(200am) CRISPR-Cas9 Plasmid DNA Delivery to Endometrial Cancer Cells for Knockout of PLAC1

Several endometrial cancers overexpress the PLAC1 protein, and overexpression has been linked to more aggressive tumors. The CRISPR-Cas9 paradigm can target the gene locus for this protein specifically and effectively in order to reduce PLAC1 expression and subsequently reduce tumor proliferation.

We have identified and developed three CRISPR-Cas9 platforms encoding for varying degrees of DNA editing at the PLAC1 site. In order to enhance delivery of these plasmids into endometrial cancer cell lines in vitro, we have formed nanoplexes by combining plasmid DNA with the cationic polymer polyethylenimine. We varied the ratio of nitrogen in PEI to phosphate in the DNA (N/P) ratio to alter the surface charge and subsequently uptake.

These nanoplexes were approximately 100 nm in diameter with a net positive surface charge. We measured the cell viability and the relative gene expression using MTS and qPCR, respectively, in three different endometrial cancer cell lines. Each of these cell lines has a different p53 mutation, thus allowing us to investigate p53 status as a compounding effect. Our cell viability results indicated that loss of function p53 cells are most susceptible to the nanoplexes. Additionally, the CRISPR sequence that encoded for the least amount of DNA editing was sufficient to reduce PLAC1 gene expression, and was more effective than those that encoded for more editing.

To the best of our knowledge, this is the first investigation into the combinatorial effects of p53 status and PLAC1 expression on endometrial cancers. This research has the potential to improve treatment options for endometrial cancer and offers an alternative to conventional treatment methods such as chemotherapy and surgery.