A literature review on AAV vector-mediated gene therapy for haemophilia B treatment

1. AAV vector-mediated gene
therapy as a treatment for
hemophilia B: A literature
review
Mark Webster

2. Introduction
What is hemophilia B?
• X-linked inherited bleeding disorder – caused by mutations in the F9
gene on the X chromosome causing human factor IX (FIX) function
deficiency
• FIX is a coagulation protein
• Sufferers tend to have increased susceptibility to hemorrhage
• Variety of mutational genotypes – third of cases due to spontaneous
mutation

3. Current prophylaxis
• Frequent intravenous injections of exogenous FIX protein
• up to 3 x week depending on frequency of bleeds
• Non-steroidal anti-inflammatory drugs (NSAIDS) should not be
administered after diagnosis
• Tranexamic acid administered during surgical procedures

4. Gene therapy as a potential treatment
• Frequent intravenous FIX administration significantly reduces patient quality of
life
• Gene therapy could remove the need for exogenous FIX
• Deliver working copy of FIX gene to haemopoietic stem cells using a viral vector
• FIX expressed in target cells
• FIX expression would reduce symptoms
• 1% increase in expression is enough to significantly reduce symptoms
• Maintained expression of FIX would reduce the need for exogenous FIX injections
• However, viral vectors can cause adverse immune response
• Increase in activity of enzymes associated with viral infection - alanine aminotransferase elevations

5. AAV vectors have become the standard choice in clinical trials
• All recent studies uses adeno-associated viral (AAV) vectors
• Replication defective and non-pathogenic to humans
• Manipulated to replace AAV coding sequence with transgene of interest (F9) under the
control of a tissue-specific promotor (liver)
• Nonintegrating vector, vector genome maintained episomally in transduced cells
• Limited transgene capacity – 4.7KB
• AAV vector is packaged into capsids and injected into patients via peripheral
venous infusion

6. Manno et al (2006)
• Used an AAV2 vector carrying FIX wild-type transgene to induce FIX expression
• Intravenous administration to hepatic artery – target hepatocytes
• Single dose of 2 x 1012 genome copies/kg
• Findings:
• Therapeutic levels of FIX expression were achieved by the dose administered
• Expression of transgene limited to just 8 weeks
• No acute or late toxicity
• Decline in FIX over time accompanied by alanine aminotransferase elevations
• Cellular immune response resulted in destruction of transduced hepatocytes
• Immunomodulation may be needed for long-term expression
Clinical success

7. Nathwani et al (2014)
• Used an AAV8 vector to induce FIX expression in 10 male patients via wild-type FIX gene transfer
• Aimed to determine efficiency of transgene expression and the level of persistent or late toxicity –
follow up period of 3 years
• Patients arranged into low, intermediate, and high dose cohorts
• Results:
• Clinically significant improvement in symptoms and FIX expression up to follow up period – 1-6% increase up to 3
years
• Increase in FIX levels is dose dependent
• No late toxic effects reported within the follow up period
• Lower incidences of cellular immune response than AAV2 vector
• Initial alanine aminotransferase elevations treated with prednisolone over course of 5 days

8. George et al (2017)
• Used an AAV5 vector consisting of a bioengineered capsid, liver specific promotor and factor IX-
R338L transgene to induce FIX expression
• 10 males who had factor IX activity of 2% or less of the normal value
• bleeding frequency and administration of exogenous FIX were evaluated after vector infusion and
were compared with baseline values
• alanine aminotransferase elevations treated with short-term prednisolone administration
• Findings:
• No cellular immune response or serious adverse reaction after FIX-R338L administration
• Prednisolone successfully reduced alanine aminotransferase elevations
• Clinically significant increase in FIX expression in all patients
• 8/10 patients no longer required exogenous FIX administration
• Significant reduction in bleeding rates – mean rate reduced from 11.1 events per year to 0.4

9. Meisbach et al (2018)
• Similar to George et al (2017); used AAV5 vector with a liver-specific promotor for wild-type FIX
gene transfer in 10 male patients with severe haemophilia B
• Single dose of 5 × 1012 (low-dose cohort) or 2 × 1013 (high dose cohort) genome copies/kg was
administered
• Patients tested for AAV5-neutralizing antibodies using a green-fluorescent protein-based assay
• Prednisolone was used to treat alanine aminotransferase elevations
• Findings:
• AAV5 vector caused “no cellular immune response and was clinically effective”
• Alanine aminotransferase elevations were asymptomatic and reduced by short-term
prednisolone treatment
• Single administration resulted in increased FIX levels and a reduction in bleeding rates up to
the follow up period (18 months) in all patients
• 8/9 patients no longer required exogenous FIX administration
• However; 4 patients in high-dose cohort tested positive for antibodies against AAV capsid

10. Limitations of gene therapy
Adverse reaction to AAV vectors
• Viral capsid proteins can activate a cellular immune response
• Poor transduction and short-lived expression
• Serotype
• Choice of serotype is dependent on likelihood of immune response
• AAV2 vectors have been shown to induce cellular immune response in most cases
• Lower rates of antibodies against AAV5 and AAV8 serotypes in general population
• Exclusion of patients with antibodies to serotype
• Patients immunoassayed for antibodies
• Positive-testing patients excluded as most susceptible to cellular immune response

11. FIX expression
• Insufficient expression
• Low transduction of transgene in target (hepatic) cells
• Lack of sustained expression
• Unknown which expression levels are optimal
• AAV5 vectors have been shown to produce high transduction and expression
rates
• Any increase in FIX expression significantly improves symptoms

12. Liver complications
• Previous viral infections – hepatitis B & C
• Safety of liver-directed gene therapy in these patients is unknown
• Use of viral vectors could cause serious adverse reactions and toxicity in hepatic tissues
• Long-term genotoxicity
• Random integration causing hepatocellular carcinoma (HCC) has been demonstrated in mice
models
• This has not been shown in human trials
• Promotors used in clinical trials are associated with very low risk
• Clinical trials using AAV vector liver-direct gene therapy have produced no significant safety
or toxicity concerns

13. Economic considerations
• Unknown how gene therapy will be priced
• Single treatment could cost more than current intravenous exogenous FIX prophylaxis – may
not be cost effective for patients and payers
• Acceptable cost for an increase in quality of life
• May be worth it to remove the need for regular intravenous FIX administration
• High application potential in developing world
• single dose treatment has been shown to have therapeutic effects up to 3 years
• Many patients do not have consistent access to exogenous FIX
• However; these patients are also those who are most unlikely to afford it

14. Conclusions
• Recent clinical trials using AAV5 vectors with liver-specific promoters have shown significant
improvements in patient symptoms and decreased the need for current prophylaxis
• Overall decrease in bleeding rates and hemorrhage events
• Significant FIX expression resulting in patient no longer needing FIX administration
• AAV5 vectors with liver-specific promotor demonstrate the most clinical success
• AAV5 resulted in lower rates of alanine aminotransferase elevations and cellular immune response
• Liver-specific promotor induces higher levels of transduction into hepatic cells
• FIX-WT suggested to be the optimal transgene
• Administration to hepatic artery results in higher transduction levels
• Lower doses are less likely to cause adverse reactions
• Limitations of gene therapy must be addressed
• is it a financially viable treatment for most patients?
• May not be a treatment option for patients who test positive for antibodies against vector serotype
and/or have previous liver infections
• FIX expression must be sufficient and long-lasting to be a successful treatment

15. Questions?