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Cancer immunotherapy has gained recent recognition as a new and clinically effective therapeutic avenue in cancer treatment. Therapeutic cancer vaccine is yet another emerging and promising direction in cancer immunotherapy. Current generation of cancer vaccines are designed to trigger a tumor specific immune response by predominantly targeting aberrantly expressed wildtype proteins in tumor cells, known as tumor-associated antigens (TAAs), oncoviral proteins from viral integration and mutant proteins from single nucleotide variations in tumors, denoted as tumor-specific antigens (TSAs). Recent clinical studies testing TAA- and TSA-based cancer vaccines has encountered several limitations in eliciting specific and robust anti-tumor immune response due to their low specificity and immunogenicity. Acknowledging the current limitations in therapeutic cancer vaccine design, we were interested in finding alternative TSAs which would be more potent than the currently used single nucleotide variants derived TSAs (SNV-TSAs). In that, we focused our attention to new frame derived antigens, abbreviated as NFDAs, which being derived from frameshift insertion/deletion mutations (not divisible by 3) and splice-site mutations, generated an alternative open reading frame (ORF), and produced C-terminal altered truncated proteins. However, they are suppressed by nonsense-mediated mRNA decay (NMD) in cells. To harness the untapped potential of NFDAs, we proposed to conduct a proof-of-concept study testing our hypothesis that NMD inhibition would allow the expression of previously suppressed NFDAs and that these antigens can further be used in cancer vaccine formulation to elicit a strong and specific immune response against tumor cells. To test our hypothesis, we identified NMD-suppressed NFDAs from whole exome sequencing data of high tumor mutation burden (TMB) mouse followed by NMD blockade through inducible shRNA knockdown and CRISPR/Cas9 knockout of essential NMD factors, Upf2 and Smg1 in these high TMB mouse cell lines. Next, the expression of natural NMD substrates and in-silico identified NMD-suppressed NFDAs was detected using qPCR assays. Our findings report the existence of NMD-suppressed NFDAs in high TMB mouse cell lines whose expression was upregulated from NMD blockage. |
