The major prerequisite for the expression of a gene transferred by baculovirus is its control by a promoter that is active in mammalian cells. This chapter describes methods for producing second generation baculovirus vectors through modification of their envelope. Envelope modified baculoviruses offer additional new applications of the system, such as their use in in vivo gene delivery, targeting, and vaccination. Methods of generating a recombinant baculovirus vector with a modified envelope and its amplification and purification, including technical scale production, are discussed. A variety of notes give clues regarding specific technical procedures. Finally, methods to analyze the virus and transduction procedures are presented.

Rapid and efficient nonviral gene delivery of CD154 to primary chronic lymphocytic leukemia cells.

PubMed

Li, L H; Biagi, E; Allen, C; Shivakumar, R; Weiss, J M; Feller, S; Yvon, E; Fratantoni, J C; Liu, L N

2006-02-01

Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporation-based gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64+/-15%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in long-term cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system.

Layer-by-layer nanoparticles as an efficient siRNA delivery vehicle for SPARC silencing.

PubMed

Tan, Yang Fei; Mundargi, Raghavendra C; Chen, Min Hui Averil; Lessig, Jacqueline; Neu, Björn; Venkatraman, Subbu S; Wong, Tina T

2014-05-14

Efficient and safe delivery systems for siRNA therapeutics remain a challenge. Elevated secreted protein, acidic, and rich in cysteine (SPARC) protein expression is associated with tissue scarring and fibrosis. Here we investigate the feasibility of encapsulating SPARC-siRNA in the bilayers of layer-by-layer (LbL) nanoparticles (NPs) with poly(L-arginine) (ARG) and dextran (DXS) as polyelectrolytes. Cellular binding and uptake of LbL NPs as well as siRNA delivery were studied in FibroGRO cells. siGLO-siRNA and SPARC-siRNA were efficiently coated onto hydroxyapatite nanoparticles. The multilayered NPs were characterized with regard to particle size, zeta potential and surface morphology using dynamic light scattering and transmission electron microscopy. The SPARC-gene silencing and mRNA levels were analyzed using ChemiDOC western blot technique and RT-PCR. The multilayer SPARC-siRNA incorporated nanoparticles are about 200 nm in diameter and are efficiently internalized into FibroGRO cells. Their intracellular fate was also followed by tagging with suitable reporter siRNA as well as with lysotracker dye; confocal microscopy clearly indicates endosomal escape of the particles. Significant (60%) SPARC-gene knock down was achieved by using 0.4 pmole siRNA/μg of LbL NPs in FibroGRO cells and the relative expression of SPARC mRNA reduced significantly (60%) against untreated cells. The cytotoxicity as evaluated by xCelligence real-time cell proliferation and MTT cell assay, indicated that the SPARC-siRNA-loaded LbL NPs are non-toxic. In conclusion, the LbL NP system described provides a promising, safe and efficient delivery platform as a non-viral vector for siRNA delivery that uses biopolymers to enhance the gene knock down efficiency for the development of siRNA therapeutics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multifunctional nanoparticles for drug/gene delivery in nanomedicine

NASA Astrophysics Data System (ADS)

Seale, Mary-Margaret; Zemlyanov, Dimitry; Cooper, Christy L.; Haglund, Emily; Prow, Tarl W.; Reece, Lisa M.; Leary, James F.

2007-02-01

Multifunctional nanoparticles hold great promise for drug/gene delivery. Multilayered nanoparticles can act as nanomedical systems with on-board "molecular programming" to accomplish complex multi-step tasks. For example, the targeting process has only begun when the nanosystem has found the correct diseased cell of interest. Then it must pass the cell membrane and avoid enzymatic destruction within the endosomes of the cell. Since the nanosystem is only about one millionth the volume of a human cell, for it to have therapeutic efficacy with its contained package, it must deliver that drug or gene to the appropriate site within the living cell. The successive de-layering of these nanosystems in a controlled fashion allows the system to accomplish operations that would be difficult or impossible to do with even complex single molecules. In addition, portions of the nanosystem may be protected from premature degradation or mistargeting to non-diseased cells. All of these problems remain major obstacles to successful drug delivery with a minimum of deleterious side effects to the patient. This paper describes some of the many components involved in the design of a general platform technology for nanomedical systems. The feasibility of most of these components has been demonstrated by our group and others. But the integration of these interacting sub-components remains a challenge. We highlight four components of this process as examples. Each subcomponent has its own sublevels of complexity. But good nanomedical systems have to be designed/engineered as a full nanomedical system, recognizing the need for the other components.

Electrosonic ejector microarray for drug and gene delivery.

PubMed

Zarnitsyn, Vladimir G; Meacham, J Mark; Varady, Mark J; Hao, Chunhai; Degertekin, F Levent; Fedorov, Andrei G

2008-04-01

We report on development and experimental characterization of a novel cell manipulation device-the electrosonic ejector microarray-which establishes a pathway for drug and/or gene delivery with control of biophysical action on the length scale of an individual cell. The device comprises a piezoelectric transducer for ultrasound wave generation, a reservoir for storing the sample mixture and a set of acoustic horn structures that form a nozzle array for focused application of mechanical energy. The nozzles are micromachined in silicon or plastic using simple and economical batch fabrication processes. When the device is driven at a particular resonant frequency of the acoustic horn structures, the sample mixture of cells and desired transfection agents/molecules suspended in culture medium is ejected from orifices located at the nozzle tips. During sample ejection, focused mechanical forces (pressure and shear) are generated on a microsecond time scale (dictated by nozzle size/geometry and ejection velocity) resulting in identical "active" microenvironments for each ejected cell. This process enables a number of cellular bioeffects, from uptake of small molecules and gene delivery/transfection to cell lysis. Specifically, we demonstrate successful calcein uptake and transfection of DNA plasmid encoding green fluorescent protein (GFP) into human malignant glioma cells (cell line LN443) using electrosonic microarrays with 36, 45 and 50 mum diameter nozzle orifices and operating at ultrasound frequencies between 0.91 and 0.98 MHz. Our results suggest that efficacy and the extent of bioeffects are mainly controlled by nozzle orifice size and the localized intensity of the applied acoustic field.

CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

PubMed

Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

2017-12-07

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

scAAV-Mediated IL-1Ra gene delivery for the Treatment of Osteoarthritis: Test of Efficacy in an Equine Model.

PubMed

Watson Levings, Rachael; Smith, Andrew D; Broome, Ted A; Rice, Brett L; Gibbs, Eric P; Myara, D Alex; Hyddmark, Viktoria; Nasri, Elham; Zarezadeh, Ali; Levings, Padraic P; Lu, Yuan; Dacanay, E Anthony; Foremny, Gregory B; Evans, Christopher H; Morton, Alison J; Winter, Mathew; Dark, Michael J; Nickerson, David M; Colahan, Patrick T; Ghivizzani, Steven Craig

2018-06-05

We are investigating self-complementary adeno-associated virus (scAAV) as a vector for intra-articular gene-delivery of IL-1Ra, and its therapeutic capacity in the treatment of osteoarthritis (OA). To model gene-transfer on a scale proportional to the human knee, a frequent site of OA incidence, we focused our studies on the joints of the equine forelimb. Using AAV2.5 capsid and equine IL-1Ra as a homologous transgene, we previously identified a functional ceiling dose of ~5 x 1012 viral genomes, which elevated the steady state levels of eqIL-1Ra in synovial fluids by more than 40-fold over endogenous production for at least 6 months. Here, using an osteochondral fragmentation model of early OA, we examined the functional capacity of scAAV.IL-1Ra gene-delivery in equine joints over a period of 12 weeks. In the disease model, transgenic eqIL-1Ra expression was several-fold higher than seen previously in healthy joints, and correlated directly with the severity of joint pathology at the time of treatment. Despite wide variation in expression, the steady-state eqIL-1Ra in synovial fluids exceeded that of IL-1 by > 400-fold in all animals, and a consistent treatment effect was observed. This included a 30-40% reduction in lameness and ~25% improvement in total joint pathology by both MRI and arthroscopic assessments, which included reduced joint effusion and synovitis, and improved repair of the osteochondral lesion. No vector-related increase in eqIL-1Ra levels in blood or urine was noted. Cumulatively our studies in the equine model indicate scAAV.IL-1Ra administration is reasonably safe and capable of sustained therapeutic IL-1Ra production intra-articularly in joints of human scale. This profile supports consideration for human testing in OA.

Topical Gene Electrotransfer to the Epidermis of Hairless Guinea Pig by Non-Invasive Multielectrode Array

PubMed Central

Guo, Siqi; Israel, Annelise L.; Basu, Gaurav; Donate, Amy; Heller, Richard

2013-01-01

Topical gene delivery to the epidermis has the potential to be an effective therapy for skin disorders, cutaneous cancers, vaccinations and systemic metabolic diseases. Previously, we reported on a non-invasive multielectrode array (MEA) that efficiently delivered plasmid DNA and enhanced expression to the skin of several animal models by in vivo gene electrotransfer. Here, we characterized plasmid DNA delivery with the MEA in a hairless guinea pig model, which has a similar histology and structure to human skin. Significant elevation of gene expression up to 4 logs was achieved with intradermal DNA administration followed by topical non-invasive skin gene electrotransfer. This delivery produced gene expression in the skin of hairless guinea pig up to 12 to 15 days. Gene expression was observed exclusively in the epidermis. Skin gene electrotransfer with the MEA resulted in only minimal and mild skin changes. A low level of human Factor IX was detected in the plasma of hairless guinea pig after gene electrotransfer with the MEA, although a significant increase of Factor IX was obtained in the skin of animals. These results suggest gene electrotransfer with the MEA can be a safe, efficient, non-invasive skin delivery method for skin disorders, vaccinations and potential systemic diseases where low levels of gene products are sufficient. PMID:24015305

Stent-mediated gene and drug delivery for cardiovascular disease and cancer: A brief insight.

PubMed

Krishnagopal, Akshaya; Reddy, Aakash; Sen, Dwaipayan

2017-05-01

This review concisely recapitulates the different existing modes of stent-mediated gene/drug delivery, their considerable advancement in clinical trials and a rationale for other merging new technologies such as nanotechnology and microRNA-based therapeutics, in addition to addressing the limitations in each of these perpetual stent platforms. Over the past decade, stent-mediated gene/drug delivery has materialized as a hopeful alternative for cardiovascular disease and cancer in contrast to routine conventional treatment modalities. Regardless of the phenomenal recent developments achieved by coronary interventions and cancer therapies that employ gene and drug-eluting stents, practical hurdles still remain a challenge. The present review highlights the limitations that each of the existing stent-based gene/drug delivery system encompasses and therefore provides a vision for the future with respect to discovering an ideal stent therapeutic platform that would circumvent all the practical hurdles witnessed with the existing technology. Further study of the improvisation of next-generation drug-eluting stents has helped to overcome the issue of restenosis to some extent. However, current stent formulations fall short of the anticipated clinically meaningful outcomes and there is an explicit need for more randomized trials aiming to further evaluate stent platforms in favour of enhanced safety and clinical value. Gene-eluting stents may hold promise in contributing new ideas for stent-based prevention of in-stent restenosis through genetic interventions by capitalizing on a wide variety of molecular targets. Therefore, the central consideration directs us toward finding an ideal stent therapeutic platform that would tackle all of the gaps in the existing technology. Copyright © 2017 John Wiley & Sons, Ltd.

Evaluation and mechanism studies of PEGylated dendrigraft poly-L-lysines as novel gene delivery vectors.

PubMed

Huang, Rongqin; Liu, Shuhuan; Shao, Kun; Han, Liang; Ke, Weilun; Liu, Yang; Li, Jianfeng; Huang, Shixian; Jiang, Chen

2010-07-02

Dendrimers have attracted great interest in the field of gene delivery due to their synthetic controllability and excellent gene transfection efficiency. In this work, dendrigraft poly-L-lysines (DGLs) were evaluated as a novel gene vector for the first time. Derivatives of DGLs (generation 2 and 3) with different extents of PEGylation were successfully synthesized and used to compact pDNA as complexes. The result of gel retardation assay showed that pDNA could be effectively packed by all the vectors at a DGLs to pDNA weight ratio greater than 2. An increase in the PEGylation extent of vectors resulted in a decrease in the incorporation efficiency and cytotoxicity of complexes in 293 cells, which also decreased the zeta potential a little but did not affect the mean diameter of complexes. Higher generation of DGLs could mediate higher gene transfection in vitro. Confocal microscopy and cellular uptake inhibition studies demonstrated that caveolae-mediated process and macropinocytosis were involved in the cellular uptake of DGLs-based complexes. Also the results indicate that proper PEGylated DGLs could mediate efficient gene transfection, showing their potential as an alternate biodegradable vector in the field of nonviral gene delivery.

Water soluble cationic dextran derivatives containing poly(amidoamine) dendrons for efficient gene delivery.

