Repair of Factor VIII by targeted RNA trans-splicing

DESCRIPTION (provided by applicant): 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 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 FVIII protein, 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. In this application we will plan to 1) optimize an adeno-associated virus (AAV) delivery system for correction of FVIII in the mouse model of hemophilia A, 2) demonstrate functional long term correction of endogenous FVIII in a knockout mouse model of hemophilia using AAV delivered PTMs and identify a lead AAV delivery system for use in clinical work, 3) identify a lead human FVIII PTM that can efficiently repair FVIII using in vitro screening models, and 4) perform biodistribution and clearance studies in mice using the lead AAV vector and lead human PTM. This work will be performed in collaboration with a key university site(Mt. Sinai School of Medicine) with extensive experience in hemophilia A biology, gene transfer and experimentation with murine and canine models of hemophilia A. The research proposed in this application will form the groundwork for a Phase I clinical trial in humans.