Circularization of Single-Stranded DNA Donor Template Unleashes the Power of Non-Viral Gene Delivery for Long-Term HSCs editing

Abstract

Over the past decade, non-viral DNA template delivery has been used with engineered nucleases to target single-stranded DNA sequences in hematopoietic stem and progenitor cells. While effective for gene therapy, this method has been limited to short DNA donor templates, restricting its applications to gene corrections. To expand its scope, we developed an editing process using kilobase-long circular single-stranded DNA donor templates and TALEN technology. Our results show that the CssDNA editing process achieves high gene insertion frequency in viable HSPCs. Compared to a conventional AAV-editing process, CssDNA-edited HSPCs show a higher propensity to engraft and maintain gene edits in a murine model. This positive outcome is partly due to higher levels of primitive edited HSPCs, a more quiescent metabolic state, and elevated expression of bone marrow niche adhesion markers. Our findings highlight the strong potential of CssDNA as a universal and efficient non-viral DNA template for gene therapy applications.