Genome Editing for FVIII and AAT Gene Therapy Cures

The CRISPR/Cas9 nuclease system is a powerful tool for manipulating genomes by DNA editing. This allows for functional knock-in for gene therapy correction. However, current limits of in vivo gene delivery methods have practically restricted clinical application. In this regard, the recombinant adeno-associated viral (AAV) vectors have been mainly applied for in vivo CRISPR/Cas9-mediated gene therapy studies. However, native AAV tropism limits the possibility of organ selective gene editing. In addition, AAV’s innate liver tropism is associated with hepatotoxicity. As an anodyne, we explored adenoviral (Ad) vector to achieve effective CRISPR/Cas9 based knock-in. The unique capacity to alter Ad tropism can address both of these limits. For alpha1-antitrypsin (AAT) deficiency lung disease, the augmentation of AAT within the lower respiratory tract is the key biologic mandate, thus we targeted Ad vector to the pulmonary endothelium. This pulmonary targeted Ad approach has been designed to accomplish gene therapy for AAT lung disease in a murine model of this disorder (SERPINA1a-e KO mice). For hemophilia A gene therapy, we have pursued an approach to limit potential hepatotoxicity. Specifically, we have sought a non-hepatic source of the corrective factor VIII (FVIII). Thus we explored a pan-endothelium delivery so that vascular endothelium can serve this goal. We designed an optimal co-delivery of CRISPR/Cas9 and FVIII therapeutic gene by a myeloid cell-binding Ad that allows effective and selective in vivo editing for endothelial target cells to achieve phenotypic correction in hemophilia A murine model. Here, we propose a highly original vectorology to traverse key barriers for efficient in vivo genome editing-based gene therapy in order to achieve long term genetic correction of hemophilia A and AAT deficiency.