160. Overcoming the Size Limitations of Gene Transfer Vectors: Using Segmental Trans-Splicing To Deliver the Coding Sequence of Von Willebrand Factor

Von Willebrand Disease (VWD), a common inherited bleeding disorder affecting 1 to 3% of the U.S. population, is caused by abnormalities in the expression and processing of von Willebrand factor (VWF), an adhesion molecule secreted by endothelial cells and platelets. Due to packaging limitations of current viral vectors, the large size of the VWF cDNA (8.6 kb) is a significant challenge to successful gene therapy for VWD, particularly when driven by the 1.7 kb native human VWF promoter. As a means to deliver VWF, we investigated "segmental trans-splicing" (STS), a strategy in which individual "donor" and "acceptor" DNA segments delivered in vitro or in vivo, generate pre-mRNAs which contain 5[variant prime] and 3[variant prime] splice signals respectively, and complementary "binding domains" through which the two pre-mRNAs bind to each other, permitting contiguous splicing into a final mature mRNA. VWF cDNA was split into two segments at the natural junction of exons 31 and 32 of the 52 exon human gene, yielding a 5.5 kb (5[variant prime]) donor and a 3 kb (3[variant prime]) acceptor coding sequence. VWF donor and acceptor constructs co-transfected into 293 cells resulted in the formation of spliced VWF mRNA containing the sequence of the mature, correct VWF mRNA. Although some cryptic splice events were also observed, these could be eliminated by site directed mutagenesis with silent alterations. These results suggest that it will be possible to package a regulated pair of VWF partial cDNAs for delivery in vivo. Since persistence of expression is also a challenge for VWF gene therapy, ex vivo gene transfer to bone marrow cells (BMC) with integrating vectors carrying partial VWF cDNAs, followed by transplantation to an ablated host could potentially achieve long term gene expression. However, it must first be established that VWF secreted from BMC would be sufficient to restore hemostasis in a VWD model system. To determine the feasibility of this approach, BMC from C57Bl/6 wild type (WT) or VWF- knockout (KO) mice were harvested 4 days after administration of 5-fluorouracil (150 mg/kg) and 2 × 10 6 BMC were transplanted intravenously into lethally irradiated (9 Gy) VWF-KO mice. Transected-tail wounds in untreated VWF-KO mice will bleed to death unless cauterized, whereas similar wounds in WT mice stop bleeding in