| Description |
- Background: A primary focus of regenerative medicine is to understand the cellular mechanisms
governing tissue regeneration across various species. Mammals generally possess a restricted
regenerative capacity compared to species like zebrafish or axolotls. Notably, all studied
mammalian species, including humans, demonstrate the ability to regenerate digit tips following
amputation at the level of the distal phalanx (P3), but not at the middle phalanx (P2). In adult
mice, this process involves intramembranous bone regrowth and predominantly scarless healing,
rendering them a valuable model for investigating mechanisms of mammalian regeneration.
Previous work from our group has identified enhancer elements that are active upon tissue injury
and return to their inactive state after healing. These tissue regeneration enhancer elements, or
TREEs, allow for spatiotemporal regulation of gene delivery with systemic administration of
adeno-associated viruses (AAVs). We asked whether AAVs driven by an injury-specific enhancer
could deliver pro-regenerative genes to injured digit tips to improve the regeneration of the distal
phalanx.
Methods: In this study, we evaluated the expression patterns of two adeno-associated virus
(AAV) constructs containing either fibroblast growth factor 8 (Fgf8) and green fluorescent
protein (GFP) or GFP alone. To target gene delivery to the injury site, we administered the cc47
AAV with an injury-specific enhancer element, LEN, upstream of the genes of interest. Wild-type
mice underwent left paw amputations at either level of the second phalanx (n=12) or the third
phalanx (n=12). The right paws served as uninjured controls. AAVs were delivered systemically
via retroorbital injection 10 days post-amputation. Within each amputation group, half received
Hsp68-LEN::Fgf8-P2A-GFP while the other half received a construct with Hsp68-LEN::GFP.
Results: Immunofluorescent imaging of the regenerating digits at 14 days post-amputation
confirmed that the viral construct allowed the successful transduction of GFP in both P2 and P3
amputated digits, but not in the uninjured control digits. Micro CT imaging of P2 amputated digits
showed similar bone length and volume between the Hsp68-LEN::Fgf8 and the Hsp68-LEN::GFP
reporter groups after 28 days. Digits with P3 amputations had similar bone length but significantly
greater bone volume in the Hsp68-LEN::Fgf8 group than in the Hsp68-LEN:GFP controls.
Conclusions: Here, we show that systemic delivery of AAVs allows for targeted, timely gene
expression in the regenerating mouse digit tip. We harnessed the LEN-AAV system for viral
delivery of pro-regenerative genes to amputated digits to enhance bone regeneration. This work
may represent a potential pathway for gene therapy applications in limb loss and tissue repair.
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