AAV gene therapy in companion dogs with severe hemophilia: Real-world long-term data on immunogenicity, efficacy, and quality of life.

The hemophilias are the most common severe inherited bleeding disorders and are caused by deficiency of clotting factor (F) VIII (hemophilia A) or FIX (hemophilia B). The resultant bleeding predisposition significantly increases morbidity and mortality. The ability to improve the bleeding phenotype with modest increases in clotting factor levels has enabled the development and regulatory approval of adeno-associated viral (AAV) vector gene therapies for people with hemophilia A and B. The canine hemophilia model has proven to be one of the best predictors of therapeutic response in humans. Here, we report long-term follow-up of 12 companion dogs with severe hemophilia that were treated in a real-world setting with AAV gene therapy. Despite more baseline bleeding than in research dogs, companion dogs demonstrated a 94% decrease in bleeding rates and 61% improvement in quality of life over a median of 4.1 years (range 2.6-8.9). No new anti-transgene immune responses were detected; one dog with a pre-existing anti-FVIII inhibitor achieved immune tolerance with gene therapy. Two dogs expressing 1%-5% FVIII post gene therapy experienced fatal bleeding events. These data suggest AAV liver-directed gene therapy is efficacious in a real-world setting but should target expression >5% and closely monitor those with levels in the 1%-5% range.

Analysis of vector genome integrations in multicentric lymphoma after AAV gene therapy in a severe hemophilia A dog.

Adeno-associated viral (AAV) vectors have traditionally been viewed as predominantly nonintegrating, with limited concerns for oncogenesis. However, accumulating preclinical data have shown that AAV vectors integrate more often than previously appreciated, with the potential for genotoxicity. To understand the consequences of AAV vector integration, vigilance for rare genotoxic events after vector administration is essential. Here, we investigate the development of multicentric lymphoma in a privately owned dog, PC9, with severe hemophilia A that was treated with an AAV8 vector encapsidating a B domain-deleted canine coagulation F8 gene. PC9 developed an aggressive B cell lineage multicentric lymphoma 3.5 years after AAV treatment. Postmortem analysis of the liver, spleen, and lymph nodes showed the expected biodistribution of the AAV genome. Integration events were found both in PC9 and a second privately owned hemophilia A dog treated similarly with canine F8 gene transfer, which died of a bleeding event without evidence of malignancy. However, we found no evidence of expanded clones harboring a single integration event, indicating that AAV genome integrations were unlikely to have contributed to PC9's cancer. These findings suggest AAV integrations occur but are mostly not genotoxic and support the safety profile of AAV gene therapy.

Factor IXa variants resistant to plasma inhibitors enhance clot formation in vivo.

Factor IXa (FIXa) plays a pivotal role in coagulation by contributing to FX activation via the intrinsic pathway. Although antithrombin (AT) and other plasma inhibitors are thought to regulate FIXa procoagulant function, the impact of FIXa inhibition on thrombin generation and clot formation in vivo remains unclear. Here, we generated FIXa variants with altered reactivity to plasma inhibitors that target the FIXa active site but maintain procoagulant function when bound to its cofactor, FVIIIa. We found that selected FIXa variants (eg, FIXa-V16L) have a prolonged activity half-life in the plasma due, in part, to AT resistance. Studies using hemophilia B mice have shown that delayed FIXa inhibition has a major impact on reducing the bleeding phenotype and promoting thrombus formation following administration of FIX protein. Overall, these results demonstrate that the regulation of FIXa inhibition contributes in a major way to the spatial and temporal control of coagulation at the site of vascular injury. Our findings provide novel insights into the physiological regulation of FIXa, enhance our understanding of thrombus formation in vivo via the intrinsic pathway, and suggest that altering FIXa inhibition could have therapeutic benefits.

Characteristics of BAY 2599023 in the Current Treatment Landscape of Hemophilia A Gene Therapy.

