Friday, May 9, 2025
5/9/2025
Modified saRNA Encoded BiTEs for Lung Cancer - PROJECT SUMMARY/ABSTRACT This proposal describes an innovative approach to treat lung cancer, the leading cause of cancer deaths in the U.S., with a novel self-amplifying ribonucleic acid (saRNA) technology encoding for a bispecific T cell engager (BiTE) antibody. Catalytic RNA technologies, such as saRNA offer substantial promise but opportunities remain for performance optimization. saRNA encodes a virally derived RNA-dependent RNA polymerase (RdRp) on the same RNA strand as the sequence for the desired protein cargo(s). After being translated, this RdRp produces more copies of the RNA template, leading to amplification of the delivered construct with resulting increased amounts and duration of the protein. Unfortunately, saRNA evokes a substantial innate immune response leading to saRNA degradation, decreased transfection capability, and adverse in vivo side effects. Although incorporation of modified nucleoside triphosphates (modNTPs) such as N1mΨU in mRNA yields a clinically functional construct, multiple attempts to use N1mΨU in saRNA have been unsuccessful. These results led to the conclusion that modNTPs are incompatible with saRNA. Contrary to these reports, we discovered that other modNTPs at 100% substitution in saRNA reduce the innate immune response and confer enhanced protein expression potency and duration. Using a functional protein expression screening assay, we identified modNTPs including 5-methylcytidine (5mC), that are transcriptionally and translationally active. 5mC saRNA significantly enhances protein expression in multiple immortalized and primary human cell lines. A single intramuscular injection of 5mC saRNA, encoding for luciferase, results in 30+ day protein expression. Further, the type I interferon response is less in transfected primary human PBMCs in vitro and after intramuscular administration in vivo compared to wild-type saRNA. We hypothesize that a 5mC modified saRNA, encoding for a BITE, will afford a functional antibody that targets tumors and engages T cells for killing, prolongs in vivo BiTE pharmacokinetics, and extends survival compared to a N1mΨU mRNA or unmodified saRNA encoding a BITE or a conventional BiTE protein therapeutic in both patient-derived xenograft (PDX) and immune competent murine models. Importantly, preliminary data support the proposed studies, well-characterized materials and rigorous experimental designs are established, and essential cross- disciplinary collaborations and expertise are in place to address this hypothesis. The specific aims of this five- year proposal are as follows. Aim 1 designs 5mC modified saRNA constructs encoding BiTE antibodies and evaluates protein expression performance in vitro. Aim 2 assess the in vitro/ in vivo bioavailability and resulting in vitro bioactivity of the BiTE antibody translated from the 5mC modified saRNA constructs. Aim 3 determines the maximum tolerated dose, pharmacokinetics, toxicity, and in vivo efficacy of 5mC modified saRNA, encoding a BiTE antibody, against lung carcinomas. Furthermore, we will establish a unique saRNA treatment strategy which will be broadly applicable to the sustained delivery of other therapeutic proteins and against other cancers.
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