Date of Award
Doctor of Philosophy (PhD)
Hemophilia B (HB) is a life-threatening inherited disease caused by mutations in the FIX gene, leading to reduced protein levels and abnormal blood clotting. Due to its monogenic nature, HB is one of the primary targets for gene therapy. Indeed, successful correction of HB has been shown in clinical trials using gene replacement approaches. However, application of these strategies to non-adult patients is limited due to high cell turnover as young patients develop, resulting in the loss of therapeutic expression. Gene editing can potentially overcome this issue by permanently inserting the corrective gene. In this study, we explored adenovirus as a platform for corrective CRISPR/Cas9-mediated gene integration in wild-type mice and in hemophilia B mice. We determined as a proof-of-principle that adenoviral delivery of CRISPR/Cas9 is capable of corrective gene addition, leading to long-term augmentation of gene expression, FIX activity, and phenotypic correction in a murine model of juvenile HB. Edited alleles were detectable throughout the time courses as were persistent vector genomes. While we found on-target error-free integration in all examined samples, some mice also contained mutations at the integration target site. Off-target analysis showed little off-target editing, although two off-target integration sites were found. Additionally, we detected adaptive immune responses against the vector and Cas9 nuclease. In total, our findings show that the adenovirus platform is suitable for gene insertion in juveniles with inherited disease and suggests this approach may be applicable to other diseases.
Chair and Committee
David Katherine T. Curiel Ponder
Sharon C. Morley, Wayne M. Yokoyama, Blair B. Madison,
Stephens, Calvin, "Adenoviral Delivery of CRISPR/Cas9 for Gene Editing-based Gene Therapy of Hemophilia B" (2018). Arts & Sciences Electronic Theses and Dissertations. 1704.
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