PubMed

Mai, Kaijin; Zhang, Shanshan; Liang, Bing; Gao, Cong; Du, Wenjun; Zhang, Li-Ming

2015-06-05

To develop new dextran derivatives for efficient gene delivery, the conjugation of poly(amidoamine) dendrons onto biocompatible dextran was carried out by a Cu(I)-catalyzed azide-alkyne cycloaddition, as confirmed by FTIR and (1)H NMR analyses. For resultant dextran conjugates with various generations of poly(amidoamine) dendrons, their buffering capacity and in vitro cytotoxicity were evaluated by acid-base titration and MTT tests, respectively. In particular, their physicochemical characteristics for the complexation with plasmid DNA were investigated by the combined analyses from agarose gel electrophoresis, zeta potential, particle size, transmission electron microscopy and fluorescence emission spectra. Moreover, their complexes with plasmid DNA were studied with respect to their transfection efficiency in human embryonic kidney (HEK293) cell lines. In the case of a higher generation of poly(amidoamine) dendrons, such a dextran conjugate was found to have much lower cytotoxicity and better gene delivery capability when compared to branched polyethylenimine (bPEI, 25kDa), a commonly used gene vector. Copyright © 2015 Elsevier Ltd. All rights reserved.

Preparation of an efficient and safe polymeric-magnetic nanoparticle delivery system for sorafenib in hepatocellular carcinoma.

PubMed

Tom, Greeshma; Philip, Sheena; Isaac, Rimal; Praseetha, P K; Jiji, S G; Asha, V V

2018-08-01

Superparamagnetic iron oxide nanoparticles (SPIONs), as drug delivery vehicles, offer to eliminate the concerns associated with hydrophobic anti-cancer agents. The current study was intended to fabricate a SPION based delivery system for sorafenib that can simultaneously enable targeted delivery of sorafenib and expand its therapeutic index against hepatocellular carcinoma (HCC). Co-precipitation and physical entrapment methods were employed for the synthesis of sorafenib loaded PVA coated SPIONs. Physicochemical characterizations were done using TEM, XRD, FTIR, Raman spectra and VSM measurements. The superior activity of nanoconjugate was demonstrated by AO/EB staining, FACS, immunofluorescence and Western blot. The safety of the sorafenib conjugated nanoparticles were verified in Wistar rats. The synthesized nanoparticles were in the size range of 5-15 nm. The adsorption of PVA to the SPIONs and the conjugation of sorafenib to the nanocarrier were confirmed by XRD, FTIR and Raman spectra analyses. VSM study ascertained the superparamagnetic nature of the nanoconjugate. Cellular uptake studies suggested its efficient entrapment in HepG2 cells. MTT assay showed that the cytotoxicity of sorafenib loaded PVA/SPIONs was comparable or higher than free sorafenib. The activation of apoptosis and autophagy pathways in HepG2 by the nanoconjugate was evidenced. Acute toxicity testing in Wistar rats supported the safe administration of the nanoconjugate and established its localization in animal tissues by Perl's Prussian Blue reaction. The novel combination of sorafenib with PVA/SPIONs showed better anticancer efficiency than free sorafenib demonstrative of its potential in cancer chemotherapy. Copyright © 2018 Elsevier Inc. All rights reserved.

New chitosan nanobubbles for ultrasound-mediated gene delivery: preparation and in vitro characterization.

PubMed

Cavalli, Roberta; Bisazza, Agnese; Trotta, Michele; Argenziano, Monica; Civra, Andrea; Donalisio, Manuela; Lembo, David

2012-01-01

The development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release. Chitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions. Based on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery.

New chitosan nanobubbles for ultrasound-mediated gene delivery: preparation and in vitro characterization

PubMed Central

Cavalli, Roberta; Bisazza, Agnese; Trotta, Michele; Argenziano, Monica; Civra, Andrea; Donalisio, Manuela; Lembo, David

2012-01-01

Background The development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release. Methods and results Chitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions. Conclusion Based on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery. PMID:22802689

Polyethylenimine-coated iron oxide magnetic nanoparticles for high efficient gene delivery

NASA Astrophysics Data System (ADS)

Nguyen, Anh H.; Abdelrasoul, Gaser N.; Lin, Donghai; Maadi, Hamid; Tong, Junfeng; Chen, Grace; Wang, Richard; Anwar, Afreen; Shoute, Lian; Fang, Qiang; Wang, Zhixiang; Chen, Jie

2018-04-01

Properties of magnetic nanoparticles (MNPs) are of notable interest in many fields of biomedical engineering, especially for gene therapy. In this paper, we report a method for synthesis and delivery of MNPs loaded with DNAs, which overcomes the drawbacks of high cost and cytotoxicity associated with current delivery techniques (chemical- and liposome-based designs). 24-nm MNPs (Fe3O4) were synthesized, functionalized and characterized by analytical techniques to understand the surface properties for DNA binding and cellular uptake. The simple surface functionalization with polyethylenimine (PEI) through glutaraldehyde linker activation gave the complex of PEI-coated MNPs, resulting in high stability with a positive surface charge of about + 31 mV. Under the guidance of an external magnetic field, the functionalized MNPs with a loaded isothiocyanate (FITC) or green fluorescent protein (GFP) will enter the cells, which can be visualized by the fluorescence of FITC or GFP. We also examined the cytotoxicity of our synthesized MNPs by MTT assay. We showed that the IC50s of these MNPs for COS-7 and CHO cells were low and at 0.2 and 0.26 mg/mL, respectively. Moreover, our synthesized MNPs that were loaded with plasmids encoding GFP showed high transfection rate, 38.3% for COS-7cells and 27.6% for CHO cells. In conclusion, we established a promising method with low cost, low toxicity, and high transfection efficiency for siRNA and gene delivery.

Gene Delivery into Plant Cells for Recombinant Protein Production

PubMed Central

Chen, Qiang

2015-01-01

Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

Gene Therapy for Osteoarthritis: Pharmacokinetics of Intra-articular scAAV.IL-1Ra Delivery in an Equine Model.

PubMed

Watson Levings, Rachael; Broome, Ted A; Smith, Andrew D; Rice, Brett L; Gibbs, Eric P; Myara, D Alex; Hyddmark, E Viktoria; Nasri, Elham; Zarezadeh, Ali; Levings, Padraic P; Lu, Yuan; Dacanay, E Anthony; Foremny, Gregory B; Evans, Christopher H; Morton, Alison J; Winter, Mathew; Dark, Michael J; Nickerson, David M; Colahan, Patrick T; Ghivizzani, Steven Craig

2018-06-05

endogenous production for at least 6 months. Here, using an osteochondral fragmentation model of early OA, we examined the functional capacity of scAAV.IL-1Ra gene-delivery in equine joints over a period of 12 weeks. In the disease model, transgenic eqIL-1Ra expression was several-fold higher than seen previously in healthy joints, and correlated directly with the severity of joint pathology at the time of treatment. Despite wide variation in expression, the steady-state eqIL-1Ra in synovial fluids exceeded that of IL-1 by > 400-fold in all animals, and a consistent treatment effect was observed. This included a 30-40% reduction in lameness and ~25% improvement in total joint pathology by both MRI and arthroscopic assessments, which included reduced joint effusion and synovitis, and improved repair of the osteochondral lesion. No vector-related increase in eqIL-1Ra levels in blood or urine was noted. Cumulatively our studies in the equine model indicate scAAV.IL-1Ra administration is reasonably safe and capable of sustained therapeutic IL-1Ra production intra-articularly in joints of human scale. This profile supports consideration for human testing in OA.

Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues

PubMed Central

Lai, Samuel K.; Wang, Ying-Ying; Hanes, Justin

2009-01-01

Mucus is a viscoelastic and adhesive gel that protects the lung airways, gastrointestinal (GI) tract, vagina, eye and other mucosal surfaces. Most foreign particulates, including conventional particle-based drug delivery systems, are efficiently trapped in human mucus layers by steric obstruction and/or adhesion. Trapped particles are typically removed from the mucosal tissue within seconds to a few hours depending on anatomical location, thereby strongly limiting the duration of sustained drug delivery locally. A number of debilitating diseases could be treated more effectively and with fewer side effects if drugs and genes could be more efficiently delivered to the underlying mucosal tissues in a controlled manner. This review first describes the tenacious mucus barrier properties that have precluded the efficient penetration of therapeutic particles. It then reviews the design and development of new mucus-penetrating particles that may avoid rapid mucus clearance mechanisms, and thereby provide targeted or sustained drug delivery for localized therapies in mucosal tissues. PMID:19133304

Site-specific gene delivery to stented arteries using magnetically guided zinc oleate-based nanoparticles loaded with adenoviral vectors

PubMed Central

Chorny, Michael; Fishbein, Ilia; Tengood, Jillian E.; Adamo, Richard F.; Alferiev, Ivan S.; Levy, Robert J.

2013-01-01

Gene therapeutic strategies have shown promise in treating vascular disease. However, their translation into clinical use requires pharmaceutical carriers enabling effective, site-specific delivery as well as providing sustained transgene expression in blood vessels. While replication-deficient adenovirus (Ad) offers several important advantages as a vector for vascular gene therapy, its clinical applicability is limited by rapid inactivation, suboptimal transduction efficiency in vascular cells, and serious systemic adverse effects. We hypothesized that novel zinc oleate-based magnetic nanoparticles (MNPs) loaded with Ad would enable effective arterial cell transduction by shifting vector processing to an alternative pathway, protect Ad from inactivation by neutralizing factors, and allow site-specific gene transfer to arteries treated with stent angioplasty using a 2-source magnetic guidance strategy. Ad-loaded MNPs effectively transduced cultured endothelial and smooth muscle cells under magnetic conditions compared to controls and retained capacity for gene transfer after exposure to neutralizing antibodies and lithium iodide, a lytic agent causing disruption of free Ad. Localized arterial gene expression significantly stronger than in control animal groups was demonstrated after magnetically guided MNP delivery in a rat stenting model 2 and 9 d post-treatment, confirming feasibility of using Ad-loaded MNPs to achieve site-specific transduction in stented blood vessels. In conclusion, Ad-loaded MNPs formed by controlled precipitation of zinc oleate represent a novel delivery system, well-suited for efficient, magnetically targeted vascular gene transfer.—Chorny, M., Fishbein, I., Tengood, J. E., Adamo, R. F., Alferiev, I. S., Levy, R. J. Site-specific gene delivery to stented arteries using magnetically guided zinc oleate-based nanoparticles loaded with adenoviral vectors. PMID:23407712

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field.

PubMed

He, Zhi-Yao; Men, Ke; Qin, Zhou; Yang, Yang; Xu, Ting; Wei, Yu-Quan

2017-05-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs (sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies. Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.

Enhanced Delivery of Plasmid Encoding Interleukin-12 Gene by Diethylene Triamine Penta-Acetic Acid (DTPA)-Conjugated PEI Nanoparticles.

PubMed

Dehshahri, Ali; Sadeghpour, Hossein; Keykhaee, Maryam; Khalvati, Bahman; Sheikhsaran, Fatemeh

2016-05-01

Recombinant therapeutic proteins have been considered as an efficient category of medications used for the treatment of various diseases. Despite their effectiveness, there are some reports on the systemic adverse effects of recombinant therapeutic proteins limiting their wide clinical applications. Among different cytokines used for cancer immunotherapy, interleukin-12 (IL-12) has shown great ability as a powerful antitumor and antiangiogenic agent. However, significant toxic reactions following the systemic administration of IL-12 have led researchers to seek for alternative approaches such as the delivery and local expression of the IL-12 gene inside the tumor tissues. In order to transfer the plasmid encoding IL-12 gene, the most extensively investigated polycationic polymer, polyethylenimine (PEI), was modified by diethylene triamine penta-acetic acid (DTPA) to modulate the hydrophobic-hydrophilic balance of the polymer as well as its toxicity. DTPA-conjugated PEI derivatives were able to form complexes in the size range around 100-180 nm with great condensation ability and protection of the plasmid against enzymatic degradation. The highest gene transfer ability was achieved by the DTPA-conjugated PEI at the conjugation degree of 0.1 % where the level of IL-12 production increased up to twofold compared with that of the unmodified PEI. Results of the present study demonstrated that modulation of the surface positive charge of PEI along with the improvement of the polymer hydrophobic balance could be considered as a successful strategy to develop safe and powerful nanocarriers.

Fenton-treated functionalized diamond nanoparticles as gene delivery system.

PubMed

Martín, Roberto; Alvaro, Mercedes; Herance, José Raúl; García, Hermenegildo

2010-01-26

When raw diamond nanoparticles (Dnp, 7 nm average particle size) obtained from detonation are submitted to harsh Fenton-treatment, the resulting material becomes free of amorphous soot matter and the process maintains the crystallinity, reduces the particle size (4 nm average particle size), increases the surface OH population, and increases water solubility. All these changes are beneficial for subsequent Dnp covalent functionalization and for the ability of Dnp to cross cell membranes. Fenton-treated Dnps have been functionalized with thionine and the resulting sample has been observed in HeLa cell nuclei. A triethylammonium-functionalized Dnp pairs electrostatically with a plasmid having the green fluorescent protein gene and acts as gene delivery system permitting the plasmid to cross HeLa cell membrane, something that does not occur for the plasmid alone without assistance of polycationic Dnp.

Nanotechnology-Based Strategies for siRNA Brain Delivery for Disease Therapy.

