Stimulating dermal cells to generate hair follicle-inducing dermal condensates

Abstract

Provided are compositions and methods for inducing formation of dermal condensates from dermal progenitor cells. The disclosed compositions are particularly suitable for increasing dermal condensate formation and promoting hair growth and regeneration. The compositions include one or more Wnt agonists, and / or one or more SHH agonists in the same or different formulations. The Wnt agonists are capable of binding to one or more components of the Wnt receptor complex, including Wnt ligands (such as Wnt3 and / or Wnt3a), the LRP5 / 6 co-receptor, Frizzled (FZD) receptors, Dishevelled 1 and / or CXXC-type zinc finger protein 5 such as DVL-CXXC5, or interfaces formed between these components.

Description

[0001] STIMULATING DERMAL CELLS TO GENERATE

[0002] HAIR FOLLICLE-INDUCING DERMAL CONDENSATES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of and priority to U.S. Provisional Application No. 63 / 746,075, filed January 16, 2025, which is specifically incorporated by reference herein in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under AR076420 awarded by National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] The disclosed invention is generally in the field of treatments for hair growth through proliferation and restoration of hair follicles in situ.

[0008] BACKGROUND OF THE INVENTION

[0009] Hair loss and hair thinning affect a substantial portion of the population and arise from a variety of causes, including androgenetic alopecia, aging, injury, inflammation, and disease. Existing therapeutic approaches are limited in their ability to restore hair follicle number, size, or cycling capacity. Many treatments focus on prolonging the growth phase of existing hair follicles or slowing follicle miniaturization, rather than regenerating new hair follicles or restoring robust follicle-inductive capacity.

[0010] Several conditions affect the health of hair follicles. The most common conditions include alopecia areata, baldness in women and people assigned female and male at birth, folliculitis, hidradenitis suppurativa, stress, telogen effluvium.

[0011] Hair follicle regeneration during development and wound repair is dependent on specialized mesenchymal cell populations, including dermal condensates and their adult counterparts, dermal papilla cells. Dermal condensates act as inductive signaling centers that coordinate hair follicle morphogenesis through tightly regulated interactions with the overlying epidermis. Consequently, strategies capable of inducing or restoring dermal condensate-like structures represent a promising avenue for promoting hair growth and reducing hair loss.

[0012] However, most therapies to date focus on transplantation to create more hair follicles, treatment for infection or inflammation, and hormonal supplementation. For example, approaches have been explored to stimulate hair growth or dermal papilla function, including growth factors, cytokines, pathway agonists, cell transplantation, and topical formulations.

[0013] 1

[0014] 45816336.1These efforts have met with limited to no success. These approaches, however, suffer from several limitations. Many existing compositions target single signaling pathways, such as Wnt, BMP, or FGF pathways, in isolation. While such pathways play roles in hair follicle biology, activation of a single pathway alone has generally proven insufficient to reliably induce dermal condensate fomiation. In some cases, excessive or unregulated pathway activation can lead to aberrant proliferation, loss of cell identity, or undesirable side effects. Also, current methods often fail to account for the precise timing, dosage, and combinatorial nature of signaling events that govern dermal condensate formation. For example, existing approaches typically do not control the transition between proliferation and differentiation, resulting in inconsistent outcomes and limited reproducibility. It is therefore, still unclear why hair follicles fail to proliferate, why they shrink in size over time, and stop or reverse this process.

[0015] Another significant contributing factor is the lack of suitable screening platforms that faithfully model dermal condensate formation and function, which has hindered the discovery and evaluation of effective compounds. Many existing screening methods rely on non-inductive dermal fibroblasts, immortalized cell lines, or bulk mesenchymal cell populations that do not possess the intrinsic capacity to undergo dermal condensate-like proliferation, cell cycle exit, and differentiation. As a result, such cells fail to recapitulate the dynamic cellular states and signaling responsiveness characteristic of dermal condensate progenitors in vivo.

[0016] In addition, commonly used screening platforms often assess pathway activation using simplified or indirect readouts, such as single-marker induction, rather than assessing whether dermal cells undergo the coordinated proliferative, differentiative, and condensateforming behaviors required for dermal condensate formation. Consequently, compounds identified through these platforms may modulate isolated molecular pathways without inducing bona fide dermal condensate formation or hair follicle-inductive activity.

[0017] Furthermore, many existing screening systems are limited in their ability to evaluate temporal and combinatorial signaling parameters, such as the order, timing, and relative concentrations of Wnt, SHH, or other morphogenetic signals that regulate dermal condensate development. In the absence of screening platforms capable of assessing these parameters in a physiologically relevant context, potentially effective compounds or combinations may be overlooked, while others may appear active under simplified or non-physiological conditions but fail to produce consistent or translatable effects in vivo.

[0018] Thus, more effective compositions and methods for stimulating dermal condensate formation, promoting hair growth, and reducing hair loss are needed. Also needed are more

[0019] 2

[0020] 45816336.1effective methods of screening and testing compounds and formulations to identify agents capable of inducing functional dermal condensates and hair follicle-inductive activity.

[0021] It is an object of the present invention to provide compounds, formulations, and methods that are useful to promote the health and regeneration of hair follicles.

[0022] It is another object of the present invention to provide compositions and methods for stimulating proliferation and differentiation of dermal progenitor cells, and formation of dermal condensates.

[0023] It is another object of the present invention to provide compounds, formulations, and methods for increasing hair growth and / or reducing hair loss.

[0024] It is another an object of the present invention to provide methods and systems for screening compounds that capable of inducing formation of dermal condensates and hair follicle regeneration and growth.

[0025] SUMMARY OF THE INVENTION

[0026] Wnt / β-catenin and Sonic Hedgehog (SHH) agonists are important and sufficient to generate hair follicle-inducing dermal populations in vivo in mouse embryonic skin. Activating Wnt / β-catenin followed by SHH activation in vitro can also induce markers of Wnt and SHH activity, respectively, and that activation of Wnt priorto SHH is important forinducing SHH responsiveness. However, it is unknown how hair follicle-inducing dermal condensates (aka dermal papillae) form. The ability to induce dermal condensates (“DC”s) in in vitro or in skin provides the opportunity to induce new hair follicle growth in adult skin or in organotypic cultures, which can be used to generate skin equivalents for drug testing and disease modeling.

[0027] A combination of single-cell RNA-sequencing combined with mouse genetic perturbation was used to identify the two signals, Wnt and SHH, that sequentially cooperate to induce DC formation in vivo during mouse skin development. Dermal Wnt activation was found to be important to prime dermal cells for SHH activation. Together, these two signals are able to induce DC formation. It was previously unknown how dermal condensates form and the signals that mediate this process. Surprisingly, although DCs do not proliferate, it was found that DC progenitors undergo a phase of Wnt-dependent proliferation prior to cell cycle exit. In the absence of Wnt activation, SHH cannot act to induce DC formation. However, if dermal cells are Wnt activated, they can receive SHH stimulation to differentiate into DC cells. It has been demonstrated that both the timing and levels of these two signals regulate the transition from an undifferentiated dermal cell into a differentiated DC cell. Therefore, not only was it observed that Wnt and SHH together are important and sufficient for DC formation, it was found

[0028] 3

[0029] 45816336.1that the levels and timing of Wnt and SHH stimulation are also important for regulating the process of DC formation.

[0030] By combining scRNA-sequencing with genetic perturbation, it was discovered that proliferative Dkk1+ progenitors transiently amplify to become quiescent dermal condensate cells by the spatiotemporal patterning of Wnt / β-catenin and sonic hedgehog (“SHH”) signaling gradients. Together, they deterministically coordinate a rapid transition from proliferation to quiescence, cell fate specification, and morphogenesis.

[0031] Provided are compositions for inducing formation of dermal condensate from dermal progenitor cells. The disclosed compositions are particularly suitable for increasing dermal condensate formation and promoting hair growth and regeneration. This staged modulation can be achieved by sequential administration of a Wnt agonist and a SHH agonist, respectively, or by use of a single formulation in which one or both agonists are present in controlled, delayed, or sustained-release form. In such forms, the relative dose, timing, and duration of Wnt and SHH pathway activation are selected to promote formation of dermal condensates of sufficient size and signaling competence to induce effective hair follicle growth and regeneration. The compositions include one or more Wnt agonists and / or one or more SHH agonists.

[0032] In some forms, the Wnt agonists can bind to one or more components of the Wnt receptor complex, including Wnt ligands (such as Wnt3 and / or Wnt3a), the LRP5 / 6 coreceptor, Frizzled (FZD) receptors, Dishevelled 1 and / or CXXC-type zinc finger protein 5 such as DVL-CXXC5, or interfaces formed between these components.

[0033] Exemplary compounds that can be used as the WNT agonist include the compounds of Formula (I) and Formula (II) described below. In one exemplary form, the WNT agonist is a compound of Formula (1):

[0034]

[0035] Formula (I)

[0036] wherein L is absent or is a linker;

[0037] wherein R2 includes a phenyl, aryl, or heteroaryl group optionally substituted with one or more substituents independently selected from C1-6 alkyl group and C1-6 alkoxy group; and

[0038] wherein Ri includes a fused bicyclic ring system containing 8 to 14 ring atoms,

[0039] 4

[0040] 45816336.1wherein the fused bicyclic ring system includes at least one ring that is a 5- or 6-membered ring containing at least one nitrogen atom;

[0041] or is a pharmaceutically acceptable salt thereof.

[0042] In exemplary forms, the Wnt agonist is a compound of Formula (II):

[0043] o

[0044]

[0045] Formula (II)

[0046] wherein Ri’ includes a pyridine ring substituted with -C(O)NR3R4

[0047] wherein R2’ includes a piperidine ring optionally substituted at the nitrogen atom with R5';

[0048] wherein R3and R4are independently selected from hydrogen and C1-6 alkyl group; wherein R5’, when present, is a C1-6 alkyl group [;

[0049] or is a pharmaceutically acceptable salt thereof.

[0050] The one or more Wnt agonists can be present in the composition at a concentration ranging from about 0.001 nM to about 200 mM, including sub-nanomolar, nanomolar, micromolar, millimolar, and high millimolar concentrations. For example, the Wnt agonist can be present in the composition at a concentration ranging from about 1 pM to about 100 mM, such as from about 50 pM. In some forms, the Wnt agonist is present in the formulation at a concentration of about 0.000001 wt% to about 10 wt%, including about 0.001 wt% to about 5 wt%, depending on formulation type, route of administration, and desired biological or cosmetic effect. For example, in rinse-off formulations, higher nominal concentrations can be used to compensate for shorter contact times, whereas in leave-on formulations lower concentrations may be used to provide sustained exposure.

[0051] The one or more SHH agonists can be present in the composition at a concentration ranging from about 0.001 nM to about 200 mM. For example, the SHH agonist is present at concentrations ranging from about 1 nM to about 100 mM, such as about 50 nM. In some forms, the SHH agonist is present in the formulation at a concentration of about 0.0000001 wt% to about 10 wt%, such as about 0.0001 wt% to about 5 wt%, depending on formulation type, route of administration, and desired biological or cosmetic effect. In some forms, when the SHH agonist is present in the same composition as a Wnt agonist, the SHH agonist is formulated for delayed release relative to the Wnt agonist. For example, release of the SHH agonist is staggered by at least about 1 day, about 2 days, or about 3 days following release of

[0052] 5

[0053] 45816336.1the Wnt agonist, thereby allowing Wnt-mediated priming of target cells prior to activation of SHH signaling.

[0054] The disclosed methods utilize biologically relevant dermal progenitor cells or dermal condensates and that permit evaluation of combinatorial and temporally regulated signaling events. This discovery and the model based on it are useful for screening to discover compounds that can be administered to hair follicles to stimulate hair growth (new hair follicles) and restore hair follicle size. These agonists of Wnt and SHH are formulated for application to skin to grow hair. The timing of application, as well as the formulation, are optimized for the site of application. In some forms, the compounds are applied topically (gel, spray, shampoo, foam, solution, transdermal patch) and incorporates at least one means for sustained or pulsed controlled delivery of the agent. In a preferred embodiment, the topical formulation includes an inert solvent, a surfactant, a viscosity modifying agent, a transdermal penetration enhancer, and soluble Wnt agonist (such as CHIR-99021) and / or SHH agonist (such as SAG dihydrochloride) either alone (CHIR 5 or 10 micromolar (pM), SAG 50, 100 or 200 nM) or together in the following combinations of these exemplary concentrations: CHIR 5 pM + either SAG 50, 100 or 200 nM and CHIR 10 pM + either SAG 50, 100, or 200 nM.

[0055] This is administered in a method for enhancing hair follicle growth. The Wnt agonist is applied first, then the SHH agonist is administered as the levels of the Wnt agonist decrease. This is achieved by either applying the agonists separately or, more preferably, formulated so that the Wnt agonist is released immediately and the SHH agonist is released thereafter, either as a delayed release formulation, or as a slow release which is timed for release of the SHH agonist as the levels of the Wnt agonist is decreasing. These concentrations of CHIR (5 pM + SAG 50 nM. combined low dose initially) can be administered topically or intradermally daily for 2-5 days to an animal such as a mouse for screening or to human skin for treatment, followed by incrementally higher dose of SAG: CHIR 10 pM +SAG (100-200 nM) based on gene responses from in vitro culture experiments daily for 2-5 days (without washout period).

[0056] BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figures 1A-1C are graphs showing induction of high levels of SHH signaling across the entire gradient of Wnt signaling prior to morphogenesis, and not just those in the peri -DC, cause every cell within that gradient to augment Wnt signaling incrementally while increasing their proliferation and expression of DC genes, resulting in more transitioning intermediates proliferating until they reach a level of Wnt signaling sufficient to cause arrest.

[0058] 6

[0059] 45816336.1Figures 1A and IB are graphs of the effect of SHH level in combination with Wnt level showing the change from no dermal condensate (“DC”) to proliferation intermediates (transition level) to arrest. Figure 1C shows this process graphically.

[0060] Figures 2A-2E are schematics of epidermis with quiescent CDs in the dermis (Figure 2A); epithelial placode with DC (Figure 2B); proliferation of DC (Figure 2C); further invagination of the epidermis as it forms a hair follicle at the site of DC (Figure 2D), and formation of the hair follicle in the epidermis (Figure 2E).

[0061] Figures 3A-3M show that dermal SHH activation is required for the rapid transition to quiescence and mature DC differentiation, Lefl levels in Bmp4+ DCs at E14.5 and E15.5 (Figure 3A). FISH showing decreased Lefl expression in Bmp4+ SHH cKO pseudo-DCs (Figure 3B). Proliferation rate of Dkk1+ peri-DC and upper dermal cells at E14.5 (Figure 3C). Pseudo-order of control and SHH cKO dermal cells at E14.5 (Figure 3D). FISH showing proliferative Bmp4+ pseudo-DCs in SHH cKO at E14.5 and E15.5 (Figure 3E). Number of Bmp4+ cells in SHH cKO and control over time ( Figure 3F). %EdU+ of Bmp4+ population over time in control and SHH cK.0 (Figure 3G). Diffusion maps of E14.5 and E15.5 control and SHH cKO dermal cells showing that SHH cKO pseudo-DCs progress only on the Wnt component by El 5.5 (Figures 3H-3K). Cartoons showing aberrant proliferation of SHH cKO Dkk1+ peri-DC cells and slow transition to quiescence (Figures 3L, 3M). Data as mean± SEM; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA. Scale bars. 50 pm.

[0062] Figures 4A-4H show that high dermal SHH activation in early Wnt-active cells deterministically reproduces events of DC genesis over more intermediates. E14.5 control and SmoM2YFP whole mount showing proliferative mutant Sox2+ clusters (Figure 4A). Number of Sox2+ cells per dermal cluster in control and SmoM2YFP at E14.5 and E15.5 (Figure 4B). Diffusion maps of E14.5 control and SrnoM2YFP cells (Figures 4C, 4D). Pseudo-order with Regions 1 and 2 demarcated (Figures 4E, 4F). FISH of E14.5 control and mutant showing virtually all Sox2+ cells co-express Ptch1 and Lefl (Figures 4G, 4H).

[0063] Figures 5A-5N show' that high dermal SHH activation in early Wnt-active cells deterministically reproduces events of DC genesis over more intermediates. El 4.5 control and SmoM2YFP whole mount showing proliferative mutant Sox2+ clusters (Figure 5A). Number of Sox2+ cells per dermal cluster in control and SmoM2YFP at El 4.5 and El 5.5 (Figure 5B). Diffusion maps of E14.5 control and SmoM2YFP cells (Figure 5C). Pseudo¬ order with regions 1 and 2 demarcated (Figure 5D). FISH of E14.5 control and mutant showing virtually all Sox2+ cells co-express Ptch1 and Lefl (Figure 5E). Pseudo-order of

[0064] 7

[0065] 45816336.1indicated genes in mutant and control (Figure 5F). E14.5 FISH showing EdU, Sox2, and Dkk1 (Figure 5G). FISH at E15.5 after EdU pulse in control and SmoM2YFP skin (Figure 5H). FISH 24 hours after EdU chase (Figure 5I). %EdU+ cells by population in E14.5 mutant and control (Figure 5J). %EdU+ of Sox2+ cells at E15.5 after EdU pulse or 24-hour chase (Figure 5K). E14.5 SmoM2;βcatfl / EX3 FISH showing Sox2+ clusters in upper and lower dermis that co-express Ptch1 and Lefl (Figure 5L). Sox2+ clusters with %EdU+ of indicated populations by condition at El 4.5 (Figure 5M). Depiction of transition rate and number of intermediates affected by modulating SHH and Wnt signaling (Figure 5N).

[0066] Figure 5O is a graph of % EdU+ for UD, Dkk1+Sox2- compared to Sox2+. Figure 5P is a diagram of the histology in cross-section of wild type, SmoM2 and SmoM2βEx3showing proliferating Dkk1+, penultimate dividing Dkk1+ and quiescent Dkk1+.

[0067] Figures 6A-6C are graphs of %DC (Figure 6A), the ratio of periDC / Perfollicular (Figure 6B) and the progression of DC differentiation from proliferative periDC (Dkkl+) to quiescent corner (Dkkl+) to DC (Dkkl-) (Figure 6C).

[0068] Figures 7 A and 7B are graphs showing dermal cells pre-treated with Wnt agonist, CHIR, significantly enhanced SHH and DC gene responses compared to without Wnt agonist pre-treatment (compare blue no pretreatment to red with Wnt pretreatment).

[0069] Figure 8 is a bar graph showing relative Lefl expression measured by qPCR in El 3.5 PDGFRa-GFP+dermal cells following treatment with Y-series compounds and pathway controls. Cells were treated with DMSO, Wnt pathway agonists (Wnt3a or CHIR99021), or individual Y-series compounds classified as SS-LRP6-Frizzled or SS-DVL-CXXC5 modulators, as indicated. Lefl expression is shown relative to DMSO controls. Bars represent mean values with error bars indicating variability across replicates, and statistical significance is denoted by asterisks.

[0070] Figure 9 is a bar graph showing relative Lefl expression measured by qPCR in El 3.5 PDGFRa-GFP+dermal cells treated with Y-series compounds alone or in combination with Wnt3a and / or the SHH agonist SAG. Treatment conditions include DMSO, Wnt3a, SAG, CHIR99021 with or without SAG, and candidate Y-series compounds, Y-0309618 (Y18), Y-0309620 (Y20), and Y-0309613 (Y13), administered alone or together with Wnt3a and / or SAG, as indicated. Lefl expression is shown relative to DMSO controls. Bars represent mean values with error bars indicating variability across replicates, and statistical significance is denoted by asterisks.

[0071] Figure 10 is a bar graph showing relative Ptch1 expression measured by qPCR in E13.5 PDGFRa-GFP+dermal cells treated with Y-series compounds in the presence or

[0072] 8

[0073] 45816336.1absence of the SHH agonist SAG and / or Wnt3a. Treatment conditions include DMSO, Wnt3a, SAG, CHIR99021 with or without SAG, and candidate Y-series compounds (Y13, Y18, or Y20), administered alone or in combination with Wnt3a and / or SAG, as indicated. Ptch1 expression is shown relative to DMSO controls. Bars represent mean values with error bars indicating variability across replicates, and statistical significance is denoted by asterisks.

[0074] Figure 11 is a bar graph showing relative Gal expression measured by qPCR in El 3.5 PDGFRa-GFP+dermal cells treated with Y-series compounds alone or in combination with Wnt3a and / or the SHH agonist SAG. Treatment conditions include DMSO, Wnt3a, SAG, CHIR99021 with or without SAG, and candidate Y-series compounds (Y13, Y18, or Y20), administered alone or together with Wnt3a and / or SAG, as indicated. Gal expression is shown relative to DMSO controls. Bars represent mean values with error bars indicating variability across replicates, and statistical significance is denoted by asterisks.

[0075] Figures 12A-12D shows cell cycle exit and molecular differentiation are tightly coupled during dermal condensate development. E13.5 and E14.5 flank skin with Sox2+ DC with lack of EdU copositivity at E14.5 were tested. Figure 12A are violin plots showing quantification of Lefl and Ptch1 levels (H-score) by dermal cell layer, %EdU+ by Lefl bin from FISH of El 3.5 wildtype skin stained for Lefl, Ptch1 or EdU after 1-hour EdU pulse. Figure 12B is a cartoon depicting graded Lefl expression spatially. Figure 12C are violin plots showing quantification of Lefl and Ptch1 levels by indicated spatial regions, %EdU+ by Lefl bin from FISH of El 4.5 skin stained for Lefl, Ptch1 or EdU. Figure 12D is a cartoon depicting Lefl and Ptch1 expression levels spatially; n=5. Data as mean± SEM (n=5 per day); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA; ns, not significant. Scale bars, 50 pm.

[0076] Figure 13A-13F shows high Wnt activity induces cell cycle exit independent of SHH.

[0077] Figure 13A is a cartoon of Wnt activity levels in E13.5 wildtype or Actpcat mutant (tamoxifen El 1.5). UMAP analysis was done to determine expression of indicated genes or cell cycle (CC) phase for El 3.5 Actpcat mutant or paired control dermal scRNA-seq data. Figure 13B are bar graphs of quantification of FISH Cdknla levels (H-score) or %EdU of Cdknla (p21)+ cells from FISH of E13.5 Lefl, Cdknla. Figure 13C is a cartoon of cell cycle and DC molecular differentiation dynamics in E13.5 Actpcat showing cells exit the cell cycle but do not express DC genes. Figure 13D is a bar graph showing FISH quantification of %EdU+ cells within indicated control or Actpcat populations for E14.5 Sox2, Cdknla. Figure 13E is a violin plot showing FISH quantification of Ptch1 level within indicated

[0078] 9

[0079] 45816336.1control or Actpcat populations for E14.5 Left, Ptch1, Sox2. Figure 13F is a cartoon of E14.5 Actpcat condition in which quiescence can occur without DC gene expression.

[0080] Figures 14A-14D show SHH cell-autonomously augments Wnt signaling to induce cell cycle exit. Figure 14A is a cartoon of experimental condition induced by expressing a constitutively activated form of Smo. Quantification of %EdU+ of Sox2+ cells at E14.5. Figures 14B and 14C show Lefl levels by Ptch1 status (Figure 14B) or %EdU by Lefl levels (bin; Figure 14C). Figure 14D is a cartoon showing that SHH activation cell-autonomously boosts Lefl levels. Data as mean± SEM; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA. Scale bars, 50 pm.

[0081] Figures 15A-15D show A Wnt-dependent gene program that corresponds to cell cycle exit is identified by GeneTrajectory. GT analysis was done of E13.5 ActSMO and paired control dermal scRNA-seq data showing four GTs and DC GT of ActSMO mutant which is not present in control. Exit GT of ActSMO colored by GT stage or indicated genes.

[0082] Figure 15A is a pathway analysis of Exit GT. UMAPs of control and ActSMO colored by indicate genes. Figure 15B are violin plots showing corresponding of quantification of Trp53in.pl or Cdknla levels from FISH showing indicated genes of E13.5 control or ActSMO mutant skin. Figure 15C is a bar graph showing corresponding quantification of %EdU positive of Trp53inpl+ cells from EdU pulse-chase experiments of E13.5-E14.5 ActSMO mutant. Figure 15D is a cartoon depicting the interpretation of cell cycle dynamics of Trp53inpl+ cells which represent cells that are immediate progeny of a division. Data as mean ± SEM, one-way ANOVA. Scale bars, 50 pm.

[0083] Figures 16A-16E shows Loss of GLI3 chromatin binding regulates cell cycle exit downstream of high Wnt activity. Figure 16A is a heat map of GLI3 binding at all transcriptional start sites (TSS) in E13.5 control or Actpcat mutant skin. Figure 16B is KEGG pathway analysis of GLI3 unbound regions in the Actpcat mutant. Figure 16C are two bar graphs showing quantification of #Sox2+ cells per DC and Cdknla transcript levels by skin population from FISH of E14.5 control and Gli3 cKO for indicated genes. Figure 16D are two bar graphs showing quantification of Cdknla transcripts or %EdU by population after 1-hour EdU pulse or after 24-hour pulse-chase from FISH of E13.5 control and Gli3 cKO with indicated genes. GT analysis of El 3.5 Gli3 cKO showing an Exit GT. Figure 16E is a volcano plot of differentially expressed genes in the Gli3 cKO vs control and those genes that overlap with GLI3 unbound genes in the Actpcat mutant (blue): genome browser tracks showing binding signal of respective antibodies at the promoter region of indicated genes.

[0084] 10

[0085] 45816336.1Data as mean± SEM; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA; ns, not significant; scale bars, 50 pm.

[0086] Figures 17A-17C show DC gene expression correlates with Wnt activity levels upon SHH activation. Figure 17A is a series of plots showing expression level of indicated DC genes by DC GT stage and UMAPS with indicated genes. Figure 17B are two plots showing Left levels by DC GT stage. Figure 17C is a cartoon showing that SHH activation cell-autonomously boosts Lef1 levels. Data as mean± SEM; *P<0.05, **P<0.01, ***P<0.001, ****p<0.0001, one-way ANOVA. Scale bars, 50 pm.

[0087] Figures 18A-18I shows coupled Wnt and SHH gradients is sufficient to synchronize cell cycle exit with molecular differentiation. Figure 18A is a cartoon of wildtype scenario showing covarying gradients and experimental scenario to induce covarying Wnt and SHH gradients by expressing SHH ligand coordinately with Wnt ligands in the epidermis. Figure 18B shows quantification of the fraction of Cdknl a+ cells that co-express DC genes (Gal) in ActSMO vs SHH OE. Figure 18C shows GT analysis of E13.5 SHH OE dermal scRNA-seq data. Figure 18D shows Lefl levels by DC G T stage; FISH image showing spatially covarying gradients of Ptch1 and Lefl that corresponds to dermal layer. Figure 18E is a heat map of GLI3 binding at all TSS in E13.5 control versus SHH OE showing global loss of binding. Figure 18F shows genome browser tracks showing binding signal of respective antibodies at the promoter region of indicated genes. Figure 18G is a bar graph showing qPCR for Lefl of E13.5 PDGFRaH2B dermal cells after SAG and / or CHIR stimulation for 48 hours (n=4). Figure 18H shows qPCR for DC genes after 48 hours of culture with CHIR and / or SAG. Figure 181 is a model of how gradient levels of Wnt and SHH interact dynamically to coordinate DC differentiation gene expression and cell cycle exit and that the covariance of the two signals is responsible for the synchronization of cell cycle exit and DC genes during DC genesis. Data as mean ± SEM, one-way ANOVA; scale bars, 50 pm.

[0088] Figure 19 is a schematic of the Wnt / p-catenin signaling pathway. When Wnt proteins bind to LRP5 / 6 and FZD, the phosphorylation and degradation of P-catenin is blocked, resulting in stabilization, accumulation and nuclear translocation of P-catenin and subsequent activation of the pathway.

[0089] Figure 20A shows the overlaid model of LRP6 with Wnt 8, Wnt 3 and Wnt 3A ligand and highlights the Wnt ligands interaction site with LRP6. Figure 20B shows the predicted binding pockets in the LRP6 and Wnt 3 / 3A ligand complex.

[0090] Figure 21 is a schematic showing FXD structure. In absence of the Wnt ligand, the destruction complex composed of Axin, APC, GSK-3, and CK1 is formed in the cytoplasm.

[0091] 11

[0092] 45816336.1By forming this complex, P-catenin is phosphorylated initially by CK1 and subsequently by GSK-3. The b-TrCP E3 linker is recruited to the phosphorylated P-catenin and P-catenin is then degraded by ubiquitin-mediated proteasomal degradation machinery. In the presence of Wnt ligand, it binds to the Frizzled / LRP5 / 6 receptor complex, leading to the dissociation of the destruction complex. Free P -catenin proteins accumulate in the cytosol and are then translocated into the nucleus for activation of TCFs / LEFs. The activation of TCFs / LEFs transcription factors induces a variety of Wnt / P -catenin signaling target genes, including those involved in skin wound healing. LEFs, lymphoid enhancing factors; TCFs, T cell factors.

[0093] DETAILED DESCRIPTION OF THE INVENTION

[0094] I. DEFINITIONS

[0095] A hair follicle is a tube-like structure (pore) that surrounds the root and strand of a hair. Hair follicles exist in the top two layers of the skin. People are born with over 5 million hair follicles in their body and over one million hair follicles on their head. As one ages, hair grows out of your hair follicles. The hair follicle is one of a few structures in the body that can stop functioning and begin functioning again (degenerate and regenerate).

[0096] Hair grows in cycles within the hair follicle:

[0097] Anagen: The first phase of hair growth takes between two to seven years. Growth begins at the root (dermal papilla) in your hair follicle, which gives the hair blood supply and the nutrients it needs to grow. The hair grows about 1 centimeter per month.

[0098] Catagen: The second phase of hair growth occurs when the hair transitions from a growing phase to a resting phase, which takes about two weeks. During this phase, the hair detaches from your blood supply.

[0099] Telogen The final phase of hair growth is the inactive phase, where your hair sheds or falls out of your hair follicle. This phase takes up to four months.

[0100] Hair follicles originate in the first and second layers of your skin (epidermis and dermis). Follicles holding your terminal hair, or the hair that grows on your scalp, eyelashes and eyebrows, extend into the first and second layer of your skin and sometimes into the third layer (subcutaneous tissue).

[0101] The hair follicle dermal condensate is the precursor to the permanent mesenchymal unit of the hair follicle, the dermal papilla, which regulates hair cycling throughout life and bears hair inductive potential. Dermal condensate morphogenesis depends on epithelial Fibroblast Growth Factor 20.

