Supramolecular nanosubstrate–mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies
Peng Yang, Shih‐Jie Chou, Jindian Li, Wenqiao Hui, Wenfei Liu, Na Sun, Ryan Y. Zhang, Yazhen Zhu, Ming‐Long Tsai, Henkie Isahwan Ahmad Mulyadi Lai, Matthew Smalley, Xinyue Zhang, Jiayuan Chen, Zulema Romero, Dahai Liu, Zunfu Ke, Chang Zou, Chin‐Fa Lee, Steven J. Jonas, Qian Ban, Paul S. Weiss, Donald B. Kohn, Kai Chen, Shih‐Hwa Chiou, Hsian‐Rong Tseng
Abstract
Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate-mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)-encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.