Elimination of proliferating cells from CNS grafts using a Ki67 promoter-driven thymidine kinase

Abstract

Pluripotent stem cell (PSC)-based cell therapy is an attractive concept for neurodegenerative diseases, but can lead to tumor formation. This is particularly relevant as proliferating neural precursors rather than postmitotic mature neurons need to be transplanted. Thus, safety mechanisms to eliminate proliferating cells are needed. Here, we propose a suicide gene approach, based on cell cycle-dependent promoter Ki67-driven expression of herpes simplex virus thymidine kinase (HSV-TK). We generated a PSC line expressing this construct and induced neural differentiation. In vitro, proliferating PSC and early neural precursor cells (NPC) were killed by exposure to ganciclovir. In vivo, transplantation of PSC led to tumor formation, which was prevented by early ganciclovir treatment. Transplanted NPC did not lead to tumor formation and their survival and neural maturation were not affected by ganciclovir. In conclusion, the cell cycle promoter-driven suicide gene approach described in this study allows killing of proliferating undifferentiated precursor cells without expression of the suicide gene in mature neurons. This approach could also be of use for other stem cell-based therapies where the final target consists of postmitotic cells.

Neurothreads: Cryogel carrier-based differentiation and delivery of mature neurons in the treatment of Parkinson’s disease

“…We observed a significant increase in the amount of cells during maturation from an NPC to a mature neuronal state (Fig, 6c). Although further analysis at longer time-points is certainly required, we consider this result as an in-vitro confirmation of the potential risk of in-vivo tumorigenesis, one of the main challenges associated with transplantation NPCs 86,87 . Our results highlight the need for tight control of proliferation and terminal differentiation.…”

“…Our results highlight the need for tight control of proliferation and terminal differentiation. This underscores the importance of emerging approaches such as the incorporation of a suicide gene for proliferating cells into the graft 86 , selection of a suitable progenitor population, expressing specific surface ventral midbrain markers 88 , or by trans-differentiation of more mature cells 89 . It also underpins the importance of approaches such as the one investigated here that enable prolonged maturation prior to implantation, offering the possibility of later quality control points.…”

Human Genomic Safe Harbors and the Suicide Gene-Based Safeguard System for iPSC-Based Cell Therapy

“…S4). Previous studies have demonstrated that the HSV‐TK/GCV suicide system ablated teratomas in an efficient and specific manner ; however, one recent study reported GCV resistance partially due to escape mutations in HSV‐TK . The experimental parameters differed between these studies, including the iPSC or embryonic stem cell (ESC) lines, cell preparation, promoter, gene integration method, HSV‐TK variant, onset and duration of GCV treatment after cell transplantation, GCV dose, route of GCV administration, and observation period.…”

Viral Vector-Based Innovative Approaches to Directly Abolishing Tumorigenic Pluripotent Stem Cells for Safer Regenerative Medicine

“…This therapy can selectively kill actively proliferating cancer cells, including undifferentiated tumorigenic cells. Lentivirus-transduced and undifferentiated hPSCs expressing the HSV-tk gene are effectively eliminated in vitro in the presence of GCV 19, 20. In addition, in vivo teratomas derived from HSV-tk- transduced hPSCs shrink following the administration of GCV.…”