Gene Therapy For Hemophilia Research Articles

Hemgenix - A Gene Therapy for Hemophilia B

Etranacogene dezaparvovec-drlb (Hemgenix – CSL Behring), an adeno-associated virus vector-based gene therapy, has been approved by the FDA for treatment of hemophilia B in adults who currently receive factor IX prophylaxis therapy or have had life-threatening or repeated, serious, spontaneous bleeding episodes.

Big stride in gene therapy for hemophilia B in China.

Big stride in gene therapy for hemophilia B in China.

Angiology and hemostasis : what's new in 2022

In this article, we have selected four topics that particularly caught our attention during the year 2022, and which are related to anticoagulation, its bleeding complications, and hemophilia. Thus, we discuss the issue of the treatment with rivaroxaban of atrial fibrillation associated with rheumatic valvulopathy, which has been studied in a randomized trial, the intensity of thromboprophylaxis in COVID outpatients and inpatients, and the bleeding risk of anticoagulation in patients with cerebral tumors. Finally, recent data on gene therapy in severe hemophilia A, an upcoming treatment, are discussed.

Infrastructural considerations of implementing gene therapy for hemophilia in the Nordic context.

Despite improvements in hemophilia care, challenges remain, including treatment burden and impaired quality of life. Gene therapy may overcome these. However, its introduction presents a challenge. To outline a function-based gene therapy working model describing critical milestones associated with gene therapy handling, administration, and follow-up to facilitate and implement an effective infrastructure for gene therapy introduction. Literature review and consensus discussion among Hemophilia Comprehensive Care centers (HCCCs) in the Nordic region. Representatives from six HCCCs sought to pinpoint milestones and key stakeholders for site readiness at the pre-, peri-, and post-infusion stages, including authority and genetically modified organism (GMO) product requirements, awareness, medical eligibility, logistics and product handling for infusion, laboratory monitoring, and follow-up. A gene therapy transit map was developed with key stakeholders identified. The approach to prepare the vector will differ between the Nordic centers, but the contracted pharmacy unit will be a key stakeholder. Therefore, a pharmacy checklist for the implementation of gene therapy was developed. For the future, Advanced Therapy Medicinal Product centers will also be implemented. Patients' expectations, commitments, and concerns need to be addressed repeatedly and education of patients and the expanded health-care professionals team will be the key to successful and optimal clinical management. Eligibility testing according to the product's summary of product characteristics and frequent follow-up and monitoring post-infusion according to the World Federation of Hemophilia chart will be crucial. The approach to deliver gene therapy in the Nordic region will differ partly between the hemophilia centers, but the defined road map with checklists for the implementation of this advanced therapy will be applicable to all. The map may also serve as a platform for the use of future GMO product options both within and outside the area of hemophilia.

The Influence Of Adeno Associated Virus Vector Receptor AAVR And Co-Receptors And Their Mutations On Tropism And Transduction Of Adeno Associated Virus During Gene Therapy A Review Tirthal Rai Usha Adiga Deepika Kamath M

Gene therapy is an experimental treatment being investigated to correct defective genes that are responsible for disease development. To treat cancer and genetic illnesses, researchers are looking into a variety of gene therapy techniques. Finding a proper vector to transfer DNA into tissues is one of the most difficult aspects of gene therapy. Some gene therapy vectors have issues with infecting both quiescent and dividing cells, provoking an immunological response, lack of indefinite expression, and reproducible high titre. The adenovirus, retrovirus, and recombinant modified adeno-associated virus are top contenders for gene therapy vectors (rAAV). The issues with gene therapy may be resolved by the adeno associated virus (AAV). Hemophilia is a condition which may be benefited by the gene therapy using AAV as vector. Aim of this review is to emphasize on the role of adeno associated virus receptors(AAVR) and co-receptors in tropism and transduction of adeno associated virus(AAV) in the gene therapy of hemophilia In Vitro as well as In Vivo.

