Compositions and methods for stimulating hair growth

Abstract

Proteins, peptides, nucleic acid molecules, compositions and methods for stimulating hair growth are provided. Described herein is a composition comprising a protein comprising a signal peptide, a CUB domain, and an EGF-like domain (SCUBE); a C-terminal peptide (SCUBE-C) thereof; the preparation method comprises the following steps: synthesizing an SCUBE peptide (SCUBE-PP); or a nucleic acid sequence encoding an SCUBE, an SCUBE-C, or an SCUBE-PP; and an optional pharmaceutically acceptable excipient. In some embodiments, the SCUBE protein is an SCUBE3 protein; a C-terminal peptide thereof (SCUBE3-C); and synthesizing the SCUBE3 peptide (SCUBE3-PP). These materials and methods can be used to treat alopecia, stimulate and accelerate hair regeneration after transplantation, and induce human hair growth.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 364,389, filed on May 9, 2022, the entire contents of which are incorporated by reference into this application.

[0002] Reference to a sequence listing

[0003] The contents of the XML file of the sequence listing named "UCI012_seq", which is 11 kb in size, was created on April 30, 2023 and is submitted electronically with this application, are incorporated herein by reference in its entirety. Background Art

[0004] Hair loss or alopecia affects a large part of the general population. For example, androgenic alopecia (hormonal baldness) affects up to 50% of women and 80% of men, and alopecia areata (autoimmune form of hair loss) affects 2% of the general population (Alessandrini et al., 2021). Baldness causes serious psychosocial distress to affected individuals. Although drug treatments for alopecia are urgently needed and generate billions of dollars worldwide each year, the 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). But all of these treatments have limited efficacy or high risk of adverse side effects. For example, local application of minoxidil lotion has the disadvantage of limited efficiency, and many patients have poor compliance because the drug must be applied twice a day and continued to take the drug to maintain its effect. Adverse reactions are the main problems 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 of great commercial interest.

[0005] Understanding the hair growth cycle provides new insights into the development of new strategies for the treatment of hair loss. Hair follicles (HFs) are complex micro-organs in the skin that maintain periodic hair regeneration during repeated hair cycles, which consist of consecutive phases of active growth (anagen), regression (catagen), and rest (telogen). The hair growth phase is powered by hair follicle stem cells (HFSCs), which are maintained and regulated by interactions with their local environment or 'niche'. Dermal papilla cells (DPs) are specialized fibroblasts embedded in the hair bulb at the base of the HF, constituting the critical HFSC niche. Signaling from the DP is required for hair development during embryogenesis and cyclical hair growth in adults. DP dysfunction can disrupt human hair growth, leading to hair loss disorders such as androgenetic alopecia (Chen et al., 2020). Given the critical necessity of the DP in hair growth and its huge transformative potential for the treatment of hair disorders, a better understanding of the molecular biology of the DP can lead to the discovery of new molecular targets for hair growth stimulation.

[0006] There remains a need for new materials and methods for stimulating hair growth. Summary of the invention

[0007] 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, stimulate and accelerate hair regeneration after transplantation, and induce human hair growth. The data presented in Example 1 below show that SCUBE3 can significantly induce new hair growth even in elderly (20 months old) mice. In addition, human data show that SCUBE3 in human scalp follicles is expressed in the same cell type as mouse dermal papilla fibroblasts and is therefore expressed in a conserved expression location. In addition, in a human-mouse xenograft model, SCUBE3 induces human hair growth.

[0008] A composition is described herein, comprising a protein containing a signal peptide, a CUB domain, and an EGF-like domain (SCUBE); a C-terminal peptide thereof (SCUBE-C); a synthetic SCUBE peptide (SCUBE-PP); or a nucleic acid sequence encoding SCUBE, SCUBE-C, or SCUBE-PP; and an optional pharmaceutically acceptable excipient. In some embodiments, the SCUBE protein is a SCUBE3 protein (SEQ ID NO: 1); a C-terminal peptide thereof (SCUBE3-C; SEQ ID NO: 2); a synthetic SCUBE3 peptide (SCUBE3-PP). In some embodiments, the composition comprises 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 number: Q8IWY4; SEQ ID NO: 3) and SCUBE2 (protein accession number: Q9NQ36; SEQ ID NO: 5). It is contemplated that 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) may be used, optionally in combination.

[0009] In some embodiments, SCUBE3-C comprises three cysteine-rich domains and a CUB domain ( repair Body protein C1r / C1s, U egf and B mp1). In some embodiments, the SCUBE3-C peptide is produced by proteolytic cleavage of SCUBE3 with 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 a cysteine-rich domain; or a CUB domain and two cysteine-rich domains.

[0010] In some embodiments, the three cysteine-rich domains have the amino acid sequence: GTKCVSCPQGTYYHGQTE QCVPCPAGTF QEREGQLSCD LCPGSDAHGP LGATNV TTCA GQCPPGQHSV DGFKPCQPCPRGTYQPEAGR TLCFPCGGGL TTKHEGAISF QDCDTKVQCS PGHYYNTSIH RCIRCAMGSY QPDFRQNFCSRCPGNTSTDF DGS TSVAQCK NRQ (SEQ ID NO: 7). In some embodiments, the CUB domain has the amino acid sequence: CGGELGE FTGYIESPNY PGNYPAGVECIWNINPPPKR KILIVV PEIF LPSEDECGDVLVMRKNSSPS SITTYETCQT YERPIAFTAR SRKLWINFKT SEANSARGFQ IPYVTYDEDY EQLVEDIVRDGRLYASENHQ EILKDKKLIK AFF EVLAHPQ NYFKYTEKHK EMLPKSFIKL LRSKVSSFLR PYK (SEQ ID NO: 2).

