Nonviral gene therapy approaches to hemophilia

The goal of hemophilia gene therapy is to obtain long-term therapeutic levels of factor VIII (FVIII) or factor IX (FIX) without stimulating an immune response against the transgene product or the vector. The success of gene therapy is largely dependent on the development of appropriate gene delivery vectors. Both viral vectors and nonviral vectors have been considered for the development of hemophilia gene therapy. In general, viral vectors are far more efficient than nonviral gene delivery approaches and resulted in long-term therapeutic levels of FVIII or FIX in preclinical animal models. However, there are several reasons why a nonviral treatment would still be desirable, particularly because some viral vectors are associated with inflammatory reactions, that render transgene expression transient, or with an increased risk of insertional oncogenesis when random integrating vectors are used. Nonviral vectors may obviate some of these concerns. Since nonviral vectors are typically assembled in cell-free systems from well-defined components, they have significant manufacturing advantages over viral vectors. The continued development of improved nonviral gene delivery approaches offers new perspectives for gene therapy of chronic diseases including hemophilia.     

Prevention of cytotoxic T lymphocyte responses to factor IX-expressing hepatocytes by gene transfer-induced regulatory T cells

Treatment of genetic disease such as the bleeding disorder hemophilia B [deficiency in blood coagulation factor IX (F.IX)] by gene replacement therapy is hampered by the risk of immune responses to the therapeutic gene product and to the gene transfer vector. Immune competent mice of two different strains were tolerized to human F.IX by hepatic gene transfer mediated by adenoassociated viral vector. These animals were subsequently challenged by systemic administration of an E1/E3-deleted adenoviral vector, which is known to induce a cytotoxic T lymphocyte response to the transgene product. Immune tolerance prevented cytotoxic T lymphocyte activation to F.IX and CD8(+) cellular infiltrates in the liver. Moreover, a sustained and substantial increase in hepatic F.IX expression from the adenoviral vector was achieved despite in vitro T cell responses to adenoviral antigens. Cytolytic responses to therapeutic and to viral vector-derived antigens had been prevented in vivo by activation of regulatory CD4(+) T cells, which mediated suppression of inflammatory lymphocyte responses to the liver. This result suggests that augmentation of regulatory T cell activation should provide new means to avoid destructive immune responses in gene transfer.     

Systemic protein delivery by muscle-gene transfer is limited by a local immune response

Adeno-associated viral (AAV) vectors have been successfully used for therapeutic expression of systemic transgene products (such as factor IX or erythropoietin) following in vivo administration to skeletal muscle of animal models of inherited hematologic disorders. However, an immune response may be initiated if the transgene product represents a neoantigen. Here, we use ovalbumin (OVA) as a model antigen and demonstrate immune-mediated elimination of expression on muscle-directed AAV-2 gene transfer. Administration to immune competent mice resulted in transient systemic OVA expression. Within 10 days, OVA-specific T-helper cells had been activated in draining lymph nodes, an inflammatory immune response ensued, and OVA-expressing muscle fibers were destroyed by a cytotoxic CD8(+) T-cell response. Use of a muscle-specific promoter did not prevent this immune response. Adoptively transferred CD4(+) cells transgenic for a T-cell receptor specific to OVA peptide-major histocompatibility complex class II showed antigen-specific, vector dose-dependent proliferation confined to the draining lymph nodes of AAV-OVA-transduced muscle within 5 days after gene transfer and subsequently participated in lymphocytic infiltration of transduced muscle. This study documents that a local immune response limits sustained expression of a secreted protein in muscle gene transfer, a finding that may have consequences for design of clinical protocols.     

Costimulatory protein B7-1 enhances the cytotoxic T cell response and antibody response to hepatitis B surface antigen.

