Biosafety of adenoviral vectors

Adenoviral vectors can efficiently transduce a broad variety of different cell types and have been used extensively in preclinical and clinical studies. However, early generation of adenoviral vectors retained residual adenoviral genes that contribute to inflammatory immune responses and toxicity. In addition, these vectors often result in transient expression of the potentially therapeutic transgene. Some clinical trials based on early generation adenoviral vectors have been discontinued because of acute inflammatory responses and toxicity and even one patient has died as a direct consequence of adenoviral toxicity. The latest generation of high-capacity adenoviral vectors is devoid of viral genes, and is having a significantly improved safety profile and yielding more prolonged transgene expression compared to early generation vectors. Nevertheless, transgene expression gradually declines even when high-capacity adenoviral vectors are used, possibly due to the gradual loss of vector genomes. Despite their improved safety, high-capacity adenoviral vectors can still trigger transient toxic effects in animals and patients. Restricting the tropism of adenoviral vectors by immunologic or genetic re-targeting may further improve their therapeutic window. The safety of adenoviral vectors has been improved further through the development of safer packaging systems that eliminate the homologous overlap between vector and helper sequences and therefore prevent formation of replication-competent adenoviruses (RCA). RCA could exacerbate inflammatory responses and act as a helper to rescue adenoviral vectors, potentially increasing the effective vector dose. Conditionally replicating adenoviruses (CRAds) have been developed for cancer gene therapy, which replicate selectively in some cancer cells. The use of CRAds in combination with chemotherapy yielded therapeutic effects in patients suffering from cancer but dose-limiting toxicity was apparent. Although there appears to be a very low theoretical risk of malignancy that is predominantly associated with the occurrence of E1-positive recombinants, no malignancies have been reported that were associated with adenoviral vectors. Nevertheless, integrating adenoviral vectors carry a greater malignancy risk due to their ability to integrate randomly into the target genomes.     

Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle

Skeletal muscle is an attractive target for somatic gene transfer of both acquired and inherited disorders. Direct injection of adenoviral vectors in the skeletal muscle leads to recombinant gene expression in a large number of muscle fibers. Transgene expression has been transient in most organs and associated with substantial inflammation when experiments are performed in adult immune competent mice. In this report, we utilize a variety of in vivo and in vitro models of T and B cell function to characterize the nature of the immune response to adenoviral vectors injected into murine skeletal muscle. Cellular immunity dependent on CD4+ and CD8+ T cells contributes to the loss of recombinant gene expression and the development of localized inflammation. Antigen specific activation of T cells occurs to both viral proteins and the reporter gene beta-galactosidase. Systemic levels of neutralizing antibody to the capsid proteins of the vector are also generated. Destructive immune responses responsible for loss of transgene expression are largely directed against beta-galactosidase in that transgene expression was stable when beta-galactosidase was eliminated as a neoantigen in mice transgenic for lacZ. A strategy to prevent the cellular and humoral immunity to this therapy was developed based on transiently ablating CD4+ T cell activation at the time of vector delivery. Encouraging results were obtained when vector was administered with one of several immune modulating agents including cyclophosphamide, mAb to CD4+ cells, and mAb to CD40 ligand. These studies indicate that cellular and humoral immune responses are elicited in the context of gene therapy directed to skeletal muscle with adenoviral vectors. Transient ablation of CD4+ T cell activation prevents the effects responses of the CD8+ T and B cells.      

