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Hemophilia Gene Therapy and the Liver

The content of this site is provided for educational purposes and is intended only for U.S. healthcare professionals.

Gene therapies for hemophilia are currently being studied to determine their safety and efficacy. No gene therapies for hemophilia have been approved for use.

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The liver plays a vital role in human metabolism and detoxification.1 In addition, it is the primary site of blood coagulation Factor VIII and Factor IX synthesis.2 The liver is, therefore, central to understanding and treating hemophilia, and is the main target tissue for adeno-associated virus (AAV)-mediated gene transfer.1

The liver exhibits various characteristics that support its role in gene therapy even beyond hemophilia. For example, the liver has a dual blood supply allowing for the rapid accumulation of vector particles following systemic administration of gene therapy.1

The liver also contains numerous cell types, including immune cells. The interaction of these different cell types with gene therapy vectors is key to understanding the potential of hemophilia gene therapy, as well as the potential barriers to be overcome for transduction.1,3

Refer to the figure below to learn more about the function and anatomy of the liver, including the various cell types that make up its composition and key properties of each.

Figure 1.2 - 1


    AAV is the most commonly used vector for hemophilia gene therapy studies.11,12 There are a number of reasons for this, including:13

    • A lack of known human pathogenicity
    • An inability to replicate in absence of viral helper
    • Potential ability to establish long-term transgene expression
    • Multiple serotypes that permit tissue / organ targeting

    Various different serotypes of AAV exist, with each one having different capsid proteins.14 These different serotypes demonstrate different levels of affinity for target cells and organs, a phenomenon known as tropism. Serotypes AAV215 AAV512 AAV615 AAV815 and AAV915,16 all exhibit liver tropism.

    The different serotypes are not, however, only liver-specific in their tropism. For example, AAV8 targets cells in the heart and pancreas alongside those in the liver.15 Bioengineering through the cross-packaging of the AAV genome between serotypes has been undertaken to produce hybrid vectors with the intention of increasing liver-specific tropism.17,18 In another approach, tissue- or cell-specific promoter DNA sequences within the transgene cassette have been employed in clinical research in an effort that aims to achieve greater control of the transgene expression in liver hepatocyte cells.19 By using a transgene cassette that includes promoters from proteins that are naturally synthesized in liver cells, the expression of F8 and F9 can be targeted specifically to the liver.20,21

    To find out more about the AAV-derived vector design and bioengineering AAV to optimize hemophilia gene therapy, click here to access the sections on Exploring the Building Blocks of Vector Capsid Design and Optimizing Transgene Expression.


    The immune system can impact gene therapy in several ways through both humoral and cellular immune responses.22–25

    In a humoral immune response, the body generates antibodies to an antigen. In the case of gene therapy for hemophilia, this antigen could be components of the AAV-based vector or the transgene. If these antibodies are ‘neutralizing’, they may prevent the vectors from delivering the gene to the target cells.22,23 In a cellular immune response, the transduced cell presents antigens, capsid particles, to circulating T cells, which then eliminate the transduced cell and, as a consequence, reduce the number of cells containing the transgene.24,25 This can result in loss of, or reduced, gene expression, and in turn impacts the level of protein produced.24,25

    Pre-existing immunity to naturally occurring AAV can cause both humoral and cellular immune responses, and this may be a barrier to the success of AAV gene transfer.26

    However, the liver has numerous unique immunological properties.27 While the liver generally exhibits a strong innate immune response,27,28 this response has been shown to be low towards AAV-based vectors.29 It also demonstrates a poor adaptive immune response, resulting in a state of relative immune unresponsiveness and immune tolerance.29,30 This is demonstrated by the lack of immune response to the large number of antigens present in the blood that flow to the liver directly from the gut.29

    This liver tolerance effect, known as being tolerogenic, can be exploited therapeutically by liver-directed gene therapy to induce immune tolerance to the transgene product; i.e. blood coagulation factor.31 Preclinical data from small and large animal models of hemophilia A with inhibitors suggest that liver-directed gene therapy may overcome pre-existing anti-FVIII antibodies, induce immune tolerance and provide sustained therapeutic FVIII expression to prevent bleeding.31

    You can explore these topics in more detail, including the specific mechanisms that underly the immune tolerance effect in the liver by clicking here to access the section on Gene Therapy and the Immune System.


