Abstract

A HIV cure is possible as demonstrated by the Berlin patient who was cured of HIV after CCR5-Δ32/Δ32 homozygous deficient bone marrow transplantations. The ease and versatility of the CRISPR/Cas9-mediated gene editing technology has spurred an immense interest in using it to edit the CCR5 gene for anti HIV human hematopoietic stem/progenitor cells (HSPCs) based gene therapy strategies.

To maximize the levels of CCR5 gene modification in HSPCs, we recently developed a thermo sensitive/replication restricted Sendai virus vector for highly efficient CRISPR-Cas9 mediated editing of the CCR5 gene (ts SeV-Cas9-CCR5). Ts SeV-Cas9-CCR5 consistently transduces human fetal liver derived and G-CSF mobilized peripheral blood CD34+HSPCs with high efficiencies (~90%) at a multiplicity of infection of 5, including the CD34+/CD38-/CD90+(Thy1+)/CD49f^high subpopulation capable of hematopoietic reconstitution by a single cell in a humanized NSG mouse. Remarkably, our SeV-Cas9-CCR5 also edits at CCR5 gene at ~80%. Shifting to 37°C resulted in rapid loss of the SeV vector. Ts SeV-Cas9-CCR5 vector transduced CD34+ HSPCs differentiate into various lineage-specific CFUs by standard in vitro colony forming assays and CD14+/CD33+ macrophages in vitro.

Our current study demonstrated unprecedented efficiency (~80%) of CCR5 gene editing in human HSPCs by a newly developed SeV CRISPR/Cas9 delivery vector. SeV is non-pathogenic in humans, has an established safety record, and has been extensively studied and modified for gene therapy applications. Our SeV-Cas9-CCR5 vector can be developed clinical applications requiring highly efficient gene editing in HSPCs.