Preclinical development and qualification of ZFN-mediated CCR5 disruption in human hematopoietic stem/progenitor cells

作者：David DiGiusto, Paula M. Cannon, Michael C. Holmes, Lijing Li, Anitha Rao, Jianbin Wang, Gary Lee, Philip D. Gregory, Kenneth A Kim, Samuel B. Hayward, Kathleen Meyer, Colin M. Exline, Evan Lopez, Jill Henley, Nancy González, Victoria Bedell, Rodica Stan, John A. Zaia · 发表于：Molecular Therapy — Methods & Clinical Development · 年份：2016 · DOI：10.1038/mtm.2016.67 · 被引用次数：116 · 研究领域：HIV Research and Treatment、CRISPR and Genetic Engineering、Cytomegalovirus and herpesvirus research

Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5) is the coreceptor required for R5-tropic HIV-1 infection of human cells. Deletion of CCR5 renders cells resistant to R5-tropic HIV-1 infection, and the potential for cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion of CCR5 in donor hematopoietic stem/progenitor cells (HSPC). The requirement for HLA-matched HSPC bearing homozygous CCR5 deletions prohibits widespread application of this approach. Thus, a strategy to disrupt CCR5 genomic sequences in HSPC using zinc finger nucleases was developed. Following discussions with regulatory agencies, we conducted IND-enabling preclinical in vitro and in vivo testing to demonstrate the feasibility and (preclinical) safety of zinc finger nucleases-based CCR5 disruption in HSPC. We report here the clinical-scale manufacturing process necessary to deliver CCR5-specific zinc finger nucleases mRNA to HSPC using electroporation and the preclinical safety data. Our results demonstrate effective biallelic CCR5 disruption in up to 72.9% of modified colony forming units from adult mobilized HSPC with maintenance of hematopoietic potential in vitro and in vivo . Tumorigenicity studies demonstrated initial product safety; further safety and feasibility studies are ongoing in subjects infected with HIV-1 (NCT02500849@clinicaltrials.gov).