Viral Vectors For Gene Delivery Research Articles

Intramuscular scAAV9-SMN Injection Mediates Widespread Gene Delivery to the Spinal Cord and Decreases Disease Severity in SMA Mice

We have recently demonstrated the remarkable efficiency of self-complementary (sc) AAV9 vectors for central nervous system (CNS) gene transfer following intravenous delivery in mice and larger animals. Here, we investigated whether gene delivery to motor neurons (MNs) could also be achieved via intramuscular (i.m.) scAAV9 injection and subsequent retrograde transport along the MNs axons. Unexpectedly, we found that a single injection of scAAV9 into the adult mouse gastrocnemius (GA) mediated widespread MN transduction along the whole spinal cord, without limitation to the MNs connected to the injected muscle. Spinal cord astrocytes and peripheral organs were also transduced, indicating vector spread from the injected muscle to both the CNS and the periphery through release into the blood circulation. Moreover, we showed that i.m. injection of scAAV9 vectors expressing "survival of motor neuron" (Smn) in spinal muscular atrophy (SMA) mice mediated high survival motor neuron (SMN) expression levels at both the CNS and the periphery, and increased the median lifespan from 12 days to 163 days. These findings represent to date the longest extent in survival obtained in SMA mice following i.m. viral vector gene delivery, and might generate a renewed interest in the use of i.m. adeno-associated viruses (AAV) delivery for the development of gene therapy strategies for MN diseases.

Co-transduction of lentiviral and adenoviral vectors for co-delivery of growth factor and shRNA genes in mesenchymal stem cells-based chondrogenic system

Gene delivery takes advantage of cellular mechanisms to express gene products and is an efficient way to deliver them into cells, influencing cellular behaviours and expression patterns. Among the delivery methods, viral vectors are applied due to their high efficiency. Two typical viral vectors for gene delivery include lentiviral vector for integrative transduction and adenoviral vector for transient episomal transduction, respectively. The selection and formulation of proper viral vectors applied to cells can modulate gene expression profiles and further impact the downstream pathways. In this study, recombinant lentiviral and adenoviral vectors were co-transduced in a synovial mesenchymal stem cells (SMSCs)-based articular chondrogenic system by which two transgenes were co-delivered - the gene for transforming growth factor (TGF)β3, to facilitate SMSC chondrogenesis, and the gene for small hairpin RNA (shRNA), targeting the mRNA of type I collagen (Col I) α1 chain to silence Col I expression and minimize fibrocartilage formation. Delivery of either gene could be achieved with either lentiviral or adenoviral vectors. Therefore, co-delivery of the two transgenes via the two types of vectors was performed to determine which combination was optimal for three-dimensional (3D) articular chondrogenesis to construct articular hyaline cartilage tissue. Suppression of Col I and expression of cartilage markers, including type II collagen, aggrecan and cartilage oligomeric matrix protein (COMP), were assessed at both the transcriptome and protein phenotypic levels. It was concluded that the combination of lentiviral-mediated TGFβ3 release and adenoviral-mediated shRNA expression (LV-T + Ad-sh) generally demonstrated optimal efficacy in engineered articular cartilage with SMSCs.

Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatography

Significant progress in the application of viral vectors for gene delivery into mammalian cells and the use of viruses as biopesticides requires downstream processing that can satisfy application-specific demands on performance. In the present work the stability and ion exchange membrane chromatography of a recombinant of Autographa californica M nucleopolyhedrovirus is studied. To adjust the degree of purification the effect of ionic conductivity or pH on the viral infectivity was assessed (0.77-78.00mS/cm, pH 3-8). Infectivity decreased rapidly by several orders of magnitude at below 5mS/cm (i.e., 0.49MPa osmotic pressure change) or at below pH 5.5 (rationalized with particle aggregation). The virus was concentrated and purified via adsorption (0.2-1.1×10(16)pfu/m(3) chromatographic bed volume, 0.6-1.1×10(12)pfu/m(2) membrane area facing the incident fluid flow) and elution at pH 6.1 and 6.35mS/cm from three strong anion exchange membranes. Virus recovery and concentration in accord with the volume reduction were obtained using a polyether sulfone-based membrane with quaternary ammonium ligands. The level of host cell protein (down to below the detection limit) and suspended DNA (below 93pg DNA per 10(6)pfu) are reported for each membrane employed, for the purpose of comparability, under equal adsorption or elution conditions respectively.

