Targeting the IL-17 Receptor Using Liposomal Spherical Nucleic Acids as Topical Therapy for Psoriasis

Abstract

The activation of T helper 17 signaling plays a critical role in psoriasis pathogenesis, and systemically-administered IL-17 inhibitors are highly effective therapy for moderate-to-severe disease. We generated topically-delivered gene-regulating nanoconstructs, comprised of spherically-arrayed antisense DNA (liposomal spherical nucleic acids [L-SNAs]), which are able to penetrate human skin to knock down cutaneous gene targets. Topically-applied L-SNAs targeting the gene encoding the mouse IL-17A receptor (Il17ra) reversed the development of psoriasis clinically, histologically, and transcriptionally in imiquimod-treated psoriasis-like mouse skin. Il17ra L-SNAs reduced the modified PASI by 74% versus controls and decreased epidermal thickness by 56%. Il17ra L-SNA reduced Il17ra protein expression by 75% and significantly decreased the mRNA expression of psoriasis markers, including Defb4, Il17c, S100a7, Pi3, Krt16, and Tnfa versus scrambled spherical nucleic acid (Scr SNA) controls. A human IL17RA L-SNA penetrates 3-dimensional cultures and normal human explants to knock down IL17RA mRNA by 63% and 66%, respectively. After topical application to psoriatic 3-dimensional rafts, anti-human IL17RA L-SNAs reduced the expression of IL17RA (by 72%) and the IL-17-induced genes IL17C (by 85%), DEFB4 (by 83%), TNFA (by 77%), and PI3 (by 65%) versus scrambled L-SNA and vehicle controls (all P < 0.001). Taken together, these data suggest that targeted suppression of IL17RA is a promising new topical treatment strategy for psoriasis.

Figure 1.. Il17ra L-SNAs improve the clinical and histological psoriatic phenotype induced by imiquimod inC57BL/6 mice.

(a) Representative clinical pictures on day 6 before harvesting of back skin. (b) Mean reduction in daily modified Psoriasis Area and Severity Index , as assessed by three blinded reviewers. (c) Routine H&E (above) and Ki67 (below) staining of day 6-harvest mouse skin. Bars = 100 μm. (d) Epidermal thickness from the stratum granulosum to the epidermal-dermal junction. (e) Number of Ki67 + cells per linear length of basal epidermis. (f) Ratio of qRT-PCR levels compared to mouse skin with only vehicle and no IMQ. (g) Representative western blot from harvested skin. Values reported are the mean ± standard error of the mean from three separate experiments. *P < 0.05, ***P < 0.001. H&E, hematoxylin and eosin; IL-17RA, IL-17A receptor; IMQ, imiquimod; qRT-PCR, quantitative real-time reverse transcriptase–PCR; Scr L-SNA, scrambled liposomal spherical nucleic acid.

Figure 2.. Il17ra L-SNA suppresses mRNA expression of inflammatory and proliferation markers and increases mRNA expression of genes promoting cell differentiation.

qRT-PCR of immune (a-f) proliferation (g) and differentiation (h, i) biomarkers associated with psoriasis. Expression of mouse 60S acidic ribosomal protein P0 and glyceraldehyde-3-phosphate dehydrogenase were averaged as a normalization control. Skin without imiquimod (IMQ) treatment was set as the comparator (no IMQ =1.0). Values are expressed as mean + standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001. IL-17RA, IL-17A receptor; IMQ, imiquimod; qRT-PCR, quantitative real-time reverse transcriptase–PCR; Scr L-SNA, scrambled liposomal spherical nucleic acid.

Figure 3.. IL17RA L-SNA knocks down IL17RA expression in human cultures, rafts, and explants.

(a, b) NHEKs were treated and harvested after 48 hrs for qRT-PCR (a) and western blots (b; siIL17RA is free small interfering RNA). (c) IL17RA knockdown in proliferating NHEKs through 96 hrs. (d) qRT-PCR of NHEK IFNA4 and IFNB1 after 24 hrs treatment with 100 nM or 500 nM IL17RA L-SNA or 1 nM poly dA/dT. (e) Immunofluorescence microscopy of 3D rafts 48 hrs after 100 nM Cy5-L-SNA application. Bars = 50μm. (f, g) IL17RA knockdown 48 hrs after 3D raft treatment. (h) Immunofluorescence microscopy 24 hrs after 30μM Cy5 L-SNA application to human explants. Bars = 50μm. (i) Dose-dependent IL17RA knockdown 24 hrs after human explant treatment. Values mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001. Red, Cy5 L-SNA; Blue, DAPI. 2D, 2-dimensional; 3D, 3-dimensional; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hrs, hours; IL17RA, IL-17A receptor; IL17RA L-SNA, IL-17A receptor liposomal spherical nucleic acid; L-SNA, liposomal spherical nucleic acid; NHEK, normal human epidermal keratinocytes; PBS, phosphate buffered saline; qRT-PCR, quantitative real-time reverse transcriptase–PCR; Scr L-SNA, scrambled liposomal spherical nucleic acid; siIL17RA, small interfering IL-17A receptor; siScr, small interfering scrambled.

Figure 4.. Human IL17RA L-SNA reduces psoriatic cytokine production by keratinocytes in 3D organotypic rafts.

3D models were treated with L-SNAs or PBS every other day starting 7 days after lifting. The cytokine mix was added 9 days after lifting, concurrent with L-SNAs (Suppl. Fig. 3d) and harvested on day 13. (a, b) Knockdown of IL17RA was determined by qRT-PCR and western blotting. Dose-dependent knockdown of psoriasis-associated genes by IL17RA L-SNAs (vs. controls) in 3D rafts: (c) PI3, (d) DEFB4, (e) IL17C, and (f) TNFA. (g) LOR, which is reduced in psoriasis, is partially rescued by IL17RA SNA treatment. Values reported as mean ± standard error of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001. 3D, 3-dimensional; IL17RA L-SNA, IL-17A receptor liposomal spherical nucleic acid; L-SNA, liposomal spherical nucleic acid; n.s, not significant; PBS, phosphate buffered saline; qRT-PCR, quantitative real-time reverse transcriptase–PCR; Scr L-SNA, scrambled liposomal spherical nucleic acid; TNF-α, tumor necrosis factor α.