[0011] A method of promoting hair growth in a subject is also described. A method of treating alopecia in a subject is additionally provided. In some embodiments, these methods comprise intradermal microinjection of a SCUBE composition as described herein. In some embodiments, these methods comprise topical skin application of a SCUBE composition as described herein. In some embodiments, topical skin application is provided after an increase in skin permeability. In some embodiments, the increase in skin permeability occurs by micro-needling.

[0012] 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 androgenic alopecia, female pattern hair loss, alopecia areata, traction alopecia, alopecia totalis, cicatricial alopecia, and / or alopecia universalis. In some embodiments, the method generates new hair follicles, accelerates hair regeneration after hair transplantation, chemotherapy, and / or increases the size of existing hair follicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figures 1A-1I. Hyperactivated hair regeneration in mice with dermal Hedgehog activation in LeprB-targeted dermal papillae. (1A) Co-expression pattern of pan-isoform Lepr mRNA and GFP reporter in dorsal LeprB-Cre;mTmG mouse skin at P50. Colocalization was observed in DP. (1B) Left: Schematic diagram of the generation of LeprB-CreER transgenic mice. Right: Whole-mount preparation showing tdTomato expression in LeprB-Cre;tdTomato dorsal skin at P55. tdTomato fluorescence was observed in nearly all DPs at all time points and in scattered vessel-like structures. (1C) Representative LeprBCre;SmoM2 between P46-84 days. + / - Time course analysis of dorsal hair growth patterns in mutant (bottom) and LeprB-Cre control mice (top). Mice were shaved after each round of photography. Hair growth was observed only in mutant mice (dark areas, marked). (1D) Unsupervised hierarchical clustering of all cells from control (k=18,496) and mutant (k=16,792) samples revealed 17 major cell types, which are color-coded and annotated at the bottom. (Color coding is shown in LiuY et al. Dev Cell. 2022 Jul 25; 57(14): 1758-1775.) Periodic EPI-periodic interfollicular keratinocytes, Krt14 + EPI–KRT14 + Interfollicular keratinocytes, Krt10 + EPI–KRT10 + Interfollicular keratinocytes, Ivl + EPI–Ivl + Interfollicular keratinocytes, upper HF – upper follicle keratinocytes, lower HF – lower follicle keratinocytes, SG – sebaceous gland cells / sebocytes, FIB – dermal fibroblasts, DP / DP-like – dermal papilla / dermal papilla-like fibroblasts, FA-FIB – fibroadipogenic fibroblasts, MUSC – muscle cells, ENDO – endothelial cells, LYM – lymphatic endothelial cells, NAEC – neuron-associated endothelial cells, IMM – immune cells, SCH – Schwann cells and MEL – melanocytes. (1E) Characterization of Scube3 expression in control vs mutant DP / DPL fibroblasts at P46. Mutant-specific DPL cells are indicated. (1F) Heatmap of activity of selected regulators in all DP subclusters, with control and mutant cells indicated. (1G) Heatmap showing Gli1 expression in all DP subclusters. Reg(+)Track plots of expression patterns of selected target genes of the regulators. Genes are annotated on the right. Gene expression levels are represented as track heights per cell and are accompanied by their specific motifs on the right. AUC – Area Under the Curve; GEX – Gene Expression. (1H) Selected Gli1 in control (top) and mutant skin (bottom) at P46. Reg(+) Expression of target gene – Scube3. DP magnification on the right. (1I) Scube3 mRNA in control (left) and mutant HF (right) at P50. Tissues were counterstained with DAPI as in 1A, 1H, 1I. Scale bars: 1A, 1H – 100 μm, 1I – 50 μm.

[0014] Figure 2A-2I .SCUBE3 induces hair growth. (2A) Expression of pan-isoform Scube3 mRNA in mouse dorsal skin at indicated postnatal time points. (2B) Representative control (top) and Scube3 mRNA between P24-30 days - / - Time course analysis of dorsal hair growth patterns in mice (bottom). Mice were shaved before each round of photography. (2C) Representative images of microinjection sites of BSA (top) and rhSCUBE3 (bottom). Affi-gel blue agarose beads are visible. Newly grown anagen HFs are stained. (2D) Quantification of induced anagen HFs in response to rhSCUBE3 at day 14. n=7; ***P<0.0002. (2E) Representative images of microinjection sites of BSA (left) and rhSCUBE3 (right) on 20-month-old mice. Affi-gel blue agarose beads are visible. Newly grown anagen HFs are stained. (2F) Quantification of induced anagen HFs in aged mice at day 14 after protein injection. n=9; ****P<0.0001. (2G) Expression of K14, KI67, and SCUBE3 mRNA in human occipital scalp HFs. SCUBE3 mRNA is specifically and highly expressed in the DP and adjacent dermal sheath cup. (2H) Representative images of microinjection sites of BSA and rhSCUBE3 in a human occipital scalp HF-SCID mouse xenograft model. (2I) Scatter plots of quantification of growing HF in humans and mice. Striped dots represent samples injected with rhSCUBE3, and solid dots represent samples injected with BSA. Tissue sections were counterstained with DAPI in 2A, 2G. Scale bars: 2A, 2G–50 μm.

