Current Gene Therapy - Volume 7, Issue 5, 2007

While based on a simple concept of introducing a functional gene to replace a non-functional sequence of a person's genome or to otherwise add a therapeutically beneficial gene, gene therapy is faced with a complex set of interactions between the gene transfer vector, the transgene, and the recipient of gene transfer. Following tremendous advances in vector development, cures have been reported in numerous animal models of human diseases, and gene therapy now holds much promise as a novel form of molecular medicine. However, complications of treatment related to insertional mutagenesis following integration of vector DNA into host chromosomes and to untoward immune responses against vectors and transgene products have merged as serious obstacles for successful translation to humans. This issue of Current Gene Therapy is dedicated to an in-depth assessment of immune responses in gene transfer. The importance of the topic is further highlighted by the recent meeting of the NIH's Recombinant DNA Advisory Committee (RAC), which discussed vector-specific T cell responses in a gene therapy trial for hemophilia on 19 June 2007, and by reports on complications of gene therapy for severe inflammatory disease. The immune system has evolved to differentiate between self and foreign molecular structures and sequences and to respond to activation signals that indicate danger to the body. Consequently, the immune system may fight off “invading” gene transfer vectors or foreign protein and nucleic acid sequences. Innate immunity provides a rapid defense system that responds to exogenous signals (e.g. pathogen-associated molecular patterns such as bacterial or viral nucleic acids) and to endogenous signals that, for example, may be derived from tissue damage during vector administration, cellular stress, or viral infection. The adaptive immune response is delayed but more specific (involving presentation of antigen to highly specific T and B cell receptors) and also generated memory B and T cells. Cytotoxic T cell and antibody responses may block gene transfer or eliminate therapeutic gene expression. The articles in this issue reflect that the potential for any of these immune responses in gene transfer depends on many factors, including the gene transfer vector (type of vector, specifics of the construct such as promoters, envelope/capsid, purity, etc.), vector dose, route of administration (ex vivo, in vivo, target organ, method of delivery), age and genetic factors of the recipient of gene therapy, and the nature of the transgene product (self, non-self, cellular localization, etc.). Nonetheless, research is well under way to overcome these hurdles. We have just begun to understand the interactions between vectors and the immune system of specific target tissues (organ-specific immunity). For example, recent research demonstrates the importance of interactions with bone marrow-derived professional antigen presenting cells. Similar to research in transplantation biology, gene therapists have uncovered strategies to manipulate the immune system, leading to sustained transgene expression in animal models. At the same time, we can take advantage of mechanisms of immune tolerance and regulation mechanisms, which have evolved to prevent autoimmunity and destructive immune responses.

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.

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.

Recent findings in a clinical trial in which an adeno-associated virus (AAV) vector expressing coagulation factor IX (F.IX) was introduced into the liver of hemophilia B subjects highlighted a new issue previously not identified in animal studies. Upon AAV gene transfer to liver, two subjects enrolled in this trial developed transient elevation of liver enzymes, likely as a consequence of immune rejection of transduced hepatocytes mediated by AAV capsid-specific CD8+ T cells. Studies in healthy donors showed that humans carry a population of antigen-specific memory CD8+ T cells probably arising from wild-type AAV infections. The hypothesis formulated here is that these cells expanded upon re-exposure to capsid, i.e. upon AAV-2 hepatic gene transfer, and cleared AAV epitope-bearing transduced hepatocytes. Other hypotheses have been formulated which include specific receptor-binding properties of AAV-2 capsid, presence of capsidexpressing DNA in AAV vector preparations, and expression of alternative reading frames from the transgene. Absence of a valid animal model has prevented an in-depth mechanistic study of the phenomenon. Several possible solutions to the problem are discussed, including the administration of a short-term anti-T cell immunosuppression regimen concomitant with gene transfer. While more studies will be necessary to further define mechanisms and risks associated with capsidspecific immune responses in humans, monitoring of these responses in clinical trials will be essential to achieving the goal of long-term therapeutic gene transfer in humans.

The first in vivo adeno-associated viral vector (AAV) gene transfer experiments were performed in murine models of muscle directed gene transfer. These studies were remarkable for stable expression of a variety of immunogenic transgenes. These findings were translated to other target organs with multiple therapeutic gene products. Technological improvements and the lessons learned from basic research have heralded an era of first-in-human clinical trials. In most settings, AAV appears to evade host immune surveillance, allowing the delivery of robust levels of genetic cargo that leads to persistent expression. However, in few experimental settings immunological responses raised following AAV mediated gene transfer have compromised vector efficacy. Parameters that determine these occurrences have been proposed to be pre-existing immunity to AAV, the route of administration, the kinetics of expression, the dose, the vector serotype and its ability to transduce antigen-presenting cells (APCs) as well as the host species and nature of the specific transgene product. Overall, the underlying mechanisms remain the topic of scientific debate. This review aims to compile, confront and critically discuss the findings in which AAV appears to be an immunogen.

Efficient gene transfer has been achieved in several animal models using different vector systems, leading to stable transgene expression. The tight control of this expression is now an important outcome for the field of gene therapy. Such regulation is likely to be required for therapeutic applications and in some instances for safety reasons. For this purpose, several regulatable systems depending on small molecules have been developed. Among these, the tetracycline and the rapamycin dependent systems have been largely used. However, if long-term regulation of the transgene has been obtained in small animal models using these inducible systems, when translational studies were initiated in larger animals, the development of an immune response against proteins involved in transgene regulation were often observed. Such immune response was especially documented when using the TetOn tetracycline regulatable system in nonhuman primates (NHP). Humoral and destructive cellular immune responses against the transactivator involved in this regulation system were documented in a large majority of NHP leading to the complete loss of the transgene regulation and expression. This review will describe the immune responses observed in these different model systems applied for transgene regulation. Focus will be finally given on future directions in which such immune responses might be surmounted, enabling longterm transgene regulation in future clinical developments of gene transfer.

