Innovating immune tolerance induction for haemophilia.

INTRODUCTION: Haemophilia A is an X-linked bleeding disorder characterized by a deficiency of coagulation protein factor VIII (FVIII). A challenging complication of therapeutic FVIII infusions is the formation of neutralizing alloantibodies against the FVIII protein defined as inhibitors. The development of FVIII inhibitors drastically alters the quality of life of the patients and is associated with tremendous increases in morbidity as well as treatment costs. AIM: Current clinical immune tolerance induction protocols to reverse inhibitors are lengthy, costly and not effective in all patients. Prophylactic protocols to prevent inhibitor formation have not yet been developed in the clinical setting. However, there has been ample progress towards this goal in recent years in preclinical studies using animal models of haemophilia. METHODS: Here, we review the mechanisms that lead to inhibitor formation against FVIII and two promising new strategies for antigen-specific tolerance induction. RESULTS: CD4+ T cells play an important role in the FVIII-specific B cell response. Immune tolerance can be induced based on transplacental delivery of FVIII domains fused to Fc or on oral delivery of leaf cells from chloroplast transgenic crop plants. CONCLUSIONS: Recent literature suggests that prophylactic tolerance induction protocols for FVIII may be feasible in haemophilia A patients.

Induction of tolerance to factor VIII by transient co-administration with rapamycin.

BACKGROUND: Formation of inhibitory antibodies is a frequent and serious complication of factor (F) VIII replacement therapy for the X-linked bleeding disorder hemophilia A. Similarly, hemophilia A mice develop high-titer inhibitors to recombinant human FVIII after a few intravenous injections. OBJECTIVE: Using the murine model, the study sought to develop a short regimen capable of inducing tolerance to FVIII. METHODS: A 1-month immunomodulatory protocol, consisting of FVIII administration combined with oral delivery of rapamycin, was developed. RESULTS: The protocol effectively prevented formation of inhibitors to FVIII upon subsequent intravenous treatment (weekly for 3.5 months). Control mice formed high-titer inhibitors and had CD4(+) T effector cell responses characterized by expression of IL-2, IL-4 and IL-6. Tolerized mice instead had a CD4(+)CD25(+)FoxP3(+) T cell response to FVIII that suppressed antibody formation upon adoptive transfer, indicating a shift from Th2 to Treg if FVIII antigen was introduced to T cells during inhibition with rapamycin. CD4(+) T cells from tolerized mice also expressed TGF-ß1 and CTLA4, but not IL-10. The presence of FVIII antigen during the time of rapamycin administration was required for specific tolerance induction. CONCLUSIONS: The study shows that a prophylactic immune tolerance protocol for FVIII can be developed using rapamycin, a drug that is already widely in clinical application. Immune suppression with rapamycin was mild and highly transient, as the mice regained immune competence within a few weeks.

Progress and prospects: immune responses to viral vectors.

Viral vectors are potent gene delivery platforms used for the treatment of genetic and acquired diseases. However, just as viruses have evolved to infect cells efficiently, the immune system has evolved to fight off what it perceives as invading pathogens. Therefore, innate immunity and antigen-specific adaptive immune responses against vector-derived antigens reduce the efficacy and stability of in vivo gene transfer. In addition, a number of vectors are derived from parent viruses that humans encounter through natural infection, resulting in preexisting antibodies and possibly in memory responses against vector antigens. Similarly, antibody and T-cell responses may be directed against therapeutic gene products that often differ from the endogenous nonfunctional or absent protein that is being replaced. As details and mechanisms of such immune reactions are uncovered, novel strategies are being developed, and vectors are being specifically engineered to avoid, suppress or manipulate the response, ideally resulting in sustained expression and immune tolerance to the transgene product. This review provides a summary of our current knowledge of the interactions between the immune system adeno-associated virus, adenoviral and lentiviral vectors, and their transgene products.

Prophylactic immune tolerance induced by changing the ratio of antigen-specific effector to regulatory T cells.