[0015] Figure 3 .Schematic representation of the Scube3 flox / flox mouse design.

[0016] Figure 4 .Schematic representation of the TetO-Scube3 mouse design.

[0017] Figure 5A-5B .SCUBE1, SCUBE2, SCUBE3 CUB domain analysis and expression. (5A) Protein BLAST analysis of the CUB domain of SCUBE1 (SEQ ID NO:4), SCUBE2 (SEQ ID NO:6), and SCUBE3 (SEQ ID NO:2) protein sequences. SCUBE1 and SCUBE2 show 83.25% and 82.20% homology with SCUBE3 in the CUB domain, respectively. (5B) Characteristic graph of Scube1, Scube2, and Scube3 expression in control (LeprB-Cre; mTmG) and mutant (LeprB-Cre; SmoM2+ / -; mTmG) fibroblasts at P46. DP / DPL fibroblasts are indicated. Scube1, Scube2, and Scube3 show similar expression patterns in fibroblasts. DETAILED DESCRIPTION

[0018] Described herein is a novel molecular strategy for treating hair loss disorders by activating SCUBE3 signaling in the hair-bearing skin of an affected subject. Target disorders include various forms of hair loss, including but not limited to androgenic alopecia and alopecia areata. In one aspect, the activator of hair growth is a naturally occurring human SCUBE3 (containing letter Peptide CUB Domain and E GF-like domain Type 3 ) protein, which is produced in the form of a pure recombinant protein. In another aspect, the hair growth activator is a purified C-terminal SCUBE3 peptide (SCUBE3-C), which is synthesized, recombinant or produced by proteolytic cleavage of native SCUBE3 with one or several matrix metalloproteinases (such as MMP2 or MMP9). SCUBE3-C consists of three cysteine-rich domains and a CUB domain ( repair Body protein C1r / C1s, U egf and Bmp1). In another aspect, the hair growth activator is a combination of pure recombinant SCUBE3 and a recombinant MMP enzyme (such as MMP2 or MMP9). In another aspect, the hair growth activator is one of several synthetic peptides (SCUBE3-PP). One such SCUBE3-PP contains the amino acid sequence of three cysteine-rich domains and one CUB domain of SCUBE3. Other SCUBE3-PPs can be: (a) SCUBE3 CUB+a cysteine-rich domain; (b) SCUBE3 CUB+two cysteine-rich domains. Also described herein is a method for intradermal microinjection of one of recombinant SCUBE3, SCUBE-C, SCUBE3+MMP or SCUBE3-PP to promote hair growth. In another aspect, a method for local skin delivery of one of recombinant SCUBE3, SCUBE-C, SCUBE3+MMP or SCUBE3-PP to promote hair growth after increased skin permeability (such as using microneedles) is described.

[0019] definition

[0020] Unless otherwise specified, all scientific and technical terms used in this application have the meanings commonly used in the art. As used in this application, the following words or phrases have the designated meanings.

[0021] As used herein, a "control" or "reference" sample means a sample that represents a normal measurement value of the corresponding marker, such as a sample obtained from a normal, healthy control subject, or a baseline amount of a marker for comparison. Typically, the baseline will be a measurement value obtained from the same subject or patient. The sample can be an actual sample used for testing, or a reference level or range based on a known normal measurement value of the corresponding marker.

[0022] As used herein, "significant difference" means a difference that can be detected in a manner that is considered reliable by one skilled in the art, such as a statistically significant difference, or a sufficiently large difference that can be detected at a reasonable level of reliability in a particular case. In one example, an increase or decrease of 10% relative to a reference sample is a significant difference. In other examples, an increase or decrease of 20%, 30%, 40% or 50% relative to a reference sample is considered a significant difference. In another example, an increase of two times relative to a reference sample is considered significant.

[0023] "Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide-nucleic acid sequences that can be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or partially synthesized by combining shorter oligonucleotide linkers, or synthesized from a series of oligonucleotides to provide a sequence capable of expressing the encoded protein.

[0024] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Preferred vectors are those capable of autonomous replication and / or expression of the nucleic acid to which they are linked. Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as "expression vectors."

[0025] When the expression control sequence controls and regulates the transcription and translation of the polynucleotide sequence, the polynucleotide sequence (DNA, RNA) is "operably linked" to the expression control 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 to allow the polynucleotide sequence to be expressed under the control of the expression control sequence and produce the desired polypeptide encoded by the polynucleotide sequence.

[0026] As used herein, the terms "complementary sequence" and "complementary" refer to the ability of two DNA molecules to base pair with each other, wherein an adenine on one DNA molecule will base pair with a thymine on a second DNA molecule, and a guanine on one DNA molecule will base pair with a cytosine on a second DNA molecule. When a nucleotide sequence in one DNA molecule can base pair with a nucleotide sequence in a second DNA molecule, the two DNA molecules are complementary to each other. For example, two DNA molecules 5'-ATGC and 5'-GCAT are complementary, and the complementary sequence of the DNA molecule 5'-ATGC is 5'-GCAT. The terms complementary sequence and complementary also include two DNA molecules, wherein one DNA molecule contains at least one nucleotide that will not base pair with at least one nucleotide present on the second DNA molecule. For example, the third nucleotide of each of the two DNA molecules 5'-ATTGC and 5'-GCTAT will not base pair, but the two DNA molecules are complementary as defined herein. Generally, if two DNA molecules hybridize under the above-mentioned standard conditions, they are complementary. Typically, two DNA molecules are complementary if they have at least about 80% sequence identity, preferably at least about 90% sequence identity.

