478. Highly Efficient Targeted Gene Addition in CD34+ Hematopoietic Stem/Progenitor Cells by Combining ZFN mRNA and AAV6 Donor Delivery

Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene correction/addition, respectively. Hematopoietic stem/progenitor cells (HSPC) are an important clinical target for gene therapy. These cells can be readily harvested from the subject, engineered using viral vectors, and re-introduced back into the patient where they give rise to all of the mature cells of the blood and immune system. Thus the ability to edit the genome of HSPCs could provide a corrective therapy for a number of monogenic diseases. However, achieving high levels of editing by HDR may be significantly more challenging than gene knockout by NHEJ. Here we report that the combination of delivering the ZFNs as mRNA and corrective donor DNA via a rAAV6 vector can induce highly efficient targeted gene addition in CD34+ HSPCs.Using CCR5-specific ZFNs and donor DNA templates as a model system we show that targeted addition of a novel restriction fragment length polymorphism (RFLP) was consistently achieved at u003e15%, while insertion of an eGFP expression cassette was achieved at u003e10% of the CCR5 loci in mobilized CD34+ cells. Furthermore, targeting the AAVS1 “safe harbor” locus with a pair of AAVS1-specific ZFNs (mRNA) and a RFLP donor (rAAV6) resulted in u003e30% of AAVS1 alleles specifically being edited to include the RFLP, suggesting this genome editing approach to achieve highly efficient targeted gene addition in CD34+ cells can be applied to different genetic loci. Edited HSPC undergo normal hematopoiesis in both in vitro differentiation assays and during engraftment of NSG mice. Using fetal liver derived cells, editing of primitive and more committed CD34+ HSPC subsets was observed at similar levels in HSPC CFU cultures, and by analysis of sorted populations of treated HSPC, including LT-HSC, and through the secondary transplantation of NSG mice.The use of ZFNs in combination with rAAV6 delivery of the donor DNA provides a significant advance in genome editing that has great potential for targeted correction of human disease-causing mutations and permanent transgene addition in human CD34+ HSPC.