The present invention relates to novel hair follicle growth factor (HFGF) proteins, genes encoding HFGFs, methods for preparing HFGF proteins and therapeutic uses of HFGF proteins. The HFGF proteins of the present invention have a characteristic reduced expression in hair follicles derived from alopecia patients and have a stimulatory effect on hair follicle cell proliferation. HFGF proteins may be used to prevent or treat alopecia and to promote or accelerate hair growth and hair follicle repair.

Description of the Invention

SUMMARY OF THE INVENTION

The present inventors have undertaken studies in an attempt to isolate a molecular factor for treating alopecia and have developed hair follicle growth factor (HFGF) proteins from human hair follicle, which have a reduced expression in hair follicles derived from alopecia patients and show stimulatory effects on hair follicle cell proliferation. The amino acid sequence of HFGF protein (SEQ ID NO: 1) is provided in FIG. 8 (see Original Patent). HFGF is considered to be an allelic form of keratinocyte growth factor-2 (KGF-2) wherein HFGF has a glutamic acid residue (Glu) at position 87, whereas KGF-2 has a lysine residue (Lys) at this position.

In one aspect, the present invention provides an isolated polypeptide having the amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO:1 or an isolated polypeptide comprising the amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO:1, wherein Glu 87 is replaced by Asp 87.

In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 or an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1, wherein Glu 87 is replaced by Asp 87.

In other aspect, the present invention provides a vector comprising the nucleic acid encoding the amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 or the amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1, wherein Glu 87 is replaced by Asp 87.

In still other aspect, the present invention provides a host cell transfected with the vector comprising a transcription promoter, a DNA encoding the amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 or the amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1, wherein Glu 87 is replaced by Asp 87, and a transcription terminator, wherein said promoter is operably linked to the DNA segment, and the DNA segment is operably linked to the transcription terminator.

In a further aspect, the present invention provides a method of producing a polypeptide comprising an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 which comprises culturing a host cell under conditions such that said polypeptide is expressed, and isolating said polypeptide from the cultures, wherein said host cell is transfected with the vector comprising a transcription promoter, a DNA encoding the amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1, and a transcription terminator, said promoter being operably linked to the DNA, and the DNA being operably linked to the transcription terminator. Also, the present invention provides a method of producing an isolated polypeptide comprising an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1, wherein Glu 87 is replaced by Asp 87, which comprises culturing a host cell under conditions such that said polypeptide is expressed and isolating said polypeptide from the cultures, wherein said host cell is transfected with the vector comprising a transcription promoter, a nucleic acid encoding the amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1, wherein Glu 87 is replaced by Asp 87, and a transcription terminator, said promoter being operably linked to the DNA, and the DNA being operably linked to the transcription terminator.

In an additional aspect, the present invention provides a pharmaceutical composition comprising the polypeptide which comprises an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 or an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 wherein Glu 87 is replaced by Asp 87, and a pharmaceutically acceptable carrier.

In still another aspect, the present invention provides a pharmaceutical composition comprising the nucleic acid molecule which comprises the nucleotide sequence encoding the amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 or the nucleotide sequence encoding an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1, wherein Glu 87 is replaced by Asp 87, and a pharmaceutically acceptable carrier.

In still another aspect, the present invention provides a method for treating, preventing or ameliorating alopecia in a subject, which comprises administering the composition containing the polypeptide comprising an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 or the polypeptide comprising an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1, wherein Glu 87 is replaced by Asp 87, and a pharmaceutically acceptable carrier, to said subject.

In still another aspect, the present invention provides a method for treating, preventing or ameliorating alopecia in a subject, which comprises administering the composition containing the nucleic acid molecule encoding an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 or the nucleic acid molecule encoding an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 wherein Glu 87 is replaced by Asp 87, and a pharmaceutically acceptable carrier, to said subject.

In another aspect, the present invention provides a method for stimulating, accelerating or inducing hair growth or hair follicle repair in a subject, which comprises administering the composition containing the polypeptide comprising an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 or the polypeptide comprising an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 wherein Glu 87 is replaced by Asp 87, and a pharmaceutically acceptable carrier to said subject.

In another aspect, the present invention provides a method for stimulating, accelerating or inducing hair growth or hair follicle repair in a subject which comprises administering the composition containing the nucleic acid molecule encoding an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 or the nucleic acid molecule encoding an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 wherein Glu 87 is replaced by Asp 87, and a pharmaceutically acceptable carrier, to said subject.