[0027] The term "modified" (or "chemically modified") refers to the derivatization of a polypeptide with one or more moieties by appending (e.g., linking by covalent or non-covalent interactions) one or more moieties to one or more amino acid residues of the polypeptide. Exemplary modifications include hydrophobic moieties (such as lipophilic moieties and fatty acid moieties), glycosylation, phosphorylation. Further exemplary modifications include hydrophilic modifications. The polypeptides used in the methods described herein may be modified, including SCUBE polypeptides. The modified polypeptides used in the methods retain one or more biological activities of the native polypeptide, and preferably also have one or more favorable physicochemical properties compared to the corresponding native and / or unmodified polypeptide. For modified or unmodified SCUBE polypeptides, biologically active fragments thereof, or other polypeptides used in the methods described herein, preferred modified polypeptides or fragments retain the biological activity of the native polypeptide, and preferably have one or more favorable physicochemical activities.

[0028] The term "append" refers to the addition of one or more moieties to an amino acid residue. The term refers to, but is not limited to, the addition of any moiety to any amino acid residue. The term includes attachment of moieties by covalent or non-covalent interactions.

[0029] The term "N-terminal amino acid residue" refers to the first amino acid residue (amino acid number 1) of a polypeptide or peptide.

[0030] 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.

[0031] In certain embodiments, the amino acids used in the applications of the present disclosure are those naturally occurring amino acids found in proteins, or naturally occurring anabolic or catabolic products of such amino acids containing an amino group and a carboxyl group and their corresponding R groups. Particularly suitable amino acid side chains include those selected from the group consisting of glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan.

[0032] The term "amino acid residue" also includes analogs, derivatives and homologues of any specific amino acid mentioned herein, as well as C-terminally or N-terminally protected amino acid derivatives (eg modified with an N-terminally or C-terminally protecting group).

[0033] When the structure of amino acid allows stereoisomer form, (D) and (L) stereoisomers of such amino acid are also included. The configuration of amino acid and amino acid residue herein is specified by appropriate symbol (D), (L) or (DL), and in addition, when no configuration is specified, amino acid or residue may have configuration (D), (L) or (DL). It is worth noting that the structure of some compounds contains asymmetric carbon atoms. It should be understood that the isomers produced by this asymmetry are also included in the scope of the present disclosure. Such isomers in substantially pure form can be obtained by classical separation techniques and by stereochemically controlled synthesis. For the purpose of the present application, unless explicitly stated otherwise, the amino acid named should be interpreted as including (D) or (L) stereoisomers.

[0034] Certain compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, their racemic mixtures, and other mixtures thereof, within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers and mixtures thereof are intended to be included in the present disclosure.

[0035] For example, if a specific enantiomer of a disclosed compound is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, in the case where the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), diastereomeric salts are formed with an appropriate optically active acid or base, followed by separation of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and the pure enantiomers are subsequently recovered.

[0036] For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th ed., 1986-87, inside cover. Likewise, for purposes of this disclosure, the term "hydrocarbon" is intended to include all permissible compounds having at least one hydrogen and one carbon atom. In a broad aspect, the permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds, which may be substituted or unsubstituted.

[0037] As used herein, "pharmaceutically acceptable carriers" or "excipients" include any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline or physiological (0.9%) saline.

[0038] Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990).

[0039] 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 typical embodiments, the subject is a human.

[0040] As used herein, "a" or "an" means at least one, unless expressly stated otherwise.

[0041] Proteins, peptides and combinations

[0042] Compositions are described herein, comprising one or more proteins or peptides for stimulating hair growth; and optional pharmaceutically acceptable excipients. In some embodiments, the composition comprises a protein containing a signal peptide, a CUB domain, and an EGF-like domain (SCUBE). In some embodiments, the composition comprises its C-terminal peptide (SCUBE-C). In some embodiments, the composition comprises a synthetic SCUBE peptide (SCUBE-PP). In some embodiments, the composition comprises a nucleic acid sequence encoding SCUBE, SCUBE-C, or SCUBE-PP. In some embodiments, the SCUBE protein is a 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, synthetic SCUBE peptides include synthetic SCUBE3 peptides (SCUBE3-PP).

[0043] In some embodiments, the composition comprises 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.

[0044] Other examples of SCUBE proteins include SCUBE1 (protein accession number: Q8IWY4; SEQ ID NO: 3) and SCUBE2 (protein accession number: Q9NQ36; SEQ ID NO: 5). SCUBE1 and SCUBE2 show more than 60% homology with SCUBE3 over their full lengths, and 83.25% and 82.20% amino acid homology in the CUB domain, respectively (Wu et al., 2004, J Biol Chem 279, 37485-37490; Figure 5A ). In particular, SCUBE1 and to a lesser extent SCUBE2 and SCUBE3 are expressed in the same hair follicle components. It is contemplated that 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) may be used, optionally in combination.

