Viral Vectors For Gene Therapy Research Articles

Common Gene Therapy Viral Vectors Do Not Efficiently Penetrate Sputum from Cystic Fibrosis Patients

Norwalk virus and human papilloma virus, two viruses that infect humans at mucosal surfaces, have been found capable of rapidly penetrating human mucus secretions. Viral vectors for gene therapy of Cystic Fibrosis (CF) must similarly penetrate purulent lung airway mucus (sputum) to deliver DNA to airway epithelial cells. However, surprisingly little is known about the rates at which gene delivery vehicles penetrate sputum, including viral vectors used in clinical trials for CF gene therapy. We find that sputum spontaneously expectorated by CF patients efficiently traps two viral vectors commonly used in CF gene therapy trials, adenovirus (d∼80 nm) and adeno-associated virus (AAV serotype 5; d∼20 nm), leading to average effective diffusivities that are ∼3,000-fold and 12,000-fold slower than their theoretical speeds in water, respectively. Both viral vectors are slowed by adhesion, as engineered muco-inert nanoparticles with diameters as large as 200 nm penetrate the same sputum samples at rates only ∼40-fold reduced compared to in pure water. A limited fraction of AAV exhibit sufficiently fast mobility to penetrate physiologically thick sputum layers, likely because of the lower viscous drag and smaller surface area for adhesion to sputum constituents. Nevertheless, poor penetration of CF sputum is likely a major contributor to the ineffectiveness of viral vector based gene therapy in the lungs of CF patients observed to date.

The influence of epileptic neuropathology and prior peripheral immunity on CNS transduction by rAAV2 and rAAV5

Adeno-associated virus (AAV) provides a promising platform for clinical treatment of neurological disorders owing to its established efficacy and lack of apparent pathogenicity. To use viral vectors in treating neurological disease, however, transduction must occur under neuropathological conditions. Previous studies in rodents have shown that AAV5 more efficiently transduces cells in the hippocampus and piriform cortex than AAV2. Using the kainic acid (KA) model of temporal lobe epilepsy and AAV2 and 5 carrying a hybrid chicken β-actin promoter driving green fluorescent protein (GFP), we found that limbic seizure activity caused substantial neuropathology and resulted in a significant reduction in subsequent AAV5 transduction. Nonetheless, this reduced transduction still was greater than AAV2 transduction in control rats. Although KA seizures compromise blood-brain barrier function, potentially increasing exposure of target tissue to circulating neutralizing antibodies, we observed no interaction between KA seizure-induced damage and immunization status on AAV transduction. Finally, while we confirmed the near total neuronal-specific transgene expression for both serotypes in control rats, AAV5-GFP expression was increasingly localized to astrocytes in seizure-damaged areas. Thus, the pathological milieu of the injured brain can reduce transduction efficacy and alter viral tropism- both relevant concerns when considering viral vector gene therapy for neurological disorders.

Development of viral vectors for gene therapy for chronic pain.

Chronic pain is a major health concern that affects millions of people. There are no adequate long-term therapies for chronic pain sufferers, leading to significant cost for both society and the individual. The most commonly used therapy for chronic pain is the application of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to addiction and may cause side effects. Further studies of the mechanisms of chronic pain have opened the way for development of new treatment strategies, one of which is gene therapy. The key to gene therapy is selecting safe and highly efficient gene delivery systems that can deliver therapeutic genes to overexpress or suppress relevant targets in specific cell types. Here we review several promising viral vectors that could be applied in gene transfer for the treatment of chronic pain and further discuss the possible mechanisms of genes of interest that could be delivered with viral vectors for the treatment of chronic pain.

