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doi: 10.1371/journal.pone.0028582. Epub 2011 Dec 12.

Development of allele-specific therapeutic siRNA in Meesmann epithelial corneal dystrophy

Affiliations

Development of allele-specific therapeutic siRNA in Meesmann epithelial corneal dystrophy

Haihui Liao et al. PLoS One.2011.

Abstract

Background: Meesmann epithelial corneal dystrophy (MECD) is an inherited eye disorder caused by dominant-negative mutations in either keratins K3 or K12, leading to mechanical fragility of the anterior corneal epithelium, the outermost covering of the eye. Typically, patients suffer from lifelong irritation of the eye and/or photophobia but rarely lose visual acuity; however, some individuals are severely affected, with corneal scarring requiring transplant surgery. At present no treatment exists which addresses the underlying pathology of corneal dystrophy. The aim of this study was to design and assess the efficacy and potency of an allele-specific siRNA approach as a future treatment for MECD.

Methods and findings: We studied a family with a consistently severe phenotype where all affected persons were shown to carry heterozygous missense mutation Leu132Pro in the KRT12 gene. Using a cell-culture assay of keratin filament formation, mutation Leu132Pro was shown to be significantly more disruptive than the most common mutation, Arg135Thr, which is associated with typical, mild MECD. A siRNA sequence walk identified a number of potent inhibitors for the mutant allele, which had no appreciable effect on wild-type K12. The most specific and potent inhibitors were shown to completely block mutant K12 protein expression with negligible effect on wild-type K12 or other closely related keratins. Cells transfected with wild-type K12-EGFP construct show a predominantly normal keratin filament formation with only 5% aggregate formation, while transfection with mutant K12-EGFP construct resulted in a significantly higher percentage of keratin aggregates (41.75%; p<0.001 with 95% confidence limits). The lead siRNA inhibitor significantly rescued the ability to form keratin filaments (74.75% of the cells contained normal keratin filaments; p<0.001 with 95% confidence limits).

Conclusions: This study demonstrates that it is feasible to design highly potent siRNA against mutant alleles with single-nucleotide specificity for future treatment of MECD.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Clinical characteristics of MECD.
(A) Ophthalmic examination of the proband from one of the families showed grey lines in the corneal epithelium, one of the characteristics of MECD. Slit lamp photography revealed multiple microcysts in the anterior epithelium (B) and uneven corneal topography secondary to damage and scarring of the underlying basement membrane (C).
Figure 2
Figure 2. Molecular genetic analysis of K12 for the MECD families.
(A) Sequence excerpt from K12 exon 1 derived from a normal person. (B) A novel heterozygous transition mutation was found in the proband: c.395T>C in exon 1 of the KRT12 gene predicting a leucine to proline amino acid substitution at codon 132 (designated p.Leu132Pro, or for brevity, Leu132Pro). (C) The mutation destroys a recognition site for the restriction enzymeMse I site, which allowed rapid exclusion of this sequence change from 50 normal, unrelated and ethnically matched control individuals byMse I digestion of KRT12 exon 1 PCR fragments. Nor = unaffected members of Family I; Aff = affected persons from Family 1. White arrowhead = filamentous keratin; blue arrowhead = keratin aggregates.
Figure 3
Figure 3. Aggregate formation in cells transfected with mutant keratins.
(A) Co-transfection of both untagged wild-type K3 and K12 cDNA constructs into Ptk2 cells resulted in production of a well-developed intermediate filament network by 24–48 hours post-transfection as demonstrated by immunofluorescence staining (antibody AE5 against K3). (B) Co-transfection of mutant versions of K12, Leu132Pro or Arg135Thr, with wild-type K3, resulted in a high percentage of K3/K12-positive cells containing dense cytoplasmic aggregates of K3/K12 protein demonstrated by the red foci. Original magnification 60×. Scale bar = 10 µm. (C) The percentage of filamentous versus aggregative-containing cells was scored from 100 cells transiently transfected with wild-type or mutant forms of K12, at 24 hours post-transfection. Error bars represent standard error of the mean of replicate experiments. Wild-type K12 showed close to 100% filamentous keratin while both mutations showed a significant difference in the percentage of cells containing aggregates, when compared to wild-type (p<0.001). The common MECD mutation Arg135Thr showed significantly less aggregate formation compared to the Leu132Pro mutant (p<0.05).
Figure 4
Figure 4. A comprehensive siRNA sequence walk for the K12 mutation Leu132Pro.
This sequence walk shows the best siRNA was at position 9 (K12-L132P-9) which was very potent against the mutant reporter (pink line on plots) even at as low as 10 pM but did not significantly inhibit wild-type (blue line on plots) at concentrations as high as 6.25 nM. siRNA inhibitor K12-L132P-13 did not affect wild-type or mutant reporter while K12-L132P-2 and K12-L132P-3 knocked down both alleles to varying degrees NSC4 is a non-specific control siRNA, (negative control). siLUC is an siRNA specific for luciferase (positive control).
Figure 5
Figure 5. Differential inhibition of mutant K12 versus wild type by allele-specific siRNAs in cultured cells.
(A) AD293 cells were transfected with wild-type or mutant K12-EGFP construct and siRNAs K12-L132P-9, 14 and 15 at a final concentration of 5 nM. Fluorescent imaging at 48 hours post-transfection showed that the expression of both wild type and mutant K12-EGFP constructs was almost equally strong for the cells treated with NSC4 while there was a significant reduction of fluorescence in the cells transfected with mutant K12-EGFP and inhibitors, although a subtle reduction of fluorescence in the cells treated with K12-L132P-9 or 15 was also observed. Original magnification 10× (except bright field image, 40×). Scale bar = 50 µm. (B) To confirm and quantify the highly differential inhibitory effect of K12-L132P-9 on mutant K12, western blot analysis was performed. AD293 cells were transiently co-transfected with either K12-L132P-EGFP expression construct and siRNA NSC4 (a non-specific control), or K12-L132P-9 at a final concentration of 5 nM. When cells were treated with K12-L132P-9, the mutant K12-EGFP was almost completely knocked down, while expression of wild type K12 construct treated with the same siRNA showed a negligible reduction compared with NSC4 treatment. Wt = wild type and mut = mutant.
Figure 6
Figure 6. Lack of off-target silencing of other keratins by lead inhibitor for K12 Leu132Pro mutation, siRNA K12-L132P-9.
SimplyBlue staining of cytoskeletal protein extracts from HaCaT cells revealed allele specific K12-L132P-9 siRNA had negligable effect on the range of endogenous keratins expressed in the HaCaT cell line. In contrast, a positive control siRNA directed against wild-type K6a demonstrated a dramatic decrease in K6a expression. Non-specific control siRNA NSC4 had no effect on keratin protein expression profile.
Figure 7
Figure 7. Therapeutic siRNA significantly reduces aggregate formation.
Ptk2 cells were co-transfected with K3 and wild type K12-EGFP or mutant K12-EGFP or a 1∶1 ratio of both wild type and mutated K12-EGFP plasmids along with wild type K3 and mutant specific siRNAs and incubated for 72 hours. Filamentous keratin was visualized by direct fluorescence of EGFP-tagged K12. There was a significant difference in aggregate formation between cells treated with siRNA K12-L132P-9 and those treated with NSC4; p<0.001. Figure 7D is representative of a cell with correct filament formation while Figures 7B and 7D illustrate the formation of keratin aggregates. Within the graph, black = filament formation; light grey = occurrence of both filaments and aggregates; and dark grey = aggregates. Original magnification 60×. Scale bar = 10 µm, white arrowhead = filaments and dark arrowhead = aggregates.
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References

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