Robert Becker, Priyanka Choudhury, Martin Oti, Youssef Fouani, Benjamin Strobel et al.
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.
Robert Becker, Priyanka Choudhury, Martin Oti, Youssef Fouani, Benjamin Strobel, Stephanie Ketterer, Simon Rumpel, John Park, Sebastian Kreuz, Udo Maier, Stefan Michelfelder, Christian Schön. STARR-CRAAVT: A platform to identify cell type-specific regulatory elements for next-generation gene therapy. 2026-03-25. https://doi.org/10.1016/j.isci.2026.115469
The PGK AAV-STARR-CRAAVT candidate library was transduced into HaCaT cells with AAV6.2 across three replicate wells at MOI 100,000 viral genomes per cell. Matched cellular DNA and RNA/cDNA counts quantify candidate representation and self-transcribed regulatory activity after 72 h.
The PGK AAV-STARR-CRAAVT candidate library was transduced into HepG2 cells with AAV6.2 across three replicate wells at MOI 100,000 viral genomes per cell. Matched cellular DNA and RNA/cDNA counts quantify candidate representation and self-transcribed regulatory activity after 72 h.
The minP AAV-STARR-CRAAVT candidate library was transduced into HepG2 cells with AAV6.2 across three replicate wells at MOI 100,000 viral genomes per cell. Matched cellular DNA and RNA/cDNA counts quantify candidate representation and self-transcribed regulatory activity after 72 h.
The minP plasmid STARR-seq candidate library was transfected into HepG2 cells with Lipofectamine 3000 and harvested after 72 h. Matched cellular DNA and RNA/cDNA counts quantify candidate representation and self-transcribed regulatory activity for comparison with AAV delivery.
The minP AAV-STARR-CRAAVT candidate library was transduced into HaCaT cells with AAV6.2 across three replicate wells at MOI 100,000 viral genomes per cell. Matched cellular DNA and RNA/cDNA counts quantify candidate representation and self-transcribed regulatory activity after 72 h.
The AFP AAV-STARR-CRAAVT candidate library was transduced into HaCaT cells with AAV6.2 across three replicate wells at MOI 100,000 viral genomes per cell. Matched cellular DNA and RNA/cDNA counts quantify candidate representation and self-transcribed regulatory activity after 72 h.
The AFP AAV-STARR-CRAAVT candidate library was transduced into HepG2 cells with AAV6.2 across three replicate wells at MOI 100,000 viral genomes per cell. Matched cellular DNA and RNA/cDNA counts quantify candidate representation and self-transcribed regulatory activity after 72 h.
The minP plasmid STARR-seq candidate library was transfected into HaCaT cells with Lipofectamine 3000 and harvested after 72 h. Matched cellular DNA and RNA/cDNA counts quantify candidate representation and self-transcribed regulatory activity for comparison with AAV delivery.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 5 files (ZIP)GSE298485_Becker2025_AllCellSamples_NormalizedCounts.txt.gzGSE298485_Becker2025_AllSamples_RawCounts.txt.gzGSE298485_family.softGSE298485_series_metadata.txtSTARR-CRAAVT_supplementary_figures_S1-S5.pdf