[0102] 12

[0103] 45816336.1As used herein, the term “small molecule’’ means a low molecular weight organic and / or inorganic compound. In general, a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not a polysaccharide. In some embodiments, a small molecule does not comprise a polysaccharide (e.g., is not a glycoprotein, proteoglycan, glycolipid, etc.).

[0104] The terms “contact,” “contacting,” or “bringing into contact” describe placement in physical association, for example, in solid and / or liquid form. For hair and scalp applications, the process may involve contacting or combining one or more Wnt pathway agonists, Sonic hedgehog (Shh) pathway agonists, or combinations thereof with a target tissue, such as the scalp, hair follicles, or hair shafts, including by topical application, transdermal delivery, or localized administration. In some forms, contacting may involve applying a formulation containing the one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof in a solution, suspension, emulsion, gel, cream, foam, serum, or patch, such that the active agent is placed in physical association with the scalp and / or hair follicles for a period sufficient to permit uptake, diffusion, or biological activity. In some forms, contacting may further involve combining the one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof with one or more carriers, excipients, penetration enhancers, stabilizers, or solvents prior to or during application, thereby facilitating delivery to hair follicles or surrounding scalp tissue.

[0105] As used herein, “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is,

[0106] 13

[0107] 45816336.1treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0108] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0109] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - 5%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - 2%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

[0110] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. It should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise.

[0111] Finally, it should be understood that all ranges refer both to the recited range as a range and as a collection of individual numbers from and including the first endpoint to and including the second endpoint. In the latter case, it should be understood that any of the individual numbers can be selected as one form of the quantity, value, or feature to which the range refers. In this way, a range describes a set of numbers or values from and including the first

[0112] 14

[0113] 45816336.1endpoint to and including the second endpoint from which a single member of the set (i.e. a single number) can be selected as the quantity, value, or feature to which the range refers. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0114] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A. B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B. and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

[0115] These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific form or combination of forms of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0116] All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or

[0117] 15

[0118] 45816336.1exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the forms and does not pose a limitation on the scope of the forms unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0119] IL COMPOSITIONS FOR INDUCING DERMAL CONDENSATE FORMATION

[0120] The hair follicle is a highly organized mini-organ composed of distinct epithelial and mesenchymal components that together regulate hair shaft formation, cycling, and regeneration. Robust and organized hair follicle growth depends on tightly coordinated heterotypic signaling between the epidermal and dermal compartments, both during embryonic development and adult regeneration. The hair shaft itself is composed of a central medulla, surrounded by the cortex, which forms the bulk of the hair fiber, and an outer cuticle composed of a single layer of flattened cells. The cuticle is encased by the inner root sheath, a three-layered structure that guides and shapes the growing hair shaft as it emerges from the follicular matrix. The inner root sheath keratinizes from the outside inward and disintegrates around the level of the isthmus. Enclosing the entire hair shaft and inner root sheath is the outer root sheath, which extends along the length of the follicle and undergoes trichilemmal keratinization near the isthmus.

[0121] Hair follicles originate at the epidermal surface and extend to variable depths depending on hair type. Terminal hair follicles penetrate into the deep dermis or subcutis, whereas vellus hair follicles extend only into the upper reticular dermis. Anatomically, the follicle is divided into three principal regions: the infundibulum, extending from the epidermal surface to the opening of the sebaceous duct; the isthmus, located between the sebaceous duct and the bulge; and the inferior segment, which includes the bulb.

[0122] The bulge region, located within the outer root sheath at the insertion site of the arrector pili muscle, serves as a critical epithelial stem cell niche and contains multipotent epidermal stem cells expressing markers such as CK19, CK15, and CD200. The inferior segment terminates in the bulb, which surrounds the dermal papilla, a specialized, vascularized mesenchymal structure containing capillaries. The dermal papilla acts as an instructive signaling center that coordinates epithelial cell proliferation, movement, and differentiation through morphogenetic cues, including Wnt / 0-catenin and Sonic Hedgehog (SHH) signaling. The dermal papilla provides essential inductive signals to the surrounding follicular matrix, which exhibits the highest mitotic activity of any tissue in the body. Matrix keratinocytes proliferate and differentiate to form the hair shaft, while melanocytes interspersed among the matrix cells supply pigmentation.

[0123] 16

[0124] 45816336.1Following establishment of the mature hair follicle structure, hair growth proceeds through a cyclical process composed of anagen, catagen, and telogen phases. The anagen phase is the active growth phase during which a new hair shaft is produced and is the only stage in which the inferior segment of the follicle is present. The duration of anagen varies by body site, lasting several years for scalp hair and only months for eyebrows or eyelashes. Entry into anagen is initiated when the dermal papilla signals multipotent epithelial stem cells within the bulge, triggering downward growth of the inferior follicle and formation of the bulb around the dermal papilla. During this process, Wnt / 0-catenin signaling is propagated in a spatially coordinated manner across populations of undifferentiated epithelial cells, such that activation in a subset of cells can drive non-cell-autonomous signaling and synchronized growth behaviors in surrounding cells. The dermal papilla then directs matrix cell proliferation, differentiation, and upward growth to generate a new hair shaft.

[0125] The catagen phase is a brief transition stage lasting only weeks, during which matrix cell proliferation ceases and the inferior follicle regresses. As regression proceeds, the dermal papilla migrates upward to reestablish contact with the bulge, and a club hair with a hardened proximal end is formed. This is followed by the telogen phase, a resting stage during which club hairs are retained for approximately 100 days on the scalp before being shed, allowing the cycle to restart with re-entry into anagen.

[0126] At any given time, approximately 85-95% of scalp hairs are in anagen, growing at an average rate of about 1 cm per month, while hair shedding is continuous, averaging roughly 100 hairs per day. Although hair follicles repeatedly generate new hair shafts throughout life, no new hair follicles are formed after birth, and long-term hair density and caliber depend on the ability of existing follicles to maintain proper cycling behavior and follicle size.

[0127] The regenerative capacity of the hair follicle is established during embryonic development, when epithelial-mesenchymal interactions give rise to the follicle unit. Around nine weeks of gestation, the fetal epidermis forms epithelial buds that migrate downward into the dermis under the influence of aggregated mesenchymal cells. These mesenchymal cells form a dermal condensate, which functions as an inductive signaling center and later matures into the dermal papilla as follicle development proceeds. The hair follicle provides an experimentally accessible and well-characterized model of stem cell-dependent tissue regeneration, allowing investigation of how morphogenetic signals are spatially disseminated to ensure robust, patterned, and compartmentalized growth. This process yields a hair follicle composed of an ectoderm-derived matrix and a mesoderm-derived dermal papilla, along with associated sebaceous glands and arrector pili muscles. In utero, lanugo hairs develop and are

[0128] 17

[0129] 45816336.1subsequently replaced by vellus or terminal hairs depending on body site and hormonal signaling. Additional details on hair anatomy and growth are found in U.S. Provisional Application No. 63 / 746,075, which is incorporated herein by reference in its entirety.

[0130] Dermal condensates are thus fundamental to both embryonic hair follicle morphogenesis and postnatal hair follicle regeneration. These highly organized clusters of specialized mesenchymal cells serve as persistent signaling hubs that regulate follicular downgrowth, architecture, size, and cycling behavior. During anagen, the dermal papilla expands in cell number, a process that is tightly linked to hair follicle growth and hair shaft size. During anagen, dermal condensates and their derivative dermal papilla structures direct hair shaft production and determine follicle caliber through tightly regulated epithelial-mesenchymal interactions. Dermal papilla expansion arises from at least two distinct progenitor populations with different temporal contributions: proliferative dermal cup cells that migrate into the dermal papilla during early anagen, and a subset of dermal papilla cells that re-enter the cell cycle during late anagen. These populations exhibit distinct spatial contributions within the dermal papilla and differential retention across hair cycles, indicating specialized roles in regeneration and follicle maintenance.

[0131] However, endogenous dermal condensate function can become diminished or dysregulated due to aging, genetic predisposition, inflammation, or environmental stress, resulting in impaired hair growth, follicular miniaturization, or progressive hair loss.

[0132] Dysregulation of the same morphogenetic signaling pathways that drive organized regeneration can also contribute to disorganized and uncontrolled growth, as observed in skin cancers such as basal cell carcinoma, which co-opts hair follicle morphogenetic programs. Dermal condensates form only during restricted developmental or regenerative windows, such as embryogenesis or anagen initiation, limiting opportunities for therapeutic intervention.

[0133] Existing approaches have struggled to distinguish molecules specifically associated with dermal condensate formation and function from the broader population of dermal or follicular signaling factors. The ability to precisely modulate signaling pathways governing dermal condensate differentiation and function, particularly Wnt and Sonic Hedgehog (SHH) signaling, is therefore important for inducing hair-inductive dermal condensates of appropriate size and activity. Controlled, dose- and time-dependent modulation of these pathways permits expansion of dermal condensate progenitors followed by induction of robust dermal condensate gene expression, thereby directly influencing hair follicle size, cycling behavior, and regenerative capacity. For example, it has been observed that chemical

[0134] 18

[0135] 45816336.1modulation of Wnt signaling using a glycogen synthase kinase-3 (GSK-3) inhibitor (e.g., CHIR99021) and modulation of SHH signaling using a Smoothened agonist (e.g., SAG) demonstrates that the timing and relative levels of Wnt and SHH pathway activation regulate distinct and sequential aspects of dermal condensate differentiation. In particular, lower levels of Wnt and SHH pathway activation promote proliferation of dermal condensate progenitor cells, resulting in an increased pool of cells capable of adopting a dermal condensate identity. In contrast, higher levels of Wnt and SHH pathway activation suppress further proliferation while inducing robust expression of dermal condensate-specific genes, thereby promoting functional maturation of the condensate.

[0136] These findings indicate that dermal condensate formation is governed by a two-step or graded signaling process, in which an initial proliferative phase expands dermal condensate progenitors, followed by a differentiation phase that establishes dermal condensate identity and signaling competence. The size and functional state of the resulting dermal condensates directly regulate hair follicle size and regenerative output, with larger and more mature dermal condensates inducing correspondingly larger and more robust hair follicles.

[0137] Additional details on the important of coordinating Wnt and SHH signaling for dermal condensate formation are found in U.S. Provisional Application No. 63 / 746,075, which is incorporated herein by reference in its entirety.

[0138] Accordingly, without a platform capable of systematically evaluating the effects of Wnt and SHH agonist dose, timing, and duration, it is not possible to rationally determine formulations or treatment regimens that reliably induce hair-inductive dermal condensates suitable for human therapeutic applications.

[0139] Therefore, compositions for facilitating dermal condensate formation and function are provided. The disclosed compositions are particularly suitable for increasing dermal condensate formation and promoting hair growth and regeneration. The compositions are configured to first promote expansion of dermal condensate progenitor cells through activation of Wnt signaling, followed by induction of dermal condensate differentiation through activation of SHH signaling. This staged modulation can be achieved by sequential administration of a Wnt agonist and a SHH agonist, respectively, or by use of a single formulation in which one or both agonists are present in controlled, delayed, or sustained-release form. In such forms, the relative dose, timing, and duration of Wnt and SHH pathway activation are selected to promote formation of dermal condensates of sufficient size and signaling competence to induce effective hair follicle growth and regeneration.

[0140] 19

[0141] 45816336.1A. WNT Agonists

[0142] The compositions contain one or more WNT agonists. The name Wnt is a portmanteau created from the names Wingless and Int-1. Wnt signaling plays an important role in regulating cell fate specification, proliferation, and migration during development and tissue regeneration. Wnt signaling drives differentiation of pluripotent stem cells into mesodermal and endodermal progenitors and supports subsequent specification into diverse lineages, including hematopoietic, endothelial, cardiac, vascular smooth muscle, neural, and germ cell lineages. Wnt signaling also regulates development of multiple tissues and organs, including the hair follicle, nervous system, gut, lung, kidney, ovary, and reproductive tissues.

[0143] In addition to directing differentiation, canonical Wnt signaling promotes cell proliferation by increasing cytoplasmic and nuclear [3-catenin, which activates transcription of cell-cycle regulators such as cyclin DI and c-Myc, thereby driving progression through the Gl / S phase transition and supporting coordinated growth of developing tissues.

[0144] Wnt signaling further regulates cell migration, a process essential for tissue patterning and morphogenesis, including convergent extension during gastrulation and later migratory events involving neural crest cells, neuroblasts, myocytes, and other cell types. Both canonical and non-canonical Wnt pathways contribute to these processes, and Wnt signaling also induces epithelial-mesenchymal transition (EMT), allowing epithelial cells to acquire migratory mesenchymal characteristics. Through these combined effects on differentiation, proliferation, and migration, Wnt signaling coordinates organized tissue development and regeneration across multiple biological systems.

[0145] Wnt signaling controls include body axis patterning, cell fate specification, cell proliferation and cell migration. These processes are important for proper formation of important tissues including bone, heart and muscle. Wnt signaling also controls tissue regeneration in adult bone marrow, skin and intestine.

[0146] Wnt includes a diverse family of secreted lipid-modified signaling glycoproteins that are 350-400 amino acids in length. The lipid modification of all Wnts is palmitoylation of a single totally conserved serine residue. Palmitoylation is important because it is required for Wnt to bind to its carrier protein Wntless (WLS) so it can be transported to the plasma membrane for secretion and it allows the Wnt protein to bind its receptor Frizzled Wnt proteins also undergo glycosylation, which attaches a carbohydrate in order to facilitate proper secretion. In Wnt signaling, these proteins act as ligands to activate the different Wnt pathways via paracrine and autocrine routes.

[0147] 20

[0148] 45816336.1Wnt proteins are highly conserved across species. Wnt signaling begins when a Wnt protein binds to the N-terminal extra-cellular cysteine-rich domain of a Frizzled (Fz) family receptor. These receptors span the plasma membrane seven times and constitute a distinct family of G-protein coupled receptors (GPCRs). However, to facilitate Wnt signaling, coreceptors may be required alongside the interaction between the Wnt protein and Fz receptor. Examples include lipoprotein receptor- related protein (LRP)-5 / 6, receptor tyrosine kinase (RTK), and ROR2. Upon activation of the receptor, a signal is sent to the phosphoprotein Dishevelled (Dsh), which is located in the cytoplasm. This signal is transmitted via a direct interaction between Fz and Dsh. Dsh proteins are present in all organisms and they all share the following highly conserved protein domains: an amino-terminal DIX domain, a central PDZ domain, and a carboxy-terminal DEP domain. These different domains are important because after Dsh, the Wnt signal can branch off into multiple pathways and each pathway interacts with a different combination of the three domains.

[0149] Three Wnt signaling pathways have been characterized: the canonical Wnt pathway, the non-canonical planar cell polarity pathway, and the non-canonical Wnt / calcium pathway. All three pathways are activated by the binding of a Wnt -protein ligand to a Frizzled family receptor, which passes the biological signal to the Dishevelled protein inside the cell. The canonical Wnt pathway involves the protein beta-catenin (P-catenin), leads to regulation of gene transcription, and is thought to be negatively regulated in part by the SPATS 1 gene. The non-canonical pathway operates independently of the protein beta-catenin (P-catenin), with the non-canonical planar cell polarity pathway regulating the cytoskeleton that is responsible for the shape of the cell, and the non-canonical Wnt / calcium pathway regulating calcium inside the cell.

[0150] Canonical Wnt Pathway

[0151] The canonical Wnt pathway (also known as the Wnt / p-catenin pathway) is characterized by an accumulation of P-catenin in the cytoplasm and its subsequent translocation to the nucleus, where it acts as a transcriptional coactivator with TCF / LEF((T-cell factor / lymphoid enhancer factor) transcription factors. Absent Wnt, P-catenin does not accumulate, as it is continuously degraded by a destruction complex composed of Axin, adenomatosis polyposis coli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase la (CKla). This complex targets P-catenin for phosphorylation, ubiquitination, and subsequent proteasomal degradation.

[0152] 21

[0153] 45816336.1Upon Wnt ligand binding to Frizzled (Fz) receptors and the co-receptors LRP5 / 6,, the destruction complex function becomes disrupted. Axin and associated components are recruited to the plasma membrane, where phosphorylation events promote Axin binding to the cytoplasmic tail of LRP5 / 6, followed by Axing de-phosphorylation and destabilization. Concurrently, Dishevelled (Dsh) is activated, and through its DIX and PDZ domains inhibits GSK3 activity, thereby preventing P-catenin degradation. Stabilized β-catenin accumulates, translocates to the nucleus, and activates gene transcription together with the TCF / LEF transcription factors.

[0154] Within the nucleus, P-catenin recruits other transcriptional coactivators, such as BCL9, Pygopus and Parafibromin / Hyrax. The P-catenin-containing transcriptional complex appears to be context dependent, with evidence from proteomics studies indicating tissuespecific interacting partners that influence target gene selection.p-catenin may be directly phosphorylated at Ser552 by Akt, promoting disassociation from adherens junctions and cytoplasmic accumulation, followed by interaction with 14-3-3ζ, which enhances its nuclear translocation. BCL9 and Pygopus have also been reported, to possess P-catenin-independent functions, indicating that their activities are not exclusively Wnt dependent.

[0155] The non-canonical planar cell polarity (PCP) pathway operates independently of P-catenin and does not utilize LRP5 / 6 as a co-receptor. Instead, it is thought to signal through alternative co-receptors such as NRH1, Ryk, PTK7, or R0R2. Binding of Wnt to Fz and its co-receptor leads to recruitment of Dsh, which forms a complex with Dishevelled-associated activator of morphogenesis 1 (DAAM1) via its PDZ and DIX domains. DAAM1 activates the small GTPase Rho, which in turn activates Rho-associated kinase (ROCK), a key regulator of cytoskeletal organization. In parallel, Dsh interacts with Rac1, promoting profilin- mediated actin binding. Rac1 activation also stimulates JNK signaling and actin polymerization, resulting in cytoskeletal reorganization important for processes such as cell polarity, migration, and gastrulation.

[0156] Non-canonical Wnt / calcium pathway

[0157] The non-canonical Wnt / calcium pathway also functions independently of P-catenin and regulates intracellular calcium signaling. Upon Wnt binding, activated Fz receptors interact with Dsh via its PDZ and DEP domains and directly couple to trimeric G-proteins, a feature that distinguishes this pathway from other Wnt cascades. Co-activation of Dsh and G-proteins can stimulate either phospholipase C (PLC) or cGMP-specific phosphodiesterase (PDE). Activation of PLC results in cleavage of PIP2 into DAG and IP3, with IP3 triggering calcium release from the endoplasmic reticulum. Elevated calcium and DAG activate protein

[0158] 22

[0159] 45816336.1kinase C (PKC) and Cdc42, which plays a role in ventral patterning. Increased calcium also activates calcineurin and CaMKII, leading to NFAT transcription factor activation, which regulates cell adhesion, migration, and tissue separation. Calcineurin additionally activates TAK1 and NLK, which can antagonize canonical TCF / -catenin signaling. In contrast, activation of PDE inhibits PKG, thereby suppressing calcium release from the ER..

[0160] Integrated Wnt Pathway

[0161] An integrated Wnt signaling model has been proposed, challenging the strict binary distinction between canonical and non-canonical pathways. Evidence from mammalian cell systems indicates that certain Wnt ligands, including Wnt5A, can simultaneously activate Wnt / p-catenin and Wnt / Ca2+signaling, suggesting convergent and context-dependent pathway integration..

[0162] Wnt signaling is tightly regulated at multiple levels to ensure proper biological function. Wnt ligands undergo palmitoylation by the enzyme porcupine, a modification required for secretion. Wnt secretion is further regulated by proteins such as GPR177 (Wntless), Evenness Interrupted, and the retromer complex.

[0163] Following secretion, ligand diffusion and receptor access can be modulated by extracellular matrix components, including heparan sulfate proteoglycans such as Dally and glypican-3 (GPC3). GPC3 regulates Wnt signaling through both its heparan sulfate chains and core protein, with specific sulfation patterns enhancing Wnt binding. A cysteine-rich domain within GPC3 forms a hydrophobic groove that directly interacts with Wnt, and blocking this interaction (e.g., with the HN3 nanobody) inhibits Wnt activation. At the Fz receptor, Wnt signaling is antagonized by multiple secreted inhibitors, including Dickkopf (Dkk), Wnt inhibitory factor- 1 (WIF-1), secreted Frizzled-related proteins (sFRPs), Cerberus, Frzb, Wise, SOST, and Naked cuticle.. Conversely, non-Wnt ligands such as Norrin and R-spondins can activate Wnt signaling in a Wnt-independent manner.

[0164] Crosstalk with other signaling pathways further modulates Wnt activity. For example, Wnt / calcium signaling can suppress canonical P-catenin-TCF signaling, while prostaglandin E2 (PGE2) enhances canonical Wnt signaling by stabilizing P-catenin via cAMP / PKA-mediated phosphorylation. PGE2 synthesis has been shown to be essential for Wnt-dependent tissue regeneration and stem cell regulation in multiple vertebrate models. Emerging evidence also implicates intrinsically disordered protein regions in fine-tuning Wnt signaling dynamics.

[0165] 23

[0166] 45816336.11. Target ligands

[0167] To identify compounds that can effectively increase Wnt pathway activity in the presence of an SHH agonist, an extensive screening effort of -50,000 compounds was undertaken to preliminarily identify -250 candidate compounds for in vitro evaluation. As demonstrated in non-limiting Example 2, these candidate compounds were selected based on binding interactions with multiple components of the Wnt signaling axis, including interactions with several Wnt ligands and associated receptor complexes, as described further below. The selected -250 compounds were subsequently tested in vitro using embryonic dermal cell-based assays to assess their ability to modulate Wnt pathway activity alone and in combination with SHH pathway activation. Compounds were prioritized based on functional readouts of Wnt pathway activation, including induction of canonical Wnt target genes (e.g., Lefl) and enhancement of ligand-dependent Wnt signaling responses, as well as their performance under combinatorial Wnt / SHH signaling conditions. From this in vitro evaluation, a subset of candidate compounds, provided below, was identified for further characterization based on their ability to potentiate Wnt ligand-dependent signaling and support downstream dermal condensate-associated gene expression (non-limiting Example 2).

[0168] In some forms, the Wnt agonists bind to one or more components of the Wnt receptor complex, such as Wnt ligands (such as Wnt3 and / or Wnt3a), the LRP5 / 6 co-receptor, Frizzled (FZD) receptors, Dishevelled 1 and / or CXXC-type zinc finger protein 5 such as DYL-CXXC5, or interfaces formed between these components.

[0169] a. Wnt 3 / Wnt3a-LRP6

[0170] In some forms, the Wnt agonist in the composition binds the proteins Wnt3 (e.g., as provided in UniProt ID: P56703), Wnt3a (e.g., as provided under UniProt ID: P56704), and / or LRP6 (Low-density lipoprotein receptor-related protein 6; as provided for example under UniProt ID: 075581), all of which are incorporated herein by reference in their entireties. In some forms, the Wnt agonist in the composition binds to a functional fragment having about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the proteins Wnt3 (e.g., as provided in UniProt ID: P56703), Wnt3a (e.g., as provided under UniProt ID: P56704), and / or LRP6 (Low-density lipoprotein receptor-related protein 6; as provided for example under UniProt ID: 075581).

[0171] In some forms, the interaction targeted for stabilization is the binding interaction between the Wnt3 and / or Wnt3a ligand and the LRP6 co-receptor (a single pass transmembrane protein), such that strengthening or prolonging this interaction promotes

[0172] 24

[0173] 45816336.1formation of an active Wnt receptor complex and enhances canonical Wnt / -catenin signaling (Figure 19). As described above, Wnt3 and Wnt3a function as canonical Wnt signaling ligands that bind to Frizzled (FZD) receptors together with the LRP5 / 6 co-receptor, triggering conformational changes that promote FZD-LRP6 oligomerization. LRP6 is a single-pass transmembrane protein that is indispensable for canonical Wnt signal transduction. Ligand-induced oligomerization of FZD and LRP6 results in recruitment of Dishevelled (DVL) (described below) to the plasma membrane and phosphorylation of the cytoplasmic tail of LRP5 / 6. Phosphorylated LRP5 / 6 binds Axin, thereby removing Axin from the P-catenin destruction complex (Axin. APC, GSK3P, CK1), which leads to disassembly of the destruction complex, stabilization and cytoplasmic accumulation of P-catenin, and subsequent nuclear translocation. Nuclear P-catenin activates transcription of canonical Wnt / p-catenin target genes that regulate cell proliferation, differentiation, and tissue morphogenesis. The structure of LRP6-Wnt3 / 3A ligand complex was obtained by overlaying the crystal structure of LRP6- Wnt8 complex PDB ID- 8CTG with PDB ID-6AHY (WNT3) and PDB ID-7DRT (WNT3A) using PymoL. The overlaid LRP6-Wnt3 and LRP6-Wnt3a complex structures were used to generate 4D grid for structure-based screening in ICM tool. Structure-based screening was performed on 50000 diverse compounds to identify candidate molecules that interact with both LRP6 and Wnt3 / 3 A ligand. The overlaid model of LRP6 with Wnt 3 and 3A ligands on crystal structure of LRP6 -Wnt8 ligand complex (PDB ID: 8CTG) as a template is shown in Figure 20A. Two different models, LRP6 -Wnt3 ligand and LRP6-Wnt3A ligand, were built for structure-based screening. Binding pockets in both the models were explored using ICM pocket finder (Figure 20B).

[0174] Stabilization of the Wnt3a-LRP6 interaction enhances productive FZD-LRP6 complex formation and is important for achieving Wnt signaling levels sufficient to drive hair follicle growth and regeneration in hair follicle- associated epithelial and mesenchymal cells.

[0175] Therefore, in some forms, the Wnt agonist functions as a positive modulator of canonical Wnt signaling by binding to Wnt ligands (e.g., Wnt3 and / or Wnt3a), the LRP6 coreceptor, FZD receptors, or interaction interfaces formed between these components. Through such binding, the Wnt agonist stabilizes ligand-receptor and / or receptor-receptor interactions, increases the affinity or residence time of the Wnt receptor complex, and promotes efficient FZD-LRP6 oligomerization. This enhanced receptor complex formation facilitates downstream signaling events, including DVL recruitment, LRP5 / 6 phosphorylation, P-catenin stabilization, nuclear translocation, and activation of Wnt-responsive gene expression,

[0176] 25

[0177] 45816336.1thereby promoting hair follicle regeneration, dermal condensate formation, and sustained hair growth.

[0178] b. DVL-CXXC5

[0179] In some forms, the Wnt agonist in the composition binds the proteins Dishevelled 1 (DVL; e.g., as provided under UniProt ID: 014640) and / or CXXC-type zinc finger protein 5 (CXXC5; e.g., as provided under UniProt ID: Q7LFL8), all of which are incorporated herein by reference in their entireties. In some forms, the Wnt agonist in the composition binds to a functional fragment having about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the proteins DVL (e.g., as provided under UniProt ID: 014640) and / or CXXC5 (e.g., as provided under UniProt ID: Q7LFL8).

[0180] In some forms, the interaction targeted for modulation is the binding interaction between DVL and CXXC5, such that attenuating, disrupting, or preventing this interaction relieves negative regulation of canonical Wnt / p-catenin signaling. As described above, DVL functions as a central cytoplasmic transducer of Wnt signaling by binding to the cytoplasmic terminus of FZD receptors, primarily through its PDZ domain, and propagating Wnt signals to downstream effectors involved in cell proliferation and differentiation (Figure 21). CXXC5 acts as a negative regulator of canonical Wnt / -catenin signaling by binding to DVL and inhibiting its ability to disrupt the P-catenin destruction complex, thereby suppressing pathway activation.

[0181] Upon Wnt ligand-induced oligomerization of FZD and LRP6, DVL is recruited to the plasma membrane to mediate downstream signaling. Binding of the C-terminal region of CXXC5 to the PDZ domain of DVL interferes with DVL function, preventing effective inhibition of the P-catenin destruction complex and maintaining repression of Wnt signaling. Disruption or attenuation of the DVL-CXXC5 interaction permits DVL to more effectively inhibit the P-catenin destruction complex, leading to stabilization and cytoplasmic accumulation of P-catenin, followed by nuclear translocation and activation of canonical Wnt / p-catenin target genes that regulate cell proliferation, differentiation, and tissue morphogenesis.

[0182] The DVL-CXXC5 interaction functions as a negative feedback mechanism that limits excessive Wnt pathway activation and has been implicated in androgen-related hair loss mechanisms. Modulation of this interaction is therefore important for achieving Wnt signaling levels sufficient to support hair follicle growth, dermal condensate formation, and regeneration in hair follicle-associated epithelial and mesenchymal cells.

[0183] 26

[0184] 45816336.1Accordingly, in some forms, the Wnt agonist functions as a positive modulator of canonical Wnt signaling by binding to DVL, CXXC5, or interaction interfaces formed between these proteins, thereby disrupting or attenuating the DVL-CXXC5 interaction. Through such binding, the Wnt agonist relieves negative feedback on DVL-mediated signal transduction, promotes disassembly of the P-catenin destruction complex, enhances -catenin stabilization and nuclear translocation, and increases activation of Wnt-responsive gene expression. In this manner, modulation of the DVL-CXXC5 interaction provides an additional mechanism for potentiating canonical Wnt signaling and promoting hair follicle regeneration, dermal condensate induction, and sustained hair growth.

[0185] 2. Exemplary Wnt Agonists

[0186] Two exemplary compounds that can be used as the WNT agonist include the compounds of Formula (I) and Formula (II) described below.

[0187] In one exemplary form, the WNT agonist is a compound of Formula (I):

[0188]

[0189] Formula (I)

[0190] wherein L is absent or is a linker;

[0191] wherein R2 includes a phenyl, aryl, or heteroaryl group optionally substituted with one or more substituents independently selected from C1-6 alkyl group and C1-6 alkoxy group; and

[0192] wherein Ri includes a fused bicyclic ring system containing 8 to 14 ring atoms, wherein the fused bicyclic ring system includes at least one ring that is a 5- or 6-membered ring containing at least one nitrogen atom;

[0193] or is a pharmaceutically acceptable salt thereof.

[0194] In some forms, the linker Lis present and is an ether linker (-O-).

[0195] In some forms, R2 is a phenyl group substituted with one or more substituents independently selected from C 1-6 alkyl group and Ci-6 alkoxy group [

[0196] In some forms, the fused bicyclic ring system includes at least one ring that is a 5- or 6-membered ring containing at least two nitrogen atoms.

