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

  • Wolfgang Miesbach
    Correspondence
    Corresponding author. Address: Medical Clinic 2 / Institute of Transfusion Medicine and Immunohaematology, University Hospital Frankfurt, Germany; Tel.: +49 69 6301-5051, fax: +49 69 6301-6738.
    Affiliations
    Medical Clinic 2 / Institute of Transfusion Medicine and Immunohaematology, University Hospital Frankfurt, Germany
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  • Graham R. Foster
    Affiliations
    Department of Hepatology, Queen Mary University of London, United Kingdom
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  • Flora Peyvandi
    Affiliations
    Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy

    Università degli Studi di Milano, Department of Pathophysiology and Transplantation, Milan, Italy
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Published:November 24, 2022DOI:https://doi.org/10.1016/j.jhep.2022.11.014
      See Article, pages xxx–xxx

      Introduction

      Close collaboration between haematologists (haemophilia treaters) and hepatologists led to the early diagnosis and treatment of viral infections, such as hepatitis C, which occurred in most individuals with haemophilia, until viral inactivated coagulation factor concentrates became available.
      • Yang X.
      • Jeong K.
      • Yabes J.G.
      • Ragni M.V.
      Prevalence and risk factors for hepatocellular carcinoma in individuals with haemophilia in the era of direct-acting antiviral agents: a national inpatient sample study.
      Now that hepatitis C virus can be eradicated, this proven collaboration should be reinitiated for people with haemophilia who are treated with therapeutically administered viruses.
      Non-pathological adeno-associated viruses (AAVs) serve as vectors for liver-targeted gene transfer for the potentially long-term prevention of bleeding in haemophilia.
      However, different immunological and cellular responses of liver cells may lead to different degrees of hepatoxicity and subsequently unsuccessful gene therapy outcomes.
      • Leebeek F.W.G.
      • Miesbach W.
      Gene therapy for hemophilia: a review on clinical benefit, limitations, and remaining issues.
      Therefore, the purpose of this statement is to provide an overview of the liver-related aspects of gene therapy in haemophilia and to highlight how the management of gene therapy could be further improved through close collaboration between haematologists and hepatologists.

      Recent developments in gene therapy for haemophilia

      Haemophilia A (HA) and haemophilia B (HB) are X-linked severe bleeding disorders characterised by an absence or reduction of coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Both FVIII and FIX are mainly synthesized in the liver.
      There are approximately 500,000 individuals with haemophilia worldwide, of whom about 50% suffer from a severe form and are at high risk of spontaneous bleeding.
      • Srivastava A.
      • Santagostino E.
      • Dougall A.
      • Kitchen S.
      • Sutherland M.
      • Pipe S.W.
      • et al.
      WFH guidelines for the management of hemophilia panelists and co-authors. WFH guidelines for the management of hemophilia, 3rd edition.
      Despite established therapy with regular intravenous substitution of clotting factors or, in some cases, regular subcutaneous injections of emicizumab, there is still an increased tendency to bleed, especially into the joints, leading to progressive joint destruction.
      To deliver the gene product into cells, the most advanced gene therapy for monogenic diseases uses recombinant AAVs (rAAV), which are characterized by high liver specificity and the ability to remain in predominantly episomal circular forms, with very low functional integration rates into genomic DNA.
      • Schnepp B.C.
      • Chulay J.D.
      • Ye G.-J.
      • Flotte T.R.
      • Trapnell B.C.
      • Johnson P.R.
      Recombinant adeno-associated virus vector genomes take the form of long-lived, transcriptionally competent episomes in human muscle.
      However, in preclinical models, liver samples from six dogs treated with canine FVIII and either an AAV8 or AAV9 vector showed 1,741 unique AAV integration events in genomic DNA and partially expanded cell clones with 44% near genes involved in cell growth. All integrated vectors were partially removed and/or restructured, and no altered liver function or tumour development was observed after a period of up to 10 years.
      • Nguyen G.N.
      • Everett J.K.
      • Kafle S.
      • Roche A.M.
      • Raymond H.E.
      • Leiby J.
      • et al.
      A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells.
      In this context, a possible association between AAV2 infection and paediatric cases of idiopathic hepatitis has recently been described and will be further evaluated.
      • de Jong Y.P.
      • Herzog R.W.
      AAV and hepatitis: cause or coincidence?.
      Clinical gene therapy trials for haemophilia involve the combination of different dosages, different AAV capsids, liver-specific promoters and gene cassettes.
      • Leebeek F.W.G.
      • Miesbach W.
      Gene therapy for hemophilia: a review on clinical benefit, limitations, and remaining issues.
      Clinical trials include adult patients with severe or moderate haemophilia who have previously been treated with clotting factor concentrates after ruling out manifest liver disease and the presence of antibodies to AAV or clotting factors. Given the prospects, but also the potential risks, of gene therapy, it is mandatory that individuals with haemophilia and their families be empowered, through transparent communication, to fully participate in the decision-making process.
      • Wang M.
      • Negrier C.
      • Driessler F.
      • Goodman C.
      • Skinner M.W.
      The hemophilia gene therapy patient journey: questions and answers for shared decision-making.
      Phase I to III studies have demonstrated therapeutic expression of FVIII and FIX, significantly reducing bleeding compared to standard treatment. Sustained expression of coagulation factors for up to 6 years has been described for HA

