Dose response results of self complementary adeno‐associated virus (AAV) vector‐mediated factor IX gene transfer in non‐human primates

Objective:  In anticipation of opening a phase I/II clinical trial of adeno‐associated virus (AAV) vector‐mediated factor IX (FIX) gene transfer in subjects with severe haemophilia B, we have recently evaluated the vector dose‐response relationship in nonhuman primates. Our approach differs from prior studies in that the vector (scAAV2/8‐LP1‐hFIXco) used, will: (i) be pseudotyped with serotype 8 capsid; (ii) be delivered by peripheral vein; and (iii) have a self‐complementary genome. Methods:  The vector scAAV2/8‐LP1‐hFIXco was produced by calcium phosphate mediated two plasmid transient transfection of 293T cells with the virus then being purified using group separation and ion exchange chromatography in our GMP facility, using exactly the same methodology that we propose to use for production of the clinical lot. The titre of the vector prep was determined by qPCR analysis. Male rhesus macaques, previously screened for the absence of serotype 8 humoral immunity, received vector via bolus infusion over 15 min through a peripheral vein. Human FIX expression in the rhesus plasma collected at serial time points following vector administration was determined by our previously validated ELISA. Summary:  We used four vector dose levels, 2 × 10 10 , 6 × 10 10 , 2 × 10 11 and 2 × 10 12 vg kg −1 body weight, in cohorts of three or four macaques each. No overt toxicity or perturbation in serum biochemistries or other laboratory values was noted in any animal. A nearly linear dose‐response relationship between vector administered and hFIX expression was observed. Mean levels of human FIX 3 weeks following vector administration, expressed as a percentage of normal (5ug mL −1 ), was as follows: 2% (2 × 10 10 ), 6% (6 × 10 10 ), 25% (2 × 10 11 ) and 200% (2 × 10 12 ) and has remained relatively stable. Of the four macaques given a dose of 2 × 10 10 vg kg −1 , one initially had a FIX level of nearly 10% that has remained at 8% for several weeks. A second animal peaked at 1% but hFIX is now undetectable. Two macaques never had detectable hFIX expression. Tissue biopsies have demonstrated by qPCR analysis that the vast majority of vector genomes are found in the liver, although they can be detected at other sites including the testis. Conclusions:  Based on these results, we plan to use 2 × 10 10 vg kg −1 as the starting dose in our upcoming clinical trial. We believe that some participants may achieve the target level of 3% hFIX expression at this dose, if we can extrapolate the information we have obtained in rhesus macaques to humans. However, we are prepared to quickly dose‐escalate if this is not the case. Our maximal clinical dose will be 2 × 10 11 vg kg −1 , 10‐fold lower than the highest dose in this preclinical study (2 × 10 12 vg kg −1 ), a dose that was found to be safe and without toxicity.