[0197] In some forms, Ri has the following structure:

[0198] 27

[0199] 45816336.1

[0200]

[0201] In some forms, L is present and is an ether linker (-O-) and R2 has the following structure:

[0202]

[0203] An exemplary compound of Formula (I) has the following structure:

[0204]

[0205] In some forms,

[0206]

[0207] is expressly excluded from Formula I.

[0208] In another exemplary form, the WNT agonist is a compound of Formula (II):

[0209] o

[0210] N N

[0211]

[0212] H H

[0213] Formula (II)

[0214] 28

[0215] 45816336.1wherein Rf includes a pyridine ring substituted with -C(O)NR3R4;

[0216] wherein R2’ includes a piperidine ring optionally substituted at the nitrogen atom with Rs’;

[0217] wherein R3and R4are independently selected from hydrogen and C1-6 alky l group; wherein Rs. when present, is a C1-6 alkyl group [;

[0218] or is a pharmaceutically acceptable salt thereof.

[0219] In some forms, R3and R4are both methyl groups. In some forms, Rs’ is present and is a tert-butyl group.

[0220] In some forms, Ri’ has the following structure:

[0221]

[0222] In some forms, R2’ has the following structure:

[0223]

[0224] An exemplary compound of Formula (II) has the following structure:

[0225]

[0226] In some forms,

[0227] 29

[0228] 45816336.1

[0229]

[0230] is expressly excluded from Formula II.

[0231] The one or more Wnt agonists are present in the composition at a concentration of about 0.001 nM to about 200 mM, about 0.01 nM to about 150 mM, about 0.1 nM to about 100 mM, about 0.5 nM to about 100 mM, about 1 nM to about 100 mM, about 5 nM to about 100 mM, about 10 nM to about 100 mM, about 25 nM to about 75 mM, about 50 nM to about 75 mM, about 100 nM to about 50 mM, about 250 nM to about 50 mM, about 500 nM to about 25 mM, about 1 pM to about 100 mM, about 2.5 pM to about 100 mM, about 5 pM to about 100 mM, about 10 pM to about 100 mM, about 15 pM to about 100 mM. about 25 pM to about 100 mM, about 50 pM to about 100 mM, about 75 pM to about 100 mM, or about 100 pM to about 100 mM.

[0232] In some forms, the Wnt agonist concentration ranges from about 1 pM to about 10 pM, about 2.5 pM to about 25 pM, about 5 pM to about 50 pM, about 10 pM to about 100 pM, about 15 pM to about 150 pM, about 25 pM to about 250 pM. about 50 pM to about 500 pM, about 75 pM to about 750 pM, about 100 pM to about 1 mM, about 250 pM to about 2.5 mM, about 500 pM to about 5 mM, about 1 mM to about 10 mM, about 2.5 mM to about 25 mM, about 5 mM to about 50 mM, about 10 mM to about 100 mM, about 25 mM to about 100 mM, or about 50 mM to about 100 mM.

[0233] In some forms, the one or more Wnt agonists are present in the formulation at a concentration of about 0.000001 wt% to about 10 wt%, about 0.000005 wt% to about 7.5 wt%, about 0.00001 wt% to about 5 wt%, about 0.00005 wt% to about 3 wt%, about 0.0001 wt% to about 2 wt%, about 0.00025 wt% to about 2 wt%, about 0.0005 wt% to about 1.5 wt%, about 0.001 wt% to about 1 wt%, about 0.0025 wt% to about 1 wt%, about 0.005 wt% to about 1 wt%, about 0.01 wt% to about 1 wt%, about 0.025 wt% to about 0.75 wt%, about 0.05 wt% to about 0.5 wt%, about 0.075 wt% to about 0.5 wt%, or about 0.1 wt% to about 0.5 wt%, based on the total weight of the formulation.

[0234] 30

[0235] 45816336.1In some forms, the Wnt agonist is present at about 0.001 wt% to about 5 wt%, about 0.0025 wt% to about 4 wt%, about 0.005 wt% to about 3 wt%, about 0.01 wt% to about 2 wt%, or about 0.05 wt% to about 1 wt%, including formulations corresponding to about 50 μM at the lower end (depending on molecular weight and vehicle density) and up to about 100 mM at the upper end (depending on solubility and vehicle capacity). For example, in rinse-off forms such as shampoos or cleansers, the Wnt agonist can be present at higher nominal concentrations, for example about 0.001 wt% to about 10 wt%, about 0.01 wt.% to about 7 wt%, or about 0.05 wt% to about 5 wt%, to compensate for shorter contact times. In another example for leave-on forms such as creams, lotions, serums, gels, or foams, the Wnt agonist can be typically present at lower concentrations, for example about 0.0001 wt% to about 6 wt%, such as about 0.0005 wt% to about 1 wt%, about 0.001 wt% to about 0.5 wt%, about 0.1 wt% to about 5 wt%, about 0.1 wt% to about 4 wt%, about 0.1 wt% to about 3 wt%, about 0.1 wt% to about 2 wt%, about 0.1 wt% to about 1 wt%, or about 0.1 wt% to about 0.5 wt%.

[0236] B. SHH Agonists

[0237] The compositions further include one or more Sonic Hedgehog (SHH) pathway agonists. As used herein, an “SHH agonist” refers to any agent capable of activating, potentiating, or enhancing Hedgehog signaling in a target cell, including activation downstream of the SHH ligand, such as through Smoothened (SMO) activation, modulation of GLI transcriptional activity, or relief of Hedgehog pathway repression.

[0238] Sonic Hedgehog Protein, also referred to as SHH, HHG1, HLP3, HPE3, MCOPCB5, SMMCI, TPT, TPTPS, sonic hedgehog, Sonic hedgehog, SHHNC, and sonic hedgehog signaling molecule. This signaling molecule is important in regulating embryonic morphogenesis in all animals. SHH controls organogenesis and the organization of the central nervous system, limbs, digits and many other parts of the body. Sonic hedgehog is a morphogen that patterns the developing embryo using a concentration gradient model has a non-uniform distribution of SHH molecules which governs different cell fates according to concentration. Sonic hedgehog still plays a role in differentiation, proliferation, and maintenance of adult tissues. Abnormal activation of SHH signaling in adult tissues has been implicated in various types of cancers including breast, skin, brain, liver, gallbladder and many more. As demonstrated in the non-limiting examples, administration of an SHH agonist facilities increased demial progenitor cell differentiation, dermal condensate maturation, and acquisition of hair follicle-inductive properties, when administered in combination with Wnt pathway modulators and / or following a Wnt-priming step. In some forms, the SHH agonists

[0239] 31

[0240] 45816336.1facilitate the transition of dermal progenitor cells from a proliferative state to a differentiated, quiescent dermal condensate state.

[0241] In some forms, the SHH agonist is a Smoothened (SMO) agonist that activates Hedgehog signaling by directly or indirectly stimulating SMO activity. Non-limiting examples of such SMO agonists include SAG (Smoothened Agonist), SAG21k, purmorphamine, Hh-Ag1.5, and sterol-based Hedgehog pathway activators, such as but not limited to oxysterols and sterol derivatives such as 20a-hydroxycholesterol, 22(S)-hydroxycholesterol, and 7-dehydrocholesterol derivatives, as well as synthetic SMO-activating small molecules and functional analogs thereof. One exemplary SAG agonist is SAG dihydrochloride. In some forms, SMO agonists are used at concentrations sufficient to induce expression of Hedgehog target genes, such as Ptch1, and to promote dermal condensate differentiation when dermal progenitor cells have been rendered competent by prior or concurrent Wnt pathway activation.

[0242] In some forms, the SHH agonist includes a native SHH ligand, a recombinant SHH protein, or a biologically active fragment, variant, or derivative thereof capable of activating Hedgehog signaling. Such ligands can include recombinant Sonic Hedgehog (SHH-N) protein, truncated or modified SHH peptides retaining Hedgehog activity, or fusion proteins comprising SHH or SHH-derived domains. These ligands can be provided in soluble form, immobilized on a substrate, incorporated into sustained- or controlled-release matrices, or added directly in cell culture media.

[0243] In some forms, the SHH agonist acts downstream of SMO by enhancing GLI transcriptional activity or by reducing GLI-mediated repression. Such agents can include small molecules that inhibit formation or activity of GLI repressors, stabilize GLI activator forms, or promote GLI nuclear localization or transcriptional activity. These downstream Hedgehog pathway agonists can be used alone or in combination with SMO agonists to modulate the magnitude, timing, or duration of Hedgehog pathway output.

[0244] In some forms, SHH agonists include agents that indirectly enhance Hedgehog signaling by modulating pathway regulators or cellular components required for pathway function. Such agents include but are not limited to inhibitors of Patched-mediated repression of SMO, modulators of primary cilium formation or function, or compounds that enhance intracellular signal propagation downstream of SMO. These agents can increase cellular responsiveness to SHH signaling without directly binding to canonical Hedgehog pathway components.

[0245] 32

[0246] 45816336.1In preferred forms, SHH agonists are used in combination with one or more Wnt pathway modulators, including Wnt ligands, Wnt agonists, or small-molecule modulators of Wnt signaling. In some forms, dermal progenitor cells are first exposed to Wnt pathway activation to induce a proliferative and competent state, followed by exposure to an SHH agonist to promote differentiation, cell cycle exit, and dermal condensate maturation. The timing, order, duration, and relative concentrations of SHH agonist administration can be varied to regulate the rate of dermal condensate formation, the size and cellular composition of dermal condensates, and the strength and stability of hair follicle-inductive signaling.

[0247] SHH agonists can be formulated in any pharmaceutically acceptable form suitable for the intended application, including topical, intradermal, or subcutaneous administration, and may be provided as solutions, suspensions, emulsions, gels, hydrogels, ointments, or controlled-release formulations. For example, the SHH agonists are formulated for localized delivery to the skin or scalp. For in vitro or ex vivo applications, SHH agonists can be included in culture media for dermal progenitor cells to induce or enhance dermal condensate formation, including for use in compound screening or testing assays.

[0248] The one or more SHH agonists are present in the composition at a concentration of about 0.001 nM to about 200 mM, about 0.01 nM to about 150 mM, about 0.1 nM to about 100 mM, about 0.5 nM to about 100 mM, about 1 nM to about 100 mM, about 5 nM to about 100 mM, about 10 nM to about 100 mM, about 25 nM to about 75 mM, about 50 nM to about 75 mM, about 75 nM to about 50 mM, about 100 nM to about 50 mM, about 250 nM to about 50 mM, about 500 nM to about 25 mM, about 1 μM to about 100 mM, about 2.5 μM to about 100 mM, about 5 μM to about 100 mM, about 10 μM to about 100 mM, about 15 μM to about 100 mM, about 25 μM to about 100 mM, about 50 μM to about 100 mM, about 75 μM to about 100 mM, or about 100 μM to about 100 mM.

[0249] In some forms, the SHH agonist concentration ranges from about 1 nM to about 100 nM, about 5 nM to about 500 nM, about 10 nM to about 1 μM, about 25 nM to about 2.5 μM, about 50 nM to about 5 μM, about 75 nM to about 7.5 μM, about 100 nM to about 10 μM, about 250 nM to about 25 μM, about 500 nM to about 50 μM, about 1 μM to about 100 μM, about 2.5 μM to about 250 μM, about 5 μM to about 500 μM, about 10 μM to about 1 mM, about 25 μM to about 2.5 mM, about 50 μM to about 5 mM, about 100 μM to about 10 mM, about 250 μM to about 25 mM, about 500 μM to about 50 mM, about 1 mM to about 10 mM, about 2.5 mM to about 25 mM, about 5 mM to about 50 mM, about 10 mM to about 100 mM, about 25 mM to about 100 mM, or about 50 mM to about 100 mM.

[0250] 33

[0251] 45816336.1In some forms, the one or more SHH agonists are present in the formulation at a concentration of about 0.0000001 wt% to about 10 wt%, about 0.0000005 wt% to about 7.5 wt%, about 0.000001 wt% to about 5 wt%, about 0.000005 wt% to about 3 wt%, about 0.00001 wt% to about 2 wt%, about 0.000025 wt% to about 2 wt%, about 0.00005 wt% to about 1.5 wt%, about 0.0001 wt% to about 1 wt%, about 0.00025 wt% to about 1 wt%, about 0.0005 wt% to about 1 wt%, about 0.001 wt% to about 1 wt%, about 0.0025 wt% to about 0.75 wt%, about 0.005 wt.% to about 0.5 wt.%, about 0.01 wt.% to about 0.5 wt.%, or about 0.05 wt% to about 0.5 wt%, based on the total weight of the formulation.

[0252] In some forms, the SHH agonist is present at about 0.0001 wt% to about 5 wt%, about 0.00025 wt% to about 4 wt%, about 0.0005 wt% to about 3 wt%, about 0.001 wt% to about 2 wt%, or about 0.005 wt% to about 1 wt%, including formulations corresponding to about 50 nM at the lower end (depending on molecular weight and vehicle density) and up to about 100 mM at the upper end (depending on solubility and vehicle capacity). For example, in rinse-off forms such as shampoos or cleansers, the SHH agonist can be present at higher nominal concentrations, for example about 0.0001 wt% to about 10 wt%, about 0.001 wt% to about 7 wt%, or about 0.005 wt% to about 5 wt%, to compensate for shorter contact times. In another example, for leave-on forms such as creams, lotions, serums, gels, or foams, the SHH agonist can be present at lower concentrations, for example about 0.00001 wt% to about 6 wt%, such as about 0.00005 wt% to about 1 wt%, about 0.0001 wt% to about 0.5 wt%, about 0.001 wt% to about 5 wt%, about 0.001 wt% to about 4 wt%, about 0.001 wt% to about 3 wt%, about 0.001 wt% to about 2 wt%, about 0.001 wt% to about 1 wt%, or about 0.001 wt% to about 0.5 wt%.

[0253] C. Additional Active Agents

[0254] The compositions can further include one or more additional active agents suitable for promoting hair re-growth, regeneration, reducing hair loss, and / or promoting general health of hair. Such additional active agents can include, without limitation, agents that stimulate hair follicle development or cycling, enhance dermal or epidermal cell function, improve follicular microenvironment health, or protect hair follicles from inflammatory, hormonal, oxidative, or age-related damage. For example, the additional active agents include but are not limited to growth factors, cytokines, peptides, or peptide mimetics that influence hair follicle morphogenesis, dermal papilla signaling, or epithelial-mesenchymal interactions. In some forms, the additional active agents include small molecules that modulate biological pathways associated with hair growth, including but not limited to BMP signaling, FGF signaling, TGF-P signaling, prostaglandin signaling, or androgen metabolism. Exemplary

[0255] 34

[0256] 45816336.1growth factors include, without limitation, fibroblast growth factors (FGFs), including FGF1, FGF2, FGF7, and FGF10; insulin-like growth factors (IGFs), including IGF-1 and IGF-2; vascular endothelial growth factor (VEGF); platelet-derived growth factors (PDGFs); epidermal growth factor (EGF); hepatocyte growth factor (HGF); transforming growth factor family members, including TGF-P modulators; and biologically active fragments, variants, or functional analogs thereof. Such growth factors may promote hair follicle development, anagen initiation or maintenance, dermal papilla function, vascularization, or follicular cell survival. Exemplary cytokines include, without limitation, interleukins and interleukin modulators (e.g., IL-6, IL-10, IL-11, IL-15), tumor necrosis factor (TNF) pathway modulators, interferons, colony- stimulating factors, and chemokines or chemokine modulators, including agents that reduce inflammatory signaling within the scalp microenvironment. In certain embodiments, cytokines or cytokine-modulating agents are selected to reduce follicular inflammation, immune-mediated hair follicle damage, or stress-induced follicle regression, thereby supporting hair growth and follicle maintenance.

[0257] Exemplary peptides or peptide mimetics include, without limitation, signaling peptides, biomimetic peptides, extracellular matrix-derived peptides, and peptides that modulate cell adhesion, migration, or survival. Such peptides include but are not limited to copper-binding peptides, collagen-derived peptides, laminin-derived peptides, fibronectin-derived peptides, keratin-associated peptides, Wnt-modulating peptides, or peptides that stimulate dermal papilla activity or hair follicle stem cell function. Peptide mimetics may include modified peptides, peptidomimetics, cyclic peptides, or stabilized peptide analogs to enhance biological activity, stability, or tissue penetration while retaining hair growth-promoting or follicle-supportive functions.

[0258] In some forms, the compositions further include agents that promote cell survival, cellular metabolism, or tissue regeneration within the scalp, including agents that enhance mitochondrial function, angiogenesis, extracellular matrix remodeling, or stem / progenitor cell maintenance. Exemplary agents include, without limitation, nicotinamide adenine dinucleotide (NAD+) precursors such as nicotinamide, nicotinamide riboside, or nicotinamide mononucleotide; coenzyme Q10; L-carnitine and acetyl-L-camitine; creatine; resveratrol and resveratrol derivatives; polyphenols; adenosine; hypoxia-mimetic agents; nitric oxide donors; and angiogenic agents such as VEGF-modulating compounds. Additional agents include extracellular matrix-modulating agents such as hyaluronic acid, glycosaminoglycans, matrix metalloproteinase modulators, or agents that promote collagen synthesis or remodeling, as

[0259] 35

[0260] 45816336.1well as compounds that support stem or progenitor cell maintenance, including lithium salts, valproic acid, or other agents known to influence progenitor cell signaling pathways.

[0261] Additional active agents also include but are not limited to anti-inflammatory agents, immunomodulatory agents, antioxidants, or agents that reduce oxidative stress, fibrosis, or microinflammation in the scalp, thereby creating a favorable environment for hair follicle regeneration and maintenance. Exemplary agents include, without limitation, corticosteroids or non-steroidal anti-inflammatory agents; salicylates; curcumin and curcumin derivatives; flavonoids; catechins; tocopherols; ascorbic acid and derivatives thereof; glutathione; N-acetylcysteine; selenium compounds; zinc compounds; botanical anti-inflammatory agents such as green tea extracts, ginseng extracts, or chamomile extracts; and agents that modulate prostaglandin signaling or inflammatory cytokine activity.

[0262] In some forms, the compositions include agents that improve hair shaft quality, hair fiber strength, or hair anchoring, or that protect existing hair follicles from miniaturization or premature cycling. Exemplary agents include, without limitation, biotin and biotin derivatives; niacinamide; panthenol; amino acids and amino acid derivatives such as cysteine, methionine, arginine, and glycine; keratin-derived peptides; silica compounds; minerals such as zinc, copper, or iron; prostaglandin analogs or modulators; anti-androgenic agents such as finasteride-like or botanical 5a-reductase inhibitors; and agents that stabilize the hair follicle extracellular matrix or follicular attachment structures. Such agents can support hair fiber integrity, anchoring of the hair shaft within the follicle, or maintenance of healthy hair cycling when used alone or in combination with the dermal condensate-stimulating compounds described herein.

[0263] In some forms, the additional active agents can include vitamins, vitamin derivatives, minerals, trace elements, botanical extracts, or bioactive natural compounds known to support hair and scalp health, provided that such agents are compatible with the compositions described herein. The additional active agents can be selected to act synergistically or additively with the compounds disclosed herein, including by promoting dermal condensate formation, supporting dermal progenitor cell function, prolonging hair follicle inductive signaling, or improving the durability of hair growth responses. The selection and inclusion of such additional active agents can be tailored depending on the desired therapeutic or cosmetic outcome, including hair re-growth, hair thickening, slowing or preventing hair loss, or maintaining overall hair and scalp health.

[0264] 36

[0265] 45816336.1D. Formulations

[0266] The disclosed compounds and compositions thereof, can be formulated in a pharmaceutical or cosmetic composition. In some forms, the pharmaceutical composition includes one or more Wnt pathway agonists and one or more Shh pathway agonists formulated together in a single composition. In such forms, the Wnt agonist and the Shh agonist can be co-formulated in a common carrier, excipient, or delivery vehicle, and be present in amounts sufficient to provide concurrent or overlapping activation of Wnt and Shh signaling in a target tissue (e.g., epidermis and / or dermis). Suitable concentrations of the one or more Wnt agonists and / or one or more SHH agonists are described above in Sections (II)(A)(2) and (II)(B), respectively.

[0267] In some forms, the one or more Wnt pathway agonists and the one or more Shh pathway agonists are formulated in separate pharmaceutical compositions. In such forms, the compositions can be administered sequentially, concurrently, or in a temporally staggered manner, and can be delivered via the same route of administration or via different routes of administration. The separate compositions can be formulated to provide distinct release profiles, dosing regimens, or tissue targeting characteristics, thereby allowing independent modulation of Wnt and Shh signaling.

[0268] In some forms, the pharmaceutical composition or compositions are formulated for oral administration, including but not limited to tablets, capsules, powders, granules, solutions, suspensions, or syrups. In such forms, the Wnt pathway agonist and the SHH pathway agonist can be co-formulated in a single oral dosage form or provided in separate oral dosage forms for coordinated or sequential administration.

[0269] In other forms, the pharmaceutical composition or compositions are formulated for topical administration, including but not limited to creams, lotions, gels, foams, ointments, serums, sprays, emulsions, patches, or transdermal delivery systems. In such forms, the Wnt pathway agonist and the Shh pathway agonist can be co-formulated in a single topical composition or provided in separate topical compositions for sequential or layered application to a target tissue. For example, in one form suitable for the treatment or management of hair loss or hair thinning, a first topical composition (e.g., hair mask) containing one or more Wnt pathway agonists is applied to the scalp to promote activation of follicular stem or progenitor cells, followed by application of a second, separate topical composition comprising one or more Shh pathway agonists to stimulate hair follicle morphogenesis, dermal papilla signaling, or progression of hair follicles into or maintenance of an anagen growth phase. The sequential application can occur within a single treatment

[0270] 37

[0271] 45816336.1session or over multiple treatment sessions and can be configured to provide temporally distinct activation of Wnt and Shh signaling. In another example, a single topical composition suitable for use in hair loss or hair regeneration applications contains both one or more Wnt pathway agonists and one or more Shh pathway agonists co-formulated in a common carrier. In such forms, the composition can be applied to the scalp as a cream, gel, foam, or serum and can be formulated to provide simultaneous or overlapping activation of Wnt and Shh signaling pathways within hair follicles, the bulge region, and / or the dermal papilla.

[0272] Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be important or desirable.

[0273] Formulations are prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The “carrier” is all components present in the phamraceutical formulation other than the active ingredient or ingredients. The term “carrier” includes but is not limited to diluents, binders, lubricants, desintegrators, fillers, and coating compositions.

[0274] “Carrier” also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. The delayed release dosage formulations may be prepared as described in references such as “Pharmaceutical dosage form tablets”, eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington -The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6thEdition, Ansel et.al., (Media, PA: Williams and Wilkins, 1995) which provides information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

[0275] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0276] Additionally, the coating material can contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Stabilizers

[0277] 38

[0278] 45816336.1are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.

[0279] Surfactants can be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tri decyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0280] Extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in “Remington - The science and practice of pharmacy” (20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000). A diffusion system typically consists of two types of devices, reservoir and matrix, and is well known and described in the art. fhe three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and carbopol 934, polyethylene oxides. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate.

[0281] Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form.

[0282] Materials to produce different release mechanisms can be combined in a final dosage form comprising single or multiple units. An immediate release portion can be added to the

[0283] 39

[0284] 45816336.1extended release system by means of either applying an immediate release layer on top of the extended release core using coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads. Extended release forms containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In a congealing method, the drug is mixed with a wax material and either spray- congealed or congealed and screened and processed. By "pulsatile" is meant that a plurality of doses are released at spaced apart intervals of time.

[0285] In one form, pulsatile release profile is achieved with dosage forms that are closed and preferably sealed capsules housing at least two drug-containing "dosage units" wherein each dosage unit within the capsule provides a different drug release profile. Control of the delayed release dosage unit(s) is accomplished by a controlled release polymer coating on the dosage unit, or by incorporation of the active agent in a controlled release polymer matrix. Alternatively, each dosage unit in the capsule may comprise a plurality of drug -containing beads, granules or particles. As is known in the art, drug-containing "beads" refer to beads made with drug and one or more excipients or polymers. Drug-containing beads can be produced by applying drug to an inert support, e.g., inert sugar beads coated with drug or by creating a "core" comprising both drag and one or more excipients. As is also known, dragcontaining "granules" and "particles" comprise drug particles that may or may not include one or more additional excipients or polymers. In contrast to drug-containing beads, granules and particles do not contain an inert support. Granules generally contains drag particles and require further processing. Generally, particles are smaller than granules, and are not further processed. Although beads, granules and particles may be formulated to provide immediate release, beads and granules are generally employed to provide delayed release.

[0286] In a further alternative form, a dosage form is provided that contains an inner drug-containing core and at least one drug-containing layer surrounding the inner core. An outer layer of this dosage form contains an initial, immediate release dose of the drug.

[0287] Alternatively, for dosage forms mimicking three times daily dosing, the dosage form has an outer layer and an inner layer free of drag.

[0288] As will be appreciated by those skilled in the art and as described in the literature, a number of methods are available for preparing drug -containing tablets, beads, granules or particles that provide a variety of drug release profiles. Such methods include, but are not limited to, the following: coating a drug or drug -containing composition with an appropriate coating material, typically although not necessarily incorporating a polymeric material;

[0289] 40

[0290] 45816336.1increasing drug particle size; placing the drug within a matrix: and forming complexes of the drug with suitable complexing agents.

[0291] Particles can be prepared entirely from a therapeutic or diagnostic agent, or from a combination of the agent and a surfactant. The particles preferably are biodegradable and biocompatible, and optionally are capable of biodegrading at a controlled rate for delivery of a therapeutic or diagnostic agent. The particles can be made of a variety of materials. Both inorganic and organic materials can be used.

[0292] Polymeric particles may be formed from any biocompatible, and preferably biodegradable polymer, copolymer, or blend. The polymers may be tailored to optimize different characteristics of the particle including: i) interactions between the agent to be delivered and the polymer to provide stabilization of the agent and retention of activity upon delivery; ii) rate of polymer degradation and, thereby, rate of drug release profiles; iii) surface characteristics and targeting capabilities via chemical modification; and iv) particle porosity.

[0293] As used herein, the term "surfactant" refers to any agent which preferentially absorbs to an interface between two immiscible phases, such as the interface between water and an organic polymer solution, a water / air interface or organic solvent / air interface. Surfactants generally possess a hydrophilic moiety and a lipophilic moiety, such that, upon absorbing to microparticles, they tend to present moieties to the external environment that do not attract similarly-coated particles, thus reducing particle agglomeration. Surfactants may also promote absorption of a therapeutic or diagnostic agent and increase bioavailability of the agent. Surfactants known in the art can be used including any naturally occurring surfactant. Other exemplary surfactants include diphosphatidyl glycerol (DPPG); hexadecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid, such as palmitic acid or oleic acid; sorbitan trioleate (Span 85); glycocholate; surfactin; a poloxomer; a sorbitan lath acid ester such as sorbitan trioleate; tyloxapol and a phospholipid.

[0294] Rapidly bioerodible polymers such as poly[lactide-co-glycolide], polyanhydrides, and polyorthoesters, whose carboxylic groups are exposed on the external surface as their smooth surface erodes, are excellent candidates for drug delivery systems. In addition, polymers containing labile bonds, such as polyanhydrides and polyesters, are well known for their hydrolytic reactivity. Their hydrolytic degradation rates can generally be altered by simple changes in the polymer backbone. Representative natural polymers include proteins, such as zein, modified zein, casein, gelatin, gluten, serum albumin, or collagen, and polysaccharides,

[0295] 41

[0296] 45816336.1such as cellulose, dextrans, polyhyaluronic acid, polymers of acrylic and methacrylic esters and alginic acid.

[0297] Shampoos typically contain water or another clarifying ingredient, Fatty alcohols such as cetyl alcohol, lauryl alcohol, Cetearyl alcohol, and stearyl alcohol as moisturizers, and thickeners such as stearic acid, gelatin, xanthan gum, carnauba wax, and stearyl alcohol.

[0298] Gel formulations typically include a gelling agent such as a carbomer, hydroxyethyl cellulose, and hydroxypropyl cellulose and solubilizers.

[0299] These formulations can be used to administer an effective amount of the agents in vitro or in vivo to induce an individual’s cells to form new hair and / or hair follicles, or to active hair formation and / or growth.

[0300] 1. Topical and Transdermal Formulations

[0301] Topical and transdermal formulations may also be prepared. These will typically be gels, ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations can include penetration enhancers.

[0302] Compositions adapted for transdermal administration may be provided as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Compositions adapted for topical administration may be provided as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical administration to the skin, mouth, eye or other external tissues, topical ointments or creams are preferably used. When formulated in an ointment, the Wnt agonist, SHH agonist, or both, may be used in either a paraffinic or water-miscible ointment base. Alternatively, the Wnt agonist, SHH agonist, or both, may be formulated in a cream with an oil-in-water base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops. In these compositions, the Wnt agonist, SHH agonist, or both, may be dissolved or suspended in a suitable carrier, e.g., an aqueous solvent.

[0303] Formulations suitable for topical administration include liquid and / or semi-liquid preparations such as, for example, solutions, lotions, oils and I or water-in-oil emulsions such as creams, ointments and / or fats, and / or solutions and / or suspensions But is not limited thereto. The topically-administrable preparation may comprise, for example, from about 1% to about 25% (w / w) Wnt agonist, SHH agonist, or both, the concentration of which can be as high as the solubility limit of the Wnt agonist. SHH agonist, or both, in the solvent.

[0304] Formulations for topical administration may include one or more of the additional ingredients mentioned herein.

[0305] 42

[0306] 45816336.1The composition can further include one or more humectants to improve scalp hydration and hair fiber moisture retention by attracting water molecules and reducing dryness of the scalp and hair shaft. Exemplary humectants include, but are not limited to, hyaluronic acid, glycerin, sorbitol, and propanediol, which complement the one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof by further promoting hydration of the scalp microenvironment and supporting hair fiber flexibility.

[0307] In some forms, the composition includes one or more emollients to help soften and condition the hair and scalp by reinforcing the scalp lipid barrier, smoothing the hair cuticle, and reducing moisture loss. Exemplary emollients include, but are not limited to, shea butter, squalane, jojoba oil, and ceramides, which support scalp barrier function and enhance delivery and retention of the one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof within hair follicles and along the hair shaft.

[0308] In some forms, the compositions further include one or more antioxidants to protect the scalp and hair follicles from oxidative stress, thereby reducing inflammation, follicular stress, and premature hair cycling. Exemplary antioxidants include, but are not limited to, vitamin C, vitamin E (tocopherol), resveratrol, coenzyme Q10 (CoQ10), and green tea extract, which support a favorable environment for activity of the one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof.

