Phenotypic Analysis Of A Gene Mutation Knock-in Mouse Model Of Epidermolvtic Palmoplanta Keratoderma And Investigation On Mutant-specific ShRNA Therapy

Background:Epidermolytic palmoplantar keratoderma（EPPK.OMIM:144200）is a relatively common autosomal-dominant inherited skin disorder and the most common subtype of palmoplantar keratodermas with high genetic heterogeneity.EPPK is caused by mutations in the keratin 9 gene,KRT9,and in a minority of cases,in KRT1.Onset is during the first weeks or months after birth,and the typical dermatological features last for a lifetime.Clinical symptoms characterized by diffuse yellowish thickening of the skin on the palms and soles with distinct erythematous borders.Many patients also combined with hyperhidrosis or bromidrosis.Some patients manefested knuckle pads,digital mutilation,and camptodactyly.So far,there is no effective mangements for EPPK.The vast majority of researches still focus on variome of the disease.Objectives:Based on the phenotypic analysis of our group’s KRT9 gene mutation knock-in mouse model,this study evaluate the feasibility and efficiency of personalized gene therapy strategy on EPPK,and estabolish foundation for the subsequent clinical gene therapy and pharmacotherapy researches.Methods:（1）In the previous studies,our group found a specific indel mutation of KRT9,c.500₅00delAinsGGCT（p.Tyr167delinsTrpLeu）,in several southern Han Chinese EPPK pedigrees.We also estabolished a Krt9/c.434delAinsGGCT（p.Tyr144delinsTrpLeu）mutant knock-in mouse model by gene targeting techniques.Here,the EPPK-like phenotype and pathology of the footpad skin from the mutant mouse were analyzed to observe the pathological changes by H&E staining,electron microscopy,etc.Molecular analysis was performed to reveal the dysfunction of K9 and its affections on the other protein expression.（2）Lentivirus expression vectors containing firefly luciferase reporter gene,pfLUC-Homo-ex1 KRT/c.500delAinsGGCT/wt（pfLUC-ex 1 KRT9-mutant/wt）for human,and pfLUC-Mus-exlKrt9-c.434delAinsGGCT/wt（pfLUC-exlKrt9-mutant/wt）for mouse,were constructed respectively.To target the mutant,16 possible siRNA molecules were designed where the mutant base was positioned according to the keratin 9 gene sequences of EPPK patient and c.434delAinsGGCT mutation mouse,respectively.A firefly luciferase reporter assay was developed to screen all 16 allele-specific siRNAs in order to identify one that knockdowned the Krt9 mutant allele specifically and effectively but had little or no effect on the wild-type allele.Based on the screened siRNA results,the lentiviral shRNA vector,lv-sh-K9mut-8,was synthesized.Then,a human immortal keratinocyte cell line,HaCat cell,was treated with lv-sh-K9mut-8 to investigate off-target effects.（3）To observe the pathological changes of EPPK-like phenotype after treatment,lv-sh-K9mut-8 was injected into the footpads of 12-week-old Krt9+/mut heterozygous mice.Histopathological and molecular analysis were carried out to test the potential relief of hyperkeratosis.The main mediators of the immune response were assessed to investigate the immunostimulatory effect of lv-sh-K9mut-8 therapy.Results:（1）A stable and distinctive phenotype was observed on the fore-and hind-paws of 11 to 12-week-old Krt9/c.434delAinsGGCT（p.Tyr144delinsTrpLeu）knock-in heterozygous mouse.H&E staining showed remarkable footpad epidermal thickening,expansion of the epidermis and massive hyperkeratosis,papillomatous hyperplasia,marked hypergranulosis and acanthosis,and vacuolated cells in the upper portion of the epidermis with atypical shapes.Ultrastructural examination by transmission electron microscopy showed cytolysis of keratinocytes and abnormal aggregation of tonofilaments in the stratum basalt of the epidermis.Immunofluorescent staining revealed an increase in the number and staining intensity of PCNA-and p63-positive cells in the footpads of K9-mutant mice;the expression of the stress-response arid wounding-healing keratins,K6 and K16,was significantly higher in Krt9+/mut and Krt9mut/mut mice than in Krt9+/+ wild-type mice;the suprabasal keratins K1 and K10 both increased significantly in response to the dysfunction of K9 in the footpads with local expansion and abnormal aggregation.Western blot analysis of K2 revealed a 60%decrease in the footpads of Krt9+/mut and Krt9mut/mut mice compared with normal epidermis.（2）Among the 16 designed siRNAs for Krt9/c.434delAinsGGCT and KRT9/c.500delAinsGGC,the optimal inhibitor for the further development was confirmed as si-K9mut-8.It could knockdown the expression of both human and mouse mutant K9 allele,while the wild-type K9 allele was little affected.Thus,the lentiviral shRNA vector（lv-sh-K9mut-8）was designed and synthesized according to the base sequence of si-K9mut-8.Then,lv-sh-K9mut-8 was transfected into HaCat cells to test the targeting inhibition efficiency.The results showed little off-target effects.（3）After Krt9+/mut mice were treated with shRNA for the first injection（day 10）and the second injection（day 10）,hyperkeratosis was relieved,indicating an ameliorated phenotype.Detection of proliferating protein markers（PCNA,p63,involucrin and filaggrin）and cytoskeletal keratin molecules（K10,K1,K6,K16,K14,K5 and K2e）verified almost normal morphology and functions of the skin,whereas the treatment caused negligible immunostimulatory response.Conclusions:Our results suggested that mutant-specific shRNA therapy could be a potential individualized gene therapy strategy for treating EPPK.This idea might be applied into the researches on other monogenic skin disorders.