[0045] In some embodiments, SCUBE3-C comprises three cysteine-rich domains and a CUB domain (complement proteins C1r / C1s, U egf and B mp1). In some embodiments, the SCUBE3-C peptide is produced by proteolytic cleavage of SCUBE3 with a protease. In some embodiments, the SCUBE3-C peptide is produced synthetically. 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 a cysteine-rich domain; or a CUB domain and two cysteine-rich domains.

[0046] In some embodiments, the three cysteine-rich domains have the amino acid sequence: GTKCVSCPQGTYYHGQTE QCVPCPAGTF QEREGQLSCD LCPGSDAHGP LGATNV TTCA GQCPPGQHSV DGFKPCQPCPRGTYQPEAGR TLCFPCGGGL TTKHEGAISF QDCDTKVQCS PGHYYNTSIH RCIRCAMGSY QPDFRQNFCSRCPGNTSTDF DGS TSVAQCK NRQ (SEQ ID NO: 7). In some embodiments, the CUB domain has the amino acid sequence: CGGELGE FTGYIESPNY PGNYPAGVECIWNINPPPKR KILIVV PEIF LPSEDECGDVLVMRKNSSPS SITTYETCQT YERPIAFTAR SRKLWINFKT SEANSARGFQ IPYVTYDEDY EQLVEDIVRDGRLYASENHQ EILKDKKLIK AFF EVLAHPQ NYFKYTEKHK EMLPKSFIKL LRSKVSSFLR PYK (SEQ ID NO: 2).

[0047] method

[0048] Methods for promoting hair growth in a subject are described herein. A method for treating alopecia in a subject is additionally provided. In some embodiments, these methods include intradermal microinjection of a SCUBE composition as described herein. In some embodiments, these methods include topical skin application of a SCUBE composition as described herein. In some embodiments, topical skin application is provided after an increase in skin permeability. In some embodiments, the increase in skin permeability occurs by micro-needling.

[0049] The SCUBE protein or peptide to be administered in the method is generally provided in a pure form, for example, in the form of a composition that is 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 (e.g., in DNA or RNA form), the composition is similarly free of non-SCUBE nucleotides. Such nucleotide-based compositions are optionally delivered in lipid formulations. In some embodiments, SCUBE proteins or peptides are provided in recombinant or synthetic form, optionally with substitutions or modifications to enhance the stability or efficacy of the peptide. SCUBE proteins or peptides in pure form may include SCUBE3, SCUBE3-C or SCUBE-3PP, substantially excluding other SCUBE proteins or peptides. In some embodiments, SCUBE proteins or peptides in pure form include a mixture of SCUBE1, SCUBE2 and SCUBE3 proteins and / or peptides.

[0050] 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 extended release.

[0051] In some embodiments, the baldness is androgenic alopecia, female pattern hair loss, alopecia areata, traction alopecia, alopecia totalis, cicatricial alopecia and / or alopecia universalis. Alopecia can be so-called male pattern baldness, or a more diffuse type of hair loss often observed in postmenopausal women. In some embodiments, the method produces new hair follicles, accelerates hair regeneration after hair transplantation, chemotherapy, and / or increases the size of existing hair follicles. For example, the SCUBE composition can be applied about one month after transplantation to accelerate the regeneration of hair while rebuilding the transplanted hair follicles. Similarly, the SCUBE composition can be applied after chemotherapy to promote the regeneration of hair.

[0052] Pill Box

[0053] Kits are described herein that include a set of agents (e.g., proteins, peptides, nucleic acid molecules, and vectors) as described herein, and optionally one or more suitable containers containing such agents. The agent may optionally include a detectable label or means for monitoring or regulating the agent. The label may be fluorescent, luminescent, enzymatic, chromogenic, or radioactive.

[0054] The kit will typically include the above container and one or more other containers containing materials desirable from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In addition, a label may be provided on the container to indicate that the composition is for a specific application, and may also indicate instructions for use, such as those described herein. Instructions and or other information may also be included on an insert accompanying the kit.

[0055] Example

[0056] The following examples are provided to illustrate the present disclosure and to aid one of ordinary skill in making and using the present invention. The examples are not intended to otherwise limit the scope of the present disclosure in any way.

[0057] Example 1: Hedgehog signaling reprograms hair follicle microenvironment fibroblasts to a hyperactivated state

[0058] This example demonstrates that SCUBE3 is a novel mesenchymal microenvironment factor that activates hair growth. The dermal papilla (DP) is a key mesenchymal microenvironment cell type that regulates the cyclical regeneration of hair follicles by coordinating paracrine signaling crosstalk with epithelial progenitor cells. We found 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 the expression of Lepr isoform B) can effectively target DP to mark and gene regulate during the growth (anagen) and resting (telogen) phases of the hair growth cycle. Using Lepr-based DP targeting, we show that activation of Hedgehog signaling in adult DP significantly accelerates the cyclical growth of hair follicles and induces the formation of new hair follicles from the sides of existing hair follicles in uninjured skin. Through single-cell RNA sequencing, we fully defined the cellular heterogeneity of DP fibroblasts, which increases upon Hedgehog activation and establishes a new gene regulatory network for the hyperactivated DP state. We also identified a novel Hedgehog-regulated signaling factor, Scube3, and revealed its role in the physiological growth of hair follicles. In normal skin, Scube3 is expressed only in the dermal papilla fibroblasts of growing hair follicles but not in those of quiescent hair follicles. Scube3-null mice showed delayed anagen onset. Microinjection of SCUBE3 protein was sufficient to induce hair growth. Furthermore, the dermal papilla-enriched expression of SCUBE3 and its growth-activating role were partially retained in human scalp hair follicles.

