Allele-Specific siRNA Silencing for the Common Keratin 12 Founder Mutation in Meesmann Epithelial Corneal Dystrophy

Allen, Eha; Atkinson, Sarah D.; Liao, Hong; Moore, Johnny; Pedrioli, Deena M. Leslie; Smith, Fjd; McLean, W.H. Irwin; Moore, Tara

doi:10.1167/iovs.12-10528

Cited by 34 publications

(31 citation statements)

References 40 publications

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“…AS gene silencing has previously been described for the treatment of Amyotrophic Lateral Sclerosis and other genetic diseases . For the cornea, AS siRNAs have been successfully developed against the mutant allele of the KRT12 gene for a severe form of Meesmann Epithelial Corneal Dystrophy . AS siRNAs represent a potential approach to treat the heterozygous dominant‐negative p63 mutations which result in EEC syndrome and corneal blindness due to LSCD.…”

Section: Discussionmentioning

confidence: 99%

Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function

et al. 2016

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Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-DNp63a-RFP and R279H-DNp63a-EGFP cell lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome. STEM CELLS 2016;34:1588-1600 SIGNIFICANCE STATEMENTThe Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare genetic disorder resulting from mutations in the p63 gene and characterized by ocular surface defects. Cell therapy treatments based on transplantation of autologous corneal stem cells are not feasible for EEC patients as both eyes are affected and corneal limbus severely compromised. Epithelial stem cells deriving from the oral mucosa can be harvested with no limitations and grafts of oral mucosa are normally used in ophthalmology to repair the ocular surface. However, being the EEC syndrome a genetic disorder, cells will have to be genetically modified. Our genetic strategy could provide a therapeutic window to correct the EEC defect and counteract the clinical downstream effects of limbal stem cell deficiency.

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Section: Discussionmentioning

confidence: 99%

Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function

et al. 2016

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“…siRNAs can be designed to specifically and potently target and silence genes that are overexpressed in disease states, or mutant alleles (including point mutations [11,[23][24][25]) whose expression causes pathology. This offers an opportunity for therapeutic intervention for a variety of skin disorders with etiologies based on specific mutations in skin-expressed proteins [4,5,26].…”

Section: Discussionmentioning

confidence: 99%

Non-Invasive Intravital Imaging of siRNA-Mediated Mutant Keratin Gene Repression in Skin

Hickerson

Speaker²,

Lara³

et al. 2015

Mol Imaging Biol

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“…Other siRNA-related approaches specifically knockdown mutant allele expression and clear mutant protein aggregation (Allen et al, 2013; Hickerson et al, 2015; Leslie Pedrioli et al, 2012). Furthermore, chaperone protein inducers and gene replacement therapy offer alternative approaches(Gentil et al, 2013).…”

Section: Current Targeted Therapeutic Approaches For If-pathiesmentioning

confidence: 99%

High-Throughput Screening for Drugs that Modulate Intermediate Filament Proteins

Sun

Groppi

Gui

et al. 2016

Methods in Enzymology

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Intermediate filament (IF) proteins have unique and complex cell and tissue distribution. Importantly, IF gene mutations cause or predispose to more than 80 human tissue-specific diseases (IF-pathies), with the most severe disease phenotypes being due to mutations at conserved residues that result in a disrupted IF network. A critical need for the entire IF-pathy field is the identification of drugs that can ameliorate or cure these diseases, particularly since all current therapies target the IF-pathy complication, such as diabetes or cardiovascular disease, rather than the mutant IF protein or gene. We describe a high throughput approach to identify drugs that can normalize disrupted IF proteins. This approach utilizes transduction of lentivirus that expresses green-fluorescent-protein-tagged keratin 18 (K18) R90C in A549 cells. The readout is drug ‘hits’ that convert the dot-like keratin filament distribution, due to the R90C mutation, to a wildtype-like filamentous array. A similar strategy can be used to screen thousands of compounds and can be utilized for practically any IF protein with a filament-disrupting mutation, and could therefore potentially target many IF-pathies. ‘Hits’ of interest require validation in cell culture then using in vivo experimental models. Approaches to study the mechanism of mutant-IF normalization by potential drugs of interest are also described. The ultimate goal of this drug screening approach is to identify effective and safe compounds that can potentially be tested for clinical efficacy in patients.

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Allele-Specific siRNA Silencing for the Common Keratin 12 Founder Mutation in Meesmann Epithelial Corneal Dystrophy

Cited by 34 publications

References 40 publications

Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function

Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function

Non-Invasive Intravital Imaging of siRNA-Mediated Mutant Keratin Gene Repression in Skin

High-Throughput Screening for Drugs that Modulate Intermediate Filament Proteins

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