PubMed

Zheng, Meng; Tao, Wei; Zou, Yan; Farokhzad, Omid C; Shi, Bingyang

2018-05-01

Small interfering RNA (siRNA)-based gene silencing technology has demonstrated significant potential for treating brain-associated diseases. However, effective and safe systemic delivery of siRNA into the brain remains challenging because of biological barriers such as enzymatic degradation, short circulation lifetime, the blood-brain barrier (BBB), insufficient tissue penetration, cell endocytosis, and cytosolic transport. Nanotechnology offers intriguing potential for addressing these challenges in siRNA brain delivery in conjunction with chemical and biological modification strategies. In this review, we outline the challenges of systemic delivery of siRNA-based therapy for brain diseases, highlight recent advances in the development and engineering of siRNA nanomedicines for various brain diseases, and discuss our perspectives on this exciting research field for siRNA-based therapy towards more effective brain disease therapy. Copyright © 2018 Elsevier Ltd. All rights reserved.

Localized gene delivery using antibody tethered adenovirus from polyurethane heart valve cusps and intra-aortic implants.

PubMed

Stachelek, S J; Song, C; Alferiev, I; Defelice, S; Cui, X; Connolly, J M; Bianco, R W; Levy, R J

2004-01-01

The present study investigated a novel approach for gene therapy of heart valve disease and vascular disorders. We formulated and characterized implantable polyurethane films that could also function as gene delivery systems through the surface attachment of replication defective adenoviruses using an anti-adenovirus antibody tethering mechanism. Our hypothesis was that we could achieve site-specific gene delivery to cells interacting with these polyurethane implants, and thereby demonstrate the potential for intravascular devices that could also function as gene delivery platforms for therapeutic vectors. Previous research by our group has demonstrated that polyurethane elastomers can be derivatized post-polymerization through a series of chemical reactions activating the hard segment amide groups with alkyl bromine residues, which can enable a wide variety of subsequent chemical modifications. Furthermore, prior research by our group investigating gene delivery intravascular stents has shown that collagen-coated balloon expandable stents can be configured with anti-adenovirus antibodies via thiol-based chemistry, and can then tether adenoviral vectors at doses that lead to high levels of localized arterial neointima expression, but with virtually no distal spread of vector. Thus, we sought to create two-device configurations for our investigations building on this previous research. (1) Polyurethane films coated with Type I collagen were thiol activated to permit covalent attachment of anti-adenovirus antibodies to enable gene delivery via vector tethering. (2) We also formulated polyurethane films with direct covalent attachment of anti-adenovirus antibodies to polyurethane hard segments derivatized with alkyl-thiol groups, thereby also enabling tethering of replication-defective adenoviruses. Both formulations demonstrated highly localized and efficient transduction in cell culture studies with rat arterial smooth muscle cells. In vivo experiments with collagen

Secure and effective gene delivery system of plasmid DNA coated by polynucleotide.

PubMed

Kodama, Yukinobu; Ohkubo, Chikako; Kurosaki, Tomoaki; Egashira, Kanoko; Sato, Kayoko; Fumoto, Shintaro; Nishida, Koyo; Higuchi, Norihide; Kitahara, Takashi; Nakamura, Tadahiro; Sasaki, Hitoshi

2015-01-01

Polynucleotides are anionic macromolecules which are expected to transfer into the targeted cells through specific uptake mechanisms. So, we developed polynucleotides coating complexes of plasmid DNA (pDNA) and polyethylenimine (PEI) for a secure and efficient gene delivery system and evaluated their usefulness. Polyadenylic acid (polyA), polyuridylic acid (polyU), polycytidylic acid (polyC), and polyguanylic acid (polyG) were examined as the coating materials. pDNA/PEI/polyA, pDNA/PEI/polyU, and pDNA/PEI/polyC complexes formed nanoparticles with a negative surface charge although pDNA/PEI/polyG was aggregated. The pDNA/PEI/polyC complex showed high transgene efficiency in B16-F10 cells although there was little efficiency in pDNA/PEI/polyA and pDNA/PEI/polyU complexes. An inhibition study strongly indicated the specific uptake mechanism of pDNA/PEI/polyC complex. Polynucleotide coating complexes had lower cytotoxicity than pDNA/PEI complex. The pDNA/PEI/polyC complex showed high gene expression selectively in the spleen after intravenous injection into mice. The pDNA/PEI/polyC complex showed no agglutination with erythrocytes and no acute toxicity although these were observed in pDNA/PEI complex. Thus, we developed polynucleotide coating complexes as novel vectors for clinical gene therapy, and the pDNA/PEI/polyC complex as a useful candidate for a gene delivery system.

Evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry.

PubMed

Shmueli, Ron B; Bhise, Nupura S; Green, Jordan J

2013-03-01

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery(1,3) . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers(4) that are hydrolytically degradable(5,6) and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)(7), mouse mammary epithelial cells(8), human brain cancer cells(9) and macrovascular (human umbilical vein, HUVECs) endothelial cells(10). A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained(11). In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi

Gene therapy for human glioblastoma using neurotropic JC virus-like particles as a gene delivery vector.

PubMed

Chao, Chun-Nun; Yang, Yu-Hsuan; Wu, Mu-Sheng; Chou, Ming-Chieh; Fang, Chiung-Yao; Lin, Mien-Chun; Tai, Chien-Kuo; Shen, Cheng-Huang; Chen, Pei-Lain; Chang, Deching; Wang, Meilin

2018-02-02

Glioblastoma multiforme (GBM), the most common malignant brain tumor, has a short period of survival even with recent multimodality treatment. The neurotropic JC polyomavirus (JCPyV) infects glial cells and oligodendrocytes and causes fatal progressive multifocal leukoencephalopathy in patients with AIDS. In this study, a possible gene therapy strategy for GBM using JCPyV virus-like particles (VLPs) as a gene delivery vector was investigated. We found that JCPyV VLPs were able to deliver the GFP reporter gene into tumor cells (U87-MG) for expression. In an orthotopic xenograft model, nude mice implanted with U87 cells expressing the near-infrared fluorescent protein and then treated by intratumoral injection of JCPyV VLPs carrying the thymidine kinase suicide gene, combined with ganciclovir administration, exhibited significantly prolonged survival and less tumor fluorescence during the experiment compared with controls. Furthermore, JCPyV VLPs were able to protect and deliver a suicide gene to distal subcutaneously implanted U87 cells in nude mice via blood circulation and inhibit tumor growth. These findings show that metastatic brain tumors can be targeted by JCPyV VLPs carrying a therapeutic gene, thus demonstrating the potential of JCPyV VLPs to serve as a gene therapy vector for the far highly treatment-refractory GBM.

Delivery of human NKG2D-IL-15 fusion gene by chitosan nanoparticles to enhance antitumor immunity.

PubMed

Yan, Chen; Jie, Leng; Yongqi, Wang; Weiming, Xiao; Juqun, Xi; Yanbing, Ding; Li, Qian; Xingyuan, Pan; Mingchun, Ji; Weijuan, Gong

2015-07-31

Nanoparticles are becoming promising carriers for gene delivery because of their high capacity in gene loading and low cell cytotoxicity. In this study, a chitosan-based nanoparticle encapsulated within a recombinant pcDNA3.1-dsNKG2D-IL-15 plasmid was generated. The fused dsNKG2D-IL-15 gene fragment consisted of double extracellular domains of NKG2D with IL-15 gene at downstream. The average diameter of the gene nanoparticles ranged from 200 nm to 400 nm, with mean zeta potential value of 53.8 ± 6.56 mV. The nanoparticles which were loaded with the dsNKG2D-IL-15 gene were uptaken by tumor cells with low cytotoxicity. Tumor cells pre-transfected by gene nanopartilces stimulated NK and T cells in vitro. Intramuscular injection of gene nanoparticles suppressed tumor growth and prolonged survival of tumor-bearing mice through activation of NK and CD8(+) T cells. Thus, chitosan-based nanoparticle delivery of dsNKG2D-IL-15 gene vaccine can be potentially used for tumor therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

Bolstering cholesteryl ester hydrolysis in liver: A hepatocyte-targeting gene delivery strategy for potential alleviation of atherosclerosis.

PubMed

He, Hongliang; Lancina, Michael G; Wang, Jing; Korzun, William J; Yang, Hu; Ghosh, Shobha

2017-06-01

Current atherosclerosis treatment strategies primarily focus on limiting further cholesteryl esters (CE) accumulation by reducing endogenous synthesis of cholesterol in the liver. No therapy is currently available to enhance the removal of CE, a crucial step to reduce the burden of the existing disease. Given the central role of hepatic cholesteryl ester hydrolase (CEH) in the intrahepatic hydrolysis of CE and subsequent removal of the resulting free cholesterol (FC), in this work, we applied galactose-functionalized polyamidoamine (PAMAM) dendrimer generation 5 (Gal-G5) for hepatocyte-specific delivery of CEH expression vector. The data presented herein show the increased specific uptake of Gal-G5/CEH expression vector complexes (simply Gal-G5/CEH) by hepatocytes in vitro and in vivo. Furthermore, the upregulated CEH expression in the hepatocytes significantly enhanced the intracellular hydrolysis of high density lipoprotein-associated CE (HDL-CE) and subsequent conversion/secretion of hydrolyzed FC as bile acids (BA). The increased CEH expression in the liver significantly increased the flux of HDL-CE to biliary as well as fecal FC and BA. Meanwhile, Gal-G5 did not induce hepatic or renal toxicity. It was also not immunotoxic. Because of these encouraging pre-clinical testing results, using this safe and highly efficient hepatocyte-specific gene delivery platform to enhance the hepatic processes involved in cholesterol elimination is a promising strategy for the alleviation of atherosclerosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cancer Imaging: Gene Transcription-Based Imaging and Therapeutic Systems

PubMed Central

Bhang, Hyo-eun C.; Pomper, Martin G.

2012-01-01

Molecular-genetic imaging of cancer is in its infancy. Over the past decade gene reporter systems have been optimized in preclinical models and some have found their way into the clinic. The search is on to find the best combination of gene delivery vehicle and reporter imaging system that can be translated safely and quickly. The goal is to have a combination that can detect a wide variety of cancers with high sensitivity and specificity in a way that rivals the current clinical standard, positron emission tomography with [18F]fluorodeoxyglucose. To do so will require systemic delivery of reporter genes for the detection of micrometastases, and a nontoxic vector, whether viral or based on nanotechnology, to gain widespread acceptance by the oncology community. Merger of molecular-genetic imaging with gene therapy, a strategy that has been employed in the past, will likely be necessary for such imaging to reach widespread clinical use. PMID:22349219

Conservation demands safe gene drive.

PubMed

Esvelt, Kevin M; Gemmell, Neil J

2017-11-01

Interest in developing gene drive systems to control invasive species is growing, with New Zealand reportedly considering the nascent technology as a way to locally eliminate the mammalian pests that threaten its unique flora and fauna. If gene drives successfully eradicated these invasive populations, many would rejoice, but what are the possible consequences? Here, we explore the risk of accidental spread posed by self-propagating gene drive technologies, highlight new gene drive designs that might achieve better outcomes, and explain why we need open and international discussions concerning a technology that could have global ramifications.

Cutaneous gene expression of plasmid DNA in excised human skin following delivery via microchannels created by radio frequency ablation.

PubMed

Birchall, James; Coulman, Sion; Anstey, Alexander; Gateley, Chris; Sweetland, Helen; Gershonowitz, Amikam; Neville, Lewis; Levin, Galit

2006-04-07

The skin is a valuable organ for the development and exploitation of gene medicines. Delivering genes to skin is restricted however by the physico-chemical properties of DNA and the stratum corneum (SC) barrier. In this study, we demonstrate the utility of an innovative technology that creates transient microconduits in human skin, allowing DNA delivery and resultant gene expression within the epidermis and dermis layers. The radio frequency (RF)-generated microchannels were of sufficient morphology and depth to permit the epidermal delivery of 100 nm diameter nanoparticles. Model fluorescent nanoparticles were used to confirm the capacity of the channels for augmenting diffusion of macromolecules through the SC. An ex vivo human organ culture model was used to establish the gene expression efficiency of a beta-galactosidase reporter plasmid DNA applied to ViaDerm treated skin. Skin treated with ViaDerm using 50 microm electrode arrays promoted intense levels of gene expression in the viable epidermis. The intensity and extent of gene expression was superior when ViaDerm was used following a prior surface application of the DNA formulation. In conclusion, the RF-microchannel generator (ViaDerm) creates microchannels amenable for delivery of nanoparticles and gene therapy vectors to the viable region of skin.

Safe motherhood voucher programme coverage of health facility deliveries among poor women in South-western Uganda.