[0059] To identify novel Cre recombinase drivers for DP cells and to enable genetic manipulation of these cells in the context of hair growth, we examined the previously reported LeprB-Cre mouse strain in which an IRES-Cre recombinase expression cassette was placed in the 3' untranslated region of Lepr, allowing Cre activity to be restricted to cells transcribing the LeprB isoform (DeFalco et al., 2001). We characterized the labeling pattern in the dorsal skin of LeprB-Cre;mTmG reporter mice. DP labeling gradually increased toward the end of the first anagen phase, became high but incomplete at P22 and P32 in the first telogen and second anagen, respectively, and finally became high and nearly complete in the second telogen by P50 ( Figure 1A RNAscope further confirmed the expression of pan-isoform Lepr in GFP+DP fibroblasts in P50LeprB-Cre;mTmG skin ( Figure 2A To achieve inducible labeling and targeting of DP based on its expressed LeprB isoform, we generated new LeprB-CreER transgenic mice 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, we treated LeprB-CreER;tdTomato mice with five daily doses of tamoxifen starting at P50. When the skin was examined after an additional day, LeprB-CreER markedly and specifically labeled the DP, with virtually no targeting of cells outside the hair follicle ( Figure 1B , right). We therefore conclude that constitutive LeprB-Cre targets the DP most efficiently in mouse dorsal skin, with labeling significantly increased during the second hair growth cycle, and that inducible LeprB-CreER allows specific and efficient DP targeting in adult mouse skin.

[0060] We asked whether inducing constitutive Hedgehog signaling in dermal cells using LeprB-Cre could affect HF growth. Normally, Hedgehog is required for embryonic 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 growth activation (Hsu et al., 2014; Oro and Higgins, 2003; Paladini et al., 2005; Sato et al., 1999). Recent studies have shown that inducing Hedgehog signaling in myofibroblasts by overexpression of constitutively active Smoothened (SmoM2) leads to the regeneration of many new HFs in small wounds in adult mice that would otherwise heal and leave a hairless scar (Lim et al., 2018). This regenerative event does not require the simultaneous activation of Hedgehog signaling in the wound epidermis. In contrast, in adult uninjured skin, simultaneous epidermal and dermal Hedgehog activation by Ptch1 deletion was reported to be required for the induction of HF neogenesis. New HFs are formed from existing HFs in the hairless paw skin and from the touch dome – a specialized sensory structure in the epidermis between hair follicles (Sun et al., 2020). Hedgehog activation by Ptch1 deletion alone in dermal cells expressing Col1a2-CreER was not sufficient to induce HF neogenesis in uninjured skin, but instead resulted in an increase in fibroblast density. We generated LeprB-Cre;SmoM2+ / - mutant mice and compared their hair phenotype with that of LeprB-Cre control mice. Hair growth was tracked non-invasively by regular hair clipping and photography as described previously (Plikus et al., 2008; Wang et al., 2017). Consistent with the relatively late onset of LeprB-Cre activation in DP fibroblasts ( Figure 1C ), the mutant mice did not show obvious differences in hair growth cycle until the second telogen. However, while no new hair growth was observed in control mice (n=8) during the entire observation period spanning days P46 to P86 of the second telogen, the mutant mice (n=8) showed significant premature hair growth ( Figure 1C ).

[0061] To identify candidate cell types and molecular events driving enhanced hair growth downstream of Hedgehog activation, we profiled the dorsal skin of LeprB-Cre;SmoM2+ / -;mTmG mutant and LeprB-Cre;mTmG control mice by scRNA-seq at P46, a time point several days prior to the onset of the mutant hair phenotype. A total of 18,496 control (n=2) and 16,792 mutant (n=2) sequenced cells met quality control filtering criteria and were processed for downstream analyses. After unsupervised clustering, all cells were classified into seventeen major clusters, and cell identities were assigned based on differential expression of signature genes. These cell types included interfollicular and follicular keratinocytes, sebaceous gland cells, dermal, fibroadipogenic, and DP fibroblasts, Schwann cells, melanocytes, immune cells, vascular, lymphoid, and neuron-associated endothelial cells, and muscle cells ( Figure 1D A significant enrichment of mutant cells was observed in fibroblasts, with some DP clusters consisting almost entirely of mutant cells ( Figure 1D , 1E This suggests that the LeprB-Cre;SmoM2+ / - hair phenotype may be driven by newly formed Hedgehog-active DP fibroblasts (DPL fibroblasts). Next, we performed differential gene expression analysis using DESeq2 to compare the mutant DP clusters (including DPL fibroblasts) with the control DP clusters. Significant changes in Scube3 expression were identified, driven by DPL cells becoming a new cellular source of the non-classical activating ligand Scube3 ( Figure 1E , 1H At P50, the morphology of LeprB-Cre;SmoM2+ / -HF ranged from telogen-like to early growth-like, showing significant Scube3 expression in DP cells, HG cells, and their derivatives ( Fig. 1I , right). In contrast, control HF remain in telogen and lack Scube3 expression ( Fig. 1I , left). We then performed gene regulatory network (GRN) analysis using pySCENIC (Van de Sande et al., 2020) to explore the regulon activity profile in mutant DPL fibroblasts. Confirming transgenic stimulation of Hedgehog signaling, mutant DPL II cells strongly activated the Gli1Reg(+) regulon ( Figure 1F Other top activated regulators include Hoxc8Reg(+), Zfp239Reg(+), Sox18Reg(+), Alx3Reg(+), and Ebf1Reg(+)( Figure 1FIn addition, several signaling genes upregulated in DPL fibroblasts, including Scube3, Bmp4, Hhip, Igf1, and Vegfa, were predicted to be located downstream of the Gli1Reg(+) regulon ( Figure 1G ). Thus, mutant DPL fibroblasts activate redundant GRNs downstream of transgene-stimulated Gli1Reg(+), including the Scube3 gene, which potentially promotes hair growth in mutant mice.

