Hemophilia Gene Therapy and the Liver

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 AAV9^15,16 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)