Compositions and Methods for Stimulating Hair Growth

Abstract

Proteins, peptides, nucleic acid molecules, compositions, and methods for stimulating hair growth are provided. Compositions are described herein that include a signal peptide, CUB domain, and EGF-like domain-containing (SCUBE) protein; its C-terminal peptide (SCUBE-C); a synthetic SCUBE peptide (SCUBE-PP); or a nucleic acid sequence encoding SCUBE, SCUBE-C, or SCUBE-PP; and optionally, a pharmaceutically acceptable excipient. In some embodiments, the SCUBE protein is the SCUBE3 protein; its C-terminal peptide (SCUBE3-C); a synthetic SCUBE3 peptide (SCUBE3-PP). These materials and methods can be used to treat alopecia, to stimulate and accelerate hair regrowth after transplantation, and to induce human hair growth.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 364,389, filed May 9, 2022, the entire content of which is incorporated herein by reference.

[0002] Reference to Sequence Listing The content of the XML file of the sequence listing named "UCI012_seq" with a size of 11 kb, created on April 30, 2023, and electronically submitted herewith with this application, is incorporated herein by reference in its entirety.

Background Art

[0003] Hair loss, i.e., alopecia, affects a large portion of the general population. For example, androgenetic alopecia (hormone-induced common baldness) affects up to 50% of women and 80% of men, and alopecia areata (autoimmune-type hair loss) affects 2% of the general population (Alesandrini et al., 2021). Alopecia causes significant psychosocial distress to affected individuals. Pharmacological treatment of alopecia is highly desired and generates billions of dollars annually worldwide, but treatment options approved by the U.S. Food and Drug Administration (FDA) are limited to oral finasteride, topical minoxidil, and topical corticosteroids (Cardoso et al., 2021). However, all of these treatments are either of limited efficacy or have a high risk of harmful side effects. For example, topical application of minoxidil lotion has the drawback of limited efficiency, and it is necessary to apply the drug twice a day and use the drug continuously to sustain its effect, so many patients have low compliance. Adverse effects are the main concerns of oral finasteride and corticosteroids, including post-finasteride syndrome and immunosuppression, respectively. Therefore, the development of new treatments for hair loss is highly sought after and has great commercial benefits.

[0004] Knowledge of the hair growth cycle sheds light on the development of new therapeutic strategies for hair loss. The hair follicle (HF) is a complex mini-organ of the skin that maintains cyclic hair regrowth while repeating a hair cycle consisting of successive phases of an active growth phase (anagen), a regression phase (catagen), and a resting phase (telogen). The anagen phase is promoted by hair follicle stem cells (HFSCs), which are maintained and regulated by their local environment, the so-called "niche". Dermal papilla cells (DPCs) are specialized fibroblasts embedded in the hair bulb at the base of the HF and constitute a crucial HFSC niche. Signaling from DPCs is required for hair development during embryogenesis and cyclic hair growth in adults. DPC dysfunction can disrupt human hair growth and lead to hair loss conditions such as androgenetic alopecia (Chen et al., 2020). Considering the critical necessity of DPCs in hair growth and its great translational potential for treating hair conditions, a better understanding of the molecular biology of DPCs could lead to the discovery of new molecular targets for hair growth stimulation.

[0005] New materials and methods for stimulating hair growth are still needed.

Summary of the Invention

[0006] The methods described herein provide proteins and peptides, as well as compositions, nucleic acid molecules, and methods for delivering them to stimulate hair growth. These materials and methods can be used to treat alopecia, to stimulate and accelerate hair regrowth after transplantation, and to induce human hair growth. The data shown in Example 1 below demonstrate that SCUBE3 can significantly induce new hair growth even in aged (20-month-old) mice. Furthermore, human data show that SCUBE3 in human scalp hair follicles is expressed in the same cell type as in mice, namely dermal papilla fibroblasts, and thus is in a conserved expression location. Additionally, in a human-on-mouse xenograft model, SCUBE3 induces human hair growth.

[0007] Compositions are described herein that contain a signal peptide, CUB domain, and EGF-like domain-containing (SCUBE) protein; its C-terminal peptide (SCUBE-C); a synthetic SCUBE peptide (SCUBE-PP); or a nucleic acid sequence encoding SCUBE, SCUBE-C, or SCUBE-PP; and optionally, a pharmaceutically acceptable excipient. In some embodiments, the SCUBE protein is the SCUBE3 protein (SEQ ID NO: 1); its C-terminal peptide (SCUBE3-C; SEQ ID NO: 2); a synthetic SCUBE3 peptide (SCUBE3-PP). In some embodiments, the composition contains a nucleic acid sequence encoding SCUBE3, SCUBE3-C, or SCUBE3-PP. In some embodiments, the nucleic acid sequence further comprises a promoter and / or other regulatory elements operably linked to the SCUBE protein or peptide coding sequence. Other examples of SCUBE proteins include SCUBE1 (protein accession: Q8IWY4; SEQ ID NO: 3) and SCUBE2 (protein accession: Q9NQ36; SEQ ID NO: 5). The use of SCUBE1, SCUBE2, and / or SCUBE3 proteins or peptides (e.g., SCUBE1-C (SEQ ID NO: 4), SCUBE1-PP, SCUBE2-C (SEQ ID NO: 6), SCUBE2-PP) is contemplated, optionally in combination.