[0062] SCUBE3 is a multifunctional protein that can enhance BMP (Lin et al., 2021) and FGF8 signaling on source cells in its membrane-tethered form (Tu et al., 2014) or TGFβ signaling in its proteolytically cleaved secreted form (Wu et al., 2011; Yang et al., 2007; Yang et al., 2020). In embryonic mouse skin, Scube3 is preferentially expressed in the DP and DS compartments of the developing HF (Haworth et al., 2007). However, the role of SCUBE3 in regulating hair growth remains enigmatic. To elucidate the role of Scube3 in periodic hair growth, we then used RNAscope to dissect the Scube3 expression pattern ( Figure 2A ). Scube3 is highly expressed in the DP of early anagen HF at P0 and P5, but then decreases significantly by P17, just before anagen termination. It is absent in the first telogen HF at P22, reappears in the second anagen HF at P32, and then shuts down again during the second telogen at P50. Scattered expression was also observed in the hair matrix, but only during the first anagen. Thus, Scube3 is a anagen-associated DP marker gene. We also examined the timing of the onset of the second anagen in the dorsal skin of germline Scube3-null mice, which are able to survive and grow approximately normal hair despite significant craniofacial defects (Lin et al., 2021). Scube3-null mice exhibited a significant (albeit slight) delay in anagen onset compared to littermate controls ( Figure 2B ), indicating that Scube3 is essential for anagen hair growth.

[0063] Considering the above patterns and its overexpression in mutant DPL fibroblasts, we asked whether SCUBE3 might play an anagen-promoting role. Microinjected intradermally for four days starting at P48, recombinant human SCUBE3 (rhSCUBE3) induced significant new hair growth compared with microinjected bovine serum albumin (BSA) at day 14 (mean difference = 18 + / - 3.44; n = 7) ( Figure 2C-D). Aging in mice is associated with HF miniaturization (Matsumura et al., 2016) and an increase in hair growth inhibitors outside the hair follicle (Chen et al., 2014). To test whether rhSCUBE3 could induce hair growth in aged skin, we repeated the protein microinjection experiments in mice older than 20 months of age. Unlike BSA, rhSCUBE3 strongly stimulated new hair growth at the injection site (mean difference = 31.78 + / - 2.40; n = 9) ( Figure 2E -F).

[0064] To begin evaluating the translational potential of SCUBE3, we first examined its expression pattern in human HF from occipital scalp of volunteer donors undergoing autologous hair transplantation. On RNAscope, SCUBE3 was expressed in DP cells and strongly expressed in the dermal sheath cup, the lowermost portion of the DS that is physically connected to the DP and surrounds the hair matrix ( Figure 2G We also xenografted human occipital scalp HFs into pigmented SCID mice and treated them with three daily doses of rhSCUBE3 or BSA at day 30 post-transplantation, when the transplanted human HFs entered the telogen phase (Oh et al., 2016). Growing human and adjacent mouse host HFs were then counted at each injection site at day 50 post-transplantation. Compared with BSA (n=5), rhSCUBE3 (n=6) accelerated the entry of the telogen phase of transplanted human HFs and stimulated the formation of new telogen phases in mouse host HFs ( Figure 2H -I). We conclude that SCUBE3 is a novel mesenchymal microenvironment-derived hair growth activator in mice, and its expression pattern and function are partially preserved in human scalp.

[0065] References

[0066] Chen, CC; et al. (2014). J Invest Dermatol 134, 2086-2096.

[0067] Chiang; C., et al. (1999). Dev Biol 205, 1-9.

[0068] DeFalco, J., et al(2001).Science 291, 2608-2613.

[0069] Gritli-Linde, A., et al. (2007). Dev Cell 12;99-112.

[0070] Haworth,K.,et al.(2007).Gene Expr Pattems 7,630-634.

[0071] Hsu,Y.C.,Li,L.,and Fuchs,E.(2014).Cell 157,935-949.

[0072] Lim,C.H.,et al.(2018).Nat Commun 9,4903.

[0073] Lin,Y.C.,et al.(2021).Am J Hum Genet 108,115-133.

[0074] Matsumura,H.,et al.(2016).Science 351,aad4395.

[0075] Mill,P.,et al(2003).Genes Dev 17,282-294.

