Poster abstracts
Poster number 136 submitted by Alejandro Tapia
Engineered Suppressor tRNAs Rescues Nonsense Mutations In TP53
Alejandro Tapia (Microbiology & Immunology), Jennifer Flora (Biochemistry & Molecular Biology), Olabode Dawodu (Biochemistry & Molecular Biology), Caitlin Specht (Biochemistry & Molecular Biology), Roland W. Herzog (Microbiology & Immunology), Jeffery Tharp (Biochemistry & Molecular Biology)
Abstract:
Background:
Over 10% of genetic diseases are caused by mutations that introduce stop codons into protein coding genes, known as premature termination codons (PTCs). mRNA transcripts harboring PTCs result in premature polypeptide cleavage, leading to loss of protein function. One promising approach for PTC-readthrough is the development of engineered suppressor transfer RNAs (sup-tRNAs), which are modified to recognize one of the 3 stop codons. Sup-tRNAs can incorporate amino acids (AAs) at PTCs, outcompeting endogenous factors that terminate translation, inducing stop codon readthrough. The application of sup-tRNAs for cancer therapeutics has not been explored. Tumor suppressor protein p53 (encoded by TP53) is one of the most mutated proteins in cancer—11% of cancer-associated mutations on TP53 are PTCs. We hypothesize that sup-tRNAs can induce translational readthrough of PTCs in the TP53 gene to restore p53 protein levels, rescue p53 function, and inhibit cancer cell proliferation.
Methods:
We developed a novel class of sup-tRNAs that efficiently suppress all 3 stop codons. We tested the ability of these sup-tRNAs to elicit translational readthrough of PTCs in cancer cells with homozygous TP53 mutations. p53 protein levels were quantified by immunoblotting. p53 activity was quantified with a combination of luciferase-based transcriptional assays and qRT-PCR.
Results:
Sup-tRNA transfection resulted in increased levels of p53 protein levels and function. Moreover, sup-tRNA-treated cells showed increased expression of several downstream genes that are transactivated by p53.
Conclusions:
Sup-tRNAs can restore functionally active p53 protein in cancer cells carrying PTC mutations in TP53. Sup-tRNAs are a potential approach to help drive tumor suppression. The use of sup-tRNAs for gene therapy can overcome current hurdles of vector transgene capacity. Further validating this therapy in animal models will help solidify its potential as a clinical therapeutic.
References:
Mort, M., et al. (2008). Human Mutation 29(8): 1037-1047.
Floquet, C., et al. (2010). Nucleic Acids Research 39(8): 3350-3362.
Albers, S., et al. (2023). Nature 618(7966): 842-848.
Biorender
Keywords: p53, sup-tRNA, PTC-readthrough