PROJECT SUMMARY Metastatic colorectal cancer (mCRC) remains highly lethal despite decades of optimizing 5-fluorouracil (5-FU)- based combination chemotherapy regimens that are central to treatment. The causes of treatment failure with 5-FU-based regimens include decreased metabolism to FdUMP, the primary active metabolite, and overexpression of thymidylate synthase (TS), the molecular target of FdUMP. To improve outcomes and overcome resistance we have developed a nanoscale FP polymer, CF10, that is comprised of FdUMP monomers linked in a single-stranded DNA backbone that releases FdUMP in a single step. CF10 displays markedly improved anti-tumor activity with low systemic toxicity relative to 5-FU and is a candidate for clinical translation. We hypothesize that nanoformulation of CF10 to increase plasma retention and actively target malignant tissue will improve anti-tumor activity and specificity. Lipid nanoparticle (LNP) formulation has proven to be a robust delivery strategy for multiple nucleic acid drugs, and in collaboration with NanoVation Therapeutics (NTx) in Aim 1 we will test two CF10:LNP formulations designed to differentially target CF10 to liver and more broadly to target metastatic tissue while protecting CF10 from degradation in plasma. In Aim 2, we will investigate PEGylation of CF10 together with active targeting to tumor tissue through conjugation with a cyclic RGD peptide that targets integrin V3 expressed specifically in malignant tissue. We will test our novel nanomaterials for improved anti- metastatic activity using: (i) a novel genetically engineered mouse model, iKAP, that forms tumors specifically in the colon with metastatic progression to the liver and lung; and (ii) a rat model of established colorectal liver metastases (CRLMs). Pharmacokinetic profiling (PK) will be evaluated using LC/MS/MS. In Aim 3, we will develop a 3rd generation FP polymer that includes 5-ethynyl-2’-deoxyuridine (EdU), a thymidine analog that is cytotoxic through complementary mechanisms to FdU. We will use the optimal LNP formulation and PEGylation strategies determined for CF10 in Aims 1 and 2 to develop a highly novel nanomaterial with exceptional potential for improved treatment of mCRC.