Successful gene therapy will revolutionize the treatment of the inherited bleeding disorders hemophilia A and B. Hemophilia A is caused by deficiency of coagulation factor VIII (FVIII) and is a prime disorder for genetic correction. The disease constitutes 80% of all hemophilia patients and is the focus of this proposal. The requirements for successful FVIII gene transfer include: the persistent expression of therapeutic levels of FVIII, the lack of significant toxicity to the gene transfer vehicle (vector), the lack of host immune response to the normal factor VIII protein and to the vector, and reduced ectopic expression of the normal product. Gene therapy through the use of Intronn's platform technology, spliceosome mediated RNA trans-splicing (SMART), can potentially circumvent some of these problems. Intronn has developed and patented constructs called pre-trans-splicing molecules (PTMs) that are capable of modifying mRNA in vivo. PTMs work by promoting trans-splicing reactions between the PTM and a targeted pre-messenger RNA. The product of a SMART reaction is a novel chimeric or composite RNA that can encode virtually any desired gene product. The product of a SMART reaction contains one or more exons of the target endogenous pre-mRNA and an exonic or cDNA sequence delivered by the PTM. We propose studies to target mutant factor VIII in cell and animal models of hemophilia A with PTMs that can perform repair of endogenous transcripts to generate full length functional FVIII. This Phase I application proposes to optimize the splice elements and binding domain of PTMs using a LacZ based repair model in 293 cells, and to subclone lead sequences into factor VIII based PTMs and to test these in a characterized mouse model of hemophilia A. Lead PTMs identified in Phase I will be further optimized in Phase II of this application using cell based medium to high throughput screens. Fully optimized PTMs in Phase II will be transferred to an AAV delivery system and tested in mouse and canine models of hemophilia A for long term correction of factor VIII. This work will be performed in collaboration with a key university site (UNC-Chapel Hill) with extensive experience in hemophilia A biology, gene transfer and experimentation with murine and canine models of hemophilia A. The research proposed in Phase I and II of this application will form the groundwork for future clinical trials in humans.
