Targeting olfactory receptor OR2AT4: An innovative aptamer-based treatment for hair growth promotion

Abstract

Non-canonical functions of olfactory receptors, one of the largest families of GPCR receptors not confined to the olfactory system, include an intriguing role on hair physiology. Previous studies using odorant agonists of the Olfactory Receptor family 2 subfamily AT member 4 (OR2AT4) showed hair growth stimulation. To develop more effective topical or systemic treatments for hair growth by specifically activating this receptor, it is crucial to identify new molecules with potent and well-defined activity. Here, we present a significant advancement in targeted therapeutic strategies for hair loss through the identification of a highly specific DNA aptamer targeting OR2AT4. The selected aptamer, designed as Ap.OR2AT4.17, effectively prolonged the anagen phase of hair growth cycle in a valuable preclinical human organ culture model. Importantly, the identified OR2AT4 aptamer was shown to be functional, leading to enhanced OR2AT4 expression in hair follicles, increased proliferation of hair matrix keratinocytes, and significant hair shaft elongation in organ-cultured human hair follicles. Notably, aptamer treatment also upregulated LL-37 and human β defensin-3 antimicrobial peptide expression, highlighting its role as a microbiota-modulating compound. These findings underscore the OR2AT4 aptamer's potential as a novel, targeted therapy for hair loss disorders, marking a promising step forward in the field.

Keywords: LL-37; MT: Oligonucleotides: Therapies and Applications; OR2AT4; alopecia; antimicrobial peptides; aptamer; olfactory receptor; β defensin-3.

Graphical abstract

Figure 1

Ap.OR2AT4.17 ssDNA aptamer was identified by cell-SELEX using 293-OR2AT4 stable transfectants for positive selection (A) OR2AT4 expression (green) by immunofluorescence in formaldehyde-fixed, saponin-permeabilized 293 and 293-OR2AT4-Clone#2 cells. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (B) Enrichment of target-specific aptamer populations during SELEX rounds 1–6, based on qPCR analysis of binding to 293-OR2AT4-Clone#2 cells. A decrease in Ct values reflects increased affinity. Data represent mean ± SEM from two independent experiments. (C) Ratio of specific binding of forward (F) and reverse (R) strand aptamers to 293-OR2AT4-Clone#2 versus 293 parental cells. Data represent the mean of two independent experiments. (D) Secondary structure prediction of the selected Ap.OR2AT4.17 aptamer using the Mfold tool. (E) Circular dichroism spectra of Ap.OR2AT4.17 recorded at three different temperatures ([DNA] = 20 μM). (F) Imino region of the NMR spectra of Ap.OR2AT4.17 in PBS buffer recorded at T = 5°C ([DNA] = 100 μM). Characteristic regions for Watson-Crick base pairs (WC) and G-tetrads (G4) are indicated. (G) Flow cytometry binding analysis of the AF647-labeled Ap.OR2AT4.17 aptamer to 293-OR2AT4-Clone#2 transfectants. The unspecific 38x(AG) aptamer (Ap.AGA) labeled with AF647 was used as a negative control. The gray histograms represent unlabeled cells. A representative experiment is shown. MFI values were plotted against varying concentrations of AF647-labeled aptamers (0–2 μM) for calculation of Kd. Each data point represents mean ± SD (n = 2), with curve fitting of the mean. (H) 293-OR2AT4-Clone#2 cells were treated with increasing concentrations of the indicated aptamers for 1 h. cAMP levels were quantified, and results are expressed as mean (SD) fold increase over basal (no treatment, set as 1) of two independent experiments with triplicate samples. Parental 293 cells were used as a control.

Figure 2

Ap.OR2AT4.17 aptamer functionally recognized OR2AT4 receptor in human ex vivo HF organ cultures Representative images showing OR2AT4 expression by immunofluorescence analyses in cryosections from microdissected human HFs after 6-day organ hair culture in the presence of ApAGA and Ap.OR2AT4.17. Increased OR2AT4 expression was observed in the suprabulbar ORS keratinocytes of HFs incubated with Ap.OR2AT4.17 as compared with Ap.AGA control group. Scale bars, 500 μm. The graph shows quantification of immunofluorescence intensities of OR2AT4 expression that was evaluated in equivalent areas (yellow rectangles) in ORS keratinocytes. Data are presented as box and whisker plots (median, first and third quartiles, minimum and maximum) (n = 15 HFs from five different donors, Mann-Whitney U test, ∗∗∗∗p < 0.0001).

