760. Optimized AAV-Mediated Human Factor VIII Gene Therapy in Hemophilia A Mice and Cynomolgus Macaques

Abstract

In an effort to optimize expression of human coagulation VIII (hFVIII) for the treatment of hemophilia A, an extensive study was performed combining liver-specific promoter and enhancer elements with a codon-optimized human B-domain-deleted hFVIII transgene. Due to the large size of the FVIII coding sequence, there is a strong requirement for gene expression control elements to be as short as possible while retaining hepatocyte-restricted transcription. Several strong liver-specific promoters were shortened and combined, with combinations of up to three liver-specific enhancer sequences, to generate 42 enhancer/promoter combinations.These 42 liver regulatory gene cassettes were packaged into the AAVrh10 capsid and were tested in FVIII KO mice. Following intravenous (IV) administration of 1010 genome copies (GC), mice were bled every two weeks to follow hFVIII activity and antibody generation to the transgene. At week 2 post-injection, mice showed a range in hFVIII activity from 0.12-2.12 IU/ml. FVIII KO mice developed antibodies to hFVIII at week 4, and by week 8, mice in most of the 42 vector groups had detectable anti-hFVIII IgG levels.Based on the FVIII KO mouse studies and a small pilot rhesus macaque study, two of the original 42 enhancer/promoter combinations were selected for further evaluation in cynomolgus macaques, using two different Clade E capsids for expression. Each of the four vector combinations were administered IV at a dose of 1.2×1013 GC/kg into five macaques per group. With one capsid plus enhancer/promoter combination, peak expression of 37% of normal FVIII levels was seen at week 2 post-vector administration, which then plateaued at 20% of normal. While antibodies to the hFVIII were detected in the majority of macaques by week 8, antibodies remained undetectable in two animals at week 30 post-vector administration.