Optimizing Liver Health Before and After Gene Therapy for Hemophilia A.

Gene therapy for severe hemophilia A employs an adeno-associated virus (AAV) vector and liver-specific promoters that depend on healthy hepatocyte function to achieve safe and long-lasting increases in FVIII activity. Thus, hepatocyte health is an essential aspect of safe and successful gene therapy. Many people living with hemophilia A have current or past chronic hepatitis C virus infection, metabolic dysfunction-associated steatosis or steatohepatitis, or other conditions that may compromise the efficacy and safety of AAV-mediated gene therapy. In addition, gene therapy may induce an immune response to transduced hepatocytes, leading to liver inflammation and reduced FVIII activity. The immune response can be treated with immunosuppression, but close monitoring of liver function tests and factor levels is necessary. The long-term risk of hepatocellular carcinoma associated with gene therapy is unknown. Routine screening by imaging for hepatocellular carcinoma, preferable every 6 months, is essential in patients at high risk and recommended in all recipients of hemophilia A gene therapy. This paper describes our current understanding of the biologic underpinnings of how liver health affects hemophilia A gene therapy, and provides practical clinical guidance for assessing, monitoring, and managing liver health both before and after gene therapy.

Integration and the risk of liver cancer-Is there a real risk?

Adeno-associated virus (AAV)-based gene therapies are in clinical development for haemophilia and other genetic diseases. Since the recombinant AAV genome primarily remains episomal, it provides the opportunity for long-term expression in tissues that are not proliferating and reduces the safety concerns compared with integrating viral vectors. However, AAV integration events are detected at a low frequency. Preclinical studies in mouse models have reported hepatocellular carcinoma (HCC) after systemic AAV administration in some settings, though this has not been reported in large animal models. The risk of HCC or other cancers after AAV gene therapy in clinical studies thus remains theoretical. Potential risk factors for HCC after gene therapy are beginning to be elucidated through animal studies, but their relevance to human studies remains unknown. Studies to investigate the factors that may influence the risk of oncogenesis as well as detailed investigation of cases of cancer in AAV gene therapy patients will be important to define the potential risk of AAV genotoxicity.

Coagulation factor VIII regulates von Willebrand factor homeostasis invivo.

BACKGROUND: Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex, but each has its distinct functions. Binding of FVIII to VWF results in a prolongation of FVIII's half-life in circulation and modulates FVIII's immunogenicity during hemophilia therapy. However, the biological effect of FVIII and VWF interaction on VWF homeostasis is not fully understood. OBJECTIVES: To determine the effect of FVIII in VWF proteolysis and homeostasis in vivo. METHODS: Mouse models, recombinant FVIII infusion, and patients with hemophilia A on a high dose FVIII for immune tolerance induction therapy or emicizumab for bleeding symptoms were included to address this question. RESULTS: An intravenous infusion of a recombinant B-domain less FVIII (BDD-FVIII) (40 and 160 µg/kg) into wild-type mice significantly reduced plasma VWF multimer sizes and its antigen levels; an infusion of a high but not low dose of BDD-FVIII into Adamts13+/- and Adamts13-/- mice also significantly reduced the size of VWF multimers. However, plasma levels of VWF antigen remained unchanged following administration of any dose BDD-FVIII into Adamts13-/- mice, suggesting partial ADAMTS-13 dependency in FVIII-augmented VWF degradation. Moreover, persistent expression of BDD-FVIII at ∼50 to 250 U/dL via AAV8 vector in hemophilia A mice also resulted in a significant reduction of plasma VWF multimer sizes and antigen levels. Finally, the sizes of plasma VWF multimers were significantly reduced in patients with hemophilia A who received a dose of recombinant or plasma-derived FVIII for immune tolerance induction therapy. CONCLUSION: Our results demonstrate the pivotal role of FVIII as a cofactor regulating VWF proteolysis and homeostasis under various (patho)physiological conditions.

Altered cleavage of human factor VIII at the B-domain and acidic region 3 interface enhances expression after gene therapy in hemophilia A mice.