[0008] In some embodiments, SCUBE3-C comprises three cysteine-rich domains and one CUB domain (complement protein C1r / C1s, Uegf, and Bmp1). In some embodiments, the SCUBE3-C peptide is generated by proteolytic cleavage of SCUBE3 by a protease. In some embodiments, the protease is one or more matrix metalloproteinase (MMP) enzymes. In some embodiments, the MMP enzyme is synthetic. In some embodiments, the composition further comprises an MMP enzyme. In some embodiments, the MMP enzyme is selected from MMP2 and MMP9. In some embodiments, SCUBE3-PP comprises three cysteine-rich domains and a CUB domain, a CUB domain and one cysteine-rich domain; or a CUB domain and two cysteine-rich domains.

[0009] In some embodiments, the three cysteine-rich domains have the amino acid sequence: GTKCVSCPQGTYYHGQTEQCVPCPAGTFQEREGQLSCDLCPGSDAHGP LGATNVTTCA GQCPPGQHSVDGFKPCQPCPRGTYQPEAGR TLCFPCGGGL TTKHEGAISF QDCDTKVQCS PGHYYNTSIH RCIRCAMGSY QPDFRQNFCS RCPGNTSTDF DGSTSVAQCK NRQ (SEQ ID NO: 7). In some embodiments, the CUB domain has the amino acid sequence: CGGELGEFTGYIESPNYPGNYPAGVECIWNINPPPKRKILIVVPEIF LPSEDECGDV LVMRKNSSPS SITTYETCQT YERPIAFTAR SRKLWINFKT SEANSARGFQ IPYVTYDEDY EQLVEDIVRD GRLYASENHQ EILKDKKLIK AFFEVLAHPQ NYFKYTEKHK EMLPKSFIKL LRSKVSSFLR PYK (SEQ ID NO: 2).

[0010] Also described is a method for promoting hair growth in a subject. Further provided is a method for treating alopecia in a subject. In some embodiments, these methods comprise intradermal microinjection of the SCUBE compositions described herein. In some embodiments, these methods comprise topical skin administration of the SCUBE compositions described herein. In some embodiments, topical skin administration is provided following an increase in skin permeability. In some embodiments, the increase in skin permeability is effected by microneedling.

[0011] In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is a human. In some embodiments, the subject suffers from alopecia. In some embodiments, the alopecia is androgenetic alopecia, female pattern hair loss, alopecia areata, traction alopecia, total alopecia, cicatricial alopecia, and / or universal alopecia. In some embodiments, the method generates new hair follicles, accelerates hair regrowth after hair transplantation or chemotherapy, and / or increases the size of existing hair follicles.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0013] A novel molecular strategy for treating hair loss is described herein by activating SCUBE3 signaling in the hairy skin of a subject suffering therefrom. Target conditions include, but are not limited to, various forms of hair loss such as androgenetic alopecia and alopecia areata. In one aspect, the hair growth activating factor is the naturally occurring human SCUBE3 (Signal Peptide, CUB Domain and EGF-like Domain Containing 3) protein produced as a pure recombinant protein. In another aspect, the hair growth activating factor is a purified C-terminal SCUBE3 peptide (SCUBE3-C) that is synthetic, recombinant, or generated by proteolytic cleavage of native SCUBE3 by one or several matrix metalloprotease enzymes such as MMP2 or MMP9. SCUBE3-C consists of three cysteine-rich domains and one CUB domain (C complement protein C1r / C1s, Uegf and Bmp1). In another aspect, the hair growth activating factor is a combination of pure recombinant SCUBE3 and one recombinant MMP enzyme, such as MMP2 or MMP9. In another aspect, the hair growth activating factor is one of several synthetic peptides (SCUBE3-PP). One such SCUBE3-PP contains the amino acid sequences of the three cysteine-rich domains and one CUB domain of SCUBE3. Other SCUBE3-PP can be (a) SCUBE3 CUB + one cysteine-rich domain; (b) SCUBE3 CUB + two cysteine-rich domains. Also described herein is a method of intradermal microinjection of one of recombinant SCUBE3, SCUBE-C, SCUBE3+MMP, or SCUBE3-PP to promote hair growth. In another aspect, a method of topical skin delivery of one of recombinant SCUBE3, SCUBE-C, SCUBE3+MMP, or SCUBE3-PP is described for promoting hair growth following increased skin permeability using microneedling or the like.

[0014] Definition All scientific and technical terms used in this application shall have the meanings commonly used in the relevant technical field, unless otherwise specified. When used in this application, the following words or phrases shall have the specified meanings.

[0015] As used herein, a "control" or "reference" sample means a sample that represents the normal measurement value of each marker, e.g., one obtained from a normal healthy control subject, or a baseline amount of the marker that can be used for comparison. Typically, the baseline is a measurement obtained from the same subject or patient. The sample can be the actual sample used for testing, or a reference level or range based on the known normal measurement value of the corresponding marker.