Parvoviruses: Structure and Infection

Parvoviruses package a ssDNA genome. Both nonpathogenic and pathogenic members exist, including those that cause fetal infections, encompassing the entire spectrum of virus phenotypes. Their small genomes and simple coding strategy has enabled functional annotation of many steps in the infectious life cycle. They assemble a multifunctional capsid responsible for cell recognition and the transport of the packaged genome to the nucleus for replication and progeny virus production. It is also the target of the host immune response. Understanding how the capsid structure relates to the function of parvoviruses provides a platform for recombinant engineering of viral gene delivery vectors for the treatment of clinical diseases, and is fundamental for dissecting the viral determinants of pathogenicity. This review focuses on our current understanding of parvovirus capsid structure and function with respect to the infectious life cycle.

Intracellular transport of recombinant adeno-associated virus vectors

Recombinant adeno-associated viral vectors (rAAVs) have been widely used for gene delivery in animal models, and are currently evaluated for human gene therapy after successful clinical trials in the treatment of inherited, degenerative or acquired diseases, such as Leber congenital amaurosis, Parkinson disease or heart failure. However, limitations in vector tropism, such as limited tissue specificity and insufficient transduction efficiencies of particular tissues and cell types, still preclude therapeutic applications in certain tissues. Wild-type adeno-associated viruses (AAVs) are defective viruses that require the presence of a helper virus to complete their life cycle. On the one hand, this unique property makes AAV vectors one of the safest available viral vectors for gene delivery. On the other, it also represents a potential obstacle because rAAV vectors have to overcome several biological barriers in the absence of a helper virus to transduce successfully a cell. Consequently, a better understanding of the cellular roadblocks that limit rAAV gene delivery is crucial and, during the last 15 years, numerous studies resulted in an expanding body of knowledge of the intracellular trafficking pathways of rAAV vectors. This review describes our current understanding of the mechanisms involved in rAAV attachment to target cells, endocytosis, intracellular trafficking, capsid processing, nuclear import and genome release with an emphasis on the most recent discoveries in the field and the emerging strategies used to improve the efficiency of AAV-derived vectors.

Viral vectors for gene delivery to the central nervous system

Viral vectors for gene delivery to the central nervous system

Targeted Gene Modification of Hematopoietic Progenitor Cells in Mice Following Systemic Administration of a PNA-peptide Conjugate

Hematopoietic stem cell (HSC) gene therapy offers promise for the development of new treatments for a variety of hematologic disorders. However, efficient in vivo modification of HSCs has proved challenging, thus imposing constraints on the therapeutic potential of this approach. Herein, we provide a gene-targeting strategy that allows site-specific in vivo gene modification in the HSCs of mice. Through conjugation of a triplex-forming peptide nucleic acid (PNA) to the transport peptide, antennapedia (Antp), we achieved successful in vivo chromosomal genomic modification of hematopoietic progenitor cells, while still retaining intact differentiation capabilities. Following systemic administration of PNA-Antp conjugates, sequence-specific gene modification was observed in multiple somatic tissues as well as within multiple compartments of the hematopoietic system, including erythroid, myeloid, and lymphoid cell lineages. As a true functional measure of gene targeting in a long-term renewable HSC, we also demonstrate preserved genomic modification in the bone marrow and spleen of primary recipient mice following transplantation of bone marrow from PNA-Antp-treated donor mice. Our approach offers a minimally invasive alternative to ex vivo gene therapy, by eliminating the need for the complex steps of stem cell mobilization and harvesting, ex vivo manipulation, and transplantation of stem cells. Therefore, our approach may provide new options for individualized therapies in the treatment of monogenic hematologic diseases such as sickle cell anemia and thalassemia.