BACKGROUND: Variants of human factor VIII (hFVIII) have been developed to further understand the structure and function of hFVIII and improve gene-based therapeutics. We have previously characterized several hFVIII variants of the furin cleavage site (1645-1648) with improved secretion. We have also identified a second cleavage site in the acidic region 3 (a3) (1657-1658) that becomes the primary hFVIII intracellular cleavage position in the absence of the furin site. We tested a hypothesis that modification of this site may confer additional functional advantages to hFVIII. OBJECTIVES: The aim of this study was to conduct the biochemical and functional characterization of hFVIII variants of the furin cleavage site, the a3 cleavage site, or in combination, both in vitro and in vivo after AAV mediated gene therapy. METHODS: Recombinant hFVIII variants of the furin cleavage site (hFVIII-Δ3), the a3 cleavage site (hFVIII-S1657P/D1658E [SP/DE]), or in combination (hFVIII-Δ3-SP/DE) were purified and characterized in vitro and in vivo. RESULTS: Recombinant hFVIII-Δ3, hFVIII-SP/DE, and hFVIII-Δ3-SP/DE variants all had comparable specific activity to B-domain deleted (BDD) hFVIII. Hemophilia A mice tolerant to hFVIII did not develop immune responses to hFVIII after protein challenge with these variants or after adeno-associated virus (AAV) delivery. Following AAV delivery, hFVIII-Δ3-SP/DE resulted in expression levels that were 2- to 5-fold higher than those with hFVIII-BDD in hemophilia A mice. CONCLUSION: The novel hFVIII-Δ3-SP/DE variant of the furin and a3 cleavage sites significantly improved secretion compared with hFVIII-BDD. This key feature of the Δ3-SP/DE variant provides a unique strategy that can be combined with other approaches to further improve factor VIII expression to achieve superior efficacy in AAV-based gene therapy for hemophilia A.

Evaluating the state of the science for adeno-associated virus integration: An integrated perspective.

On August 18, 2021, the American Society of Gene and Cell Therapy (ASGCT) hosted a virtual roundtable on adeno-associated virus (AAV) integration, featuring leading experts in preclinical and clinical AAV gene therapy, to further contextualize and understand this phenomenon. Recombinant AAV (rAAV) vectors are used to develop therapies for many conditions given their ability to transduce multiple cell types, resulting in long-term expression of transgenes. Although most rAAV DNA typically remains episomal, some rAAV DNA becomes integrated into genomic DNA at a low frequency, and rAAV insertional mutagenesis has been shown to lead to tumorigenesis in neonatal mice. Currently, the risk of rAAV-mediated oncogenesis in humans is theoretical because no confirmed genotoxic events have been reported to date. However, because insertional mutagenesis has been reported in a small number of murine studies, there is a need to characterize this genotoxicity to inform research, regulatory needs, and patient care. The purpose of this white paper is to review the evidence of rAAV-related host genome integration in animal models and possible risks of insertional mutagenesis in patients. In addition, technical considerations, regulatory guidance, and bioethics are discussed.

Preclinical assessment of an optimized AAV-FVIII vector in mice and non-human primates for the treatment of hemophilia A.

Extensive clinical data from liver-mediated gene therapy trials have shown that dose-dependent immune responses against the vector capsid may impair or even preclude transgene expression if not managed successfully with prompt immune suppression. The goal of this preclinical study was to generate an adeno-associated viral (AAV) vector capable of expressing therapeutic levels of B-domain deleted factor VIII (FVIII) at the lowest possible vector dose to minimize the potential Risk of a capsid-mediated immune response in the clinical setting. Here, we describe the studies that identified the investigational agent SPK-8011, currently being evaluated in a phase 1/2 study (NCT03003533) in individuals with hemophilia A. In particular, the potency of our second-generation expression cassettes was evaluated in mice and in non-human primates using two different bioengineered capsids (AAV-Spark100 and AAV-Spark200). At 2 weeks after gene transfer, primates transduced with 2 × 1012 vg/kg AAV-Spark100-FVIII or AAV-Spark200-FVIII expressed FVIII antigen levels of 13% ± 2% and 22% ± 6% of normal, respectively. Collectively, these preclinical results validate the feasibility of lowering the AAV capsid dose for a gene-based therapeutic approach for hemophilia A to a dose level orders of magnitude lower than the first-generation vectors in the clinic.