Figure 3

Ap.OR2AT4.17 aptamer induced hair growth and HF pigmentation in human HF organ cultures (A) Human hair cycle staging was evaluated by quantitative morphometry. The percentage of follicles that remained in anagen after 6 days in culture was significantly greater in Ap.OR2AT4.17-treated follicles in comparison with control groups. Data are expressed as mean (SD), and compared using ANOVA followed by Tukey’s multiple comparison test (n = 12–25 HFs for each condition, from 3 to 5 different donors; ∗∗∗∗p < 0.0001; ns, not significant). (B) Representative images showing IGF-1 immunoreactivity after 6-day aptamer treatment. Scale bars, 100 μm. Equivalent areas in the ORS (yellow rectangles) were selected for quantification purposes. Graph shows quantification of the fluorescence intensity. Results are expressed as mean (SD) (n = 25 HFs for each condition, from five different donors; Student’s t test with Welch’s correction; ∗∗∗∗p < 0.0001). (C) Representative images showing TGFβ2 immunoreactivity after 6-day aptamer treatment. Scale bars, 100 μm. Equivalent areas in the ORS (yellow rectangles) were selected for quantification purposes. Graph shows quantification of the fluorescence intensity. Results are expressed as mean (SD) (n = 25 HFs for each condition, from five different donors; Student’s t test with Welch’s correction; ∗∗∗∗p < 0.0001). (D) A representative image of human HFs after 6-day organ culture showing a significant increase in hair shaft length in the Ap.OR2AT4.17-treated follicle in comparison with control groups. Scale bars, 1.2 mm. Graph shows final percentage of hair shaft elongation after 7 days of culture as compared to day 0. Data are presented as box and whisker plots (median, first and third quartiles, minimum and maximum) (n = 12–25 HFs for each condition, from 3 to 5 different donors). p values were determined by ANOVA followed by Kruskal-Wallis test (∗∗∗∗p < 0.0001). (E) Representative images of Ki-67 staining in the bulb region. Scale bars, 100 μm. Graph shows quantified data of Ki-67+ matrix keratinocytes in selected areas marked in yellow below the Auber’s line (white dotted line), which were normalized to the number of DAPI-stained cells. Results are expressed as mean (SD) (n = 25 HFs for each condition, from five different donors; Student’s t test; ∗∗∗∗p < 0.0001). (F) Representative images of Masson-Fontana histochemistry showing increased hair shaft and HM melanin content in the Ap.OR2AT4.17-treated follicle. Scale bars, 100 μm. Graph shows quantified data of melanin deposition in equivalent areas of the hair bulb (red rectangles). Data expressed as mean (SD) (n = 20 HFs for each condition, from five different donors; Student’s t test; ∗∗p < 0.01). (G) Representative images show increased number of gp100+ melanocytes in the HM of Ap.OR2AT4.17-treated follicle in comparison with control. Scale bars, 100 μm. Graph in the middle panel shows quantification of the number of gp100+ cells in equivalent areas of the hair bulb (yellow rectangles), which were normalized by the total number of DAPI-stained cells. Data are presented as mean (SD) (n = 20 HFs for each condition, from five different donors; Student’s t test; ∗∗p < 0.01). Graph in the right panel shows quantification of the level of expression (fluorescence intensity), where data are presented as box and whisker plots (median, first and third quartiles, minimum and maximum) (n = 20 HFs for each condition, from five different donors; Mann-Whitney U test; ∗p < 0.05).

Figure 4

Ap.OR2AT4.17 aptamer enhanced LL-37 and hBD-3 protein expression in human HF organ cultures (A) Representative images showing immunofluorescence staining of LL-37 (green) and hBD-3 (red) on frozen sections of microdissected full-length scalp HFs. Scale bars, 200 μm. (B) Representative images showing LL-37 immunoreactivity after 6-day aptamer treatment. Scale bars, 100 μm. Graph shows quantification of the fluorescence intensity that was evaluated in equivalent areas of the hair bulb (yellow rectangles). Data are presented mean (SD) (n = 15 HFs for each condition, from three different donors; Student’s t test with Welch’s correction; ∗∗∗p < 0.001). (C) Representative images showing hBD-3 immunoreactivity after 6-day aptamer treatment. Scale bars, 100 μm. Graph shows quantification of the fluorescence intensity that was evaluated in equivalent areas of the hair bulb (yellow rectangles). Data are presented as box and whisker plots (median, first and third quartiles, minimum and maximum) (n = 18 HFs for each condition, from three different donors; Student’s t test with Welch’s correction; ∗∗∗∗p < 0.0001).