Efficacy of Asymmetric siRNA Targeting Androgen Receptors for the Treatment of Androgenetic Alopecia

Abstract

Asymmetric small interfering RNAs (asiRNAs) that mediate RNA interference have been investigated for therapeutic use in various tissues, including skin tissue. Androgenetic alopecia (AGA) is caused by a combination of genetic factors, resulting in sensitivity to dihydrotestosterone (DHT), which binds to the androgen receptor (AR) to mediate a series of biomolecular changes leading to hair loss. This study aimed to evaluate the therapeutic potential of a cell-penetrating, AR-targeting asiRNA (cp-asiAR) for AGA treatment, which was designed to silence the AR gene. AGA mouse models were developed by stimulation with DHT, and ex vivo human scalp tissues were also used for analysis. Cp-asiAR-mediated changes in mRNA expression and protein levels of AR were assessed along with the examination of phenotypic improvements in mouse model of AGA. We also assessed downstream signaling associated with AR in primary human dermal papilla (DP) cells. Several cp-asiARs were screened for selecting the optimal sequence of AR using cell lines in vitro. A cholesterol-conjugated, chemically modified cp-asiAR candidate was optimized under passive uptake conditions in vitro. Intradermal cp-asiAR injection efficiently reduced mRNA and protein levels corresponding to AR in mouse models. Moreover, cp-asiAR injection promoted hair growth in mouse models with DHT-induced AGA. In ex vivo human hair follicle culture, the proportion of telogen hair decreased, and the mean hair bulb diameter increased in the cp-asiAR-treated group. In isolated primary human DP cells, AR expression was effectively downregulated by cp-asiAR. Furthermore, cp-asiAR attenuated DHT-mediated increases in interleukin-6, transforming growth factor-β1, and dickkopf-1 levels. No significant toxicity was observed in DP cells after cp-asiAR treatment. Cp-asiAR treatment showed effective downregulation of AR expression and prevention of DHT-mediated alterations in the hair cycle and hair diameter, indicating its potential as a novel therapeutic option for AGA.

Keywords: androgen receptor; androgenetic alopecia; dermal papilla; dihydrotestosterone; hair follicle; siRNA.

Figure 1

In vitro validation of the efficacy of cell-penetrating, AR-targeting asiRNA (cp-asiAR) knockdown. (A) A549 cells were treated with cp-asiARs at indicated concentrations by passive uptake. mRNA levels of AR observed 24 h after treatment were normalized using tubulin mRNA expression. AR mRNA levels relative to those of vehicle control (VC) are presented as mean and standard deviation (n = 6). AR protein levels at 48 h after treatment are shown below. β-actin protein level was considered as a loading control. (B) Knockdown efficacy was validated in T98G cells. Representative images are shown. NTC denotes nontargeting control siRNA treatment at a concentration of 10 μM. Statistical significance was calculated using a t-test with VC (*, p < 0.05, **, p < 0.01, ***, p < 0.001).

Figure 2

In vivo validation of cp-asiAR KD efficacy. cp-asiAR was intradermally injected into the dorsal skin of mice, and the mRNA (A) or protein (B) level of AR in the injection site was measured after 24 h (n = 3). Protein level was quantitated using image analysis of western blotting (B, inset). RPL32 and β-actin were used as references for the relative mRNA and protein levels of AR, respectively. (C) cp-asiAR was injected at three doses, and skin biopsy was performed at the indicated times after injection. AR protein level normalized by β-actin was measured in the biopsy tissue. AR protein levels relative to the vehicle control are shown with the standard deviations (n = 4). Statistical significance was calculated using the t-test with the vehicle control (VC) (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 3

In vivo validation of cp-asiAR therapeutic efficacy in an AGA mouse model. (A) All mice were injected daily with DHT (25 mg/kg) during the entire experiment to mimic AGA-like hair loss conditions. They were injected intraperitoneally with flutamide (200 mg/kg) daily or intradermally at different doses of cp-asiAR on days 1, 8, and 15. On day 21, the mice were sacrificed, and hair regrowth was imaged (top, n = 5). The dorsal skin was isolated for histology and stained with H&E (bottom). Notable promotion of hair growth caused by cp-asiAR treatment at all tested doses was observed on day 21. (B) Regrown hair was shaved and weighed using a microscale. cp-asiAR treatment resulted in significant promotion of hair regrowth compared with the vehicle control. The mean and standard deviation are shown in the figure. (C) Presumptive anagen and telogen hair follicles were quantified from the H&E, and the ratio is presented as mean and standard deviation. Statistical significance was calculated using a t-test with the vehicle control (*, p < 0.05; **, p < 0.01).

Figure 4

Ex vivo validation of cp-asiAR therapeutic efficacy in human hair follicles. Hair follicles were isolated from the human scalp and incubated with DHT (100 nM) to mimic AGA-like hair loss conditions. (A) Each group of hair follicles was co-treated with flutamide or cp-asiAR at the indicated doses and observed on days three and six after incubation (n = seven-nine/group). (Top) Images were obtained on day six, and telogen hair was identified based on morphological characteristics as described previously. Tapered, elongated bulb shape, and dissociation of very compact papilla were considered as the key features of telogen hair. (Bottom) Hair follicles from each group were isolated on day two after incubation. Frozen sections of the tissue were hybridized with the AR riboprobe to visualize the mRNA levels of AR in the hair follicle. (B) Fluorescence intensity in the DP area was analyzed using ImageJ, and the AR levels relative to the untreated control are presented as mean and standard deviation. Statistical significance was calculated using a t-test with the vehicle control (**, p < 0.01; ***, p < 0.001). (C) Telogen ratio in each treatment group was quantified based on the number of anagen and telogen hair follicles. Statistical significance was calculated using the Chi-square test with the vehicle control. (D) Effect of cp-asiAR on the hair follicle diameter following DHT treatment. A total of 130 hair follicles from 13 patients (8 with AGA and 5 without AGA) were collected, and the hair follicle diameter was measured on days three and six after incubation with the indicated treatment. Statistical significance was calculated using the t-test with the vehicle control of the same day (*, p < 0.05, **, p < 0.01, ***, p < 0.001).

Figure 5

AR knockdown and downstream signaling inhibition in human DP cells. Primary human DP cells were cultured and incubated with DHT to mimic AGA-like conditions. The cells were co-treated with flutamide or cp-asiAR at the indicated concentration. Statistical significance was calculated using the t-test with the vehicle control (*, p < 0.05). (A) The mRNA level of AR was measured using qPCR 24 h after treatment (n = 3). (B) Protein expression profile was obtained using western blotting 24 h after treatment. Treatment with cp-asiAR effectively downregulated the expression of AR, dickkopf-1 (DKK-1), interleukin (IL)-6, and transforming growth factor (TGF)-β1. (C) Effects of cp-asiAR treatment on DP cell viability were tested using the modified MTT assay. No significant decrease in DP cell viability was observed.