PubMed

Kanya, Lucy; Obare, Francis; Warren, Charlotte; Abuya, Timothy; Askew, Ian; Bellows, Ben

2014-07-01

There has been increased interest in and experimentation with demand-side mechanisms such as the use of vouchers that place purchasing power in the hands of targeted consumers to improve the uptake of healthcare services in low-income settings. A key measure of the success of such interventions is the extent to which the programmes have succeeded in reaching the target populations. This article estimates the coverage of facility deliveries by a maternal health voucher programme in South-western Uganda and examines whether such coverage is correlated with district-level characteristics such as poverty density and the number of contracted facilities. Analysis entails estimating the voucher coverage of health facility deliveries among the general population and poor population (PP) using programme data for 2010, which was the most complete calendar year of implementation of the Uganda safe motherhood (SM) voucher programme. The results show that: (1) the programme paid for 38% of estimated deliveries among the PP in the targeted districts, (2) there was a significant negative correlation between the poverty density in a district and proportions of births to poor women that were covered by the programme and (3) improving coverage of health facility deliveries for poor women is dependent upon increasing the sales and redemption rates. The findings suggest that to the extent that the programme stimulated demand for SM services by new users, it has the potential of increasing facility-based births among poor women in the region. In addition, the significant negative correlation between the poverty density and the proportions of facility-based births to poor women that are covered by the voucher programme suggests that there is need to increase both voucher sales and the rate of redemption to improve coverage in districts with high levels of poverty. Published by Oxford University Press in association with The London School of Hygiene and Tropical Medicine © The Author

Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery

PubMed Central

2011-01-01

Background Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes. Methods In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector. Results The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo. Conclusion The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin. PMID:21255430

Conservation demands safe gene drive

PubMed Central

Esvelt, Kevin M.

2017-01-01

Interest in developing gene drive systems to control invasive species is growing, with New Zealand reportedly considering the nascent technology as a way to locally eliminate the mammalian pests that threaten its unique flora and fauna. If gene drives successfully eradicated these invasive populations, many would rejoice, but what are the possible consequences? Here, we explore the risk of accidental spread posed by self-propagating gene drive technologies, highlight new gene drive designs that might achieve better outcomes, and explain why we need open and international discussions concerning a technology that could have global ramifications. PMID:29145398

Safe Sleep Practices of Kansas Birthing Hospitals

PubMed Central

Ahlers-Schmidt, Carolyn R.; Schunn, Christy; Sage, Cherie; Engel, Matthew; Benton, Mary

2018-01-01

Background Sleep-related death is tied with congenital anomalies as the leading cause of infant mortality in Kansas, and external risk factors are present in 83% of these deaths. Hospitals can impact caregiver intentions to follow risk-reduction strategies. This project assessed the current practices and policies of Kansas hospitals with regard to safe sleep. Methods A cross-sectional survey of existing safe sleep practices and policies in Kansas hospitals was performed. Hospitals were categorized based on reported delivery volume and data were compared across hospital sizes. Results Thirty-one of 73 (42%) contacted hospitals responded. Individual survey respondents represented various hospital departments including newborn/well-baby (68%), neonatal intensive care unit (3%) and other non-nursery departments or administration (29%). Fifty-eight percent of respondents reported staff were trained on infant safe sleep; 44% of these held trainings annually. High volume hospitals tended to have more annual training than low or mid volume birth hospitals. Thirty-nine percent reported a safe sleep policy, though most of these (67%) reported never auditing compliance. The top barrier to safe sleep education, regardless of delivery volume, was conflicting patient and family member beliefs. Conclusions Hospital promotion of infant safe sleep is being conducted in Kansas to varying degrees. High and mid volume birth hospitals may need to work more on formal auditing of safe sleep practices, while low volume hospitals may need more staff training. Low volume hospitals also may benefit from access to additional caregiver education materials. Finally, it is important to note hospitals should not be solely responsible for safe sleep education. PMID:29844848

Microfluidic electroporation for delivery of small molecules and genes into cells using a common DC power supply.

PubMed

Wang, Hsiang-Yu; Lu, Chang

2008-06-15

Electroporation is an efficient method of introducing foreign impermeant molecules such as drugs and genes into cells. Conventional electroporation has been based on the application of short electrical pulses (electropulsation). Electropulsation requires specialized equipment and cannot be integrated easily with techniques such as electrophoresis which is based on constant voltage. Here we demonstrate the delivery of small molecules and genes into cells, using a microfluidic electroporation technique based on constant direct current (DC) voltage that we developed earlier. We demonstrate the delivery of two molecules into Chinese hamster ovary (CHO-K1) cells: a membrane impermeable nucleic acid dye (SYTOX Green) and a plasmid vector carrying the gene for green fluorescent protein (pEGFP-C1). Our devices can exert field variations to flowing cells that are analogous to the application of single or multiple pulses by having different geometries. We investigate the effects of the electrical parameters and different geometries of the device on the transfection efficiency and cell viability. Our technique provides a simple solution to electroporation-based drug and gene delivery by eliminating the need for a pulse generator. We envision that these simple microscale electroporation devices will have the potential to work in parallel on a microchip platform and such technology will allow high-throughput functional screening of drugs and genes. (c) 2008 Wiley Periodicals, Inc.

Gemini surfactants mediate efficient mitochondrial gene delivery and expression.

PubMed

Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Cardoso, Ana L; Silva, Sandra G; do Vale, M Luísa; Marques, Eduardo F; Pedroso de Lima, Maria C; Jurado, Amália S

2015-03-02

Gene delivery targeting mitochondria has the potential to transform the therapeutic landscape of mitochondrial genetic diseases. Taking advantage of the nonuniversal genetic code used by mitochondria, a plasmid DNA construct able to be specifically expressed in these organelles was designed by including a codon, which codes for an amino acid only if read by the mitochondrial ribosomes. In the present work, gemini surfactants were shown to successfully deliver plasmid DNA to mitochondria. Gemini surfactant-based DNA complexes were taken up by cells through a variety of routes, including endocytic pathways, and showed propensity for inducing membrane destabilization under acidic conditions, thus facilitating cytoplasmic release of DNA. Furthermore, the complexes interacted extensively with lipid membrane models mimicking the composition of the mitochondrial membrane, which predicts a favored interaction of the complexes with mitochondria in the intracellular environment. This work unravels new possibilities for gene therapy toward mitochondrial diseases.

PEGylation of polypropylenimine dendrimers: effects on cytotoxicity, DNA condensation, gene delivery and expression in cancer cells.

PubMed

Somani, Sukrut; Laskar, Partha; Altwaijry, Najla; Kewcharoenvong, Paphitchaya; Irving, Craig; Robb, Gillian; Pickard, Benjamin S; Dufès, Christine

2018-06-20

Diaminobutyric polypropylenimine (DAB) dendrimers have been shown to be highly efficient non-viral gene delivery systems for cancer therapy. However, their cytotoxicity currently limits their applications. To overcome this issue, PEGylation of DAB dendrimer, using various PEG molecular weights and dendrimer generations, has been attempted to decrease the cytotoxicity and enhance the DNA condensation, size and zeta potential, cellular uptake and transfection efficacy of these dendriplexes. Among all the PEGylated dendrimers synthesized, generation 3- and generation 4-DAB conjugated to low molecular weight PEG (2 kDa) at a dendrimer: DNA ratio of 20:1 and 10:1 resulted in an increase in gene expression on almost all tested cancer cells lines (by up to 3.2-fold compared to unmodified dendrimer in A431 cells). The highest level of β-galactosidase gene expression (10.07 × 10 -3  ± 0.09 × 10 -3  U/mL) was obtained following treatment of B16F10-Luc cells with G4-dendrimer PEGylated with PEG2K at a dendrimer: DNA ratio of 20:1. These delivery systems significantly decreased cytotoxicity on B16F10-Luc cells, by more than 3.4-fold compared to unmodified dendrimer. PEGylated generations 3- and 4-DAB dendrimers are therefore promising gene delivery systems for cancer therapy, combining low cytotoxicity and high transfection efficacy.

Intracochlear Drug Delivery Systems

PubMed Central

Borenstein, Jeffrey T.

2011-01-01

Introduction Advances in molecular biology and in the basic understanding of the mechanisms associated with sensorineural hearing loss and other diseases of the inner ear, are paving the way towards new approaches for treatments for millions of patients. However, the cochlea is a particularly challenging target for drug therapy, and new technologies will be required to provide safe and efficacious delivery of these compounds. Emerging delivery systems based on microfluidic technologies are showing promise as a means for direct intracochlear delivery. Ultimately, these systems may serve as a means for extended delivery of regenerative compounds to restore hearing in patients suffering from a host of auditory diseases. Areas covered in this review Recent progress in the development of drug delivery systems capable of direct intracochlear delivery is reviewed, including passive systems such as osmotic pumps, active microfluidic devices, and systems combined with currently available devices such as cochlear implants. The aim of this article is to provide a concise review of intracochlear drug delivery systems currently under development, and ultimately capable of being combined with emerging therapeutic compounds for the treatment of inner ear diseases. Expert Opinion Safe and efficacious treatment of auditory diseases will require the development of microscale delivery devices, capable of extended operation and direct application to the inner ear. These advances will require miniaturization and integration of multiple functions, including drug storage, delivery, power management and sensing, ultimately enabling closed-loop control and timed-sequence delivery devices for treatment of these diseases. PMID:21615213

Low molecular weight chitosan conjugated with folate for siRNA delivery in vitro: optimization studies

PubMed Central

Fernandes, Julio C; Qiu, Xingping; Winnik, Francoise M; Benderdour, Mohamed; Zhang, Xiaoling; Dai, Kerong; Shi, Qin

2012-01-01

The low transfection efficiency of chitosan is one of its drawbacks as a gene delivery carrier. Low molecular weight chitosan may help to form small-sized polymer-DNA or small interfering RNA (siRNA) complexes. Folate conjugation may improve gene transfection efficiency because of the promoted uptake of folate receptor-bearing cells. In the present study, chitosan was conjugated with folate and investigated for its efficacy as a delivery vector for siRNA in vitro. We demonstrate that the molecular weight of chitosan has a major influence on its biological and physicochemical properties, and very low molecular weight chitosan (below 10 kDa) has difficulty in forming stable complexes with siRNA. In this study, chitosan 25 kDa and 50 kDa completely absorbed siRNA and formed nanoparticles (≤220 nm) at a chitosan to siRNA weight ratio of 50:1. The introduction of a folate ligand onto chitosan decreased nanoparticle toxicity. Compared with chitosan-siRNA, folate-chitosan-siRNA nanoparticles improved gene silencing transfection efficiency. Therefore, folate-chitosan shows potential as a viable candidate vector for safe and efficient siRNA delivery. PMID:23209368

Vector systems for prenatal gene therapy: principles of retrovirus vector design and production.

PubMed

Howe, Steven J; Chandrashekran, Anil

2012-01-01

Vectors derived from the Retroviridae family have several attributes required for successful gene delivery. Retroviral vectors have an adequate payload size for the coding regions of most genes; they are safe to handle and simple to produce. These vectors can be manipulated to target different cell types with low immunogenicity and can permanently insert genetic information into the host cells' genome. Retroviral vectors have been used in gene therapy clinical trials and successfully applied experimentally in vitro, in vivo, and in utero.

Gene Therapy for Neuropathic Pain through siRNA-IRF5 Gene Delivery with Homing Peptides to Microglia.

PubMed

Terashima, Tomoya; Ogawa, Nobuhiro; Nakae, Yuki; Sato, Toshiyuki; Katagi, Miwako; Okano, Junko; Maegawa, Hiroshi; Kojima, Hideto

2018-06-01

Astrocyte- and microglia-targeting peptides were identified and isolated using phage display technology. A series of procedures, including three cycles of both in vivo and in vitro biopanning, was performed separately in astrocytes and in M1 or M2 microglia, yielding 50-58 phage plaques in each cell type. Analyses of the sequences of this collection identified one candidate homing peptide targeting astrocytes (AS1[C-LNSSQPS-C]) and two candidate homing peptides targeting microglia (MG1[C-HHSSSAR-C] and MG2[C-NTGSPYE-C]). To determine peptide specificity for the target cell in vitro, each peptide was synthesized and introduced into the primary cultures of astrocytes or microglia. Those peptides could bind to the target cells and be selectively taken up by the corresponding cell, namely, astrocytes, M1 microglia, or M2 microglia. To confirm cell-specific gene delivery to M1 microglia, the complexes between peptide MG1 and siRNA-interferon regulatory factor 5 were prepared and intrathecally injected into a mouse model of neuropathic pain. The complexes successfully suppressed hyperalgesia with high efficiency in this neuropathic pain model. Here, we describe a novel gene therapy for the treatment neuropathic pain, which has a high potential to be of clinical relevance. This strategy will ensure the targeted delivery of therapeutic genes while minimizing side effects to non-target tissues or cells. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Developing a Novel Gene-Delivery Vector System Using the Recombinant Fusion Protein of Pseudomonas Exotoxin A and Hyperthermophilic Archaeal Histone HPhA

PubMed Central

Zhang, Ling; Feng, Yan; Li, Zehong; Wu, GuangMou; Yue, Yuhuan; Li, Gensong; Cao, Yu; Zhu, Ping

