RESUMO
With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessively and dominantly inherited monogenic disorders. In this study, we have corrected a causal hotspot mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double-nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-delivery into EBS keratinocytes of a Cas9 D10A nickase (Cas9n), a predicted single guide RNA pair specific for intron 7, and a minicircle donor vector harboring the homology donor template resulted in a recombination efficiency of >30% and correction of the mutant KRT14 allele. Phenotypic correction of EBS-gen sev keratinocytes was demonstrated by immunofluorescence analysis, revealing the absence of disease-associated K14 aggregates within the cytoplasm. We achieved a promising safety profile for the CRISPR/Cas9 double-nicking approach, with no detectable off-target activity for a set of predicted off-target genes as confirmed by next generation sequencing. In conclusion, we demonstrate a highly efficient and specific gene-editing approach for KRT14, offering a causal treatment option for EBS.
Assuntos
Sistemas CRISPR-Cas , Epidermólise Bolhosa Simples/terapia , Edição de Genes/métodos , Queratina-14/genética , Queratinócitos/metabolismo , Reparo de DNA por Recombinação , Sequência de Bases , Células Cultivadas , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Epidermólise Bolhosa Simples/genética , Epidermólise Bolhosa Simples/metabolismo , Epidermólise Bolhosa Simples/patologia , Éxons , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Íntrons , Queratina-14/metabolismo , Queratinócitos/patologia , Queratinócitos/transplante , Terapia de Alvo Molecular , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia de Cinetoplastídeos/genéticaRESUMO
A knot polymer, poly[bis(2-acryloyl)oxyethyl disulphide-co-2-(dimethylamino) ethyl methacrylate] (DSP), was synthesized, optimized and evaluated as a non-viral vector for gene transfection for skin cells, keratinocytes. With recessive dystrophic epidermolysis bullosa keratinocytes (RDEBK-TA4), the DSP exhibited high transfection efficacy with both Gaussia luciferase marker DNA and the full length COL7A1 transcript encoding the therapeutic type VII collagen protein (C7). The effective restoration of C7 in C7 null-RDEB skin cells indicates that DSP is promising for non-viral gene therapy of recessive dystrophic epidermolysis bullosa (RDEB).