Immune responses to adenovirus and adeno-associated vectors used for gene therapy of brain diseases: the role of immunological synapses in understanding the cell biology of neuroimmune interactions

Researchers have conducted numerous pre-clinical and clinical gene transfer studies using recombinant viral vectors derived from a wide range of pathogenic viruses such as adenovirus, adeno-associated virus, and lentivirus. As viral vectors are derived from pathogenic viruses, they have an inherent ability to induce a vector specific immune response when used in vivo. The role of the immune response against the viral vector has been implicated in the inconsistent and unpredictable translation of pre-clinical success into therapeutic efficacy in human clinical trials using gene therapy to treat neurological disorders. Herein we thoroughly examine the effects of the innate and adaptive immune responses on therapeutic gene expression mediated by adenoviral, AAV, and lentiviral vectors systems in both pre-clinical and clinical experiments. Furthermore, the immune responses against gene therapy vectors and the resulting loss of therapeutic gene expression are examined in the context of the architecture and neuroanatomy of the brain immune system. The chapter closes with a discussion of the relationship between the elimination of transgene expression and the in vivo immunological synapses between immune cells and target virally infected brain cells. Importantly, although systemic immune responses against viral vectors injected systemically has thought to be deleterious in a number of trials, results from brain gene therapy clinical trials do not support this general conclusion suggesting brain gene therapy may be safer from an immunological standpoint.     

Tissue-specific targeting for cardiovascular gene transfer. Potential vectors and future challenges

The introduction of genes to cardiovascular cells in vivo remains the major challenge for current gene therapy modalities. However, recent developments in retargeting adenoviral vectors are promising to improve transduction efficiency in the cardiovascular cells. After systemic application, most adenoviral vectors are trapped by the liver, hampering delivery to target cardiovascular tissues. Furthermore, a majority of vectors for vascular gene transfer utilizes strong heterologous viral promoters, such as CMV. A potential side effect related to the use of such vectors is the systemic organ toxicity resulting from unrestricted transgene expression. These vectors have the additional problem of being frequently shut-down in vivo. Therefore, both retargeting adenoviral vectors and the use of tissue-specific promoter-driven vectors offer an enhanced safety profile by reducing ectopic expression in vital organs including the liver and lung. However, the limiting factor for the use of tissue-specific promoters is the low-level of expression compared with their viral counterparts. Both the development of efficient and strong vectors using cell-specific regulatory elements and the production of therapeutic proteins at sufficient levels is urgently needed to inhibit vasculoproliferative disorders. This review will focus on some of the recent achievements in vector development relevant to the delivery of vascular gene therapies targeted to the vascular endothelium, smooth muscle cells and macrophages during arterial remodelling.     

Immune response to helper dependent adenoviral mediated liver gene therapy: challenges and prospects

Adenovirus-mediated gene therapy holds significant potential especially for applications requiring high levels of target tissue transduction. While significant advances in clinical adenoviral gene therapy applications have been made in cancer, the clinical translation of adenoviral gene replacement therapy for genetic disease has lagged. Encouragingly, advances in vector production have led to the development of Helper-Dependent ("gutted" or "high capacity") adenoviral vectors (HDV) deleted of all viral coding genes. HDV significantly reduces the chronic toxicity associated with early generation adenoviral vectors that has been most significant after systemic administration in both small and large animal models. However, the field remains confounded by innate immune responses inherent to adenovirus, and more generally, to the adaptive immune response to transgene. Together they decrease the effective therapeutic index for any particular treatment. This review summarizes the current advances toward understanding the decisive cell and molecular mechanisms underlying the acute toxicity to systemic HDV administration. We focus on the complex immune response and consequences of systemic vector delivery in the context of liver-directed monogenic disease therapy. Future development of interventions to avoid the innate immune response, including vector and pharmacologic manipulations, should further contribute to minimizing vector toxicity while maximizing the efficacy of systemic HDV gene transfer.     

Advances in helper-dependent adenoviral vector research

The immunogenicity and cytotoxicity associated with early generations of adenoviral vectors provided a strong incentive for the development of helper-dependent adenovirus, a last generation of adenoviral vectors that is devoid of all viral coding sequences. These vectors have shown to mediate longer high-level transgene expression in vivo with reduced toxicity and thus offer enormous potential for human gene therapy. In addition, they possess a considerably larger cloning capacity than conventional adenoviral vectors making the transfer of large cDNAs, multiple transgenes and longer tissue-specific or regulable promoters possible. In this article, we review the progress made with helper-dependent adenoviral vectors. The development and optimization of scalable production processes and strategies for helper removal will be presented. Current chromatography options available for vector purification and the new challenges facing researchers for the separation of empty particles and/or helper viruses will be discussed. Finally, we will describe recent advances made in our understanding of their interaction with the immune system and their potential as gene delivery vehicles in vivo for the treatment of diseases affecting liver, skeletal muscle and brain.     