AAV-mediated photoreceptor transduction of the pig cone-enriched retina

Recent success in clinical trials supports the use of adeno-associated viral (AAV) vectors for gene therapy of retinal diseases caused by defects in the retinal pigment epithelium (RPE). In contrast, evidence of the efficacy of AAV-mediated gene transfer to retinal photoreceptors, the major site of inherited retinal diseases, is less robust. In addition, although AAV-mediated RPE transduction appears efficient, independently of the serotype used and species treated, AAV-mediated photoreceptor gene transfer has not been systematically investigated thus so far in large animal models, which also may allow identifying relevant species-specific differences in AAV-mediated retinal transduction. In the present study, we used the porcine retina, which has a high cone/rod ratio. This feature allows to properly evaluate both cone and rod photoreceptors transduction and compare the transduction characteristics of AAV2/5 and 2/8, the two most efficient AAV vector serotypes for photoreceptor targeting. Here we show that AAV2/5 and 2/8 transduces both RPE and photoreceptors. AAV2/8 infects and transduces photoreceptor more efficiently than AAV2/5, similarly to what we have observed in the murine retina. The use of the photoreceptor-specific rhodopsin promoter restricts transgene expression to porcine rods and cones, and results in photoreceptor transduction levels similar to those obtained with the ubiquitous promoters tested. Finally, immunological, toxicological and biodistribution studies support the safety of AAV subretinal administration to the large porcine retina. The data presented here on AAV-mediated transduction of the cone-enriched porcine retina may affect the development of gene-based therapies for rare and common severe photoreceptor diseases.

Reducing Immune Response against Lentiviral Vectors: Lentiviral Vector Presentation of CD47, The ‘Marker of Self’

Immune response to viral gene therapy vectors and their transgene products is a significant problem in the field of gene therapy. Viral vectors, because they are derived from viruses, can induce an immune response. This makes gene delivery inefficient and can pose a significant danger to patients. Macrophages act as immunological gatekeepers at the interface of tissue and lymph. They take up antigens from the extracellular environment and then present them to the immune system. Macrophage uptake has been shown to be inhibited by CD47 interaction with SIRP alpha. Viral vectors presenting CD47 on their surface should show reduced levels of phagocytosis by macrophages, and thus reduced presentation and clearance by the immune system. In this work, HEK 293T cells were transduced, using a lentiviral vector, to over-express CD47 with green fluorescent protein (GFP) at the C-terminus. These transduced cells were then transfected to produce a second set of lentiviral vectors. Since the lentivirus takes a piece of the cell membrane to make its envelope when it buds from the cell, these vectors express CD47 on their envelope. The main goal of this work is to qualitatively and quantitatively characterize the presentation of CD47 by these lentiviral vectors. Fluorescent microscopy of equilibrium density gradient fractions indicates that these lentiviral vectors present CD47-GFP. The fluorescence intensity of individual and aggregated viral vectors was quantified. This will be the first step in using CD47 expression as a method to reduce immune response to lentiviral vectors in order to increase the efficacy and safety of lentiviral vector mediated gene therapy.

Expression of therapeutic proteins after delivery of chemically modified mRNA in mice

Current viral vectors for gene therapy are associated with serious safety concerns, including leukemogenesis, and nonviral vectors are limited by low gene transfer efficiency. Here we investigate the therapeutic utility of chemically modified mRNA as an alternative to DNA-based gene therapy. A combination of nucleotide modifications abrogates mRNA interaction with Toll-like receptor (TLR)3, TLR7, TLR8 and retinoid-inducible gene I (RIG-I), resulting in low immunogenicity and higher stability in mice. A single intramuscular injection of modified murine erythropoietin mRNA raises the average hematocrit in mice from 51.5% to 64.2% after 28 days. In a mouse model of a lethal congenital lung disease caused by a lack of surfactant protein B (SP-B), twice weekly local application of an aerosol of modified SP-B mRNA to the lung restored 71% of the wild-type SP-B expression, and treated mice survived until the predetermined end of the study after 28 days.

