Safety and Efficacy of an Adeno Associated Virus-Based Vector Carrying the Human Clotting Factor IX Gene (AAV5-hFIX) As Studied in Non-Human Primates
Authors: Nijmeijer Bart , Petry Harald , Spronck Lisa , Van Der Kruijssen Corina , Petersen Jorgen , Salmon Florence 
Published: October 2019

Abstract
Restoration of clotting Factor IX (FIX) expression through gene transfer is a promising option for the treatment of Hemophilia B. We have developed an Adeno-Associated Virus serotype 5-based vector containing the human coagulation factor IX gene (AAV5-hFIX), in our fully scalable GMP-compliant baculovirus-based production platform. To evaluate safety and efficacy of AAV5-hFIX in a relevant preclinical model, 4 groups of 3 cynomolgus macaques were dosed intravenously with 5x1011, 5x1012, 2.5x1013 and 0.93x1014 vector genome copies (gc) per kg body weight, respectively. Blood was sampled periodically, from 4 weeks pre-trial until necropsy at 26 weeks after dosing. Clinical chemistry and inflammation markers were assessed using standard techniques. Circulating human FIX protein levels were assessed by hFIX-specific ELISA. At necropsy, full histopathological examination was performed, and selected tissues were assessed for the presence of vector DNA and -RNA by quantitative PCR and RT-PCR, respectively. No signs of adverse reactions were observed in any of the animals throughout the in-life period. Clinical chemistry and inflammatory markers were unaffected by treatment with AAV5-hFIX. At necropsy, there were no macroscopic or microscopic tissue findings that could be attributed to the vector. Infusion of AAV5-hFIX resulted in dose-dependent circulating levels of hFIX protein. In the highest-dose group, hFIX levels peaked to 30% of normal human levels seven days after administration, and stabilized around 10-15% of normal human levels over the remaining observation period of 6 months. In the lowest-dose group, hFIX levels did not increase above 1%. Analysis of tissues after necropsy revealed dose-dependent vector DNA delivery to the liver, and concurrent dose-dependent transgene expression levels. No significant off-target expression was observed. In one animal, circulating hFIX levels decreased to baseline one month after vector administration. This decrease was shown to be caused by the development of xenoreactive hFIX-specific antibodies. This immune response was limited to sequestration of hFIX from the circulation, and did not entail T cell reactivity towards transduced hepatocytes as the transgene expression levels in the liver of this animal were comparable to those observed in its group mates. Based on the observed expression levels, and by extrapolating the dose one-on-one between non-human primates and humans, the Minimum Anticipated Biological Effect Level (MABEL) in man is 5x1012 gc/kg. In conclusion, administration of AAV5-hFIX to non-human primates was well tolerated without any noticeable adverse effects, and resulted in (i) dose-dependent transgene delivery to the liver, (ii) liver-specific transgene expression, and (iii) circulating human FIX protein to levels that are expected to be of significant clinical benefit in the setting of hemophilia B.

Disclosures
Nijmeijer: uniQure BV: Employment. Petry:uniQure B. V,: Employment. Spronck:uniQure BV: Employment. Van Der Kruijssen:uniQure BV: Employment. Petersen:uniQure: Consultancy. Salmon:uniQure B. V.: Employment.