Introduction to the issues: viral vectors and potential problems in their use.
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Animals↗
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[Recombinant adeno-associated viral vector 2-mediated gene transfer of VEGF165 improved porcine chronic myocardial ischemia].
OBJECTIVE: Vectors commonly used for therapeutic angiogenesis such as adenovirus and plasmid had their own limitations. Adenovirus-associated virus (AAV) is a relatively new but probably more ideal vector as it is safe and efficient. We will study the efficiency of recombinant AAV-2 mediated vascular endothelial growth factor165 gene transfer in inducing angiogenesis and arteriogenesis, in improving blood flow and myocardium function in a porcine chronic myocardial ischemic model. METHODS: Chinese experimental minipigs underwent placement of a left circumflex artery aneroid constrictor. Five weeks later, electrocardiogram, coronary angiography and magnetic resonance imaging were performed to confirm occlusion of LCX or ischemia of myocardium in LCX territory. Coronary blood flow, myocardium perfusion and left ventricular wall function were also evaluated. Then the animals were randomized to treatment with rAAV2-VEGF(165) (1 x 10(12) virus genome) or administration of PBS, both by direct myocardial injection. Three and six months after therapy, the animals were evaluated with regard to expression of VEGF(165) Capillary density and arteriole density of the ischemic myocardium, coronary angiography, myocardial perfusion and left ventricular function were also assessed six months after therapy. RESULTS: Five weeks after aneroid occluder implantation, all the animals demonstrated complete or nearly complete occlusion of LCX and perfusion deficiency in LCX territory. Three months after therapy, expression of VEGF(165) mRNA and protein were higher in the VEGF than control group. The difference between the two groups diminished after six months. There was significant increase in capillary density (1404.06 +/- 250.48/mm(2) vs 976.88 +/- 344.79/mm(2), P < 0.05) and arteriole density (167.81 +/- 36.29/mm(2) vs 116.56 +/- 34.48/mm(2), P < 0.05) in VEGF group compared with control. Comparison of myocardial perfusion demonstrated marked differences between the two groups with significant improvement in animals treated with rAAV2-VEGF(165). No significant improvement in left ventricular function was seen in either the VEGF or control group. CONCLUSIONS: Transmyocardial delivery of rAAV2-VEGF(165) resulted in VEGF gene expression for at least three months and stimulated angiogenesis and arteriogenesis in porcine model of chronic myocardial ischemia. Myocardial perfusion was also improved after VEGF gene delivery.
Adenoviridae↗
Viral vectors as therapeutic agents for glioblastoma.
Established treatments such as surgery, radiation and chemotherapy have not altered the median survival of glioblastoma, the most common malignant brain tumor. Since these failures reflect the highly invasive nature of glioblastoma, as well as the fact that few cells are actively replicating at any given point in time, therapies need to act in areas of the brain distant from the site of tumor origin and for long after their introduction. Over the past decade, laboratory studies and early clinical trials have raised hope that these therapeutic requirements may be fulfilled by gene therapy using non-replicating transgene-bearing viruses, oncolytic viruses or migratory stem cells to deliver tumoricidal transgenes. The principles behind these approaches and their initial results are reviewed.
Adenoviridae↗
[Dogs recover their sight thanks to viral vectors that could be used in humans].
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Animals↗
Safety testing programs for gene therapy viral vectors.
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Adenoviridae Infections↗
Alveolar stem cell transduction by an adeno-associated viral vector.
In inherited disorders such as surfactant protein deficiencies or cystic fibrosis (CF), where lung damage develops progressively after birth, gene replacement is best accomplished in the neonatal period. We use the adeno-associated virus (AAV) as a vector for gene transfer in the newborn rabbit lung where stem cells are activated for lung growth and differentiation. AAV-mediated gene transfer as assayed by lacZ gene expression occurred preferentially in alveoli in the alveolar epithelial progenitor cell, the type II cell, and in the large airway tracheobronchial basal and ciliated cells. Cell proliferation was confirmed by 5-bromo-deoxyuridine (BRDU) labeling in regions undergoing alveolarization and airway branch points. Regions of cell proliferation coincided with areas of significant lacZ expression. Thus, dividing and differentiating cells can be targeted by AAVlacZ delivery to newborn lung.