Hemophilia A, a single gene disorder leading to deficient Factor VIII (FVIII), is a suitable candidate for gene therapy. The aspiration is for single administration of a genetic therapy that would allow the production of endogenous FVIII sufficient to restore hemostasis and other biological processes. This would potentially result in reliable protection from bleeding and its associated physical and emotional impacts. Gene therapy offers the possibility of a clinically relevant improvement in disease phenotype and transformational improvement in quality of life, including an opportunity to engage in physical activities more confidently. Gene therapy products for hemophilia A in advanced clinical development use adeno-associated viral (AAV) vectors and a codon-optimized B-domain deleted FVIII transgene. However, the different AAV-based gene therapies have distinct design features, such as choice of vector capsid, enhancer and promoter regions, FVIII transgene sequence and manufacturing processes. These, in turn, impact patient eligibility, safety and efficacy. Ideally, gene therapy technology for hemophilia A should offer bleed protection, durable FVIII expression, broad eligibility and limited response variability between patients, and long-term safety. However, several limitations and challenges must be overcome. Here, we introduce the characteristics of the BAY 2599023 (AAVhu37.hFVIIIco, DTX 201) gene therapy product, including the low prevalence in the general population of anti-AAV-hu37 antibodies, as well as other gene therapy AAV products and approaches. We will examine how these can potentially meet the challenges of gene therapy, with the ultimate aim of improving the lives of patients with hemophilia A.

Immune complications and their management in inherited and acquired bleeding disorders.

Disorders of coagulation, resulting in serious risks for bleeding, may be caused by autoantibody formation or by mutations in genes encoding coagulation factors. In the latter case, antidrug antibodies (ADAs) may form against the clotting factor protein drugs used in replacement therapy, as is well documented in the treatment of the X-linked disease hemophilia. Such neutralizing antibodies against factors VIII or IX substantially complicate treatment. Autoantibody formation against factor VIII leads to acquired hemophilia. Although rare, antibody formation may occur in the treatment of other clotting factor deficiencies (eg, against von Willebrand factor [VWF]). The main strategies that have emerged to address these immune responses include (1) clinical immune tolerance induction (ITI) protocols; (2) immune suppression therapies (ISTs); and (3) the development of drugs that can improve hemostasis while bypassing the antibodies against coagulation factors altogether (some of these nonfactor therapies/NFTs are antibody-based, but they are distinct from traditional immunotherapy as they do not target the immune system). Choice of immune or alternative therapy and criteria for selection of a specific regimen for inherited and autoimmune bleeding disorders are explained. ITI serves as an important proof of principle that antigen-specific immune tolerance can be achieved in humans through repeated antigen administration, even in the absence of immune suppression. Finally, novel immunotherapy approaches that are still in the preclinical phase, such as cellular (for instance, regulatory T cell [Treg]) immunotherapies, gene therapy, and oral antigen administration, are discussed.

Why is AAV FVIII gene therapy not approved by the US Food and Drug Administration yet?

The prospect of a clinical strategy using an adeno-associated virus (AAV) vector for expression of therapeutic levels of factor VIII (FVIII) has been highly desirable. This was initially anticipated by promising data from clinical studies on AAV5-FVIII in men with severe hemophilia A. However, long-term follow-up showed a unique efficacy concern on the sustainability and durability derived from a continuous decline in the FVIII transgene levels starting 1 year after vector injection through year 5. Additional follow-up of early-phase studies and outcomes of an ongoing phase 3 study will likely provide evidence on the feasibility of this approach. Here, the potential underlying mechanisms of the FVIII declining levels, together with the revision of several unique early and late onset findings, are discussed. The lack of long-term preclinical studies in large animal models prevents the firm conclusion that FVIII levels decline was unexpected. It is possible that the combination of vector manufacturing platform and dose, accompanied with ectopic expression of supraphysiologic levels of FVIII at short-term follow-up, may all contribute to the sustainability and durability of the transgene levels. Notably, vector readministration to further improve the FVIII levels is not feasible at this time. Thus, the need of a one-and-done AAV strategy to achieve sustain FVIII levels of expression is sine qua non to impact favorably the disease phenotype.