2015-01-01

Non-viral gene delivery system with many advantages has a great potential for the future of gene therapy. One inherent obstacle of such approach is the uptake by endocytosis into vesicular compartments. Receptor-mediated gene delivery method holds promise to overcome this obstacle. In this study, we developed a receptor-mediated gene delivery system based on a combination of the Pseudomonas exotoxin A (PE), which has a receptor binding and membrane translocation domain, and the hyperthermophilic archaeal histone (HPhA), which has the DNA binding ability. First, we constructed and expressed the rPE-HPhA fusion protein. We then examined the cytotoxicity and the DNA binding ability of rPE-HPhA. We further assessed the efficiency of transfection of the pEGF-C1 plasmid DNA to CHO cells by the rPE-HPhA system, in comparison to the cationic liposome method. The results showed that the transfection efficiency of rPE-HPhA was higher than that of cationic liposomes. In addition, the rPE-HPhA gene delivery system is non-specific to DNA sequence, topology or targeted cell type. Thus, the rPE-HPhA system can be used for delivering genes of interest into mammalian cells and has great potential to be applied for gene therapy. PMID:26556098

Regulated release of a novel non-viral gene delivery vector from electrospun coaxial fiber mesh scaffolds

NASA Astrophysics Data System (ADS)

Saraf, Anita

The development of novel strategies for tissue engineering entails the evolution of biopolymers into multifunctional constructs that can support the proliferation of cells and stimulate their differentiation into functional tissues. With that in mind, biocompatible polymers were fabricated into a novel gene delivery agent as well as three dimensional scaffolds that act as reservoirs and controlled release constructs. To fabricate a novel gene delivery agent a commercially available cationic polymer, poly(ethylenimine), PEI, was chemically conjugated to a ubiquitous glycosaminoglycan, hyaluronic acid (HA). The novel polymer, PEI-HA, had significantly reduced toxicity and improved transfection efficiency with multipotent human mesenchymal stem cells. This transfection efficiency could further be modulated by changing the concentration of sodium chloride and temperature used to assemble PEI-HA/DNA complexes. To facilitate the regulated delivery of these complexes in the context of tissue engineering, an emerging technology for scaffold fabrication, coaxial electrospinning was adapted to include PEI-HA and plasmid DNA within the scaffold fibers. Initially, a factorial design was employed to assess the influence of processing parameters in the absence of gene delivery vectors and plasmids. The study elucidated the role of sheath polymer concentration and core polymer concentration and molecular weight and the presence of sodium chloride on fiber diameters and morphologies. Subsequently, PEI-HA and plasmid DNA were entrapped within the sheath and core compartments of these fibers and the influence of processing parameters was assessed in the context of fiber diameter, release kinetics and transfection efficiency over a period of 60 days. The release of PEI-HA was found to be dependent upon the loading dose of the vector and plasmid. However, the transfection efficiency correlated to the core polymer properties, concentration and molecular weight. The processing

Gene Therapy to Cure HIV: Where to from Here?

PubMed

Johnston, Rowena

2016-12-01

A variety of approaches are being tested to cure HIV, but with the exception of the Berlin patient case, none has been successful. The Berlin patient, positive for both HIV and acute myeloid leukemia (AML), received two stem cell transplants from a donor homozygous for the CCR5delta32 mutation. In the 8 years since his second transplant, he has remained free of both HIV and AML. This case provides strong proof-of-principle that a cure for HIV is possible and might be achieved through gene therapy. Several technological barriers must be resolved and are discussed here, including the safe delivery of the intervention throughout the body of the infected person, increased efficiency of gene editing, and avoidance of resistance to the therapy. Delivery of a gene therapy intervention to HIV-infected people around the world will also be a considerable challenge.

Design of PEI-conjugated bio-reducible polymer for efficient gene delivery.

PubMed

Nam, Joung-Pyo; Kim, Soyoung; Kim, Sung Wan

2018-07-10

The poly(cystaminebis(acrylamide)-diaminohexane) (poly(CBA-DAH)) was designed previously as a bio-reducible efficient gene delivery carrier. However, the high weight ratio required to form the polyplexes between poly(CBA-DAH) with pDNA is still a problem that needs to be addressed. To solve this problem and increase the transfection efficiency, poly(ethylenimine) (PEI, 1.8 kDa) was conjugated to poly(CBA-DAH) via disulfide bond. The PEI conjugated poly(CBA-DAH) (PCDP) can bind with pDNA at a very low weight ratio of 0.5 and above, like PEI 25 kDa, and form the polyplexes with nano-size (102-128 nm) and positive surface charge (27-34 mV). PCDP and PCDP polyplexes had negligible cytotoxicity and indicated similar or better cellular uptake than the comparison groups such as PEI 25 kDa and Lipofectamine® polyplexes. To confirm the transfection efficiency, the plasmid DNA (pDNA) encoded with the luciferase reporter gene (gWiz-Luc) and green fluorescent protein reporter gene (GFP) were used and treated with PCDP into the A549, Huh-7, and Mia PaCa-2 cells. PCDP/pDNA polyplexes showed highest transfection efficiency in all tested cell lines. In the luciferase assay, PCDP polyplexes showed 10.2 times higher gene transfection efficiency than Lipofectamine® polyplexes in mimic in vivo conditions (30% FBS, A549 cells). The VEGF siRNA expressing plasmid (pshVEGF), which is constructed as a therapeutic gene by our previous work, was delivered by PCDP into the cancer cells. The VEGF gene expression of PCDP/pshVEGF polyplexes was dramatically lower than control and the VEGF gene silencing efficiencies of PCDP/pshVEGF (w/w; 10/1) polyplexes were 54% (A549 cells), 77% (Huh-7 cells), and 66% (Mia PaCa-2 cells). In addition, PCDP/pshVEGF had reduced cell viability rates of about 31% (A549 cells), 39% (Huh-7 cells), and 42% (Mia PaCa-2 cells) and showed better results than all comparison groups. In the transfection efficiency and VEGF silencing assay, PCDP polyplexes showed

Supplemental oxygen: ensuring its safe delivery during facial surgery.

PubMed

Reyes, R J; Smith, A A; Mascaro, J R; Windle, B H

1995-04-01

Electrosurgical coagulation in the presence of blow-by oxygen is a potential source of fire in facial surgery. A case report of a patient sustaining partial-thickness facial burns secondary to such a flash fire is presented. A fiberglass facial model is then used to study the variables involved in providing supplemental oxygen when an electrosurgical unit is employed. Oxygen flow, oxygen delivery systems, distance from the oxygen source, and coagulation current levels were varied. A nasal cannula and an adapted suction tubing provided the oxygen delivery systems on the model. Both the "displaced" nasal cannula and the adapted suction tubing ignited at a minimum coagulation level of 30 W, an oxygen flow of 2 liters/minute, and a linear distance of 5 cm from the oxygen source. The properly placed nasal cannula did not ignite at any combination of oxygen flow, coagulation current level, or distance from the oxygen source. Facial cutaneous surgery in patients provided supplemental oxygen should be practiced with caution when an electrosurgical unit is used for coagulation. The oxygen delivery systems adapted for use are hazardous and should not be used until their safety has been demonstrated.

[Application of ultrasound-enhanced gene and drug delivery to the ocular tissue].

PubMed

Sonoda, Shozo; Yamashita, Toshifumi; Suzuki, Ryo; Maruyama, Kazuo; Sakamoto, Taiji

2013-01-01

Visual images provide an immensely rich source of information about the external world. Eye has characteristic structure sensory cells are arranged along the eye wall, and is filled inside with vitreous body. In recent years, intravitreal injection of anti-vascular endothelial growth factor (VEGF) agent had widely spread, and numerous number of patients who suffered ocular angiogenic disease such as diabetic retinopathy, age-related macular degeneration and retinal vascular occlusion for the disease, were treated and spared the blindness. Vitreous cavity was regarded as reservoir of drug, intravitreal injection is thought a sort of drug delivery. However, with regard to the administration of a selective drug deliver, it has not yet been solved. Our aim is to establish a new method of gene transfer, drug delivery using low-energy ultrasound to the eye, to date, we confirmed drug and gene deliver to the ocular tissue such as cornea, conjunctiva and retina with high efficiency. In addition, tissue damage was minimal. We have also shown that ultrasound irradiation with combination of a microbubbles or bubble liposome could be introduced drug and gene more effectively. Based on these knowledge, we will focus on development of a new device for intraocular ultrasound exposure and potential for therapeutic application of ultrasound to humans retinal disease such as retinal artery obstruction.

In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs.

PubMed

Bez, Maxim; Sheyn, Dmitriy; Tawackoli, Wafa; Avalos, Pablo; Shapiro, Galina; Giaconi, Joseph C; Da, Xiaoyu; David, Shiran Ben; Gavrity, Jayne; Awad, Hani A; Bae, Hyun W; Ley, Eric J; Kremen, Thomas J; Gazit, Zulma; Ferrara, Katherine W; Pelled, Gadi; Gazit, Dan

2017-05-17

More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 ( BMP - 6 ) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation. Copyright © 2017, American Association for the Advancement of Science.

In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs

PubMed Central

Bez, Maxim; Sheyn, Dmitriy; Tawackoli, Wafa; Avalos, Pablo; Shapiro, Galina; Giaconi, Joseph C.; Da, Xiaoyu; Ben David, Shiran; Gavrity, Jayne; Awad, Hani A.; Bae, Hyun W.; Ley, Eric J.; Kremen, Thomas J.; Gazit, Zulma; Ferrara, Katherine W.; Pelled, Gadi; Gazit, Dan

2017-01-01

More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro–computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchy-mal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation. PMID:28515335

Water-soluble fullerene (C60) derivatives as nonviral gene-delivery vectors.

PubMed

Sitharaman, Balaji; Zakharian, Tatiana Y; Saraf, Anita; Misra, Preeti; Ashcroft, Jared; Pan, Su; Pham, Quynh P; Mikos, Antonios G; Wilson, Lon J; Engler, David A

2008-01-01

A new class of water-soluble C60 transfecting agents has been prepared using Hirsch-Bingel chemistry and assessed for their ability to act as gene-delivery vectors in vitro. In an effort to elucidate the relationship between the hydrophobicity of the fullerene core, the hydrophilicity of the water-solubilizing groups, and the overall charge state of the C60 vectors in gene delivery and expression, several different C60 derivatives were synthesized to yield either positively charged, negatively charged, or neutral chemical functionalities under physiological conditions. These fullerene derivatives were then tested for their ability to transfect cells grown in culture with DNA carrying the green fluorescent protein (GFP) reporter gene. Statistically significant expression of GFP was observed for all forms of the C60 derivatives when used as DNA vectors and compared to the ability of naked DNA alone to transfect cells. However, efficient in vitro transfection was only achieved with the two positively charged C60 derivatives, namely, an octa-amino derivatized C60 and a dodeca-amino derivatized C60 vector. All C60 vectors showed an increase in toxicity in a dose-dependent manner. Increased levels of cellular toxicity were observed for positively charged C60 vectors relative to the negatively charged and neutral vectors. Structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized C60 compounds, the C60 vector/DNA complexes, their physical attributes (aggregation, charge) and their transfection efficiencies. Recently, similar Gd@C60-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging (MRI). Thus, the successful demonstration of intracellular DNA uptake, intracellular transport, and gene expression from DNA using C60 vectors suggests the possibility of developing analogous Gd@C60-based vectors to serve simultaneously as both

Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery

PubMed Central

Towne, Chris; Pertin, Marie; Beggah, Ahmed T; Aebischer, Patrick; Decosterd, Isabelle

2009-01-01

Background Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. Results Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependant manner. More than 90% of transduced cells were small and medium sized neurons (< 700 μm2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (≈ 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell

Self-Immolative Polycations as Gene Delivery Vectors and Prodrugs Targeting Polyamine Metabolism in Cancer

PubMed Central

2015-01-01

Polycations are explored as carriers to deliver therapeutic nucleic acids. Polycations are conventionally pharmacological inert with the sole function of delivering therapeutic cargo. This study reports synthesis of a self-immolative polycation (DSS-BEN) based on a polyamine analogue drug N1,N11-bisethylnorspermine (BENSpm). The polycation was designed to function dually as a gene delivery carrier and a prodrug targeting dysregulated polyamine metabolism in cancer. Using a combination of NMR and HPLC, we confirm that the self-immolative polycation undergoes intracellular degradation into the parent drug BENSpm. The released BENSpm depletes cellular levels of spermidine and spermine and upregulates polyamine catabolic enzymes spermine/spermidine N1-acetyltransferase (SSAT) and spermine oxidase (SMO). The synthesized polycations form polyplexes with DNA and facilitate efficient transfection. Taking advantage of the ability of BENSpm to sensitize cancer cells to TNFα-induced apoptosis, we show that DSS-BEN enhances the cell killing activity of TNFα gene therapy. The reported findings validate DSS-BEN as a dual-function delivery system that can deliver a therapeutic gene and improve the outcome of gene therapy as a result of the intracellular degradation of DSS-BEN to BENSpm and the subsequent beneficial effect of BENSpm on dysregulated polyamine metabolism in cancer. PMID:25153488

[Proliferation of hepatocytes after delivery of exogenous hepatocyte growth factor gene].