[0016] As used herein, "significant difference" means a difference that can be detected by a method considered reliable by those skilled in the art, such as a statistically significant difference, or a difference of sufficient magnitude that can be detected with a reasonable level of confidence in the circumstances. In one example, an increase or decrease of 10% compared to the reference sample is a significant difference. In other examples, an increase or decrease of 20%, 30%, 40%, or 50% compared to the reference sample is considered a significant difference. In yet another example, an increase of two-fold compared to the reference sample is considered significant.

[0017] "Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide-nucleic acid sequences that can provide a sequence capable of expressing the encoded protein, which can be assembled from smaller fragments, isolated from larger fragments, or newly chemically synthesized or partially synthesized by combining shorter oligonucleotide linkers or a series of oligonucleotides.

[0018] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. Preferred vectors are those capable of autonomous replication and / or expression of the nucleic acids to which they are ligated. A vector capable of directing the expression of a gene to which it is operably linked is referred to herein as an "expression vector".

[0019] A polynucleotide sequence (DNA, RNA) is "operably linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence. The term "operably linked" includes having an appropriate start signal (e.g., ATG) before the polynucleotide sequence to be expressed, and maintaining the correct reading frame so as to permit expression of the polynucleotide sequence under the control of the expression control sequence and production of the desired polypeptide encoded by the polynucleotide sequence.

[0020] As used herein, the terms "complement" and "complementary" refer to the ability of two DNA molecules to base pair with each other, where adenine on one DNA molecule base pairs with thymine on the other DNA molecule, and guanine on one DNA molecule base pairs with cytosine on the other DNA molecule. Two DNA molecules are complementary to each other if the nucleotide sequence within one DNA molecule can base pair with the nucleotide sequence within the other DNA molecule. For example, the two DNA molecules 5'-ATGC and 5'-GCAT are complementary, and the complement of the DNA molecule 5'-ATGC is 5'-GCAT. The terms "complementary" and "complementary" also encompass two DNA molecules where one DNA molecule contains at least one nucleotide that does not base pair with at least one nucleotide present on the other DNA molecule. For example, the third nucleotide of each of the two DNA molecules 5'-ATTGC and 5'-GCTAT does not base pair, but these two DNA molecules are complementary as defined herein. Typically, two DNA molecules are complementary if they hybridize under the standard conditions referred to above. Typically, two DNA molecules are complementary if they have at least about 80% sequence identity, preferably at least about 90% sequence identity.

[0021] The term "modified" (or "chemically modified") refers to derivatizing a polypeptide with one or more moieties by adding one or more moieties to one or more amino acid residues of the polypeptide (e.g., attaching via a covalent or non-covalent interaction). Exemplary modifications include hydrophobic moieties such as lipophilic and fatty acid moieties, glycosylation, phosphorylation. Further exemplary modifications include hydrophilic modifications. The polypeptides for use in the methods described herein, including SCUBE polypeptides, can be modified. The modified polypeptides for use in the methods retain one or more of the biological activities of the native polypeptide and preferably further have one or more advantageous physiochemical properties as compared to the corresponding native and / or unmodified polypeptide. For modified or unmodified SCUBE polypeptides, bioactive fragments, or other polypeptides for use in the methods described herein, preferred modified polypeptides or fragments retain the biological activity of the native polypeptide and preferably have one or more advantageous physiochemical activities.

[0022] The term "added" refers to the addition of one or more moieties to an amino acid residue. This term refers to the addition of any moiety to any amino acid residue, including, but not limited to, the attachment of a moiety via a covalent or non-covalent interaction.

[0023] The term "N-terminal amino acid residue" refers to the first amino acid residue (amino acid number 1) of a polypeptide or peptide.

[0024] The term "C-terminal amino acid residue" refers to the last amino acid residue (amino acid number n, where n = the total number of residues in the peptide or polypeptide) of a polypeptide or peptide.

[0025] In certain embodiments, the amino acids used in the applications of the present disclosure are the naturally occurring amino acids found in proteins, or the naturally occurring catabolic or anabolic products of such amino acids that contain amino and carboxyl groups, as well as their corresponding R groups. In particular, as the official amino acid side chains, the side chains selected from the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan are mentioned.

[0026] The term "amino acid residue" further includes analogs, derivatives, and congeners of any particular amino acid referred to herein, as well as C-terminal or N-terminal protected amino acid derivatives (e.g., those modified with N-terminal or C-terminal protecting groups).

[0027] Also, when the structure of an amino acid permits stereoisomeric forms, the (D) and (L) stereoisomers of such amino acids are also included. The configuration of the amino acids and amino acid residues herein is designated with the appropriate symbols (D), (L), or (DL), and further, when the configuration is not specified, the amino acid or residue may have a (D), (L), or (DL) configuration. It should be noted that some structures of the compounds contain asymmetric carbon atoms. Thus, it is understood that the isomers resulting from such asymmetry are included within the scope of the present disclosure. Such isomers may be obtained in substantially pure form by classical resolution techniques and by stereocontrolled synthesis. For the purposes of the present application, unless specifically stated to the contrary, the designated amino acids are to be construed as including both (D) and (L) stereoisomers.