Intracellular Trafficking Mechanism and Cytosolic Protein Interactions of a Non Viral Gene Delivery Vector: Studies Based on Transferrin Conjugated Pullulan-PEI

Compared to viral vectors the intracellular barriers faced by non-viral vectors are high, which lead to lesser transfection efficiency. The various limiting steps include endocytosis, endosomal escape, intracellular stability and mobility, unpacking and nuclear localization. The understanding of these mechanisms for overcoming these barriers is essential for developing gene delivery vectors which can really be used for appropriate clinical/medical applications. Transferrin conjugated pullulan-PEI (PPETf) was developed for neuronal cell targeted gene delivery which was evaluated for its transfection efficiency. The stability of the polymer-DNA nanoplexes in the presence of plasma and cytosolic proteins was evaluated by agarose gel electrophoresis. Time dependent uptake of the nanoplexes by using labeled polymer and YOYO tagged plasmid was employed to understand the uptake mechanism and the sequence of unpacking of DNA from the vector. The uptake pathway, endosomal escape, trafficking etc. are evaluated in the presence of endocytic and microtubilin inhibitors to understand the mechanism of nanoplexes trafficking into and within the cell. Keywords: Gene delivery, hemocompatibility, intracellular trafficking, polyethyleneimine, pullulan, Cytosolic Protein Interactions, Transferrin

Limb girdle muscular dystrophies

This review is an up-to-date analysis of the genetic diagnosis and therapeutic strategies for limb girdle muscular dystrophies (LGMDs). LGMDs are an example of both clinical and genetic heterogeneity. Clinically, by the description of non-LGMD phenotypes associated with LGMD genes and of LGMD phenotypes associated with originally non-LGMD disease genes; and genetically, by the description of new LGMD genes that further increase the diagnostic complexity. Moreover, new powerful approaches for DNA analysis, such as exome sequencing, promise to revolutionize the field of heterogeneous genetic diseases, also providing information about the true penetrance of LGMD mutations. The recent inputs on novel pathogenic mechanisms and pathways in LGMD will suggest novel therapeutic approaches and future clinical trials. In addition, therapeutic approaches of gene and cell delivery into animal models show promising results that will be translated into clinical trials. The genetic diagnosis of LGMD from the present home-made algorithms will move toward high-throughput diagnostic strategies based on next-generation sequencing (NGS) technologies. As therapy, new powerful drug approaches based on recent pathogenetic findings will be pushed to clinical trials. In addition, novel more efficient and safer viral vectors for gene delivery will be proposed.

In vivo transfection study of chitosan-DNA-FAP-B nanoparticles as a new non viral vector for gene delivery to the lung

Gene therapy targeted at the respiratory epithelium holds therapeutic potential for diseases such as cystic fibrosis and lung cancer. We recently reported that Chitosan-DNA-FAP-B nanoparticles are good candidates for targeted gene delivery to fibronectin molecules (FAP-B receptors) of lung epithelial cell membrane. In this study Chitosan-DNA-FAP-B nanoparticles were nebulized to mice using air jet nebulizer. The effect of nebulization on size, zeta potential and DNA binding ability of nanoparticles were studied. The level of gene expression in the mice lungs was evaluated. Nebulization did not affect the physicochemical properties of nanoparticles. Aerosol delivery of Chitosan-DNA-FAP-B nanoparticles resulted in 16-fold increase of gene expression in the mice lungs compared with Chitosan-DNA nanoparticles. This study suggested that Chitosan-FAP-B nanoparticle can be a promising carrier for targeted gene delivery to the lung.

Assessment of Hippocampal Adeno-Associated Viral Vector Gene Delivery via Frameless Stereotaxis in a Nonhuman Primate

Background/Aims: Expression of the neuropeptide galanin in hippocampal neurons reduces seizures in the kainic acid rodent model of epilepsy. In order to translate these findings into a human clinical trial, the safety and feasibility of hippocampal adeno-associated viral (AAV) vector expression must be demonstrated in a nonhuman primate model. Methods: The Stealth Frameless Stereotactic System and Navigus Biopsy Appliance (Medtronic) were used to inject self-complementary AAV2 carrying the gene for green fluorescent protein (GFP) into monkey hippocampi. Using a single occipital trajectory per side (n = 8 trajectories), multiple injections spaced by 5 mm were delivered to each hippocampus. Results: GFP was expressed in both neuronal and glial cells. Injections led to nonhomogeneous gene expression, suggesting closer spacing of injections may lead to more gene expression. Increasing injection volumes entailed a general increase in volume of expression, but there was no overlap of expression within the 5-mm injection interval. Efforts to avoid the occipital horn failed to prevent leaking of vector into the ventricle, and resulted in deviation of the trajectory at proximal points from the hippocampus. Conclusion: Using the occipital approach, adequate cannulation of the monkey hippocampus will require transventricular trajectories.

