Brief report on gene therapy for severe hemophilia

Patients affected by severe hemophilia A and B must be treated with plasmaderived or recombinant factor replacement with significant improvement in morbidity and mortality, as well as in their quality of life. Nowadays prophylaxis represents the golden standard for the management of hemophilia, using fixed doses of factor VIII/factor IX (FVIII/FIX) concentrates at fixed timing of infusions. However, such treatment is extremely costly and often complicated by life-threatening bleeding and/or by the onset of inhibitors against FVIII and FIX. These considerations prompted efforts to develop novel approaches for treatment of hemophilia using viral vector-mediated gene transfer.

Early gene therapy studies focused on the treatment of hemophilia B are in progress. Ongoing efforts have also accelerated with respect to the development of vectors suitable for the treatment of hemophilia A. Gene therapy for hemophilia is based on gene transfer with recombinant adeno-associated viruses (rAAV), which derive from many serotypes of AAV that exist in nature and that are thought to be nonpathogenic.

Two clinical trials were completed with limited results exploiting rAAV vectors for treatment of hemophilia B. The first trial involved 8 men, who were treated by intramuscular injections at multiple sites of rAAV encoding hFIX in a classic dose-escalation study. The injections were well tolerated, but the expression was minimal, because only one individual treated with low dose showed to have detectable hFIX, that persisted over a period of more than 10 years.

A second clinical trial targeted the liver that was thought as the natural site for hFIX synthesis and expression of the transgene. Moreover liver was judged to be a less immunogenic site than intramuscolar injections for developing neutralizing antibodies against hFIX. Seven patients with severe hemophilia B received rAAV2 vector into the liver via hepatic artery injection. Only individuals treated at the highest dose (2×10¹² vg/kg) demonstrated measurable FIX levels in blood. However, after several weeks, the levels of FIX declined. As cytotoxic T-lymphocytes appeared into the blood stream the hFIX-expressing hepatocytes were destroyed. Among multiple serotypes AAV8 seems to be more efficient in transducing hepatocytes. Another favorable factor for the AAV8 serotype is that the genome is rapidly uncoated, leading to a prompt expression. The incidence of neutralizing antibodies with AAV8 is about 20%, even though recent studies report a higher percentage. Two studies have shown that transient immunosuppression may facilitate transduction of the liver in vivo in animal models. Preclinical studies were carried out in mice showing to achieve a dose-dependent increase in FIX up to 100% in animals receiving the highest dose. In these studies the liver uptake of vector by peripheral injection was equivalent to that given by portal vein infusion. Moreover scAAV2/8 vector particles were found to be much more effective at transducing hepatocytes if compared with ssrAAV2/8. Also preclinical studies on non-human primate hepatocytes resulted successfully using a peripheral vein injection. Dispite the observation in mice,  the two serotypes seemed more equivalent in non-human primates and in the clinical trials.

Twelve individuals partecipated to a clinical trial designed as a classic phase I/II dose-escalation study. The initial dose was 2×10¹¹ vector genomes (vg)/kg, with an intermediate dose of 6×10¹¹ vg/kg and a higher dose of 2×10¹²vg/kg respectively. Results regarding the first 10 subjects, who were followed 3 years or more after the single vector injection,  demonstrated measurable levels of FIX. A stable production of hFIX in 2 patients more recently treated was observed. The average FIX level was 5.1% ± 1.7% in the initial six patients who received the highest dose, with each having a production of ≥2%. FIX expression resulted in a significant decrease of FIX replacement therapy. The majority of patients suffered from a mild elevation in transaminases level, which was resolved giving prednisolone as soon as an increase of 50% or greater of basal value appeared. Corticosteroid was given for one or two weeks or withdrawn when  transaminases returned to normal value. Steroid therapy allowed maintenance of  FIX levels in each patient. Other ongoing studies on the use F9 cDNA, incorporating the Padua mutation, which enhances up to 8-fold the expression of FIX, confirm the results of AAV-mediate gene transfer following systemic administration of the vector. No adverse events in any studies have been observed. The best data emerge from a trial using a novel engineered AAV capsid and a codon-optimized, and a gain-offunction Padua variant. Data show that the low dose (5×10¹¹vg/kg) of their vector produce stable FIX activity levels between 12 and 63% of normal value,  giving a single injection of SPK-9001 at the initial dose level of the study.

The human FVIII coding sequence (7 kb in length) is too large to be packaged into an AAV capsid. FVIII has three functional domains. The B domain can be deleted and a 226-amino-acid spacer was included in its place in the construction of the vector. Within this spacer an amino acid triplets, which function as glycosylation sites, were also included so that the resulting 5.2-kb vector was packaged after its coding sequences were codon-optimized. Nine subjects with severe hemophilia A have been treated at doses ranging from 6×10¹² to 6×10¹³vg/kg, using an AAV5 containing the S (serine) Q (glutamine) linker codon-optimized  FVIII expression in a study. FVIII levels in the seven patients treated at a dose of 6×10¹³vg/kg have consistently been within the normal range of 40-150% beyond 12 weeks after gene transfer, showing a 91% drop in the mean of annual bleeding rate and 98% reduction of prophylactic infusions.

The future of gene therapy in hemophilia seems to be very stimulating. However many unsolved criticisms still remain:

1. CD8+ T-cell response to AAV capsid
2. transient immunosuppression
3. the lowest therapeutic dose of vector
4. the best AAV serotype to use
5. the safe approach in pediatric patients
6. mantainance of transgene expression mediated by AAV in children
7. the frequency of doses’ administration
8. long-term expression of normal FVIII/FIX
9. useful of chronic transgene synthesis of FVIII/FIX by liver on iduction of immune-tolerance in hemophiliacs with inhibitor.

Suggested reading

• Nienhuis AW, Nathwani AC, Davidoff AM. (2017). Gene therapy for Hemophilia. Mol Ther.  3;25(5):1163-1167
• Clement, N., and Grieger, J.C. (2016). Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol. Ther. Methods Clin. Dev. 3, 16002.
• Lheriteau, E., Davidoff, A.M., and Nathwani, A.C. (2015). Haemophilia gene therapy:Progress and challenges. Blood Rev. 29, 321–328.
• Gao, G.P., Alvira, M.R., Wang, L., Calcedo, R., Johnston, J., and Wilson, J.M. (2002).Novel adeno-associated viruses from rhesusmonkeys as vectors for human gene therapy. Proc. Natl. Acad. Sci. USA 99, 11854–11859.