[0028] Certain compounds of the present disclosure may exist in specific geometric forms or stereoisomeric forms. The present disclosure contemplates that all such compounds, including cis and trans isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, their racemic mixtures, as well as other mixtures thereof, are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, and mixtures thereof, are intended to be included in the present disclosure.

[0029] For example, if a particular enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or by induction with a chiral auxiliary, where the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to obtain the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group such as an amino or an acidic functional group such as a carboxyl, diastereomeric salts are formed using an appropriate optically active acid or base, and subsequently, the diastereomers so formed are resolved by fractional crystallization or chromatographic means well known in the art, and the subsequent pure enantiomers are recovered.

[0030] For the purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986 - 87. Also, for the purposes of the present disclosure, the term "hydrocarbon" is intended to include all acceptable compounds having at least one hydrogen atom and one carbon atom. In a broad aspect, acceptable hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic compounds, which may be substituted or unsubstituted.

[0031] As used herein, "pharmaceutically acceptable carrier" or "excipient" includes any material which, when combined with an active ingredient, retains the biological activity of that ingredient and does not react with the immune system of the subject. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as oil / water emulsions, and various types of wetting agents. Diluents preferred for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.

[0032] Compositions containing such carriers are formulated by well-known conventional methods (see, e.g., Remington’s Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990).

[0033] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects. In a typical embodiment, the subject is a human.

[0034] As used herein, "a" or "an" means at least one, unless expressly indicated otherwise.

[0035] Proteins, Peptides, and Compositions Compositions are described herein that include one or more proteins or peptides for stimulating hair growth; and optionally, a pharmaceutically acceptable excipient. In some embodiments, the composition includes a signal peptide, CUB domain and EGF-like domain-containing (SCUBE) protein. In some embodiments, the composition includes its C-terminal peptide (SCUBE-C). In some embodiments, the composition includes a synthetic SCUBE peptide (SCUBE-PP). In some embodiments, the composition includes a nucleic acid sequence encoding SCUBE, SCUBE-C, or SCUBE-PP. In some embodiments, the protein is the SCUBE3 protein. In some embodiments, the SCUBE3 protein has the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the C-terminal peptide of SCUBE is SCUBE3-C having the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the synthetic SCUBE peptide includes a synthetic SCUBE3 peptide (SCUBE3-PP).

[0036] In some embodiments, the composition includes a nucleic acid sequence encoding SCUBE3, SCUBE3-C, or SCUBE3-PP. In some embodiments, the nucleic acid sequence further includes a promoter and / or other regulatory elements operably linked to the SCUBE protein or peptide coding sequence.

[0037] Other examples of SCUBE proteins include SCUBE1 (protein accession: Q8IWY4; SEQ ID NO: 3) and SCUBE2 (protein accession: Q9NQ36; SEQ ID NO: 5). SCUBE1 and SCUBE2 show more than 60% homology with SCUBE3 over its entire length and show amino acid homologies of 83.25% and 82.20% respectively in the CUB domain (Wu et al., 2004, J Biol Chem 279, 37485-37490; FIG. 5A). In particular, SCUBE1, and to a lesser extent SCUBE2, are expressed in the same hair follicle components as SCUBE3. The use of SCUBE1, SCUBE2, and / or SCUBE3 proteins or peptides (e.g., SCUBE1-C (SEQ ID NO: 4), SCUBE1-PP, SCUBE2-C (SEQ ID NO: 6), SCUBE2-PP) is contemplated, optionally in combination.

[0038] In some embodiments, SCUBE3-C comprises three cysteine-rich domains and one CUB domain (complement protein C1r / C1s, Uegf and Bmp1). In some embodiments, the SCUBE3-C peptide is produced by proteolytic cleavage of SCUBE3 by a protease. In some embodiments, the SCUBE3-C peptide is produced synthetically. In some embodiments, the protease is one or more matrix metalloprotease (MMP) enzymes. In some embodiments, the MMP enzyme is synthetic. In some embodiments, the composition further comprises an MMP enzyme. In some embodiments, the MMP enzyme is selected from MMP2 and MMP9. In some embodiments, SCUBE3-PP comprises three cysteine-rich domains and a CUB domain, a CUB domain and one cysteine-rich domain; or a CUB domain and two cysteine-rich domains.

[0039] In some embodiments, the three cysteine-rich domains have the amino acid sequence: GTKCVSCPQGTYYHGQTEQCVPCPAGTFQEREGQLSCDLCPGSDAHGP LGATNVTTCA GQCPPGQHSVDGFKPCQPCPRGTYQPEAGR TLCFPCGGGL TTKHEGAISF QDCDTKVQCS PGHYYNTSIH RCIRCAMGSY QPDFRQNFCS RCPGNTSTDF DGSTSVAQCK NRQ (SEQ ID NO: 7). In some embodiments, the CUB domain has the amino acid sequence: CGGELGEFTGYIESPNYPGNYPAGVECIWNINPPPKRKILIVVPEIF LPSEDECGDV LVMRKNSSPS SITTYETCQT YERPIAFTAR SRKLWINFKT SEANSARGFQ IPYVTYDEDY EQLVEDIVRD GRLYASENHQ EILKDKKLIK AFFEVLAHPQ NYFKYTEKHK EMLPKSFIKL LRSKVSSFLR PYK (SEQ ID NO: 2).