Pre-existing anti-factor VIII immunity alters therapeutic platelet-targeted factor VIII engraftment following busulfan conditioning through cytotoxic CD8 T cells

BackgroundWe previously demonstrated that busulfan preconditioning enabled sustained therapeutic platelet-derived factor VIII (FVIII) expression in naïve FVIIInull mice transplanted with 2bF8-transduced Sca-1+ cells. However, in mice with pre-existing inhibitors, platelet-FVIII expression was lost. ObjectiveIn this study, we aimed to describe the mechanism of this platelet-FVIII loss. MethodsWe monitored platelet-FVIII expression in FVIIInull mice that were immunized with rhFVIII to induce inhibitors and subsequently conditioned with busulfan before whole bone marrow transplantation or Sca-1+ hematopoietic stem cell transplantation (HSCT) from 2bF8 transgenic (2bF8Tg) mice. Busulfan with or without antithymocyte globulin or anti-CD8 antibody was employed before 2bF8Tg HSCT. Interferon gamma-ELISpot assay was used to assess which subset of cells was the target in platelet-FVIII loss. B-cell–deficient homozygous mutant mice were used to determine whether platelet-FVIII loss in FVIII-primed mice was mediated by antibody-dependent cellular cytotoxicity. ResultsPlatelet-FVIII expression was sustained in 2bF8Tg bone marrow transplantation but not in 2bF8Tg HSCT recipients. CD8 T-cell depletion in addition to busulfan preconditioning restored platelet-FVIII expression in 2bF8Tg-HSCT recipients. ELISpot analyses showed that FVIII-primed CD8 T cells were efficiently restimulated by 2bF8Tg-Sca-1+ cells and secreted interferon gamma, but were not stimulated by 2bF8Tg platelets/megakaryocytes, suggesting that 2bF8Tg-Sca-1+ cells are targets for FVIII-primed CD8 T cells. When 2bF8Tg-Sca-1+ cells were transplanted into FVIII-primed homozygous mutant mice preconditioned with busulfan, no FVIII expression was detected, suggesting that antibody-dependent cellular cytotoxicity was not the mechanism of platelet-FVIII loss in FVIII-primed mice. ConclusionPre-existng immunity can alter the engraftment of 2bF8Tg-Sca-1+ cells through the cytotoxic CD8 T-cell–mediated pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet gene therapy in hemophilia A with inhibitors.

Gene therapy of Hemophilia: Recommendations from the German, Austrian, and Swiss Society for Thrombosis and Haemostasis Research (GTH)

Gene therapy has recently become a realistic treatment perspective for patients with hemophilia. Reviewing the literature and our personal experience from clinical trials, we discuss key aspects of hemophilia A and B gene therapy with vectors derived from adeno-associated virus, including predictable results, risks, adverse events, and patient-reported outcomes. Patient selection, informed consent, administration, and monitoring of gene therapy as well as data collection are explained. We also discuss the need for interdisciplinary cooperation with hepatology and other specialties. We emphasize structural and organizational requirements for treatment centers according to the hub-and-spoke model and recommend the use of electronic diaries to ensure safe and timely collection and exchange of data. Electronic diaries will play a key role as a primary source of data for pharmacovigilance, postmarketing clinical studies, national and international registries, as well as health technology and benefit assessment. Reimbursement aspects and the future of gene therapy in adolescents and children are also considered. In a rapidly evolving scientific environment, these recommendations aim to support treatment providers and payers to prepare for the implementation of gene therapy following marketing authorization.

Factor VIII and Factor IX Activity Measurements for Hemophilia Diagnosis and Related Treatments.

Accurate measurement of clotting factors VIII (FVIII) or IX (FIX) is vital for comprehensive diagnosis and management of patients with hemophilia A or B. The one-stage activated partial thromboplastin time (aPTT)-based clotting assay is the most commonly used method worldwide for testing FVIII or FIX activities. Alternatively, FVIII and FIX chromogenic substrate assays, which assess the activation of factor X, are available in some specialized laboratories. The choice of reagent or methodology can strongly influence the resulting activity. Variation between one-stage FVIII or FIX activities has been reported in the measurement of some standard and extended half-life factor replacement therapies and gene therapy for hemophilia B using different aPTT reagents. Discrepancy between one-stage and chromogenic reagents has been demonstrated in some patients with mild hemophilia A or B, the measurement of some standard and extended half-life factor replacement therapies, and the transgene expression of hemophilia A and B patients who have received gene therapy. Finally, the measurement of bispecific antibody therapy in patients with hemophilia A has highlighted differences between chromogenic assays. It is imperative that hemostasis laboratories evaluate how suitable their routine assays are for the accurate measurement of the various hemophilia treatment therapies.

