Stevens Christian S, Carmichael Jillian C, Watkinson Ruth, Kowdle Shreyas, Reis Rebecca A, Hamane Kory, Jang Jason, Park Arnold, Pernet Olivier, Khamaikawin Wannisa, Hong Patrick, Thibault Patricia, Gowlikar Aditya, An Dong Sung, Lee Benhur
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
UCLA School of Nursing, Los Angeles, California, USA.
J Virol. 2024 Dec 17;98(12):e0083224. doi: 10.1128/jvi.00832-24. Epub 2024 Nov 4.
The therapeutic potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and less immunogenic Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types with limited induction of an innate immune response. ts SeV demonstrates high transduction efficiency in human CD34 hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34/CD38/CD45RA/CD90(Thy1)/CD49f stem cell enriched subpopulation. The frequency of editing exceeded 90% and bi-allelic editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14 monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help further expand the possibilities in personalized medicine and the treatment of genetic disorders.
Gene editing has the potential to be a powerful tool for the treatment of human diseases including HIV, β-thalassemias, and sickle cell disease. Recent advances have begun to overcome one of the major limiting factors of this technology, namely delivery of the CRISPR-Cas9 gene editing machinery, by utilizing viral vectors. However, gene editing therapies have yet to be implemented due to inherent risks associated with the DNA viral vectors typically used for delivery. As an alternative strategy, we have developed an RNA-based Sendai virus CRISPR-Cas9 delivery vector that does not integrate into the genome, is temperature sensitive, and does not induce a significant host interferon response. This recombinant SeV successfully delivered CRISPR-Cas9 in primary human CD14+ monocytes resulting in a high level of CCR5 editing and inhibition of HIV infection.
基因编辑技术的治疗潜力取决于安全有效的递送方法的开发。在本研究中，我们开发了一种温度敏感且免疫原性较低的仙台病毒（ts SeV），作为用于CRISPR-Cas9的新型递送载体，并用于在先天免疫反应诱导有限的敏感人类细胞类型中进行高效基因编辑。ts SeV在人类CD34造血干细胞和祖细胞（HSPCs）中显示出高转导效率，包括对富含CD34/CD38/CD45RA/CD90（Thy1）/CD49f干细胞的亚群的转导。编辑频率超过90%，双等位基因编辑超过70%，导致原代人CD14单核细胞中HIV-1感染受到显著抑制。这些结果证明了ts SeV平台作为当前基因编辑工具递送方法的一种安全、高效且灵活的补充的潜力，这可能有助于进一步扩大个性化医学和遗传疾病治疗的可能性。
基因编辑有潜力成为治疗人类疾病（包括HIV、β-地中海贫血和镰状细胞病）的强大工具。最近的进展已开始通过利用病毒载体克服该技术的一个主要限制因素，即CRISPR-Cas9基因编辑机制的递送。然而，由于与通常用于递送的DNA病毒载体相关的固有风险，基因编辑疗法尚未得到实施。作为一种替代策略，我们开发了一种基于RNA的仙台病毒CRISPR-Cas9递送载体，该载体不整合到基因组中，对温度敏感，并且不会诱导显著的宿主干扰素反应。这种重组SeV成功地在原代人CD14+单核细胞中递送了CRISPR-Cas9，导致高水平的CCR5编辑和HIV感染的抑制。