[0040] Method Methods for promoting hair growth in a subject are described herein. Further provided are methods for treating alopecia in a subject. In some embodiments, these methods include intradermal microinjection of an SCUBE composition described herein. In some embodiments, these methods include topical skin administration of an SCUBE composition described herein. In some embodiments, the topical skin administration is provided following an increase in skin permeability. In some embodiments, the increase in skin permeability is caused by microneedling.

[0041] The SCUBE protein or peptide administered by the present method is typically provided in a pure form, for example, as a composition substantially free of other proteins or peptides. In some embodiments, the composition is at least 90% SCUBE protein or peptide, or at least "90% pure". In some embodiments, the composition is at least 95%, 96%, 97%, 98%, or 99% pure SCUBE protein or peptide. Similarly, when administered in nucleic acid form, for example as DNA or RNA, the composition is likewise free of non-SCUBE nucleotides. Such nucleotide-based compositions are optionally delivered in a lipid preparation. In some embodiments, the SCUBE protein or peptide is provided in recombinant or synthetic form and optionally has substitutions or modifications to improve peptide stability or efficacy. The pure form of the SCUBE protein or peptide may include SCUBE3, SCUBE3-C, or SCUBE-3PP, substantially excluding other SCUBE proteins or peptides. In some embodiments, the pure form of the SCUBE protein or peptide includes a mixture of SCUBE1, SCUBE2, and SCUBE3 proteins and / or peptides.

[0042] In some embodiments, the SCUBE protein or peptide is delivered in the form of beads or other known delivery vehicles. In some embodiments, the SCUBE protein or peptide is administered via a wearable patch that provides long-term release.

[0043] In some embodiments, alopecia is androgenetic alopecia, female pattern hair loss, alopecia areata, traction alopecia, total alopecia, cicatricial alopecia, and / or universal alopecia. The alopecia can be that known as male pattern hair loss, or the diffuse hair loss often observed in postmenopausal women. In some embodiments, the method generates new hair follicles, accelerates hair regrowth after hair transplantation or chemotherapy, and / or increases the size of existing hair follicles. For example, the SCUBE composition can be administered about 1 month after transplantation to accelerate hair regrowth while the transplanted hair follicles are being re-established. Similarly, the SCUBE composition can be administered after chemotherapy to promote hair regrowth.

[0044] Kit A kit is described herein that includes a set of agents described herein, such as proteins, peptides, nucleic acid molecules, and vectors, and optionally, one or more suitable containers containing such agents. The agents can optionally include a detectable label, or means for monitoring or modulating the agent. The label can be fluorescent, luminescent, enzymatic, chromogenic, or radioactive.

[0045] Typically, the kit comprises the above container(s) and one or more other containers containing materials desirable from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, and an instructional leaflet with instructions for use. Additionally, a label can be presented on the container to indicate that the composition is to be used for a particular application, and can also indicate instructions for use, such as those described above. The instructions and / or other information can also be included in the instructional leaflet included with the kit.

Examples

[0046] The following examples illustrate the disclosure and are presented to assist one of ordinary skill in the art in making and using the disclosure. The examples are in no way intended to otherwise limit the scope of the disclosure.

[0047] Example 1: Hedgehog signaling reprograms dermal papilla niche fibroblasts into a hyperactivated state This example demonstrates that SCUBE3 is a new mesenchymal niche factor that activates hair growth. The dermal papilla (DP) is an important mesenchymal niche cell type that regulates the cyclic regeneration of hair follicles by modulating paracrine signaling crosstalk with epithelial progenitor cells. We show that the leptin receptor (Lepr) is a robust marker gene for adult DP fibroblasts in mice, and that constitutive Cre and inducible CreER genetic tools, whose activity follows Lepr isoform B expression, can efficiently target the DP for labeling and gene regulation between both the growth (anagen) and resting (telogen) phases of the hair cycle. Using Lepr-based DP targeting, we show that activation of Hedgehog signaling activity in adult DP significantly accelerates the cyclic growth of hair follicles and induces the formation of new hair follicles from the sides of existing hair follicles in unwounded skin. By single-cell RNA sequencing, we fully define the cellular heterogeneity of DP fibroblasts that increases upon Hedgehog activation and establish a new gene regulatory network for the hyperactivated DP state. We also identify a new Hedgehog-modulating signaling factor, Scube3, and clarify its role in the physiological growth of hair follicles. In normal skin, SCube3 is expressed only in dermal papilla fibroblasts of growing hair follicles and not in resting hair follicles. SCube3 null mice showed a delay in anagen onset. SCUBE3 protein microinjection was sufficient to induce hair growth. Furthermore, the abundant expression of SCUBE3 in the dermal papilla and its growth-activating effect are partially conserved in human scalp hair follicles.