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.

Gene-modified T cells were the first gene therapy tool used in clinical gene transfer trials. After the first applications in immunodeficiency diseases, T cell gene therapy has been extended to HIV infection and cancer. The primary obstacle to successful T cell gene therapy has proven to be the robust immune responses elicited by the gene-modified T cells even in severely immunosuppressed patients. The potent antibody and cytotoxic immune responses have interfered with the expression and persistence of the therapeutic transgene. In this review we will address each of the components of T cell gene therapy - culture conditions, vector, and transgene - that have elicited these immune responses and the strategies used to minimize them.

Tolerance must be maintained to prevent deleterious immune responses. Thus, when tolerance is lost, autoimmunity can result. A number of novel approaches to (re-) induce tolerance for potential clinical applications have been developed in the last decade. Our lab has implemented an immunoglobulin-based gene therapy approach, which may have powerful implications for the treatment of human conditions. These include a variety of autoimmune diseases, transplantation, and the immune response to therapeutic proteins (as in the treatment of hemophilia A) or gene therapy per se. We clone the target (immunogenic) protein in frame with an immunoglobulin heavy chain and deliver it via retrovirus to an activated B cell. In our system, we observe tolerance to multiple epitopes of the protein cloned. An important advantage of this regimen is that identification of the precise peptide epitopes of a target protein is not necessary since selection and presentation by the host's own antigen presenting cells (APC's) eliminates the issue of HLA polymorphism. Additionally, our data indicate that these tolerogenic B cells are stimulating an endogenous population of regulatory T cells, which are effective at suppressing the immune response in both naïve and primed hosts. Thus, this approach has potential for future clinical therapy.

Induction and maintenance of immune tolerance to therapeutic transgene products are key requirements for successful gene replacement therapies. Gene transfer may also be used to specifically induce immune tolerance and thereby augment other types of therapies. Similarly, gene therapies for treatment of autoimmune diseases are being developed in order to restore tolerance to self-antigens. Regulatory T cells have emerged as key players in many aspects of immune tolerance, and a rapidly increasing body of work documents induction and/or activation of regulatory T cells by gene transfer. Regulatory T cells may suppress antibody formation and cytotoxic T cell responses and may be critical for immune tolerance to therapeutic proteins. In this regard, CD4+CD25+ regulatory T cells have been identified as important components of tolerance in several gene transfer protocols, including hepatic in vivo gene transfer. Augmentation of regulatory T cell responses should be a promising new tool to achieve tolerance and avoid immune-mediated rejection of gene therapy. During the past decade, it has become obvious that immune regulation is an important and integral component of tolerance to self-antigens and of many forms of induced tolerance. Gene therapy can only be successful if the immune system does not reject the therapeutic transgene product. Recent studies provide a rapidly growing body of evidence that regulatory T cells (Treg) are involved and often play a crucial role in tolerance to proteins expressed by means of gene transfer. This review seeks to provide an overview of these data and their implications for gene therapy.

Successful gene therapy protocols rely on the hypo-responsiveness of the immune system to transgene products generated from gene transfer vectors. In order to prevent cytotoxic lymphocyte or antibody formation induced by transgene expression, various strategies derived from recent advances in immune tolerance induction protocols have been tested in gene therapy model systems. Current immunosuppressive drugs were used to nonspecifically target T-cell activation, clonal expansion, and differentiation into effector cells. Central tolerance can be induced from intrathymic deletion of T cells with thymically expressed antigens or generation of hematopoietic mixed chimerism. Peripheral tolerance to transgenes may be achieved by several different pathways including deletion of activated/effector T cells by depleting antibodies, generation of T cell apoptosis or anergy by costimulation blockade, and active suppression by T regulatory cells. This review outlines the development of these strategies using various immune modulation regimens and protocols to induce long-term immune tolerance specific to the transgene product.

Gene therapy could result in the permanent correction or amelioration of the clinical manifestations of many genetic diseases. However, immune responses to the therapeutic protein pose a significant hurdle for successful gene therapy. Problematic immune responses can include the development of a cytotoxic T lymphocyte (CTL) response that results in the destruction of genetically-modified cells and/or the formation of antibodies directed against the therapeutic protein. One approach to avoid an immune response is to perform gene therapy in newborns, which takes advantage of the fact that the immune system is relatively immature at birth. This approach has been highly effective in mice, and has resulted in stable expression without antibody formation for proteins that are highly immunogenic after transfer to adults. High levels of expression after neonatal gene therapy were more effective at inducing tolerance than low levels of expression in mice, which suggests that high antigen levels are more efficient at inducing tolerance. A criticism of this approach is that the murine immune system is less mature at birth than the immune systems of larger animals. Indeed, neonatal gene therapy to cats with mucopolysaccharidosis I resulted in a CTL response that destroyed expressing cells. Nevertheless, the immune system was still relatively immature, as transient administration of a single immunosuppressive agent at the time of neonatal gene therapy resulted in stable expression. Neonatal administration can reduce, but not eliminate, immune responses after gene therapy.