      Laffan M, Rangarajan S, Lester W, Symington E, Madan B, Hart D, et al. Hemostatic results for up to 6 years following treatment with valoctocogene roxaparvovec, an AAV5-hFVIII-SQ gene therapy for severe hemophilia A [abstract]. https://abstracts.isth.org/abstract/hemostatic-results-for-up-to-6-years-following-treatment-with-valoctocogene-roxaparvovec-an-aav5-hfviii-sq-gene-therapy-for-severe-hemophilia-a/.

      and up to 8 years for HB.
      • Nathwani A.C.
      • Reiss U.
      • Tuddenham E.
      • Chowdary P.
      • McIntosh J.
      • Riddell A.
      • et al.
      Adeno-associated medi- ated gene transfer for hemophilia B:8 Year follow up and impact of removing “empty viral particles” on safety and efficacy of gene transfer.
      However, after administration of gene therapy, an increase in certain liver parameters, particularly alanine aminotransferease (ALT), has been observed in many clinical studies and has been partially associated with loss of transgene expression. Elevated ALT levels may be accompanied by elevated aspartate aminotransferase levels, but bilirubin levels or laboratory markers of liver synthesis are not affected.
      According to recently reported phase III trials results, the number of cases of ALT elevation and the consecutive need for immunosuppression is higher in gene therapy for HA than in gene therapy for HB. An increase in liver enzymes with the need for immunosuppression was present in 85% of patients receiving therapy with valoctocogene roxaparvovec for the treatment of HA. The median duration of treatment with immunosuppressants was 230 (22-551) days. Of patients treated with glucocorticoids, 71.8% developed steroid-typical side effects.
      • Ozelo M.C.
      • Mahlangu J.
      • Pasi K.J.
      • Giermasz A.
      • Leavitt A.D.
      • Laffan M.
      • et al.
      GENEr8-1 trial group. Valoctocogene roxaparvovec gene therapy for hemophilia A.
      Among patients with HB treated with etranacogene dezaparvovec, 17% had an increase in liver enzymes. The mean duration of steroid treatment was 79 days and was completed by week 26 in all patients.

      Miesbach, W., Leebeek F.W.G., Recht M., Key N.S., Lattimore S., Castaman G., et al. Final analysis from the pivotal phase 3 HOPE-B Gene Therapy trial: Stable steady-state efficacy and Safety of etranacogene dezaparvovec in adults with severe or moderately severe haemophilia B [abstract EAHAD 2022 and CSL Behring Press Release 4th Feb 2022]. 22.07.2022]; Available from: https://eahad.org/eahad-2022-latest-clinical-trial-results-session-press-release/; https://www.cslbehring.com/newsroom/2022/hope-b-data-presented-at-eahad2022.

      In the long term, a hypothetical risk of malignancy remains due to integration of the vector into the genome, although recent data from liver biopsies show no liver toxicity or malignancy.
      • Fong S.
      • Yates B.
      • Sihn C.R.
      • Mattis A.N.
      • Mitchell N.
      • Liu S.
      • et al.
      Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A.
      Recently, the occurrence of hepatocellular carcinoma in a >65-year-old patient with a history of hepatitis C (eradicated 3 years prior to enrolment), hepatitis B and evidence of non-alcoholic fatty liver changes was reported. The histopathological examination revealed no connection with the gene therapy.

      Schmidt, M.R., Foster G.R., Coppens M., Thomsen H., Cooper D., Dolmetsch R., et al. Liver Safety Case Report from the Phase 3 HOPE-B Gene Therapy Trial in Adults with Hemophilia B [abstract, ISTH 2021]. 2021 22.07.2022]; Available from: https://abstracts.isth.org/abstract/liver-safety-case-report-from-the-phase-3-hope-b-gene-therapy-trial-in-adults-with-hemophilia-b/.