There is a need for more effective therapy for chronic virus infections. A principle natural mechanism for elimination of virus-infected host cells is activation of viral antigen-specific cytotoxic T lymphocytes (CTL). In an effort to develop methods of inducing virus-specific CTL responses that might be utilized in therapy of virus infections, we have investigated the effect of B7, a costimulatory factor for T-cell activation. In this study we show that delivery of genes encoding human B7-1 and a viral antigen in the same recombinant viral vector to cells of mice induces a greater viral antigen-specific CTL response than does similar delivery of the viral antigen gene alone. Two recombinant adenovirus vectors were constructed with the foreign genes inserted in the early region 3. One of them (Ad1312) directed expression of the surface antigen gene of hepatitis B virus (HBS); the other (Ad1310) directed coexpression of HBS and human B7-1 (CD80) by means of an internal ribosomal entry site placed between the two coding sequences. When inoculated into BALB/c mice, both vectors induced a viral surface antigen-specific CTL response. The response induced by Ad1310 was stronger than that by Adl312 as measured by a chromium release assay for CTL activity and limiting dilution analysis for CTL precursor frequency, indicating that the B7-1 gene co-delivered with the HBS gene had an enhancing effect on the CTL response against surface antigen. Ad1310 also induced a higher titer of antibody against surface antigen than did Ad1312. This result suggests that expression of a costimulatory protein and a viral antigen in the same cells in vivo induces stronger immune responses than expression of the antigen alone. This could be a novel strategy for development of both preventive and therapeutic vaccines against infectious agents.     

Induction and role of regulatory CD4+CD25+ T cells in tolerance to the transgene product following hepatic in vivo gene transfer

Gene replacement therapy is complicated by the risk of an immune response against the therapeutic transgene product, which in part is determined by the route of vector administration. Our previous studies demonstrated induction of immune tolerance to coagulation factor IX (FIX) by hepatic adeno-associated viral (AAV) gene transfer. Using a regulatory T-cell (T(reg))-deficient model (Rag-2(-/-) mice transgenic for ovalbumin-specific T-cell receptor DO11.10), we provide first definitive evidence for induction of transgene product-specific CD4(+)CD25(+) T(regs) by in vivo gene transfer. Hepatic gene transfer-induced T(regs) express FoxP3, GITR, and CTLA4, and suppress CD4(+)CD25(-) T cells. T(regs) are detected as early as 2 weeks after gene transfer, and increase in frequency in thymus and secondary lymphoid organs during the following 2 months. Similarly, adoptive lymphocyte transfers from mice tolerized to human FIX by hepatic AAV gene transfer indicate induction of CD4(+)CD25(+)GITR(+) that suppresses antibody formation to FIX. Moreover, in vivo depletion of CD4(+)CD25(+) T(regs) leads to antibody formation to the FIX transgene product after hepatic gene transfer, which strongly suggests that these regulatory cells are required for tolerance induction. Our study reveals a crucial role of CD4(+)CD25(+) T(regs) in preventing immune responses to the transgene product in gene transfer.     

Distinct immune responses to transgene products from rAAV1 and rAAV8 vectors

Recently developed serotypes of recombinant adeno-associated virus (rAAV) vectors have significantly enhanced the use of rAAV vectors for gene therapy. However, host immune responses to the transgene products from different serotypes remain uncharacterized. In the present study, we evaluated the differential immune responses to the transgene products from rAAV1 and rAAV8 vectors. In non-obese diabetic (NOD) mice, which have a hypersensitive immunity, rAAV serotype 1 vector (rAAV1-hAAT) induced high levels of both humoral and cellular responses, while rAAV8-hAAT did not. In vitro studies showed that rAAV1, but not rAAV8 vector transduced dendritic cells (DCs) efficiently. In vivo studies indicated that vector transduction of DCs was essential for the immune responses; while the presence of a transgene product (or foreign gene product produced by host cells) was not immunogenic. Intriguingly, preimmunization with rAAV8-hAAT vector or with serum of hAAT transgenic NOD mouse induced immune tolerance to rAAV1-hAAT injection. These results demonstrate the immunogenic differences of rAAV1 and rAAV8 and imply tremendous potential for these vectors in different applications, where an immune response to transgene is to be either elicited or avoided.     