BACKGROUND: Gene and protein replacement therapies for inherited protein deficiencies such as hemophilia or lysosomal storage disorders are limited by deleterious immune responses directed against their respective therapeutic proteins. Therefore, the development of protocols preventing such responses is key to providing successful long-term therapy. OBJECTIVES: We sought to develop a protocol, utilizing a drug/peptide cocktail, that would effectively shift the antigen-specific CD4+ T-cell population, tipping the balance from effector T cells (Teffs) towards regulatory T cells (Tregs). METHODS: Treg-deficient (DO11.10-tg Rag2(-/-)) BALB/c mice were used to screen for an optimal protocol addressing the aforementioned goal and to study the mechanisms underlying in vivo changes in T-cell populations. Muscle-directed gene transfer to hemophilia B mice was also performed in order to test the optimal protocol in a therapeutically relevant setting. RESULTS: Specific antigen administration (4-week repeated dosing) combined with rapamycin and interleukin-10 led to substantial reductions in Teffs, via activation-induced cell death, and induced CD4+CD25+FoxP3+ Tregs to a large extent in multiple organs. The proportion of apoptotic T cells also increased over time, whereas Teffs and Tregs were differentially affected. When applied to a model of protein deficiency (gene therapy for hemophilia B), the protocol successfully prevented inhibitor formation, whereas non-specific immunosuppression was only marginally effective. CONCLUSIONS: It is feasible to provide a short-term, prophylactic protocol allowing for the induction of immune tolerance. This protocol may provide a marked advance in efforts seeking to improve clinical outcomes in disorders involving therapeutic protein replacement.

Role of regulatory T cells in tolerance to coagulation factors.

The immune response to coagulation factors VIII or IX, in particular formation of inhibitory antibodies, complicates treatment of hemophilia. Therefore, a number of recent studies in animal models have explored novel approaches toward induction of immune tolerance in protein or gene replacement therapy. Strong evidence has emerged that regulatory T cells (Treg) are an important component of the mechanism by which tolerance is maintained and inhibitor formation, a T help dependent response, is prevented. Limited data in patients also support this concept. In particular, CD4+ CD25+ FoxP3+ Treg, whether naturally occurring or induced, have been invoked in suppression of antibody and of cytotoxic T lymphocyte responses to the therapeutic clotting factor. This review summarizes the data on this emerging concept of Treg-mediated regulation of the immune response in treatment of hemophilia, strategies and mechanisms of Treg induction and function, and the implications for development of immune tolerance protocols.

A viable mouse model of factor X deficiency provides evidence for maternal transfer of factor X.

BACKGROUND: Activated factor X (FXa) is a vitamin K-dependent serine protease that plays a pivotal role in blood coagulation by converting prothrombin to thrombin. There are no reports of humans with complete deficiency of FX, and knockout of murine F10 is embryonic or perinatal lethal. OBJECTIVE: We sought to generate a viable mouse model of FX deficiency. METHODS: We used a socket-targeting construct to generate F10-knockout mice by eliminating F10 exon 8 (knockout allele termed F10(tm1Ccmt), abbreviated as '-'; wild-type '+'), and a plug-targeting construct to generate mice expressing a FX variant with normal antigen levels but low levels of FX activity [4-9% normal in humans carrying the defect, Pro343-->Ser, termed FX Friuli (mutant allele termed F10(tm2Ccmt), abbreviated as F)]. RESULTS: F10 knockout mice exhibited embryonic or perinatal lethality. In contrast, homozygous Friuli mice [F10 (F/F)] had FX activity levels of approximately 5.5% (sufficient to rescue both embryonic and perinatal lethality), but developed age-dependent iron deposition and cardiac fibrosis. Interestingly, F10 (-/F) mice with FX activity levels of 1-3% also showed complete rescue of lethality. Further study of this model provides evidence supporting a role of maternal FX transfer in the embryonic survival. CONCLUSIONS: We demonstrate that, while complete absence of FX is incompatible with murine survival, minimal FX activity as low as 1-3% is sufficient to rescue the lethal phenotype. This viable low-FX mouse model will facilitate the development of FX-directed therapies as well as investigation of the FX role in embryonic development.

Major role of local immune responses in antibody formation to factor IX in AAV gene transfer.

The risk of an immune response to the coagulation factor IX (F.IX) transgene product is a concern in gene therapy for the X-linked bleeding disorder hemophilia B. In order to investigate the mechanism of F.IX-specific lymphocyte activation in the context of adeno-associated viral (AAV) gene transfer to skeletal muscle, we injected AAV-2 vector expressing human F.IX (hF.IX) into outbred immune-competent mice. Systemic hF.IX levels were transiently detected in the circulation, but diminished concomitant with activation of CD4+ T and B cells. ELISPOT assays documented robust responses to hF.IX in the draining lymph nodes of injected muscle by day 14. Formation of inhibitory antibodies to hF.IX was observed over a wide range of vector doses, with increased doses causing stronger immune responses. A prolonged inflammatory reaction in muscle started at 1.5-2 months, but ultimately failed to eliminate transgene expression. By 1.5 months, hF.IX antigen re-emerged in circulation in approximately 70% of animals injected with high vector dose. Hepatic gene transfer elicited only infrequent and weaker immune responses, with higher vector doses causing a reduction in T-cell responses to hF.IX. In summary, the data document substantial influence of target tissue, local antigen presentation, and antigen levels on lymphocyte responses to F.IX.