[0076] Oh,J.W.,et al.(2016).J Invest Dermatol 136,34-44.

[0077] Oro,A.E.,and Higgins,K.(2003).Dev Biol 255,238-248.

[0078] Paladini,R.D.,et al.(2005).J Invest Dermatol 125,638-646.

[0079] Plikus,M.V.,et al.(2008).Nature 451,340-344.

[0080] Sato,N.,Leopold,P.L,and Crystal,R.G.(1999).J Clin Invest 104,855-864.

[0081] St-Jacques,B.,et al.(1998).Curr Biol 8,1058-1068.

[0082] Sun,X.,et al.(2020).Elife 9.

[0083] Tu, CF, et al. (2014). J Biol Chem 289, 18928-18942.

[0084] Van de Sande, B., et al. (2020). Nat Protoc 15, 2247-2276.

[0085] Wang, Q., et al.(2017).Elife 6.

[0086] Woo, WM, Zhen, HH, and Oro, A, E. (2012). Genes Dev 26, 1235-1246.

[0087] Wu, YY, et al. (2011). Oncogene 30, 3682-3693.

[0088] Yang, HY, et al. (2007). Cardiovasc Res 75, 139-147.

[0089] Yang, X., et al. (2020). Cancer Biother Radiopharm 35, 120-128.

[0090] Example 2: Verification of the effect of SCUBE3 on hair growth in vivo

[0091] Scube3 flox / flox and Tet0-Scube3 mouse strains were generated to verify the effects of tissue-specific Scube3 deletion / overexpression on hair growth in vivo. The mouse design is shown in Figure 3 (Scube3 flox / flox mice) and Figure 4 (Tet0-Scube3 mice). We will cross the novel 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). By tamoxifen induction in those mice, LeprB-CreER; Scube3 flox / floxDP-specific Scube3 deletion in (flox mutant) mice and Scube3 overexpression in LeprB-Cre; TetOn-Scube3 (TetOn mutant) mice. We will evaluate and compare the hair growth patterns of flox and TetOn mutant mice and their control littermates using a well-established non-invasive hair cycle tracking assay, a hair plucking-induced hair growth model, and immunostaining for skin hair growth markers, including proliferation markers. This will further validate our finding that SCUBE3 can induce hair growth in vivo. Examples supporting the predicted relationship between in vivo overexpression / deletion and local injection / local application / delivery can be found in Oshimori N et al. Cell Stem Cell. 2012 Jan 6; 10(1):63-75.

[0092] Throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0093] Those skilled in the art will recognize that the concepts and specific embodiments disclosed in the foregoing description can be easily used as the basis for modifying or designing other embodiments for achieving the same purpose of the present disclosure. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the present invention as set forth in the appended claims.

Claims

1. A composition comprising a type 3 (SCUBE3) protein containing a signal peptide, a CUB domain and an EGF-like domain; a C-terminal peptide thereof (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 of claim 1, wherein the SCUBE3-C comprises three cysteine-rich domains and a CUB domain (complement proteins C1r / C1s, U egf and B mp1).

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

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

5. The composition of claim 1, further comprising a MMP enzyme.

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

7. The composition of claim 1, wherein the SCUBE3-PP comprises: (a) Three cysteine-rich domains and the CUB domain, (b) a CUB domain and a cysteine-rich domain; or (c) CUB domain and two cysteine-rich domains.

8. The composition of claim 7, wherein the three cysteine-rich domains of (a) have the amino acid sequence: GTKCVSCP QGTYYHGQTE QCVPCPAGTF QEREGQLSCD LCPGSDAHGP LGATNVTTCAGQCPPGQHSV DGFKPCQPCP RGTYQPEAGR TLCFPCGGGL TTKHEGAISF QDCDTKVQCS PGHYYNTSIHRCIRCAMGSY QPDFRQNFCS RCPGNTSTDF DGSTSVAQCK NRQ (SEQ ID NO: 7).

9. The composition of claim 7, wherein the CUB domain has the amino acid sequence: CGGELGEFTGYIESPNY PGNYPAGVECIWNINPPPKR KILIVVPEIF LPSEDECGDV LVMRKNSSPS SITTYETCQTYERPIAFTAR SRKLWINFKT SEANSARGFQ IPYVTYDEDY EQLVEDIVRD GRLYASENHQ EILKDKKLIKAFFEVLAHPQ NYFKYTEKHK EMLPKSFIKL LRSKVSSFLR PYK (SEQ ID NO: 2).

10. A method for promoting hair growth in a subject, the method comprising intradermal microinjection of the composition of any one of claims 1 to 9.

11. A method of promoting hair growth in a subject, the method comprising topically applying to the skin the composition of any one of claims 1-9.

12. The method of claim 11, wherein the topical skin application is provided after skin permeability has increased.

13. The method of claim 12, wherein the increase in skin permeability occurs via microneedles.

14. The method of any one of claims 10-13, wherein the subject is a mammalian subject.

15. The method of claim 14, wherein the subject is a human.

16. The method of claim 15, wherein the subject suffers from alopecia.

17. The method of claim 16, wherein the alopecia is androgenic alopecia, female pattern hair loss, alopecia areata, traction alopecia, alopecia totalis, cicatricial alopecia and / or alopecia universalis.

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

19. The composition or method of any preceding claim, wherein the SCUBE3-PP is chemically modified.

Images