Objective: To increase the safety of future cell-based therapy in Parkinson’s disease, we generated human induced pluripotent stem cells (hiPSCs) possessing a safety switch via the CRISPR/Cas9 techniques.

Background: Cell-based therapy using hiPSCs is an upcoming treatment in Parkinson’s disease. Although the manipulation of hiPSCs has been improved in order to meet clinical requirement, transplantation of somatic cells obtained from hiPSCs still has concerns of adverse events including tumorigenesis. One strategy to solve this issue is the use of a suicide gene, and Herpes simplex virus thymidine kinase (HSV-TK) have shown a promise as a safety switch in hiPSCs. However, previous studies adopted retroviral random integration of the HSV-TK gene, thus transgenes could suffer position effect and in worst case transduction itself could dysregulate cancer-related genes. To overcome this limitation, we herein genetically modified hiPSCs using the clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated protein (Cas) system and inserted HSV-TK into an extragenic safe harbor.

Methods: We selected an extragenic safe harbor located far from any gene or microRNA loci so as not to dysregulate nearby gene expression. We employed double nicking strategy and truncated single-guide RNAs for reducing CRISPR/Cas9 off-target effect. Precise integration of a suicide gene was confirmed in isolated iPSCs colonies. Efficacy of HSV-TK in an extragenic safe harbor was analyzed in vitro and in vivo.

Results: We confirmed CRISPR/Cas9-mediated knock-in of HSV-TK in HEK293T and iPSCs. Six out of 15 (33%) isolated iPS colonies possessed the knock-in allele. HSV-TK expression was detected in established iPSC lines and ganciclovir treatment was effective to eliminate genetically modified iPSCs.

Conclusions: A suicide gene, HSV-TK, in an extragenic safe harbor could be a safe bet in future cell-based therapy. Furthermore, this strategy can be applicable in therapeutic gene transfer for future regenerative medicine.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/generation-of-human-induced-pluripotent-stem-cells-carrying-a-safety-bet-for-cell-based-therapy-in-parkinsons-disease/