Optimized production of high-titer recombinant adeno-associated virus in roller bottles.

Adeno-associated viral (AAV) vectors are used for in vivo gene transfer in a number of preclinical models of genetic diseases (including large-animal models) and are currently being tested in clinical trials for treatment of hemophilia B and cystic fibrosis. Protocols for production of AAV vectors in a helper virus-free system are available and are based on transient transfection of HEK-293 cells with multiple plasmids. Scale-up of vector production has been labor intensive and inefficient because of a lack of larger culture vessels suitable for growth of adherent cells, large-scale transfection, and vector production. Here we report efficient production of AAV vector in roller bottles, which represents a 10-fold scale-up from the conventional flask or plate method. Optimized production yielded greater than 10(13) vector genomes per bottle and was as cost effective as published protocols using plates. Successful vector production by this method was dependent on optimization of transfection by calcium phosphate precipitation, of monitoring of cell growth (by measurement of glucose consumption), of cell culture conditions, and CO2/air exchange with the culture vessel.

Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males.

A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.

Muscle-directed gene transfer and transient immune suppression result in sustained partial correction of canine hemophilia B caused by a null mutation.

The X-linked bleeding disorder hemophilia B is caused by absence of functional blood coagulation factor IX (F9) and can be treated by adeno-associated viral (AAV) mediated gene transfer to skeletal muscle. The safety of this approach is currently being evaluated in a phase I clinical trial. Efficacy of this and several other gene therapy strategies has been addressed in hemophilia B dogs, an important preclinical model of the disease. While previously published data demonstrated sustained expression of canine F9 in dogs with a missense mutation in the gene F9, we show here that AAV-mediated canine F9 gene transfer to skeletal muscle of hemophilia B dogs carrying a null mutation of F9 (causing an early stop codon and an unstable mRNA) results in induction of inhibitory anti-canine F9 at comparable vector doses (1 x 10(12) vector genomes/kg). Thus, the risk of inhibitor formation following AAV-mediated F9 gene therapy may be influenced by the nature of the underlying mutation in F9. Transient immune suppression with cyclophosphamide at the time of vector administration blocked formation of anti-canine F9 antibodies in the one animal treated with this approach. Treatment with this combination of gene transfer and transient immune modulation has resulted in sustained expression (>8 months) of canine F9 at levels sufficient for partial correction of coagulation parameters.

Risk and prevention of anti-factor IX formation in AAV-mediated gene transfer in the context of a large deletion of F9.

The safety of several gene therapy approaches for treatment of the severe, X-linked bleeding disorder hemophilia is currently being evaluated in early phase clinical trials. One strategy seeks to correct deficiency of functional coagulation factor IX (hemophilia B) by intramuscular (IM) administration of an adeno-associated viral (AAV) vector. A potentially serious complication of any treatment for hemophilia is formation of inhibitory antibodies against the coagulation factor protein, a risk that increases in the setting of null mutations in the factor IX gene (F9). Here, we describe hemophilia B mice with a large F9 deletion that form inhibitors within 1 to 2 months after IM administration of an AAV vector expressing mouse F9 or after repeated intravenous infusion of mouse F9 concentrate. In both cases, inhibitors are primarily IgG1 immunoglobulins representing a Th2-driven humoral immune response. We further demonstrate that anti-mouse F9 antibody formation in the gene-based approach can be reduced by transient immune modulation at the time of vector administration. Moreover, this maneuver resulted in complete absence of anti-mouse F9 and sustained expression of functional mouse F9 in some hemophilia B mice, particularly in those animals treated with the immunosuppressive drug cyclophosphamide. These data have direct relevance for design of clinical trials and strategies aimed at avoiding immune responses against a secreted transgene product.

Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice.

Inbred immunocompetent C57BL/6 mice have been a favored strain to study transgene expression of human blood coagulation factor IX (hF.IX) from viral vectors because systemic expression of the secreted protein is not limited by antibody responses following intravenous (i.v.) injection of vector. For example, i.v. injection of an adenoviral (Ad) vector results in sustained expression of hF.IX in normal or hemophilic C57BL/6 mice, while anti-hF.IX antibodies rapidly emerge in other strains (Gene Therapy 4: 473; Blood 91: 784). To investigate these observations further, we injected naive C57BL/6 mice and C57BL/6 mice with pre-existing anti-hF.IX with Ad-hF.IX vector via peripheral vein. All mice expressed hF.IX antigen without detectable anti-hF.IX, even when challenged with hF.IX in different immunogenic settings at later time points. Moreover, in mice with pre-existing immunity, anti-hF.IX titers diminished to undetectable levels after i.v. administration of Ad-hF.IX. Lymphocytes from mice that had received Ad-hF.IX i.v. failed to proliferate when stimulated with hF.IX in vitro after the animals had been repeatedly challenged with hF.IX protein formulated in complete Freund's adjuvant. Thus, absence of anti-hF.IX in C57BL/6 mice after i.v. injection of Ad vector is not due to ignorance to the foreign transgene product. Similar experiments in other strains showed that immune tolerance to hF.IX does not correlate with the strain haplotype or expression of IL-10 cytokine. Given the well-documented immunogenicity of the first-generation adenoviral vector, data from C57BL/6 mice may therefore grossly underestimate immunological consequences in certain gene therapy protocols.