Laying the foundations for gene therapy in Italy for patients with haemophilia A: A Delphi consensus study.

Current treatment for haemophilia A involves factor VIII replacement or non-replacement (emicizumab) therapies, neither of which permanently normalise factor VIII levels. Gene therapy using adeno-associated viral (AAV) vectors is an emerging long-term treatment strategy for people with severe haemophilia A (PwSHA) that is likely to be available for clinical use in the near future. This article proposes practical guidelines for the assessment, treatment, and follow-up of potential PwSHA candidates for AAV-based gene therapy. Using the Delphi method, a working group of Italian stakeholders with expertise in and knowledge of the care of adults with haemophilia A analysed literature for AAV-based gene therapy and drafted a list of statements that were circulated to a panel of Italian peers. During two rounds of voting, panel members voted on their agreement with each statement to reach a consensus. The Delphi process yielded 40 statements regarding haemophilia A gene therapy, across five topics: (1) organisational model; (2) multidisciplinary team; (3) patient engagement; (4) laboratory surveillance; and (5) patient follow-up and gene therapy outcomes. The consensus was reached for all 40 statements, with the second round of voting needed for five statements. Use of the hub-and-spoke organisational model and multidisciplinary teams are expected to optimise patient selection for gene therapy, as well as the management of dosing and patient follow-up, patient engagement, laboratory surveillance, and patient expectations regarding outcomes. This approach should allow the benefits of AAV-based gene therapy for haemophilia A to be maximised.

Gene therapy for hemophilia: looking beyond factor expression.

Hemophilia A (factor VIII [FVIII] deficiency) and hemophilia B (factor IX [FIX] deficiency) are the X-linked recessive bleeding disorders that clinically manifest with recurrent bleeding, predominantly into muscles and joints. In its severe presentation, when factor activity is less than 1% of normal, hemophilia presents with spontaneous musculoskeletal bleeds and may progress to debilitating chronic arthropathy. Management of hemophilia has changed profoundly in the past decades. From on-demand to prophylactic factor concentrate replacement, the treatment goal shifted from controlling bleeds to preventing bleeds and improving quality of life. In this new scenario, gene therapy has arisen as a paradigm-changing therapeutic option, a one-time treatment with the potential to achieve sustained coagulation FVIII or FIX expression even within the normal range. This review discusses the critical impact of adeno-associated virus (AAV) gene transfer in hemophilia care, including the recent clinical outcomes, changes in disease perceptions, and its treatment burden. We also discuss the challenging scenario of the AAV-directed immune response in the clinical setting and potential strategies to improve the long-lasting efficacy of hemophilia gene therapy efficacy.

FDA approves 2 therapeutic firsts

In a span of less than a week, the US Food and Drug Administration approved two new therapies, each of which is a first-in-class treatment for its respective disease. The first to receive the nod was Provention Bio’s teplizumab, an antibody that can delay the onset of type 1 diabetes in patients 8 years and older. Days later came approval for CSL Behring’s Hemgenix, the first gene therapy for hemophilia B. The one-time treatment restores peoples’ ability to produce proteins that help with blood clot formation.

Interspecies normalization of dose-response relationship for adeno-associated virus-mediated haemophilia gene therapy-Application to human dose prediction.

