STARR-CRAAVT: A platform to identify cell type-specific regulatory elements for next-generation gene therapy

Designing enhancer-promoter combinations that enable potent and precise transgene expression is a promising strategy for advancing gene therapy mediated by adeno-associated virus (AAV). We introduce STARR-CRAAVT (self-transcribing active regulatory region sequencing of captured libraries for rAAV gene therapy), a STARR-Seq-based platform using in silico tailored sequence libraries to screen for enhancers in AAVs and assess relevant applications and critical screening parameters in an in vitro proof-of-concept study. Candidate libraries were generated by integrating epigenetic data, followed by the capture of candidate-containing genomic fragments and packaging into AAVs. Using STARR-CRAAVT, we identified enhancers driving cell type-specific expression and showed that the promoter type is a key determinant for the ability of candidates to act as enhancers. Notably, we also found that switching the candidate position in the AAV genome can significantly influence enhancer activity. Our study yields insights into enhancer function and describes a blueprint for the implementation of STARR-CRAAVT to identify cell type-specific enhancers for AAV-based gene therapy.