[0309] In some forms, the compositions further include one or more peptides to support hair follicle function, dermal papilla signaling, and hair fiber strength. Exemplary peptides include, but are not limited to, palmitoyl tripeptide- 1, palmitoyl tetrapeptide-7, acetyl hexapeptide-8, and copper peptides, which enhance the bioactivity of the formulation comprising the one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof for hair follicle support and scalp health.

[0310] In some forms, the compositions further include one or more vitamins to provide additional benefits, including anti-inflammatory effects, improved scalp condition, and support of normal hair growth and cycling. Exemplary vitamins include, but are not limited to, niacinamide (vitamin B3), retinol (vitamin A), and panthenol (pro-vitamin B5), which further support the conditioning and follicle-supportive effects of the one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof.

[0311] In some forms, the compositions further include one or more botanical extracts to provide soothing, anti-inflammatory, and scalp-conditioning properties that support a healthy hair growth environment in the presence of the one or more Wnt pathway agonists, Shh pathway

[0312] 43

[0313] 45816336.1agonists, or combinations thereof. Exemplary botanical extracts include, but are not limited to, aloe vera, chamomile, licorice root extract, and Centella asiatica.

[0314] A “gel” is a colloid in which the dispersed phase has combined with the continuous phase to produce a semisolid material, such as jelly. A “gel” is a semisolid system containing dispersions of small or large molecules in a liquid vehicle that is rendered semisolid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle. The liquid may include a lipophilic component, an aqueous component or both. Some emulsions may be gels or otherwise include a gel component. Some gels, however, are not emulsions because they do not contain a homogenized blend of immiscible components.

[0315] Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose; Carbopol homopolymers and copolymers; and combinations thereof. Suitable solvents in the liquid vehicle include, but are not limited to, diglycol monoethyl ether; alklene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol. The solvents are typically selected for their ability to dissolve the drug. Other additives, which improve the skin feel and / or emolliency of the formulation, may also be incorporated. Examples of such additives include, but are not limited, isopropyl myristate, ethyl acetate, C12-C15 alkyl benzoates, mineral oil, squalane, cyclomethicone, capric / caprylic triglycerides, and combinations thereof.

[0316] An “oil” is a composition containing at least 95% wt of a lipophilic substance.

[0317] Examples of lipophilic substances include but are not limited to naturally occurring and synthetic oils, fats, fatty acids, lecithin, triglycerides and combinations thereof.

[0318] A “continuous phase” refers to the liquid in which solids are suspended or droplets of another liquid are dispersed, and is sometimes called the external phase. This also refers to the fluid phase of a colloid within which solid or fluid particles are distributed. If the continuous phase is water (or another hydrophilic solvent), water-soluble or hydrophilic drugs will dissolve in the continuous phase (as opposed to being dispersed). In a multiphase formulation (e.g., an emulsion), the discreet phase is suspended or dispersed in the continuous phase.

[0319] “Emollients” are an externally applied agent that softens or soothes skin and are generally known in the art and listed in compendia, such as the “Handbook of Pharmaceutical Excipients”, 4thEd., Pharmaceutical Press, 2003. These include, without limitation, almond oil, castor oil, ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin,

[0320] 44

[0321] 45816336.1glycerin monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium-chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof. In one embodiment, the emollients are ethylhexylstearate and ethylhexyl palmitate.

[0322] An “ointment” is a semisolid preparation containing an ointment base and optionally one or more active agents. Examples of suitable ointment bases include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); water-removable bases (e.g., hydrophilic ointment), and water-soluble bases (e.g., polyethylene glycol ointments). Pastes typically differ from ointments in that they contain a larger percentage of solids.

[0323] Pastes are typically more absorptive and less greasy that ointments prepared with the same components.

[0324] “Surfactants” are surface-active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product. Suitable non-ionic surfactants include emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof. In one embodiment, the non-ionic surfactant is stearyl alcohol.

[0325] An “emulsion” is a composition containing a mixture of non-miscible components homogenously blended together. In particular forms, the non-miscible components include a lipophilic component and an aqueous component. An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water-in-oil emulsion. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.

[0326] 45

[0327] 45816336.1“Emulsifiers” are surface active substances which promote the suspension of one liquid in another and promote the formation of a stable mixture, or emulsion, of oil and water. Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds. Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, selfemulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gum and combinations thereof. In one embodiment, the emulsifier is glycerol stearate.

[0328] In some forms, the compositions may be in the form of ointments, pastes, creams, lotions, gels, foams, powders, solutions, sprays, or patches, including formulations suitable for application to the scalp and hair-bearing skin. In some forms, formulations of the compositions can be creams, which may further contain saturated or unsaturated fatty acids such as stearic acid, palmitic acid, oleic acid, palmitoleic acid, cetyl alcohol, or oleyl alcohol, stearic acid being particularly preferred.

[0329] Foams contain an emulsion in combination with a gaseous propellant. The gaseous propellant consists primarily of hydrofluoroalkanes (HFAs). Suitable propellants include HFAs such as 1,1, 1,2- tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or may become approved for medical use are suitable. The propellants preferably are not hydrocarbon propellant gases which can produce flammable or explosive vapors during spraying. Furthermore, the compositions preferably contain no volatile alcohols, which can produce flammable or explosive vapors during use.

[0330] A “lotion” is a low- to medium-viscosity liquid formulation. A lotion can contain finely powdered substances that are in soluble in the dispersion medium through the use of suspending agents and dispersing agents. Alternatively, lotions can have as the dispersed phase liquid substances that are immiscible with the vehicle and are usually dispersed by means of emulsifying agents or other suitable stabilizers. In one embodiment, the lotion is in the form of an emulsion having a viscosity of between 100 and 1000 centistokes. The

[0331] 46

[0332] 45816336.1fluidity of lotions permits rapid and uniform application over a wide surface area. Lotions are typically intended to dry on the skin leaving a thin coat of their medicinal components on the skin’s surface.

[0333] A “cream" is a viscous liquid or semi-solid emulsion of either the “oil-in-water” or “water-in-oil type". Creams may contain emulsifying agents and / or other stabilizing agents. In one embodiment, the formulation is in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams are often time preferred over ointments as they are generally easier to spread and easier to remove.

[0334] Creams may also contain a non-ionic surfactant, for example polyoxy -40- stearate, to promote uniform dispersion and stability of the active agents. In some embodiments, the one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof are admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives. stabilizers, or buffers as may be required for topical administration to the scalp. In some embodiments, the compositions are formulated to facilitate delivery to hair follicles, including penetration into the follicular infundibulum, bulge region, or dermal papilla. In further embodiments, the compositions are formulated as leave-on or rinse-off hair and scalp products, including but not limited to serums, lotions, foams, or sprays, suitable for repeated application. Additionally, the use of transdermal or topical patches is contemplated, which may provide controlled or sustained delivery of the one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof to a localized region of the scalp. Such dosage forms can be made by dissolving or dispersing the one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof in an appropriate medium or matrix suitable for topical or transdermal application.

[0335] A sub-set of emulsions are the self-emulsifying systems. These drug delivery systems are typically capsules (hard shell or soft shell) composed of the drug dispersed or dissolved in a mixture of surfactant(s) and lipophilic liquids such as oils or other water immiscible liquids. When the capsule is exposed to an aqueous environment and the outer gelatin shell dissolves, contact between the aqueous medium and the capsule contents instantly generates very small emulsion droplets. These typically are in the size range of micelles or nanoparticles. No mixing force is required to generate the emulsion as is typically the case in emulsion formulation processes.

[0336] The basic difference between a cream and a lotion is the viscosity, which is dependent on the amount / use of various oils and the percentage of water used to prepare the formulations. Creams are typically thicker than lotions, may have various uses and often one

[0337] 47

[0338] 45816336.1uses more varied oils / butters, depending upon the desired effect upon the skin. In a cream formulation, the water-base percentage is about 60-75 % and the oil-base is about 20-30 % of the total, with the other percentages being the emulsifier agent, preservatives and additives for a total of 100 %.

[0339] Buffers are used to control pH of a composition. Preferably, the buffers buffer the composition from a pH of about 4 to a pH of about 7.5, more preferably from a pH of about 4 to a pH of about 7, and most preferably from a pH of about 5 to a pH of about 7. In a preferred embodiment, the buffer is triethanolamine.

[0340] Preservatives can be used to prevent the growth of fungi and microorganisms.

[0341] Suitable antifungal and antimicrobial agents include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal.

[0342] Additional agents that can be added to the formulation include penetration enhancers. In some embodiments, the penetration enhancer increases the solubility of the drug, improves transdermal delivery of the drug across the skin, in particular across the stratum corneum, or a combination thereof. Some penetration enhancers cause dermal irritation, dermal toxicity and dermal allergies. However, the more commonly used ones include urea, (carbonyldiamide), imidurea, N, N-diethylformamide, N-methyl-2-pyrrolidone, 1-dodecal-azacyclopheptane-2-one, calcium thioglycate, 2-pyrrolidone, N, N-diethyl-m- toluamide, oleic acid and its ester derivatives, such as methyl, ethyl, propyl, isopropyl, butyl, vinyl and glycerylmonooleate, sorbitan esters, such as sorbitan monolaurate and sorbitan monooleate, other fatty acid esters such as isopropyl laurate, isopropyl myristate, isopropyl palmitate, diisopropyl adipate, propylene glycol monolaurate, propylene glycol monooleatea and nonionic detergents such as Brij® 76 (stearyl poly(10 oxyethylene ether), Brij® 78 (stearyl poly(20)oxyethylene ether), Brij® 96 (oleyl poly(10)oxyethylene ether), and Brij® 721 (stearyl poly (21) oxyethylene ether) (ICI Americas Inc. Corp.). Chemical penetrations and methods of increasing transdermal drug delivery are described in Inayat, et al., Tropical Journal of Pharmaceutical Research, 8(2): 173- 179 (2009) and Fox, et al., Molecules, 16: 10507-10540 (2011). In some embodiments, the penetration enhancer is, or includes, an alcohol such ethanol, or others disclosed herein or known in the art.

[0343] Delivery of drugs by the transdermal route has been known for many years.

[0344] Advantages of a transdermal drug delivery compared to other types of medication delivery such as oral, intravenous, intramuscular, etc., include avoidance of hepatic first pass

[0345] 48

[0346] 45816336.1metabolism, ability to discontinue administration by removal of the system, the ability to control drug delivery for a longer time than the usual gastrointestinal transit of oral dosage form, and the ability to modify the properties of the biological barrier to absorption.

[0347] Controlled-release topical or transdermal devices rely for their effect on delivery of a known flux of one or more hair-active agents to the scalp for a prolonged period of time, generally a day, several days, or a week. Two mechanisms may be used to regulate the flux of the one or more active agents: either the active agent is contained within a drug reservoir, which is separated from the scalp of the wearer by a synthetic membrane through which the active agent diffuses; or the active agent is held dissolved or suspended in a polymer matrix, through which the active agent diffuses to the scalp and underlying hair follicles.

[0348] Devices incorporating a reservoir will deliver a steady flux of the active agent across the membrane as long as excess undissolved active agent remains in the reservoir. Matrix or monolithic devices are typically characterized by a falling flux with time, as the matrix layers closer to the scalp are depleted of the active agent. Usually, reservoir patches include a porous membrane covering the reservoir of active agent which can control release, while heat-melting thin layers of active agent embedded in the polymer matrix (e.g., within an adhesive layer) can control release of the active agent from matrix or monolithic devices. Accordingly, one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof can be released from a patch in a controlled fashion without necessarily being formulated as a controlled-release composition.

[0349] Patches can include a release liner which protects the patch during storage and is removed prior to use; one or more active agent layers comprising the one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof in direct contact with the release liner; an adhesive layer which serves to adhere the components of the patch together and to secure the patch to the scalp; one or more membranes, which can separate other layers and control release of the active agent from a reservoir or multilayer patch; and a backing layer which protects the patch from the external environment.

[0350] Common types of transdermal or topical patches suitable for application to the scalp include, but are not limited to, single-layer dmg-in-adhesive patches, wherein the adhesive layer contains the active agent and serves both to adhere the patch to the scalp and to release the active agent; multi-layer drug-in-adhesive patches, which include multiple layers, for example, a layer configured for immediate release of the active agent and another layer configured for controlled release; reservoir patches, wherein the active agent layer is a liquid or semi-liquid compartment containing a solution or suspension of the active agent separated

[0351] 49

[0352] 45816336.1by an adhesive layer; matrix patches, wherein the active agent is dispersed in a semisolid polymer matrix that is surrounded and partially overlaid by the adhesive layer; and vapor patches, wherein the adhesive layer serves both to adhere the patch to the scalp and to release a volatile or vapor-phase active agent. Methods for making transdermal patches are described in U. S. Patent Nos. 6,461,644; 6,676,961; 5,985,311; and 5,948,433.

[0353] In some forms, the disclosed compositions are provided in or on an occlusive cap, such as a hair treatment cap, or a hair mask cap, configured for application to the hair and scalp. In such forms, the occlusive cap includes an inner surface that is coated, impregnated, or otherwise loaded with one or more of the disclosed compositions, including compositions containing a Wnt pathway agonist, a Shh pathway agonist, or combinations thereof. Upon placement of the cap over the hair and scalp, the coated inner surface comes into direct or indirect contact with the scalp and hair, thereby transferring the composition to the target area. The occlusive cap e.g., hair treatment cap creates an occlusive environment that reduces evaporation, maintains hydration, and enhances penetration of the composition into the scalp and hair follicles. In some forms, the cap is configured to provide sustained or controlled release of the composition from the inner surface over a defined period of time, thereby prolonging contact between the composition and the scalp. The occlusive cap can be disposable or reusable and can be formed from polymeric, elastomeric, silicone, or multilayer materials. In some forms, the cap is worn for a defined treatment duration, such as minutes to hours, and may be used alone or in combination with additional topical applications to support hair follicle activation, regeneration, or cosmetic improvement of hair and scalp condition.

[0354] The topical and / or transdermal formulations can include immediate release, delayed release, and / or extended-release forms of the compositions as disclosed herein. In some forms, the formulations are configured to provide rapid initial delivery of one or more active agents upon application, followed by sustained or controlled release over an extended period of time to maintain activity at the scalp or skin surface. In other forms, the formulations are designed to delay release of one or more active agents until a predetermined time after application, thereby enabling temporal separation of biological effects.

[0355] In some forms, the formulation includes both a Wnt pathway agonist and a Shh pathway agonist, wherein the Wnt pathway agonist is formulated for immediate release and sustained release, such that Wnt signaling is initiated upon application and maintained continuously or intermittently over a defined priming period e.g., about 1 day, about 2 days, about 3 days, up to about 1 week, while the Shh pathway agonist is formulated for delayed

[0356] 50

[0357] 45816336.1release relative to the Wnt pathway agonist. In such forms, release of the Shh pathway agonist occurs only after Wnt signaling has been established, thereby providing sequential activation of Wnt signaling followed by Shh signaling within hair follicle-associated cells. The delayed release of the Shh pathway agonist may occur after a defined delay period, such as hours or days following application, for example, about 1 day, about 2 days, about 3 days, up to about 1 week, and can be combined with extended release of the Wnt pathway agonist to sustain Wnt-mediated priming prior to Shh pathway activation. Further description of delayed and sustained release forms are provided below for enteral formulations, which are applicable to topical and transdermal formulations.

[0358] 2. Enteral Formulations

[0359] Enteral formulations are prepared using pharmaceutically acceptable carriers.

[0360] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Thus, the composition can be formulated as a solid or liquid. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can be prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.

[0361] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name Eudragit® (Roth Pharma, Westerstadt, Germany), Zein, shellac, and polysaccharides.

[0362] Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.

[0363] Optional pharmaceutically acceptable excipients present in the drug-containing tablets, beads, granules or particles include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also termed "fillers," are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powder sugar.

[0364] 51

[0365] 45816336.1Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, amino alkyl methacrylate copolymers, polyacrylic acid / poly-methacrylic acid and polyvinylpyrrolidone.

[0366] Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.

[0367] Disintegrants are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as crosslinked PVP (Polyplasdone XL from GAF Chemical Corp).

[0368] Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.

[0369] Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tri decyl ether, polypropylene glycol butyl ether, POLOXAMER® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples

[0370] 52

[0371] 45816336.1of amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0372] If desired, the tablets, beads granules or particles may also contain minor amount of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents, and preservatives.

[0373] a. Extended release dosage forms

[0374] The extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in “Remington, The science and practice of pharmacy” (21st ed., Lippincott Williams & Wilkins, Baltimore, MD, 2006). A diffusion system typically consists of two types of devices, reservoir and matrix, and is well known and described in the art. The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and Carbopol 934, polyethylene oxides. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl Tri stearate. Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.

[0375] The devices with different drug release mechanisms described above could be combined in a final dosage form comprising single or multiple units. Examples of multiple units include multilayer tablets, capsules containing tablets, beads, granules, etc.

[0376] An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.

[0377] Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry granulation processes. Their formulations usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inert powdered substances such as any of many different kinds of starch, powdered cellulose, especially

[0378] 53

[0379] 45816336.1crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

[0380] Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In a congealing method, the drug is mixed with a wax material and either spray- congealed or congealed and screened and processed.

[0381] b. Delayed release dosage forms

[0382] Delayed release formulations are created by coating a solid dosage form with a film of a polymer that is insoluble in the acid environment of the stomach, and soluble in the neutral environment of small intestines.

[0383] The delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug -containing composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core" dosage form, or a plurality of drug -containing beads, particles or granules, for incorporation into either a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolysable, gradually water-soluble, and / or enzymatically degradable polymers, and may be conventional "enteric" polymers. Enteric polymers, as will be appreciated by those skilled in the art, become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate,

[0384] 54

[0385] 45816336.1cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and / or ethyl methacrylate, and other methacrylic resins that are commercially available under the tradename EUDRAGIT®.

[0386] (Rohm Pharma; Westerstadt, Germany), including EUDRAGIT®. L30D-55 and L100-55 (soluble at pH 5.5 and above), EUDRAGIT®. L-100 (soluble at pH 6.0 and above), EUDRAGIT®. S (soluble at pH 7.0 and above, as a result of a higher degree of esterification), and EUDRAGITS®. NE, RL and RS (water-insoluble polymers having different degrees of permeability and expandability); vinyl polymers and copolymers such as polyvinyl pyrrolidone, vinyl acetate, vinyl acetate phthalate, vinyl acetate crotonic acid copolymer, and ethylene- vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials may also be used. Multi-layer coatings using different polymers may also be applied.

[0387] The preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies.

[0388] The coating composition may include conventional additives, such as plasticizers, pigments, colorants, stabilizing agents, glidants, etc. A plasticizer is normally present to reduce the fragility of the coating, and will generally represent about 10 wt. % to 50 wt. % relative to the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil and acetylated monoglycerides. A stabilizing agent is preferably used to stabilize particles in the dispersion. Typical stabilizing agents are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. % to 100 wt. % of the polymer weight in the coating solution. One effective glidant is talc. Other glidants such as magnesium stearate and glycerol monostearates may also be used. Pigments such as titanium dioxide may also be used. Small quantities of an anti-foaming agent, such as a silicone (e.g., simethicone), may also be added to the coating composition.

[0389] 55

[0390] 45816336.1Methods of manufacturing

[0391] As will be appreciated by those skilled in the art and as described in the pertinent texts and literature, a number of methods are available for preparing drug-containing tablets, beads, granules or particles that provide a variety of drug release profiles. Such methods include, but are not limited to, the following: coating a drug or drug-containing composition with an appropriate coating material, typically although not necessarily incorporating a polymeric material, increasing drug particle size, placing the drug within a matrix, and forming complexes of the drug with a suitable complexing agent.

[0392] The delayed release dosage units may be coated with the delayed release polymer coating using conventional techniques, e.g., using a conventional coating pan, an airless spray technique, fluidized bed coating equipment (with or without a Wurster insert). For detailed information concerning materials, equipment and processes for preparing tablets and delayed release dosage forms, see Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989), and Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, 11th Ed. (Media, PA: Williams & Wilkins, 2017).

[0393] A preferred method for preparing extended release tablets is by compressing a drugcontaining blend, e.g., blend of granules, prepared using a direct blend, wet-granulation, or dry-granulation process. Extended release tablets may also be molded rather than compressed, starting with a moist material containing a suitable water-soluble lubricant. However, tablets are preferably manufactured using compression rather than molding. A preferred method for forming extended release drug-containing blend is to mix drug particles directly with one or more excipients such as diluents (or fillers), binders, disintegrants, lubricants, glidants, and colorants. As an alternative to direct blending, a drug-containing blend may be prepared by using wet-granulation or dry-granulation processes. Beads containing the active agent may also be prepared by any one of a number of conventional techniques, typically starting from a fluid dispersion. For example, a typical method for preparing drug-containing beads involves dispersing or dissolving the active agent in a coating suspension or solution containing pharmaceutical excipients such as polyvinylpyrrolidone, methylcellulose, talc, metallic stearates, silicone dioxide, plasticizers or the like. The admixture is used to coat a bead core such as a sugar sphere (or so-called "non-pareil") having a size of approximately 60 to 20 mesh.

[0394] An alternative procedure for preparing drug beads is by blending drug with one or more pharmaceutically acceptable excipients, such as microcrystalline cellulose, lactose, cellulose, polyvinyl pyrrolidone, talc, magnesium stearate, a disintegrant, etc., extruding the

[0395] 56

[0396] 45816336.1blend, spheronizing the extrudate, drying and optionally coating to form the immediate release beads.

[0397] III. IDENTIFICATION AND TUNING OF COMPOSITIONS FOR INDUCING DC FORMATION AND HAIR GROWTH AND REGENERATION

[0398] The signals that coordinate the events that lead to DC genesis have been identified. Data indicate that sufficient levels of SHH signaling determine when cells begin the transition to DC status, and the length of transition is delimited by a threshold level of Wnt signaling and quiescence. This transition phase is characterized by proliferation, augmented Wnt signaling, and acquisition of DC markers followed by cell cycle exit. The spatiotemporal distance between threshold levels of SHH and Wnt signaling determines the number of transitioning intermediates and a cell’s probability to undergo DC commitment.

[0399] Dermal SHH and Wnt signaling coordinate events of DC genesis within a short window of time and space. DCs form by the local migration of quiescent cells to form a cluster. The work shows that a local pool of quiescent cells is generated by a selectively proliferative population. These proliferative progenitors express Dkk1 and are located in a defined transition zone in the peri-DC region.

[0400] Studies have demonstrated the role of SHH in differentiated DC cells, establishing its function in DC maintenance. However, the function for dermal SHH signaling in DC differentiation and DC expansion is difficult to examine, as the progenitors and cellular processes that lead to DC differentiation have been largely unknown. By leveraging scRNA-seq methods, a previously unrecognized role for SHH in cooperating with and augmenting Wnt signaling to drive the pre-DC-to-DC transition was identified. SHH functions in Wnt-active DC progenitors to: 1) promote their proliferation, 2) induce expression of mature DC genes, 3) stimulate autonomous morphogenetic events and cell fate patterning, and 4) cause their timely arrest by augmenting Wnt signaling. In support of studies showing that high levels of Wnt signaling promote Cdkn1a expression and cell cycle exit, Wnt signaling levels correlated with quiescence across all mutants examined, including SHH cKO mutants.

[0401] Notably, genetic co-activation of SHH and Wnt signaling induces these events independent of FGF20, indicating that FGF20 likely functions upstream to modulate these two principal signals.

[0402] Figures 1A-1C are graphs showing induction of high levels of SHH signaling across the entire gradient of Wnt signaling prior to morphogenesis, and not just those in the peri-DC, cause every cell within that gradient to augment Wnt signaling incrementally while increasing their proliferation and expression of DC genes, resulting in more transitioning

[0403] 57

[0404] 45816336.1intermediates proliferating until they reach a level of Wnt signaling sufficient to cause arrest. Figures 1 A and IB are graphs of the effect of SHH level in combination with Wnt level showing the change from no dermal condensate (“DC”) to proliferation intermediates (transition level) to arrest. Figure 1C shows this process graphically.

[0405] The tight coupling of threshold levels of SHH and Wnt signaling in peri-DC cells should result in few transitioning intermediates prior to arrest, partly explaining why this transition is difficult to capture. Inducing high levels of SHH signaling across the entire gradient of Wnt signaling prior to morphogenesis, and not just those in the peri-DC, would cause every cell within that gradient to augment Wnt signaling incrementally while increasing their proliferation and expression of DC genes, resulting in more transitioning intermediates proliferating until they reach a level of Wnt signaling sufficient to cause arrest. Effectively, the SmoM YFP trajectory represents a “transition eigenvector” with more intermediates to resolve the process in “slow-motion”.

[0406] In SmoM2YFP;βcatfl / EX3mutants, both SHH and Wnt signaling levels are simultaneously induced at or above threshold levels, and mutant cells undergo that transition faster and more similar to the wildtype condition. Thus, two mitogenic signals can cooperate to regulate the length of transition between proliferation and arrest if they covary in a feed forward fashion, and one reins in this proliferation to cause arrest at a threshold level. This work supports a mechanism by which differentiation is coordinated with proliferation and cell cycle exit, a phenomenon utilized by other somatic stem cells, including muscle and neuronal progenitors. The data indicates the length of transition may depend upon the spatiotemporal distance between signal thresholds. This distance may define the number of divisions or duration that transit-amplifying cells divide before undergoing terminal differentiation.

[0407] Based on the SmoM2YFP trajectory, SHH and Wnt signaling cooperate to deterministically drive DC differentiation, and the spatial pattern follows this order.

[0408] Differentiation coupled to morphogenesis may have evolved to ensure that DC cells condense upon commitment to provide rapid and precise local feedback to the epidermis to regulate placode size or the spatial segregation of placode cell fates. Spatially segregated placode populations follow branching trajectories. Time and space are necessarily coupled to establish a committed pool of quiescent DC cells. Placode Wnt and SHH signaling antagonize each other to segregate placode cell fates, while the reciprocal convergence of these two signals in dermal cells results in accelerated signaling and DC differentiation. The factors that direct

[0409] 58

[0410] 45816336.1cell context-specific molecular interactions in the epidermis and dermis are unclear but indicate that signal segregation regulates cell fate trajectories.

[0411] Dermal SHH signaling promotes adult HF neogenesis following wounding. At the same time, forced activation of dermal P-catenin in adult skin inhibits HF neogenesis. The heterogeneous cell populations involved in wound healing likely require dermal Wnt activity to be tightly regulated. Signals have been identified which are sufficient to reproduce a major aspect of DC genesis as well as the tunable parameters that regulate the rate of DC genesis and DC size, which can help guide biomimetic methods to recreate inductive mesenchymal condensates.

[0412] These signals, using agonists and / or antagonists of Wnt and SSH, can be applied to skin to induce HF neogenesis and / or to restore HF size.

[0413] DCs form by the local migration of quiescent cells to form a cluster. A local pool of quiescent cells is generated by a selectively proliferative population. These proliferative progenitors express Dkk1 and are located in a defined transition zone in the peri-DC region. Previous seminal studies demonstrated the role of SHH in differentiated DC cells, establishing its function in DC maintenance (Figures 2A-2E; Figures 3A-3M).

[0414] Dermal SHH signaling is important to induce Region 2 changes, including accelerated dermal Wnt signaling. It was found that SHH cKO embryos showed expanded Wnt-active (Lef1+) placode cells at E14.5, indicating that placode Wnt ligands were preserved, but underlying dermal cells were unclustered and expressed much lower Lef1 levels than control DC cells (FIGs 3A-B). By E15.5, a small cluster of dermal cells was seen in SHH cKO embryos with Lef1 levels now reaching levels similar to E14.5 control DC cells, reflecting a slower rate of increase in Lefl expression. Using Bmp4 as a DC marker previously shown to be retained in SHH cKO DCs, significantly fewer Bmp4+ cells were found in SHH cKO DCs than control DCs (FIGs 3D-F). Also, while control Bmp4+ DCs were quiescent (G0 / G1 fraction=1) and surrounded by proliferating Dkk1+ cells, a significant proportion of SHH cKO Bmp4+ cells were aberrantly proliferating and co-expressed Dkk1 (FIGs 3D-E, 3G). At the same time, SHH cKO Dkk1+ / Bmp4- peri-DC cells showed a lower rate of proliferation than control Dkk1+ peri-DC cells, suggesting a defect in the proliferation of Dkk1+ peri-DC cells as well as their transition into quiescence (FIG 3C).

[0415] The SHH cKO Bmp4+ population did not increase in size over time (FIG. 3F).

[0416] Eventually SHH cKO Bmp4+ cells formed small quiescent clusters, showing that SHH cKO DC-like (pseudo-DC) cells undergo delayed entry into quiescence (FIGs 3D-G). These results were corroborated using the in vivo Fucci2 cell cycle reporter in which G1 / G0 cells

[0417] 59

[0418] 45816336.1express mCherry (Abe et al., Development 140(l):237-246 (2013)). Only by E15.5, small quiescent mCherry + / Ki-67-dermal clusters were observed underneath SHH cKO placodes.

[0419] To confirm that the defects in DC differentiation were not indirectly due to a loss of epidermal SHH signaling, the transducer of SHH signaling, Smo, was ablated in epidermal cells (K14Cre; Smofl / fl) (Long et al., Development 128(24):5099-5108 (2001)). At E14.5, mutant placode cells lacked SHH activity but were associated with Sox2+ DCs that showed an unexpected modest increase in the number of Sox2+ cells per DC. While K14Cre; Smofl / fl placodes were stalled in growth due to the lack of epidermal SHH activation, their corresponding DCs expanded at a greater rate than control DCs. Mutant DCs remained in close approximation to the epidermis with a persistently high Lefl+ / Ptch1+ copositive peri-DC population, while controls showed a diminishing Lefl+ / Ptch1-i- co-positive peri-DC population that corresponded to the downward displacement of the DC from the epidermis. Taken together with previous work (Gritli-Linde et al., Dev. 301(2):309-326 (2007); Woo et al. Genes & Dev. 26, 1235-1246 (2012)) for dermal SHH signaling in DC genesis this supports the model that SHH / Wnt co-activation promotes DC differentiation.

[0420] Diffusion maps of El 5.5 SHH cKO and control dermal populations showed that mutant cells progressed on a Wnt component but not a DC component. Specifically, E15.5 SHH cKO cells formed a divergent pseudo-DC branch that differed from controls by their lack of genes associated with the DC component, while their expression pattern was aligned with an attenuated Wnt component that terminates with quiescence. Consistent with this, SHH cKO pseudo-DCs express many genes found in Wnt-active dermal cells prior to DC formation (Gupta et al., Dev. Cell 48(1):1731 (2019)). Collectively, these results reveal a previously unrecognized mechanism by which dermal SHH signaling governs DC genesis by promoting the proliferation of Wnt-active peri-DC cells and their rapid entry into quiescence and mature DC differentiation.