Human JC virus-like particles as a gene delivery vector

Introduction: As a viral gene delivery vector, the recombinant JC virus-like particles (VLPs) can be easily generated in large quantities and at low cost. Exogenous genes of interest can be packaged by the VLP without the involvement of viral genetic material and then delivered into any tissue susceptible to JC virus (JCV) to allow gene transduction. Therefore, it should be possible in the future to develop a gene delivery vector using the human JC VLPs that will allow gene therapy.Areas covered: Development of a gene delivery vector using the polyomavirus VLPs is reviewed in this article. The advantages and disadvantages of using JC VLP for gene delivery are discussed.Expert opinion: Human JC VLPs are readily produced and can be engineered with ease; they allow specific targeting without the presence of any viral genetic material. For therapeutic purposes, gene(s) of interest or other compounds can be packaged into the VLP and delivered to JCV-susceptible cells at high efficiency.

Gene therapy for glaucoma

Glaucoma is a chronic progressive disease for which ideal treatment would provide a localized long-lasting therapy with minimal side-effects. A gene therapy approach in which a mutated gene is replaced or inactivated, or in which a new gene is introduced, could provide a novel and more effective way of targeting the disease. The aim of this review is to provide an update on recent advances in the field of gene therapy for the treatment of glaucoma. Both viral and nonviral vector gene delivery systems have been used to target specific tissues involved in the pathogenesis of glaucoma. These tissues include the trabecular meshwork, ciliary body, ciliary epithelium, Müller cells, and retinal ganglion cells. Recent studies in large animal models have demonstrated effective long-term gene expression in the trabecular meshwork following intracameral delivery of adeno-associated viral vectors and lentiviral vectors with limited effect on surrounding ocular tissues. Other promising studies have focused on vector-mediated expression of neurotrophic factors and have demonstrated a neuroprotective effect following intravitreal delivery of vectors in glaucomatous animal models. Given the recent laboratory studies utilizing gene therapy techniques to lower intraocular pressure and to provide neuroprotection, and the continued development of tissue-specific vectors, it seems that we are well poised for a new generation of treatments for glaucoma.

α-Galactosidase A Expressed in the Salivary Glands Partially Corrects Organ Biochemical Deficits in the Fabry Mouse Through Endocrine Trafficking

Fabry disease is caused by an X-linked deficiency of the lysosomal enzyme α-galactosidase A (GLA) and has been treated successfully with enzyme replacement therapy (ERT). Gene therapy has been proposed as an alternative to ERT due to the presumed advantages of continuous, endogenous production of the therapeutic enzyme. GLA production in the liver and its therapeutic efficacy in the Fabry mouse have been demonstrated previously with various viral vector systems. In consideration of the potential advantages of using the salivary glands as endogenous GLA biosynthesis sites, we explored the feasibility of this approach in the Fabry mouse. GLA -/0 or -/- mice received an adenoviral vector (2 × 10(10) or 1 × 10(9) viral particles) expressing GLA to the right submandibular gland via oral cannulation of the submandibular duct. Four days later, animals were sacrificed; saliva, plasma, kidney, liver, and brain were collected and assayed using ELISA, Western blot, and a GLA enzymatic activity assay using both traditional fluorescence methods and isotope dilution mass spectrometry by following the U.S. EPA Method 6800. GLA activity was significantly elevated in the serum and liver of both treatment groups, and improvement in the kidney was marginally significant (P < 0.069) in the high-dose group. Notably, we found that liver and salivary gland produce different glycoforms of the GLA transgene. Only small numbers of adenoviral genomes were observed in the livers of treated animals, but in four of 14 in the high-dose groups, liver levels of adenovirus exceeded 20 copies/μg, indicating that the sequestration in the salivary gland was imperfect at high doses. Taken together, these results indicate that the salivary gland-based gene therapy for Fabry disease is promising, and further studies with advanced viral vector gene delivery systems (e.g., adeno-associated virus) for long-term treatment appear to be warranted.