Immune responses to lentiviral vectors

Efficient delivery and sustained expression of a therapeutic gene into human tissues are the requisite to accomplish the high expectations of gene therapy. A major challenge has concerned development of gene transfer systems capable of efficient cell transduction and transgene expression without harm to the recipient. A lot of work has been done to demonstrate the efficacy of gene therapy in animal models that mimic situations in humans. Use of lentiviral vectors (LVs) offers multiple advantages for gene replacement therapy, because they combine efficient delivery, ability to transduce proliferating and resting cells, capacity to integrate into the host chromatin to provide stable long-term expression of the transgene, absence of any viral genes in the vector and absence of interference from preexisting viral immunity. However, one of the major barriers to stable gene transfer by LVs and other viral vectors is the development of innate and adaptive immune responses to the delivery vector and the transferred therapeutic transgene. Since this greatly hinders the therapeutical benefits of gene therapy by LVs, developing strategies to overcome the host immune response to the transfer vector and the transgene is a matter of current investigation.     

Adenoviral vector-mediated gene transfer for human gene therapy

Human gene therapy promises to change the practice of medicine by treating the causes of disease rather than the symptoms. Since the first clinical trial made its debut ten years ago, there are over 400 approved protocols in the United States alone, most of which have failed to show convincing data of clinical efficacy. This setback is largely due to the lack of efficient and adequate gene transfer vehicles. With the recent progress in elucidating the molecular mechanisms of human diseases and the imminent arrival of the post genomic era, there are increasing numbers of therapeutic genes or targets that are available for gene therapy. Therefore, the urgency and need for efficacious gene therapies are greater than ever. Clearly, the current fundamental obstacle is to develop delivery vectors that exhibit high efficacy and specificity of gene transfer. Recombinant adenoviruses have provided a versatile system for gene expression studies and therapeutic applications. Of late, there has been a remarkable increase in adenoviral vector-based clinical trials. Recent endeavors in the development of recombinant adenoviral vectors have focused on modification of virus tropism, accommodation of larger genes, increase in stability and control of transgene expression, and down-modulation of host immune responses. These modifications and continued improvements in adenoviral vectors will provide a great opportunity for human gene therapy to live up to its enormous potential in the second decade.     

Adenoviral vectors for gene replacement therapy

Adenovirus-based vectors are promising vehicles for gene replacement therapy due to their ability to efficiently transduce a wide variety of proliferating and non-proliferating cells. Over the past decade, different versions of adenoviral vectors (Ads) have been developed. These vectors can be classified into two major categories, based on whether the viral coding sequences are partially (first or second-generation Ads) or completely deleted (helper-dependent or gutted Ads). Both types of Ads have been tested in a variety of gene delivery studies, and major obstacles to their clinical application have been identified. Currently, innate and adaptive host immune responses to Ads remain major challenges, limiting both the initial viral dose and the effectiveness of subsequent administrations. Recent developments in vector design and delivery methods have improved the potential of Ads for successful gene therapy application.     

