Project Summary Specific Aims Solid tumors account for nearly 90% of adult cancers and are challenging to eliminate. Surgery and adjuvant therapy (e.g., radiation, chemotherapy, and immunotherapy) are powerful methods for treating solid tumors. Still, they are not universally effective or curative, and would benefit significantly from some means of augmenting the tumor-killing response. Local delivery of cytotoxic, immunostimulatory, and gene therapy agents, especially in combinations, has proven very effective in promoting solid tumor clearance. Given this potential, it would be powerful to have a platform that sustainably and simultaneously delivers multiple immunostim- ulatory and gene-therapy payloads directly at the tumor, thus promoting its clearance. While current strategies, such as local drug delivery, lentivirus, and mRNA delivery methods, could generate such modifica- tions, they suffer limitations for broader use such as permanent modifications and off-target effects (lentivirus), transient gene expression (mRNA) and drug release, and limited payload. Self-amplifying RNA (saRNA) has shown great promise for prolonged and non-integrative therapeutic gene expression and coupled with lipid na- noparticles (LNPs) could serve as a viable method for local gene delivery and tumor modification. Until recently the potent early immune response triggered upon entry of saRNA into the cell has severely constraining the potency of saRNA, with standard strategies for lowering RNA immunogenicity such as incorporating the modified nucleoside N1-methyl-pseudouridine (found in all mRNA COVID vaccine) yielding non-functional saRNA. How- ever, we have recently discovered that complete substitution of saRNA with the 5 methylcytidine (5mC) drasti- cally reduces immune recognition of saRNA while preserving saRNA replication and gene expression capabili- ties, leading to elevated and prolonged transgene expression. This powerful discovery empowers the appli- cation of saRNA for other therapeutic applications and provides an opportunity to optimize these thera- pies for efficient, multiplex gene delivery. To achieve this, I propose studies for optimizing the saRNA platform by two approaches. The first approach (aim 1) will involve screening both saRNA and LNP formulation to opti- mize their gene delivery across a panel of tumor types, using our best-performing saRNA to express an innova- tive gene drive cassette that would selectively enrich the expression of a suicide gene in the tumor and overcome drug resistance. The second approach (Aim 2) will focus on maximizing transgene cargo in saRNA, screening 1) how many genes can be encoded in a single saRNA by implementing RNA sequence elements, and 2) the maximum possible length of saRNA that can be successfully delivered. This work will establish new bench- marks for RNA therapeutics, providing a platform for efficiently delivering the next generation of gene- therapies for solid tumor treatment.