A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells.

Nine dogs with hemophilia A were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9-11.3% of normal FVIII levels. In two of nine dogs, levels of FVIII activity increased gradually starting about 4 years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of the integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A, while emphasizing the importance of long-term monitoring for potential genotoxicity.

Functionalized lipid-like nanoparticles for in vivo mRNA delivery and base editing.

Messenger RNA (mRNA) therapeutics have been explored to treat various genetic disorders. Lipid-derived nanomaterials are currently one of the most promising biomaterials that mediate effective mRNA delivery. However, efficiency and safety of this nanomaterial-based mRNA delivery remains a challenge for clinical applications. Here, we constructed a series of lipid-like nanomaterials (LLNs), named functionalized TT derivatives (FTT), for mRNA-based therapeutic applications in vivo. After screenings on the materials, we identified FTT5 as a lead material for efficient delivery of long mRNAs, such as human factor VIII (hFVIII) mRNA (~4.5 kb) for expression of hFVIII protein in hemophilia A mice. Moreover, FTT5 LLNs demonstrated high percentage of base editing on PCSK9 in vivo at a low dose of base editor mRNA (~5.5 kb) and single guide RNA. Consequently, FTT nanomaterials merit further development for mRNA-based therapy.

Origins and organization of the NHLBI State of the Science Workshop: Generating a national blueprint for future research on factor VIII inhibitors.

INTRODUCTION: The major complication of protein replacement therapy for haemophilia A is the development of anti-FVIII antibodies or inhibitors that occur in 25%-30% of persons with severe haemophilia A. Alternative therapeutics such as bypassing agents or immune tolerance induction protocols have additional challenges and are not always effective. AIM: Assemble a National Heart, Lung and Blood Institute (NHLBI) State of the Science (SOS) Workshop to generate a national blueprint for research on inhibitors to solve the problem of FVIII immunogenicity. METHODS: An Executive Steering Committee was formed in October 2017 to establish the scientific focus and Scientific Working Groups for the SOS Workshop in May 2018. Four working groups were assembled to address scientific priorities in basic, translational and clinical research on inhibitors. RESULTS: Working Group 1 was charged with determining the scientific priorities for clinical trials to include the integration of non-intravenous, non-factor therapeutics including gene therapy into the standard of care for people with haemophilia A with inhibitors. Working Group 2 established the scientific priorities for 21st-century data science and biospecimen collection for observational inhibitor cohort studies. The scientific priorities for acquiring an actionable understanding of FVIII immunogenicity and the immunology of the host response and FVIII tolerance were developed by Working Group 3. Working Group 4 designed prospective pregnancy/birth cohorts to study FVIII immunogenicity, inhibitor development and eradication. CONCLUSION: The NHLBI SOS Workshop generated a focused summary of scientific priorities and implementation strategies to overcome the challenges of eradicating and preventing inhibitors in haemophilia A.

Infused factor VIII-expressing platelets or megakaryocytes as a novel therapeutic strategy for hemophilia A.

B-domainless factor VIII (FVIII) ectopically expressed in megakaryocytes (MKs) is stored in α granules of platelets (pFVIII) and is capable of restoring hemostasis in FVIIInull mice, even in the presence of circulating inhibitors. However, our prior studies have shown that this ectopically expressed pFVIII can injure developing MKs. Moreover, the known risks of prolonged thrombocytopenia after bone marrow transplantation are significant challenges to the use of this strategy to treat individuals with severe hemophilia A and particularly those with intractable clinically relevant inhibitors. Because of these limitations, we now propose the alternative therapeutic pFVIII strategy of infusing pFVIII-expressing MKs or platelets derived from induced pluripotent stem cells (iPSCs). pFVIII-expressing iPSC-derived MKs, termed iMKs, release platelets that can contribute to improved hemostasis in problematic inhibitor patients with hemophilia A. As proof of principle, we demonstrate that hemostasis can be achieved in vitro and in vivo with pFVIII-expressing platelets and show prolonged efficacy. Notably, pFVIII-expressing platelets are also effective in the presence of inhibitors, and their effect was enhanced with recombinant FVIIa. Human pFVIII-expressing iMKs improved hemostasis in vitro, and derived platelets from infused human pFVIII-expressing iMKs improved hemostasis in FVIIInull mice. These studies indicate the potential therapeutic use of recurrent pFVIII-expressing MK or platelet infusions with prolonged hemostatic coverage that may be additive with bypassing agents in hemophilia A patients with neutralizing inhibitors.