Emerging adenoviral vectors for stable correction of genetic disorders

Recent drawbacks in treating patients with severe combined immunodeficiency disorders with retroviral vectors underline the importance of generating novel tools for stable transduction of mammalian cells. Substantial progress has been made over the recent years which may offer important steps towards stable and more importantly safer correction of genetic diseases. This article discusses recent advances for stable transduction of target cells based on adenoviral gene transfer. There is accumulating evidence that recombinant adenoviral vectors (AdVs) based on various human serotypes with a broad cellular tropism and adenoviruses (Ads) from different species will play an important role in future gene therapy applications. In combination with recombinant AdVs for somatic integration these gene transfer vectors offer high transduction efficiencies with potentially safer integration patterns. Other approaches for persistent transgene expression include excision of stable episomes from the adenoviral vector genome, but also long-term persistence of the complete adenoviral vector genome as an episomal DNA molecule was demonstrated and exemplified by the treatment of various genetic diseases in small and large animal models. This review displays advantages but also limitations of these Ad based vector systems. This is the perfect time to pursue such approaches because alternative strategies for stable transduction of mammalian cells undergoing many cell divisions are urgently needed. Looking into the future, we believe that a combination of different components from different viral vectors in concert with non-viral vector systems will be successful in designing significantly optimized transfer vehicles for a broad range of different genetic diseases.     

Genetically modified dendritic cells for cancer immunotherapy

The ability to grow and differentiate dendritic cells (DC) ex vivo has allowed their genetic manipulation to enhance immune activation against tumor antigens. Gene engineering of DC can be achieved with a variety of physical methods and using different viral vectors. RNA or DNA transfection, either alone (naked), coated with liposomes or using electroporation or gene guns leads to T cell activation while transgene expression is frequently undetectable. Adenoviral and retroviral vectors have proven to be highly efficient in DC genetic modification, and have been widely used in preclinical models. Other vectors like lentivirus, poxvirus, herpes virus and adeno-associated virus (AAV) can also lead to foreign transgene expression in DC leading to immune cell activation. DC have been genetically engineered to provide constitutive and high level of tumor antigen expression or to introduce genes that further enhance their immune stimulatory ability. The promising results from preclinical animal models and from in vitro human immune cell culture systems have provided a strong rationale to initiate pilot clinical trials. Recently published or communicated clinical experiences and ongoing trials have used defined tumor antigen RNA transfection for prostate carcinoma and melanoma, liposome-encoated DNA transfection for breast or pancreatic cancer, adenoviral vector tumor antigen gene modification for melanoma and small cell lung cancer, and poxvirus-mediated expression of costimulatory molecules for colon carcinoma. These preliminary experiences suggest that genetically modified DC can safely induce T cell responses but few clinical responses.     

Gene therapy using an adenovirus vector for apoptosis-related genes is a highly effective therapeutic modality for killing glioma cells

Preclinical studies in animal models and human clinical trials have evaluated the safety and efficacy of adenoviral vectors for cancer gene therapy. These studies have indicated that gene delivery via adenoviral vectors, including p53 gene therapy, represents a promising therapeutic modality for many types of human cancers. This review focuses on novel strategies to induce apoptosis in glioma cells by transduction with adenoviral vectors carrying a variety of apoptosis-related genes, including Fas ligand, Fas, FADD, caspase-8, p53, p33ING1, p73alpha, Bax, Apaf-1, caspase-9, IkappaBdN, caspase-3, Bcl-2, and Bcl-X(L). We conclude that adenoviral vector-mediated delivery of apoptosis-related genes other than p53 is a potentially useful gene therapy approach toward the treatment of human brain tumors.     

Adenovirus as an integrating vector

Recombinant adenoviral vectors have served as one of the most efficient gene delivery vehicles in vivo thus far. Multiply attenuated or completely gutless adenoviral vectors have been developed to achieve long-term gene expression in animal models by overcoming cellular immunity against de novo synthesized adenoviral proteins. However, since adenovirus lacks native integration machinery, the goal of gene therapy obtaining permanent expression cannot be realized with current adenoviral vector systems. Recent studies have shown that replication-incompetent adenoviral vectors randomly integrate into host chromosomes at frequencies of 0.001-1% of infected cells. To improve the integration frequencies of adenoviral vectors, a variety of hybrid vectors combining the highly efficient DNA delivery of adenovirus with the integrating machinery of retroviruses, adeno-associated viruses, and transposons, have been emerging. These hybrid vectors have shown promise, at least in in vitro systems. Furthermore, adenoviral vectors have shown potential as gene targeting vectors. These developments should eventually lead to more effective gene therapy vectors that can transduce a myriad of cell types stably in vivo.     

