Enhancer-based Gene Therapy For Digit Regeneration: Identification Of Precision AAV Control Elements With Starr-seq

PURPOSE: Amputations of digits and limbs are common, life-altering injuries, emphasizing the need for therapies that improve healing or even restore lost tissues. Mouse digit tip (MDT) regeneration serves as a unique model of partial limb regrowth, exhibiting comparable anatomic stages to other species capable of complete limb regeneration. Gene expression during digit regeneration is regulated in part by tissue regeneration enhancer elements (TREEs). Identifying TREE sequences holds great promise for understanding the regenerative process and for use as tools in enhancer-based gene therapy for limb regeneration. METHODS: To find regeneration-associated enhancers, we profiled chromatin accessibility in MDTs 14 days post-amputation (dpa) versus uninjured digits using ATAC-seq. Candidates were refined by linking differentially accessible regions to nearby genes with concordant injury-responsive expression from single-cell RNA-seq and spatial transcriptomics.To test hundreds of potential elements in vivo at once, we implemented an AAV-delivered STARR-seq-style MPRA: ~725-bp genomic fragments were cloned into the 3′ UTR of a self-transcribing reporter in a self-complementary AAV backbone (pscAAV[Exp]-SCP1>EGFP), so active enhancers drive their own barcoded transcripts (Figure 1). The enhancer library was packaged into AAV, administered systemically at 10 dpa, and regenerating digit tips were collected at 14 and 21 dpa. Total RNA and genomic DNA were co-isolated, the variable region PCR-amplified and sequenced, and enhancer activity quantified by modeling RNA output normalized to DNA input with multiple-testing correction. Elements were prioritized by statistical significance and consistency across time points. RESULTS: ATAC-seq identified hundreds of noncoding loci with increased accessibility during regeneration, enriched near promoters and distal regulatory regions of injury-responsive genes. Integrating single-cell and spatial data yielded 568 TREE candidates associated with blastema, nail matrix, or regeneration epidermis. Serotype screening indicated AAV9 provided the most effective in vivo enhancer activation (Figure 2).The in vivo AAV-MPRA resolved subsets of elements with robust activity and clear temporal specificitydistinct profiles at 14 dpa (blastema formation) versus 21 dpa (tissue remodeling) - with a set of elements active at both stages (Figure 3). Consistently active candidates were linked to genes implicated in appendage repair, including Ror2, Fscn1, Pecam1, and Traf6. Ongoing studies will deploy AAV-TREE vectors to drive pro-regenerative effectors and test how enhancer-guided delivery augments or accelerates MDT regeneration. CONCLUSION: Our results to date demonstrate a novel screening method for TREEs during MDT regeneration that couples chromatin accessibility profiling, single-cell/spatial expression maps, and in vivo AAV-based MPRA to discover and functionally rank TREEs. The resulting catalog and top-ranked elements clarify enhancer-mediated regulation in mammalian appendage regeneration and provide a toolkit for precise enhancer-mediated regulation, laying a foundation for stage-targeted AAV therapeutics.© 2026. Plastic Surgery Research Council | All rights reserved |*Source: https://ps-rc.org/meeting/Program/2026/38.cgi*