Overexpression of factor VIII after AAV delivery is transiently associated with cellular stress in hemophilia A mice.

Factor VIII (FVIII) is a large glycoprotein that is challenging to express both in vitro and in vivo. Several studies suggest that high levels of FVIII expression can lead to cellular stress. After gene transfer, transgene expression is restricted to a subset of cells and the increased FVIII load per cell may impact activation of the unfolded protein response. We sought to determine whether increased FVIII expression in mice after adeno-associated viral liver gene transfer would affect the unfolded protein response and/or immune response to the transgene. The FVIII gene was delivered as B-domain deleted single chain or two chain (light and heavy chains) at a range of doses in hemophilia A mice. A correlation between FVIII expression and anti-FVIII antibody titers was observed. Analysis of key components of the unfolded protein response, binding immunoglobulin protein (BiP), and C/EBP homologous protein (CHOP), showed transient unfolded protein response activation in the single chain treated group expressing >200% of FVIII but not after two chain delivery. These studies suggest that supraphysiological single chain FVIII expression may increase the likelihood of a cellular stress response but does not alter liver function. These data are in agreement with the observed long-term expression of FVIII at therapeutic levels after adeno-associated viral delivery in hemophilia A dogs without evidence of cellular toxicity.

Minimal modification in the factor VIII B-domain sequence ameliorates the murine hemophilia A phenotype.

Recombinant canine B-domain deleted (BDD) factor VIII (FVIII) is predominantly expressed as a single-chain protein and exhibits greater stability after activation compared with human FVIII-BDD. We generated a novel BDD-FVIII variant (FVIII-RH) with an amino acid change at the furin cleavage site within the B domain (position R1645H) that mimics the canine sequence (HHQR vs human RHQR). Compared with human FVIII-BDD, expression of FVIII-RH protein revealed a 2.5-fold increase in the single-chain form. Notably, FVIII-RH exhibited a twofold increase in biological activity compared with FVIII-BDD, likely due to its slower dissociation of the A2-domain upon thrombin activation. Injection of FVIII-RH protein in hemophilia A (HA) mice resulted in more efficacious hemostasis following vascular injury in both the macro- and microcirculation. These findings were successfully translated to adeno-associated viral (AAV)-based liver gene transfer in HA mice. Expression of circulating FVIII-RH was approximately twofold higher compared with AAV-FVIII-BDD-injected mice. Moreover, FVIII-RH exhibits superior procoagulant effects compared with FVIII-BDD following a series of hemostatic challenges. Notably, the immunogenicity of FVIII-RH did not differ from FVIII-BDD. Thus, FVIII-RH is an attractive bioengineered molecule for improving efficacy without increased immunogenicity and may be suitable for both protein- and gene-based strategies for HA.

Light chain of factor VIII is sufficient for accelerating cleavage of von Willebrand factor by ADAMTS13 metalloprotease.