Animals↗
[Selective transfection of animal cells by non viral vectors: polylysine carrying recognition signals].
A process for the selective transfer of genes using glycosylated polylysine was established. Glycosylated polylysines and plasmid DNA form complexes which are taken up by cells expressing surface lectins recognizing the sugar moieties of glycosylated polylysines. Modifications made to this process allow for efficient and specific transfer in in vitro animal cell models.
Animals↗
Non-replicating viral vector-based AIDS vaccines: interplay between viral vectors and the immune system.
During the past 20 years, the development of HIV vaccines has come a long way. The focus has progressively changed from the traditional protein-based HIV vaccines that induce humoral immunity to the live recombinant viral vector-based HIV vaccines capable of eliciting both cellular and humoral immune responses. These new viral vector-based vaccines encoding multiple HIV antigens, delivered either alone or in heterologous prime-boost modalities elicited antigen-specific CTL responses in immunized hosts and protected animals from disease. The viral vector-based vaccines have proven to be potent vaccines in pre-clinical studies and foster the hope to put an end to the ever-increasing threat of the AIDS epidemic. Several unique features of viral vector-based HIV vaccines have contributed to their success, including their intrinsic immune-modulating properties, high transduction efficiency, and in vivo production of immunogens within the cell mimicking a natural infection without the associated health risks. In this review, we will discuss the characteristics of non-replicating viral vectors most commonly used for HIV vaccines with a particular focus on immune responses elicited by the vector particles alone and their effect on the potency of viral vector-based HIV vaccines.
AIDS Vaccines↗
Cationic lipid vectors for plasmid DNA delivery.
Successful gene therapy depends on efficient gene transfer vectors. Viral vectors and non-viral vectors have been investigated extensively. Cationic lipids are non-viral vectors, which resemble traditional pharmaceuticals, display little immunogenicity, and have no potential for viral infection. However, toxicity and low transfection efficiency are two barriers limiting the clinical applications of cationic lipids. Over the last decade, hundreds of cationic lipids have been synthesized to address these problems. In this brief review, we summarized recent research results concerning the structures of DNA/liposomes complexes, some important strategies used to design different classes of cationic lipids, and use of disulfide cationic lipids in plasmid DNA delivery.
Animals↗
Efficient expression of foreign proteins in roots from tobravirus vectors.
Viral vectors were constructed from infectious cDNA clones of each of the three tobraviruses, tobacco rattle virus (TRV), pea early-browning virus (PEBV), and pepper ringspot virus (PepRSV). RNA2 of each of the three viruses was modified to carry an additional coat protein subgenomic promoter and was used to express green fluorescent protein (GFP) when inoculated to plants. The tobravirus expression vectors have a wide host range and were able to express GFP in, for example, Nicotiana species, tomato, pea, arabidopsis, and sugar beet. The TRV vector was able to invade and express GFP very efficiently in roots, whereas the widely used PVX vector was not.
Biotechnology↗
DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors.
Viral vectors based on adeno-associated virus (AAV) preferentially transduce cells in S phase of the cell cycle. We recently found that DNA-damaging agents increased the transduction of nondividing cells. However, the optimal concentrations were toxic to cells. Here we show that the transduction of normal human fibroblasts by AAV vectors is increased by prior exposure to DNA synthesis inhibitors, such as aphidicolin or hydroxyurea, and topoisomerase inhibitors, such as etoposide or camptothecin. Transduction efficiencies could be increased > 300-fold in stationary cultures at concentrations that did not affect cell viability or proliferative potential. Both S-phase and non-S-phase cells were affected, suggesting that cellular functions other than replicative DNA synthesis may be involved. Applying these methods to gene transfer protocols should improve prospects for gene therapy by AAV vectors.
Aphidicolin↗
Efficient gene transfer into human hepatocytes by baculovirus vectors.
