In vivoexpansion of gene-targeted hepatocytes through transient inhibition of an essential gene

Abstract

Homology Directed Repair (HDR)-based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. Here, we tested ″Repair Drive″, a novel method for improving targeted gene insertion in the liver by selectively expanding correctly repaired hepatocytesin vivo. Our system consists of transient conditioning of the liver by knocking down an essential gene, and delivery of an untargetable version of the essential genein ciswith a therapeutic transgene. We show that Repair Drive dramatically increases the percentage of correctly targeted hepatocytes, up to 25%. This resulted in a five-fold increased expression of a therapeutic transgene. Repair Drive was well-tolerated and did not induce toxicity or tumorigenesis in long term follow up. This approach will broaden the range of liver diseases that can be treated with somatic genome editing.

AAV-mediated genome editing is influenced by the formation of R-loops

“…On the other hand, researchers aiming to target the Alb locus by CRISPR/Cas9-mediated HR discovered secondary off-target genes in Tfr and Afp 44 . More recently, De Giorgi et al, used CRISPR/Cas9-mediated HR to perform genome editing in the hepatic Apoa1 locus and found integrated AAV vectors again in Alb , Afp , and Errfi1 loci 45 . Interestingly, the genes that are prone to off-target AAV integration have high R-loop levels (Fig.5a), although, the most frequent “off-target” gene in the liver is Alb which is also the most highly transcribed locus (Fig.…”

“…5) . Of note, the Alb locus was the most common 'off-target' genome region where the vector was found integrated in all the studies taken into consideration [42][43][44][45] . Thus, we speculate that a combination of high transcription (Alb is the highest transcribed gene in liver) and R-loop levels might promote AAV vector genome integration, without ruling out the possibility that this combination of events could also lead to increased DNA damage which in turn could also favor AAV integration 49 .…”

“…However, this view has been challenged with reports showing low frequencies of random AAV vector genome integration upon in vivo liver gene transfer in different species 8,47,48 . Moreover, independent studies described how AAVs have been found within multiple genes throughout the murine liver genome [42][43][44][45] . Interestingly, in our DRIP-seq experiments in mouse liver we found out that most of these 'off-target' integrations genes exhibit high levels of R-loops (Fig.…”

Gene editing-based targeted integration for correction of Wiskott-Aldrich syndrome

“…Recent studies have revealed complex mis-integration profiles in CRISPR KI experiments at the Cas9 on-target cut-site, including incomplete integration, asymmetric HDR (defined as repair events showing a proper HDR junction at one of the two homology arms, while repair at the other homology arm resolves through a non-HDR mechanism), AAV capture and AAV concatemer capture. 28 , 29 Although primarily maintained in an episomal state, AAV genome can be integrated into the host genome at CRISPR on-target and OT cut-sites. 30 Currently no single tool can detect all the complex integration outcomes, especially events occurring with very low frequencies.…”