339. FIV Lentiviral Vector Gene Transfer for Hemophilia A

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Hemophilia A is an X-linked genetic disorder resulting in deficiencies in coagulation Factor VIII and a risk of sustained bleeding after trauma or injury. Hemophilia A is an attractive candidate for treatment by gene transfer because only partial correction is required for clinically relevant effects. We previously reported that gene transfer to the liver of E16 FVIII deficient mice using a lentiviral vector derived from feline immunodeficiency virus (FIV) resulted in sustained partial correction of the bleeding disorder. We are now investigating the impact of several vector and transgene modifications on gene transfer to hepatocytes using baculovirus GP64 pseudotyped FIV. The goals of the project are to improve transgene expression through several vector modifications and to test the optimized vector's effects on protein expression. Modifications include rendering the lentiviral vector |[ldquo]|self-inactivating|[rdquo]| by deleting the U3 region of the 3’ LTR, inserting the FIV central polypurine tract (cPPT) element upstream of the internal promoter, and incorporating the woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) downstream of a luciferase reporter gene or the FVIII cDNA. Furthermore, two sites were mutated, a major splice donor and the start codon of the partial Gag sequence, preventing production of undesired product. In addition, the post- transcriptional control element from the 5’ LTR of spleen necrosis virus (SNVU5) was incorporated into the delivery vector. The liver- specific mouse albumin enhancer and human alpha-1-antitrypsin promoter (Alb/HAAT) are being used to direct firefly luciferase, B- domain deleted human FVIII (BDD-FVIII), and a FVIII cDNA engineered to include several asparagine-linked oligosaccharides within a short B-domain spacer (F226aa/N6) in wild type and E16 FVIII deficient mice on a congenic C57/BL6 background. Findings from these ongoing experiments will be reported.