      A similar approach with careful histopathological work-up will be required in future cases of malignancies.
      Currently, there is limited data on the long-term effects of gene therapy on the liver and long-term data collection is necessary. In the clinical trial of gene therapy for haemophilia with the longest follow-up, no liver toxicities or hepatocellular carcinomas associated with gene therapy were observed after 15 years.
      • George L.A.
      • Ragni M.V.
      • Rasko J.E.J.
      • Raffini L.J.
      • Samelson-Jones B.J.
      • Ozelo M.
      • et al.
      Long-term follow-up of the first in human intravascular delivery of AAV for gene transfer: AAV2-hFIX16 for severe hemophilia B.
      Based on knowledge and experience with gene therapy, it has been proposed that gene therapy for haemophilia can be delivered by multidisciplinary haemophilia treatment centres (HTCs) collaborating in a hub-and-spoke model, for example.
      • Miesbach W.
      • Chowdary P.
      • Coppens M.
      • Hart D.P.
      • Jimenez-Yuste V.
      • Klamroth R.
      • et al.
      Delivery of AAV-based gene therapy through haemophilia centres-A need for re-evaluation of infrastructure and comprehensive care: a Joint publication of EAHAD and EHC.
      In line with the changing role of HTCs in supporting gene therapy, there will also be new demands on the various members of the HTCs, for example in physiotherapy to restore joint health or even in some cases when psychological support is needed. Collaboration with a hepatology centre is a prerequisite for effective and safe delivery of gene therapy, including for assessment of liver health before and after gene therapy and management of ALT elevation.

      Assessment of liver health before and after gene therapy

      The inclusion and exclusion criteria of the clinical gene therapy trials provide useful information to better identify the target population and to avoid gene therapy-induced liver reactions.
      The following liver-related exclusion criteria have been established:
      • Pre-existing liver disease (cirrhosis/advanced fibrosis/malignancy)
      • Active/chronic viral infections with hepatitis B or hepatitis C virus
      • Hepatotoxic medication (e.g., HIV medication)
      Transaminase levels should be consistently within the normal range prior to therapy. A slight increase in bilirubin is not always a criterion for exclusion. Specific risk estimation is needed when including patients who have recovered from hepatitis B or hepatitis C virus infection.
      It is recommended that liver imaging by abdominal ultrasound and FibroScan be evaluated by an experienced hepatologist. No evidence of cirrhosis should be detected and the FibroScan value should not exceed 11.5 kPa to rule out advanced fibrosis and cirrhosis. Even with borderline FibroScan values between 6 and 8 kPA and definitely above, a highly individualised approach should be chosen.
      After gene therapy, certain lifestyle factors and adherence to follow-up visits should be assured to maintain liver health:
      • Alcohol abstinence for at least 6 months after administration of gene therapy
      • Weight management
      • Not taking hepatotoxic medications
      • No excessive physical activity
      • Participation in follow-up examinations
      In addition to regular laboratory measurement of transaminases after gene therapy, ultrasound examination of the liver should be performed every 6 months to rule out hepatotoxicity and liver-related malignancy. All patients should be included in national and international registries to ensure lifelong follow-up.
      • Konkle B.
      • Pierce G.
      • Coffin D.
      • Naccache M.
      • Clark R.C.
      • George L.
      • et al.
      ISTH subcommittee on Factor VIII, Factor IX, rare bleeding disorders. Core data set on safety, efficacy, and durability of hemophilia gene therapy for a global registry: communication from the SSC of the ISTH.

