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ABE8e adenine base editor precisely and efficiently corrects a COL7A1 nonsense mutation

ABSTRACT: Base editing introduces precise single-nucleotide edits in genomic DNA and has the potential to treat genetic diseases such as the blistering skin disease recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by mutations in the COL7A1 gene and type VII collagen (C7) deficiency. Adenine base editors (ABEs) convert A-T base pairs to G-C base pairs without requiring double-stranded DNA breaks or donor DNA templates. Here, we use ABE8e, a recently evolved ABE, to correct primary RDEB fibroblasts harboring the recurrent RDEB nonsense mutation c.5047 C>T (p.Arg1683Ter) in exon 54 of COL7A1 and use a next generation sequencing workflow to interrogate post-treatment outcomes. Electroporation of ABE8e mRNA into a bulk population of RDEB patient fibroblasts resulted in remarkably efficient (94.6%) correction of the pathogenic allele, restoring COL7A1 mRNA and expression of C7 protein in western blots and in 3D skin constructs. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. Taken together, we have established a highly efficient pipeline for gene correction in primary fibroblasts with a favorable safety profile. This work lays a foundation for developing therapies for RDEB patients using ex vivo or in vivo base editing strategies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE211876 | GEO | 2022/08/25

REPOSITORIES: GEO

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Project description:Recessive dystrophic epidermolysis bullosa (RDEB) is a genodermatosis characterized by fragile skin forming blisters that heal invariably with scars. It is due to mutations in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils connecting the cutaneous basement membrane to the dermis. Identical COL7A1 mutations often result in inter- and intra-familial disease variability, suggesting that additional modifiers contribute to RDEB course. Here, we studied a monozygotic twin pair with RDEB presenting markedly different phenotypic manifestations, while expressing similar amounts of collagen VII. Genome-wide expression analysis in twins' fibroblasts showed differential expression of genes associated with TGF-β pathway inhibition. In particular, decorin, a skin matrix component with anti-fibrotic properties, was found to be more expressed in the less affected twin. Accordingly, fibroblasts from the more affected sibling manifested a profibrotic and contractile phenotype characterized by enhanced α-smooth muscle actin and plasminogen activator inhibitor 1 expression, collagen I release and collagen lattice contraction. These cells also produced increased amounts of proinflammatory cytokines interleukin 6 and monocyte chemoattractant protein-1. Both TGF-β canonical (Smads) and non-canonical (MAPKs) pathways were basally more activated in the fibroblasts of the more affected twin. The profibrotic behaviour of these fibroblasts was suppressed by decorin delivery to cells. Our data show that the amount of type VII collagen is not the only determinant of RDEB clinical severity, and indicate an involvement of TGF-β pathways in modulating disease variability. Moreover, our findings identify decorin as a possible anti-fibrotic/inflammatory agent for RDEB therapeutic intervention. Primary fibroblast cultures from biopsies from two twins affected by recessive dystrophic epidermolysis bullosa were analyzed. Each hybridization was performed in biological triplicate and in technical duplicate.

Project description:The gain-of-function mutation of c.G6055A (p.G2019S) in Leucine-rich repeat kinase 2 (LRRK2) gene is the most prevalent genetic cause of Parkinson’s disease (PD). Although clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing has been used to generate isogenic control lines, there are risks of creating indels and genomic instability together with a lower efficiency in non-dividing cells. The recent advance of adenine base editors (ABEs) could convert targeted A•T base pairs to G•C base pairs without double-strand DNA breaks or donor DNA templates and function in post-mitotic cells. Here, we demonstrate a complete correction of an induced pluripotent stem cell (iPSC) line derived from a PD patient having LRRK2 p.G2019S mutation using variable genome editing methods, including CRISPR/Cas9-based homology-directed repair (HDR) and ABEs. The corrected isogenic iPSCs derived dopaminergic neurons showed a down-regulated LRRK2 kinase activity, decreased phospho-a-synuclein accumulations, reduced apoptosis and restored neurite shrinkage phenotypes. The mutation correction efficacy, off-target and indels rates between CRISPR/Cas9-HDR and ABE were compared. Among the 47 clones generated by HDR, 3 clones (6.4%) were on-targeted corrected, while ABE showed a much higher correction rate (13 of 53 clones, 24.5%). Whole genome sequencing analysis revealed that there are 27 clones of HDR (57.4%) having deletions but none in clones of ABE, albeit ABE created 14 clones (26.4%) having off-target missense mutations. RNA sequencing and proteomic analysis of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of PD relevant pathways, including calcium ion dependent exocytosis, synaptic transports well as potential novel targets relevant to PD pathophysiology. These results envision that ABE could directly correct the pathogenic PD mutation in iPSCs for exploring the earliest events in PD pathophysiology and providing dopaminergic neurons for future cell therapies.

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ABE8e adenine base editor precisely and efficiently corrects a COL7A1 nonsense mutation

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