The overall goal of this project is to develop an in vivo gene transfer approach for the treatment of hemophilia A (factor VIII deficiency). Towards this end, we are using a lentiviral vector based on the non-primate feline immunodeficiency virus (FIV) expressing the human FVIII (FVIII) cDNA to correct the phenotype in a mouse model of the disorder. We demonstrated previously that intravenous delivery of an FIV vector encoding FVIII produced sustained protein expression and phenotypic correction in hemophilia A mice. We recently made a series of modifications to the FIV vector constructs, generating a self-inactivating vector containing several additional cis-acting regulatory/boundary elements. Furthermore, we found that the baculovirus GP64 glycoprotein directs efficient gene transfer to hepatocytes. Therefore, we hypothesize that the FVIII expression following in vivo gene transfer will be enhanced by pseudotyping the FIV vector with the GP64 glycoprotein. Aim 1 will investigate the efficacy of the extensively modified, baculovirus GP64 pseudotyped FIV vector in correcting FVIII deficiency in vivo. Studies proposed in Aim 2 seek to identify the cellular receptor(s) for GP64 pseudotyped FIV. Identification of viral receptors lends the ability to predict which cell types that can be transduced and allows a better understanding of vector/host cell biology. We will apply a cDNA microarray-based technique that was recently successfully used to identify the receptor for AAV5 to identify the receptor(s) for GP64/FIV. Aim 3 will investigate the FIV vector integration events in the liver following in vivo delivery. Because persistent expression from an integrated transgene is the desired outcome for this approach, documenting integration and mapping sites of integration are important first steps in understanding the fate of the vector following in vivo gene transfer. Completion of these aims will move us closer to clinical gene transfer application of FIV for the treatment of hemophilia A. [unreadable] [unreadable] [unreadable]