Intra-arterial Targeted Islet-specific Expression of Sirt1 Protects β Cells From Streptozotocin-induced Apoptosis in Mice

Gene therapy provides a promising approach to curing diabetes. However, an effective route for islet-specific targeting has yet to be established. Toward this end, the pancreatic blood circulation system in Balb/c mice was determined by the injection of rhodamine-containing beads. The efficiency of islet targeting was then measured by the injection of adenoviral vectors carrying a green fluorescence gene via the celiac trunk (C.T.). The results showed that >95% of islets and about 60% of β cells within the pancreatic body and tail could be labeled 3 days after surgery. α-Cell labeling was not as efficient, whereas labeling of nonendocrine tissues was barely detectable. For proof of principle, adenoviral vectors carrying a Sirtuin transgene were injected similarly to test the islet protection effect in the streptozotocin (STZ)-induced type 1 diabetic model. The results demonstrated that overexpression of Sirtuin in STZ-treated mice reduced the level of β-cell death and extent of glucose intolerance. This study reports on efficient islet-specific targeting by using adenoviral injection. This procedure could be invaluable to the treatment of diabetes and the study of islet biology.

Improvement of adenoviral vector-mediated gene transfer to airway epithelia by folate-modified anionic liposomes

Despite remarkable progress in the development of both viral and nonviral gene delivery vectors for airway disease treatment, poor gene transfer efficiency to the airway epithelium is a major obstacle in clinical application. To take advantage of the unique features of viral and nonviral vectors, we have developed complexes of adenovirus vector and anionic liposomes (AL-Ad5) by the calcium-induced phase change method. In the current study, based on the fact that there are overexpressed folate receptors on the surface of airway epithelia, we further modified the AL-Ad5 complexes with folate (F-AL-Ad5) to improve the transduction ability of Ad5 in airway epithelia. The transduction efficiencies of the obtained F-AL-Ad5 and AL-Ad5 complexes were assessed in primary-cultured airway epithelia in vitro. Our results indicated that compared with naked adenovirus vector, both AL-Ad5 and F-AL-Ad5 could significantly enhance the gene transduction efficiency of adenovirus vector in primary-cultured airway epithelial cells. Moreover, the enhancement mediated by F-AL-Ad5 was more dramatic than that by AL-Ad5. These results suggested that F-AL-Ad5 may be a useful strategy to deliver therapeutic genes to the airway epithelia and is promising in clinical application.

Cationic nanoemulsions as non-viral vectors for plasmid DNA delivery

Non-viral gene carriers have been extensively investigated as alternatives to viral vectors for therapeutic gene delivery. Many cationic lipid carriers including liposomes, emulsions, and solid lipid nanoparticles are used to transfer plasmid DNA. Stable nanoemulsions were prepared and modified by conjugating fatty acids with cationic amino acids including lysine, arginine, and histidine with the help of carbodiimide. Concentrations of crosslinker and amino acids were optimized to obtain the maximal surface potential. The zeta potential and size distribution of the cationic nanoemulsions were measured using photon correlation spectroscopy. The morphology of nanoemulsion-DNA complexes was examined by transmission electron microscopy. The transfection efficiencies and cytotoxicity of three cationic nanoemulsions were evaluated using 3T3 fibroblast cells. The maximal zeta potentials of lysine-, arginine-, and histidine-modified nanoemulsions were 50, 43, and 7 mV, respectively. The transfection efficiencies of amino acid-modified nanoemulsions were in the order of lysine > arginine > histidine. Low cytotoxicities of these three amino acid-modified nanoemulsions were observed. A facile and inexpensive in situ modification for producing cationic nanoemulsions was developed. The results show the potential of amino acid-modified cationic nanoemulsions as non-viral vectors for gene delivery.