PubMed

Lin, Yong; Xie, Wei fen; Chen, Wei-zhong; Zhang, Xin; Zeng, Xin; Chen, Yue-xiang; Yang, Xiu-jiang; Zhang, Zhong-bing

2003-06-01

To explore the proliferation of primary cultured rats hepatocytes after delivery of exogenous hepatocyte growth factor (HGF) gene which was inserted into the genome of replication-deficient recombinant adenovirus vector. The recombinant adenovirus-AdHGF which could express HGF was generated by homologous recombination. After the HGF gene was delivered into the hepatocytes, the expression of both HGF and c-met/HGF receptor mRNA in the cells was detected by RT-PCR and the level of HGF in the culture supernatant was also assayed by ELISA. On the other hand, cell proliferation was compared between before and after delivery of the HGF gene by MTS assay and the percentages of cell cycles were analyzed by flow cytometry. In addition, the expression of proliferating cell nuclear antigen (PCNA) was determined by immunocytofluorescent stain. 4 x 10(10) efu/ml titer of AdHGF was obtained after recombination, RT-PCR indicated that the expression of HGF mRNA in hepatocytes increased on the third day after infected by the viruses and c-met/HGF receptor mRNA was also up-regulated. The HGF level in the culture supernatant assayed by ELISA was (5,939.0+/-414.39) pg/ml, which was much higher than that in the control (208.1pg/ml+/-37.20pg/ml, F=13.661, P<0.01). In addition, the proliferation of hepatocytes infected with AdHGF increased significantly according to MTS method (F>or=15.158, P<0.01) and more hepatocytes in G0/G1 stages changed into S stage (chi2=41.616, P<0.01), accordingly, PCNA index increased from 6.42+/- 1.88 to 14.56+/-2.85 (F=42.122, P<0.01). show that HGF gene delivered into hepatocytes by AdHGF can be expressed with high efficiency in the cells, which can stimulate hepatocytes proliferation. It may be an effective tool for hepatocyte transplantation by gene modified donor hepatocytes.

Zein-based Nanocarriers as Potential Natural Alternatives for Drug and Gene Delivery: Focus on Cancer Therapy.

PubMed

Elzoghby, Ahmed; Freag, May; Mamdouh, Hadeer; Elkhodairy, Kadria

2017-01-01

Protein nanocarriers possess unique merits including minimal cytotoxicity, numerous renewable sources, and high drug-binding capability. In opposition to delivery carriers utilizing hydrophilic animal proteins, hydrophobic plant proteins (e.g, zein) have great tendency in fabricating controlled-release particulate carriers without additional chemical modification to stiffen them, which in turn evades the use of toxic chemical crosslinkers. Moreover, zein is related to a class of alcohol-soluble prolamins and generally recognized as safe (GRAS) carrier for drug delivery. Various techniques have been adopted to fabricate zein-based nanoparticulate systems including phase separation coacervation, spray-drying, supercritical anti-solvent approach, electrospinning and self-assembly. This manuscript reviews the recent advances in the zein-based colloidal nano-carrier systems such as nanospheres, nanocapsules, micelles and nanofibers with a special focus on their physicochemical characteristics and drug delivery applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

In Vivo Bio-distribution and Efficient Tumor Targeting of Gelatin/Silica Nanoparticles for Gene Delivery

NASA Astrophysics Data System (ADS)

Zhao, Xueqin; Wang, Jun; Tao, SiJie; Ye, Ting; Kong, Xiangdong; Ren, Lei

2016-04-01

The non-viral gene delivery system is an attractive alternative to cancer therapy. The clinical success of non-viral gene delivery is hampered by transfection efficiency and tumor targeting, which can be individually overcome by addition of functional modules such as cell penetration or targeting. Here, we first engineered the multifunctional gelatin/silica (GS) nanovectors with separately controllable modules, including tumor-targeting aptamer AGRO100, membrane-destabilizing peptide HA2, and polyethylene glycol (PEG), and then studied their bio-distribution and in vivo transfection efficiencies by contrast resonance imaging (CRI). The results suggest that the sizes and zeta potentials of multifunctional gelatin/silica nanovectors were 203-217 nm and 2-8 mV, respectively. Functional GS-PEG nanoparticles mainly accumulated in the liver and tumor, with the lowest uptake by the heart and brain. Moreover, the synergistic effects of tumor-targeting aptamer AGRO100 and fusogenic peptide HA2 promoted the efficient cellular internalization in the tumor site. More importantly, the combined use of AGRO100 and PEG enhanced tumor gene expression specificity and effectively reduced toxicity in reticuloendothelial system (RES) organs after intravenous injection. Additionally, low accumulation of GS-PEG was observed in the heart tissues with high gene expression levels, which could provide opportunities for non-invasive gene therapy.

Controlled superficial assembly of DNA-amorphous calcium phosphate nanocomposite spheres for surface-mediated gene delivery.

PubMed

Oyane, Ayako; Araki, Hiroko; Nakamura, Maki; Shimizu, Yoshiki; Shubhra, Quazi T H; Ito, Atsuo; Tsurushima, Hideo

2016-05-01

Surface-mediated gene delivery systems have many potential applications in tissue engineering. We recently fabricated an assembly consisting of DNA-amorphous calcium phosphate (DNA-ACP) nanocomposite spheres on a polymer substrate via coprecipitation in a labile supersaturated calcium phosphate (CaP) solution and demonstrated the assembly's high gene delivery efficacy. In this study, we conducted a detailed investigation of the coprecipitation process in solution and revealed that the negatively charged DNA molecules were immobilized in the ACP spheres during the initial stage of coprecipitation and functioned as both sphere-dispersing and size-regulating agents. As a result, the DNA-ACP nanocomposites grew into size-regulated submicrospheres in solution and assembled onto the substrate via gravity sedimentation. The assembled nanocomposite spheres were chemically anchored to the substrate surface through an intermediate layer of CaP-based nanoparticles that was formed heterogeneously at the substrate surface. The coprecipitation conditions, i.e., coprecipitation time and Ca and P concentrations in solution, greatly affected the state of assembly of the nanocomposite spheres, thereby influencing the gene expression level of the cells cultured on the substrate. Increasing the number density and decreasing the size of the nanocomposite spheres did not always increase the assembly's gene delivery efficacy (per surface area of the substrate) due to adverse effects on cellular viability. As demonstrated herein, controlling the coprecipitation conditions is important for designing a cell-stimulating and biocompatible scaffold surface consisting of an assembly of DNA-ACP nanocomposite spheres. Copyright © 2016 Elsevier B.V. All rights reserved.

Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium.

PubMed

Manunta, Maria D I; McAnulty, Robin J; Tagalakis, Aristides D; Bottoms, Stephen E; Campbell, Frederick; Hailes, Helen C; Tabor, Alethea B; Laurent, Geoffrey J; O'Callaghan, Christopher; Hart, Stephen L

2011-01-01

Gene therapy mediated by synthetic vectors may provide opportunities for new treatments for cystic fibrosis (CF) via aerosolisation. Vectors for CF must transfect the airway epithelium efficiently and not cause inflammation so they are suitable for repeated dosing. The inhaled aerosol should be deposited in the airways since the cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed predominantly in the epithelium of the submucosal glands and in the surface airway epithelium. The aim of this project was to develop an optimised aerosol delivery approach applicable to treatment of CF lung disease by gene therapy. The vector suspension investigated in this study comprises receptor-targeting peptides, cationic liposomes and plasmid DNA that self-assemble by electrostatic interactions to form a receptor-targeted nanocomplex (RTN) of approximately 150 nm with a cationic surface charge of +50 mV. The aerodynamic properties of aerosolised nanocomplexes produced with three different nebulisers were compared by determining aerosol deposition in the different stages of a Next Generation Pharmaceutical Impactor (NGI). We also investigated the yield of intact plasmid DNA by agarose gel electrophoresis and densitometry, and transfection efficacies in vitro and in vivo. RTNs nebulised with the AeroEclipse II BAN were the most effective, compared to other nebulisers tested, for gene delivery both in vitro and in vivo. The biophysical properties of the nanocomplexes were unchanged after nebulisation while the deposition of RTNs suggested a range of aerosol aerodynamic sizes between 5.5 µm-1.4 µm cut off (NGI stages 3-6) compatible with deposition in the central and lower airways. RTNs showed their ability at delivering genes via nebulisation, thus suggesting their potential applications for therapeutic interventions of cystic fibrosis and other respiratory disorders.

Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery.

PubMed

Gaj, Thomas; Staahl, Brett T; Rodrigues, Gonçalo M C; Limsirichai, Prajit; Ekman, Freja K; Doudna, Jennifer A; Schaffer, David V

2017-06-20

Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA oligonucleotide. However, combining RNPs with transgene-containing donor templates for targeted gene addition has proven challenging, which in turn has limited the capabilities of the RNP-mediated genome editing toolbox. Here, we demonstrate that combining RNP delivery with naturally recombinogenic adeno-associated virus (AAV) donor vectors enables site-specific gene insertion by homology-directed genome editing. Compared to conventional plasmid-based expression vectors and donor templates, we show that combining RNP and AAV donor delivery increases the efficiency of gene addition by up to 12-fold, enabling the creation of lineage reporters that can be used to track the conversion of striatal neurons from human fibroblasts in real time. These results thus illustrate the potential for unifying nuclease protein delivery with AAV donor vectors for homology-directed genome editing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Development of a Microfluidics-Based Intracochlear Drug Delivery Device

PubMed Central

Sewell, William F.; Borenstein, Jeffrey T.; Chen, Zhiqiang; Fiering, Jason; Handzel, Ophir; Holmboe, Maria; Kim, Ernest S.; Kujawa, Sharon G.; McKenna, Michael J.; Mescher, Mark M.; Murphy, Brian; Leary Swan, Erin E.; Peppi, Marcello; Tao, Sarah

2009-01-01

Background Direct delivery of drugs and other agents into the inner ear will be important for many emerging therapies, including the treatment of degenerative disorders and guiding regeneration. Methods We have taken a microfluidics/MEMS (MicroElectroMechanical Systems) technology approach to develop a fully implantable reciprocating inner-ear drug-delivery system capable of timed and sequenced delivery of agents directly into perilymph of the cochlea. Iterations of the device were tested in guinea pigs to determine the flow characteristics required for safe and effective delivery. For these tests, we used the glutamate receptor blocker DNQX, which alters auditory nerve responses but not cochlear distortion product otoacoustic emissions. Results We have demonstrated safe and effective delivery of agents into the scala tympani. Equilibration of the drug in the basal turn occurs rapidly (within tens of minutes) and is dependent on reciprocating flow parameters. Conclusion We have described a prototype system for the direct delivery of drugs to the inner ear that has the potential to be a fully implantable means for safe and effective treatment of hearing loss and other diseases. PMID:19923811

Gene therapy for Parkinson's disease: Disease modification by GDNF family of ligands.

PubMed

Kirik, Deniz; Cederfjäll, Erik; Halliday, Glenda; Petersén, Åsa

2017-01-01

Gene transfer is a promising drug delivery method of advanced therapeutic entities for Parkinson's disease. One advantage over conventional therapies, such as peripheral delivery of the dopamine pre-cursor l-DOPA, is site-specific expression of proteins with regenerative, disease-modifying and potentially neuroprotective capacity. Several clinical trials have been performed to test the capacity of glial-cell line derived neurotrophic factor and neurturin to rescue degenerating dopaminergic neurons in the substantia nigra and their axon terminals in the striatum by delivery of these neurotrophic factors either as purified protein or by means of viral vector mediated gene delivery to the brain. Although gene therapy approaches tested so far have been shown to be safe, none met their primary endpoints in phase II clinical trials designed and powered to test the efficacy of the intervention. Within the scope of this review we aim to describe the state-of-the-art in the field, how different technical parameters were translated from pre-clinical studies in non-human primates to clinical trials, and what these trials taught us regarding important factors that may pave the way to the success of gene therapy for the treatment of Parkinson's disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Progranulin gene delivery protects dopaminergic neurons in a mouse model of Parkinson's disease.

PubMed

Van Kampen, Jackalina M; Baranowski, David; Kay, Denis G

2014-01-01

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by tremor, rigidity and akinesia/bradykinesia resulting from the progressive loss of nigrostriatal dopaminergic neurons. To date, only symptomatic treatment is available for PD patients, with no effective means of slowing or stopping the progression of the disease. Progranulin (PGRN) is a 593 amino acid multifunction protein that is widely distributed throughout the CNS, localized primarily in neurons and microglia. PGRN has been demonstrated to be a potent regulator of neuroinflammation and also acts as an autocrine neurotrophic factor, important for long-term neuronal survival. Thus, enhancing PGRN expression may strengthen the cells resistance to disease. In the present study, we have used the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD to investigate the possible use of PGRN gene delivery as a therapy for the prevention or treatment of PD. Viral vector delivery of the PGRN gene was an effective means of elevating PGRN expression in nigrostriatal neurons. When PGRN expression was elevated in the SNC, nigrostriatal neurons were protected from MPTP toxicity in mice, along with a preservation of striatal dopamine content and turnover. Further, protection of nigrostriatal neurons by PGRN gene therapy was accompanied by reductions in markers of MPTP-induced inflammation and apoptosis as well as a complete preservation of locomotor function. We conclude that PGRN gene therapy may have beneficial effects in the treatment of PD.