[0421] SHH functions in Wnt-active DC progenitors to: 1) promote their proliferation, 2) induce expression of mature DC genes, 3) stimulate autonomous morphogenetic events and cell fate patterning, and 4) cause their timely arrest by augmenting Wnt signaling.

[0422] However, the essential function for dermal SHH signaling in DC differentiation and DC expansion was difficult to examine, as the progenitors and cellular processes that lead to DC differentiation were largely unknown. By leveraging scRNA-seq methods, a previously unrecognized role for SHH in cooperating with and augmenting Wnt signaling to drive the pre-DC-to-DC transition was discovered.

[0423] 60

[0424] 45816336.1In support of studies showing that high levels of Wnt signaling promote Cdknla expression and cell cycle exit, Wnt signaling levels correlated with quiescence across all mutants examined, including SHH cKO mutants. Notably, genetic co-activation of SHH and Wnt signaling induces these events independent of FGF20, indicating that FGF20 likely functions upstream to modulate these two principal signals.

[0425] Fig. 4A-4H show that high dermal SHH activation in early Wnt-active cells deterministically reproduces events of DC genesis over more intermediates. E14.5 control and SmoM2YFP whole mount showing proliferative mutant Sox2+ clusters (FIG. 4A). Number of Sox2+ cells per dermal cluster in control and SmoM2YFP at E14.5 and E15.5 (FIG. 4B). Diffusion maps of E14.5 control and SmoM2YFP cells (FIG. 4C, 4D). Pseudoorder with Regions 1 and 2 demarcated (FIG. 4E, 4F). FISH of E14.5 control and mutant showing virtually all Sox2+ cells co-express Ptch1 and Lefl (FIG. 4G, 4H).

[0426] Pseudo-order of indicated genes in mutant and control (FIG. 4F, 4G). E14.5 FISH showing EdU, Sox2, and Dkk1 (FIG. 4G). FISH at E15.5 after EdU pulse in control and SmoM2YFP skin (FIG. 4H). FISH 24 hours after EdU chase (FIG. 41). %EdU+ cells by population in E14.5 mutant and control (FIG. 41). %EdU+ of Sox2+ cells at E15.5 after EdU pulse or 24-hour chase (FIG. 4K). E14.5 SmoM2;0catfl / EX3 FISH showing Sox2+ clusters in upper and lower dermis that co-express Ptch1 and Lefl (FIG. 4L). Sox2+ clusters with %EdU+ of indicated populations by condition at E14.5 (FIG. 4M). Depiction of transition rate and number of intermediates affected by modulating SHH and Wnt signaling (FIG. 4N).

[0427] It is believed that the tight coupling of threshold levels of SHH and Wnt signaling in peri-DC cells will result in few transitioning intermediates prior to arrest, partly explaining why this transition is difficult to capture. Inducing high levels of SHH signaling across the entire gradient of Wnt signaling prior to morphogenesis, and not just those in the peri-DC, will cause every cell within that gradient to augment Wnt signaling incrementally while increasing their proliferation and expression of DC genes, resulting in more transitioning intermediates proliferating until they reach a level of Wnt signaling sufficient to cause arrest. Effectively, the SmoM2YFP trajectory represents a “transition eigenvector’’ with more intermediates to resolve the process in “slow-motion”. In SmoM2YFP;

[0428]

[0429] mutants, both SHH and Wnt signaling levels are simultaneously induced at or above threshold levels, and mutant cells undergo that transition faster and more similar to the wildtype condition. Thus, two mitogenic signals can cooperate to regulate the length of transition between proliferation and arrest if they covary in a feed forward fashion, and one reins in this proliferation to cause arrest at a threshold level.

[0430] 61

[0431] 45816336.1This supports a mechanism by which differentiation is coordinated with proliferation and cell cycle exit, a phenomenon utilized by other somatic stem cells including muscle and neuronal progenitors. The data indicates that the length of transition may depend upon the spatiotemporal distance between signal thresholds. This distance may define the number of divisions or duration that transit-amplifying cells divide before undergoing terminal differentiation.

[0432] Based on the SmoM2YFP trajectory, SHH and Wnt signaling cooperate to deterministically drive DC differentiation, and the spatial pattern follows this order.

[0433] Coupling of differentiation to morphogenesis may have evolved to ensure that DC cells condense upon commitment to provide rapid and precise local feedback to the epidermis to regulate placode size or the spatial segregation of placode cell fates. Spatially segregated placode populations follow branching trajectories. Time and space are necessarily coupled to establish a committed pool of quiescent DC cells. Placode Wnt and SHH signaling antagonize each other to segregate placode cell fates, while the reciprocal convergence of these two signals in dermal cells results in accelerated signaling and DC differentiation. The factors that direct cell context- specific molecular interactions in the epidermis and dermis are unclear but suggest that signal segregation may regulate cell fate trajectories.

[0434] The data support a mechanism to explain how dermal SHH signaling promotes adult HF neogenesis following wounding. At the same time, forced activation of dermal β-catenin in adult skin inhibits HF neogenesis. The heterogeneous cell populations involved in wound healing likely require dermal Wnt activity to be tightly regulated. Studies demonstrate that the signals sufficient to reproduce a major aspect of DC genesis as well as the tunable parameters that regulate the rate of DC genesis and DC size can help guide biomimetic methods to recreate inductive mesenchymal condensates.

[0435] Studies demonstrate that the DC differentiation trajectory was defined by Wnt and DC components. The data show that the combination of these two eigenvectors represents a significant aspect of DC genesis.

[0436] The following two-stage model was proposed for DP expansion. In the first stage, the DP itself is quiescent and dermal cup cells surrounding the DP proliferate to expand the DP. In the second stage, the DP starts proliferating to expand itself. This indicates that DP cells are not terminally differentiated but rather have self-renewing capacity. The results reveal a previously unknown potential for DP self-renewal. This provides a way to understand signals that regulates DP regeneration, which can help to expand DP cells in vitro for clinical applications.

[0437] 62

[0438] 45816336.1To determine the dose response of dermal fibroblasts to Wnt and / or SHH stimulation, two molecular signals required and sufficient for DC formation in mouse embryonic skin, varying doses of soluble Wnt agonist (CHIR-99021) and / or SHH agonist (SAG dihydrochloride) were added to cultured primary embryonic dermal fibroblasts. Specifically, dorslateral skin was micro dissected from mouse embryos in which all dermal cells genetically express nuclear GFP (PDGFRaH2BGFP). Skin is collected in 4%FCS / PBS wash buffer and dissociated into a single-cell suspension using 0.25% trypsin (Gibco, Life Technologies) for 20 minutes at 37° C. Cells are then washed in 4%FCS / PBS before filtering through a 70 mm strainer and FACS. All live GFP+ dermal cells are FACS sorted and collected in wash buffer. Cells are collected by centrifugation at 300g for 10 min. Cells are resuspended in media (10% FCS / DMEM) at 0.5x106per well supplemented with varying doses of vehicle alone (DMSO), CHIR (5 and 10 mM), or SAG (50 or 200 nM) or both CHIR and SAG. Cells are incubated at 37°C / 5% CO2 for 24, 48 or 96 hours. At each time point, adherent cells are collected by trypsinization (0.05% Trypsin) and RNA is isolated using a Qiagen RNeasy column. RNA is quantified and reverse-transcribed into cDNA (Superscript III, Invitrogen) before analyzing relative mRNA levels by qPCR with Fast SYBR Green Master Mix and primers specific for selected marker genes of Wnt activation (e.g. Lefl, Tcf7, Axin2), SHH activation (e.g. Glil, Ptch1) or DC differentiation (e.g. FoxDl, Soxl8) to assess gene expression responses to the above culture conditions.

[0439] To assess the functional ability of the above cultured dermal fibroblasts to induce hair follicle formation, a standard hair follicle patch assay is used Here, 2x106cultured dermal cells (PDGFRaH2BGFP+) from the above conditions are mixed in PBS with 1x106neonatal primary keratinocytes isolated from P0-P1 neonatal mice. Immunodeficient host mice (Nude) mice are injected with this mixture of dermal and epidermal cells subcutaneously (3 replicate grafts per culture condition) into the back skins of host mice. Mice are sacrificed after 4-5 weeks and grafts are harvested from skin. Under a stereomicroscope, regenerated hair follicles can be visualized volumetrically and quantified for number of hair follicles / graft, number of DC cells per hair follicle and hair fiber length and size.

[0440] There is currently no cell platform that responds to Wnt and SHH agonists to induce hair inductive DC genes. This is resolved with the use of embryonic dermal cells allows for robust and tunable Wnt and SHH gene expression responses as well as DC gene responses (when both Wnt and SHH are combined).

[0441] Using this regimen, Axin2CreER; SmoM2YFP embryos were examined at E14.5 and a lack of placodes noted as evidenced by the lack of epidermal Edar or Ptch1 transcripts. Sox2+

[0442] 63

[0443] 45816336.1dermal clusters were found in the upper dermis that were bigger than control DCs at both E14.5 and E15.5 and that expressed eYFP and Ptch1. These clusters usually lacked a direct connection to the epidermis but were confined to the upper dermis and occasionally abutted one another.

[0444] Sox9 was also expressed by control and mutant dermal clusters, while the mutant epidermis largely lacked Sox9+ cells normally found in SHH-active placodes. Thus, high dermal SHH activation is sufficient to induce Sox2+ dermal clusters in the Wnt-active upper dermis independent of placodes.

[0445] As high SHH activation is sufficient to induce DC differentiation in early Wnt-active cells, it was hypothesized that molecular changes of Region 2 are caused by sufficient levels of SHH signaling. scRNA-seq data from E14.5 control and SmoM2YFP embryos confirmed that neither placode markers nor eYFP was detected in the epidermal population of mutants. Due to the global transcriptional differences between wildtype (eYFP-) and mutant (eYFP+) dermal populations, dermal diffusion maps of mutant cells were analyzed separately. The SmoM2YFP trajectory was represented by one eigenvector, while other eigenvectors were either not significantly correlated with DC changes or contained minimal variance. Similar to Region 2 of controls, the mutant pseudo-order showed that Lefl levels increased monotonically, and Sox2+ cells were concentrated near the terminus. Further, Lefl and Ptch1 levels co-varied at a nearly constant rate along the SmoM2YFP pseudo-order.

[0446] By the SmoM2YFP trajectory, SHH / Wnt co-activation functions as a deterministic component that correlates with expression of DC and Wnt target genes. Notably, SmoM2YFP cells transition across more Dkkl + proliferative intermediates that gradually acquire DC genes before quiescence. To verify these inferences, it was found that virtually all Sox2+ cells in mutant embryos coexpressed Ptch1 and Lefl, supporting the notion that Wnt and SHH coactivation is required for DC differentiation. As dermal Wnt signaling requires epidermal Wnt ligands, all mutant Sox2 clusters were located in the upper dermis. At E14.5, it was also found that many mutant Sox2+ cells were proliferating and coexpressed Dkkl-i-, but by E15.5, most Sox2+ cells were quiescent, lacked Dkkl expression, and were surrounded by a ring of proliferating Dkkl+ cells similar to control DCs. Further, E15.5 mutant cells pulsed with EdU at El 4.5 showed that many quiescent Sox2+ cells originated from E14.5 proliferating cells in contrast to control DCs that expanded from a quiescent pool.

[0447] These results show that Wnt and SHH co-activation reproduces DC changes without placodes represented by a longer or “slower” continuum of proliferative Dkkl + intermediate states that progressively gain DC markers prior to quiescence.

[0448] 64

[0449] 45816336.1Recognizing that FGF20 is a placode signal previously shown to be essential for DC differentiation and morphogenesis, the role of FGF20 in this process was assessed.

[0450] SmoM2YFP expression in Axin2CreER; SmoM2YFP; FGF201acZ / lacZ embryos that lack FGF20 (Huh et al., 2012) was measure and it was found that mutant Sox2 clusters formed similar to SmoM2YFP embryos. FGF201acZ / lacZ embryos largely lacked Sox2+ DCs at E14.5, indicating that SHH may act downstream of FGF20. Consistent with this, SHH cKO placodes showed intact Fgf20 expression. Further, there was a marked reduction in the number of Ptch1+ and Lefl+ cells in the dermis underlying FGF201acZ / lacZ placodes, and Sox2+ cells that were seen co-expressed Ptch1 and Lefl. These data indicate that FGF20 modulates DC differentiation upstream of the two principal drivers of this process.

[0451] The spatial patterning of SHH and Wnt signaling gradients regulates the number of transitioning intermediates. The data show that inducing uniformly high SHH activation across a gradient of Wnt signaling results in a gradient response of DC differentiation. Thus, DC genes that are normally restricted to the distal region of the wildtype DC trajectory are now distributed across the entire gradient of Wnt signaling and decoupled from terminal quiescent cells. Based on these observations, inducing high Wnt signaling in SmoM2YFP cells could bypass earlier intermediate states. Both β-catenin and Smo were activated in Axin2CreER; SmoM2YFP; catfl / EX3 (SmoM2YFP; catfl / EX3) embryos (Harada et al., 1999). In contrast to SmoM2YFP embryos, Sox2+ clusters were now seen in the upper and lower dermis and surrounded by proliferating Dkkl+ cells in SmoM2YFP; catfl / EX3 embryos. Additionally, most Sox2+ clusters in SmoM2YFP; catfl / EX3 embryos were quiescent similar to control DCs, indicating a faster transition to quiescence concurrent with molecular differentiation and morphogenesis (Figures 5A-5N; 6A-6B).

[0452] Figures 7A and 7B are graphs showing dermal cells pre-treated with Wnt agonist, CHIR, significantly enhanced SHH and DC gene responses compared to without Wnt agonist pre-treatment (compare blue no pretreatment to red with Wnt pretreatment). Figures 7A and 7B show that pre-treating dermal cells with Wnt agonist, CHIR (2 micromolar for 24 hours) followed by SAG (10 nanomolar for 48 hours) significantly enhanced SHH and DC gene responses compared to without Wnt agonist pre-treatment (compare no pretreatment with vehicle (DMSO alone) to Wnt pretreatment). This indicates that Wnt activation (low dose) primes cells to respond more robustly to SHH activation.

[0453] 65

[0454] 45816336.1IV. METHODS OF USE

[0455] A. Methods of Generating Dermal Condensates

[0456] Provided are methods for generating, inducing, enhancing, or maintaining dermal condensates in skin or hair-bearing tissue. The methods disclosed herein promote dermal condensate formation and / or function by modulating key signaling pathways, including the Wnt signaling pathway and the Sonic hedgehog (Shh) signaling pathway, using compositions containing one or more Wnt pathway agonists, one or more Shh pathway agonists, or combinations thereof, as described above.

[0457] In some forms, the methods include contacting skin, scalp, or hair-bearing tissue with an effective amount of a composition comprising one or more Wnt pathway agonists. In such forms, Wnt pathway activation promotes mesenchymal cell aggregation, condensation, and acquisition of a dermal condensate phenotype, including changes in cell adhesion, spatial organization, and signaling activity. In some forms, Wnt pathway activation increases responsiveness of dermal cells to epithelial-derived signals that initiate dermal condensate formation.

[0458] In some forms, the methods include contacting skin, scalp, or hair-bearing tissue with an effective amount of a composition comprising one or more Shh pathway agonists. In such forms, Shh pathway activation promotes stabilization, maturation, or functional activity of dermal condensates, including support of dermal papilla precursor development, maintenance of mesenchymal signaling competence, and reinforcement of epithelial-mesenchymal interactions necessary for hair follicle induction and growth.

[0459] In some forms, the methods include administering both one or more Wnt pathway agonists and one or more Shh pathway agonists to generate dermal condensates. In such forms, the agonists can be administered together in a single composition or separately in distinct compositions. When administered together, the composition can provide simultaneous or overlapping activation of Wnt and Shh signaling to promote coordinated dermal condensate formation and function. When administered separately, the agonists can be delivered sequentially or in a staged manner, for example by first administering a Wnt pathway agonist to initiate mesenchymal condensation, followed by administration of a Shh pathway agonist to promote condensate stabilization, maturation, or signaling output.

[0460] In some forms, the methods include a temporal regimen comprising an induction phase and, optionally, a maintenance phase. During the induction phase, one or more Wnt pathway agonists and / or Shh pathway agonists may be administered at a frequency or concentration sufficient to induce dermal condensate formation. During the maintenance

[0461] 66

[0462] 45816336.1phase, administration may be continued at a reduced frequency or concentration to preserve dermal condensate integrity, dermal papilla activity, or ongoing hair follicle regeneration.

[0463] In some forms, dermal condensates are generated in vivo by topical, transdermal, intradermal, or localized administration of the disclosed compositions to the scalp or other hair-bearing skin. In such forms, the compositions are formulated to facilitate penetration into the dermis and interaction with dermal fibroblasts, dermal sheath cells, or dermal papilla precursor cells.

[0464] In some forms, dermal condensates are generated ex vivo or in vitro, for example by contacting isolated dermal cells, dermal fibroblasts, dermal papilla cells, or mesenchymal progenitor cells with one or more Wnt pathway agonists and / or Shh pathway agonists under conditions that promote cell aggregation and condensate formation. The resulting dermal condensates can be used for research purposes, screening applications, or, in some embodiments, for subsequent implantation or application to skin or scalp tissue.

[0465] Also provided are methods for screening a test compound for activity in promoting dermal condensate formation. An exemplary method includes the following steps:

[0466] (i) contacting dermal progenitor cells in vitro with the test compound in the presence or absence of the Wnt pathway agonist and / or the Shh pathway agonist; and

[0467] (ii) assessing formation of dermal condensate cells by detecting cell aggregation, dermal condensate morphology, expression of dermal condensate markers, or combinations thereof, wherein increased dermal condensate formation relative to a control identifies the test compound as promoting dermal condensate formation.

[0468] In some forms, assessing formation of dermal condensate cells comprises quantifying cell aggregation size, cell number, cell compactness, or a combination thereof. For example, assessing formation of dermal condensate cells includes detecting expression of one or more dermal condensate-associated markers. The control can include dermal progenitor cells cultured in the absence of the test compound.

[0469] B. Methods of Treatment

[0470] Methods of treatment are also provided and can be used alone or in combination with other methods disclosed herein, such as methods for promoting hair regeneration, preventing or reducing hair loss or hair thinning, improving scalp condition, and monitoring treatment response. The methods typically include administering to a subject in need thereof, an effective amount of a disclosed composition, e.g., a composition containing one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof, to treat the subject.

[0471] 67

[0472] 45816336.1This is particularly true where the condition is characterized by impaired hair follicle activity, reduced follicular stem or progenitor cell signaling, shortened anagen phase, follicular miniaturization, or an unfavorable scalp microenvironment. Additionally, or alternatively, particularly where scalp inflammation, oxidative stress, fibrosis, or microvascular dysfunction is present, the compositions can be used alone or in combination with additional agents that support scalp health.

[0473] Activation of Wnt and Shh signaling pathways can be directed toward hair follicles and surrounding scalp tissue to promote hair follicle regeneration, re-entry into anagen, maintenance of hair growth, and prevention of premature hair cycling. Similarly, the Wnt pathway agonists and Shh pathway agonists can be administered in combination or sequentially to provide coordinated modulation of signaling pathways involved in hair follicle morphogenesis and cycling.

[0474] In cases where hair follicle cycling is disrupted or where follicles exhibit features of domrancy or miniaturization, the use of topical, transdermal, or localized delivery of the compositions can be used alone or in combination with systemic or adjunctive approaches to enhance follicular responsiveness.

[0475] An exemplary method includes treating a subject (e.g., a human) having hair loss, hair thinning, scalp dysfunction, or a related condition by administering to the subject an effective amount of a pharmaceutical composition comprising one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof, optionally formulated for topical, transdermal, or oral administration.

[0476] In some forms, the methods administer a composition comprising one or more Wnt pathway agonists and / or Shh pathway agonists to a subject (e.g., a human) having a condition associated with reduced hair growth or impaired scalp health, in an amount effective to promote hair regeneration, increase hair density or thickness, or prevent further hair loss. For example, in some forms, the methods treat a condition associated with reduced Wnt or Shh signaling within hair follicles by administering to the subject an effective amount of a pharmaceutical composition comprising one or more Wnt pathway agonists and / or Shh pathway agonists.

[0477] For example, in some forms, the methods treat a subject having hair loss or scalp dysfunction by administering to the subject an effective amount of a pharmaceutical composition comprising one or more Wnt pathway agonists, one or more Shh pathway agonists, or both, wherein the composition is applied to the scalp or hair-bearing skin. The

[0478] 68

[0479] 45816336.1compositions may be administered as a single formulation or as separate formulations administered sequentially or concurrently.

[0480] In some forms, the methods further include administering additional agents that support hair follicle function or scalp health, including but not limited to anti-inflammatory agents, antioxidants, peptides, vitamins, botanical extracts, or penetration enhancers, optionally as part of the same composition or as a separate composition.

[0481] 1. Conditions or Diseases to be Treated

[0482] The compositions and methods disclosed herein are useful for the treatment, prevention, management, or amelioration of conditions, disorders, or diseases associated with hair loss, hair thinning, impaired hair growth, or scalp dysfunction. Hair growth rate and hair density naturally decline with age, and disruption of normal hair follicle cycling, stem cell activity, or scalp homeostasis can result in progressive hair loss (alopecia). In some forms, the disclosed compositions and methods promote hair follicle regeneration, activation of dormant or miniaturized hair follicles, prolongation of the anagen phase, restoration of normal hair cycling, and / or improvement of the scalp microenvironment through modulation of Wnt and Shh signaling pathways.

[0483] In some forms, the conditions or diseases to be treated include alopecia, including but not limited to androgenetic alopecia, female pattern hair loss, male pattern hair loss, alopecia areata, alopecia totalis, alopecia universalis, traction alopecia, telogen effluvium, anagen effluvium, scarring alopecia, and non-scarring alopecia. Alopecia can arise from a variety of underlying causes, including hormonal imbalance, altered mitotic activity, growth cycle abnormalities, nutritional factors, pharmacologic exposure, inflammation, or age-related decline in follicular regenerative capacity. In such forms, the compositions and methods can be used to reduce hair shedding, increase hair density or thickness, slow or prevent progression of hair loss, and / or promote regrowth of terminal hairs.

[0484] In some forms, the conditions or diseases to be treated include hair follicle miniaturization, follicular dormancy, or reduced follicular stem or progenitor cell activity, including conditions characterized by diminished Wnt signaling, impaired Shh signaling, or dysregulated crosstalk between epithelial and mesenchymal components of the hair follicle. For example, in androgenetic alopecia, increased dihydrotestosterone signaling shortens successive anagen cycles, leading to progressive follicle shrinkage and production of thinner, vellus-like hairs. The disclosed compositions can be used to stimulate follicular stem cell activation, dermal papilla signaling, or re-entry of hair follicles into an active growth phase.

[0485] 69

[0486] 45816336.1In some forms, the conditions or diseases to be treated include scalp conditions that adversely affect hair growth or hair retention, including but not limited to scalp inflammation, microinflammation, oxidative stress, fibrosis, reduced vascularization, seborrheic conditions, dry scalp, itching, scalp irritation, or disruption of the scalp barrier. Such conditions may be identified through clinical evaluation methods including, but not limited to, physical examination, hair pull testing, daily hair counts, part-width assessment, trichograms, hair pluck tests, scalp biopsy, hormonal evaluation, or microbiological analysis. In such forms, treatment can improve scalp health, reduce inflammatory or stress-related signals that impair hair growth, and create a microenvironment conducive to hair follicle regeneration and maintenance.

[0487] In some forms, the disclosed compositions and methods are used to treat or prevent age-related hair thinning or hair loss, including conditions associated with aging-related decline in hair follicle cycling, stem cell exhaustion, or reduced responsiveness to endogenous growth signals. Because durable tissue regeneration depends on the coordinated behavior of adult stem cells residing within specialized niches, failure to maintain or appropriately activate these stem cell populations can result in tissue dysfunction and loss. In such forms, the compositions can be administered to restore or enhance signaling pathways involved in youthful hair growth patterns.

[0488] In some forms, the conditions or diseases to be treated include hair shaft disorders or conditions associated with reduced hair fiber quality, including decreased hair thickness, reduced tensile strength, brittleness, or increased breakage, particularly where such conditions are associated with impaired follicular function or scalp health.

[0489] In some forms, the disclosed compositions and methods are used in prophylactic or maintenance settings, including to prevent the onset or progression of hair loss in individuals at risk of developing alopecia or scalp dysfunction, or to maintain hair density and scalp health following successful treatment. Such use may be particularly beneficial in individuals with early-stage follicular miniaturization or subclinical alterations in hair cycling that precede overt hair loss.

[0490] In some forms, the conditions or diseases to be treated may be cosmetic in nature, therapeutic in nature, or both, depending on the formulation, route of administration, and intended use. In some forms, the compositions are used to improve the appearance, fullness, thickness, or coverage of hair, without requiring diagnosis of a medical condition. In other forms, the compositions are used to treat clinically recognized hair or scalp disorder. Accurate characterization of the underlying hair or scalp condition may guide selection of appropriate

[0491] 70

[0492] 45816336.1treatment regimens, as therapeutic outcomes can depend on the biological basis of hair loss or dysfunction.

[0493] In some forms, the disclosed compositions and methods are be used alone or in combination with other treatments or interventions, including topical, oral, procedural, or lifestyle-based approaches, to address one or more of the foregoing conditions or diseases. Such combination approaches can be informed by screening methods developed to identify compounds capable of restoring hair follicle size, functionality, and regenerative signaling in embryonic or adult skin-derived stem or progenitor cells.

[0494] In some forms, the disclosed compositions and methods are intended for non-therapeutic, cosmetic use, including uses that do not involve the treatment, prevention, or diagnosis of a disease or medical condition. In such forms, the compositions containing one or more Wnt pathway agonists, Shh pathway agonists, or combinations thereof are used to improve the cosmetic appearance, condition, or perceived health of hair and / or the scalp, including enhancing hair fullness, thickness, density, strength, shine, manageability, or overall visual coverage, and / or to support the appearance of normal hair growth. In some forms, the cosmetic compositions are formulated and marketed as over-the-counter (OTC) consumer products, including but not limited to shampoos, conditioners, serums, lotions, foams, sprays, tonics, masks, or leave-on treatments intended for routine personal use. In such forms, the compositions are cosmetically acceptable, suitable for repeated application, and configured to improve hair and scalp appearance without systemic effects or therapeutic claims. In other forms, the cosmetic compositions are formulated for professional or salon use, including use by licensed cosmetologists, hair care professionals, or aesthetic practitioners. In such forms, the compositions can be provided as concentrated formulations, treatment kits, or multi-step regimens, optionally applied during professional hair or scalp treatments to enhance hair appearance, support scalp condition, or improve the cosmetic outcome of hair styling, coloring, or restorative procedures. Such professional or salon-use embodiments remain non-therapeutic and are not intended to treat, cure, or prevent a disease. In some forms, the cosmetic compositions and methods are used for maintenance, enhancement, or preventative aesthetic care, including maintaining the appearance of healthy hair, mitigating the visible effects of aging, stress, or environmental exposure on hair and scalp appearance, or prolonging the cosmetic benefits of prior hair treatments.

[0495] 2. Treatment / Application Regimen

[0496] The methods disclosed herein can be carried out using a variety of treatment or application regimens depending on the desired outcome, the condition being addressed, the

[0497] 71

[0498] 45816336.1formulation used, and the subject’s hair and scalp characteristics. In some forms, the treatment regimen includes administering one or more Wnt pathway agonists, one or more Shh pathway agonists, or combinations thereof, according to a defined schedule that provides coordinated modulation of hair follicle signaling pathways.

[0499] In some forms, the one or more Wnt pathway agonists and the one or more Shh pathway agonists are administered together as part of a single composition. In such forms, the composition can be applied to the scalp or hair-bearing skin in a single treatment step, providing simultaneous or overlapping activation of Wnt and Shh signaling within hair follicles, including within the bulge region, dermal papilla, or other follicular compartments. Co-administration in a single formulation may be advantageous for simplifying treatment, improving compliance, or achieving synergistic effects on hair follicle activation and cycling.

[0500] In forms in which a single composition is administered to the subject, in which both a Wnt pathway agonist and a Shh pathway agonist, the Wnt pathway agonist is formulated for immediate release, while the Shh pathway agonist is formulated for delayed or sustained release. In such forms, the Wnt pathway agonist is released upon application and initiates Wnt signaling within the hair follicle, while release of the Shh pathway agonist is delayed such that the Wnt pathway agonist is present and active for a defined priming period prior to Shh pathway activation. In some forms, the delayed release of the Shh pathway agonist occurs after at least about 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, about 80 hours, about 90 hours, about 4 days, about 5 days, about 10 days, about 2 weeks, or more, following application, thereby allowing accumulation or sustained exposure to relatively low levels of Wnt signaling prior to Shh pathway activation. For example, the delayed release is configured such that the Wnt pathway agonist is released and sustained for at least about 1 day, about 2 days, or about 3 days before substantial release of the Shh pathway agonist occurs.

[0501] In some forms, the one or more Wnt pathway agonists and the one or more Shh pathway agonists are administered separately according to a sequential or staged regimen. In such forms, a Wnt pathway agonist may be administered first to promote activation of follicular stem or progenitor cells, followed by administration of a Shh pathway agonist to promote hair follicle morphogenesis, anagen entry, or maintenance of hair growth. The interval between administrations can range from minutes to hours, days, or longer, depending on the formulation, route of administration, and desired biological effect.

[0502] For example, the Wnt pathway agonist is administered at least about 1 hour, about 6 hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hours prior to

[0503] 72

[0504] 45816336.1administration of the Shh pathway agonist. In some forms, the Wnt pathway agonist is administered once or repeatedly over a priming period of about 1 day to about 7 days, about 2 days to about 5 days, or about 3 days to about 4 days prior to administration of the Shh pathway agonist, thereby promoting accumulation of Wnt signaling activity before initiation of Shh signaling. In some forms, the Wnt pathway agonist is administered daily, twice daily, or every other day during the priming period, followed by one or more administrations of the Shh pathway agonist.