Posttranslational modifications of recombinant myotube-synthesized human factor IX.

Recent data demonstrate that the introduction into skeletal muscle of an adeno-associated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical gamma-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity. Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and gamma-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX.

Gene therapy for hereditary hematological disorders.

The year 2000 saw the first successful treatment of a genetic disorder by gene therapy. Pediatric patients with X-linked severe combined immunodeficiency disorder (SCID-X1) received autologous CD34+ hematopoietic cells following ex vivo gene transfer using a retroviral vector, with subsequent demonstration of improved immune responses. A number of preclinical and clinical studies have been conducted with the aim of developing gene therapy for hemophilia, Fanconi anemia, sickle cell disease, beta-thalassemia, chronic granulomatous disease, and other inherited hematological disorders. The greatest advances in novel approaches toward treatment of hematological disorders have been made in hemophilia, with 3 current phase I clinical trials ongoing. Two trials are investigating the safety and feasibility of utilizing either an ex vivo, non-viral gene transfer technique or an intravenous infusion of a retroviral vector to treat adults with severe hemophilia A (factor VIII deficiency). The third study involves intramuscular administration of an adeno-associated viral (AAV) vector for expression of factor IX in adult patients with hemophilia B. Results from this study and from preclinical studies preceding the trial demonstrate that it is possible to safely administer high doses of a viral vector in vivo.

Role of vector in activation of T cell subsets in immune responses against the secreted transgene product factor IX.

Defining immune responses against the secreted transgene product in a gene therapy setting is critical for treatment of genetic diseases such as hemophilia B (coagulation factor IX deficiency). We have previously shown that intramuscular administration of an adeno-associated viral (AAV) vector results in stable expression of therapeutic levels of factor IX (F.IX) and may be associated with humoral immune responses against F.IX. This study demonstrates that intramuscular injection of an AAV vector expressing F.IX fails to activate F.IX-specific cytotoxic T lymphocytes (CTLs) in hemostatically normal or in hemophilia B mice, so that there is an absence of cellular immune responses against F.IX. However, transgene-derived F.IX can cause B cell responses characterized by production of T helper cell-dependent antibodies (predominantly IgG1, but also IgG2 subclasses) resulting from activation of CD4+ T helper cells primarily of the Th2 subset. In contrast, administration of an adenoviral vector efficiently activated F.IX-specific CTLs and T helper cells of both Th1 and Th2 subsets, leading to inflammation and destruction of transduced muscle tissue and activation of B cells as well. Therefore, vector sequences fundamentally influence T cell responses against transgene-encoded F.IX. In conclusion, activation of the immune system in AAV-mediated gene transfer is restricted to pathways mediated by F.IX antigen presentation through MHC class II determinants resulting in T and B cell responses that are more comparable to responses in the setting of protein infusion rather than of viral infection/gene transfer.

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (FIX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2 x 10(11) vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of FIX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.

Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/promoter.

Hemophilia B is caused by the absence of functional coagulation factor IX (F.IX) and represents an important model for treatment of genetic diseases by gene therapy. Recent studies have shown that intramuscular injection of an adeno-associated viral (AAV) vector into mice and hemophilia B dogs results in vector dose-dependent, long-term expression of biologically active F.IX at therapeutic levels. In this study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 x 10(11) vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished through the use of an improved expression cassette that uses the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.2-kilobase portion of human skeletal actin promoter. These results correlated with enhanced levels of F.IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes. Systemic F.IX expression from constructs containing the CMV enhancer/promoter alone was 120 to 200 ng/mL in mice injected with 1 x 10(11) vector genomes. Muscle-specific promoters performed poorly for F.IX transgene expression in vitro and in vivo. However, the incorporation of a sequence from the alpha-skeletal actin promoter containing at least 1 muscle-specific enhancer and 1 enhancer-like element further improved muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of F.IX expression with lower vector doses, thus enhancing efficacy and safety of the protocol. (Blood. 2000;95:2536-2542)

Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.

Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5x10(12) vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.