Cellular and genetic therapies for haemophilia

Summary.  Haemophilia continues to be a prime target for a variety of gene and cell-based therapies. Pre-clinical successes in both mouse and dog models of the disease have been documented with a variety of approaches over the past decade, and there have now been six small clinical trials of gene transfer in haemophilia. To date, the only significant adverse events documented in these trials have been related to host immune responses, indicating that immunologic barriers continue to represent the major obstacle to achieving success of gene transfer in humans. Despite these challenges, new strategies are being explored with novel serotypes of viral vectors and with the use of transient periods of immunosuppression to attenuate the immune response to the vector and transgene product following gene delivery. Two new clinical trials, both using AAV vectors, will likely start within the next year, and additional large animal pre-clinical studies using other viral vector-mediated approaches for gene transfer are expected in the near future.     

A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase.

Adenoviral vector-mediated gene transfer offers significant potential for gene therapy of many human diseases. However, progress has been slowed by several limitations. First, the insert capacity of currently available adenoviral vectors is limited to 8 kb of foreign DNA. Second, the expression of viral proteins in infected cells is believed to trigger a cellular immune response that results in inflammation and in only transient expression of the transferred gene. We report the development of a new adenoviral vector that has all viral coding sequences removed. Thus, large inserts are accommodated and expression of all viral proteins is eliminated. The first application of this vector system carries a dual expression cassette comprising 28.2 kb of nonviral DNA that includes the full-length murine dystrophin cDNA under control of a large muscle-specific promoter and a lacZ reporter construct. Using this vector, we demonstrate independent expression of both genes in primary mdx (dystrophin-deficient) muscle cells.     

Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver.

First-generation recombinant adenoviruses that lack E1 sequences have shown tremendous promise in animal and human models of gene therapy. Important limitations of these vectors are that recombinant gene expression is transient and inflammation occurs at the site of gene transfer. Our hypothesis for generating vectors with increased persistence is that present recombinant adenoviruses express viral proteins that stimulate cellular immune responses leading to destruction of the infected cells and repopulation of the organ with non-transgene-containing cells. This model predicts that further crippling of the virus will improve persistence and diminish pathology. We describe in this report second-generation recombinant adenoviruses harboring a beta-galactosidase-expressing transgene in which a temperature-sensitive mutation has been introduced into the E2A gene of an E1-deleted recombinant. At nonpermissive temperature, this virus fails to express late gene products, even when E1 is expressed in trans. The biology of this recombinant was studied in vivo in the context of mouse liver, a setting that is permissive for adenovirus type 5 replication. Animals that received the second-generation virus expressed the transgene for at least 70 days, whereas expression of the first-generation virus was no longer than 14 days. In addition, the inflammatory response, as measured by infiltration of CD8+ T cells, was blunted and delayed in livers infected with second-generation virus. These studies illustrate that modifications that disrupt structural protein expression in recombinant adenoviruses may be useful in enhancing their utility for gene therapy.     

Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.

An important limitation that has emerged in the use of adenoviruses for gene therapy has been loss of recombinant gene expression that occurs concurrent with the development of pathology in the organ expressing the transgene. We have used liver-directed approaches to gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-generation recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the recombinant viral genome express the transgene as desired; however, low-level expression of viral genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransgene-containing hepatocytes. These findings suggest approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes.     

