Thursday, September 19, 2024
9/19/2024
Project Summary: The strategy of selective killing cancer cells while sparing healthy cells holds great promise for cancer therapy, but its effective implementation remains a formidable challenge for numerous cancer types. Targeted therapies have so far primarily focused on inhibiting or modulating the activity of proteins that help cancer cells survive or proliferate, rather than directly targeting the mutations in driver genes. This proposal seeks to bridge this critical void by introducing and evaluating a novel approach. Its primary goal is to assess a method harnessing engineered catalytic RNA molecules, known as ribozymes, for the specific killing of cells expressing mRNA of cancer genes with certain cancer driver mutations. In addition, it will test our recently developed combinatorial method (RABADOCS) for optimizing ribozyme sequences by sampling millions of variants to identify the most specific and sufficiently efficient ones. We will use group I intron ribozymes from the species Tetrahymena thermophila that can be re-engineered to specifically recognize a splice site on a targeted mRNA and replace the 3'-portion of that RNA with a sequence provided by the ribozyme. These ribozymes require a U (uridine) at the target’s splice site and are inactive when this U is absent. This will allow the ribozymes to differentiate between mutant and wild type target mRNA and ultimately between cancer and healthy cells. Our ribozymes will mediate cell killing by replacing the 3'-portion of the target mRNA with a sequence encoding a cytotoxic peptide in-frame. Translation of spliced mRNAs will then induce death for cells expressing the mutant mRNA. As a proof of concept, we will develop and optimize trans-splicing ribozymes that kill cells containing the JAK2 mutation V617F (c.1849G>T), which is the most common driver mutation in classical myeloproliferative neoplasms (MPNs). Aim 1 will optimize the ribozyme’s trans-splicing efficiency and specificity independent of any toxin sequence. The ribozyme’s external guide sequence will be optimized by employing ribozyme libraries and selecting the best variants using RABADOCS. In parallel, aim 2 will test several toxin peptides that are translated from constructs with the same sequence as ribozyme splice products. The results will identify ribozyme-mediated toxicities that maximize the desired killing while minimizing off-target killing. Lastly, in Aim 3 the most promising ribozymes and toxin sequences will be tested in murine hematopoietic stem-cell like cells (HPC-7) expressing wild-type JAK2 and/or JAK2V617F. We hypothesize that our combinatorial approach will yield ribozymes with high target sensitivity and specificity, providing the proof of concept that ribozyme-mediated targeted killing of cancer cells can be achieved. As a by- product, our study may provide the groundwork for a novel therapeutic strategy for JAK2V617F-driven MPNs.
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