Viral Vectors For Gene Delivery Research Articles

Synthesis, and Characterization, and Evaluation of Cellular Effects of the FOL‐PEG‐g‐PEI‐GAL Nanoparticles as a Potential Non‐Viral Vector for Gene Delivery

In this manuscript, we synthesized the potential non viral vector for gene delivery with proper transfection efficiency and low cytotoxicity. Polyethylenimine (PEI) is a well‐known cationic polymer which has high positive surface charge for condensing plasmid DNA. However; it is highly cytotoxic in many cell lines because of the high surface charge, non‐biodegradability and non‐biocompatibility. To enhance PEI biodegradability, the graft copolymer “PEG‐g‐PEI” was synthesized. To target cancer liver cells, two targeting ligands folic acid and galactose (lactobionic acid) which are over expressed on human hepatocyte carcinoma were attached to graft copolymer and “FOL‐PEG‐g‐PEI‐GAL” copolymer was synthesized. Composition of this grafted copolymer was characterized using 1H‐NMR and FTIR spectra. The molecular weight and zeta potential of this copolymer was compared to PEI. The particle size and zeta potential of FOL‐PEG‐g‐PEI‐GAL/DNA complexes at various N/P ratio were measured using dynamic light scattering (DLS). Cytotoxicity of the copolymer was also studied in cultured HepG2 human hepatoblastoma cell line. The FOL‐PEG‐g‐PEI‐GAL/DNA complexes at various N/P ratios exhibited no cytotoxicity in HepG2 cell line compared to PEI 25K as a control. The novel copolymer showed enhanced biodegradability in physiological conditions in compared with PEI and targeted cultured HepG2 cells. More importantly, significant transfection efficiency was exhibited in cancer liver cells. Together, our results showed that “FOL‐PEG‐g‐PEI‐GAL” nanoparticals could be considered as a useful non‐viral vector for targeted gene delivery.

Anionic Liposomes Increase the Efficiency of Adenovirus-Mediated Gene Transfer to Coxsackie-Adenovirus Receptor Deficient Cells

Despite remarkable progress in the research of both viral and nonviral gene delivery vectors, the drawbacks in each delivery system have limited their clinical applications. Therefore, one of the concepts for developing novel vectors is to overcome the limitations of individual vectors by combining them. In the current study, adenoviral vectors were formulated with anionic liposomes to protect them from neutralizing antibodies and to improve their transduction efficiency in Coxsackievirus-adenovirus receptor (CAR) deficient cells. A calcium-induced phase change method was applied to encapsulate adenovirus 5 (Ad5) into anionic liposomes to formulate the complexes of Ad5 and anionic liposomes (Ad5-AL). Meanwhile, the complexes of Ad5 and cationic liposomes (Ad5-CL) were also prepared as controls. LacZ gene expression in CAR overexpressing cells (A549) and CAR deficient cells (CHO and MDCK) was measured by either qualitative or quantitative detection. Confocal laser scanning microscopy was performed to determine intracellular location of Ad5 after their infection. Human sera with a high titer of antiadenovirus antibody were used to assess the neutralizing antibody protection ability of the complexed vectors. Accompanying the enhanced gene expression, a high ability to introduce Ad5 into cytoplasm and nucleus mediated by Ad5-AL was also observed in CAR deficient cells. Additionally, antibody neutralizing assay indicated that neutralizing serum inhibited naked Ad5 and Ad5-CL at rather higher dilution than Ad5-AL, which demonstrated Ad5-AL was more capable of protecting Ad5 from neutralizing than Ad5-CL. In conclusion, anionic liposomes prepared by the calcium-induced phase change method could significantly enhance the transduction ability of Ad5 in CAR deficient cells.

Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.

461. Engineering Adeno-Associated Viral Vectors for Glial-Targeted Gene Delivery

461. Engineering Adeno-Associated Viral Vectors for Glial-Targeted Gene Delivery

Generation of iPS cells using defined factors linked via the self-cleaving 2A sequences in a single open reading frame