We previously demonstrated that coagulation factor VIII (FVIII) accelerates proteolytic cleavage of von Willebrand factor (VWF) by A disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS13) under fluid shear stress. In this study, the structural elements of FVIII required for the rate-enhancing effect and the biological relevance of this cofactor activity are determined using a murine model. An isolated light chain of human FVIII (hFVIII-LC) increases proteolytic cleavage of VWF by ADAMTS13 under shear in a concentration-dependent manner. The maximal rate-enhancing effect of hFVIII-LC is ∼8-fold, which is comparable with human full-length FVIII and B-domain deleted FVIII (hFVIII-BDD). The heavy chain (hFVIII-HC) and the light chain lacking the acidic (a3) region (hFVIII-LCΔa3) have no effect in accelerating VWF proteolysis by ADAMTS13 under the same conditions. Although recombinant hFVIII-HC and hFVIII-LCΔa3 do not detectably bind immobilized VWF, recombinant hFVIII-LC binds VWF with high affinity (K(D), ∼15 nM). Moreover, ultra-large VWF multimers accumulate in the plasma of fVIII(-/-) mice after hydrodynamic challenge but not in those reconstituted with either hFVIII-BDD or hFVIII-LC. These results suggest that the light chain of FVIII, which is not biologically active for clot formation, is sufficient for accelerating proteolytic cleavage of VWF by ADAMTS13 under fluid shear stress and (patho) physiological conditions. Our findings provide novel insight into the molecular mechanism of how FVIII regulates VWF homeostasis.

Muscle Gene Therapy for Hemophilia.

Muscle-directed gene therapy for hemophilia is an attractive strategy for expression of therapeutic levels of clotting factor as evident from preclinical studies and an early phase clinical trial. Notably, local FIX expression by AAV-mediated direct intramuscular injection to skeletal muscle persists for years. Development of intravascular delivery of AAV vector approaches to skeletal muscle resulted in vector in widespread areas of the limb and increased expression of FIX in hemophilia B dogs. The use of FIX variants with improved biological activity may provide the opportunity to increase the efficacy of these approaches. Studies for hemophilia A are less developed at this point, but utilizing transgenes that improve hemostasis independent of FIX and FVIII has potential therapeutic application for both hemophilia A and B. Continuous monitoring of humoral and T cell responses to the transgene and AAV capsid in human trials will be critical for the translation of these promising approaches for muscle gene therapy for hemophilia.

Animal models of hemophilia.

The X-linked bleeding disorder hemophilia is caused by mutations in coagulation factor VIII (hemophilia A) or factor IX (hemophilia B). Unless prophylactic treatment is provided, patients with severe disease (less than 1% clotting activity) typically experience frequent spontaneous bleeds. Current treatment is largely based on intravenous infusion of recombinant or plasma-derived coagulation factor concentrate. More effective factor products are being developed. Moreover, gene therapies for sustained correction of hemophilia are showing much promise in preclinical studies and in clinical trials. These advances in molecular medicine heavily depend on availability of well-characterized small and large animal models of hemophilia, primarily hemophilia mice and dogs. Experiments in these animals represent important early and intermediate steps of translational research aimed at development of better and safer treatments for hemophilia, such a protein and gene therapies or immune tolerance protocols. While murine models are excellent for studies of large groups of animals using genetically defined strains, canine models are important for testing scale-up and for long-term follow-up as well as for studies that require larger blood volumes.

Efficacy and safety of long-term prophylaxis in severe hemophilia A dogs following liver gene therapy using AAV vectors.

Developing adeno-associated viral (AAV)-mediated gene therapy for hemophilia A (HA) has been challenging due to the large size of the factor VIII (FVIII) complementary DNA and the concern for the development of inhibitory antibodies to FVIII in HA patients. Here, we perform a systematic study in HA dogs by delivering a canine FVIII (cFVIII) transgene either as a single chain or two chains in an AAV vector. An optimized cFVIII single chain delivered using AAV serotype 8 (AAV8) by peripheral vein injection resulted in a dose-response with sustained expression of FVIII up to 7% (n = 4). Five HA dogs administered two-chain delivery using either AAV8 or AAV9 via the portal vein expressed long-term, vector dose-dependent levels of FVIII activity (up to 10%). In the two-chain approach, circulating cFVIII antigen levels were more than fivefold higher than activity. Notably, no long-term immune response to FVIII was observed in any of the dogs (1/9 dogs had a transient inhibitor). Long-term follow-up of the dogs showed a remarkable reduction (>90%) of bleeding episodes in a combined total of 24 years of observation. These data demonstrate that both approaches are safe and achieve dose-dependent therapeutic levels of FVIII expression, which supports translational studies of AAV-mediated delivery for HA.