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. 2016 Jan;136(1):45-51.
doi: 10.1038/JID.2015.375.

Correction of Hair Shaft Defects through Allele-Specific Silencing of Mutant Krt75

Ying Liu  1 , Elizabeth R Snedecor  2 , Xu Zhang  1 , Yanfeng Xu  1 , Lan Huang  1 , Evan C Jones  3 , Lianfeng Zhang  1 , Richard A Clark  3 , Dennis R Roop  4 , Chuan Qin  5 , Jiang Chen  6
Affiliations

Correction of Hair Shaft Defects through Allele-Specific Silencing of Mutant Krt75

Ying Liu et al. J Invest Dermatol. 2016 Jan.

Abstract

Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele-specific small interfering RNAs capable of selectively suppressing the expression of a mutant Krt75, which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75-specific short hairpin RNA (shRNA) persistently suppressed this phenotype. The phenotypic correction was associated with a significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of mutant keratin genes.

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Conflict of interest statement

CONFLICT OF INTEREST

None to declare.

Figures

Figure 1
Figure 1. Mutant Krt75-specific siRNA
(a) Sequences of wild-type and c.545_547del (p.N159del) Krt75 and candidate siRNAs for mutant Krt75. (b) Relative expression levels of wild-type and mutant Krt75 in HEK293T cells co-transfected with wild-type and mutant Krt75 and siRNA (15 nM) by quantitative RT-PCR. Blank, cells transfected with Krt75 expression plasmids without siRNA; NC, cells transfected with negative control (a fragment of inverted beta-galactosidase sequence) siRNA; Krt75-siRNA, a commercially available siRNA against Krt75. (c, d) Relative expression levels of wild-type and mutant Krt75 in cells transfected with 0 − 15 nM siN159D-5 and siN159D-6 siRNAs, as normalized to GAPDH. All experiments were carried out in triplicates in a minimum of three independent experiments.
Figure 2
Figure 2. Phenotypes of hair regenerated with shRNA-modified homozygous mutant Krt75 keratinocyte progenitor cells
(a–c) Representative gross appearance, low and high power images of hair regenerated with non-infected cells. Control (a), shN159D-6 lentiviral vector infected cells (b), and scrambled (shN159D-6S) lentiviral vector infected cells (c) at one month of grafting. Arrows point to bulbous lesions (blebs) along the hair shaft. (d) Quantification of hair shafts containing blebs. Asterisk, P < 0.05. Scale bar: 250 μm.
Figure 3
Figure 3. Gene expression in skin grafts regenerated with shRNA modified homozygous mutant Krt75 keratinocyte progenitor cells
(a) Relative expression level of mutant Krt75 (Krt75-N159del) in grafts regenerated with ex vivo cultured cells (as described in Figure 2) by qRT-PCR one month after grafting . (b) Relative expression levels of Krt1, Krt5, Krt6a, Krt14, and Krt17 by qRT-PCR in skin grafts described in (a). All experiments were carried out in triplicates in a minimum of three independent experiments. Asterisk, P < 0.05.
Figure 4
Figure 4. Phenotype and gene expression in skin grafts regenerated with shRNA-modified heterozygous mutant Krt75 keratinocyte progenitor cells
(a–c) Representative hair phenotypes of hair regenerated with control and lentiviral vector-infected cells (as described in Figure 2) at one month of grafting. Arrows point to bulbous lesions (blebs) along the hair shaft. (d) Quantification of hair shafts containing blebs. (e) Relative expression level of wild-type (Krt75-WT) and mutant Krt75 (Krt75-N159Del) in skin grafts described in (a). Asterisk, P < 0.05. Scale bar: 250 μm.
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References

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