Therapeutic Efficacy Of Antisense Oligonuleotides And SiRNA Targeting Keratin 17 On Psoriasis In SCID-hu Mouse Model

Psoriasis is a common chronic inflammatory skin disease. It is increasingly being recognized that psoriasis is a T-cell-mediated autoimmune disease linked to certain genetic backgroud and preceding streptococcal infections. Besides the increased proliferation of keratinocytes observed in psoriasis there are distinct changes in the expression of various keratins, where keratin 17 (K17), a Mr 46 000 type I keratin is mainly involved. K17 is only detected in hair follicle of growth phase and sweat gland in normal human skin, but highly expressed in psoriatic lesional epidermis. It has been found that K17 shares similar antigenic epitopes with the M protein of streptococcus. There are several T-activating epitopes on the surface of K17. Both K17 and M protein have the capacity of activating T cells and promoting the release of interferonγ(IFNγ), while inversely, IFNγhas been proved to be able to increase the expression of K17 in epithelial cells. Accordingly, a positive feedback loop-IFNγ/K17 autoimmune loop is established and leads to persistent inflammatory response and abnormal proliferation within psoriatic lesions. Because K17 plays a very key role in the pathogenesis of psoriasis, we define K17 as a new target to treat psoriasis.Our previous studies have revealed that both anti-keratin polyclonal auto-antibodies and the monoclonal antibody against K17 are capable of inhibiting the proliferation of keratinocytes and improving the psoriasis phenotypes in vitro and in vivo. Furthermore, we transfected the K17 antisense oligonucleotides (K17- ASODN) and siRNA (K17-siRNA) into cultured KCs by liposome. As a result, both of them inhibited the proliferation and induced the apoptosis of KCs in a dose-dependent manner.The purpose of our present study is to comfirm the efficacy of ASODN and siRNA therapies targeting K17 on psoriasis using animal model. A SCID-hu xenogeneic transplantation model of psoriasis was developed and K17-ASODN and K17-siRNA were delivered topically by liposome. The clinical and histopathological changes of grafts were observed, and subsequently, the levels of K17 mRNA and protein in the psoriatic lesions were analyzed by RT-PCR and immunohistochemistry, respectively. The main experimental procedures and results are described as follows:1. The production of the SCID-hu xenogeneic transplantation model Skin samples were taken from patients with chronic plaque-type psoriasis in our department. None of them underwent any treatment before admission. Informed consent was obtained from all patients. SCID mice (6-8-week-old) were purchased from ShangHai Silaike experiment animal center and kept in SPF environment.Spindle-shaped skin tissues were obtained from involved psoriatic skin. After sterilization with alcohol, involved psoriatic skin (2–3 cm2) was obtained from each patient under local anesthesia with 0.5% xylocaine. The harvest skin was prepared for transplantation by removing subcutaneous fat, kept in sterilized saline solution and used for grafting within 1-2 h. The full-thickness skin specimens were dissected into pieces 8 mm in diameter and then transplanted onto the back of the SCID mice, each mouse carrying one plant. Ten days after the transplantation, Con-A-stimulated PBMCs (approximately 1×106 cells in 0.1 ml of RPMI 1640 medium for each mouse) were injected subcutaneously under the grafted skin. The result showed that most of the grafts maintained psoriatic characteristics on the SCID mice up to 15 days after transplantation with the average lesion score of 8.042±0.838, where the typical lesion score in psoriatic patients is between 4.0~10.0 according to Baker score system.Immunohistochemistry study revealed that K17-positive cells could be detected in the epidermis except for the basal layer. It is therefore indicated that the SCID-human skin chimeric model with clinical, histological and immunological characteristics of psoriasis was successfully constructed.2. The effects of K17 antisense oligonucleotides on psoriasis in animal model The synthesis of K17 antisense oligonucleotidesWe prepared for ASODN directly against the mRNA sequence of K17 according to our previous methods. The anti-sense sequence is 5'GCCAGCAGCCCATCAACA 3'and its sense sequence, which served as a control in this study, is 5'TGTTGATGGGCTGCTGGC 3'. The ASODN and SODN had been chemically modified phosphorothiately and been synthesized by ShangHai bio-technique company.TreatmentThe experimental mice were divided into 3 groups. In the treatment group, K17-ASODN was delivered transcutaneously by liposome at a dose of 15μg for each mouse (n=4). K17-SODN treated and corresponding vehicle-treated mice served as controls. Every mouse received 5 times of the topical preparation, applied once every 3 days.ResultsThe psoriatic lesions were improved gradually by K17-ASODN treatment. After 5 times of the topical application, the grafts on K17-ASODN treated mice achieved marked improvement while no significant improvement was noticed in K17-SODN treated and vehicle-treated mice. Histological appearance of grafts showed the thining of the epidermis and reducing of lymphocyte infiltration in the dermis in K17-ASODN group. In contrast, the epidermis was still thick and the features of psoriasis including hyperkeratosis, acanthosis with elongation of rete pegs and lymphocytic infiltration were still observed in control groups, although the appearance of the granular layer concomitant with the disappearance of parakeratotic changes was notable in some of the lesions.Results of RT-PCR showed that with no remarkable difference at theβ-actin expression, high level expression of K17 could be seen in the control groups, while the K17 expression was remarkably reduced in K17-ASODN treatment group.Immunostaining of grafts for K17 demonstrated that positive staining was observed in the epidermis except for the basal layer. It was markedly reduced in the psoriatic skin grafts in K17-ASODN treatment group compared with controls.3. The effects of K17-siRNA on psoriasis in animal model Large-scale preparation of plasmidThe express vector of K17-siRNA , which we named as psilencer3.1/K17, was constructed previously. An unrelated dsRNA vector - psilencer3.1/neg was used as control. Plasmid DNA of the vectors were extracted by using QIAGEN plasmid maxi kit, and proved to be correct by enzyme digestion.TreatmentThe experimental mice were divided into 3 groups. In the treatment group, K17- siRNA was delivered transcutaneously by liposome at a dose of 15μg for each mouse (n=4). Every mouse received 5 times of the treatment, once every 3 days. In control groups, unrelated plasmid or corresponding vehicle were delivered topically with the same dosage and method.ResultsAfter exposure to K17-siRNA, erythema and scale nearly disappeared. The thining of the epidermis and reducing of lymphocyte infiltration were observed by HE staining, and histological score was decreased significantly. In control group, erythema was still observed in the grafts and the pathological changes of psoriasis were not improved. K17 expression was markedly reduced in K17-siRNA treated mice, whereas high levels of K17 mRNA and protein were still detected in control mice by RT-PCR and immunohistochemistry, respectively.In conclusion, we have successfully established a psoriasis mouse model by transplanting psoriatic lesions onto SCID mice. By using this mouse model, excellent therapeutic efficacy of both K17-ASODN and K17-siRNA was achived. No adverse reaction to the treatments was observed. We therefore validated K17 as a novel target for the anti-psoriasis therapy. Furthermore, K17-ASODN and K17-siRNA are hopeful to be developed as new clinical therapies for psoriasis in the near future.