Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy.

Yang Yang; Yang Liu; Min Chen; Shuangpeng LI; Xuan LU; Yu HE; Kun Zhang; Qingjian Zou

Return

Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy.

Author: Yang YANG ¹ ; Yang LIU ² ; Min CHEN ³ ; Shuangpeng LI ³ ; Xuan LU ³ ; Yu HE ⁴ ; Kun ZHANG ¹ ; Qingjian ZOU ³
Author Information

1. School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, Guangdong, China.
2. School of Life Sciences, University of Science and Technology of China, Hefei 230026, Anhui, China.
3. Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen 529020, Guangdong, China.
4. National Drug Clinical Trial Institution, Jiangmen Central Hospital, Jiangmen 529000, Guangdong, China.
Publication Type:Journal Article
Keywords: AAVS1; caspase 9; homodimerization; human induced pluripotent stem cells (hiPSCs); rapamycin
MeSH: Humans; Induced Pluripotent Stem Cells; Sirolimus/metabolism*; Caspase 9/metabolism*; RNA, Guide, CRISPR-Cas Systems; Pluripotent Stem Cells/metabolism*; Cell Differentiation; Puromycin/metabolism*
From: Chinese Journal of Biotechnology 2023;39(10):4098-4107
CountryChina
Language:Chinese
Abstract: Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.