Patterns of transgene expression and viral clearance from the transplanted liver following ex vivo adenovirus-mediated gene transfer

BACKGROUND/AIMS: In the rat liver transplant model, the liver graft can be transduced ex vivo by adenovirus encoding CTLA-4Ig (AdCTLA-4Ig) to achieve high level of immunosuppression in the liver after transplantation. To characterize the pattern of transgene expression following ex vivo gene transfer to the liver and examine whether immunosuppression would promote adenovirus persistence, we followed the life span of vector DNA and transgene expression in the transplanted liver. METHODS: Rat liver grafts were perfused ex vivo with adenovirus carrying the reporter gene beta-galactosidase (AdlacZ). The period of transgene expression was assessed at predetermined intervals after transplantation into syngeneic, allogeneic or nude (athymic) recipients. Clearance of vector DNA was assessed by PCR analysis of liver DNA after transplantation. RESULTS: Graft transduction with AdCTLA-4Ig or systemic cyclosporine treatment effectively abrogated the alloimmune response but did not result in sustained lacZ expression. The course of viral DNA clearance from the liver was also unaffected by immunosuppression as was the implied nucleolytic cleavage of viral DNA. CONCLUSIONS: In the transplant setting, local expression of CTLA-4Ig or systemic immunosuppression does not solve the problem of viral clearance from the liver. Non-adaptive immune mechanisms may have a significant role in the host response to adenovirus after liver transplantation.     

Induction of antigen-specific CD4+ T-cell anergy and deletion by in vivo viral gene transfer

Immune responses to the therapeutic gene product are a potentially serious complication in treatment of genetic disease by gene therapy. Induction and maintenance of immunologic hypo-responsiveness to the therapeutic antigen is therefore critical to the success of gene-based treatment of inherited protein deficiency. Here, we demonstrate induction of antigen-specific CD4+ T-cell tolerance to a secreted transgene product (ovalbumin, ova) in ova-specific T-cell receptor (TCR) transgenic mice by hepatic adeno-associated virus (AAV)-mediated gene transfer. Transduced mice maintained stable circulating ova levels without evidence of an immune response. Lymph node cells and splenocytes were hypo-responsive to ova as early as day 10 after gene transfer. Numbers of TCR+CD4+ cells were reduced in secondary lymphoid organs and in the thymus by 1 to 2 months after vector administration. The remaining TCR+CD4+ cell population was anergic to ova antigen in vitro and enriched for CD25+ cells. These data provide direct evidence that transgene expression following in vivo viral gene transfer can induce CD4+ T-cell tolerance to the transgene product, involving anergy and deletion mechanisms.     

Intrahepatic injection of adenovirus reduces inflammation and increases gene transfer and therapeutic effect in mice

Recombinant adenoviruses (Ad) are among the most extensively used vectors for liver gene transfer. One of the major limitations for the clinical application of these vectors is the inflammatory immune response associated with systemic administration of high dose of virus. We evaluated the effect of Ad administration route on the inflammatory immune response and liver transgene expression. We compared direct intrahepatic injection (IH) with the systemic administration via tail vein (IV). IH injection of Ad resulted in a lower inflammatory response and a higher transgene expression. When a relatively low dose of virus was used, IV administration resulted in no detectable protein expression but production of proinflammatory cytokines. In contrast, IH administration induced high levels of transgene expression and no inflammation, although we detected a transient hypertransaminemia, which fully resolved within days. Furthermore, IH injection resulted in a faster protein expression being more intense at the site of injection, whereas IV administration caused slower but diffuse liver expression. IH injection also reduced the spreading of the virus to other organs. Independently of the route, depletion of Kupffer cells significantly enhanced the transduction efficiency of Ad. This effect was stronger when using IV injection, indicating that IH injection partially overcomes Kupffer cell phagocytic activity. Moreover, the antitumor efficacy of high-capacity-Ad encoding murine interleukin-12 (IL-12) was significantly greater when the vector was administered by IH injection than when given IV. In conclusion, IH injection of adenovirus represents a safe and efficient administration route for clinical applications of gene therapy targeting the liver.     