In another aspect, the present invention provides a method for transplanting hair in a subject which comprises supplementing scalp hair follicles or grafts with the polypeptide comprising an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 or the polypeptide comprising an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1, wherein Glu 87 is replaced by Asp 87 and transplanting the supplemented hair grafts or follicles with the polypeptide to the bald or thinning area of said subject. Also, the present invention provides a method for transplanting hair in a subject which comprises supplementing scalp hair follicles or grafts with the nucleic acid molecule encoding an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 or the nucleic acid molecule encoding an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1, wherein Glu 87 is replaced by Asp 87.

In another aspect, the present invention provides a method for diagnosing alopecia in a subject comprising collecting a blood or tissue sample from said subject and detecting HFGF proteins in said sample.

DETAILED DESCRIPTION OF THE INVENTION

The full amino acid sequence and nucleotide sequence of Keratinocyte Growth Factor-2 (KGF-2) are known in the art. The polypeptide of the present invention shown in SEQ ID NO: 1 has an amino acid sequence containing a glutamic acid residue at position 87, instead of a lysine residue at position 87. This polypeptide was designated Hair Follicle Growth Factor and is referred to herein as HFGF or HFGF protein. HFGF has a characteristic reduced expression in hair follicles derived from alopecia patients and shows a stimulatory effect on hair follicle cell proliferation. According to the present invention, an amino acid sequence of Ser 69 to Ser 208 having a glutamic acid residue at position 87 as shown in SEQ ID NO: 1 is found to be important to effecting hair follicle cell proliferation. The polypeptides comprising at least an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 are nearly equal to HFGF in their stimulatory activity on hair follicle cell proliferation. In particular, the amino acid residue at position 89, i.e., glutamic acid (Glu 89), is found to have a strong influence on hair follicle cell proliferation. Accordingly, the polypeptides of the present invention include the polypeptides further comprising at least one contiguous sequence of amino acids Met 1 to Ala 39 of SEQ ID NO: 1 at the N-terminus of said polypeptide. In addition, the polypeptides of the present invention include the polypeptides having substitutions, deletions and/or insertions of one, two, three, four or more amino acid residues in the region of Met 1 to Ala 39 of SEQ ID NO: 1.

The polypeptides according to the present invention include another group of polypeptides comprising an amino acid sequence in glutamic acid at position 37 is replaced by aspartic acid (Asp). Likewise, this group of the polypeptides having an aspartic acid residue at position 87 includes the polypeptides further comprising at least one contiguous sequence of amino acids Met 1 to Ala 39 of SEQ ID NO:. 1 at the N-terminus of said polypeptide and the polypeptides having substitutions, deletions and/or insertions of one, two, three, four or more amino acid residues in the region of Met 1 to Ala 39 of SEQ ID NO: 1. As with Glu 87, Asp 87 plays an important role in proliferation of hair follicle cells.

The isolated polypeptides as defined above are sometimes collectively referred to herein as "HFGF proteins". Therefore, examples of HFGF proteins are the polypeptide having Met 1 to Ser 208 of the amino acid sequence shown in SEQ ID NO: 1, the polypeptide having Leu 40 to Ser 208 of the amino acid sequence shown in SEQ ID NO: 1, and the polypeptide having Ser 69 to Ser 208 of the amino acid sequence shown in SEQ ID NO: 1.

Additionally, the polypeptides of the present invention may further comprise a Met residue at the N-terminus of any of said amino acid sequences. Moreover, the polypeptides of the present invention may be mature proteins. These polypeptides are also included in HFGF proteins of the present invention.

In the broadest aspect, the present invention therefore provides an isolated polypeptide comprising an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 or an isolated polypeptide comprising an amino acid sequence of Ser 69 to Ser 208 of SEQ ID NO: 1 wherein Glu 87 is replaced by Asp 87.

In one embodiment, the present invention is directed to a HFGF that is newly isolated from hair follicles of human scalp skin and is a variant or allelic form of known KGF-2. To isolate HFGF of the present invention from hair follicles of human scalp skin, total mRNA was extracted from hair follicles and cDNA was obtained from total RNA by performing RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). After the nucleotide sequences of cDNA produced by the above RT-PCR were identified, the amino acid sequences corresponding to the nucleotide sequences of said cDNA were deduced and determined. One of the deduced amino acid sequences was identified as an amino acid sequence wherein glutamic acid replaces lysine at position 87 of the KGF-2 protein. The amino acid sequence of HFGF protein is shown in SEQ ID NO: 1.

In another embodiment, the present invention is directed to a variant or allelic form of HFGF wherein Asp 87 replaces Glu 87. It was found by the inventors that a negatively charged amino acid at position 87 of KGF-2 increases the hair follicle cell proliferation activity in comparison to wild type KGF-2.

