Sustained factor VIII expression after in utero gene therapy

Objective. FVIII deficiency is an excellent gene therapy model because it is a single gene defect with a broad therapeutic window. One 5% correction of FVIII levels can prevent spontaneous bleeding episodes, and a number of experimental animal models exist. Current gene therapy approaches are limited by immune reaction to the gene product and viral proteins, and the need for multiple administrations. In this study, we investigate whether in utero administration of AAV or lentiviral vectors containing the Factor VIII gene would overcome these obstacles and allow sustained expression. Methods. AAV and lentiviral vectors containing a canine Factor VIII construct with an upstream liver-specific promoter were generated. Fourteen-day gestation fetuses of Factor VIII deficient mice were injected intraperitoneally or intravenously with AAV2/8 serotype vector or VSV-g pseudotyped lentiviral vector. Canine Factor VIII levels in plasma have now been assessed at monthly intervals up to 8 months after in utero treatment. FVIII levels are expressed as the percentage of canine plasma Factor VIII activity in knockout mouse serum. Results. In utero injections with AAV and lentiviral constructs resulted in 33 and 68% survival rates to weaning, respectively, with the differences related to known technical issues. Approximately 45% (5/11) of mice injected with the AAV vector had greater than 1% FVIII at 4 weeks, with a mean of 3.8 ± 2.0%. These levels remained stable for up to 8 months after injection. In addition 53% (9/17) of mice given the lentiviral construct showed FVIII levels greater than 1% with a mean of 1.0 ± 0.1%, with levels remaining stable for 6 months. Conclusions. In utero gene delivery of Factor VIII of AAV or Lentiviral vectors results in durable, postnatal plasma levels of Factor VIII protein in a murine model of Hemophilia A. Further optimization of dosage, titer, route of administration (intravenous versus intraperitoneal), and viral constructs are being investigated to achieve higher levels of expression. However, the sustained expression demonstrated in this study supports the promise of prenatal gene transfer.