The aim of this study is to develop an approach to predict human dose-response relationship and first-in-human (FIH) dose for adeno-associated virus (AAV)-mediated haemophilia gene therapy. Preclinical dose-response relationships of 7 AAV vectors were normalized to a species-invariant scale using an exponent of 0.25, and the normalized dose-response relationship was then used for FIH dose prediction. The performance of this dose-response normalization approach for FIH dose prediction was compared to that of direct body weight-based dose (vg/kg) conversion and allometric scaling approaches. A power regression analysis of normalized factor VIII (FVIII) or factor IX (FIX) showed a moderate-to-strong correlation between FVIII or FIX and vector dose across 3 species (R2 ranged 0.59 to 0.89), indicating it was feasible to normalize dose-response in multiple species to a species-invariant scale. When the mean values of normalized FVIII or FIX at each dose level were used for regression, the R2 values of the 7 vectors were improved to be >0.84. The FIH doses predicted by the 3 approaches were ranked as allometric scaling > dose-response normalization > direct vg/kg conversion for all vectors except for rAAV2-hAAT-FIX and scAAV2/8-LP1-hFIXco. Among the 7 vectors, dose-response normalization, direct vg/kg conversion and allometric scaling generated accurate FIH dose predictions for 2, 2 and 3 vectors, respectively. This study first demonstrated that it is feasible to normalize dose-response relationship of AAV-mediated haemophilia gene therapy in multiple species to a species-invariant scale. The normalized dose-response relationship from preclinical species was successfully extrapolated to patients for FIH dose prediction.

Liver-related aspects of gene therapy for haemophilia: Call to action for collaboration between haematologists and hepatologists

Liver-related aspects of gene therapy for haemophilia: Call to action for collaboration between haematologists and hepatologists

Transient expression of factor VIII and a chronic high-fat diet induces ER stress and late hepatocyte oncogenesis

Liver-directed gene therapy using adeno-associated virus (AAV) for delivery of therapeutic transgenes has posed particular challenges over the past 25 years, and there are no FDA-approved products to date. Reasons include lack of efficacy or toxicity or both, despite acceptable efficacy and toxicology results in animals, including non-human primates. The experience with AAV-mediated gene therapy for hemophilia is a case in point. The clotting factor VIII gene was cloned in 1984, but production of the full-length recombinant protein is challenging.

Hemophilia “A” gene therapy: Lost in translation

Successful gene therapy for hemophilia A had been considered as one of the “holy grails” in the gene therapy field. An important milestone toward realizing this goal had been achieved (https://www.ema.europa.eu/en/news/first-gene-therapy-treat-severe-haemophilia) following the first conditional marketing authorization approval in the European Union for an adeno-associated virus type 5 (AAV5)-based gene therapy strategy.1 Most of the treated patients remained off prophylaxis and bleed-free 2 years after dosing and up to 5 years in a supporting trial.

Efficacy and Safety of Viral Vector Based Gene Therapy in Patients with Hemophilia a: A Systematic Review

Background: Hemophilia A results from a mutation in the gene for coagulation factor VII and serious disease can cause bleeding in soft tissues and joints. These patients rely on exogenous factor VIII. However, frequent infusions are needed due to the short half-life of factor VIII which can lead to non-compliance to therapy and poor quality of life. Gene therapy aims to safely impart the expression of stable factor VIII. In this review, we will assess the efficacy and safety of gene therapy in hemophilia A. Methods: We searched PubMed, Web of Science, and clinicaltrials.gov with mesh terms, "hemophilia A" and "genetic therapy" from the inception of data till May 30, 2022. Out of 6,699 articles, 4 clinical trials (N=173) were included based on inclusion criteria measuring the efficacy and safety of gene therapy in hemophilia A. Case reports, review articles, systematic reviews, meta-analyses, pre-clinical studies, and clinical trials with irrelevant treatment or population were excluded. Results: In 4 clinical trials, 173 adult patients (<18 years old) were treated with vector-based gene therapy. Adeno-associated virus serotype 5 (AAV5) vector was used in 155 patients while SPK-8011 was used in 18 patients. 146 patients were treated at a high dose of 6x1013 vg/kg. All patients had a factor VIII activity of <1 IU/dl before therapy. In 2 clinical trials (N=139), mean factor VIII activity significantly increased to 45.42 IU/dl on treatment with 6x1013 vg/kg of AAV5-hFVIII-SQ. However, the activity level was ≤2 IU/dl after treatment at lower doses. On three years follow-up by Pasi et al. (N=7), the mean factor VIII activity level was 31 IU/dl. The mean annual bleeding rate decreased from 5.95 (N=139) to 0.95 after therapy. Mean consumption of factor VIII decreased from 4130.8 (N=139) to 21.84 IU/kg after treatment with 6x1013 vg/kg of AAV5-hFVIII-SQ. In one clinical trial by George et al., 18 patients were treated with SPK-8011-FVIII-SQ and the median annual bleeding rate decreased from 8.5 to 0.3. Mean factor VIII activity was 8 at a dose of 2x1012. ALT elevations, adrenal insufficiency, pyrexia, diarrhea, rectal hemorrhage, and progression of arthropathy were among the serious side effects reported in patients with gene therapy. Table 1. Conclusion: Gene therapy was well tolerated by most of the patients and ALT elevation was the most common adverse effect reported. AAV5-hFVIII-SQ and SPK-8011-FVIII-SQ significantly improved the endogenous production of factor VIII, bleeding rate, and factor VIII concentrate consumption, especially at doses of 6x1013 and 2x1012 vg/kg, respectively. AAV5-hFVIII-SQ was safe and effective in patients with hemophilia A on long-term follow-up. More randomized double-blind clinical trials are needed to confirm these results. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