Generation of induced pluripotent stem (iPS) cells from somatic cells has been achieved successfully by simultaneous viral transduction of defined reprogramming transcription factors (TFs). However, the process requires multiple viral vectors for gene delivery. As a result, generated iPS cells harbor numerous viral integration sites in their genomes. This can increase the probability of gene mutagenesis and genomic instability, and present significant barriers to both research and clinical application studies of iPS cells. In this paper, we present a simple lentivirus reprogramming system in which defined factors are fused in-frame into a single open reading frame (ORF) via self-cleaving 2A sequences. A GFP marker is placed downstream of the transgene to enable tracking of transgene expression. We demonstrate that this polycistronic expression system efficiently generates iPS cells. The generated iPS cells have normal karyotypes and are similar to mouse embryonic stem cells in morphology and gene expression. Moreover, they can differentiate into cell types of the three embryonic germ layers in both in vitro and in vivo assays. Remarkably, most of these iPS cells only harbor a single copy of viral vector. This system provides a valuable tool for generation of iPS cells, and our data suggest that the balance of expression of transduced reprogramming TFs in each cell is essential for the reprogramming process. More importantly, when delivered by non-integrating gene-delivery systems, this re-engineered single ORF will facilitate efficient generation of human iPS cells free of genetic modifications.

Gene therapy in epilepsy

Results from animal models suggest gene therapy is a promising new approach for the treatment of epilepsy. Several candidate genes such as neuropeptide Y and galanin have been demonstrated in preclinical studies to have a positive effect on seizure activity. For a successful gene therapy-based treatment, efficient delivery of a transgene to target neurons is also essential. To this end, advances have been made in the areas of cell transplantation and in the development of recombinant viral vectors for gene delivery. Recombinant adeno-associated viral (rAAV) vectors in particular show promise for gene therapy of neurological disorders due to their neuronal tropism, lack of toxicity, and stable persistence in neurons, which results in robust, long-term expression of the transgene. rAAV vectors have been recently used in phase I clinical trials of Parkinson's disease with an excellent safety profile. Prior to commencement of phase I trials for gene therapy of epilepsy, further preclinical studies are ongoing including evaluation of the therapeutic benefit in chronic models of epileptogenesis, as well as assessment of safety in toxicological studies.

Designing Heart Performance by Gene Transfer

The birth of molecular cardiology can be traced to the development and implementation of high-fidelity genetic approaches for manipulating the heart. Recombinant viral vector-based technology offers a highly effective approach to genetically engineer cardiac muscle in vitro and in vivo. This review highlights discoveries made in cardiac muscle physiology through the use of targeted viral-mediated genetic modification. Here the history of cardiac gene transfer technology and the strengths and limitations of viral and nonviral vectors for gene delivery are reviewed. A comprehensive account is given of the application of gene transfer technology for studying key cardiac muscle targets including Ca(2+) handling, the sarcomere, the cytoskeleton, and signaling molecules and their posttranslational modifications. The primary objective of this review is to provide a thorough analysis of gene transfer studies for understanding cardiac physiology in health and disease. By comparing results obtained from gene transfer with those obtained from transgenesis and biophysical and biochemical methodologies, this review provides a global view of cardiac structure-function with an eye towards future areas of research. The data presented here serve as a basis for discovery of new therapeutic targets for remediation of acquired and inherited cardiac diseases.

Avian Adeno-Associated Virus-Based Expression of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein for Poultry Vaccination

The avian adeno-associated virus (AAAV) is a replication-defective nonpathogenic virus member of the family Parvoviridae that has been proved to be useful as a viral vector for gene delivery. The use of AAAV for transgenic expression of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein and its ability to induce immunity in chickens were assessed. Proposed advantages of this system include no interference with maternal antibodies, diminished immune response against the vector, and the ability to accommodate large fragments of genetic information. In this work the generation of recombinant AAAV virions expressing the HN protein (rAAAV-HN) was demonstrated by electron microscopy, immunocytochemistry, and western blot analysis. Serological evidence of HN protein expression after in ovo or intramuscular inoculation of the recombinant virus in specific-pathogen-free chickens was obtained. Serum from rAAAV-HN-vaccinated birds showed a systemic immune response evidenced by NDV-specific enzyme-linked immunosorbent assay and hemagglutination inhibition testing. Positive virus neutralization in embryonated chicken eggs and indirect immunofluorescence detection of NDV infected cells by serum from rAAAV-HN vaccinated birds is also reported. A vaccine-challenge experiment in commercial broiler chickens using a Venezuelan virulent viscerotropic strain of NDV was performed. All unvaccinated controls died within 5 days postchallenge. Protection up to 80% was observed in birds vaccinated in ovo and revaccinated at 7 days of age with the rAAAV-HN. The results demonstrate the feasibility of developing and using an AAAV-based gene delivery system for poultry vaccination.