Evolutionary insights into coagulation factor IX Padua and other high-specific-activity variants.

The high-specific-activity factor IX (FIX) variant Padua (R338L) is the most promising transgene for hemophilia B (HB) gene therapy. Although R338 is strongly conserved in mammalian evolution, amino acid substitutions at this position are underrepresented in HB databases. We therefore undertook a complete 20 amino acid scan and determined the specific activity of human (h) and canine (c) FIX variants with every amino acid substituted at position 338. Notably, we observe that hFIX-R338L is the most active variant and cFIX-R338L is sevenfold higher than wild-type (WT) cFIX. This is consistent with the previous identification of hFIX-R338L as a cause of a rare X-linked thrombophilia risk factor. Moreover, WT hFIX and cFIX are some of the least active variants. We confirmed the increased specific activity relative to FIX-WT in vivo of a new variant, cFIX-R338I, after gene therapy in an HB dog. Last, we screened 232 pediatric subjects with thromboembolic disease without identifying F9 R338 variants. Together these observations suggest a surprising evolutionary pressure to limit FIX activity with WT FIX rather than maximize FIX activity.

B cell-activating factor modulates the factor VIII immune response in hemophilia A.

Inhibitors of factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell-activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to those of noninhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 antibody-mediated B cell depletion. In naive HA mice, prophylactic anti-BAFF antibody therapy prior to FVIII immunization prevented inhibitor formation and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI.

Factor IX assay discrepancies in the setting of liver gene therapy using a hyperfunctional variant factor IX-Padua.

BACKGROUND: Limited information exists regarding the factor IX (FIX) coagulant activity (FIX:C) measured by different assays following FIX-Padua gene therapy. OBJECTIVE: Assess for the first time FIX:C in five commonly used coagulation assays in plasma samples from hemophilia B subjects receiving FIX-Padua gene transfer. METHODS: FIX:C was compared between central (n = 1) and local laboratories (n = 5) in the study, and across four commonly used FIX:C one-stage assays and one FIX:C chromogenic assay. For comparison, samples of pooled congenital FIX-deficient plasma spiked with purified recombinant human FIX (rHFIX)-Padua protein or rHFIX (nonacog alfa) to obtain FIX:C concentrations from ~20% to ~40% were tested. RESULTS: FIX:C results at local laboratories strongly correlated with central laboratory results. However, absolute values at the central laboratory were consistently lower than those at local laboratories. Across five different FIX:C assays, a consistent pattern of FIX:C was observed for subjects receiving fidanacogene elaparvovec-expressed gene transfer. Use of Actin FSL activated partial thromboplastin time (APTT) reagent in the central laboratory resulted in lower FIX:C values compared with other APTT reagents tested. The chromogenic assay determined lower FIX:C than any of the one-stage assays. The rHFIX-Padua protein-spiked samples showed similar results. In contrast, FIX:C results for rHFIX-nonacog alfa measured within 25% of expected for all one-stage assays and below 25% in the chromogenic assay. CONCLUSIONS: Assay-based differences in FIX:C were observed for fidanacogene elaparvovec transgene product and rHFIX-Padua protein, suggesting the variable FIX:C determined with different assay reagents is inherent to the FIX-Padua protein and is not specific to gene therapy-derived FIX-Padua.

Gene Therapy for Inherited Bleeding Disorders.

Decades of preclinical and clinical studies developing gene therapy for hemophilia are poised to bear fruit with current promising pivotal studies likely to lead to regulatory approval. However, this recent success should not obscure the multiple challenges that were overcome to reach this destination. Gene therapy for hemophilia A and B benefited from advancements in the general gene therapy field, such as the development of adeno-associated viral vectors, as well as disease-specific breakthroughs, like the identification of B-domain deleted factor VIII and hyperactive factor IX Padua. The gene therapy field has also benefited from hemophilia B clinical studies, which revealed for the first time critical safety concerns related to immune responses to the vector capsid not anticipated in preclinical models. Preclinical studies have also investigated gene transfer approaches for other rare inherited bleeding disorders, including factor VII deficiency, von Willebrand disease, and Glanzmann thrombasthenia. Here we review the successful gene therapy journey for hemophilia and pose some unanswered questions. We then discuss the current state of gene therapy for these other rare inherited bleeding disorders and how the lessons of hemophilia gene therapy may guide clinical development.