AAV8 Gene Therapy for Hemophilia A Patients from China

Background Treatments of hemophilia A patients by adeno-associated virus (AAV) based delivery of coagulation factors VIII (FVIII) have shown great promise, with multiple clinical trials be conducted currently. However, to ensure enough expression level of FVIII, the dosages of AAV capsids are relatively high (up to 6E13vg/kg) in these trials. High dosages of AAV capsid carry the potential risk of toxicity, raise the possibilities of host immune response and increase the economic burden for patients. Besides, the sustainability of efficacy is also a challenge. Current gene therapies showed that the level of FVIII gradually decreased over time. Hence, it is necessary to explore a gene therapy with high expression level of FVIII and sustained efficacy. GS001, an rAAV8 vector expressing B domain deleted FVIII was developed. In the preclinical study, high level of FVIII activity (FVIII:C) (155 IU/dl) was achieved when the hemophilia A mice received a single infusion of GS001 at a dose of 1.1E11 vg/kg. Toxicity studies in monkeys showed good tolerance at the dose of 2E13 vg/kg. Based on these preclinical data, GS001 was investigated in clinical trial for hemophilia A gene therapy. The purpose is to evaluate the safety and efficacy of GS001 in Chinese hemophilia A patients and obtain clinical data for further study. Method We enrolled Chinese hemophilia A patients (> 18 years) whose baseline FVIII:C was < 1 IU/dl without FVIII inhibitor. All subjects had low titer of neutralizing antibodies (Nab) (≤1:5) against the vector capsid. Eligible participants received a single intravenously infusion of GS001 with a dose of 2E12vg/kg after one week of prophylactic prednisone treatment or prednisone and tacrolimus treatment. The primary endpoint was safety including treatment-related adverse events (AEs), change of alanine aminotransferase (ALT) and antibodies against the vector capsid. Enrollment is now ongoing. This study was registered at ClinicalTrials.gov, NCT04728841. Results Six patients were enrolled. The first three patients (patients 1 to 3) were infused with GS001 after one week of prophylactic prednisone treatment. The mean follow-up time were 397±52 days. FVIII:C level reached the peak around seven weeks post infusion. After that, the FVIII:C decreased with the elevation of ALT, and the decrease trend was improved after treatment with tacrolimus. Patients 4 to 6 were infused with GS001 after one week of prophylactic prednisone and tacrolimus treatment. The mean follow-up time were 49±8 days. The FVIII:C level quickly reached peak level in the first week compared to the first three participants. No rising trend of ALT was found. The mean annual bleeding rates (ABR) and the factor VIII usage for all the six participants decreased dramatically after GS001 infusion. There were totally 55 AEs, with 54 AEs were grade 1(54 AE) and only one was grade 2 which was not related to GS001. Twenty-two (40%) AEs were possibly related to the immunosuppressant therapy, only 12.7% of the AEs (7/55) was possibly related to GS001. No severe adverse events have been observed. None of the six participants had developed a FVIII inhibitor. Conlcusions This pilot study demonstrated the safety and tolerance of GS001. Vector derived FVIII:C level was sufficient to prevent bleeding events and minimize the need for replacement therapy. Prophylaxis treatment of tacrolimus combined with prednisone could be an effective immunosuppressive regimen against the vector capsid in gene therapy for hemophilia A patients. These findings support further studies.