Viral vectors are the most efficient tools for gene delivery, and the search for tissue-specific infecting viruses is important for the development of in vivo gene therapy strategies. The baculovirus Autographa californica nuclear polyhedrosis virus is widely used as a vector for expression of foreign genes in insect cells, and its host specificity is supposed to be restricted to arthropods. Here we demonstrate that recombinant A. californica nuclear polyhedrosis virus is efficiently taken up by human hepatocytes via an endosomal pathway. High-level reporter gene expression from heterologous promoters was observed in human and rabbit hepatocytes in vitro. Mouse hepatocytes and some other epithelial cell types are targeted at a considerably lower rate. The efficiency of gene transfer by baculovirus considerably exceeds that obtained by calcium phosphate or lipid transfection. These properties of baculovirus suggest a use for it as a vector for liver-directed gene transfer but highlight a potential risk in handling certain recombinant baculoviruses.
Animals↗
Neuroprotection with herpes simplex vectors expressing virally derived anti-apoptotic agents.
A large body of literature dealing with neurotoxicity has focused on trying to define the exact nature of cell death following a neurological insult. While there is some debate in the field, it has been shown that a number of neurons in a given population can respond to an acute insult stimulus by activating the apoptotic cascade. To what extent, however, these insults result in the classical manifestations of either apoptosis or necrosis, or whether a mixture of the two results, is highly controversial, in part dependent on the particular system utilized. In this paper, we investigate the role of particular apoptotic signals in cultured rat hippocampal neurons, following acute excitotoxicity, metabolic poisoning, and heat stress. In particular, we examine these effects by utilizing a modified herpes simplex viral vector to specifically deliver viral anti-apoptotic genes. We have selected a battery of viral genes (crmA, p35, gamma34.5, KsBcl-2) that have evolved to suppress suicidal host responses to infection. We examine these inhibitors in the face of the above classes of insults and report that each viral agent tested has a unique profile in its ability to protect hippocampal neurons following acute neurological insults. Specifically, the effects of domoic acid excitotoxicity can be alleviated only with crmA, p35 and gamma34.5 whereas all genes tested can protect against heat stress. Conversely, no genes tested can protect against metabolic poisoning by cyanide. Such a study helps us to further understand the nature of apoptotic signals in different insults.
Animals↗
Gene transfer using nonviral delivery systems.
In peritoneal dialysis, loss of peritoneal function is a major factor in treatment failure. The alterations in peritoneal function are related to structural changes in the peritoneal membrane, including peritoneal sclerosis with increased extracellular matrix. Although peritoneal sclerosis is considered reversible to some extent through peritoneal rest, which improves peritoneal function and facilitates morphological changes, there has been no therapeutic intervention and no drug against the development and progression of peritoneal sclerosis. Using recent biotechnological advances in genetic engineering, a strategy based on genetic modification of the peritoneal membrane could be a potential therapeutic maneuver against peritoneal sclerosis and peritoneal membrane failure. Before this gene therapy may be applied clinically, a safe and effective gene delivery system as well as the selection of a gene therapy method must be established. There are presently two kinds of gene transfer vectors: viral and nonviral. Viral vectors are used mainly as a gene delivery system in the field of continuous ambulatory peritoneal dialysis research; however, they have several problems such as immunogenicity and toxicity. On the other hand, nonviral vectors have several advantages over viral vectors. We review here gene transfer using nonviral vector systems in the peritoneum: electroporation, liposomes, and cationized gelatin microspheres. In the field of peritoneal dialysis, gene therapy research using nonviral vectors is presently limited. Improvement in delivery methods together with an intelligent design of targeted genes has brought about large degrees of enhancement in the efficiency, specificity, and temporal control of nonviral vectors.
Gene Transfer Techniques↗
Challenges and strategies: the immune responses in gene therapy.
The host immune responses, including T lymphocytes mediated immune response and humoral immune responses are the important parts of the challenges in gene therapy. There are some potential immunostimulants in gene delivery systems, such as viral and non-viral vectors. Viral gene products, transgene products, viral proteins derived from viral particles required by dead-end infection, and CpG DNA in plasmid may play important roles in inducing the host immune responses when foreign genes are transferred into the targeted tissues. The immune responses should lead to many problems in gene therapy: transient expression of therapeutic gene, non-efficient re-administration of the same vectors, and severe side-effects in clinical trials. Although RNAi may act as gene therapeutic agent for suppression of specific gene expression, little attention has been given to the potential non-specific effects that might be induced. It was reported that small interfering RNAs (siRNAs) can induce the host interferon response following transfected to mammalian cells. Facing these challenges, a number of studies have been focused on taking measures to solve them, such as immunosuppression, selection of different administration routes and dose of the vectors, using the tissue-specific promoters and modifying the vectors.