Peptides Used in the Delivery of Small Noncoding RNA

PubMed Central

2015-01-01

RNA interference (RNAi) is an endogenous process in which small noncoding RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), post-transcriptionally regulate gene expressions. In general, siRNA and miRNA/miRNA mimics are similar in nature and activity except their origin and specificity. Although both siRNAs and miRNAs have been extensively studied as novel therapeutics for a wide range of diseases, the large molecular weight, anionic surface charges, instability in blood circulation, and intracellular trafficking to the RISC after cellular uptake have hindered the translation of these RNAs from bench to clinic. As a result, a great variety of delivery systems have been investigated for safe and effective delivery of small noncoding RNAs. Among these systems, peptides, especially cationic peptides, have emerged as a promising type of carrier due to their inherent ability to condense negatively charged RNAs, ease of synthesis, controllable size, and tunable structure. In this review, we will focus on three major types of cationic peptides, including poly(l-lysine) (PLL), protamine, and cell penetrating peptides (CPP), as well as peptide targeting ligands that have been extensively used in RNA delivery. The delivery strategies, applications, and limitations of these cationic peptides in siRNA/miRNA delivery will be discussed. PMID:25157701

Pseudotyped Lentiviral Vectors for Retrograde Gene Delivery into Target Brain Regions

PubMed Central

Kobayashi, Kenta; Inoue, Ken-ichi; Tanabe, Soshi; Kato, Shigeki; Takada, Masahiko; Kobayashi, Kazuto

2017-01-01

Gene transfer through retrograde axonal transport of viral vectors offers a substantial advantage for analyzing roles of specific neuronal pathways or cell types forming complex neural networks. This genetic approach may also be useful in gene therapy trials by enabling delivery of transgenes into a target brain region distant from the injection site of the vectors. Pseudotyping of a lentiviral vector based on human immunodeficiency virus type 1 (HIV-1) with various fusion envelope glycoproteins composed of different combinations of rabies virus glycoprotein (RV-G) and vesicular stomatitis virus glycoprotein (VSV-G) enhances the efficiency of retrograde gene transfer in both rodent and nonhuman primate brains. The most recently developed lentiviral vector is a pseudotype with fusion glycoprotein type E (FuG-E), which demonstrates highly efficient retrograde gene transfer in the brain. The FuG-E–pseudotyped vector permits powerful experimental strategies for more precisely investigating the mechanisms underlying various brain functions. It also contributes to the development of new gene therapy approaches for neurodegenerative disorders, such as Parkinson’s disease, by delivering genes required for survival and protection into specific neuronal populations. In this review article, we report the properties of the FuG-E–pseudotyped vector, and we describe the application of the vector to neural circuit analysis and the potential use of the FuG-E vector in gene therapy for Parkinson’s disease. PMID:28824385

Enhancement of therapeutic drug and DNA delivery into cells by electroporation* Enhancement of therapeutic drug and DNA delivery into cells by electroporation

NASA Astrophysics Data System (ADS)

Rabussay, Dietmar; Dev, Nagendu B.; Fewell, Jason; Smith, Louis C.; Widera, Georg; Zhang, Lei

2003-02-01

The effectiveness of potentially powerful therapeutics, including DNA, is often limited by their inability to permeate the cell membrane efficiently. Electroporation (EP) also referred to as `electropermeabilization' of the outer cell membrane renders this barrier temporarily permeable by inducing `pores' across the lipid bilayer. For in vivo EP, the drug or DNA is delivered into the interstitial space of the target tissue by conventional means, followed by local EP. EP pulses of micro- to millisecond duration and field strengths of 100-1500 V cm-1 generally enhance the delivery of certain chemotherapeutic drugs by three to four orders of magnitude and intracellular delivery of DNA several hundred-fold. We have used EP in clinical studies for human cancer therapy and in animals for gene therapy and DNA vaccination. Late stage squamous cell carcinomas of the head and neck were treated with intratumoural injection of bleomycin and subsequent EP. Of the 69 tumours treated, 25% disappeared completely and another 32% were reduced in volume by more than half. Residence time of bleomycin in electroporated tumours was significantly greater than in non-electroporated lesions. Histological findings and gene expression patterns after bleomycin-EP treatment indicated rapid apoptosis of the majority of tumour cells. In animals, we demonstrated the usefulness of EP for enhanced DNA delivery by achieving normalization of blood clotting times in haemophilic dogs, and by substantially increasing transgene expression in smooth muscle cells of arterial walls using a novel porous balloon EP catheter. Finally, we have found in animal experiments that the immune response to DNA vaccines can be dramatically enhanced and accelerated by EP and co-injection of micron-sized particles. We conclude that EP represents an effective, economical and safe approach to enhance the intracellular delivery, and thus potency, of important drugs and genes for therapeutic purposes. The safety and pharmaco

Gene Silencing by Gold Nanoshell-Mediated Delivery and Laser-Triggered Release of Antisense Oligonucleotide and siRNA

PubMed Central

Huschka, Ryan; Barhoumi, Aoune; Liu, Qing; Roth, Jack A.; Ji, Lin; Halas, Naomi J.

2013-01-01

The approach of RNA interference (RNAi)- using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein- is very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly(L)lysine peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), in vitro. Light-triggered delivery resulted in ∼ 47% and ∼49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay. PMID:22862291

Selective Inhibition of Histone Deacetylation in Melanoma Increases Targeted Gene Delivery by a Bacteriophage Viral Vector.

PubMed

Campbell, Samuel; Suwan, Keittisak; Waramit, Sajee; Aboagye, Eric Ofori; Hajitou, Amin

2018-04-21

The previously developed adeno-associated virus/phage (AAVP) vector, a hybrid between M13 bacteriophage (phage) viruses that infect bacteria only and human Adeno-Associated Virus (AAV), is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the α ν integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC). We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP.

Transdermal Drug Delivery: Opportunities and Challenges for Controlled Delivery of Therapeutic Agents Using Nanocarriers.

PubMed

Kurmi, Balak Das; Tekchandani, Pawan; Paliwal, Rishi; Paliwal, Shivani Rai

2017-01-01

Transdermal drug delivery represents an extremely attractive and innovative route across the skin owing to the possibility for achieving systemic effect of drugs. The present scenario demands a special focus on developing safe medicine with minimized toxic adverse effects related to most of the pharmacologically active agents. Transdermal drug delivery would be a focal paradigm which provides patient convenience, first-pass hepatic metabolism avoidance, local targeting and reduction in toxic effect related to various categories of drugs like, analgesics, antiinflammatory, antibiotics, antiviral, anaesthetic, anticancer etc. Even this route has challenges due to highly organized structure of skin which acts as a main barrier to penetration of drug via the skin. Several alternative possible strategies are available which overcome these barriers, including use of penetration enhancer, eletroporation, iontophoresis and various nanotechnologically developed nanocarrier systems. The latest one includes employing liposome, dendrimers, nanoparticles, ethosome, carbon nanotube and many more to avoid associated limitations of conventional formulations. Numerous transdermal products such as Estrasorb, Diractin, VivaGel®, Daytrana®, Aczone, Sileryst® are available in the market having a novel strategy to achieve higher penetration of drugs. This encourages formulation fraternity to develop structurally deformable and stable nanocarriers as an alternative approach for controlled and reliable drug delivery across the skin barrier. In this review, we will discuss nanocarriers mediated approaches that come-up with the solutions to the different challenges towards transdermal drug delivery, its clinical importance and latest insight to research in it. The reports presented in this review confirm the wide application of nanocarriers for transdermal delivery of drug/gene. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Selective Gene Transfection of Individual Cells In Vitro with Plasmonic Nanobubbles

PubMed Central

Lukianova-Hleb, Ekaterina; Samaniego, Adam P.; Wen, Jianguo; Metelitsa, Leonid; Chang, Chung-Che; Lapotko, Dmitri

2011-01-01

Gene delivery and transfection of eukaryotic cells is widely used for research and for developing gene cell therapy. However, the existing methods lack selectivity, efficacy and safety when heterogeneous cell systems must be treated. We report a new method that employs plasmonic nanobubbles (PNBs) for delivery and transfection. A PNB is a novel, tunable cellular agent with a dual mechanical and optical action due to the formation of the vapor nanobubble around a transiently heated gold nanoparticle upon its exposure to a laser pulse. PNBs enabled the mechanical injection of the extracellular cDNA plasmid into the cytoplasm of individual target living cells, cultured leukemia cells and human CD34+CD117+ stem cells and expression of a green fluorescent protein (GFP) in those cells. PNB generation and lifetime correlated with the expression of green fluorescent protein in PNB-treated cells. Optical scattering by PNBs additionally provided the detection of the target cells and the guidance of cDNA injection at single cell level. In both cell models PNBs demonstrated a gene transfection effect in a single pulse treatment with high selectivity, efficacy and safety. Thus, PNBs provided targeted gene delivery at the single cell level in a single pulse procedure that can be used for safe and effective gene therapy. PMID:21315120

Coating barium titanate nanoparticles with polyethylenimine improves cellular uptake and allows for coupled imaging and gene delivery

PubMed Central

Dempsey, Christopher; Lee, Isac; Cowan, Katie; Suh, Junghae

2015-01-01

Barium titanate nanoparticles (BT NP) belong to a class of second harmonic generating (SHG) nanoprobes that have recently demonstrated promise in biological imaging. Unfortunately, BT NPs display low cellular uptake efficiencies, which may be a problem if cellular internalization is desired or required for a particular application. To overcome this issue, while concomitantly developing a particle platform that can also deliver nucleic acids into cells, we coated the BT NPs with the cationic polymer polyethylenimine (PEI) – one of the most effective nonviral gene delivery agents. Coating of BT with PEI yielded complexes with positive zeta potentials and resulted in an 8-fold increase in cellular uptake of the BT NPs. Importantly, we were able to achieve high levels of gene delivery with the BT-PEI/DNA complexes, supporting further efforts to generate BT platforms for coupled imaging and gene therapy. PMID:23973999

Polyethylenimine functionalized magnetic nanoparticles as a potential non-viral vector for gene delivery.

PubMed

Zhou, Yangbo; Tang, Zhaomin; Shi, Chunli; Shi, Shuai; Qian, Zhiyong; Zhou, Shaobing

2012-11-01

Polyethylenimine (PEI) functionalized magnetic nanoparticles were synthesized as a potential non-viral vector for gene delivery. The nanoparticles could provide the magnetic-targeting, and the cationic polymer PEI could condense DNA and avoid in vitro barriers. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, dynamic light scattering measurements, transmission electron microscopy, vibrating sample magnetometer and atomic force microscopy. Agarose gel electrophoresis was used to asses DNA binding and perform a DNase I protection assay. The Alamar blue assay was used to evaluate negative effects on the metabolic activity of cells incubated with PEI modified magnetic nanoparticles and their complexes with DNA both in the presence or absence of an external magnetic field. Flow cytometry and fluorescent microscopy were also performed to investigate the transfection efficiency of the DNA-loaded magnetic nanoparticles in A549 and B16-F10 tumor cells with (+M) or without (-M) the magnetic field. The in vitro transfection efficiency of magnetic nanoparticles was improved obviously in a permanent magnetic field. Therefore, the magnetic nanoparticles show considerable potential as nanocarriers for gene delivery.

Development of a Targeted anti-HER2 scFv Chimeric Peptide for Gene Delivery into HER2-Positive Breast Cancer Cells.

PubMed

Cheraghi, Roya; Nazari, Mahboobeh; Alipour, Mohsen; Majidi, Asia; Hosseinkhani, Saman

2016-12-30

Chimeric polymers are known as suitable carriers for gene delivery. Certain properties are critical for a polymer to be used as a gene delivery vector. A new polymer was designed for the targeted delivery of genes into breast cancer cell lines, based on MPG peptide. It is composed of different functional domains, including HIV gp41, nuclear localization sequence of SV40 T-antigen, two C-terminus repeats of histone H1, and the scFv of anti-HER2 antibody. The results demonstrated that the vector can effectively condense plasmid DNA into nanoparticles with an average size of 250nm. Moreover, fusion of the scFv portion to the carrier brought about the specific recognition of HER2. Overall, the transfection efficiency of the vector demonstrated that it could deliver the desired gene into BT-474 HER2-positive breast cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthesis and characterization of N-(2-hydroxy)propyl-3-trimethyl ammonium chitosan chloride for potential application in gene delivery.

PubMed

Xiao, Bo; Wan, Ying; Wang, Xiaoyu; Zha, Qichen; Liu, Haoming; Qiu, Zhiye; Zhang, Shengmin

2012-03-01

A series of N-(2-hydroxy)propyl-3-trimethyl ammonium chitosan chloride (HTCC) samples with various degrees of quaternization ranging from 12.4 to 43.7% was synthesized. The structures and properties of HTCC were investigated by FT-IR, (1)H NMR, conductometric titration and XRD analysis. It was found that HTCC had a more amorphous structure than chitosan. HTCC samples showed significantly lower cytotoxicity than polyethyleneimine in HepG2 and HeLa cell lines. The samples spontaneously formed complexes with pGL3 luciferase plasmid. These complexes had desirable particle sizes (160-300 nm) and zeta potentials (10.8-18.7 mV) when the weight ratios of HTCC to plasmid altered in the range of 3:1-20:1. In vitro gene transfection results indicated that HTCC had significantly high transfection efficiency compared with chitosan for delivering pGL3 luciferase plasmid to HeLa cells. The results suggest that HTCC could be a promising non-viral vector for safe and efficient DNA delivery. Copyright © 2011 Elsevier B.V. All rights reserved.