Durability of Factor IX Activity and Bleeding Rate in People with Severe or Moderately Severe Hemophilia B after 5 Years of Follow-up in the Phase 1/2 Study of AMT-060, and after 3 Years of Follow-up in the Phase 2b and 2 Years of Follow-up in the Phase 3 Studies of Etranacogene Dezaparvovec (AMT-061)

Introduction: Gene therapy for people with hemophilia B appears to have a durable response, with data presented over 5 years (Samelson-Jones. Blood 2021; 138_suppl 1: 3975) and 8 years (Nathwani. Blood 2018; 132_suppl 1: 491) post-administration. Here we review the durability of AMT-060 and etranacogene dezaparvovec (formerly AMT-061) investigational gene therapies for hemophilia B, defined by sustained factor IX (FIX) activity levels and hemostatic protection. AMT-060 was developed as a precursor to etranacogene dezaparvovec. Both gene therapies are almost identical, comprising an adeno-associated virus serotype 5 (AAV5) vector containing a FIX transgene under the control of a liver-specific promoter; the transgene encodes wild-type FIX in AMT-060 and the highly active Padua variant of FIX in etranacogene dezaparvovec, differing by only one amino acid. Therefore, durability of response is expected to be similar for AMT-060 and etranacogene dezaparvovec. Aim: Outline the observed durability of AMT-060 and etranacogene dezaparvovec in people with severe or moderately severe hemophilia B. Methods: Three clinical trials are ongoing in adults with severe or moderately severe hemophilia B. Data have been captured over 5 years in the Phase 1/2 study assessing AMT-060 (N=10; Cohort 1: n=5, 5x1012 gc/kg ; Cohort 2: n=5, 2x1013 gc/kg; NCT02396342), 3 years in the Phase 2b study assessing etranacogene dezaparvovec (N=3, 2x1013 gc/kg; NCT03489291) and 2 years in the Phase 3 HOPE-B study assessing etranacogene dezaparvovec (N=54, 2x1013 gc/kg; NCT03569891). Study designs have been reported previously. All three studies assessed FIX activity levels and bleeding events. Results: In the Phase 1/2 AMT-060 study, FIX activity levels remained stable in both cohorts 5 years after dosing. Mean FIX activity was 4.4% for Cohort 1 at 52 weeks and 6.9% for Cohort 2 at 26 weeks; mean FIX activity after 5 years was 5.2% and 7.4%, for Cohorts 1 and 2, respectively. Greater FIX activity levels were achieved with etranacogene dezaparvovec. After 3 and 2 years of follow-up in the Phase 2b and 3 studies, respectively, mean FIX activity levels remained in the near-normal range (Table). In the Phase 2b study, mean FIX activity was 23.4% at Week 3 (n=3); this increased to 36.9% at Year 3 (n=2). A sustained mean FIX activity level was seen in the Phase 3 study: 38.95% at Month 6 (n=51) and 36.66% at Year 2 (n=50). This was observed in both the full analysis set (n=54) and in the modified intent-to-treat population (n=52) that excluded two non-responders (one with the highest AAV5 neutralizing antibody titer of 3212; one that received only a partial dose; Table). With regards to hemostatic protection, in Year 1 post-AMT-060 administration, the mean annualized bleeding rate (ABR) was 7.30 in Cohort 1, and this decreased to 6.40 at Year 5. In Cohort 2, mean ABR decreased from 1.58 in Year 1 to 0.20 in Year 5. The etranacogene dezaparvovec studies showed a similar low bleeding rate. In the Phase 2b study, no bleeding episodes occurred between 2.5-3 years of follow-up. Overall, the ABR over 3 years was 0.22. Two bleeding episodes (one very mild, spontaneous bleed; one traumatic) occurred in one participant within 18 months of dosing; the participant received FIX product replacement therapy (1700 IU) after each bleed. In the Phase 3 study, ABR (all bleeds) achieved at Months 7-18 post-administration was 1.51 (reduced from 4.18 at baseline); this was maintained through to 2 years in the full analysis set (n=54; Table). A similar trend was seen when assessing the modified intent-to-treat population (n=52), with a reduction in ABR from 4.00 at baseline to 0.95 after 2 years of follow-up. Reductions in ABR for spontaneous, joint, and traumatic bleeds were also maintained through 2 years of follow-up. No new safety signals have been identified after 5 years of follow-up for AMT-060 and up to 3 years of follow-up for etranacogene dezaparvovec. Conclusions: Gene therapy for hemophilia B appears to have a durable response. Across the Phase 1/2 study of AMT-060 and the Phase 2b and Phase 3 studies of etranacogene dezaparvovec, FIX activity levels are sustained throughout the observation period. Reductions in bleeding events also remained stable across the course of the three studies. Similar durable responses have been observed in published literature for hemophilia B gene therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long-Term Real-World Safety and Efficacy of Liver-Directed AAV Gene Therapy for Severe Hemophilia with and without Inbibitors in Privately Owned Dogs