[0048] To identify new Cre recombinase drivers for DP cells and to enable genetic manipulation of these cells in the context of hair growth, the inventors investigated the previously reported LeprB-Cre mouse strain. In this mouse strain, an IRES-Cre recombinase expression cassette is placed in the 3' untranslated region of Lepr, thereby restricting Cre activity to cells that transcribe the LeprB isoform (DeFalco et al., 2001). The inventors characterized the labeling pattern in the dorsal skin of LeprB-Cre;mTmG reporter mice. DP labeling gradually increased until late anagen 1, peaked at P22 and P32 during telogen 1 and anagen 2, respectively, but was incomplete, and finally peaked at P50 by telogen 2 and was nearly complete (Figure 1A). RNAscope was used to further confirm the expression of pan-isoform Lepr in GFP+ DP fibroblasts of P50 LeprB-Cre;mTmG skin (Figure 2A). To enable inducible labeling and targeting of DP based on the expression of the LeprB isoform, the inventors generated a new LeprB-CreER transgenic mouse in which the IRES-iCreERT2 construct was inserted after the exon 19 stop codon of the Lepr gene (Figure 1B, left). To test its specificity and efficacy, LeprB-CreER;tdTomato mice were treated with tamoxifen starting at P50 and administered daily for 5 days. Examination of the skin 1 day later revealed that LeprB-CreER labeled DP prominently and specifically, and there was substantially no targeting of cells outside the hair follicle (Figure 1B, right). Therefore, the inventors concluded that in the dorsal skin of mice, constitutive LeprB-Cre targets DP most efficiently, with a marked increase in labeling during the second hair growth cycle, and that inducible LeprB-CreER may enable specific and efficient DP targeting in the skin of adult mice.

[0049] We explored whether inducing constitutive Hedgehog signaling in skin cells using LeprB-Cre could affect HF growth. Normally, Hedgehog is required for fetal hair development (Chiang et al., 1999; Gritli-Linde et al., 2007; Mill et al., 2003; St-Jacques et al., 1998; Woo et al., 2012) and proper hair follicle activation (Hsu et al., 2014; Oro and Higgins, 2003; Paladini et al., 2005; Sato et al., 1999). Recent studies have demonstrated that inducing Hedgehog signaling in myofibroblasts by overexpressing constitutively active Smoothened (SmoM2) results in the regeneration of numerous new HFs in small wounds of adult mice that normally heal with hairless scars (Lim et al., 2018). In this regeneration event, co-activation of Hedgehog signaling in the wound epidermis is not required. In contrast, in adult unwounded skin, it has been reported that co-activation of epidermal and dermal Hedgehog by Ptch1 deletion is necessary to induce HF neogenesis. New HFs are formed from existing HFs in hairless foot skin and from touch domes (special sensory structures of the interfollicular epidermis) (Sun et al., 2020). Activation of Hedgehog by Ptch1 deletion alone in Col1a2-CreER-expressing skin cells was not sufficient to induce HF neogenesis in unwounded skin and instead resulted in an increase in fibroblast density. We generated LeprB-Cre;SmoM2+ / - mutant mice and compared their hair phenotypes to those of LeprB-Cre control mice. As previously described, hair growth was non-invasively tracked via regular shaving and photography (Plicus et al., 2008; Wang et al., 2017). Consistent with the relatively late onset of LeprB-Cre activation in DP fibroblasts (Figure 1C), mutant mice did not show obvious differences in the hair growth cycle until the second telogen.However, in the control mice (n = 8), no new hair growth was observed during the entire observation period covering P46 to P86 corresponding to the second telogen, but the mutant mice (n = 8) showed significantly earlier hair growth (Figure 1C).

[0050] To identify candidate cell types and molecular events that promote enhanced hair growth downstream of Hedgehog activation, we profiled the dorsal skin of LeprB-Cre;SmoM2+ / -;mTmG mutant mice and LeprB-Cre;mTmG control mice by scRNA-seq at P46, a few days before the appearance of the mutant hair phenotype. A total of 18,496 sequenced control (n = 2) and 16,792 mutant (n = 2) cells passed quality control filtering metrics and were processed for downstream analysis. During unsupervised clustering, all cells were classified into 17 main clusters, and cell identities were assigned according to the differential expression of signature genes. These cell types included interfollicular and follicular keratinocytes, sebocytes, dermis, fibroadipogenic and DP fibroblasts, Schwann cells, melanocytes, immune cells, blood vessels, lymphatic and nerve-associated endothelial cells, and muscle cells (Figure 1D). Pronounced enrichment of mutant cells was observed in fibroblasts, and several DP clusters were almost entirely composed of mutant cells (Figure 1D, 1E). This indicated that the LeprB-Cre;SmoM2+ / - hair phenotype might be driven by newly formed Hedgehog-activated DP fibroblasts (DPL fibroblasts). Next, we performed differential gene expression analysis using DESeq2 to compare mutant DP clusters (including DPL fibroblasts) with control DP clusters. A significant change in SCube3 expression was identified, which was driven by DPL cells serving as a new cellular source of the non-canonical activating ligand Scube3 (Figure 1E, 1H). At P50, LeprB-Cre;SmoM2+ / - HFs with a telogen-like to early anagen-like morphology showed prominent Scube3 expression in DP cells, HG cells, and their derivatives (Figure 1I, right). In contrast, control HFs remained in telogen and had no SCube3 expression (Figure 1I, left). We then used pySCENIC (Van de Sande et al., 2020) to perform gene regulatory network (GRN) analysis to investigate the regulon activity profile in mutant DPL fibroblasts.We confirmed the transgenic stimulation of hedgehog signaling, and mutant DPL II cells strongly activated the Gli1Reg(+) legron (Figure 1F). Other highly activated legons included Hoxc8Reg(+), Zfp239Reg(+), Sox18Reg(+), Alx3Reg(+), and EbF1Reg(+) (Figure 1F). Furthermore, several signaling genes upregulated in DPL fibroblasts, including Scute3, Bmp4, Hip, Igf1, and Vegfa, were predicted to be downstream of the Gli1Reg(+) legron (Figure 1G). Thus, mutant DPL fibroblasts activate a redundant GRN downstream of transgenic-stimulated Gli1Reg(+) that potentially promotes hair growth in mutant mice and includes the SCube3 gene.