[0505] In some forms, the treatment regimen includes alternating administration of the Wnt pathway agonist and the Shh pathway agonist over a defined treatment period. For example, the Wnt pathway agonist can be applied minutes, until one or more hours prior to application of the composition containing the Shh agonist. For example, alternating administration occurs on a multi-day schedule, such as administration of the Wnt pathway agonist on one or more consecutive days followed by administration of the Shh pathway agonist on a subsequent day, or repeated cycles in which Wnt pathway agonist administration precedes Shh pathway agonist administration by about 1 day, about 2 days, about 3 days, about 4 days, or about 7 days. Such staggered regimens may be repeated weekly, biweekly, monthly, or according to individualized treatment cycles. In some forms, the compositions disclosed herein may be administered once or multiple times, including through repeated or periodic administration over a defined treatment period.

[0506] In some forms, the Wnt agonist, SHH agonist, or combination thereof is reapplied periodically to maintain continuous or nearly continuous presence of the compound(s) in dermal condensate and / or dermal progenitor cells. For example, in some forms, a single application / per day or every other day is used. In other forms, two, three, four, five, or more applications per day is used. In some forms, the compound(s) is / are reapplied after washing or sweating.

[0507] In some forms, the treatment regimen includes an initial induction phase followed by a maintenance phase. During the induction phase, the one or more Wnt pathway agonists and / or Shh pathway agonists may be administered more frequently or at higher concentrations to stimulate hair follicle activation or re-entry into anagen. During the maintenance phase, the frequency or concentration of administration may be reduced to maintain hair growth, prevent regression, or preserve cosmetic benefits.

[0508] In some forms, the treatment regimen involves daily, semi-weekly, weekly, or intermittent application, and can be adjusted based on observed response, tolerability, or user

[0509] 73

[0510] 45816336.1preference. The duration of treatment may range from days to weeks, months, or longer, including long-term or continuous use for maintenance of hair growth or scalp condition.

[0511] In some forms, the Wnt pathway agonist and the Shh pathway agonist are delivered via the same route of administration, such as topical or transdermal delivery to the scalp. In other forms, the Wnt pathway agonist and the Shh pathway agonist are delivered via different routes, for example, topical administration of one agonist and oral administration of the other, to achieve combined local and systemic effects.

[0512] In some forms, the treatment regimen further includes adjustment of dosing, timing, or formulation based on factors such as age, sex, extent of hair loss, scalp condition, or responsiveness to treatment. In some forms, the regimen is customized for cosmetic use, therapeutic use, OTC products, or professional or salon-based treatments.

[0513] In some forms, the treatment regimen is be used alone or in combination with additional agents or interventions that support hair or scalp health, provided that such combinations do not interfere with the coordinated timing and activity of the Wnt and Shh pathway agonists.

[0514] As used herein, an “effective amount” refers to an amount of a Wnt agonist and an amount of a SHH agonist that, when administered to a subject in need thereof, either alone or in combination and sequentially or concurrently as described herein, produces a detectable improvement in one or more parameters associated with hair follicle regeneration, hair growth, or hair follicle maintenance.

[0515] In some forms, the effective amount of the Wnt agonist is sufficient to induce or prime Wnt signaling activity in cells of the epidermis, dermis, or hair follicle unit, thereby rendering the tissue responsive to subsequent SHH agonist exposure. In some forms, the effective amount of the SHH agonist is sufficient to augment or amplify Wnt-driven signaling pathways involved in hair follicle morphogenesis, dermal papilla activation, or initiation of anagen.

[0516] In some forms, the Wnt agonist is present in the composition at any concentration described above, such as ranging from about 0.001 pM to about 1,000 pM, about 0.01 pM to about 500 pM, about 0.1 pM to about 100 pM, or about 1 pM to about 50 pM. In some forms, the Wnt agonist is administered in an amount corresponding to an in vitro effective concentration of at least about 10 pM, at least about 25 pM, or at least about 50 pM, with adjustment for topical, transdermal, or localized delivery in vivo.

[0517] In some forms, the SHH agonist is present in the composition at a concentration ranging from about 0.001 nM to about 1,000 nM, about 0.01 nM to about 500 nM, about 0.1

[0518] 74

[0519] 45816336.1nM to about 100 nM, or about 1 nM to about 50 nM. In certain forms, the SHH agonist is administered in an amount corresponding to an in vitro effective concentration of at least about 5 nM, at least about 10 nM, at least about 15 nM, up to at least about 200 nM with adjustment for in vivo administration.

[0520] In some forms, the Wnt agonist and SHH agonist are administered sequentially, such that the Wnt agonist is administered first for a priming period prior to administration of the SHH agonist. In such forms, the priming period may range from about 1 hour to about 14 days, about 6 hours to about 7 days, or about 1 day to about 5 days, prior to SHH agonist administration. In some forms, the Wnt agonist is reapplied one or more times during the priming period to maintain Wnt signaling activity.

[0521] In other forms, the Wnt agonist and SHH agonist are administered concurrently using a formulation configured to provide delayed or sustained release of the SHH agonist relative to the Wnt agonist, such that functional sequential signaling is achieved in vivo.

[0522] An effective amount may be determined based on one or more observable or measurable parameters associated with hair growth or regeneration. Non-limiting examples of such parameters include increases in hair follicle density, induction of anagen phase follicles, increases in hair shaft thickness or length, increased dermal papilla cell number or activity, increased expression of hair follicle-associated genes, or formation of new hair follicles.

[0523] In some forms, efficacy of the disclosed compositions is assessed as measured by one or more of: phototrichogram analysis, trichoscopy, scalp photography with image analysis, hair count per unit area, hair shaft diameter measurements, histological analysis of skin biopsies, immunohistochemical detection of hair follicle markers, or molecular assays measuring expression of Wnt- or SHH-responsive genes. In some forms, improvement is determined as measured by an increase of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 50%, or at least about 60% in one or more of the foregoing hair growth parameters relative to a baseline or control.

[0524] In some forms, the effective amount produces partial or complete regeneration of miniaturized hair follicles, prolongation of the anagen phase, delayed entry into catagen, or maintenance of existing terminal hair follicles. For example, an effective amount of the composition is sufficient to produce a clinically observable improvement in hair coverage, hair density, and / or hair quality in the treated area.

[0525] It will be understood that the effective amount can vary depending on factors including, but not limited to, the identity of the Wnt agonist and SHH agonist, formulation

[0526] 75

[0527] 45816336.1type, route of administration, frequency of administration, treatment duration, and the condition and characteristics of the subject being treated.

[0528] Examples

[0529] Example 1: Interaction of Wnt and SHH Gradients Synchronizes Cell Cycle Exit and Differentiation

[0530] Materials and Methods

[0531] Tamoxifen induction of mice

[0532] Embryos were staged as days post coitum, with embryonic day E0.5 considered as noon of the day a vaginal plug was detected after overnight mating. Pregnant dams were given one dose of Tamoxifen dissolved in corn oil (20mg / ml, Sigma) at 40-60 µg / gm body weight by oral gavage at El 1.5 as indicated.

[0533] EdU Incorporation Assay

[0534] To assess active proliferation, EdU was administered to pregnant mice intraperitoneally (25 µg / gm) and embryos were harvested after 1.5 hour. For pulse-chase experiments, 25 µg / gm EdU was administered to pregnant mice at indicated times and embryos were harvested at later times. EdU incorporation was assessed using the Click-it EdU Imaging kit Alexa 555 or Alexa 488 (Life technologies, cl0338) according to manufacturer’s instructions. Briefly, skin explants were treated with a mixture of IX Click-iT reaction buffer, CuSO4, Alexa Fluor azide dye and IX reaction buffer additive, all provided in the Click-it EdU Imaging Kit, for 30 minutes at RT before washing in PBS.

[0535] Histology

[0536] 10% formalin-fixed paraffin embedded (FFPE) whole embryos were used for histological analysis. FFPE embryos were sectioned at 10 pm thickness for FISH.

[0537] Whole mount immunofluorescence

[0538] Dorsolateral skin from embryos were micro-dissected and placed on nucleopore filters (VWR, WHA 800281) and fixed overnight with 4%PFA at 4° C. Skin explants were blocked for 6 hours in 5% normal donkey serum, 1% bovine serum albumin and 0.5% Triton-X100 at RT. Explants were incubated overnight at 4° C in the following primary antibodies: rabbit anti-Sox2 (1:400, Abeam, ab97959), chicken anti-GFP (1:500, Abeam, abl3970), rabbit anti-RFP (1:500, Rockland, 600-401-379). Explants were then washed in 0.2% Tween20 / PBS for 4-6 hours on a rotator and then incubated with the following secondary antibodies: Alexa Fluor 568-donkey anti-rabbit (1:200, Invitrogen, A10042), Alexa Fluor 488-donkey antirabbit (1:200, Invitrogen, A21206), Alexa Fluor 488-goat anti-chicken (1:200, Invitrogen, A11039) overnight at RT. Washes were carried out for 6 hours with 0.2% Tween20 / PBS.

[0539] 76

[0540] 45816336.1Hoechst (1:750 in PBS) was used for 45 min before mounting on slides with SlowFade Gold Antifade Mountant (Invitrogen, S36936).

[0541] In-situ hybridization

[0542] The RNAscope Multiplex Fluorescent Detection Kit v2 (ACDBio, 323110) was used for single-molecule fluorescence in situ hybridization (FISH) according to the manufacturer’s protocol. Briefly, sections were deparaffinized and permeabilized with hydrogen peroxide followed by antigen retrieval and protease treatment before probe hybridization. After hybridization, amplification and probe detection were done using the Amp 1-3 reagents. Probe channels were targeted using the provided HRP-C1-3 reagents and tyramide signal amplification VIVID fluorophores - 650 and 570 (ACD biotechne, 323271). EdU staining was done using the Click-it EdU Imaging Kit Alexa 488 (Life Technologies, C10338) according to the manufacturer’s instructions. Nuclear counter-stain was done using Hoechst 33342 (Invitrogen, H3570) before mounting with SlowFade Mountant. RNA scope probes used (ACDBio) - Mm-Lefl (441861), Mm-Ptch1 (402811), Mm-Dkkl (402521), Mm-Sox2 (401041), Mm-Gal (400961), Mm-Gli3 (400961) and Mm-Trp53inpl (1161531).

[0543] Microscopy

[0544] FISH paraffin-embedded images were acquired using the Leica Stellaris 8 DMi8 confocal microscope with a x40 oil immersion (Numerical Aperture 1.3) objective lens, scanned at 5 pm thickness, 1, 024 x 1, 024 pixel width, 400Hz. Whole mount explants were imaged in 3 dimensions using the LaVision TriM Scope II (LaVision Biotec) microscope equipped with a Chameleon Vision II (Coherent) two-photon laser (810-1000 nm) to acquire z-stack images ranging from 50-120 pm (2 pm serial optical sections) using a 20X water immersion lens (NA 1.0; Olympus), scanned with a field of view of 0.3-0.5 mm2 at 800 Hz.

[0545] Single-cell dissociation

[0546] Embryonic dorsolateral / flank skin was micro-dissected and pooled after genotyping (3 embryos per condition) and dissociated into a single-cell suspension using 0.25% trypsin (Gibco, Life Technologies) for 14 minutes at 37° C. Single-cell suspensions were then stained with DAPI (Fisher Scientific, NBP2-31156) just prior to fluorescence-activated cell sorting.

[0547] Fluorescence-activated cell sorting

[0548] DAPI-excluded live skin cells were sorted on a BD FACS Aria II (Biosciences) sorter with a 100 pm nozzle. Cells were sorted in bulk and submitted for 10X Genomics library preparation at 0.75-1.0xl06 / mL concentration in 4% FCS / PBS solution.

[0549] 77

[0550] 45816336.1Single-cell RNA sequencing and library preparation

[0551] Chromium Single cell 3’ Library and Gel Bead Kit v2 Chromium Single Cell 3' Library & Gel Bead Kit v2 (PN-120237), Chromium Single Cell 3' Chip kit v2 (PN-120236) and Chromium i7 Multiplex Kit (PN- 120262) were used according to the manufacturer’s instructions in the Chromium Single Cell 3' Reagents Kits V2 User Guide. After cDNA libraries were created, they were subjected to Novaseq 6000 (Illumina) sequencing.

[0552] 10X scRNA-seq data pipeline to matrix

[0553] The transcriptomes of live single cells from E13.5 or E14.5 mouse skin samples under different biological conditions were sequenced. Raw 10X sequencing data was processed into a matrix employing the standard 10X CellRanger pipeline. Briefly, base call files were fastq format which were aligned to the mmlO reference genome followed by nUMI and barcode counting, constructing the nUMI count matrices. nUMI matrices were filtered, centered and normalized using Seurat@1. Briefly, for each original run condition (e.g., El 3.5 wildtype), cells with > 1,000 detected genes, mitochondrial content < 25%, and ribosomal content > 5% (calculated via PercentageFeatureSet in Seurat) were retained. Doublets were identified and removed using scDblFinder@2. Data were then log-normalized by total UMI count.

[0554] Principal Component Calculation and UMAP Visualization

[0555] Principal components were computed using Seurat’s RunPCA on the top 2,000 variable genes. The number of PCs retained was determined from the elbow plot@7, and used for downstream analysis, including dimensionality reduction for visualization and clustering.

[0556] Cell type specification

[0557] For each original run, UMAP was performed on log -normalized, centered, and scaled UMI count matrices using the retained PCs. Unsupervised clustering was then conducted using Seurat’s FindClusters function with a resolution of 0.5. Clusters expressing Collal were defined as dermal, while clusters expressing Lefl or Dkkl were defined as upper-dermal.

[0558] Gene trajectory inference

[0559] GeneTrajectory@3 was applied to each sample to identify multiple, independent gene dynamic processes. For each sample, the recommended guidelines were followed. Selected were the top 2,000 highly variable genes (identified using Seurat’s FindVariableFeatures) that were expressed in >1% and <50% of cells as input. The top 20PCs were used to compute a diffusion map, and the top 10 diffusion components were used to construct a cell k-nearest neighbor graph (k = 10).

[0560] 78

[0561] 45816336.1The number of gene trajectories and the time steps for gene inclusion within each trajectory were determined interactively by inspecting the top three or more gene embeddings. For the samples analyzed in this study, the following gene trajectories and associated time steps (t) were identified:

[0562] (i) E14.5 wild type: 3 trajectories (t = 13, 10, 5)

[0563] (ii) E13.5 SmoM2: 4 trajectories (t = 1, 3, 2, 1); control: 3 trajectories (t = 3, 2, 2) (iii) E13.5 TG: 3 trajectories (t = 6, 3, 3); control: 3 trajectories (t = 5, 3, 3)

[0564] (iv) E13.5 EX3: 4 trajectories (t = 3, 3, 2, 2); control: 3 trajectories (t = 1, 3, 3) (v) E13.5 GH3-KO: 4 trajectories (t = 8, 7, 6, 1); control: 3 trajectories (t = 4, 3, 1) Gene bins and cell stages

[0565] Following GeneTrajectory guidelines, each trajectory from mutant samples was divided into 5 or 6 gene bins. For each bin, a gene bin score was computed for both mutant and control samples to compare gene expression dynamics across the cell embedding. Cell involved in each trajectory were stratified into the same number of stages, based on whether they expressed more than 50% of the genes in the corresponding gene bin. Cells assigned to multiple stages were resolved by assigning them to the stage closer to the terminal end of the trajectory (i.e., farther from the branching point).

[0566] Exit GT Score

[0567] Exit GT scores for each sample were calculated using Seurat’s AddModuleScore, based on all genes from the exit gene trajectory identified in E13.5 SmoM2.

[0568] Pathway enrichment analysis

[0569] Reactome pathway enrichment analysis was performed using ReactomePA@4, with pathways showing p < 0.05 considered significantly enriched.

[0570] Cell cycle estimation of scRNA-seq data

[0571] Seurat’s cell cycle scoring was used48. Briefly, averaged relative expression of those cell cycle related genes were used to calculate G2 / M and S scores, which were used for binning cells into G2 / M, S and G1 / G0 bins.

[0572] Data merge and normalization

[0573] For comparative analysis, upper-dermal cell matrices from each pair of conditions separately (i.e. El 3.5 control and Gli3-KO) were merged, followed by standard preprocessing and normalization using the workflow provided by Seurat.

[0574] Differential abundance and Differential gene expression analysis Differential abundant cells were identified using DA-seq@6, with a DA score threshold > 0.8. Differential gene expression analysis between DA cells from E13.5 Gli3-K0

[0575] 79

[0576] 45816336.1and all other upper-dermal cells from both E13.5 Gli3-KO and control was performed using Seurat’s FindMarkers without prefiltering. Significant DEGs for visualization were defined using a combined threshold of padj < le-5 and log2FC > 0.23.

[0577] CUT& RUN experiments

[0578] CUT& RUN was performed with the Epicypher CUT ANA™ ChIC / CUT& RUN Kit (14-1048) according to the manufacturer’s specifications with the following conditions: 500,000 cells per sample were prepared first and nuclei were extracted according to the Epicypher CUT& RUN Manual Appendix. Nuclei were incubated with activated ConA beads. The following antibodies (0.5 ug of antibody per reaction) were used: IgG Control antibody: CUTANA™ Kit Rabbit IgG CUT& RUN Negative Control Antibody, EpiCypher Rabbit anti-H3K27Ac antibody, Millipore Mouse anti-Flag antibody, Antibodies-online Rabbit anti-CTNNB1 antibody. Libraries were prepared using the Epicypher CUTANA™ CUT& RUN Library Prep Kit (14-1002) according to the manufacturer’s specifications. Library quality was analyzed using Agilent Tapestation D1000 High Sensitivity Tapes (#5067-5584).

[0579] Sequencing was performed with 5 million 150bp paired-end reads per sample on an Illumina NovaSeq 6000.

[0580] CUT& RUN data analysis

[0581] CUT& RUN reads were analyzed for quality using FastQC and adaptors trimmed using Trimmomatic. Reads were then aligned to mm9 by Bowtie2 version 2.3.1. SAMTools version 1.11 was then used to convert to BAM format, index, isolate uniquely mapped paired reads, and remove duplicates. MACS2 version 2.2.8 was used to call peaks with IgG as input control. Read counts across genomic intervals and peak visualization were performed using deepTools version 3.5 and bigwig files were visualized using the Integrative Genomics Viewer. Deseq2 was used to identify differentially bound peaks between conditions, DiffBind used to plot heatmaps of accessible / bound regions and HOMER to identify de novo motifs most enriched in the differentially bound regions and annotate associated genes.

[0582] Western blot

[0583] Cells were lysed in RIPA buffer (Thermo Fisher Scientific, Cat# 89900) supplemented with protease inhibitors (Thermo Fisher Scientific, Cat# 87786) for 30 minutes at 4 °C. The lysates were clarified by centrifugation at 10,000 x g for 20 minutes at 4 °C, and protein concentrations were determined using Bradford Protein Assay Kit (Bio-Rad, Cat# 5000006). Equal amounts of protein (10 pg) were denatured at 95 °C for 5 minutes and loaded onto 4-12% precast polyacrylamide gels (Bio-Rad, Cat# 3450125) for electrophoresis. Proteins were transferred to PVDF membranes (Thermo Fisher Scientific, Cat# IB24001)

[0584] 80

[0585] 45816336.1using an iBlot2 transfer device (Invitrogen, Cat# IB21001). Membranes were blocked with 5% BSA (vol / vol) in lx TBST (AmericanBio, Cat# AB 14330-01000) at room temperature, incubated with primary antibodies overnight at 4 °C, washed three times with TBST for 5 minutes each, incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature, and washed again three times with TBST.

[0586] Membranes were developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Cat# 34577) and imaged on a ChemiDoc imaging system (Bio-Rad). The following antibodies were used: mouse anti-Flag (Millipore, Cat# F1804), rabbit anti-Actin (Cell Signaling Technology, Cat# 4970), HRP-conjugated goat antirabbit IgG (Cell Signaling Technology, Cat# 7074), and HRP-conjugated goat anti-mouse IgG (Bio-Rad, Cat# STAR207P, 1:10,000).

[0587] Dermal cell culture

[0588] E13.5 PDGFRaH2BGFP skin cells were sorted based on their GFP status. GFP+ cells were collected and pelleted (300 x g, 10 min, 4°C), resuspended at 2 x 106cells / mL in complete growth medium (DMEM + 10% FBS, Penicillin-Streptomycin, and L-Glutamine), and viability was assessed by trypan blue exclusion. Cells were plated at 3 x 106cells / well in 6- well plates in 2 mL total volume. For chemical perturbation assays, cells were mixed 1:1 with a 2x agonist solution. For example, 10 pM CHIR99021 (Sigma, Cat# SML1046) was prepared in DMSO and diluted to a working concentration of 5 pM in culture. Similarly, SAG (Sigma, Cat# SML1314) was prepared as a 50 pM stock and diluted as needed. Media with agonist was used fresh or stored at 4°C for up to two days. Cells were cultured in 48-72 hours.

[0589] qPCR

[0590] Total RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen, Cat# 74134) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from 500 ng-1 pg of total RNA using the SuperScript™ III First-Strand Synthesis System (Invitrogen, Cat# 18080051) with random hexamer primers. Quantitative PCR was performed using the PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, Cat# 4364344) on a Viia7 Real-Time PCR System (Applied Biosystems) in 384-well optical plates. Reactions were run in technical triplicates using gene-specific primers, and threshold cycle (Ct) values were normalized to Gapdh as a housekeeping gene. Relative expression was calculated using the AACt method.

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[0592] 45816336.1Quantification and Statistical Analysis

[0593] Image analysis

[0594] Raw image stacks acquired from whole-mount explants were imported into Fiji or Adobe Photoshop for analysis. ggplot2 and cowplot R libraries were used for graphical representation of the scRNA-seq data. Diffusion maps were generated as described above.

[0595] Quantification

[0596] For quantification of DC density, 3-dimensional whole-mount tiled mosaics of skin explants with a 1000 x 1000 pm field of view and a z-depth of 60-100 pm were used for n=3-4 embryos. For volumetric quantification of DC cell number, EdU-i- or Tom+ percentage of DC or UD, 3-dimensional whole mount mosaics stained with Sox2 and EdU or RFP were used to manually count positive cells, using ImageJ (Fiji) software using planar (XY) and orthogonal (YZ, XZ) views. For volumetric quantification of cell cycle phases, 3 -dimensional whole mount mosaics stained with markers for G0 / G1 cells (based on Fucci2 reporter) were done manually by counting nuclear positive cells using ImageJ (Fiji) software. For quantification of upper dermal cells, a distance of 10 um below the epidermis was considered upper dermis. For quantification of the peri-DC region by whole-mount, cells two cell layers out from the Sox2+ DC were counted. For quantification based on FISH, cells with 4 5 dots were considered positive (according to the RNAScope manufacturer’s instructions) and sections from a total of n=4 different embryos were examined. To measure RNA expression levels, H-scores were calculated according to ACDBio manufacturer’s instructions: a cell with 0 dot is scored 0, 1-3 dots score 1, 4-9 dots score 2, 10-15 dots and / or less than 10% clustered dots score 3, and more than 15 dots and / or more than 10% clustered dots score 4; then the final H-score of a given cell type A is calculated by summing the (% cells scored B within all cells in A)*B for score B in 0-4. For quantification of upper dermal cells, a distance of 10-20 pm below the epidermis was considered as upper dermis. For quantification of cells in the peri-DC region Dkkl +Sox2 was considered.

[0597] Statistical analysis

[0598] All statistical values are expressed as mean ± SEM. An unpaired Student’s r-test was used to analyze data sets with two groups and *P <0.05 to, **P<0.01, ***p< 0.001 and **** / ><□ 00001 indicated a significant difference. When comparing more than two groups, P values were determined by one-way ANOVA with Tukey’s HSD test performed as the post hoc analysis. Statistical calculations were performed using Prism software package (GraphPad). For categorical data, a chi-square test was employed to estimate a P value.

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[0600] 45816336.1Results

[0601] Cell cycle exit and molecular differentiation are tightly coupled during dermal condensate development

[0602] To examine how Wnt and SHH signaling regulate the pre-DC-to-DC transition, where and when quiescence and DC gene expressions occur in relationship to Wnt and SHH activity were first characterized. During mouse skin development, dermal condensates (DCs) emerge around El 4.0-E14.5 underneath hair follicle epithelial placodes (data not shown). DC cells express cell type markers, including Sox2, Soxl8, and Gal. Cells expressing these markers correspond to cells that are quiescent as shown by a very low rate of EdU nucleotide incorporation (data not shown). DC marker expression also corresponds to expression of the in vivo G1 / G0 Fucci2 reporter (data not shown)23’29, and virtually all Sox2+ cells coexpress the cell cycle inhibitor, p21(Cdknla) with only rare cells expressing Cdknla but not Sox2 (data not shown). Thus, markers of quiescence largely colocalize with cells that express DC marker genes. Using genetic lineage tracing and EdU pulse-chase experiments, it was observed that while DC cells are quiescent, they are immediate progeny of highly proliferative Dkkl+ progenitors that are located in the upper dermis at El 3.5 and later surrounding the growing DC (data not shown)24. It was observed that these Dkkl + proliferative progenitors undergo a short phase of proliferation early during morphogenesis (between approximately E13.5-E14.5) to give rise to the vast majority of arrested DC cells. Thus, cell cycle exit and molecular DC differentiation occur synchronously after a phase of high proliferation (data not shown).

[0603] It was next asked where and when Wnt and SHH activity are expressed during DC formation. As SHH ligand is exclusively expressed by hair follicle epithelial placodes, SHH is not active at E13.5. However at this time point, Wnt signaling is activated broadly across the upper dermis (UD) in response to Wnt ligands secreted from the epidermis. This Wnt activity is required for DC and hair follicle initiation25,30. Although Wnt activity alone is not sufficient for DC gene expression, it was tested whether Wnt activity alone may promote quiescence independent of SHH. Lefl was used as a canonical marker of Wnt activity, whose expression was observed to highly correlate with other known Wnt target genes

[0604] (e.g., Axin2, Wifi, Tcf7; data not shown)2425.

[0605] It was found that Lefl expression gradually decreases over the first four upper dermal cell layers with the most superficial dermal layer (closest to the epidermis) showing the highest average Lefl expression (Figures 12A and 12B). Prior to SHH, no significant correlation was observed between the rate of EdU incorporation and Lefl expression levels

[0606] 83

[0607] 45816336.1(Figure 12A). By E14.5, SHH is secreted by placode epithelial cells, resulting in SHH activation in the placode epithelium and underlying dermis (Figure 12C). Using Ptch1 as a canonical marker of SHH activity, it was found that SHH activity correlates with Wnt signaling levels at E14.5, and both correlate spatially with pre-DC-to-DC cell states, with highest levels of Ptch1 and Lefl seen in cells that express DC markers. Al this point, increasing levels of both Wnt and SHH activity correlate with decreasing rates of EdU incorporation and quiescence. Notably, Wnt signaling is amplified after El 3.5, and the highest levels of Wnt and SHH activity now correspond with cell cycle exit and DC gene expression (Figure 12D). Hence, prior to SHH (E13.5), neither DC genes nor quiescence appear to be regulated by Wnt signaling levels. When epithelial placodes upregulate Wnt and SHH ligands at E14.5, both dermal Wnt and SHH activity are concurrently upregulated during the pre-DC-to-DC transition. During this transition, cell cycle exit and DC gene expression occur synchronously.

[0608] High Wnt activity induces cell cycle exit independent of SHH

[0609] As high levels of both Wnt and SHH activity seen at El 4.5 correspond to cell cycle exit and DC gene expression, it was tested whether high levels of Wnt and / or SHH activity regulate these events. It was observed that expression of an activated form of P-catenin in the dermis induces uniformly high Wnt activity30,31. This results in larger DCs where placodes normally form (data not shown), suggesting that cells with high levels of Wnt signaling are primed to undergo DC differentiation upon SHH expression. To assess if increasing Wnt activity to a level higher than that seen at E13.5 (when there are no detectable DC changes) can promote pre-DC changes prior to SHH expression, activated P-catenin was expressed in the dermis (Axin2CreER; caf / EX3or Actpcat, Figure 13A) by administering tamoxifen at El 1.5. To profile molecular changes induced by high Wnt activation, scRNA-seq analysis was performed of dermal populations obtained from microdissected dorsolateral skin from E13.5 mutant and paired control embryos. Dermal cells were analyzed after filtering cells based on known dermal markers (e.g., Coll al a) as previously described. The mutant dermal population showed increased expression of Wnt target genes, including Lefl, Axin2 and Tcf7, but lacked DC markers (e.g., Sox2) or SHH target genes (data not shown ). Differential gene expression analysis was done between E13.5 control and Actpcat dermal cells (Supp table 1). Despite upregulation of Wnt target genes in mutant dermal cells, DC markers such as Sox2 and Soxl 8 were not significantly expressed.

[0610] However, Cdknla, a cell cycle inhibitor expressed by DC cells at E14.5, was among the most significantly upregulated genes in the Actpcat dermis, suggesting that high Wnt

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[0612] 45816336.1activation alone may induce cell cycle exit independent of DC gene expression (data not shown). By FISH, the lack of GUI or Sox2 expression in the ActPcat dermis was verified. However, significant expression of Cdknla was detected in the mutant but not in the control dermis (data not shown, Figure 13B). These Cdknla+ cells showed high levels

[0613] of Lefl expression and correlated with a low rate of EdU incorporation, indicating a low rale of proliferation (Figure 13B). These data demonstrate that constitutive activation of Wnt signaling is sufficient to induce expression of Cdknla, independent of SHH activation (Figure 13C). By E14.5, Cdknla was highly upregulated in both control and mutant DCs. However, in contrast to controls in which Cdknla expression almost perfectly overlaps with Sox2 expression, many cells surrounding Sox2+ clusters and within the upper dermis also expressed high levels of Cdknla but not DC markers in the ActPcat mutant. They also showed a low rate of EdU incorporation (Figure 13D). Cdknla+ cells that coexpressed Sox2 in the ActPcat mutant were spatially located nearest to the placode and corresponded to cells with both high Wnt and high SHH activity. Notably, cells that expressed Sox2 in the ActPcat mutant showed highest Ptch1 expression, suggesting a threshold level of SHH activity may be required for DC gene expression even if high levels of Wnt activity are induced. To assess Wnt activity levels caused by expression of activated P-catenin, Lefl levels were quantified by quantitative FISH. As expected, E13.5 ActPcat mutant dermal cells showed higher levels of Lefl expression than control E13.5 dermal cells. By E14.5, higher levels of Lefl are also observed in the mutant dermis compared to controls but were never greater than those seen in control DC cells, indicating that dermal expression of stabilized P-catenin does not induce levels above those seen physiologically in forming DCs (data not shown). Taken together, these results suggest that increased Wnt signaling can induce quiescence independent of SHH, while DC gene expression appears to require Wnt activity as well as high levels of SHH activation (Figures 2F and 2G). Hence, Wnt and SHH interact to induce cell cycle exit or DC gene expression through different mechanisms during DC genesis.