Multiple applications for replication-defective herpes simplex virus vectors

Herpes simplex virus (HSV) is a neurotropic DNA virus. The viral genome is large (152 kb), and many genes are dispensable for viral function, allowing insertion of multiple or large transgene expression cassettes. The virus life cycle includes a latent phase, during which the viral genome remains as a stable episomal element within neuronal nuclei for the lifetime of the host, without disturbing normal function. We have exploited these features of HSV to construct a series of nonpathogenic gene therapy vectors that efficiently deliver therapeutic and experimental transgenes to neural and non-neural tissue. Importantly, transgene expression may be sustained long term; reporter gene expression has been demonstrated for over a year in the nervous system. This article discusses the generation of replication-defective HSV vectors and reviews recent studies investigating their use in several animal models of human disease. We have demonstrated correction or prevention of a number of important neurological phenotypes, including neurodegeneration, chronic pain, peripheral neuropathy, and malignancy. In addition, HSV-mediated transduction of non-neurological tissues allows their use as depot sites for synthesis of circulating and locally acting secreted proteins. New applications for this vector system include the genetic modification of stem cell populations; this may become an important means to direct cellular differentiation or deliver therapeutic genes systemically. Replication-defective HSV vectors are an effective and flexible vehicle for the delivery of transgenes to numerous tissues, with multiple applications.     

Modifying adenoviral vectors for use as gene-based cancer vaccines

The past decade has produced significant advances in our understanding of antigen-presenting cells, tumor antigens, and other components of the immune response to cancer. Gene-based vaccination is emerging as one of the more promising approaches for loading dendritic cells (DC) with tumor-associated antigens. In this respect, it is proposed that adenoviral (AdV) vectors can deliver high antigen concentrations, promote effective processing and MHC expression, and stimulate potent cell-mediated immunity. While AdV vectors have performed well in pre-clinical vaccine models, their application to patient care has limitations. The in vivo administration of AdV vectors is associated with both innate and adaptive host responses that result in tissue inflammation and injury, viral neutralization, and premature clearance of AdV-transduced cells. A variety of strategies have been developed to address these limitations. The ideal vaccine would avoid vector-related immune responses, have relative specificity for transducing DC, and induce high levels of transgene expression. This review describes the range of host responses to AdV vaccines, identifies strategies to reduce viral recognition and enhance transgene antigen expression, and suggests future approaches to vector development and administration. There is every reason to believe that safer and more effective forms of AdV-based vaccines can be developed and applied to patient therapy.     

Genomic context vectors and artificial chromosomes for cystic fibrosis gene therapy

Cystic fibrosis (CF) is caused by mutations of the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP dependent chloride channel whose expression is finely tuned in space and time. Gene therapy approaches to CF lung disease have demonstrated partial efficacy and short-lived CFTR expression in the airways. Drawbacks in the use of classical gene transfer vectors include immune response to viral proteins or to unmethylated CpG motifs contained in bacterially-derived vector DNA, and shut-off of viral promoters. These limitations could be overcome by providing stable maintenance and expression of the CFTR gene inside the defective cells. This strategy makes use of large fragments of DNA of various sizes containing the CFTR transgene and its relevant regulatory regions, (genomic context vectors [GCVs], reaching ultimate complexity in the form of an artificial chromosome [AC]) as vector for the transgene. Appropriate regulation in space and time would be achieved by the presence of the endogenous promoter and other control elements, while retention in daughter cells could be ensured by the presence of sequences which guarantee episomal replication. In this review, we describe recent advances in GCVs and ACs and the technology underlying their construction. These vectors have been shown to be suitable for delivery and expression of therapeutically relevant genes, including CFTR. The major issue which now limits their routine use is delivery inefficiency. Once this issue is resolved, we will be closer to achieving the goal of regulated gene therapy for CF.