Abstract: Adult teleost fish and urodele amphibians can regenerate entire amputated appendages, whereas this ability is restricted to the tip of the distal phalanx in humans and mice. Though modest in comparison to its zebrafish and salamander counterparts, mouse digit tip (MDT) regeneration exhibits similar anatomic stages and proceeds with intramembranous bone regrowth and mostly scarless healing, even in adults. By studying shared principles from organisms with elevated regenerative potential and applying them to mammalian models, we can gain valuable insight into strategies to augment human tissue repair and improve the care of limb loss patients. The catalogue of defined molecular factors in tissue regeneration is expanding and it has become critical to determine how genes involved in regenerative events are engaged upon injury. We previously identified a new class of regulatory sequences we named tissue regeneration enhancer elements (TREEs) that contain sequence information necessary for precise control of regeneration genes. We also reported that TREEs of zebrafish origin can be engineered in viral gene therapy constructs to target expression of pro-regenerative factors to mammalian injury sites and enhance tissue repair. In preliminary collaborative studies between the Poss and Brown groups, we have identified a new set of candidate TREEs from chromatin samples of regenerating MDTs. We have also performed comparative studies guided by single-cell transcriptome analysis of regenerating zebrafish fins and complemented by molecular genetics in mice, to implicate a conserved family of transcription factors in control of digit tip regeneration. Concurrently, we have identified TREEs of zebrafish origin and an effective adeno- associated virus (AAV) capsid variant that can transduce limb tissue and enable selective expression of gene cargoes at an injury site. Our preliminary studies thus unveil a pipeline of TREE control element identification, novel candidate pro-regenerative factor identification, and application of gene therapy methodology. Here, we propose to: 1) identify distal regulatory elements that control key programs in MDT regeneration; 2) define transcriptional mechanisms by conserved factors; and 3) enhance MDT regeneration by spatiotemporal delivery of pro-regenerative factors. This work will increase understanding of transcriptional regulation during mammalian digit tip regeneration and provide important perspective for comprehending, and perhaps changing, existing limitations in the regenerative capacity of human limbs.