Efficient Gene Delivery by EGF-Lipoplexes In Vitro and In Vivo

In this work, we have evaluated the ability of targeted lipoplexes to enhance transgene expression in EGF receptor (EGFR) overexpressing tumor cells by using lipoplexes. We prepared DOTAP/cholesterol liposomes modified with EGF at 0.5/1, 1/1, 2/1 and 5/1 lipid/DNA (+/-) charge ratio by sequentially mixing the liposomes with the ligand and adding the reporter or the therapeutic plasmid gene, pCMVLuc (pVR1216) or pCMVIL12, respectively. HepG2, DHDK12proB and SW620 cells were used for in vitro experiments, which were performed in the presence of 60% serum. The characterization of EGF-lipoplexes indicated a size close to 300 nm and a variable net surface charge as a function of the amount of EGF associated to the cationic liposomes. EGF-lipoplexes, which showed an increased transfection activity, were positively charged, noncytotoxic and highly effective in protecting DNA from DNase I attack. Transfection activity in vitro resulted in an enhancement in the luciferase and IL-12 expression by EGF-lipoplexes compared with those without ligand (plain-lipoplexes) and to naked DNA. The results observed in SW620 cells, which are deficient in EGFR, confirmed that DNA uptake was predominantly via EGFR-mediated endocytosis. In vivo transfection activity was confirmed by luciferase imaging in living mice. Bioluminiscence could be detected mainly in the lung with a maximum signal 24 h after application. The resulting EGF-lipoplexes significantly increased the level of gene expression in mice compared with control or naked DNA. These findings indicate that these nanovectors may be an adequate alternative to viral vectors for gene therapy.

Viral vectors for cystic fibrosis gene therapy: What does the future hold?

Viral vectors for cystic fibrosis gene therapy: What does the future hold? Uta Griesenbach1, Makoto Inoue2, Mamoru Hasegawa2, Eric WFW Alton11Department of Gene Therapy, Imperial College London, UK; The UK Cystic Fibrosis Gene Therapy Consortium; 2DNAVEC Corporation, Tsukuba, JapanAbstract: Gene transfer to the airway epithelium has been more difficult than originally anticipated, largely because of significant extra- and intracellular barriers in the lung. In general, viral vectors are more adapted to overcoming these barriers than nonviral gene transfer agents and are, therefore, more efficient in transferring genes into recipient cells. Viral vectors derived from adenovirus, adeno-associated virus, and Sendai virus, which all have a natural tropism for the airway epithelium, have been evaluated for cystic fibrosis (CF) gene therapy. Although these vectors transduce airway epithelial cells efficiently, gene expression is transient and repeated administration is inefficient. They are, therefore, unlikely to be suitable for CF gene therapy. More recently, lentiviruses (LV) have been assessed for lung gene transfer. In contrast to retroviruses, they transduce nondividing cells and randomly integrate into the genome. However, LVs do not have a natural tropism for the lung, and a significant amount of effort has been put into pseudotyping these vectors with proteins suitable for airway gene transfer. Several studies have shown that LV-mediated transduction leads to persistent gene expression (for the lifetime of the animal) in the airways and, importantly, repeated administration is feasible. Thus, appropriately pseudotyped LV vectors are promising candidates for CF gene therapy. Here, we will review preclinical and clinical research related to viral CF gene therapy.Keywords: cystic fibrosis, gene therapy, adenovirus, AAV, lentivirus, Sendai virus

AAV plasmid DNA simplifies liver‐directed in vivo gene therapy: comparison of expression levels after plasmid DNA‐, adeno‐associated virus‐ and adenovirus‐mediated liver transfection

Successful liver gene therapy depends on efficient gene transfer techniques and long-lasting gene expression after successful transfer. Over the last decades, important progress has been made with the introduction of viral vectors using animal models, although their use is hampered by a complex and costly preparation compared to the simple and cost-effective preparation of plasmid DNA. These problems become even more critical when considering the application of viral vectors in human gene therapy and gene therapy trials. In a previous study, we were able to show that the hydrodynamics-based gene transfer of plasmid-DNA, containing the adeno-associated-virus specific inverted terminal repeats (AAV-ITR), prolongs gene expression in the liver, although it remained unclear whether plasmid gene transfer could achieve similar expression levels compared to viral-vector gene transfer. Rat livers were transfected in-vivo with AAV-ITR-containing plasmid-DNA using a modified hydrodynamics-based procedure. Expression levels were monitored thereafter and compared with expression levels after viral-vector gene transfer. A high and stable long-term expression was achieved after in vivo transfection of rat livers with AAV-ITR-containing plasmids. The expression course resembled that after AAV-mediated gene transfer, and the expression was at least as high, and lasted as long, compared to recombinant AAV-mediated gene transfer. We consider AAV-ITR-containing plasmids as a simple and cost-effective alternative to recombinant viral vectors, especially for liver-directed gene therapy in rodents. With ongoing progress in gene transfer methods for naked DNA, these plasmids may also become a successful alternative to recombinant viral vectors in human gene therapy.

Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver

shRNA overexpression from viral gene therapy vectors can trigger cytotoxicity leading to organ failure and lethality in mice and rats. This process likely involves saturation of endogenous cellular RNAi factors including exportin-5 (Xpo-5). Here, we have shown that Xpo-5 overexpression enhanced shRNA efficiency in the liver of adult mice but increased hepatotoxicity. We identified the 4 members of the human Argonaute (Ago) protein family as downstream factors involved in saturation of endogenous cellular RNAi, all of which were able to interact with shRNAs in cells and mice. In Ago/shRNA coexpression studies, Ago-2 (Slicer) was the primary rate-limiting determinant of both in vitro and in vivo RNAi efficacy, toxicity, and persistence. In adult mice, vector-based Ago-2/Xpo-5 coexpression enhanced U6-driven shRNA silencing of exogenous and endogenous hepatic targets, reduced hepatotoxicity, and extended RNAi stability by more than 3 months. Use of weaker RNA polymerase III promoters to minimize shRNA expression likewise alleviated in vivo toxicity and permitted greater than 95% persistent knockdown of hepatitis B virus and other transgenes in mouse liver for more than 1 year. Our studies substantiate that abundant small RNAs can overload the endogenous RNAi pathway and reveal possible strategies for reducing hepatotoxicity of short- and long-term clinical gene silencing in humans.

Analysis of adsorption of a baculovirus bioreaction bulk on an ion-exchange surface by surface plasmon resonance

The binding and elution of the key components of a bioreaction bulk for the production of recombinant baculoviruses—a promising viral vector for gene therapy and vaccination—on a model ion-exchange surface have been successfully measured and interpreted by surface plasmon resonance (SPR) spectroscopy. The micro-scaled, ion-exchange surface was produced by immobilizing a typical ion-exchange ligand, diethylaminoethyl, onto commercially available planar gold sensor chip surfaces, which were pre-derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid. Each isolated analyte was injected into the SPR cell at defined operating conditions of salt and solute concentrations to determine the adsorption equilibrium plateau, and then eluted at the same salt concentration, upon which a well-defined, residual desorption equilibrium plateau was observed. From the analysis of the binding and elution curves and equilibrium plateaus for seven key biomolecules, it is possible to determine the adsorption isotherms over a broad range of equilibrium conditions for the three main cuts of the baculovirus bioreaction bulk: the product (the infective baculovirus), the main product-related impurities, and the main process-related impurities. A model that quantitatively explains the SPR-derived adsorption/desorption data was successfully developed for this complex biological system.

Affinity recovery of lentivirus by diaminopelargonic acid mediated desthiobiotin labelling

Desthiobiotin-tagged lentiviral vectors have been metabolically produced by DBL producer cells in a 7,8-diaminopelargonic acid (7-DAPA) dependent manner for envelope independent, single-step affinity purification. 7-DAPA, which has little or no affinity for avidin/streptavidin, was synthesised and verified by NMR spectroscopy and mass spectrometry. By expressing the biotin acceptor, biotin ligase and desthiobiotin synthase bioD, DBL cells converted exogenous 7-DAPA into membrane-bound desthiobiotin. Desthiobiotin on the DBL cell surface was visualised by confocal microscopy and the desthiobiotin density was quantified by HABA-avidin assay. Desthiobiotin was then spontaneously incorporated onto the surface of lentiviral vectors produced by the DBL cells. It has been demonstrated by flow cytometry that the desthiobiotinylated lentiviruses were captured from the crude 7-DAPA-containing viral supernatant by Streptavidin Magnespheres ® and eluted by biotin solution efficiently whilst retaining infectivity. The practical, high yielding virus purification using Pierce monomeric avidin coated columns indicates a highly efficient biotin-dependent recovery of infectious lentiviruses at 68%. The recovered lentiviral vectors had a high purity and the majority were eluted within 45 min. This 7-DAPA mediated desthiobiotinylation technology can be applied in scalable production of viral vectors for clinical gene therapy.