[0614] SHH cell-autonomously augments Wnt signaling to induce cell cycle exit Based on these results, how high SHH affects DC gene expression and cell cycle exit in the absence of high Wnt activity was examined. Uniformly high SHH activity was induced in the dermis by expressing a constitutively activated form of Smoothened Axin2CreER; RosaLSL-Smom2YFP or ActSMO) at E13.5 when Wnt signaling levels are low (tamoxifen El 1.5; Figure 14A). Whole-mount analysis of E14.5 skin showed that high SHH activation in Wnt-active upper dermal cells induced large DCs that formed autonomously

[0615] 85

[0616] 45816336.1without placodes (Figure 14A). Interestingly, Sox2+ cells in the center of these dermal clusters lacked EdU incorporation, indicating that cell cycle exit still occurred. At the same time, many Sox2+ cells in the periphery were actively proliferating as shown by Sox2+ / EdU+ double positive cells (Figure 14A, data not shown)24. The dermal clusters in the ActSMO mutant were also diffuse and less tightly aggregated, suggesting that the coordination of DC gene expression with quiescence may be important for DC formation. This result suggests that high SHH can induce both DC genes and cell cycle exit asynchronously.

[0617] How quiescence can occur if high SHH was induced in cells with low Wnt activity and in the absence of placodes that are typically thought to be the source of high Wnt ligands was tested. Wnt activity was first measured in the E13.5 ActSMO mutant and surprisingly found that Lefl levels in the ActSMO mutant were significantly higher than in E13.5 controls and were similar to that seen in wildtype DC cells (Figure 14B, data not shown). Further similar to the Actpcat mutant, these cells co-expressed high levels of Cdknla and corresponded with lower rates of EdU incorporation (Figure 14C). Notably, only cells that expressed Ptch1 (i.e., cells expressing the ActSMO mutation) showed

[0618] elevated Lefl expression while neighboring wildtype Ptch1 -negative cells (Cre nonrecombined) in the upper dermis showed levels similar to the control upper dermis (Figure 14B). Thus, Lefl expression is amplified cell-autonomously by SHH activation and possibly independent of epidermal Wnt ligands. It was found that many cells with the

[0619] highest Lefl transcript levels frequently were not spatially closest to the epidermis where Wnt ligands are expressed (data not shown). Collectively, these results show that SHH activation cell-autonomously accelerates Wnt activity. High Wnt activity, in turn, induces cell cycle exit, These data also suggest that high levels of dermal Wnt activity seen at E14.5 may be caused by SHH activation rather than by placode Wnt ligands (Figure 14D).

[0620] DC gene expression and cell cycle exit are off sync upon high SHH activation. It was next sought to use this model to dissect out how each process was mediated. Here, employed an innovative computational approach, GeneTrajectory (GT), was used to unbiasedly separate out co-occurring independent biological gene programs from whole transcriptome scRNA-seq data. Briefly, GT is a computational method developed to unbiasedly detect independent gene dynamics from whole transcriptome data in which each GT represents a distinct biological process (data not shown). Traditional cell trajectory inference methods first organize cells into lineages and assign a one-dimensional pseudotime and then gene dynamics are assessed with respect to those cell lineages. A major limitation of these

[0621] 86

[0622] 45816336.1approaches arises when multiple biological processes coexist within the same group of cells (e.g., when cell cycle is coupled with differentiation). Here, the inferred cell pseudotime often conflates the effects of distinct processes, making it difficult to disentangle their respective gene programs. By contrast, GT does not require cell trajectory inference or predefining an initial and terminal cell state. It directly constructs gene trajectories and allows each cell to participate in multiple concurrent biological processes. This is done by computing pairwise optimal transport distances between gene expression distributions over the cell-cell graph from which gene programs, and their pseudo temporal gene order is extracted based on their sequence of activation along the inl'crrcd biological processes. This separates apart coexisting biological processes that are entangled at the cell level into independent gene programs with distinct sequential gene expression patterns data not shown).

[0623] GT was first used to examine E14.5 wildtype dermal scRNA-seq data, which identified three GTs that correspond to cell cycle (CC), DC differentiation and lower dermal (LD) differentiation processes, respectively (data not shown, data not shown). Here, the DC G identifies an order of gene activity in which DC genes are expressed at the terminus along with Cdknla (data not shown). This method was benchmarked against other cell trajectory inference methods, showing superior accuracy in delineating the order of genes involved in the DC differentiation process based on biological validaton28. Different cell stages were then defined along the DC differentiation process by partitioning the DC GT into gene bins, with each bin representing marker genes for a specific stage. For each cell, a gene bin score was computed representing the proportion of genes from a given bin that were expressed. Based on these scores, cells were exclusively assigned to a stage or left unassigned (Method - Gene bin and cell stages, data not shown). Canonical DC genes, including Gal, Soxl8 and Sox2, were largely expressed at the last stage 6 (data not shown). Similarly, the CC GT represents an independent process in which cells are colored according to their expression of genes along a progression from S and G2 / M phases (data not shown). As shown, cells expressing genes along the DC GT also simultaneously express genes that correspond to an independent cell cycle process (proliferation). Measuring the fraction of cells in G1 / G0 phase by DC GT stages showed that cells at stage 6 are quiescent with a G1 fraction near 1.0 (data not shown). At the same time, both Lefl and Ptch1 covary and show increasing levels across DC GT stages with highest levels of both observed at stage 6 (data not shown). It was observed that Wnt activity is coupled to SHH activation in the wildtype condition, and cell cycle exit and molecular DC differentiation occur simultaneously at the terminus (data not shown).

[0624] 87

[0625] 45816336.1A gene program that corresponds to cell cycle exit is identified by GeneTrajectory Based on the observation that there were proliferating Sox2+ cells surrounding a quiescent center in the ActSMO condition, this suggests that cell cycle exit does not always occur simultaneously with DC gene expression. It was asked if GT can be used to separate out the two processes of cell cycle exit and DC gene expression in the ActSMO condition. scRNA-seq data was obtained from El 3.5 ActSMO and paired control skin to exclude any possible confounding effects of endogenous SHH activation and DC formation. As expected, the ActSMO mutant showed uniformly high Ptch1 expression in dermal cells, while Wnt target genes remained localized to cells that express UD markers (data not shown). Interestingly, GT identified four GTs in the ActSMO mutant: one corresponded to CC (S / G2 / M) and another to LD differentiation, which were similar to the control (data not shown, data not shown). As expected, one GT contained DC genes as well as Wnt target genes, which was not detected in the El 3.5 control. Additionally, a new GT was detected in the ActSMO mutant that was not present in the El 3.5 control (data not shown). To characterize this new gene module, pathway analysis was performed. It was found that genes belonging to this GT were most highly associated with regulation of TP53, which is involved in tumor suppression and inhibits cell cycle progression (Figure 15 A). These genes included cell cycle regulators such as Trp53inpl and Plk2, which overlap with Cdknla expression (Figure 15A, data not shown). Cells expressing this gene program were predominantly Wnt-active and restricted to Gl / GO phase (data not shown). Based on these findings, this GT was defined as a cell cycle exit GT (Exit GT). By FISH, both Cdknla and Trp53inpl were significantly upregulated in the E13.5 ActSMO dermis compared to E13.5 control dermis, which showed negligible expression of these genes ( Figure 15B). Wnt activity was first measured in the E13.5 ActSMO mutant by measuring Lefl levels in Trp53inpl+ cells. Consistent with the ability of SHH activation to cell-autonomously upregulate Lefl levels ( Figure 15 A), Trp53inpl+ cells showed significantly higher Lefl transcript levels than El 3.5 control upper dermal cells with some Trp53inpl+ cells showing levels similar to E14.5 wildtype DC cells ( Figure 15B). In contrast to E14.5 wildtype embryos in which Trp53inpl and Cdknla are largely restricted to Sox2+ DC cells, these genes were expressed by both Sox2+ and surrounding Sox2- cells in the El 3.5 ActSMO mutant. This indicates that Exit GT genes are expressed not exclusive in cells that express terminal DC genes in the ActSMO mutant.

[0626] It was examined if this gene program can be detected in the El 3.5 Actpcat mutant, which also induced cell cycle exit but not DC genes. GT method also identified a similar GT in the E13.5 Actpcat dermis, which shared several genes with the E13.5 ActSMO Exit GT,

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[0628] 45816336.1including Trp53inpl along with Cdknla, which was also upregulated in cells expressing this GT (data not shown). To further compare these GTs across conditions, a module projection strategy (i.e., label transfer) was applied to assess if a gene program is preserved in a subset of cells under different conditions. The module score was calculated of the gene set in the other condition and visualized local neighborhoods of cells that exhibited a high module score, indicating that the average expression of the gene program is retained in that cell population. This allowed the identification of shared or condition-specific cell states across samples without requiring sample integration, thus avoiding potential biases introduced by batch correction methods that may obscure true biological differences. Label transfer of the E13.5 ActSMO Exit GT onto the E13.5 Act0cat and paired control showed that the two mutants share a similar gene module and molecular cell state, which was not detected in the control sample (data not shown). Moreover, scRNA-seq data was obtained from El 3.5 Wntless cKO (KI4Cre;\Vl "'fi) embryos, which ablates epidermal Wnt ligands, resulting in decreased dermal Wnt signaling and a lack of DCs or hair follicles (data not shown)30,32. Here, an Exit gene program could not be detected upon label transfer (data not shown), indicating that this Exit GT is Wnt-dependent. These results show that high Wnt activity alone is sufficient to induce genes that regulate cell cycle exit independent of DC gene expression, and this Exit gene program is Wnt-dependent. Furthermore, despite the decreased rate of proliferation seen in the Wntless cKO, an Exit GT was not detected, indicating that this Exit gene program does not simply reflect prolonged G1 phase caused by slower rates of proliferation.

[0629] Although some Sox2+ cells are proliferating in ActSMO dermal clusters at E14.5, most Sox2+ cells are quiescent by E15.5, similar to wildtype E14.5 DC cells (data not shown). It was possible that the proliferating cells in E14.5 ActSMO dermal clusters represent cells that will immediately exit the cell cycle upon cell division similar to wildtype transitioning cells but continuously over days. To assess if the Exit GT represents a gene program expressed by cells that are actively exiting the cell cycle, EdU pulse-chase experiments were performed. First, it was observed that most cells in the ActSMO mutant that express Trp53inpl at both E13.5 and E14.5 show a low rate of EdU incorporation similar to quiescent DC cells ( Figure 15C). It was tested if these quiescent Trp53inpl+ cells are recent progeny of a cell division by pulsing embryos 24 hours prior to harvest. At both El 3.5 and E14.5, there was a significant proportion of Trp53inpl+ cells that retained EdU nucleotide, similar to wildtype E14.5 DC cells, indicating Trp53inpl+ cells are quiescent but many are progeny of a recent cell division. Interestingly, an accumulation of Trp53inpl+

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[0631] 45816336.1cells over days was not observed ( Figure 15C), suggesting that its expression may be downregulated after a cell has undergone cell cycle exit ( Figure 15C). Thus, in the ActSMO condition, Trp53in.pl + cells represent cells that are undergoing cell cycle exit continuously ( Figure 15C). These data also suggest that the Exit GT reflects a process in which cells enter quiescence as immediate progeny of a cell division similar to the cell cycle dynamics characteristic of the normal pre-DC-to-DC transition ( Figure 15D, data not shown).

[0632] Loss of GLI3 chromatin binding regulates cell cycle exit downstream of high Wnt activity

[0633] To next examine how elevated Wnt signaling promotes cell cycle exit, genes differentially bound by CTNNB1 in the E13.5 Act0cat mutant versus control skin were first profiled. Although CTNNB 1 was more bound at Wnt target genes

[0634] (e.g., Sp5, Axin2, Lefl, Twist2, Tcf7), binding to Cdknla or any Exit GT genes were not detected, nor significant binding to genes associated with cell cycle inhibition (data not shown, data not shown). These results are in keeping with other work showing that Wnt signaling is important for upper dermal cell proliferation and indirectly

[0635] inhibits Cdknla expression30. Given the paradoxical finding that high Wnt activity promotes cell cycle exit and that this high Wnt signaling is induced by SHH activation ( data not shown, Figure 14B)24, it was asked if this cell cycle exit program is specifically induced in the context of SHH pathway mediators. It was reasoned that high Wnt activity and cell cycle exit normally is coupled to high SHH activity and DC gene expression during DC formation. Hence, SHH effectors may cooperate with Wnt signaling to induce cell cycle exit. Yet cell cycle exit is observed in the Actflcat mutant at El 3.5 and independent of SHH. As GUI is only expressed upon SHH expression, Gli 1 activator would not likely mediate this effect seen in the Act0cat condition. By contrast, Wnt signaling is known to promote GU3 expression independent of SHH in the neural tube26. Similarly, GU3 was also expressed in Wnt-active cells in both control and Acl[kal dermal cells at El 3.5 prior to SHH (data not shown). GLI3 acts as a SHH transcription repressor in the absence of SHH and binds to a broad range of SHH target genes, including cell cycle regulators in the limb bud33,34. To profile genes that GLI3 binds and that may be regulated in the Act[3cat condition, CUT& RUN was performed to identify GLI3-bound genomic regions at E13.5. Surprisingly, it was found reproducible global reduction of GLI3 binding to chromatin in the Aclf>cal condition, indicated by decreased GLI3-bound read density at transcriptional start sites ( Figure 16A). GLI motifs were the most enriched in the differentially bound regions based on HOMER de novo motif analysis35(data not shown). KEGG pathway analysis also showed that those unbound regions

[0636] 90

[0637] 45816336.1are enriched for genes involved in SHH signaling despite the fact that most canonical SHH target genes are not significantly expressed (Figure 16B). This is consistent with findings that GLI3 binding prior to SHH is actually inert at many GLI3-bound regions and does not actually repress them36. It was also found that loss of GLI3 binding did not derepress many canonical SHH target genes, including Ptch1 or GUI (data not shown). By western blot analysis, it was confirmed that loss of GLI3 binding in the ActPcat condition was not due to decreased protein expression (data not shown). Additionally, Gli3 remained largely expressed in its cleaved repressor form in both the control and mutant, suggesting other mechanisms (e.g., other post-translational modifications or cofactors) likely regulate GLI3 binding. These results show that high Wnt signaling alone unexpectedly induces loss of GLI3 binding at many genomic regions independent of SHH but without derepression of many of those unbound genes.

[0638] Even though many GLI3 -unbound regions were not derepressed in the ActPcat mutant, it was questioned whether a subset of unbound regions may be sufficient to induce cell cycle exit downstream of high Wnt activity. If so, loss of Gli3 itself should be sufficient to induce cell cycle exit. GU3 was genetically ablated in the dermis ( / \xin2CreKR;(ili3ll / l1or Gli3 cKO; tamoxifen El 1.5). At E14.5, DCs of Gli3 cKO embryos were smaller than control DCs (Figure 16C, data not shown). Additionally, Dkkl+ peri-DC progenitors showed aberrant upregulation of Cdknla with lower rates of EdU incorporation compared to controls in which most Dkkl+ cells do not express Cdknla and are proliferative (Figure 16C, data not shown). Examination of El 3.5 Gli3 cKO dermis prior to DC formation also showed that Cdknla was highly upregulated similar to E13.5 ActPcat embryos and correlated with a lower rate of EdU incorporation (Figure 16D). scRNA-seq data was obtained from E13.5 Gli3 cKO embryos and paired controls. Examination of the Gli3 cKO dermal population showed that GH3 gene expression was diminished, but expression of Wnt target genes was not affected, indicating that Gli3 acts downstream of high Wnt activity (data not shown). Also, neither canonical SHH target genes nor DC genes (e.g., Soxl8, Gal) were induced upon loss of GH3, confirming that GLI3 binding does not repress many SHH target genes (data not shown, data not shown). Interestingly, GT identified four GTs in the Gli3 cKO dermal scRNA-seq data (data not shown). Three GTs corresponded to those found in the paired E13.5 control dermal data (UD, LD, CC). An additional GT identified in the mutant data and not found in the control revealed that several of the genes contained in this GT overlapped with those found in the Exit GT of the ActPcat mutant, including Trp53inpl ( data not shown). Similarly, most cells within this GT were confined to G1 / G0 phase and aberrantly

[0639] 91

[0640] 45816336.1expressed Cdknla ( data not shown). Next, genes that were unbound by GLI3 in the E13.5 Act[3cat mutant were intersected to genes differentially expressed in the E13.5 Gli3 cKO versus control. Genes involved in cell cycle inhibition, including Mxd.4 and Efna5, were upregulated and unbound in the Gli3 cKO and contained more active promoter marks (H3K4me3, Figure 16E). It was also found Exit GT genes, Baiap2 and Cptlc, were unbound by GLI3 and upregulated.

[0641] The Exit GT label was also transferred from the ActSMO mutant to the Gli3 cKO, which identified the same Exit GT population ( data not shown), indicating that this gene program is shared among all three mutants (ActSMO, Actpcat, Gli3 cKO). Similar to the ActSMO condition, Cdknla-high cells in the Gli3 cKO showed a low rate of active EdU incorporation, while pulsing embryos with EdU 24 hours prior showed that a significant proportion of Cdknl -high cells are progeny of a recent division (Figure 16D). These data show that both P-catenin and Gli3 function in a common pathway that causes cell cycle exit. As DC genes were not expressed in the Gli3 cKO dermis, these data suggest that high Wnt signaling (downstream of SHH co-activation) induces loss of GL13 binding specifically to regulate cell cycle exit ( data not shown).

[0642] DC gene expression correlates with Wnt activity levels upon SHH activation The E13.5 ActSMO GT that corresponded to DC genes were examined (data not shown). This DC GT showed that the gene order correlated with known DC genes,

[0643] including Gal, Soxl8 and Sox2, which interestingly, were sequentially activated over DC GT stages in a manner that correlated with increasing Lefl levels (Figures 17A and 17B). This was in contrast to the El 4.5 wildtype DC GT in which DC genes were simultaneously activated at the last stage when both Wnt and SHH activity are highest. As SHH target genes are uniformly expressed across the dermis and no longer progresses with DC differentiation, canonical SHH pathway genes were not detected in the ActSMO DC GT. These data show that high SHH activity can induce DC genes in cells with varying levels of Wnt activity and not only in cells that have high levels of Wnt signaling (Figure 17C). However, the level of DC genes expressed corresponds with the level of Wnt activity. This implies that the synchronization of cell cycle exit with high DC gene expression seen in the wildtype scenario may occur by restricting high SHH activity to cells with high Wnt signaling. As cell cycle exit occurs in cells that do not express high DC genes in the ActSMO condition, the level of Wnt signaling important for cell cycle exit would be lower than that required to express high DC genes (Figure 17C).

[0644] 92

[0645] 45816336.1Coupled Wnt and SHH gradients is sufficient to synchronize cell cycle exit with molecular differentiation

[0646] Based on the above results, cell cycle exit and expression of high DC genes are only seen in cells with coupled high Wnt and high SHH activity induced by placode SHH ligand. It was asked if inducing SHH ligand expression across the epidermis is sufficient to accelerate Wnt activity and to couple high levels of Wnt signaling with high SHH activity without placodes. Here, a covarying gradient of Wnt and SHH activation were genetically induced in the upper dermis by expressing SHH ligand uniformly across the epidermis where Wnt ligands are normally expressed prior to hair follicle morphogenesis K14Cre; Rosa-LSL-SHH or SHH OE; Figure 18A). ScRNA-seq analysis of E13.5 SHH overexpressing (SHH OE) mutant and paired control dermal cells showed that both Left and Ptch1 were expressed in an overlapping covarying pattern over the UMAP cell embedding in the mutant (data not shown). Cells with the highest levels of Left and Ptch1 also expressed DC genes

[0647] (e.g., Gal, Soxl8, Sox ). Additionally, Cdknla was largely localized to cells expressing DC genes in contrast to ActSMO dermal cells in which many Cdknla+ cells do not express many DC genes (data not shown, Figure 18B). Notably, GT analysis showed that only three GTs were identified that corresponded to similar gene programs identified in E14.5 wildtype dermal cells (i.e., CC, LD, DC; Figure 18C, data not shown). A separate Exit GT was not detected. Similar to the E 14.5 wildtype DC GT (Figure 13B), the El 3.5 SHH OE DC GT showed a gene order in which Wnt and SHH levels correlate with each other and with DC genes, which were restricted to the last stage (Figure 18C). Cdknla was also activated together with DC genes (Figure 18C). Additionally, label transfer of the ActSMO Exit GT did not detect a separate population in the SHH OE or control dermis (data not shown). Lefl expression was accelerated in the SHH OE compared to the E13.5 control, with the highest levels corresponding to arrest and DC genes (Figure 18D, data not shown). Taken together, these data suggest that covarying levels of Wnt and SHH activity synchronize cell cycle exit with DC genes and recapitulate the wildtype pre-DC-to-DC transition.

[0648] Quantification of Lefl and Ptch1 levels by spatial dermal layer showed that both genes covary with higher Lefl levels in the first two layers of the dermis in the SHH OE compared to controls, consistent with SHH accelerating Lefl levels (Figure 18D). By FISH, it was found DC genes were restricted to the most superficial dermal layers where

[0649] both Lefl and Ptch1 levels were highest and where proliferation is low (Figure 18D, data not shown). GLI3 CUT& RUN was next performed in the mutant and control and found global loss of GLI3 binding at genomic regions similar to the ActPcat mutant (Figure 18G, data not

[0650] 93

[0651] 45816336.1shown), indicating that loss of GLI3 binding represents an endogenous molecular event during DC genesis and not simply an artifact of genetically stabilizing P-catenin. Loss of GLI3 binding was observed at SHH target genes (Ptch1, GUI) but now with increased transcriptional activity as marked by increased H3K4Me3 signal at these genes. This suggests that Gli activator may be required for expression of most SHH target genes and that GLI3 may function to prime those sites for GLI activator upon SHH expression. Loss of binding at cell cycle regulators, Mxd4 and Efna5 similar to the Actpcat mutant was also detected (Figure 18F).

[0652] It was determined whether Wnt and SHH levels can be modulated to recapitulate the interaction between these signals and their regulated biological processes by establishing an in vitro dermal culture system. Previous attempts to culture dermal papilla cells used neonatal dermal cells that contain preformed DC / dermal papilla cells3738. However, they show limited ability to maintain DC / DP markers and immediately lose Wnt, SHH, and DC gene expression. Further, these cells show poor responses to Wnt or SHH agonists. Similar results were found using neonatal dermal cells with only a mild increase in Lefl or Ptch1 expression upon Wnt agonism with CHIR99021 (CHIR) or SHH agonism (SAG), respectively (data not shown It was observed that HD AC inhibitors incrementally augmented CHIR Lefl responses but had no effect on SAG responses (data not shown). Next cultured were dermal cells FACS isolated from El 3.5 PDGFRaH2BGFP embryos. In contrast to neonatal fibroblasts, El 3.5 dermal cells showed robust dose-dependent target gene responses to either CHIR or SAG after 48-72 hours of culture (data not shown).

[0653] Using this model, it was determined whether SHH activation can augment Wnt signaling by assessing the effect of SAG on CHIR-induced Lefl gene expression. It was found that SAG treatment boosted Lefl expression in CHlR-treated cells (Figure 18G). Moreover, the fold increase in Lefl expression was significantly more in cells treated with higher levels of CHIR (5 pM vs 2 pM) upon SAG treatment, suggesting that the amount of Wnt acceleration depends on the initial levels of Wnt activity (Figure 18G). Consistent with in vivo results, high levels of CHIR (5 pM) alone are sufficient to induce Cdknla expression independent of SHH activation (data not shown), while co-treatment with CHIR and SAG induced DC genes such as Gal and Soxl8 (Figure 18H). Importantly, consistent with the in vivo E14.5 Actpcat results (data not shown), DC gene induction was SAG-dependent, indicating that higher levels of SHH activation is essential for DC gene expression.

[0654] Altogether, these results demonstrate that levels of Wnt and SHH activation can be tuned to

[0655] 94

[0656] 45816336.1coordinate cell cycle exit and DC gene expression and can be used to recapitulate these events in vitro.

[0657] Discussion

[0658] To make functional organs, stem and progenitor cells must precisely coordinate proliferation with differentiation, so that distinct cell types assemble into organized populations of the correct size, location, and relative cell proportions. In many tissues, cell cycle exit is associated with terminal differentiation1. In certain scenarios such as in mouse or human embryonic stem cells, G1 phase represents a permissive state in which cells are more responsive to cell type transcription factors that induce differentiation genes2 4. Here, cells may asynchronously differentiate upon entering G1 / G0 phase, and the signals that regulate cell cycle arrest may be independent of those that induce differentiation. In other scenarios such as during cortical neurogenesis, cell cycle regulators may have a dual function in regulating cell type differentiation such that differentiation occurs synchronously with cell cycle phase5,6. During development and regeneration, cells can also progress through intermediate states of differentiation (e.g., transit- amplifying progenitors) before undergoing cell cycle exit and terminal differentiation7 11. Here, intermediate states of differentiation are coordinated with proliferation. At the same time, failure to coordinate these events can lead to pathological states such as cancer. Therefore, understanding how these processes are coupled is important for efforts to promote proper regeneration and to mitigate uncontrolled and autonomous growth.

[0659] A challenge to examining this question during development or regeneration is that different cell types dynamically undergo a mixture of unique and shared biological processes as they progress along a lineage trajectory. This makes it challenging to accurately delineate the progression of cell states within a lineage and the biological processes that govern transitions between cell states. Knowledge of a cell’s past and future cell state has been largely dependent on biased tools that durably label specific cell types or cell states. Live imaging has given the ability to track individual cells over time in the same tissue allowing for real-time analysis of cellular differentiation12-15. These tools have been instrumental to the understanding of lineage trajectories and the signals that regulate cell fate decisions. Still, dissecting out the specific mechanisms of how cell proliferation and differentiation are coordinated remains limited by the lack of tools and biomarkers to track dynamic changes in these biological processes and to interrogate them individually, especially when they occur concurrently. With the emergence of single-cell RNA-sequencing (scRNA-seq) technology, the ability to profile molecular differences between cells has provided a useful tool to infer

[0660] 95

[0661] 45816336.1signals that regulate cell type differentiation. Still, cell-based trajectory methods often fail to accurately delineate independent processes when they co-occur in the same group of cells, as they collapse cells into a single pseudo temporal path that mixes their distinct dynamics. Thus, the molecular programs that coordinate tissue growth and cell type patterning remain poorly defined in many tissues and poses a major barrier to efforts aimed at recapitulating organ formation in vitro or in adult animals.

[0662] In this study, this question was examined using a defined biological system that represents the first differentiated population in mammalian dermis and heralds the onset of dermal lineage diversification16 18. In mouse embryos, the hair follicle dermal condensate (DC) emerges at around E14.0-E14.5 in association with overlying epithelial placodes19 23. DCs are clusters of specialized quiescent dermal cells that are essential for hair follicle formation. A major obstacle to delineating the sequence of steps that lead to DC genesis is the fact that DC cells form through an acute cell fate transition with few observable intermediate states to identify and track DC progenitors using conventional methods. To address this issue, scRNA-seq was used to infer events and signals that precede DC commitment24,26. Combined with in vivo lineage tracing, it was shown that DC cells are immediate quiescent progeny of a highly proliferative population (Dkk / -ex pressing) that divides over a short time frame before DC gene expression and quiescence. It was shown that dermal Wnt / p-catenin and SHH signaling, together, are required and sufficient to induce DC genesis. In other biological systems (e.g., hair follicle epithelial placode, neural tube), Wnt and SHH signaling antagonize each other13,26,27. It was shown that in the dermis, they unexpectedly cooperate to induce DC genesis. However, it remains unknown how Wnt and Shh act together intracellularly to coordinate a cell fate transition in which cell cycle exit is tightly coupled with molecular DC differentiation, especially as Wnt and SHH antagonize each other in other systems. As such, the DC system provides a unique opportunity to understand how these two key signals can be differentially combined to regulate diverse cell fate patterns and transitions across tissues.

[0663] One problem is that both Wnt and SHH ligands are secreted from a common epidermal source, resulting in covarying and overlapping Wnt and SHH signaling gradients in which both signals are simultaneously upregulated across the transition. Furthermore, both cell cycle exit and molecular DC differentiation occur synchronously across the pre-DC-to-DC transition, making it difficult to separate them apart and to identify their respective molecular mediators. As neither Wnt nor SHH activation alone is sufficient to induce the transition, an approach was tested to dissect out how these two signals regulate the pre-DC-

[0664] 96

[0665] 45816336.1to-DC transition. Here, an innovative computational method recently developed specifically to deconvolve co-occurring biological processes from whole-transcriptome scRNA-seq data was used28. In contrast to standard cell trajectory inference methods, GeneTrajectory (GT), identifies parallel gene trajectories, allowing independent biological processes to be resolved. Using this method and genetic models that modulate Wnt or SHH activity, it was shown that while cell cycle exit and DC gene expression occur concurrently, they are not actually regulated by shared downstream effectors as is the case in other tissues (e.g. neurogenesis). Rather, they are synchronized by the mere coupling of upstream Wnt and SHH co-activation, which when combined, result in a sharp cell fate transition. In particular, it was shown that Wnt and SHH signaling induce cell cycle exit and molecular differentiation through distinct downstream effectors, which revealed a previously unknown role for Gli3 in cell cycle exit. By genetically decoupling these two graded morphogens cell cycle exit and DC differentiation occur asynchronously, resulting in a gradual transition across time. Notably, it was shows that the observed overlapping levels of Wnt and SHH during normal DC formation is caused by their ability to modulate each other, thereby facilitating their paired activity and the synchronization of cell cycle arrest and DC differentiation. This study provides a model to understand how other tissues may differentially combine morphogen signals to balance proliferation with differentiation during patterned tissue growth.

