This study will address a novel tumor cell killing approach designed to ablate solid tumors with a low proliferative index and the attendant neoplastic progenitor cells. Our laboratory has pursued 2-Fluoroadenine (F-Ade), a purine base that disrupts DNA, RNA, and protein synthesis, as an anticancer compound. Here, we utilize F-araAMP, a clinically approved drug that otherwise has negligible activity against solid tumors, as an F- Ade precursor. F-Ade is generated from fludarabine by intratumoral expression of the E. coli purine nucleoside phosphorylase (PNP) gene. The strategy has demonstrated significant efficacy in comprehensive nonclinical studies and a recently completed, first-in-human trial of head and neck squamous cell carcinoma (HNSCC). The scientific objectives of the current proposal are to establish strong and safe tumor regressions based on activity across multiple HNSCC tumor types in vitro and in vivo, and to augment delivery and safety of the PNP gene (Specific Aims 1 and 2). The experiments will evaluate mechanism of action (nucleoside cleavage, PNP transgene activity, disruption of the noncycling tumor cell compartment) (Specific Aim 3). Our experimental plan is multidisciplinary (biomedical engineering, nanoparticle chemistry, DNA/RNA delivery technology, nucleoside metabolism and enzymology), mutually reinforcing, and directed by a senior investigator trained in oncology (Sorscher) and a biomedical engineering scientist beginning his research career (Dahlman). Translational potential is high, as indicated by a successful Phase 1 trial of the technology, formal orphan drug designation, and a Phase 2 study approved by FDA.