Modeling electrostatic interactions of baculovirus vectors for ion-exchange process development

Product-related impurities constitute a major burden in the production of recombinant viral vectors for gene therapy and vaccination; it impairs not only the biological efficacy of the preparation but the process yield/productivity. Recombinant baculovirus was used as an enveloped virus model to address this issue. Given that ion-exchange chromatography is a process of choice for purification of viral vectors, the analysis of the electrostatic behavior can be instrumental for the improvement of impurity removal. The main species, product (infective virus particle) and product-derived impurities (dsDNA-, glycoprotein-, and envelope-deprived baculovirus particles), were isolated and correspondent ζ potentials were analyzed through dynamic light scattering. A model of the virus based on the viral components critical for biological function is proposed. The contribution of these viral components to the overall particle electrostatic interaction energy profile (calculated between the particle and a putative ion-exchange surface) was assessed as a function of ionic strength and pH. This resulted in a deterministic tool capable of distinguishing the electrostatic properties of the infective virus particle from the major virus-related impurities. Within an ion-exchange bind-elute process, this knowledge helps narrow the optimization space in early stage process development for viral vectors by predicting the best selectivity conditions.

Gene delivery by lipoplexes and polyplexes

Gene therapy has emerged as a promising approach for the treatment or prevention of acquired and genetic diseases. At the present time, major somatic gene transfer approaches employ either viral or non-viral vectors. Viral vectors show high gene transfer efficiency, but are deficient in several areas, including the induction of a host inflammatory and immune response. Some of these problems can be circumvented by employing non-viral vehicles, such as cationic liposomes or polymers. The complexes they form with DNA are defined as "lipoplexes" or "polyplexes, respectively, and constitute the most promising alternative to the use of viral vectors for gene therapy. Here we review the interactions between the vectors and DNA leading to complex formation, the supramolecular structures of lipoplexes and polyplexes, and their mechanisms of DNA transfer. Our objective is to provide a framework for the future design and synthesis of optimal non-viral vectors for gene therapy. The structure, charge and formulation of these vehicles are also related to the stability, and consequently to the efficiency of gene transfection. While lipids that facilitate transformation of lipoplexes to non-bilayer phases mediate high transfection activity in vitro, lipids, like cholesterol, that confer stability in serum, are more suitable for gene delivery in vivo. The efficiency of polyplex-mediated transfection depends on the ability of the polymer to condense DNA, while allowing it to dissociate once inside the cell.

Endoplasmic reticulum chaperone gp96 is essential for infection with vesicular stomatitis virus

The envelope glycoprotein of vesicular stomatitis virus (VSV-G) enables viral entry into hosts as distant as insects and vertebrates. Because of its ability to support infection of most, if not all, human cell types VSV-G is used in viral vectors for gene therapy. However, neither the receptor nor any specific host factor for VSV-G has been identified. Here we demonstrate that infection with VSV and innate immunity via Toll-like receptors (TLRs) require a shared component, the endoplasmic reticulum chaperone gp96. Cells without gp96 or with catalytically inactive gp96 do not bind VSV-G. The ubiquitous expression of gp96 is therefore essential for the remarkably broad tropism of VSV-G. Cells deficient in gp96 also lack functional TLRs, which suggests that pathogen-driven pressure for TLR-mediated immunity maintains the broad host range of VSV-G by positively selecting for the ubiquitous expression of gp96.

