A Dual-Locus-Targeting Strategy to Enhance CRISPR/Cas9-mediated CFTR Replacement via Helper-Dependent Adenoviral vector in porcine genome
Abstract
Gene therapy has been the subject of extensive research following the advent of gene-editing technologies. Genetic disorders with difficult-to-target tissues, such as cystic fibrosis (CF), still face many challenges in developing efficacious gene therapy. The potential universal approach of gene replacement involves inserting a functional CFTR gene after generating DNA double strand breaks using gene editors such as CRISPR/Cas9. However, this strategy has not achieved clinical significance, as CRISPR/Cas9-mediated integration of CFTR is limited primarily by the infrequent activity of the homology-directed repair (HDR) pathway. To circumvent this limitation and improve CFTR transgene integration and expression, we explored a method of adding a second integration site, which we termed the dual-locus-targeting method. Using a helper-dependent adenoviral vector (HDAd)-delivered CRISPR/Cas9 system in porcine epithelial cells, we found that sequential delivery of two vectors, one targeting the CFTR locus and the other the genomic safe harbour site GGTA1, enhanced the integration efficiency of lacZ and CFTR donor genes to 16.5% and 3.4%, respectively. These results demonstrated a potential strategy to improve the efficacy of CFTR replacement for the development of a universal and permanent gene therapy treatment for CF lung disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/731381v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@14c8583org.highwire.dtl.DTLVardef@d1862eorg.highwire.dtl.DTLVardef@1c0594eorg.highwire.dtl.DTLVardef@1271c10_HPS_FORMAT_FIGEXP M_FIG C_FIG
Full-text reasoning
From the deep-tier full-text analysis of this preprint.
Reasoning review — 4 key claims, 1 well-supported, 3 with gaps
- supported Sequential delivery of two vectors targeting CFTR and GGTA1 achieved integration efficiencies of 16.5% (lacZ) and 3.4% (CFTR) in porcine epithelial cells.
- partial The dual-locus-targeting method is a potential strategy to improve CFTR replacement efficacy for universal, permanent CF gene therapy. gap: This claim over-generalizes from in vitro porcine cell data to a "universal and permanent gene therapy treatment for CF lung disease" without in vivo or clinical evidence.
- partial The dual-locus-targeting method produces more undesired edits and an increased number of potential off-target sites, mostly at low frequencies. gap: The conclusion about "more undesired edits" and "increased number" is stated without direct comparative evidence against a single-locus method within this study, and the off-target analysis presented is limited.
- unsupported The results enhance understanding of HDR-mediated integration mechanisms, guiding optimization of future gene editing experiments. gap: The text does not provide specific details or analysis of how the study's results enhance the understanding of HDR-mediated integration mechanisms.
Claims and gaps are read from the full text by a language model, shown for transparency; they do not affect ranking or selection.
Lifecycle
- biorxiv v1 2026-06-11 source ↗
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bioRxiv Genetics @biorxiv-genetic.bsky.social · 5965 followers neutral
A Dual-Locus-Targeting Strategy to Enhance CRISPR/Cas9-mediated CFTR Replacement via Helper-Dependent Adenoviral vector in porcine genome https://www.biorxiv.org/content/10.64898/2026.06.10.731381v1
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bioRxivpreprint @biorxivpreprint.bsky.social · 8895 followers neutral
A Dual-Locus-Targeting Strategy to Enhance CRISPR/Cas9-mediated CFTR Replacement via Helper-Dependent Adenoviral vector in porcine genome https://www.biorxiv.org/content/10.64898/2026.06.10.731381v1