[0666] The results fit with a scenario in which cell cycle exit is tightly coupled to differentiation. The decision to exit the cell cycle may be made in G2 / M phase or in G1 phase. To explain this coupling, previous work has proposed a direct model in which cell cycle regulators have a dual role in regulating cell cycle progression and cell type differentiation (e.g. activating cell type-specific transcription factors). Here, an unexpected mechanism is revealed in which these two processes are independently mediated but are coupled to each other due to the mere coupling of Wnt and SHH activity. In the DC system, Wnt activity levels are mechanistically dependent on SHH activation and explain the synchronization of cell cycle exit and molecular DC differentiation, yet the processes themselves are separable because the threshold level of Wnt activity required for cell cycle exit is less than that required for expressing maximal DC genes (Figure 6). This contrasts with other models in which the processes are coupled to each other by co-dependent downstream mediators. The results also demonstrate that two covarying gradients do not merely reflect overlapping independent signals induced by a common ligand source. Rather, the spatial gradients seen during DC formation, in part, reflect the dynamic interaction between Wnt and SHH to regulate each other and to coordinate distinct biological processes

[0667] 97

[0668] 45816336.1over a cell fate transition. Specifically, SHH is sufficient to cell-autonomously amplify Wnt activity, and the high levels of Lefl normally seen in DC cells may be caused by SHH coactivation rather than by local extrinsic placode Wnt ligands. In the wild-type scenario, only a subset of cells that have sufficient pre-existing Wnt activity (dermal layer 1 or peri-DC cells) will induce cell cycle exit and expression of all DC genes synchronously once their Wnt activity is accelerated by SHH co-activation (Figure 7L). Therefore, the coordination of Wnt and SHH signaling levels in pre-DC progenitors is likely due to the spatial proximity of these cells to epidermal cells that produce both SHH and Wnt ligands. However, the acceleration of Wnt signaling that initiates the pre-DC-to-DC transition is independent of epidermal Wnt ligands. How SHH boosts Wnt activity in dermal cells is unclear, but it is likely indirect, as GLI transcription factors were not found to bind to Wnt target genes, including Lefl. This model may also help explain how other scenarios can time proliferation with differentiation. For example, hair follicle epithelial stem cells rely on Wnt and SHH activation during anagen phase and give rise to transit- amplifying cells that undergo proliferation and differentiation before they terminally arrest into different spatially organized cell types. It is possible that relative levels of Wnt and SHH (or other signals) regulate the coordination of these two processes to time when a progenitor exits the cell cycle and the specific cell type genes it expresses. From a different perspective, when distinct biological processes of a cell fate transition occur synchronously, there may be few intermediate states to capture the transition, giving the appearance that a population emerged from “thin air.”

[0669] In the ActSMO condition, uniformly high SHH activation was induced in cells with varying Wnt activity, and the number and expression level of DC genes correlated with Wnt signaling levels. However, in the wild-type condition, DC marker genes are only expressed in cells that show both high Wnt and high SHH activity. This suggests that a threshold level of SHH activity, which is induced in the ActSMO mutant, is required for DC gene expression. It is plausible that high Wnt activity can reciprocally accelerate SHH activity to ensure that only cells with high Wnt activity activate high SHH signaling to induce DC genes. Consistent with this, cells in the peri-DC region of the E14.5 Act0cat mutant exhibited higher levels of Wnt activity than control peri-DC cells but also higher Ptch1 levels than in the control, suggesting that Wnt activity levels may regulate SHH activity. Such an intricate feedback mechanism between Wnt and SHH signaling may deterministically drive a rapid sequence of cell cycle exit and DC gene expression.

[0670] How is cell cycle exit induced upon high Wnt activity? Wnt signaling is known to promote Gl-S transition and is typically associated with sternness and proliferation.

[0671] 98

[0672] 45816336.1Consistent with this, loss of dermal Wnt activity prior to SHH has been shown to result in decreased dermal cell proliferation with concomitant loss of dermal cell density. Inducing high Wnt activity, above that seen prior to SHH, results in cell cycle exit. This high Wnt signaling unexpectedly results in a global reduction in GLI3 binding to chromatin, and loss of GU3 is sufficient to mediate cell cycle exit. Interestingly, canonical SHH target genes such as Ptch1 or GUI were not derepressed despite loss of GLI3 binding at those sites in the ActPcat mutant. These genes become expressed upon SHH, suggesting that GUI activator (e.g., GIJ1) is required to activate those GLI sites. It remains unclear how high Wnt signaling causes GLI3 unbinding. Other transcription cofactors or GLI3 post-translational modifications may regulate GLI3 binding. Prior to SHH, Wnt signaling may promote GLI3 binding at canonical and non-canonical SHH target genes. It is possible that upon SHH, cell cycle exit is immediately induced by GLI3 unbinding (after SHH-induced Wnt amplification), while DC and SHH target genes may be activated soon after upon GUI expression and GLI1 binding to those unbound regions. The global loss of GLI3 binding upon acceleration of Wnt signaling could serve as a mechanism to couple high Wnt activity with efficient upregulation of SHH activity. This would allow that DC genes are only expressed coincident with or just after cell cycle exit. In the ActSMO condition, molecular DC differentiation occurs while cells are still proliferating. These dermal clusters are diffuse with fuzzy borders and largely fail to contact the epidermis and do not induce placodes. The clustering of DC cells and their ability to communicate with the epidermis may rely on their ability to stop dividing and to form stable dense clusters. It will be interesting to assess whether cell-cell adhesion genes or extracellular matrix genes are affected by cell cycle regulators.

[0673] This study also underscores the utility of computational methods to provide mechanistic insight into complex cell fate transitions by separating independent gene programs. This approach was useful in identifying GH3 as a molecular mediator that regulates a specific subpopulation of G1 / G0 phase cells, which represent immediate quiescent progeny of a division. The possibility of distinguishing heterogeneity within G1 / G0 phase cells during lineage specification holds the potential to uncover mechanisms that dictate if and when a cell will undergo cell cycle arrest, as well as how differentiation is balanced with cell divisions (e.g., asymmetric cell divisions in stem cells and transit-amplifying cells). From a translational perspective, this study sheds light on how distinct biological processes of DC formation can be tuned to grow and differentiate DC cells in vitro, an important step toward generating DC organoids and organotypic hair follicles to model hair growth and disease.

[0674] 99

[0675] 45816336.1References:

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[0723] Example 2: Identification of Exemplary Small-Molecule Modulators of DC Formation and DC-Associated Signaling Using an Exemplary Screening Platform as Disclosed Materials and Methods

[0724] Additional experimental results, particularly data figures, not presented in this example can be found in Li, et al., (2025), “Interaction of Wnt and SHH gradients synchronizes cell cycle exit and differentiation, bioRxiv, 660399, the contents of which is incorporated herein by reference in its entirety.

[0725] Isolation and Culture of Embryonic Dermal Cells

[0726] Embryonic day 13.5 (El 3.5) mouse skin was harvested, and dermal cells were isolated using established dissociation procedures. PDGFRa-GFP -positive dermal cells were enriched by fluorescence-activated cell sorting (FACS) and plated for in-vitro culture. Cells were maintained under standard culture conditions and allowed to recover prior to treatment. Cultures were maintained for 48 hours following being treated with the Y-compound, unless otherwise indicated.

[0727] Small-Molecule Compounds and Ligands

[0728] A series of Y-series small molecules were evaluated as exemplary modulators of Wnt and Hedgehog (SHH) pathway activity. Compounds were prepared as stock solutions in DMSO and diluted into culture media immediately prior to use.

[0729] 101

[0730] 45816336.1Control treatments included DMSO vehicle control, CHIR99021 (Wnt pathway activation control), Recombinant Wnt3a (80 ng / mL), Smoothened agonist (SAG; SHH pathway activation control), and Combined Wnt3a + SAG conditions.

[0731] Compounds were tested alone and in combination with Wnt3a and / or SAG as indicated in each experiment.

[0732] Candidate compounds were identified based on their ability to modulate Lefl expression alone or in combination with Wnt3a and / or SAG, as well as their capacity to induce Ptch1 and Gal expression under combinatorial signaling conditions. Compounds exhibiting precipitation or solubility limitations upon dilution were noted for further testing and adjustments. The Y-series compounds that were tested were diluted from 100 mM to 50 pM for the in vitro testing. The candidate compounds included Y-0309618 (Y18), Y- 0309620 (Y20), and Y-0309613 (Y13). The structures for these compounds are provided below

[0733]

[0734] Wnt Agonist 1 - N-[6-(2-methoxy-4-methylphenoxy)pyridin-3-yl]-lH,2H,3H,4H- pyrrolo [ 1,2-a] pyrazine-6-carboxamide

[0735]

[0736] Wnt Agonist 2 - 4-{[(l-tert-butylpiperidin-4-yl)carbamoyl]amino}-N, N- dimethylpyridine-2-carboxamide

[0737] 102

[0738] 45816336.1Experimental Design and Treatment Conditions

[0739] Dermal cells were treated with individual Y-series compounds in the presence or absence of Wnt3a and / or SAG to assess pathway-specific and combinatorial signaling responses. Treatments were performed in parallel with appropriate pathway controls to allow comparison across experimental conditions. Each treatment condition was assessed after 48 hours of exposure.

[0740] RNA isolation and Quantitative PCR (qPCR)

[0741] Total RNA was extracted from treated dermal cell cultures using standard RNA isolation methods. cDNA was synthesized using reverse transcription. qPCR was performed to measure expression of pathway-responsive and dermal condensate-associated genes. The following markers were analyzed: Lefl as a readout of canonical Wnt signaling, Ptch1 as a readout of SHH pathway activation, and Gal as a marker associated with dermal condensate differentiation. Gene expression levels were normalized to internal reference genes and expressed relative to DMSO-treated controls.

[0742] Results

[0743] Lefl induction in E13.5 dermal GFP+cells treated with Y-series compounds Quantitative PCR analysis of Lefl expression was performed in E13.5 PDGFRa-GFP+dermal cells following 48-hour treatment with Y-series compounds and pathway controls. Treatment with the Wnt pathway agonists Wnt3a and CHIR99021 resulted in increased Lefl expression relative to DMSO-treated controls (Figure 8).

[0744] Among the Y-series compounds, those classified as SS-LRP6-Frizzled modulators (orange bars) elicited variable but, in several cases, robust Lefl induction. Y20 produced the strongest Lefl response across all Y-compound conditions, exceeding the induction observed with CHIR99021 (Figure 8). Y18 also significantly increased Lefl expression relative to DMSO, albeit to a lesser extent than Y20 (Figure 8). Y13 induced a moderate increase in Lefl expression, positioning it between the strongest and weakest SS-LRP6-Frizzled responders (Figure 8).

[0745] These results identify Y20 as the most potent Lefl -inducing compound in this assay, with Y18 and Y13 showing intermediate activity, and demonstrate that distinct classes of Wnt modulators differentially regulate the magnitude of Wnt target gene activation in embryonic dermal cells.

[0746] 103

[0747] 45816336.1\ nt3a-dependent Lefl induction by Y-series compounds

[0748] Relative Lefl expression was quantified by qPCR in El 3.5 PDGFRa-GFP+dermal cells treated with Y-series compounds alone or in combination with Wnt3a and / or the SHH agonist SAG. The positive control, CHIR99021, robustly induced Lefl expression, whereas the negative control alone, SAG, did not substantially increase Lefl levels (Figure 9).

[0749] Among the Y-series compounds tested, Y20 exhibited minimal Lefl induction when administered alone but showed a pronounced and statistically significant increase in Lefl expression when combined with Wnt3a, resulting in the highest Lefl levels observed across all experimental conditions, including CHIR99021 (Figure 9). This enhancement was Wnt3a-dependent, as the addition of SAG to Y20 did not further augment Lefl expression.

[0750] In contrast, Y 18 demonstrated low Lefl expression across all conditions tested, including in the presence of Wnt3a or SAG (Figure 9). Y13 induced moderate Lefl expression relative to DMSO and showed a further increase when combined with Wnt3a and SAG (Figure 9), indicating a weaker but detectable responsiveness that may depend on combined pathway stimulation.

[0751] Collectively, these results identify Y20 as a potent amplifier of Wnt ligand-dependent Lefl expression, while Y13 exhibits moderate Lefl induction under combined Wnt / SHH conditions and Y18 displays minimal activity in this assay.

[0752] Ptch1 activation by Y-series compounds in the presence or absence of SAG Relative Ptch1 expression, a canonical readout of Hedgehog pathway activation, was measured by qPCR in El 3.5 PDGFRa-GFP+dermal cells treated with the candidate Y-series compounds alone or in combination with the SHH agonist SAG and / or Wnt3a. SAG robustly induced Ptch1 expression compared with DMSO controls, whereas Wnt3a alone did not substantially activate Ptch1 (Figure 10).

[0753] The induction of Ptch1 by SAG was modestly dampened in the context of Wnt pathway activation, as evidenced by reduced Ptch1 levels in conditions combining SAG with Wnt agonists relative to SAG alone. Treatment with CHIR99021 in combination with SAG similarly resulted in attenuated Ptch1 induction compared with SAG alone, indicating that elevated Wnt signaling can partially suppress SHH transcriptional output (Figure 10).

[0754] Among the Y-series compounds, Y20 and Y13 exhibited increased Ptch1 expression when combined with SAG, with Y20 + Wnt3a + SAG producing the strongest Ptch1 response among the Y-compound conditions tested. In contrast, Y18 showed minimal Ptch1 induction across all treatment conditions, including in the presence of SAG (Figure 10).

[0755] 104

[0756] 45816336.1Together, these data demonstrate that Ptch1 induction in dermal cells is primarily SAG-dependent and is modestly attenuated by concurrent Wnt activation, while select Y-series compounds, particularly Y20 and Y13, can support SHH pathway activation under combinatorial signaling conditions.

[0757] Gal induction by Y-series compounds under combined Wnt and SHH signaling Relative expression of Gal, a marker associated with dermal condensate (DC) differentiation, was measured by qPCR in El 3.5 PDGFRa-GFP+dermal cells treated with Y-series compounds alone or in combination with Wnt3a and the SHH agonist SAG. Gal expression was low under basal conditions and modestly induced by Wnt3a or SAG alone, with higher induction observed under combined Wnt and SHH pathway activation (Figure 11).

[0758] Among the Y-series compounds tested, Y-0309620 (Y20) in combination with Wnt3a and SAG produced the strongest Gal induction observed across all conditions, exceeding Gal expression levels induced by the CHIR99021 + SAG (Figure 11). This indicates that Y20 robustly promotes DC-associated gene expression under combinatorial Wnt / SHH signaling conditions.

[0759] Y13 also exhibited significant Gal induction when combined with Wnt3a and SAG, although the magnitude of induction was lower than that observed with Y20 (Figure 11). In contrast, Y18 showed minimal Gal expression across all treatment conditions, including in the presence of Wnt3a and SAG (Figure 11).

[0760] Overall, these results demonstrate that Y20 is the most effective inducer of DC marker expression in this assay, with Y13 displaying moderate activity under combined Wnt / SHH stimulation and Y18 exhibiting little to no activity, consistent with their relative effects on Wnt and SHH pathway readouts described above.

[0761] 105

[0762] 45816336.1

Claims

1. We claim:

1. A method of producing dermal condensate (DC) cells, comprising:(a) contacting dermal progenitor cells in vitro with an effective amount of a Wnt pathway agonist and an effective amount of a Sonic hedgehog (Shh) pathway agonist, wherein the contacting in (a) induces the dermal progenitor cells to form dermal condensate cells, andwherein the Wnt pathway agonist is a compound of Formula (I):Formula (I)wherein L is absent or is a linker;wherein R2 comprises a phenyl, aryl, or heteroaryl group optionally substituted with one or more substituents independently selected from C1-6 alkyl group and C1-6 alkoxy group; andwherein Ri comprises a fused bicyclic ring system comprising 8 to 14 ring atoms, wherein the fused bicyclic ring system comprises at least one ring that is a 5- or 6-membered ring containing at least one nitrogen atom; or a pharmaceutically acceptable salt thereof; orwherein the Wnt pathway agonist is a compound of Formula (II):R-A H H'RI'Formula (II)wherein Rf comprises a pyridine ring substituted with -C(O)NR3R4; wherein R2’ comprises a piperidine ring optionally substituted at the nitrogen atom with Rs’:wherein R3and R4are independently selected from hydrogen and C1-6 alkyl group;wherein R5’, when present, is a C1-6 alkyl group;10645816336.1or is a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the dermal progenitor cells are contacted with the Wnt pathway agonist and the Shh pathway agonist simultaneously, orwherein the dermal progenitor cells are contacted with the Wnt pathway agonist prior to contacting the dermal progenitor cells with the Shh pathway agonist.

3. A method of inducing formation of dermal condensate cells comprising:(a) contacting dermal progenitor cells with an effective amount of a Wnt / p-catenin pathway agonist to induce Wnt-dependent priming and proliferation of the dermal progenitor cells; and(b) contacting Wnt-primed dermal progenitor cells from step (a) with an effective amount of a Sonic hedgehog (SHH) pathway agonist to induce differentiation of the dermal progenitor cells into dermal condensate cells.

4. A method of inducing formation of dermal condensate cells comprising inducing dermal condensate progenitor cells to undergo a phase of Wnt-dependent proliferation by contacting the cells an effective amount of Wnt agonist, followed by inducing the progenitor cells to differentiate into dermal condensate cells by contacting the cells with an effective amount of Shh agonist, optionally wherein the Wnt agonist and Shh agonist are administered to the skin of human subject at the same or different times.

5. The method of any one of claims 1-4, wherein the Wnt pathway agonist is a compound of Formula (I):Formula (I)wherein L is absent or is a linker;wherein R2 comprises a phenyl, aryl, or heteroaryl group optionally substituted with one or more substituents independently selected from Ci-6 alkyl group and Ci-6 alkoxy group; andwherein Ri comprises a fused bicyclic ring system comprising 8 to 14 ring atoms, wherein the fused bicyclic ring system comprises at least one ring that is a 5- or 6- membered ring containing at least one nitrogen atom;10745816336.1or is a pharmaceutically acceptable salt thereof.

6. The method of claim 5, wherein the linker L is present and is an ether linker (-O-) and / or wherein R2 is a phenyl group substituted with one or more substituents independently selected from C1-6 alkyl group and C1-6 alkoxy group.

7. The method of claim 5, wherein the fused bicyclic ring system comprises at least one ring that is a 5- or 6-membered ring containing at least two nitrogen atoms.

8. The method of claim 5, wherein Ri has the following structure:

9. The method of claim 5, wherein L is present and is an ether linker (-O-) and R2 has the following structure:

10. The method of claim 5, wherein the compound has the following structure:

11. The method of any one of claims 1-4, wherein the Wnt pathway agonist is a compound of Formula (II):oFormula (II)wherein Rf comprises a pyridine ring substituted with -C(O)NR3R4;10845816336.1wherein R2’ comprises a piperidine ring optionally substituted at the nitrogen atom with Rs’;wherein R3and R4are independently selected from hydrogen and C1-6 alkyd group; wherein R5’, when present, is a C1-6 alky l group;or is a pharmaceutically acceptable salt thereof.

12. The method of claim 11, wherein R3and R4are both methyl groups.

13. The method of claim 11, wherein R5’ is present and is a tert-butyl group.

14. The method of claim 11, wherein RT has the following structure:

15. The method of claim 11, wherein R2’ has the following structure:

16. The method of claim 11, wherein the compound has the following structure:

17. The method of claim 5 or 11. wherein the concentration of the Wnt pathway agonist ranges from 0.001 nM to 200 mM, optionally from about 0.1 pM to about 100 pM, optionally about 50 pM18. The method of any one of claims 1-4, wherein the one or more Shh pathway agonists are selected from SAG (Smoothened agonist), purmorphamine, Hh-Ag1.5, SAG21k, GSA-10, oxysterols, 20(S)-hydroxycholesterol, 22(S)-hydroxycholesterol, and combinations thereof.10945816336.

119. The method of any one of claims 1-4, wherein the concentration of the Shh pathway agonist ranges from 0.01 to 200 mM, optionally from about 0.1 nM to about 100 pM, optionally about 50 nM20. The method of any one of claims 1-4, wherein the dermal progenitor cells are human, rat, or mouse dermal progenitor cells.

21. The method of any one of claims 1-4, wherein contact between the dermal progenitor cells and the Wnt pathway agonist and / or Shh pathway agonist comprises culturing the dermal progenitor cells in the presence of the Wnt pathway agonist and / or Shh pathway agonist.

22. The method of claim 21,wherein culturing the dermal progenitor cells comprises culturing the cells in a growth medium comprising a basal medium supplemented with serum and one or more antibiotics, andwherein the dermal progenitor cells are cultured for a period sufficient to induce dermal condensate formation.

23. The method of claim 22, wherein the basal medium comprises DMEM supplemented with fetal bovine serum (FBS), L-glutamine, and penicillin-streptomycin.

24. The method of claim 21, wherein the dermal progenitor cells are cultured at a density from 1 x 10sto 5 x 106cells per well.

25. The method of claim 21, wherein the dermal progenitor cells are cultured in the presence of the Wnt pathway agonist and the Shh pathway agonist for 24 to 96 hours.

26. A method of treating or preventing a hair- or scalp-related condition in a subject in need thereof, comprising:(i) administering to the subject, an effective amount of a composition comprising one or more Wnt pathway agonists and one or more Sonic hedgehog (Shh) pathway agonists, wherein the Wnt pathway agonist is a compound of Formula (I):Formula (I)wherein L is absent or is a linker;11045816336.1wherein R2 comprises a phenyl, aryl, or heteroaryl group optionally substituted with one or more substituents independently selected from C1-6 alkyl group and C1-6 alkoxy group; andwherein Ri comprises a fused bicyclic ring system comprising 8 to 14 ring atoms, wherein the fused bicyclic ring system comprises at least one ring that is a 5- or 6- membered ring containing at least one nitrogen atom; or a pharmaceutically acceptable salt thereof; or wherein the Wnt pathway agonist is a compound of Formula (11):oFormula (11)wherein Ri’ comprises a pyridine ring substituted with -C(O)NR3R4; wherein R2’ comprises a piperidine ring optionally substituted at the nitrogen atom with R5';wherein R3and R4are independently selected from hydrogen and Cue alkyl group;wherein R5’, when present, is a C1-6 alkyl group, or is a pharmaceutically acceptable salt thereof,wherein the administering increases hair follicle activity, hair regeneration, or maintenance of hair growth.

27. The method of claim 26, wherein the hair- or scalp-related condition is selected from androgenetic alopecia, female pattern hair loss, male pattern hair loss, alopecia areata, telogen effluvium, hair thinning, hair follicle miniaturization, or scalp dysfunction, or a combination thereof.

28. A method of increasing dermal condensate cells in a subject in need thereof, comprising:(i) administering to the subject, an effective amount of a composition comprising one or more Wnt pathway agonists and one or more Sonic hedgehog (Shh) pathway agonists, wherein the Wnt pathway agonist is a compound of Formula (I):11145816336.1Formula (I)wherein L is absent or is a linker;wherein R2 comprises a phenyl, aryl, or heteroaryl group optionally substituted with one or more substituents independently selected from C1-6 alkyl group and C1-6 alkoxy group; andwherein Ri comprises a fused bicyclic ring system comprising 8 to 14 ring atoms, wherein the fused bicyclic ring system comprises at least one ring that is a 5- or 6-membered ring containing at least one nitrogen atom; or a pharmaceutically acceptable salt thereof; orwherein the Wnt pathway agonist is a compound of Formula (II):R-A H H'RI'Formula (II)wherein Rf comprises a pyridine ring substituted with -C(O)NR3R4; wherein R2’ comprises a piperidine ring optionally substituted at the nitrogen atom with R5’;wherein R3and R4are independently selected from hydrogen and C1-6 alkyl group;wherein R5’, when present, is a C1-6 alkyl group, or is a pharmaceutically acceptable salt thereof,wherein the administering induces formation, expansion, or maintenance of dermal condensate cells in skin or hair-bearing tissue of the subject.

29. The method of claim 28, wherein the subject is has or is at risk of developing androgenetic alopecia, female pattern hair loss, male pattern hair loss, alopecia areata, telogen effluvium, hair thinning, hair follicle miniaturization, or scalp dysfunction, or a combination thereof.11245816336.

130. The method of claim 26 or 28, wherein:the one or more Wnt pathway agonists and the one or more Shh pathway agonists are administered together in a single formulation; orthe one or more Wnt pathway agonists are administered prior to the one or more Shh pathway agonists.

31. The method of claim 26 or 28, wherein the Wnt pathway agonist is a compound of Formula (I):Formula (I)wherein L is absent or is a linker;wherein R2 comprises a phenyl, aryl, or heteroaryl group optionally substituted with one or more substituents independently selected from C1-6 alkyl group and C1-6 alkoxy group; andwherein Ri comprises a fused bicyclic ring system comprising 8 to 14 ring atoms, wherein the fused bicyclic ring system comprises at least one ring that is a 5- or 6-membered ring containing at least one nitrogen atom;or is a pharmaceutically acceptable salt thereof.

32. The method of claim 31, wherein the linker L is present and is an ether linker (-O-).

33. The method of claim 31, wherein R2 is a phenyl group substituted with one or more substituents independently selected from C1-6 alkyl group and C1-6 alkoxy group34. The method of claim 31, wherein the fused bicyclic ring system comprises at least one ring that is a 5- or 6-mcmbcrcd ring containing at least two nitrogen atoms.

35. The method of claim 31, wherein Ri has the following structure:

36. The method of claim 31, wherein L is present and is an ether linker (-O-) and R2 has the following structure:11345816336.1— o37. The method of claim 31, wherein the compound has the following structure:N / •NH38. The method of claim 26 or 28, wherein the Wnt pathway agonist is a compound of Formula (II):oFormula (II)wherein R comprises a pyridine ring substituted with -C(O)NR3R4; wherein R2’ comprises a piperidine ring optionally substituted at the nitrogen atom with Rs’;wherein R3and R4are independently selected from hydrogen and C1-6 alkyd group; wherein Rs’, when present, is a C1-6 alkyl group;or is a pharmaceutically acceptable salt thereof.

39. The method of claim 38, wherein R3and R4are both methyl groups.

40. The method of claim 38, wherein Rs’ is present and is a tert-butyl group.

41. The method of claim 38, wherein Ri’ has the following structure:11445816336.

142. The method of claim 38, wherein R2’ has the following structure:

43. The method of claim 38, wherein the compound has the following structure:

44. The method of claim 31 or 38, wherein the concentration of the Wnt pathway agonist ranges from 0.001 nM to 200 mM, optionally from about 0.1 pM to about 100 pM, optionally about 50 pM.

45. The method of claim 26 or 28, wherein the one or more Shh pathway agonists are selected from SAG (Smoothened agonist), purmorphamine, Hh-Ag1.5, SAG21k, GSA-10, oxysterols, 20(S)-hydroxy cholesterol, 22(S)-hydroxy cholesterol, and combinations thereof.

46. The method of claim 45, wherein the concentration of the Shh pathway agonist ranges from 0.01 to 200 mM, optionally from about 0.1 nM to about 100 pM, optionally about 50 nM47. A composition comprising a Wnt agonist, optionally a sonic hedgehog (SHH) agonist; and optionally one or more pharmaceutically or cosmetically acceptable excipients, in a carrier;wherein the Wnt agonist is a compound of Formula (I):Formula (1)wherein L is absent or is a linker;11545816336.1wherein R2 comprises a phenyl, aryl, or heteroaryl group optionally substituted with one or more substituents independently selected from C1-6 alkyl group and C1-6 alkoxy group; andwherein Ri comprises a fused bicyclic ring system comprising 8 to 14 ring atoms, wherein the fused bicyclic ring system comprises at least one ring that is a 5- or 6-membered ring containing at least one nitrogen atom;or is a pharmaceutically acceptable salt thereof.

48. The composition of claim 47, wherein the linker L is present and is an ether linker (-O-).

49. The composition of claim 47, wherein R2 is a phenyl group substituted with one or more substituents independently selected from C1-6 alkyl group and C1-6 alkoxy group 50. The composition of claim 47, wherein the fused bicyclic ring system comprises at least one ring that is a 5- or 6-membered ring containing at least two nitrogen atoms.

51. The composition of claim 47, wherein Ri has the following structure:

52. The composition of claim 47, wherein L is present and is an ether linker (-O-) and R2 has the following structure:11645816336.

153. The composition of claim 47, wherein the compound has the following structure:N- Ck / / if54. A composition comprising a Wnt agonist, optionally a sonic hedgehog (SHH) agonist; and optionally one or more pharmaceutically or cosmetically acceptable excipients, in a carrier;wherein the Wnt agonist is A compound of Formula (II):Formula (II)wherein Ri’ comprises a pyridine ring substituted with -C(O)NR3R4; wherein R2’ comprises a piperidine ring optionally substituted at the nitrogen atom with R5’;wherein R3and R4are independently selected from hydrogen and C1-6 alkyl group; wherein R5’, when present, is a C1-6 alkyl group;or is a pharmaceutically acceptable salt thereof.

55. The composition of claim 54, wherein R3and R4are both methyl groups.

56. The composition of claim 54, wherein R5’ is present and is a tert-buty l group.

57. The composition of claim 54, wherein Rf has the following structure:11745816336.

158. The composition of claim 54, wherein R2' has the following structure:

59. The composition of claim 54, wherein the compound has the following structure:

60. The composition of claim 47 or 54. wherein the concentration of the Wnt pathway agonist ranges from 0.001 nM to 200 mM, optionally from about 0.1 pM to about 100 pM, optionally about 50 pM61. The composition of claim 47 or 54, wherein the one or more Shh pathway agonists are selected from SAG (Smoothened agonist), purmorphamine, Hh-Ag1.5, SAG21k, GSA-10, oxysterols, 20(S)-hydroxy cholesterol, 22(S)-hydroxy cholesterol, and combinations thereof.

62. The composition of claim 61. wherein the concentration of the Shh pathway agonist ranges from 0.01 to 200 mM, optionally from about 0.1 nM to about 100 pM, optionally about 50 nM63. The composition of claim 47 or 54, further comprising one or more additional agents selected from the group consisting of hair care agents, scalp care agents, cosmetic agents, and therapeutic agents, wherein the one or more additional agents are selected from humectants, emollients, conditioners, antioxidants, vitamins, peptides, botanical extracts, antiinflammatory agents, penetration enhancers, preservatives, surfactants, polymers, filmformers, rheology modifiers, sunscreens, antimicrobials, antifungals, sebum-regulating agents, vasodilators, grow th-supporting agents, and combinations thereof.

64. The composition of claim 47 or 54, wherein the carrier is selected from aqueous carriers, alcoholic carriers, hydroalcoholic earners, oil-based carriers, emulsions,11845816336.1microemulsions, liposomal carriers, lipid-based carriers, polymeric carriers, gel-based carriers, foam carriers, silicone-based carriers, and combinations thereof.

65. The composition of claim 47 or 54, wherein the composition is a topical formulation.

66. The composition of claim 65, wherein the topical formulation is selected from a cream, gel, foam, lotion, serum, spray, ointment, occlusive cap, or patch.11945816336.1

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