Prevalence of Pre-Existing Neutralizing Antibodies Against Adeno-Associated Virus Serotypes 1, 2, 5, 6, 8, and 9 in Sera of Different Pig Strains.

Pre-existing neutralizing antibodies (NAb) to adeno-associated virus (AAV) may diminish the efficacy of AAV-based therapies depending on the titer. To support gene therapy studies in pigs, the seroprevalence of NAb to AAV1, 2, 5, 6, 8, and 9 serotypes were assessed in the sera of 3 different strains of pigs consisting of 60 Norsvin Topigs-20 strain, 22 Gottingen minipigs, and 40 Yucatan minipigs. Cell-based NAb assays were developed for various AAV serotypes. The sera were tested for NAb in a Lec-2 cell line for AAV9 vector and in a COS-7 cell line for the other AAV serotypes. In the 60 Topigs-20 strain 2 to 4 years of age, 100% were positive for AAV2 NAb, 45% positive for AAV6 NAb, and ∼20% positive for each of AAV1, 5, 8, and 9 NAb. These data showed that ∼80% of Norsvin Topigs-20 pigs evaluated were seronegative for pre-existing NAb to the AAV1, 5, 8, and 9 serotypes, respectively. In 22 Gottingen minipigs at 5-6 months of age, serum AAV serotype-specific NAb coexisted with that of various other AAV serotypes at 32% to 46% between two serotypes. These results suggested that coexisting NAb resulted either from multiple AAV serotype coinfection or from one (or more) serotypes that can crossreact with other AAV serotypes in some minipigs. Among the 40 Yucatan minipigs, 20 of the minipigs were <3 months old and were all negative for NAb against AAV5, 8, and 9, and only one of these 20 pigs was positive to AAV1 and 6. We further determined the titers in those positive pigs and found most Gottingen minipigs had low titer at 1:20, whereas some of Topigs-20 pigs had titers between 1:80 and 1:320, and some of Yucatan pigs had titers between 1:160 and 1:640. These results suggested that the majority of the pigs in the three strains would be amenable to gene therapy study using AAV1, AAV5, AAV8, and AAV9 and that prescreening on circulating AAV antibodies could be helpful before inclusion of pigs into studies.

Enhanced Factor IX Activity following Administration of AAV5-R338L "Padua" Factor IX versus AAV5 WT Human Factor IX in NHPs.

Gene therapy for severe hemophilia B is advancing and offers sustained disease amelioration with a single treatment. We have reported the efficacy and safety of AMT-060, an investigational gene therapy comprising an adeno-associated virus serotype 5 capsid encapsidating the codon-optimized wild-type human factor IX (WT hFIX) gene with a liver-specific promoter, in patients with severe hemophilia B. Treatment with 2 × 1013 gc/kg AMT-060 showed sustained and durable FIX activity of 3%-13% and a substantial reduction in spontaneous bleeds without T cell-mediated hepatoxicity. To achieve higher FIX activity, we modified AMT-060 to encode the R338L "Padua" FIX variant that has increased specific activity (AMT-061). We report the safety and increased FIX activity of AMT-061 in non-human primates. Animals (n = 3/group) received intravenous AMT-060 (5 × 1012 gc/kg), AMT-061 (ranging from 5 × 1011 to 9 × 1013 gc/kg), or vehicle. Human FIX protein expression, FIX activity, and coagulation markers including D-dimer and thrombin-antithrombin complexes were measured. At equal doses, AMT-060 and AMT-061 resulted in similar human FIX protein expression, but FIX activity was 6.5-fold enhanced using AMT-061. Both vectors show similar safety and transduction profiles. Thus, AMT-061 holds great promise as a more potent FIX replacement gene therapy with a favorable safety profile.