In 2021, over 15 million people were living with a spinal cord injury. For treatment, researchers have turned to neuronal engraftment, the transplant of neuronal cells, as a possible remedy. However, this option remains limited due to the lack of access to primary neuronal cells, which do not regenerate. Cellular reprogramming offers a potential solution by converting accessible cell types, such as mouse embryonic fibroblasts (MEFs), into induced motor neurons (iMNs). This can be achieved by overexpressing the transcription factors (TFs) Lhx3, Isl1, and Ngn2; nevertheless, reprogramming rates are low unless the cells are in a hyperproliferative state. This state is normally restricted by p53, which halts fast-cycling cells to prevent genomic instability. The p53 dominant-negative mutant p53DD overcomes this barrier and improves reprogramming rates. Its effects are further enhanced when paired with the proliferative oncogene HRASG12V; this combination is known as DDR. However, current DDR delivery relies on viral genomic integration, raising concerns about integrated oncogenes and tumorigenesis. The small-molecule PMA has been explored as a substitute for HRASG12V, but no replacement has been identified for the virally delivered p53DD. This project aims to address that gap by delivering p53DD via self-amplifying RNA (saRNA), a platform capable of sustained expression without genomic integration. In Spring 2026, we designed constructs combining p53DD and saRNA elements. These plasmids were cloned and transcribed in vitro, then introduced into HEK293T cells and MEFs to evaluate successful saRNA delivery. In the upcoming academic year, we plan to assess reprogramming efficiency, cellular viability, and the effects of saRNA on the reprogramming process. Ultimately, this work seeks to evaluate whether combining p53DD with saRNA can provide a more efficient platform for generating reprogrammed iMNs.

I decided to participate in the SuperUROP program to expand my research skills. I wanted to learn how to write a technical paper and communicate my results. While I do have experience in these areas, I want to further improve myself as a researcher and engineer. I am excited about my project and hope to enhance cellular reprogramming via saRNA technology!