Low Current-driven Micro-electroporation Allows Efficient In Vivo Delivery of Nonviral DNA into the Adult Mouse Brain

Viral gene transfer or transgenic animals are commonly used technologies to alter gene expression in the adult brain, although these approaches lack spatial specificity and are time consuming. We delivered plasmid DNA locally into the brain of adult C57BL/6 mice in vivo by voltage- and current-controlled electroporation. The low current-controlled delivery of unipolar square wave pulses of 125 µA with microstimulation electrodes at the injection site gave 16 times higher transfection rates than a voltage-controlled electroporation protocol with plate electrodes resulting in currents of about 400 mA. Transfection was restricted to the target region and no damage due to the electric pulses was found. Our current-controlled electroporation protocol indicated that the use of very low currents resulting in applied voltages within the physiological range of the membrane potential, allows efficient transfection of nonviral plasmid DNA. In conclusion, low current-controlled electroporation is an excellent approach for electroporation in the adult brain, i.e., gene function can be influenced locally at a high level with no mortality and minimal tissue damage.

Surface immobilization of hexa-histidine-tagged adeno-associated viral vectors for localized gene delivery

Adeno-associated viral vectors, which are undergoing broad exploration in clinical trials, have significant promise for therapeutic gene delivery due to their safety and delivery efficiency. Gene delivery technologies capable of mediating localized gene expression may further enhance AAV’s potential in a variety of therapeutic applications by reducing spread outside of a target region, which may thereby reduce off-target side effects. We have genetically engineered an AAV variant capable of binding to surfaces with high affinity via a hexahistidine-metal binding interaction. This immobilized AAV vector system mediates high efficiency delivery to cells that contact the surface and thus may have promise for localized gene delivery, which may aid numerous applications of AAV delivery to gene therapy.

A First Step in Viral Gene Therapy for Muscular Dystrophy

Muscular dystrophies are genetically diverse disorders clinically characterized by progressive weakness of muscles of limbs, cranium, or both and by histologic degeneration and regeneration of muscle without abnormal storage of metabolic products. Advances in molecular genetics have revolutionized our understanding of muscular dystrophies, beginning in 1987 with the identification of mutations leading to dystrophin deficiency in Duchenne's muscular dystrophy [1]. Since then, at least 20 genetically distinct forms of limb-girdle muscular dystrophy (LGMD) have been identified [2]. Four subtypes of autosomal recessive LMGD are the result of mutations in genes encoding sarcoglycans (SGs) α, β, γ, and δ, which form a subcomplex of the dystrophin–glycoprotein complex linked to muscle membranes. The LGMD variants due to SG mutations are LGMD2C (δ-SG), LGME2D (α-SG), LGMD2E (β-SG), and LGMD2F (γ-SG) [3]. Although molecular genetics has been a powerful tool in unraveling the pathogenesis of muscular dystrophies, harnessing the therapeutic potential of molecular biology for these devastating muscle diseases has been technically challenging. For autosomal recessive diseases with loss of functional protein (e.g., sarcoglycanopathies and dystrophinopathy), gene replacement therapy is a promising approach. Viral delivery of genes was first performed successfully in children with severe combined immunodeficiency by using retroviruses to deliver normal genes to the patients' bone marrow stem cells, which were removed from the children, infected with the virus (tranduced), and injected into the bloodstream, where they engrafted and restored normal immune cells [4, 5]. Unfortunately, 5 of 20 children treated with retroviral gene therapy developed leukemias because vector insertion into the patients' DNA activated an oncogene, thus exposing one of the potential pitfalls of gene therapy [6]. Other technical obstacles to gene therapy include inefficient gene delivery, immune rejection, and other vector toxicities. To develop gene therapy for muscular dystrophy, viral and nonviral vectors have been used to deliver genes in experimental cellular and animal models [7]. Adeno-associated viruses (AAVs) are promising viral vectors for gene delivery because they are not known to cause human disease; however, AAVs have a small DNA-carrying capacity and therefore cannot be used to carry large genes such as dystrophin. In contrast, the α-SG gene (SGCA) is relatively small and can be packaged into AAVs; therefore, AAV delivery of SGCA to patients with LGMD2D is a potentially achievable goal that Mendell and colleagues are pursuing.