Inhibitors-Recent insights.

The development of inhibitory antibodies to therapeutic factor VIII (FVIII) in haemophilia A (HA) patients is the major complication in treatment/prevention of haemorrhages. The reasons some HA patients develop inhibitors while others do not remain unclear. This review briefly summarizes our understanding of anti-FVIII immune responses, the roles of T cells, both effector and regulatory, and generally discusses the interplay between FVIII and the immune system, both in factor replacement therapy and gene therapy, with some comparisons to factor IX and haemophilia B therapies. Notably, we propose that the prevailing observed active tolerance to FVIII in both HA and non-HA individuals rests to greater or lesser extents on peripherally induced immune tolerance. We also propose that the immune systems of inhibitor-negative HA patients do not merely ignore therapeutic FVIII, but rather have immunologically assessed and actively tolerized the patients to exogenous FVIII. Induction of such peripheral immune tolerance may further be triggered in HA patients who failed to tolerize upon initial FVIII exposure by 'appropriate' stimulation of their immune system, eg by immune tolerance induction therapy via intensive FVIII therapy, by oral administration of FVIII, by cellular therapies or by gene therapy directed to immuno-tolerogenic sites such as the liver.

Gene Therapy for Hemophilia: Facts and Quandaries in the 21st Century.

Therapy for hemophilia has evolved in the last 40 years from plasma-based concentrates to recombinant proteins and, more recently, to non-factor therapeutics. Along this same timeline, research in adeno-associated viral (AAV) based gene therapy vectors has provided the framework for early phase clinical trials initially for hemophilia B (HB) and now for hemophilia A. Successive lessons learned from early HB trials have paved the way for current advanced phase trials. Nevertheless, questions linger regarding 1) the optimal balance of vector dose to transgene expression, 2) amount and durability of transgene expression required, and 3) long-term safety. Some trials have demonstrated unique findings not seen previously regarding transient elevation of liver enzymes, immunogenicity of the vector capsid, and loss of transgene expression. This review will provide an update on the clinical AAV gene therapy trials in hemophilia and address the questions above. A thoughtful and rationally approached expansion of gene therapy to the clinics would certainly be a welcome addition to the arsenal of options for hemophilia therapy. Further, the global impact of gene therapy could be vastly improved by expanding eligibility to different patient populations and to developing nations. With the advances made to date, it is possible to envision a shift from the early goal of simply increasing life expectancy to a significant improvement in quality of life by reduction in spontaneous bleeding episodes and disease complications.

Timing of Intensive Immunosuppression Impacts Risk of Transgene Antibodies after AAV Gene Therapy in Nonhuman Primates.

Adeno-associated virus (AAV) vector gene therapy is a promising treatment for a variety of genetic diseases, including hemophilia. Systemic administration of AAV vectors is associated with a cytotoxic immune response triggered against AAV capsid proteins, which if untreated can result in loss of transgene expression. Immunosuppression (IS) with corticosteroids has limited transgene loss in some AAV gene therapy clinical trials, but was insufficient to prevent loss in other studies. We used a nonhuman primate model to evaluate intensive T cell-directed IS combined with AAV-mediated transfer of the human factor IX (FIX) gene. Early administration of rabbit anti-thymocyte globulin (ATG) concomitant with AAV administration resulted in the development of anti-FIX antibodies, whereas delayed ATG by 5 weeks administration did not. The anti-FIX immune response was associated with increases in inflammatory cytokines, as well as a skewed Th17/regulatory T cell (Treg) ratio. We conclude that the timing of T cell-directed IS is critical in determining transgene-product immunogenicity or tolerance. These data have implications for systemically administered AAV gene therapy being evaluated for hemophilia A and B, as well as other genetic diseases.