      Characteristics and management of ALT elevation

      ALT elevation has been observed in all studies of gene therapy for haemophilia, peaking 1.5- to 2-fold above the upper normal limit in most cases. It occurs in up to 86% of patients,
      • Ozelo M.C.
      • Mahlangu J.
      • Pasi K.J.
      • Giermasz A.
      • Leavitt A.D.
      • Laffan M.
      • et al.
      GENEr8-1 trial group. Valoctocogene roxaparvovec gene therapy for hemophilia A.
      most commonly at weeks 7-14 post infusion, and is more common in studies of HA gene therapy than in HB gene therapy, which is also likely to depend on the dosage of the vector but not on the capsid, genome, or promoter used or the manufacturing method.
      • Srivastava A.
      • Santagostino E.
      • Dougall A.
      • Kitchen S.
      • Sutherland M.
      • Pipe S.W.
      • et al.
      WFH guidelines for the management of hemophilia panelists and co-authors. WFH guidelines for the management of hemophilia, 3rd edition.
      In all patients, ALT elevation is asymptomatic and transient but may be associated with a reduction or loss of transgene expression.
      The circumstances of ALT elevation and its influence on the decline in transgene expression are still unclear, indicating critical gaps and the need for further studies. After initial treatment with immunosuppressive agents, the initial elevated transgene level could not be restored in some patients. Some studies have also investigated the prophylactic use of immunosuppressants,
      • Chowdary P.
      • Shapiro S.
      • Makris M.
      • Evans G.
      • Boyce S.
      • Talks K.
      • et al.
      Phase 1-2 trial of AAVS3 gene therapy in patients with hemophilia B.
      but consensus on the dosage, type and duration of immunosuppressive treatment has not yet been reached. An unpredictable T-cell-induced immune response against transduced liver cells could be responsible for the presentation of capsid fragments of the viral vector on their surface, but the results of the different studies are not consistent.
      • Leebeek F.W.G.
      • Miesbach W.
      Gene therapy for hemophilia: a review on clinical benefit, limitations, and remaining issues.
      Oxidative stress, other immune responses, and concomitant use of drugs with hepatotoxic effects are also considered as possible alternative mechanisms. In the future, liver biopsies could help clarify this issue.
      To date, all ALT elevations were transient and could be successfully treated by tapering doses of immunosuppressants, mainly glucocorticoids. However, in some patients, the initial elevated transgene level could not be restored. Some studies have also investigated the prophylactic use of immunosuppressants, but consensus on the dosage, type and duration of immunosuppressive treatment has not yet been reached.

      Close monitoring and treatment of ALT elevation

      Close monitoring of ALT and coagulation factors is important for the early initiation of treatment, to monitor the effect of treatment, and to allow for timely tapering of immunosuppressive treatment.
      There is further controversy as to what ALT change should be considered relevant. In many studies, an increase of 1.5-fold from baseline is the threshold at which immunosuppression is initiated. In addition, creatine kinase levels should be measured to rule out ALT elevation due to physical activity.
      It has been shown that early initiation of immunosuppression can contribute to the preservation of transgene expression. During the first year after gene therapy, it may be necessary to repeat liver function tests several times a week to every two months and every three months thereafter. More than one year after treatment, ALT elevation is less closely linked to the reduction or loss of transgene expression.
      The strategy of immune suppression includes initiation of high-dose prednisolone treatment to control the immune response, but also rapid dose reduction to minimise toxicities. In many studies, prednisolone is used at a dosage of 60 mg daily, followed by tapering of the dosage.
      This should be combined with additional therapy for protection of gastric ulcers and osteoporosis. Alternative agents include budenoside, mycophenolate, or calcineurin inhibitors such as tacrolimus, but experience is limited.

      Conclusion

      Various liver-related factors are involved in the different phases of gene therapy, such as assessment of liver health in the pre-treatment period, patient selection and follow-up, maintenance of liver health after gene therapy and management of potential short- and long-term adverse events. An increase in ALT is a common adverse event that requires rapid evaluation and an immunosuppressive approach.
      It is therefore important that haemophilia treaters and hepatologists collaborate at all stages of gene therapy to assess potential safety issues and ensure the long-term success of gene therapy. Special attention should be given to the management of immunosuppressive therapy (indications and handling) and to patients with ill-defined conditions, e.g. patients with some degree of liver fibrosis or fatty liver disease, patients with a history of hepatitis C and hepatitis B infection, patients with HIV infection and those taking medications that may affect liver function.

      Financial support

      The authors received no financial support to produce this manuscript.

      Conflicts of interests

      WM received honoraria from Bayer, Biomarin, Biotest, CSL Behring, Chugai, Freeline, LFB, Novo Nordisk, Octapharma, Pfizer, Regeneron, Roche, Sanofi, sobi, Takeda/Shire, uniQure. GRF: Abbve, Biomarin, Gilead, GSK, MSD, uniQure. FP received honoraria as speaker at the educational symposia by Grifols, Roche, Sanofi, Sobi and Takeda; she is member of the advisory board of Biomarin, Roche, Sanofi, Sobi and Takeda.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      This manuscript has been written and approved by all authors.

      Supplementary data

      The following are the supplementary data to this article:

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