Cellular Endocytosis and Gene Delivery

Endocytosis is the process by which cells take up macromolecules from the surrounding medium. The best-characterized process is the so-called clathrin-dependent endocytosis, although much is also currently known about clathrin-independent endocytic processes such as those involving caveolae and lipid rafts. An understanding of endocytosis and the cellular trafficking that occurs thereafter has a great deal of relevance to current molecular medicine. Gene therapy, which is presently being investigated for its therapeutic potential in treating immunodeficiency and metabolic diseases, cancer and heart disease, employs a variety of viral and nonviral vectors, which can be delivered to the target cells of the body and are subsequently endocytosed and dissembled. A variety of vectors can be used to deliver genes to organs in vivo or cells ex vivo. Various routes of vector delivery have been investigated. The mechanisms by which vectors such as adenoviruses, adeno-associated viruses, retroviruses and liposomes enter the cell are increasingly being investigated as the effort to increase the efficiency of gene therapy continues. This review focuses on mechanisms of endocytosis and how they relate to the internal trafficking of viral and nonviral vectors in gene therapy.

Endocytic uptake of fluorescence labelled DNA in yeast

After dispiriting results using viral vectors in gene therapy, by which a number of patients acquired cancer as a result of the use of retroviral vector constructs, the percentage of non-viral approaches has increased over recent years. To elucidate potential bottlenecks in the non-viral transfection process we here introduce a novel method to directly visualize endocytic non-viral DNA uptake in a transfection approach. This novel method allows for the first time to monitor the location of DNA which is taken up by endocytosis in yeast (Saccharomyces cerevisiae) wild type and mutant strains. More specifically it enables drawing conclusions about conditions favouring non-viral gene transfection.

Research Highlights

Research Highlights

Mechanism of Reduction in Titers From Lentivirus Vectors Carrying Large Inserts in the 3′LTR

Self-inactivating (SIN) lentiviruses flanked by the 1.2-kb chicken hypersensitive site-4 (cHS4) insulator element provide consistent, improved expression of transgenes, but have significantly lower titers. The mechanism by which this occurs is unknown. Lengthening the lentiviral (LV) vector transgene cassette by an additional 1.2 kb by an internal cassette caused no further reduction in titers. However, when cHS4 sequences or inert DNA spacers of increasing size were placed in the 3'-long terminal repeat (LTR), infectious titers decreased proportional to the length of the insert. The stage of vector life cycle affected by vectors carrying the large cHS4 3'LTR insert was compared to a control vector: there was no increase in read-through transcription with insertion of the 1.2-kb cHS4 in the 3'LTR. Equal amount of full-length viral mRNA was produced in packaging cells and viral assembly/packaging was unaffected, resulting in comparable amounts of intact vector particles produced by either vectors. However, LV vectors carrying cHS4 in the 3'LTR were inefficiently processed following target-cell entry, with reduced reverse transcription and integration efficiency, and hence lower transduction titers. Therefore, vectors with large insertions in the 3'LTR are transcribed and packaged efficiently, but the LTR insert hinders viral-RNA (vRNA) processing and transduction of target cells. These studies have important implications in design of integrating vectors.

Pharmaceutical Strategies Enhancing Cell Penetration Efficiencies of Non-Viral Gene Delivery Systems

To achieve therapeutic effects, gene transportation or gene transfection becomes the key technique of gene therapy. Since viral vectors for gene therapy have shown serious problems, including immunogenicity and other adverse effects, non-viral carriers have attracted a great deal of attention, despite the fact that the transfection efficiencies are lower than those by viral carriers. Therefore, the major challenge of gene delivery is to improve the transfection efficiencies of the non-viral carriers, and first of all, the cell penetration efficiencies. This review focuses on recent approaches in the development of new non-viral gene delivery vectors, with great respect to those constructed based on cell penetration pathways of intracellular process considering transfection efficiency and cell penetration efficiency. Further development of sophisticated delivery systems is prospected for the non-viral vectors application in gene therapy.