Translational Potential of Immune Tolerance Induction by AAV Liver-Directed Factor VIII Gene Therapy for Hemophilia A.

Hemophilia A (HA) is an X-linked bleeding disorder due to deficiencies in coagulation factor VIII (FVIII). The major complication of current protein-based therapies is the development of neutralizing anti-FVIII antibodies, termed inhibitors, that block the hemostatic effect of therapeutic FVIII. Inhibitors develop in about 20-30% of people with severe HA, but the risk is dependent on the interaction between environmental and genetic factors, including the underlying F8 gene mutation. Recently, multiple clinical trials evaluating adeno-associated viral (AAV) vector liver-directed gene therapy for HA have reported promising results of therapeutically relevant to curative FVIII levels. The inclusion criteria for most trials prevented enrollment of subjects with a history of inhibitors. However, preclinical data from small and large animal models of HA with inhibitors suggests that liver-directed gene therapy can in fact eradicate pre-existing anti-FVIII antibodies, induce immune tolerance, and provide long-term therapeutic FVIII expression to prevent bleeding. Herein, we review the accumulating evidence that continuous uninterrupted expression of FVIII and other transgenes after liver-directed AAV gene therapy can bias the immune system toward immune tolerance induction, discuss the current understanding of the immunological mechanisms of this process, and outline questions that will need to be addressed to translate this strategy to clinical trials.

Gene therapy for hemophilia: Progress to date and challenges moving forward.

Over the past decades hemophilia has been transformed from a debilitating disease to a manageable condition. However, the current treatment options are expensive, complex, and inaccessible to a large portion of the global population. Moreover, the development of antibodies to replacement factors, termed inhibitors, is a common complication that not only renders conventional prophylaxis regimens ineffective but also increases the annual bleeding rate in affected patients. Fortunately, much progress has been made toward developing a curative gene therapy treatment for hemophilia and these efforts have led to a series of human trials with promising results. This review seeks to address some of the new issues raised by recent progress in the field, including the differences between available recombinant adeno-associated viral (rAAV) vectors, the etiology of transaminitis following vector administration, and techniques to induce long-term factor expression. We also address other unresolved questions, including strategies to overcome pre-existing neutralizing antibodies to AAV, approaches that can make vector re-administration possible, and whether gene therapy can be used to induce factor tolerance and treat inhibitors. Finally, we discuss logistical and ethical issues related to hemophilia gene therapy including how to accurately measure therapeutic outcomes, when to consider treatment of pediatric patients, and how to equitably price the medication to ensure fair compensation while maximizing accessibility. As the field marches forward from clinical trials towards clinical application, answers to these questions will determine the future of gene therapy for hemophilia.

Hyperactivity of factor IX Padua (R338L) depends on factor VIIIa cofactor activity.

Adeno-associated-viral (AAV) vector liver-directed gene therapy (GT) for hemophilia B (HB) is limited by a vector-dose-dependent hepatotoxicity. Recently, this obstacle has been partially circumvented by the use of a hyperactive factor IX (FIX) variant, R338L (Padua), which has an eightfold increased specific activity compared to FIX-WT. FIX-R338L has emerged as the standard for HB GT. However, the underlying mechanism of its hyperactivity is undefined; as such, safety concerns of unregulated coagulation and the potential for thrombotic complications have not been fully addressed. To this end, we evaluated the enzymatic and clotting activity as well as the activation, inactivation, and cofactor-dependence of FIX-R338L relative to FIX-WT. We observed that the high-specific-activity of FIX-R338L requires factor VIIIa (FVIIIa) cofactor. In a novel system utilizing emicizumab, a FVIII-mimicking bispecific antibody, the hyperactivity of both recombinant FIX-R338L and AAV-mediated-transgene-expressed FIX-R338L from HB GT subjects is ablated without FVIIIa activity. We conclude that the molecular regulation of activation, inactivation, and cofactor-dependence of FIX-R338L is similar to FIX-WT, but that the FVIIIa-dependent hyperactivity of FIX-R338L is the result of a faster rate of factor X activation. This mechanism helps mitigate safety concerns of unregulated coagulation and supports the expanded use of FIX-R338L in HB therapy.