Background Hemophilia A (HA) and hemophilia B (HB) are inherited bleeding disorders resulting from deficiency of coagulation factors VIII (FVIII) or factor IX (FIX) activity, respectively. HA and HB both occur naturally in dogs. The bleeding phenotype of severe canine HA and HB is analogous to the human patient experience with frequent spontaneous and trauma-induced bleeding. Previous studies of dogs from HA and HB colonies at research institutes have been highly informative about the safety and efficacy of adeno-associated virus (AAV) liver-directed gene therapy for HA and HB. However, these animals are housed in highly controlled environments; in contrast, pet dogs with HA or HB are exposed to real-world challenges that likely affect their bleeding phenotype and the efficacy of novel therapeutics. In this regard, privately owned dogs may better recapitulate the human patient experience. Aims To determine the real-world efficacy of liver-directed AAV gene therapy for HA/HB in privately owned canines with and without inhibitors. Methods Eleven dogs with severe HA and one dog with severe HB were treated with liver-directed AAV gene therapy with canine (c) FVIII or cFIX, as detailed in Table 1. Pre-existing neutralizing antibodies to AAV8 were detected using a luciferase-based transduction inhibition assay. Pre- and post-gene therapy annualized bleeding rates were calculated by cumulative bleeding events divided by the age at gene therapy (pre) or duration of follow-up (post). FVIII activity was determined using a chromogenic assay and FIX activity using a one-stage assay. Results All dogs had AAV8 neutralizing antibody titers ≤ 1:5 at enrollment. The HA dogs (10 males and 1 female) were treated with either dual chain vectors of cFVIII-SQ (n = 2, total dose of 5 x 1013 vg/kg) or single-chain vectors encapsidating high-expression cFVIII variants: cFVIII-ΔF (n = 2, 6x1012 vg/kg) or cFVIII-ΔF/V3 (n = 7, 5.4-9 x 1012 vg/kg). F8 genetic testing revealed intron 22 inversions (n=7), small deletion (n=2), missense (n=1) and complex mutation (n=1) all compatible with severe HA. Of note, one HA dog (PC3) had a pre-existing FVIII inhibitor which was eradicated following gene therapy. In all dogs with available data (n=10), FVIII activity is > 1% with 5 in the moderate hemophilia range and 4 in the mild range with a median FVIII activity of 3.2% (range 1.1-13.8%) after a median duration of 4.1 years (range 2.6-8.9 years). In HA dogs, the ABR post-gene therapy declined by an average of 93%. The sole HB dog carried two missense F9 mutations and was treated with wildtype cFIX at 3 x 1012 vg/kg and has been followed for 4.5 years with FIX activity of 5.7% and has had no bleeding episodes since gene therapy. Three animals have died during follow up. The previously inhibitor positive dog (PC3) died approximately 2 years after gene therapy from an unrelated cause. An HA dog (PC1) treated with dual chain vector with expression in the ~2% normal range died from a bleeding episode at 8.8 years after gene therapy. Notably, many of his bleeds were associated with real-world activities including having his legs being pulled by a toddler. A third HA dog (PC9) was euthanized 3.5 years post-gene therapy after being diagnosed with widely disseminated cancer; the details of the vector integration analysis of his tumor will be reported elsewhere. Conclusion AAV gene therapy was successful at substantially ameliorating the bleeding phenotype in both male and females dogs with > 1 year of follow-up. However, some dogs continued to have bleeding, especially animals with <5% normal cFVIII activity. One animal developed cancer after gene therapy; there were no other safety concerns. As we have described previously, liver-directed AAV gene therapy also induced immune tolerance in an HA dog with a pre-existing inhibitors. We show that AAV liver-directed gene therapy is safe and efficacious in a real-world experience of privately owned dogs with severe HA and HB. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Vector Analysis of Multicentric Lymphoma in a Severe Hemophilia Α Dog after AAV Gene Therapy

