The use of CRISPR/DCAS9 engineered mscs to prevent cisplatin-induced cellular damage in human granulosa cells

Chemotherapy-induced ovarian failure (COF) can cause ovarian dysfunction diseases in which the ovaries temporarily or permanently stop functioning due to chemotherapy. Low estrogen serum levels and amenorrhea are infertility signs may contribute the clinical appearance of COF. Cisplatin is a platinum-based anticancer chemotherapeutic drug that has a gonadotoxic effect on all ovarian cells. Stem cells, especially mesenchymal stem cells (MSCs) obtained from different adult tissues, have been tried as an adjunct therapy for COF. CRISPR genome editing technology is the most broadly used gene editing system, and dCas9 (dead Cas9) is a targeted CRISPR activation tool that lacks the nuclease effect. A synergistic activation mediator (SAM) is a type of CRISPR/dCas9 activation system composed of the dCas9-VP64 fusion protein integrated with a modified sgRNA, including two MS2 aptamers. MS2 binding protein is fused with two other activator domains, p65 and HSF1, and this fusion protein (MS2-p65-HSF1) interacts with MS2 aptamers. Exosomes are extracellular vesicles that range between 20–200 nm and can be isolated from biological fluids. They contain mRNA, long non-coding RNA, microRNAs, proteins, and lipids. Exosomes play an essential role in intercellular communication, in delivering gene and protein expression signals of cells. The aim of this research is to produce dCas9 genome edited MSCs and their exosomes and to test whether these cells are more resistant or chemotherapeutic, cisplatin, or not. Cisplatin-resistant mesenchymal stem cells were obtained by activating the CD99 gene with the SAM activator system. Then, exosomes were obtained from normal and resistant mesenchymal stem cells separately. Later, spheroids were formed with these cells. Co-cultures of MSCs were established with the experimental treatment groups at with hGRC1 (human granulosa cell line) cells in the insert using the Transwell aid. After the co-cultures were established, the cells were exposed to cisplatin. After that, hGRC1 cells were harvested at 24, 48, 72, and 96 hours, respectively. Firstly, a cell viability assay was performed on the collected cells. Then, by qPCR the expression of inflammatory or apoptotic markers as IL-10, IL-1Ra, Bax, and Bcl-2 genes was analyzed. As a result, it was observed that there were more viable cells in all experimental groups compared to the control group, and inflammatory and apoptosis values decreased in each hour group. And it was also discovered that resistant cells have provided a more effective protection than the non-resistant cells. The group that provides the most significant protection compared to all other experimental groups is the group that uses resistant mesenchymal stem cells and exosomes isolated from these cells, and this finding is important as it can be interpreted that the use of resistant mesenchymal cells with their exosomes can be an alternative adjunct therapy to protect ovarian granulosa cells during the use of cisplatin like chemotherapeutics for cancer. Keywords: Mesenchymal Stem Cells, Cisplatin, CRISPR/dCas9, Exosomes, Cancer, Infertility