Gene therapy for inherited lung disorders: an insight into pulmonary defence

This review summarizes the latest developments in viral and nonviral gene delivery systems to the lung, and the problems that have to be overcome. Gene delivery has the potential to offer effective treatment to patients with life-threatening lung diseases such as cystic fibrosis and alpha(1)-antitrypsin deficiency, and could modify gene-environment relationships in asthma and other respiratory diseases. Phase I clinical trials conducted in the early 1990s showed that in principle gene transfer to the lung was safe. Although the preliminary results gave encouraging laboratory data, gene expression from viral or nonviral gene delivery systems was too inefficient or transient to offer clinical benefit. Initial optimism gave way to the realization that gene therapy to the lung was unlikely to be straightforward. The host innate and acquired immune system, which protects against infection from inhaled bacteria and viruses, represents a major barrier to successful gene transfer to the lung. A better understanding of the immunological barriers which exist in the lung may allow the development of pharmacological and/or immunological agents that modulate the host immune system to allow for a more continuous and regulated level of gene expression following gene transfer.     

In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance

Liver gene transfer is a highly sought goal for the treatment of inherited and infectious diseases. Lentiviral vectors (LVs) have many desirable properties for hepatocyte-directed gene delivery, including the ability to integrate into nondividing cells. Unfortunately, upon systemic administration, LV transduces hepatocytes relatively inefficiently compared with nonparenchymal cells, and the duration of transgene expression is often limited by immune responses. Here, we investigated the role of innate antiviral responses in these events. We show that administration of LVs to mice triggers a rapid and transient IFNalphabeta response. This effect was dependent on functional vector particles, and in vitro challenge of antigen-presenting cells suggested that plasmacytoid dendritic cells initiated the response. Remarkably, when LVs were administered to animals that lack the capacity to respond to IFNalphabeta, there was a dramatic increase in hepatocyte transduction, and stable transgene expression was achieved. These findings indicate that, even in the setting of acute delivery of replication-defective vectors, IFNs effectively interfere with transduction in a cell-type-specific manner. Moreover, because disabling a single component of the innate/immune network was sufficient to establish persistent xenoantigen expression, our results raise the hope that the immunologic barriers to gene therapy are less insurmountable than expected.     

Human Papillomaviruses; Epithelial Tropisms,and the Development of Neoplasia

Papillomaviruses have evolved over many millions of years to propagate themselves at specific epithelial niches in a range of different host species. This has led to the great diversity of papillomaviruses that now exist, and to the appearance of distinct strategies for epithelial persistence. Many papillomaviruses minimise the risk of immune clearance by causing chronic asymptomatic infections, accompanied by long-term virion-production with only limited viral gene expression. Such lesions are typical of those caused by Beta HPV types in the general population, with viral activity being suppressed by host immunity. A second strategy requires the evolution of sophisticated immune evasion mechanisms, and allows some HPV types to cause prominent and persistent papillomas, even in immune competent individuals. Some Alphapapillomavirus types have evolved this strategy, including those that cause genital warts in young adults or common warts in children. These strategies reflect broad differences in virus protein function as well as differences in patterns of viral gene expression, with genotype-specific associations underlying the recent introduction of DNA testing, and also the introduction of vaccines to protect against cervical cancer. Interestingly, it appears that cellular environment and the site of infection affect viral pathogenicity by modulating viral gene expression. With the high-risk HPV gene products, changes in E6 and E7 expression are thought to account for the development of neoplasias at the endocervix, the anal and cervical transformation zones, and the tonsilar crypts and other oropharyngeal sites. A detailed analysis of site-specific patterns of gene expression and gene function is now prompted.     

Endocrine aspects of cancer gene therapy

The field of cancer gene therapy is in continuous expansion, and technology is quickly moving ahead as far as gene targeting and regulation of gene expression are concerned. This review focuses on the endocrine aspects of gene therapy, including the possibility to exploit hormone and hormone receptor functions for regulating therapeutic gene expression, the use of endocrine-specific genes as new therapeutic tools, the effects of viral vector delivery and transgene expression on the endocrine system, and the endocrine response to viral vector delivery. Present ethical concerns of gene therapy and the risk of germ cell transduction are also discussed, along with potential lines of innovation to improve cell and gene targeting.