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Baboon Envelope Pseudotyped "Nanoblades" Carrying Cas9/gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34(+) Cells and Knock-in of AAV6-Encoded Donor DNA in CD34(+) Cells.
Front Genome Ed, 3:604371.
Programmable nucleases have enabled rapid and accessible genome engineering ineukaryotic cells and living organisms. However, their delivery into human bloodcells can be challenging. Here, we have utilized "nanoblades," a new technology thatdelivers a genomic cleaving agent into cells. These are modified murine leukemiavirus (MLV) or HIV-derived virus-like particle (VLP), in which the viral structuralprotein Gag has been fused to Cas9. These VLPs are thus loaded with Cas9 proteincomplexed with the guide RNAs. Highly efficient gene editing was obtained in celllines, IPS and primary mouse and human cells. Here, we showed that nanoblades wereremarkably efficient for entry into human T, B, and hematopoietic stem andprogenitor cells (HSPCs) thanks to their surface co-pseudotyping with baboonretroviral and VSV-G envelope glycoproteins. A brief incubation of human T and Bcells with nanoblades incorporating two gRNAs resulted in 40 and 15% edited deletionin the Wiskott-Aldrich syndrome (WAS) gene locus, respectively. CD34(+) cells(HSPCs) treated with the same nanoblades allowed 30-40% exon 1 drop-out in the WASgene locus. Importantly, no toxicity was detected upon nanoblade-mediated geneediting of these blood cells. Finally, we also treated HSPCs with nanoblades incombination with a donor-encoding rAAV6 vector resulting in up to 40% of stableexpression cassette knock-in into the WAS gene locus. Summarizing, this newtechnology is simple to implement, shows high flexibility for different targetsincluding primary immune cells of human and murine origin, is relatively inexpensiveand therefore gives important prospects for basic and clinical translation in thearea of gene therapy.
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