70. Enhancer-Based Gene Therapy for Digit Regeneration

PURPOSE: An important goal of regenerative medicine is to manipulate endogenous mechanisms of tissue regeneration. Digit tip regeneration is a unique physiological aspect of mammalian wound healing and represents a model for complex tissue regeneration, as well as an important foundation for long-term efforts to restore lost fingers and limbs. In this study, we sought to develop a systemically delivered, injury site-directed gene therapy platform to enhance bone regeneration in the mouse digit tip. METHODS: A recently discovered capsid variant of the adeno-associated virus 9 vector (AAV9) was shown to effectively transduce a variety of mouse tissues, including limb. Viral vectors were constructed using both a ubiquitous promoter and a distal regulatory element derived from zebrafish (LEN), which has known activity at injury sites. Viral payloads were comprised of reporter genes, allowing spatial localization of viral expression, as well as the pro-regenerative factor FGF8. RESULTS: Reporter vectors demonstrated robust viral transduction throughout the mesenchyme of regenerating digits. Bioluminescent imaging confirmed sustained expression over at least 10 days after amputation. Constructs driven by the LEN enhancer showed injury-site localization to the distal digit tip after systemic injection. Viral over-expression of FGF8 with the LEN enhancer accelerated distal phalangeal regeneration in wild-type mice and partially rescued the impaired digit regeneration observed in FGF8 pathway knockout mice, as measured by micro-CT. CONCLUSION: AAV vectors allow widespread transduction throughout the regenerating digit, opening a pathway to controlling gene expression through viral gene therapy. Use of the LEN enhancer allows injury site specific gene delivery, which may limit off target effects of the virus and allow for more precise control of effector genes such as FGF8. Ongoing work with this platform aims to enhance regeneration from more proximal limb amputation sites.