Abstract
Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.
Highlights
Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined
AAV vector sequences are detected at CRISPR-induced double-strand breaks (DSBs)
In analyzing next-generation sequencing (NGS) data from an in vivo CRISPR gene therapy approach targeting the Tmc1Beethoven mutation in the inner ear, we observed AAV inverted terminal repeat (ITR) sequences within CRISPR indels[22]. To confirm this observation and expand these findings to other genes, we first analyzed AAV vector integration in cultured cortical neurons derived from wild type (WT) C57BL/6 mice
Summary
Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. We observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Uppsala University, Department of Public Health and Caring Sciences, Geriatrics, Uppsala, Sweden. Delivery of CRISPR/Cas[9] nucleases by AAV vectors has shown therapeutic benefit in multiple preclinical models of diseases[6,7,8,9,10] and this modality is moving quickly towards clinical trials[11]. We analyze integration of AAV vectors genomewide and into CRISPR-induced DSBs in vivo focusing on therapeutically relevant target genes in differentiated cells of the nervous system (APPSW, Mecp[2], Dnmt3b), muscle (Dmd) and the inner ear (Tmc[1]). Outside of the CRISPR target region, genome-wide AAV integration rates are not different between an AAV control vector and AAV carrying Cas[9] and gRNA, suggesting that Cas[9] does not lead to widespread genotoxic effects in the brain
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