Adjuvants, Immunologic↗
Immunotherapeutic potential of DISC-HSV and OX40L in cancer.
Several vectors, viral and bacterial, have been developed over the past few years for means of generating an effective antitumor immune response. We have developed and studied a "model for immunotherapy" using a viral vector disabled infectious single cycle-herpes simplex virus (DISC-HSV), which efficiently transduces various tumor cell lines and offers a useful vehicle for the further development of cell-based vaccines. The immunotherapeutic potential of DISC-HSV encoding granulocyte macrophage colony stimulating factor (GM-CSF) was demonstrated in a number of murine carcinoma models, leading to complete regression of well-established tumors in up to 70% of the mice. Moreover, the therapeutic potential of DISC-HSV-GM-CSF was significantly enhanced when used in combination therapy with either OX40L or dendritic cells (DC), even in a poorly immunogenic tumor model. The ability of this vector to accept large gene inserts, its good safety profile, its ability to undergo only a single round of infection, the inherent viral immunostimulatory properties and its ability to infect various tumor cell lines efficiently, make DISC-HSV an ideal candidate vector for immunotherapy. The DISC- CT-26 tumor model was used to investigate the mechanisms associated with immunotherapy induced tumor rejection. Although CTL induction, was positively correlated with regression, MHC class I down regulation and accumulation of immature Gr1+ myeloid cells were shown to be the main immuno-suppressor mechanisms operating against regression and associated with progressive tumor growth. The CTL response was associated with the immuno-dominant AH-1 peptide of the retroviral glycoprotein gp70. This model of immunotherapy has provided an opportunity to dissect further the immunological events associated with tumor-rejection and escape. Since other antigens may be important in initiating tumor rejection, we have investigated the expression of MTA-1, an antigen that appears to be expressed widely in human and murine tumors. The immunogenicity of MTA-1 was studied and its potential as a tumor rejection antigen is under investigation.
Animals↗
[Quality control of gene therapy products: approach of the French Agency for the Safety of Health Products].
Gene therapy is a new therapeutic strategy which can constitute in some diseases a true alternative or a complement to the "classical treatments". Regarding the innovative features, the complexity and the extreme diversity of the gene therapy products (naked DNA, synthetic vectors, viral vectors, genetically modified cells), these new products presently in clinical trials have to be precisely evaluated and controlled for their medicine quality as well as their biological origin and/or their specific characteristics of genetically modified organisms. The French Agency for the Safety of Health Products engaged an in-depth scientific review concerning the control of this very heterogeneous class of potential therapeutics through the creation of a working group. The objectives of this group were to determine the testings to be performed by a national authority for each type of gene therapy products and to select the appropriate techniques or methods to be developed. Controls considered as essential are listed and include the verification of the identity, the purity, the transfer and expression efficiency as well as the microbiological and viral safety of the products. This implies the development of diverse techniques of molecular biology, cellular biology, physico-chemistry, animal testing, histology and microbiology. Finally, in order to define the basis of testings of these emerging products, the marketing of which should be effective for some of them in the next years, it appears extremely important to harmonize the quality, efficiency and safety criteria, to develop specific references and standards and to create specific guidelines for the control of gene therapy products.
France↗
Stimuli-responsive polymers in gene delivery.
Recent interest in clinical therapy has been directed to deliver nucleic acids (DNA, RNA or short-chain oligonucleotides) that alter gene expression within a specific cell population, thereby manipulating cellular processes and responses, which in turn stimulate immune responses or tissue regeneration, or blocks expression at the level of transcription or translation for treatment of several diseases. Both ex vivo and in vivo gene delivery can be achieved mostly by using a delivery system (vector). Viral vectors exhibit high gene expression, but also have very significant side effects. Mainly cationic polymeric systems are used as nonviral vectors, although usually with low levels of transfection. Through the use of stimuli-responsive polymers as novel vectors for gene delivery, two benefits can be obtained: high gene expression efficiency and more selective gene expression.
Gene Expression↗