Increased virus replication in mammalian cells by blocking intracellular innate defense responses

The mammalian innate immune system senses viral infection by recognizing viral signatures and activates potent antiviral responses. Besides the interferon (IFN) response, there is accumulating evidence that RNA silencing or RNA interference (RNAi) serves as an antiviral mechanism in mammalian cells. Mammalian viruses encode IFN antagonists to counteract the IFN response in infected cells. A number of IFN antagonists are also capable of blocking RNAi in infected cells and therefore serve as RNA-silencing suppressors. Virus replication in infected cells is restricted by these innate antiviral mechanisms, which may kick in earlier than the viral antagonistic or suppressor protein can accumulate. The yield of virus vaccines and viral gene delivery vectors produced in mammalian producer cells may therefore be suboptimal. To investigate whether blocking of the innate antiviral responses in mammalian cells leads to increased viral vector production, we expressed a number of immunity suppressors derived from plant and mammalian viruses in human cells. We measured that the yield of infectious human immunodeficiency virus-1 particles produced in these cells was increased 5- to 10-fold. In addition, the production of lentiviral and adenoviral vector particles was increased 5- to 10-fold, whereas Sindbis virus particle production was increased approximately 100-fold. These results can be employed for improving the production of viral gene transfer vectors and viral vaccine strains.

Evolving vaccine choices for the continuously evolving avian influenza viruses.

Abstract Avian influenza viruses (AIVs) are a continuously evolving group of RNA viruses with segmented genomes. Although AIV is thought to originate from wild bird populations, the capacity for ready mutation and adaptation to mammalian hosts makes AIV both a potential agricultural and public health risk. Worldwide spread of AIV is inevitable because of the ever-presence of infection in wild, especially migratory, bird reservoirs. Therefore, vaccine development is of high priority. The potential for genetic shift (reassortment) and drift (point mutations) is a concern for dependency on live, attenuated vaccine strategies, which were the first vaccines of wide use in poultry. Inactivated whole virus and subunit protein vaccines provide limited immune responses. The evolution of vaccine strategies is the result of creative applications of genetic engineering to develop plasmid and viral gene delivery vectors. Gene vaccines that include plasmid DNA and viral vector delivery of AIV genes allow for endogenous, in vivo amplification of proteins within cells. The viral vectors are Nature's specialists for cellular delivery of genetic information and, if replication-competent, also for the spread from host cell to cell, furthering opportunities for induction of immunity. Viral proteins that induce neutralizing antibodies are preferred to subunit vaccines. The replication-competent fowlpox virus vectors have been licensed and proved to be efficacious even in field situations. Several avian respiratory viruses, with DNA or RNA genomes, are being developed with promising practical application for poultry. Maximum exploitation of vectored vaccines may incorporate enhancing immune molecules. Future vaccines must also allow for differentiation of vaccinated birds from naturally infected birds.

Adhesion Receptors Mediate Efficient Non-viral Gene Delivery

For a variety of reasons, including production limitations, potential unanticipated side effects, and an immunological response upon repeated systemic administration, virus-based vectors are as yet not ideal gene delivery vehicles, justifying further research into alternatives. Unlike viral vectors, non-viral vectors pose minimal health risks, but to meet therapeutic requirements their efficacy needs major improvement. This goal may be accomplished by better defining the mechanism of non-viral gene delivery and exploiting specific cellular properties. Here we demonstrate that transfection of epithelial cells with lipoplexes is almost exclusively mediated by the beta1 integrin cell surface receptor. More important, we show that in general, adhesion receptors can be exploited by lipoplexes to gain access to cells, including difficult-to-transfect primary neural stem cells and suspension cells, thereby leading to productive transfection. We propose that adhesion receptors serve as "natural" receptors for lipoplexes. As no natural cellular receptors for lipoplexes have previously been identified, our results are an important step forward in understanding the mechanisms of non-viral gene delivery. Moreover, the finding that adhesion receptors mediate efficient non-viral gene delivery paves the way for the optimization of (standard) transfection procedures as well as ex vivo gene therapy protocols using non-viral vectors.