Background Adeno-associated viral (AAV) vector gene therapy for severe hemophilia A (HA) is approaching a clinical reality with multiple successful clinical trials. Despite this success, questions remain about the long-term safety of AAV vectors, especially regarding genomic integration and resulting oncogenesis. Studies of neonatal mice have demonstrated AAV integrations that drive hepatocellular carcinoma formation (Donsante et al. Science, 2007). Studies in HA canine models have also demonstrated AAV integrations and clonal expansion of hepatocytes with integrated vector genomes, but no evidence of tumor formation (Nguyen et al. Nat Biotechnol, 2021). Additionally, three tumors have been reported in AAV gene therapy recipients with HA or hemophilia B in clinical trials, without evidence of vector integrations driving oncogenesis. Though overall reassuring, these data highlight that the field needs to remain vigilant about the possibility of AAV integrations driving cancer development. Thus, the assessment of tumors in gene therapy recipients remains important for the AAV field. Aims To determine the role of AAV vector in the development of multicentric lymphoma in a severe HA dog that received liver-directed gene therapy. Methods Vector genome copy numbers in relevant tissues were determined by real-time PCR with primers situated in the 3'-end of the promoter and the 5'- end of the transgene. The vector copy number was calculated by comparing the analyte amplification signal against a standard curve of the plasmid DNA. Results A privately owned mixed-breed dog (PC9) with a history of recurrent spontaneous bleeding events presented with canine factor VIII (cFVIII) levels <1% normal, consistent with severe HA. At 9-months of age, he received liver-directed AAV8 gene therapy with a high-expressing canine factor FVIII (cFVIII-ΔF/V3) variant transgene at a dose of 6 x 1012 vg/kg via intravenous administration. Following gene therapy, his cFVIII levels increased to 2-3% normal and remained stable through duration of follow-up. His annualized bleeding rate decreased from 8 pre-gene therapy to 0 post-gene therapy. At 3.5 years after vector administration, PC9 was diagnosed with a widely disseminated cancer and euthanized due to complications of the disease. At autopsy, he was diagnosed with multicentric lymphoma involving peripheral and visceral lymph nodes, spleen, liver, and lungs. The average age of lymphoma diagnosis in dogs is between 6-9 years of age (Edwards et al. Vet Comp Oncol, 2003), so the development of cancer at an early age in a gene therapy recipient warranted additional study. Vector copy number analysis showed that the liver had the highest levels of AAV vector genomes, as expected. Liver samples showed a heterogenous distribution of vector genomes, consistent with previous studies of AAV8 transduction (Bell et al. Hum Gene Ther, 2011). Splenic samples also had a high level of AAV vector genomes, which again is consistent with known targeting of AAV8. Importantly, the lymph nodes had no detectable AAV vector genomes present, suggesting that vector integration could not contribute to lymphoma development. cFVIII levels remained stable before and after diagnosis of lymphoma, supporting lack of vector integration in the malignant cells. Conclusions AAV gene therapy resulted in a 100% reduction in the annualized bleeding rate in this pet dog with severe HA. Furthermore, the multicentric lymphoma that developed 3.5 years after liver-directed gene therapy is unlikely related to the AAV vector. We are unable to rule out other factors that may have contributed, including environmental influences or genetic predispositions for lymphoma. These results support the ongoing efforts to treat HA with AAV-based gene therapy and reinforce the safety profile of AAV vectors outside of a laboratory environment.