Repair of von Willebrand Factor (vWF) Gene Defect by Targeted RNA Trans-Splicing.

RNA repair based on spliceosome-mediated RNA trans-splicing allows for direct repair or reprogramming of endogenous genetic defects at the pre-mRNA level. Trans-splicing has significant therapeutic and safety advantages over traditional gene therapy including the ability to correct defects in large gene. Progress has been made during the past few years toward developing trans-splicing for the treatment of variety of hematological diseases. von Willebrand disease (vWD) is the most common inherited bleeding disorder. Due to the large size of von Willebrand factor (vWF) gene (approximately 180kb, cDNA 9kb) and packaging size restrictions of most gene delivery vectors, methods for genetic correction of vWD are limited. We developed a spliceosome-mediated RNA trans-splicing strategy to correct a murine model of vWD. The vWD knockout mice were created with a neomycin gene insertion at vWF gene intron 5. We designed pre-mRNA trans-splicing molecules (PTMs) that bind to intron 6 and carry the normal vWD gene sequence from exon 1–5. The 5′ trans-splicing of the pre-mRNA generated from the PTM is designed to hybridize with the endogenous vWD pre-mRNA at intron 6. Before testing in animals, we developed several PTMs with different binding domains to murine vWF intron 6. These PTMs carry a GFP hemi-reporter that includes the 5′ half of the GFP coding region followed by a spacer sequence and a binding domain sequence that is complementary to vWF gene intron-6. The target for these PTMs was a murine vWD mini-gene expressing pre-mRNA containing vWF gene intron-6 followed by sequences encoding 3′GFP. The expression of both PTMs and mini-gene target were driven by CMV IE promoter/enhancer. Both PTM and mini-gene target were co-transfected into 293 cells. Trans-splicing in this in vitro model produces an intact GFP gene easily measured by fluorescence. Two of the 15 PTMs restored full-length GFP mRNA as measured RT-PCR, FACS and sequence analysis of splice junction. No positive results were obtained in controls (either PTM or target alone). These results encouraged us to design murine vWF gene specific PTMs that encode for vWF exons 1–6. A new vWF mini-gene target was also generated by replacing 3′GFP coding region of hemi-reporter mini-gene with murine vWF coding sequences from exon 7–51. To assess specific trans-splicing between 5′ splice donor of vWF PTM and 3′ splice acceptor of vWF mini-gene target, both PTMs and target were co-delivered into 293 cells by transient transfection. Total RNA of transfected cells was isolated and a 411bp PCR fragment representing specific exon 6 and 7 splice junction trans-spliced mRNA was detected by RT-PCR and confirmed by sequencing. The level of trans-splicing was estimated to be at 0.1–1%. Our results demonstrate the repair of defective murine vWF mini-gene pre-mRNA was achieved in our in vitro model established to mimic the genetic defect in vWF−/− KO mice. The use of 2 selected PTMs in vWD KO mice is now ongoing. The fact that only a few rational designed PTMs (2 out 15) were functional also suggested that a systemic and versatile approach is needed in order to identify the most efficient PTM with high specificity. Such an effort is on going in our lab. Overall, These data and our previous results for identifying optimal PTM provide strong evidence to support the use of trans-splicing to correct vWF pre-mRNA in vWF−/− KO mice.