AN OPEN LABEL DOSE-ESCALATION STUDY OF A SELF COMPLEMENTARY AAV VECTOR FOR GENE THERAPY OF HAEMOPHILIA B

In the proposed study we wish to test a new approach called gene therapy for the treatment of patients with haemophilia B. This inherited disorder in which life threatening bleeding occurs without trauma results from an absence or defect of a blood clotting protein called Factor IX (FIX) that arises due to mutations in the FIX gene. The goal of our gene therapy approach, therefore, is to treat the disease by transferring to the patient s liver, a normal copy of the FIX gene so that normal FIX protein can be continuously produced by the patient s own cells. To this end we have developed a novel vector based on adeno-associated virus (scAAV2/8-LP1-FIXco) which is highly efficient at transferring the normal FIX gene to the liver, its natural site of synthesis. Importantly, AAV has the best safety profile among gene transfer vectors of viral origin. In murine and nonhuman primate models we have consistently achieved long-term expression of human FIX at levels that would be sufficient to prevent spontaneous life threatening bleeding in haemophilia B patients following a single injection of scAAV2/8-LP1-FIXco. The vector is simply infused into a peripheral vein, a highly convenient route of administration that dispenses with the need for surgical intervention for efficient transfer of the FIX gene to the liver. Based on these encouraging preclinical results we wish to evaluate this vector system in a small number of adults with severe haemophilia B. Eligible subjects, drawn from the worldwide pool of haemophiliacs, will receive explicit and detailed instructions about the risks and merits of this study prior to obtaining their consent for enrolment. Our primary aim is to establish the safety of peripheral vein administration of scAAV2/8-LP1-hFIXco over three different dosage levels. This study, which will be overseen by an international panel of medical experts, will involve a comprehensive plan of investigations to monitor safety, with particular emphasis on immune response to the virus and normal FIX protein. An additional objective is to determine the dose of vector that is required to achieve stable expression of human FIX at or above 5% of normal levels which would be sufficient to prevent spontaneous bleeding. Success with our approach could significantly impact on a wide variety of life threatening genetic disorders including alpha-1 antitrypsin deficiency, lysosomal storage and urea cycle disorders.

Our overriding objective is to develop a safe and effective gene therapy strategy for monogenetic disorders affecting the liver. Using haemophilia B (HB), an X-linked bleeding disorder, as our model system we have developed a novel vector system that is highly efficient at transducing the liver. Based on adeno-associated virus (AAV) which has an excellent safety profile, this vector contains a small liver-restricted human factor IX (hFIX) expression cassette which can be packaged as complementary dimers within individual vector particles. After uncoating these self-complementary (sc) genomes rapidly anneal to form stable, transcriptionally active double-stranded molecules. This unique property results in an almost 3-log improvement in hFIX expression in mice over comparable conventional single stranded AAV vectors. Administration of only 1x1010 scAAV particles resulted in hFIX expression at supraphysiological levels (8IU/ml) and correction of the bleeding diathesis is HB knockout mice without any toxicity. In nonhuman primates a single peripheral vein administration of a low dose of scAAV (1x1012 vector particles[vg]/kg) resulted in mean stable hFIX levels of 20% of normal without any toxicity, even in animals with pre-existing immunity to AAV. This is significantly above hFIX levels required for amelioration of bleeding in HB patients. Based on these encouraging preclinical data, we wish to perform a Phase I/II dose escalation study in adults with severe HB patients who have no evidence of active hepatitis or anti hFIX antibodies. This study is distinct as it involves peripheral vein administration of scAAV8 pseudotyped vectors and will have primary and secondary endpoints of safety and efficacy respectively. Starting with the lowest vector dose, three doses levels (5.0x1011, 1.6x1012 and 5.0x1012vg/kg) will be sequentially assessed in a minimum of two subjects. Enrolment of each subject will proceed only after the previous subject has been observed for at least 42 days. During this time they will undergo a comprehensive array of investigations to assess safety with a particular focus on immune response to hFIX and AAV as well as detection of the vector genomes in semen. A dose will be judged to have exceeded the maximum tolerated dose if Grade III toxicity is observed in two subjects or Grade IV toxicity in one subject, recruited to that dose. A therapeutic vector dose is one that mediates hFIX expression at !Y5% of normal for a minimum of 90 days. Success with this approach will impact on other disorders including lysosomal storage diseases and urea cycle disorders.