Engineering Adeno-Associated Virus for One-Step Purification via Immobilized Metal Affinity Chromatography

Adeno-associated virus (AAV) is a promising vehicle for gene therapy, which will rely on the generation of high-titer, high-purity recombinant vectors. However, numerous purification protocols can involve challenging optimization or scalability issues, and most AAV serotypes do not bind heparin or sialic acid, used for AAV2/3 or AAV4/5 purification, requiring the development of new chromatography strategies. Immobilized metal affinity chromatography (IMAC) allows for robust protein purification via affinity tags such as the hexahistidine (His(6)) sequence. Through the combination of a diverse AAV2 library and rational peptide insertions, we have located an optimal His(6) tag insertion site within the viral capsid. This mutant and a related AAV8 variant can be purified from clarified cell lysate in a single gravity column step at infectious particle yields exceeding 90%. Furthermore, injection of IMAC-purified vector into the brain demonstrates that it mediates high-efficiency gene delivery in vivo, equivalent to that of wild-type capsid, with minimal immune cell activation. This affinity chromatography method may offer advantages in ease of purification, final vector purity, and process scalability. Moreover, a combined rational design and high-throughput library selection approach can aid in the design of enhanced viral gene delivery vectors.

Functional Recovery in a Friedreich's Ataxia Mouse Model by Frataxin Gene Transfer Using an HSV-1 Amplicon Vector

Functional Recovery in a Friedreich's Ataxia Mouse Model by Frataxin Gene Transfer Using an HSV-1 Amplicon Vector

Targeted retroviral gene delivery using ultrasound

Achieving specificity of delivery represents a major problem limiting the clinical application of retroviral vectors for gene therapy, whilst lack of efficiency and longevity of gene expression limit non-viral techniques. Ultrasound and microbubble contrast agents can be used to effect plasmid DNA delivery. We therefore sought to evaluate the potential for ultrasound/microbubble-mediated retroviral gene delivery. An envelope-deficient retroviral vector, inherently incapable of target cell entry, was combined with cationic microbubbles and added to target cells. The cells were exposed to pulsed 1 MHz ultrasound for 5 s and subsequently analysed for marker gene expression. The acoustic pressure profile of the ultrasound field, to which transduction efficiency was related, was determined using a needle hydrophone. Ultrasound-targeted gene delivery to a restricted area of cells was achieved using virus-loaded microbubbles. Gene delivery efficiency was up to 2% near the beam focus. Significant transduction was restricted to areas exposed to > or = 0.4 MPa peak-negative acoustic pressure, despite uniform application of the vector. An acoustic pressure-dependence was demonstrated that can be exploited for targeted retroviral transduction. The mechanism of entry likely involves membrane perturbation in the vicinity of oscillating microbubbles, facilitating fusion of the viral and cell membranes. We have established the basis of a novel retroviral vector technology incorporating favourable aspects of existing viral and non-viral gene delivery vectors. In particular, transduction can be controlled by means of ultrasound exposure. The technology is ideally suited to targeted delivery following systemic vector administration.

Verification of functional AAV-mediated neurotrophic and anti-apoptotic factor expression

The use of viral vectors for gene delivery offer many advantages for both basic research and therapeutic application through the continuous expression of a gene product within a target region. It is vital however that any gene product is correctly expressed in a biologically active form, and this should be confirmed prior to large scale in vivo studies. Using adeno-associated viral (AAV) vectors to direct the expression of either a neurotrophic factor or an anti-apoptotic protein, we have developed a range of in vitro assays to verify functional transgenic protein expression. Brain-derived neurotropic factor (BDNF) activity was confirmed by demonstrating enhanced generation of GABAergic neurons in embryonic (E15) striatal cultures and AAV-mediated glial-derived neurotrophic factor (GDNF) function using an assay for dopaminergic differentiation of embryonic (E14) ventral mesencephalic cultures. To assess functional anti-apoptotic factor expression we designed cell-survival assays, using embryonic cortical cultures to confirm Bcl-x L activity and the HT1080 cell-line for X-linked inhibitor of apoptosis protein (XIAP) activity following AAV-mediated expression. This study demonstrates that the use of functional assays provides valuable confirmation of desired biotherapeutic expression prior to extensive investigation with new gene delivery vectors.

Virus-based gene therapy strategies for bone regeneration

Gene therapy has emerged as a promising strategy for the repair and regeneration of damaged musculoskeletal tissues. Application of this paradigm to bone healing has shown enhanced efficacy in preclinical animal studies compared to conventional bone grafting approaches. This review discusses current and emerging virus-based genetic engineering strategies for the delivery of therapeutic molecules which promote skeletal regeneration. Viral gene delivery vectors are discussed in the context of bone repair in order to illustrate the challenges and applications of these methods with tissue-specific examples. Moreover the concepts discussed can be broadly applied to promote healing in a wide range of tissues. We also present important considerations involved in the application of these gene therapy techniques to a variety of osteogenic (e.g. bone marrow-derived cells) and non-osteogenic (e.g. fibroblasts and skeletal myoblasts) cell types. Criteria for the selection of regenerative molecules with soluble versus intracellular modes of action and emerging combinatorial approaches are also discussed. Overall, gene transfer technologies have the potential to overcome limitations associated with existing bone grafting approaches and may enable investigators to design therapies which more closely mimic the complex spatial and temporal cascade of proteins involved in endogenous bone development and repair.

Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity

A common viral immune evasion strategy involves mutating viral surface proteins in order to evade host neutralizing antibodies. Such immune evasion tactics have not previously been intentionally applied to the development of novel viral gene delivery vectors that overcome the critical problem of anti-vector immunity. Recombinant, replication-incompetent adenovirus serotype 5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens have proved highly immunogenic in preclinical studies but will probably be limited by the high prevalence of pre-existing anti-Ad5 immunity in human populations, particularly in the developing world. Here we show that rAd5 vectors can be engineered to circumvent anti-Ad5 immunity. We constructed novel chimaeric rAd5 vectors in which the seven short hypervariable regions (HVRs) on the surface of the Ad5 hexon protein were replaced with the corresponding HVRs from the rare adenovirus serotype Ad48. These HVR-chimaeric rAd5 vectors were produced at high titres and were stable through serial passages in vitro. HVR-chimaeric rAd5 vectors expressing simian immunodeficiency virus Gag proved comparably immunogenic to parental rAd5 vectors in naive mice and rhesus monkeys. In the presence of high levels of pre-existing anti-Ad5 immunity, the immunogenicity of HVR-chimaeric rAd5 vectors was not detectably suppressed, whereas the immunogenicity of parental rAd5 vectors was abrogated. These data demonstrate that functionally relevant Ad5-specific neutralizing antibodies are focused on epitopes located within the hexon HVRs. Moreover, these studies show that recombinant viral vectors can be engineered to circumvent pre-existing anti-vector immunity by removing key neutralizing epitopes on the surface of viral capsid proteins. Such chimaeric viral vectors may have important practical implications for vaccination and gene therapy.

Kunjin virus replicons: an RNA-based, non-cytopathic viral vector system for protein production, vaccine and gene therapy applications

The application of viral vectors for gene expression and delivery is rapidly evolving, with several entering clinical trials. However, a number of issues, including safety, gene expression levels, cell selectivity and antivector immunity, are driving the search for new vector systems. A number of replicon-based vectors derived from positive-strand RNA viruses have recently been developed, and this paper reviews the current knowledge on the first flavivirus replicon system, which is based on the Australian flavivirus Kunjin (KUN). Like most replicon systems, KUN replicons can be delivered as DNA, RNA or virus-like particles, they replicate their RNA in the cytoplasm and direct prolonged high-level gene expression. However, unlike most alphavirus replicon systems, KUN replicons are non-cytopathic, with transfected cells able to divide, allowing the establishment of cell lines stably expressing replicon RNA and heterologous genes. As vaccine vectors KUN replicons can induce potent, long-lived, protective, immunogen-specific CD8+ T cell immunity, a feature potentially related to extended production of antigen and double-stranded RNA-induced ‘danger signals’. The identification of KUN replicon mutants that induce increased levels of IFN-α/β has also spawned investigation of KUN replicons for use in cancer gene therapy. The unique characteristics of KUN replicons may thus make them suitable for specific protein production, vaccine and gene therapy applications.

480. Gene Transfer to Gut K Cells for Physiologic Insulin Replacement in Diabetes

We have previously demonstrated the feasibility of using gut K- cells as surrogate cells for insulin production. Transgenic mice expressing human insulin specifically in K-cells remained normoglycemic and glucose tolerant following destruction of their pancreatic |[beta]|-cells. However, a vector system is needed to transfer effective amount of the therapeutic insulin gene to duodenal epithelial cells in vivo. Previous efforts using viral and non-viral gene delivery vector resulted in some success. However, persistent transgene expression and detectable circulating proteins were not observed. Accordingly, this study is to identify a suitable viral vector and an optimal delivery methodology to achieve long-term gene transfer to the duodenum.

Gene Therapy Progress and Prospects: Viral trafficking during infection

The intracellular steps involved in viral infection, namely cytoplasmic trafficking and nuclear import, are critical events in the viral life cycle that have lagged behind other areas of viral research. This review examines recent advances in our understanding of these steps for viruses commonly employed as viral gene delivery vectors. Steps governing the cytoplasmic trafficking and nuclear import of Herpes Simplex virus, Human Immunodeficiency virus and Adenovirus are reviewed in this article.