    Liver-directed gene therapy has the potential for sustained therapeutic benefit for people with hemophilia, although it is not yet clear how long the effects of gene therapy may last.1,13 It is recognized that there is much to be understood about this therapeutic approach and a number of considerations to be taken into account.13

    AAV vectors can induce immune responses

    AAVs are naturally occurring and so people with hemophilia may have been naturally exposed to AAVs during their lifetime.13,32 This exposure can result in the development of AAV antibodies, which may limit the effectiveness of gene therapy by preventing the transduction of AAV vectors into target cells.22,23

    Liver growth in childhood might impact the durability of hemophilia gene therapy

    Transgenes delivered to liver cells via AAV-derived vectors exist as episomes in the nucleus of transduced target cells.22,33 Unlike the adult liver, in which hepatocytes are post-mitotic and long-lived, cell division is rapid during childhood34 with liver weight doubling at 4 months, 16 months, 6 years, and 12 years.22 As a result of this growth from cell division, a dilutive effect of the transgenes may occur due to an unaccompanied concurrent replication of the episomes.35 However, as the rate of hepatocyte proliferation transitions from a high to low rate towards adulthood, current gene therapy strategies may be considered in the adolescent population in the future.35

    Liver toxicity

    Hemophilia gene therapy trials to date have excluded patients with active liver conditions, such as current hepatitis C infection.11,31 The safety of gene therapy in people with hemophilia who also have liver conditions is, therefore, currently unknown.36

    There are a number of aspects of liver-directed gene therapy for hemophilia that remain unknown. These ‘known unknowns’ include:

    • The potential for long-term effects resulting from low-level AAV integration11
    • The impact of alcohol consumption on sustained transgene expression37
    • Whether targeting hepatocytes limits the impact of gene therapy on hemophilia A (FVIII is normally produced in liver sinusoid epithelial cells38)



1. Kattenhorn LM, et al. Hum Gene Ther 2016;27:947–61. 2. VandenDriessche T, et al. Hum Gene Ther 2017;28:1013–23. 3. High KA. Hum Gene Ther 2014;25:915–22. 4. Zanolini D, et al. Haematologica 2015:100:881–92. 5. Burroughs A, Senzolo M. (Accessed April 2021). 6. Trefts E, et al. Current Biol 2017:27;1141–55. 7. Kalra A, et al. Physiology, Liver. In: StatPearls [Internet]. Treasure Island (FL), StatPearls Publishing, 2020. 8. Racanelli V, et al. Hepatology 2006;43:554–62. 9. Nguyen TH, et al. Nat Gene Therapy 2004:11;76–84. 10. Lautt WW. Hepatic Circulation: Physiology and Pathophysiology. San Rafael (CA), Morgan & Claypool Life Sciences, 2009. 11. Doshi BS, et al. Ther Adv Hematol 2018;9:273–93. 12. George LA. Blood Adv 2017;1:2591–9. 13. Mingozzi F, et al. Blood 2013;122:23–36. 14. Holehonnur R, et al. BMC Neuro 2014;15:28–42. 15. Wu Z, et al. Mol Ther 2006;14:316–27. 16. Mingozzi F, et al. J Virol 2002;76:10497–502. 17. Balakrishnan B, Jayandharan GR. Curr Gene Ther 2014;14:1–15. 18. Colella P, et al. Mol Ther Methods Clin 2018;8:87–104. 19. Powel SK, et al. Discov Med 2015;19(102):49–57. 20. Li C, Samulski RJ. Nat Rev Genet 2020;21(4):255–72. 21. Ohmori T. Int J Hematol 2020;111:31–41. 22. Baruteau J, et al. J Inherit Metab Dis 2017;40:497–517. 23. Rapti K, et al. Mol Ther 2012;20:73–83. 24. Pien GC, et al. J Clin Invest 2009;119:1688–95. 25. Kotterman MA, Schaffer DV. Nat Rev Genetics 2014;15:445–51. 26. Verdera HC, et al. Mol Ther 2020;28:723–46. 27. Gao B. Cell Mol Immunol 2016;13:265–66. 28. Horst AK, et al. Cell Mol Immunol 2016;13:277–92. 29. Sack BK, et al. Mol Ther 2014;1:14013. 30. Kubes P, Jenne C. Ann Rev Immuno 2018;36:247–77. 31. Samelson-Jones BJ, Arruda VR. Front Immunol 2020;11:618. 32. Scallan CD, et al. Blood 2006;107:1810–7. 33. Naso MF, et al. BioDrugs 2017;31:317–34. 34. HemAware: Pushing the Boundaries of Treatment for Hemophilia A and B. (Accessed April 2021). 35. Pipe S. Haemophilia 2020;27;114–21. 36. George LA. Hematology Am Soc Hematol Educ Program 2017;587–94. 37. Sidonio Jr RF, et al. Blood Rev 2020;Nov 9:100759. doi: 10.1016/j.blre.2020.100759. Online ahead of print. 38. Arruda V. Haematologica 2015;100:849–50. 39. Lheriteau E, et al. Blood Rev 2015;29:321–8.

Date of preparation: April 2021

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