Synergistically combined gene delivery for enhanced VEGF secretion and anti-apoptosis

PubMed Central

Won, Young-Wook; Lee, Minhyung; Kim, Hyun Ah; Nam, Kihoon; Bull, David A.; Kim, Sung Wan

2013-01-01

With current pharmacological treatments, preventing the remodeling of the left ventricle and the progression to heart failure is a difficult task. Gene therapy is considered to provide a direct treatment to the long-term complications of ischemic heart diseases. Although current gene therapies that use single molecular targets seem potentially possible, they have not achieved a success in the treatment of ischemic diseases. With an efficient polymeric gene carrier, PAM-ABP, we designed a synergistically combined gene delivery strategy to enhance vascular endothelial growth factor (VEGF) secretion and prolong anti-apoptotic effects. A hypoxia-inducible plasmid expressing both hypoxia-inducible heme oxygenase-1 (HO-1) and the Src homology domain-2 containing tyrosine phosphatase-1 microRNA (miSHP 1) and a hypoxia-responsive VEGF plasmid were combined in this study. The positive feedback circuit between HO-1 and VEGF, and the negative regulatory role of SHP-1 in angiogenesis enhance VEGF secretion synergistically. The synergy in VEGF secretion as a consequence of the gene combination and the prolonged HO-1 activity was confirmed in hypoxic cardiomyocytes and cardiomyocyte apoptosis under hypoxia, and was decreased synergistically. These results suggest that the synergistic combination of VEGF, HO-1, and miSHP-1 may be promising for the clinical treatment of ischemic diseases. PMID:24007285

Degradable self-assembling dendrons for gene delivery: experimental and theoretical insights into the barriers to cellular uptake.

PubMed

Barnard, Anna; Posocco, Paola; Pricl, Sabrina; Calderon, Marcelo; Haag, Rainer; Hwang, Mark E; Shum, Victor W T; Pack, Daniel W; Smith, David K

2011-12-21

This paper uses a combined experimental and theoretical approach to gain unique insight into gene delivery. We report the synthesis and investigation of a new family of second-generation dendrons with four triamine surface ligands capable of binding to DNA, degradable aliphatic-ester dendritic scaffolds, and hydrophobic units at their focal points. Dendron self-assembly significantly enhances DNA binding as monitored by a range of experimental methods and confirmed by multiscale modeling. Cellular uptake studies indicate that some of these dendrons are highly effective at transporting DNA into cells (ca. 10 times better than poly(ethyleneimine), PEI). However, levels of transgene expression are relatively low (ca. 10% of PEI). This indicates that these dendrons cannot navigate all of the intracellular barriers to gene delivery. The addition of chloroquine indicates that endosomal escape is not the limiting factor in this case, and it is shown, both experimentally and theoretically, that gene delivery can be correlated with the ability of the dendron assemblies to release DNA. Mass spectrometric assays demonstrate that the dendrons, as intended, do degrade under biologically relevant conditions over a period of hours. Multiscale modeling of degraded dendron structures suggests that complete dendron degradation would be required for DNA release. Importantly, in the presence of the lower pH associated with endosomes, or when bound to DNA, complete degradation of these dendrons becomes ineffective on the transfection time scale-we propose this explains the poor transfection performance of these dendrons. As such, this paper demonstrates that taking this kind of multidisciplinary approach can yield a fundamental insight into the way in which dendrons can navigate barriers to cellular uptake. Lessons learned from this work will inform future dendron design for enhanced gene delivery. © 2011 American Chemical Society

Expression of monellin in a food-grade delivery system in Saccharomyces cerevisiae.

PubMed

Liu, Jun; Yan, Da-zhong; Zhao, Sheng-jun

2015-10-01

Genetically modified (GM) foods have caused much controversy. Construction of a food-grade delivery system is a desirable technique with presumptive impact on industrial applications from the perspective of bio-safety. The aim of this study was to construct a food-grade delivery system for Saccharomyces cerevisiae and to study the expression of monellin from the berries of the West African forest plant Dioscoreophyllum cumminsii in this system. A food-grade system for S. cerevisiae was constructed based on ribosomal DNA (rDNA)-mediated homologous recombination to enable high-copy-number integration of the expression cassette inserted into the rDNA locus. A copper resistance gene (CUP1) was used as the selection marker for yeast transformation. Because variants of transformants containing different copy numbers at the CUP1 locus can be readily selected after growth in the presence of elevated copper levels, we suggest that this system would prove useful in the generation of tandemly iterated gene clusters. Using this food-grade system, a single-chain monellin gene was heterologously expressed. The yield of monellin reached a maximum of 675 mg L(-1) . This system harbors exclusively S. cerevisiae DNA with no antibiotic resistance genes, and it should therefore be appropriate for safe use in the food industry. Monellin was shown to be expressed in this food-grade delivery system. To our knowledge, this is the first report so far on expression of monellin in a food-grade expression system in S. cerevisiae. © 2014 Society of Chemical Industry.

Vector delivery technique affects gene transfer in the cornea in vivo.

PubMed

Mohan, Rajiv R; Sharma, Ajay; Cebulko, Tyler C; Tandon, Ashish

2010-11-27

This study tested whether controlled drying of the cornea increases vector absorption in mouse and rabbit corneas in vivo and human cornea ex vivo, and studied the effects of corneal drying on gene transfer, structure and inflammatory reaction in the mouse cornea in vivo. Female C57 black mice and New Zealand White rabbits were used for in vivo studies. Donor human corneas were used for ex vivo experiments. A hair dryer was used for drying the corneas after removing corneal epithelium by gentle scraping. The corneas received no, once, twice, thrice, or five times warm air for 10 s with a 5 s interval after each 10 s hair dryer application. Thereafter, balanced salt solution (BSS) was topically applied immediately on the cornea for 2 min using a custom-cloning cylinder. The absorbed BSS was quantified using Hamilton microsyringes. The adeno-associated virus 8 (AAV8) vector (1.1×10(8) genomic copies/µl) expressing marker gene was used to study the effect of corneal drying on gene transfer. Animals were sacrificed on day 14 and gene expression was analyzed using commercial staining kit. Morphological changes and infiltration of inflammatory cells were examined with H & E staining and immunocytochemistry. Mice, rabbit or human corneas subjected to no or 10 s drying showed 6%-8% BSS absorption whereas 20, 30, or 50 s corneal drying showed significantly high 14%-19% (p<0.001), 21%-22% (p<0.001), and 25%-27% (p<0.001) BSS absorption, respectively. The AAV8 application on mouse cornea after 50 s drying showed significantly higher transgene delivery (p<0.05) in vivo with mild-to-moderate changes in corneal morphology. The 30 s of drying also showed significantly (p<0.05) high transgene delivery in mouse stroma in vivo without jeopardizing corneal morphology whereas 10 or 20 s drying showed moderate degree of gene transfer with no altered corneal morphology. Corneas that underwent 50 s drying showed high CD11b-positive cells (p<0.01) compared to control corneas whereas 20

Increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes.

PubMed

Arangoa, M A; Düzgüneş, N; Tros de Ilarduya, C

2003-01-01

A novel lipidic vector composed of DOTAP/Chol liposomes, asialofetuin (AF), protamine sulfate and DNA has been developed. The resulting protamine-AF-lipoplexes improved significantly the levels of gene expression in cultured cells and in the liver upon i.v. administration. Lipoplexes containing the optimal amount of AF (1 microg/microg DNA) showed a 16-fold higher transfection activity in HepG2 cells than non-targeted (plain) complexes. The uptake by cells having asialoglycoprotein receptors (ASGPr) on their plasma membrane was decreased by the addition of free AF, indicating that AF-lipoplexes were taken up specifically by cells via ASGPr-mediated endocytosis. Results from transfections performed in cells defective in ASGPr, ie HeLa cells, confirmed this mechanism. By addition of the condensing peptide, protamine sulfate, smaller complexes were obtained, which enhanced even more the uptake of AF-complexes in HepG2 cells and in the liver. The optimal amount of protamine was 0.4 microg/mcirog DNA, and gene expression was about 5-fold over that obtained with AF-lipoplexes in the absence of the peptide, and 75-fold higher than that with plain conventional lipoplexes. Protamine-AF-lipoplexes increased by a factor of 12 luciferase gene expression in the liver of mice administered systemically via the tail vein, compared to plain complexes. In summary, our findings extend the scope of previous studies where AF-lipoplexes were used to introduce DNA into hepatocytes. The combination of targeting and protamine condensation obviated the need for partial hepatectomy, commonly required to obtain efficient gene delivery in this organ. Since protamine sulfate has been proven to be non-toxic in humans, the novel liver-specific vector described here may be useful for the delivery of clinically important genes to this organ.

Charge-reversal Lipids, Peptide-based Lipids, and Nucleoside-based Lipids for Gene Delivery

PubMed Central

LaManna, Caroline M.; Lusic, Hrvoje; Camplo, Michel; McIntosh, Thomas J.; Barthélémy, Philippe; Grinstaff, Mark W.

2013-01-01

Conspectus Twenty years after gene therapy was introduced in the clinic, advances in the technique continue to garner headlines as successes pique the interest of clinicians, researchers, and the public. Gene therapy’s appeal stems from its potential to revolutionize modern medical therapeutics by offering solutions to a myriad of diseases by tailoring the treatment to a specific individual’s genetic code. Both viral and non-viral vectors have been used in the clinic, but the low transfection efficiencies when utilizing non-viral vectors have lead to an increased focus on engineering new gene delivery vectors. To address the challenges facing non-viral or synthetic vectors, specifically lipid-based carriers, we have focused on three main themes throughout our research: 1) that releasing the nucleic acid from the carrier will increase gene transfection; 2) that utilizing biologically inspired designs, such as DNA binding proteins, to create lipids with peptide-based headgroups will improve delivery; and 3) that mimicking the natural binding patterns observed within DNA, by using lipids having a nucleoside headgroup, will give unique supramolecular assembles with high transfection efficiency. The results presented in this Account demonstrate that cellular uptake and transfection efficacy can be improved by engineering the chemical components of the lipid vectors to enhance nucleic acid binding and release kinetics. Specifically, our research has shown that the incorporation of a charge-reversal moiety to initiate change of the lipid from positive to negative net charge during the transfection process improves transfection. In addition, by varying the composition of the spacer (rigid, flexible, short, long, and aromatic) between the cationic headgroup and the hydrophobic chains, lipids can be tailored to interact with different nucleic acids (DNA, RNA, siRNA) and accordingly affect delivery, uptake outcomes, and transfection efficiency. Introduction of a peptide

'Safe passage': pregnant Iranian Kurdish women's choice of childbirth method.

PubMed

Shahoei, Roonak; Riji, Haliza Mohd; Saeedi, Zhila Abed

2011-10-01

This article is a report of a grounded theory study of the influence of emotions on women's selection of a method of childbirth. There is substantial evidence to indicate that a pregnant woman's emotions play an important role in the decision-making process of selecting a child delivery method. Despite this, however, there is a notable lack of research about the relationship between pregnant women's emotions and their choice of a childbirth method in developing countries. A qualitative study using the grounded theory approach was conducted. The data were collected from 22 Iranian Kurdish pregnant women in their third trimester using semi-structured interviews. Concurrent data collection and analysis took place between 2008 and 2009. A cumulative process of theoretical sampling and constant comparison was used to identify concepts and then expand, validate, and clarify them. The substantive grounded theory that was identified from data analysis was 'safe passage'. 'Safe passage' involved five phases that were not mutually exclusive in their occurrence. The five phases of the 'safe passage' theory that were identified from the data analysis were: 'safety of baby', 'fear', 'previous experience', 'social support' and 'faith'. The goal of 'safe passage' was to achieve a healthy delivery and to ensure the health of the newborn. 'Safe passage' was a process used to determine how the emotions of pregnant Iranian Kurdish women influenced their choice of the mode of child delivery. More research is needed in this field to develop a body of knowledge beneficial to midwifery education and practice. © 2011 Blackwell Publishing Ltd.

Sweet Vector for Gene Delivery: the Sugar Decoration of Polyplexes Reduces Cytotoxicity with a Balanced Effect on Gene Expression.

PubMed

Albuquerque, Lindomar J C; Alavarse, Alex C; Carlan da Silva, Maria C; Zilse, Morgana S; Barth, Maitê T; Bellettini, Ismael C; Giacomelli, Fernando C

2018-02-01

The use of sugar-functionalized polyplexes as a nonviral gene delivery vector with lower cytotoxicity than the well-known polymeric carrier branched polyethyleneimine (BPEI) is investigated. The substitution of primary amine groups in the BPEI chains with lactose residues leads to larger polyplexes, presumably due to the higher amount of polymer required to complete DNA condensation. Nevertheless, the sugar functionalization substantially reduces the cytotoxicity of the assemblies. The nanocomplexes are taken up by the cells to a greater extent, whereas the levels of gene expression are maintained compared to those obtained using BPEI, which is known for its excellent transfection efficiency. Accordingly, the preparation of lower-cytotoxicity polyplexes while maintaining gene expression, which is highly relevant to the field, is demonstrated. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.