[0051] SCUBE3 is a multifunctional protein that can enhance BMP (Lin et al., 2021) and FGF8 signaling (Tu et al., 2014) on source cells in its membrane-bound form, or TGFβ signaling (Wu et al., 2011; Yang et al., 2007; Yang et al., 2020) in its proteolytically cleaved secreted form. In fetal mouse skin, SCube3 is preferentially expressed in the DP and DS compartments that generate HFs (Haworth et al., 2007). However, the role of SCUBE3 in regulating hair growth remains unclear. To explain the role of SCube3 in cyclic hair growth, we next profiled the expression pattern of SCube3 using RNAScope (Figure 2A). SCube3 was highly expressed in the DP of HFs at the early anagen stage of P0 and P5, but then decreased significantly by P17, before the end of anagen. SCube3 was absent in the first telogen HFs at P22, reappeared in the second anagen HFs at P32, and then disappeared again during the second telogen at P50. Scattered expression was also seen in the hair matrix, but only during the first anagen phase. Therefore, Scube3 is an anagen-phase-related DP marker gene. We also investigated the timing of the onset of the second anagen in the dorsal skin of germline SCube3 null mice that are viable and develop an overall normal coat despite having distinct craniofacial defects (Lin et al., 2021). Compared with littermate controls, Scube3 null mice showed a mild but distinct delay in the onset of anagen (Figure 2B), suggesting that SCube3 is essential for hair growth during anagen.

[0052] Considering the above patterns and their overexpression in mutant DPL fibroblasts, we explored whether SCUBE3 could exert an anagen-promoting effect. When intradermal microinjections were performed starting at P48 for 4 days, recombinant human SCUBE3 (rhSCUBE3) significantly induced new hair growth compared to bovine serum albumin (BSA) microinjected on day 14 (mean difference = 18+ / -3.44; n = 7) (Figs. 2C-D). Aging in mice is associated with miniaturization of the HF (Matsuura et al., 2016) and an increase in factors inhibiting hair growth outside the hair follicle (Chen et al., 2014). To test whether rhSCUBE3 could induce hair growth in aged skin, we repeated the protein microinjection experiment in mice over 20 months of age. Unlike BSA, rhSCUBE3 strongly stimulated new hair growth at the injection site (mean difference = 31.78+ / -2.40; n = 9) (Figs. 2E-F).

[0053] To initiate the evaluation of the translatability of SCUBE3, first, we examined its expression pattern in anagen human HFs from the occipital scalp of volunteer donors undergoing autologous hair transplantation. By RNAscope, SCUBE3 was expressed in DP cells and strongly expressed in the outer root sheath of the hair bulb, the lowest part of the DS that physically connects to the DP and surrounds the hair matrix (Fig. 2G). Also, human occipital scalp HFs were xenografted into SCID mice that were colored, and on day 30 after transplantation when the transplanted human HFs entered telogen, they were treated with rhSCUBE3 or BSA daily for 3 days (OH et al., 2016). Then, anagen-phase human and adjacent mouse host HFs were counted at each injection site on day 50 after transplantation. Compared to BSA (n = 5), rhSCUBE3 (n = 6) accelerated the transition of transplanted human HFs to anagen and stimulated new anagen in mouse host HFs (Figs. 2H-I). We conclude that SCUBE3 is a novel mesenchymal niche-derived hair growth activator in mice and that its expression pattern and function are partially conserved in human skin.

[0054] References

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[0058] Gritli - Linde, A., et al. (2007). Dev Cell 12, 99 - 112.

[0059] Haworth, K., et al. (2007). Gene Expr Patterns 7, 630 - 634.

[0060] Hsu, Y.C, Li, L., and Fuchs, E. (2014). Cell 157, 935 - 949.

[0061] Lim, C.H., et al. (2018). Nat Commun 9, 4903.

[0062] Lin, Y.C., et al. (2021). Am J Hum Genet 108, 115 - 133.

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[0066] Oro, A.E., and Higgins, K. (2003). Dev Biol 255, 238 - 248.

[0067] Paladini, R.D., et al. (2005). J Invest Dermatol 125, 638 - 646.