Protein-Engineered Coagulation Factors for Hemophilia Gene Therapy.

Hemophilia A (HA) and hemophilia B (HB) are X-linked bleeding disorders due to inheritable deficiencies in either coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Recently, gene therapy clinical trials with adeno-associated virus (AAV) vectors and protein-engineered transgenes, B-domain deleted (BDD) FVIII and FIX-Padua, have reported near-phenotypic cures in subjects with HA and HB, respectively. Here, we review the biology and the clinical development of FVIII-BDD and FIX-Padua as transgenes. We also examine alternative bioengineering strategies for FVIII and FIX, as well as the immunological challenges of these approaches. Other engineered proteins and their potential use in gene therapy for hemophilia with inhibitors are also discussed. Continued advancement of gene therapy for HA and HB using protein-engineered transgenes has the potential to alleviate the substantial medical and psychosocial burdens of the disease.

Gene therapy for hemophilia: what does the future hold?

Recent phase I/II adeno-associated viral vector-mediated gene therapy clinical trials have reported remarkable success in ameliorating disease phenotype in hemophilia A and B. These trials, which highlight the challenges overcome through decades of preclinical and first in human clinical studies, have generated considerable excitement for patients and caregivers alike. Optimization of vector and transgene expression has significantly improved the ability to achieve therapeutic factor levels in these subjects. Long-term follow-up studies will guide standardization of the approach with respect to the combination of serotype, promoter, dose, and manufacturing processes and inform safety for inclusion of young patients. Certain limitations preclude universal applicability of gene therapy, including transient liver transaminase elevations due to the immune responses to vector capsids or as yet undefined mechanisms, underlying liver disease from iatrogenic viral hepatitis, and neutralizing antibodies to clotting factors. Integrating vectors show promising preclinical results, but manufacturing and safety concerns still remain. The prospect of gene editing for correction of the underlying mutation is on the horizon with considerable potential. Herein, we review the advances and limitations that have resulted in these recent successful clinical trials and outline avenues that will allow for broader applicability of gene therapy.

Padua FIXa resistance to Protein S and a potential therapy for hyperactive FIXa.

INTRODUCTION: Abnormalities in the levels and functions of proteins that maintain hemostasis can cause thrombosis. Factor IX (FIX) R338L, i.e., Factor IX Padua, is a hyperactive clotting factor that promotes thrombosis. The R338L mutation increases the clotting rate by 8-fold despite increasing the Factor IXa enzymatic activity by only 2-fold. Protein S (PS) is a natural anticoagulant that directly inhibits FIXa. Because individuals affected by the R338L mutation have normal concentrations of PS, we speculated that the Padua hypercoagulation phenotype is due to decreased inhibition of FIXa R338L by PS. METHODS: We measured the ability of PS to inhibit FIX R338L, and we assessed the ability of PS to mitigate the prothrombotic effect FIX R338L. RESULTS: Plasma clotting assays demonstrated that 3-fold more PS was required to inhibit FIXa R338L compared with inhibition of wild type FIXa. Thrombin generation assays with Padua patient plasma recapitulated this biochemical consequence of the R338L mutation. Importantly, the less efficient inhibition of FIXa R338L was reversed by increasing PS concentration. Binding and co-immunoprecipitation studies revealed that the decrease in the inhibition of FIXa R338L by PS was caused by a 3- to 4-fold reduction in FIXa R338L affinity for PS. CONCLUSION: In summary, the resistance of FIXa R338L to inhibition by PS likely contributes to the unexpectedly high clotting rate in Padua individuals. Moreover, PS-mediated reversal of the pathological properties of FIXa R338L suggests that PS administration may be a novel and effective means to mitigate thrombophilia caused by any source of elevated FIXa activity.