siRNA Silencing of the Mutant Keratin 12 Allele in Corneal Limbal Epithelial Cells Grown From Patients With Meesmann's Epithelial Corneal Dystrophy

Courtney, David; Atkinson, Sarah D.; Allen, E.; Moore, Johnny; Walsh, Colum P.; Pedrioli, Deena M. Leslie; MacEwen, C J; Pellegrini, Graziella; Maurizi, Eleonora; Serafini, Chiara; Fantacci, Monica; Liao, Hong; Irvine, Alan D.; McLean, Whi; Moore, Carrie

doi:10.1167/iovs.13-12957

Cited by 30 publications

(17 citation statements)

References 37 publications

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“…Cas9/sgRNA constructs were delivered to the mouse cornea by intrastromal injection, as previously described (Courtney et al . 8 ). Both a guide targeted to Luc2 (sgLuc2 - right eye) and a non-specific control guide (sgNSC - left eye) were injected intrastromally in a total volume of 4 µl of PBS at a concentration of 500ng/µl.…”

Section: Methodsmentioning

confidence: 99%

Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders

Christie

Courtney

DeDionisio³

et al. 2017

Sci Rep

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CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at “off-target” sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated.

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Section: Methodsmentioning

confidence: 99%

Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders

Christie

Courtney

DeDionisio³

et al. 2017

Sci Rep

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“…Additional K12 expression constructs previously described 7 were used to assess allele specificity and potency. Firefly luciferase plasmids with the full mRNA sequence for either K12-WT or K12-L132P inserted 3′ of the stop codon, hereafter named as K12WT-Luc and K12LP-Luc, respectively, 7 and expression plasmids for mature haemagglutinin (HA)-tagged K12-WT and K12-L132P protein, 10 with plasmids hereafter known as K12WT-HA and K12LP-HA, respectively, were used. An expression construct for Renilla luciferase (pRL-CMV, Promega, Southampton, UK) was used for the dual-luciferase assay to normalize transfection efficiency.…”

Section: Methodsmentioning

confidence: 99%

“…Using the same cDNA samples assessed by quantitative reverse transcriptase-PCR, pyrosequencing was carried out to determine the ratio of remaining K12-L132P mRNA to K12-WT mRNA, exactly as described previously. 10 …”

Section: Methodsmentioning

confidence: 99%

“…Through use of techniques adapted from our previous research identifying therapeutic allele-specific short interfering RNAs, 7 , 9 , 10 , 11 this Cas9-based gene-editing system was found to be potent and allele specific. This study constitutes the first description of allele-specific CRISPR/Cas9 gene editing at a novel PAM created by a disease-causing heterozygous SNP.…”

Section: Introductionmentioning

confidence: 99%

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CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting

et al. 2015

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CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

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“…Therapeutic benefit was now demonstrated in cells from patients and animal models, and promising results of the first phase Ib clinical trial using siRNA-based allele-specific therapy were reported in pachyonychia congenita, an inherited skin disorder due to dominant mutations in the keratin 6 gene [31]. The allele-specific siRNA silencing of the mutant keratin 12 allele was also applied in corneal limbal epithelial cells grown from patients with Meesmann's epithelial corneal dystrophy [32,33]. It has also been shown that modified siRNAs conferring allele-specific silencing against disease-causing ALK2 mutants found in fibrodysplasia ossificans progressiva, without affecting normal ALK2 allele [34].…”

Section: Allele-specific Rnaimentioning

confidence: 99%

siRNA-Induced RNAi Therapy in Acute Kidney Injury

Yang¹,

Yang²

2016

RNA Interference

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AbstractsiRN" therapy has great potential in humans, and its applications have been significantly improved. The kidney is a comparatively easy target organ of siRN" therapy due to its unique structural and functional characteristics. Here, we reviewed recent achievements in the design, delivery, and utilization of RN"i with a focus on kidney diseases, in particular acute kidney injury. In addition, the perspectives and challenges of siRN" therapy such as increasing its serum stability and immune tolerance, targeting single/double/ multiple genes, cell/allele-specific delivery, time-controlled silencing, and siRN"-modified stem cell therapy were also discussed. Finally, selecting target genes and therapeutic time windows were addressed.

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siRNA Silencing of the Mutant Keratin 12 Allele in Corneal Limbal Epithelial Cells Grown From Patients With Meesmann's Epithelial Corneal Dystrophy

Abstract: Combined with an effective delivery vehicle this siRNA approach represents a viable treatment option for prevention of the MECD pathology observed in K12-Leu132Pro heterozygous individuals.

Cited by 30 publications

References 37 publications

Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders

Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders

CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting

siRNA-Induced RNAi Therapy in Acute Kidney Injury

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