Generation of Hutat2:Fc Knockin Primary Human Monocytes Using CRISPR/Cas9

Generation of Hutat2:Fc Knockin Primary Human Monocytes Using CRISPR/Cas9

May 20, 2019

Abstract

The ability of monocytes to travel through the bloodstream, traverse tissue barriers, and aggregate at disease sites endows these cells with the attractive potential to carry therapeutic genes into the nervous system. However, gene editing in primary human monocytes has long been a challenge. Here, we applied the CRISPR/Cas9 system to deliver the large functional Hutat2:Fc DNA fragment into the genome of primary monocytes to neutralize HIV-1 transactivator of transcription (Tat), an essential neurotoxic factor that causes HIV-associated neurocognitive disorder (HAND) in the nervous system. Following homology-directed repair (HDR), ∼10% of the primary human monocytes exhibited knockin of the Hutat2:Fc gene in the AAVS1 locus, the “safe harbor” locus of the human genome, without selection. Importantly, the release of Hutat2:Fc by these modified monocytes protected neurons from Tat-induced neurotoxicity, reduced HIV replication, and restored T cell homeostasis. Moreover, compared with lentiviral transfection, CRISPR-mediated knockin had the advantage of maintaining the migrating function of monocytes. These results establish CRISPR/Cas9-mediated Hutat2:Fc knockin monocytes and provide a potential method to cross the blood-brain barrier for HAND therapy.

Authors: Bowen Wang, Jiahui Zuo, Wenzhen Kang, Qianqi Wei, Jianhui Li, Chunfu Wang, Zhihui Liu, Yuanan Lu, Yan Zhuang, Bianli Dang, Qing Liu, Wen Kang, Yongtao Sun

Influence Factor: 6.392

Citation: Mol Ther Nucleic Acids 11, 130-141 (2018).

Read entire article

Share:

Back to top