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[0079] Example 2: Verification of the effect of SCUBE3 on hair growth in vivo The SCube3 flox / flox and TetO - Scute3 mouse lines were generated for use in verifying the impact of tissue - specific Scute3 deletion / overexpression on hair growth in vivo. The mouse designs are shown in Figure 3 (Scute3flox / flox mice) and Figure 4 (TetO - Scute3 mice). We crossed the new flox and Tet - On transgenic mice with LeprB - CreER mice (generated in our recent study Liu Y, et al. Dev Cell. 2022 Jul 25;57(14):1758 - 1775; PMID: 35777353). LeprB - CreER;SCube3 flox / flox (flox mutant) mice for DP - specific Scube3 deletion and LeprB - Cre;TetOn - SCube3 (TetOn mutant) mice for SCube3 overexpression are achieved by tamoxifen induction in these mice. We used a well - established non - invasive hair cycle tracking assay, a plucking - induced hair growth model, and skin immunostaining for hair growth markers including proliferation markers to evaluate and compare the hair growth patterns of flox and TetOn mutant mice and their control littermates. This further verifies our finding that SCUBE3 can induce hair growth in vivo. Examples supporting the predicted relationship between overexpression / deletion in vivo and local injection / local application / delivery can be found in Oshimori N, et al. Cell Stem Cell. 2012 Jan 6;10(1):63 - 75.

[0080] Throughout this application, various publications are referenced. For the purpose of more fully describing the state of the art to which this application pertains, the disclosures of these publications are hereby incorporated by reference in their entirety into this specification.

[0081] Those skilled in the art will understand that the concepts and specific embodiments disclosed in the foregoing description can be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of this application. Those skilled in the art will also understand that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

1. A composition comprising: a signal peptide, a CUB domain, and an EGF-like domain-containing 3 (SCUBE3) protein; its C-terminal peptide (SCUBE3-C); a synthetic SCUBE3 peptide (SCUBE3-PP); or a nucleic acid sequence encoding SCUBE3, SCUBE3-C, or SCUBE3-PP; and optionally, a pharmaceutically acceptable excipient.

2. The composition according to claim 1, wherein SCUBE3-C comprises three cysteine-rich domains and one CUB domain (complement protein C1r / C1s, Uegf, and Bmp1).

3. The composition according to claim 2, wherein the SCUBE3-C peptide is produced by the proteolytic cleavage of SCUBE3 by a protease.

4. The composition according to claim 3, wherein the protease is one or more matrix metalloproteinase (MMP) enzymes.

5. The composition according to claim 1, further comprising an MMP enzyme.

6. The composition according to claim 4 or 5, wherein the MMP enzyme is selected from MMP2 and MMP9.

7. The aforementioned SCUBE3-PP is (a) Three cysteine-rich domains and CUB domains, (b) A CUB domain and one cysteine-rich domain, or (c) The composition according to claim 1, comprising a CUB domain and two cysteine-rich domains.

8. The composition according to claim 7, wherein the three cysteine-rich domains in (a) have the amino acid sequence: GTKCVSCP QGTYYHGQTE QCVPCPAGTF QEREGQLSCD LCPGSDAHGP LGATNVTTCA GQCPPGQHSV DGFKPCQPCP RGTYQPEAGR TLCFPCGGGL TTKHEGAISF QDCDTKVQCS PGHYYNTSIH RCIRCAMGSY QPDFRQNFCS RCPGNTSSTDF DGSTSVAQCK NRQ (Sequence ID 7).

9. The composition according to claim 7, wherein the CUB domain has the amino acid sequence: CGGELGE FTGYIESPNY PGNYPAGVE C IWNINPPPKR KILIVVPEIF LPSEDECGDV LVMRKNSSSPS SITTYETCQT YERPIAFTAR SRKLWINFKT SEANSARGFQ IPHYVTYDEDY EQLVEDIVRD GRLYASENHQ EILKDKKLIK AFFEVLAHPQ NYFKYTEKHK EMLPKSFIKL LRSKVSSFLR PYK (Sequence ID 2).

10. A composition according to claim 1 for use in a method for promoting hair growth in a subject, wherein the method comprises intradermal microinjection of the composition.

11. A composition according to claim 1 for use in a method for promoting hair growth in a subject, wherein the method comprises topical skin administration of the composition.

12. The composition according to claim 11, wherein the topical skin administration is provided following an increase in skin permeability.

13. The composition according to claim 12, wherein the increase in skin permeability is caused by microneedling.

14. The composition according to any one of claims 10 to 13, wherein the subject is a mammal.

15. The composition according to claim 14, wherein the subject is a human.

16. The composition according to claim 15, wherein the subject is suffering from alopecia.

17. The composition according to claim 16, wherein the alopecia is androgenic alopecia, female pattern alopecia, alopecia areata, traction alopecia, complete alopecia, scarring alopecia, and / or generalized alopecia.

18. The composition according to claim 15, wherein the method generates new hair follicles, accelerates hair regrowth after hair transplantation or chemotherapy, and / or increases the size of existing hair follicles.

19. The composition according to claim 1, wherein the SCUBE3-PP is chemically modified.

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