The HFGF proteins of the present invention can be readily made by a conventional recombinant DNA technique. The coding region for BFGF proteins can be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library"), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.). Polymerase chain reaction (PCR) can be used to amplify DNA sequences encoding HFGF proteins in a genomic or cDNA library. Synthetic oligonucleotides may be utilized as primers to amplify by PCR sequences from a source (RNA or DNA), preferably a cDNA library. The DNA being amplified can include cDNA or genomic DNA from any human. After successful isolation or amplification of a segment of HFGF, that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone.

Alternatives to isolating the coding regions for HFGF proteins include, but are not limited to, chemically synthesizing the gene sequence itself from the proposed sequence. Other methods are possible and within the scope of the invention. The above methods are not meant to limit the following general description of methods by which HFGF proteins can be obtained.

The identified and isolated gene can be inserted into an appropriate cloning vector for amplification of the gene sequence. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR 322 or pUC plasmid derivatives or the BLUESCRIPT vector (Stratagene). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and gene may be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.

In an alternative method, the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment of the desired gene, for example, by size fractionation, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinant DNA molecules that comprise the gene encoding HFGF protein, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA. Copies of the gene are used in mutagenesis experiments to study the structure and function of HFGF proteins.

The mutations present in HFGF proteins of the present invention can be produced by various methods known in the art. The manipulations which result in their production can be produced at the gene or protein level. For example, the cloned coding region of the KGF-2 protein can be modified by any of numerous strategies known in the art (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd. Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. Additionally, the nucleic acid sequences encoding the HFGF proteins can be mutated in vitro or in vivo, to create variations in desired coding regions (e.g., amino acid residue 87 substitution), and/or to create and/or destroy translation, initiation, and/or termination sequences, and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem 253:6551), PCR-based overlap extension (Ho et al., 1989, Gene 77:51-59), PCR-based megaprimer mutagenesis (Sarkar et al., 1990, Biotechniques, 8:404-407), etc. Mutations can be confirmed by double stranded dideoxy DNA sequencing.

Manipulations of the mutant sequence may also be made at the protein level. Included within the scope of the invention are HFGF proteins which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to another cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBH.sub.4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

In a specific embodiment, HFGF of the present invention was isolated from hair follicles of human scalp skin and showed a different level of expression in hair follicles of alopecia patients in comparison to hair follicles of persons that do not have alopecia. To investigate the expression level of HFGF, hair follicles with a morphological structure characteristic of anagen were obtained from human scalp skin of alopecia patients and persons that do not have alopecia. Total mRNA was then extracted from said hair follicles and cDNA was obtained from the total RNA by RT-PCR and analyzed on agarose gels.

The results of agarose gel analysis showed that cDNA encoding HFGF was detected in persons that do not have alopecia while cDNA corresponding to mRNA encoding HFGF in alopecia patients was not detected. In contrast, cDNA of HFGF receptor was detected in both (see FIG. 3 (see Original Patent)), demonstrating that HFGF may be a molecular factor that regulates hair loss.

In another specific embodiment, the present invention also provides a gene or isolated nucleic acid molecule encoding HFGF protein (herein referred to as "HFGF gene"). A HFGF gene encodes a protein having the 2.sup.nd to the 208.sup.th amino acids of SEQ ID NO: 1, wherein lysine 87 is replaced by glutamic acid. The HFGF gene may further comprise an initiation codon at the 5'-end of said gene.

In additional specific embodiment, mutant HFGF genes of the present, invention may comprise the 4.sup.th to the 627.sup.th nucleotides or the 118.sup.th to the 627.sup.th nucleotides of SEQ ID NO: 2, wherein the codon corresponding to the amino acid at position 87 of the amino acid sequence encodes negatively charged amino acid, i.e., glutamic acid or aspartic acid. These mutants may further comprise an initiation codon at the 5'-end of said nucleotide sequences.

The present invention provides methods for producing HFGF proteins. Methods of the present invention include subcloning, for example, HFGF gene into a vector, transforming host cells with said vector and culturing said transformants, wherein said HFGF gene encodes the 2.sup.nd to the 208.sup.th amino acids of SEQ ID NO:1, and may further comprise an initiation codon at the 5'-end of the nucleic acid sequences. A HFGF gene of the present invention may be a gene fusion in which additional nucleotide sequences are joined to a HFGF gene.

The present invention also provides pharmaceutical compositions containing HFGF proteins, or genes encoding said proteins, as an active component. A specific embodiment uses HFGF comprising the 2.sup.nd to the 208.sup.th amino acids of SEQ ID NO:1. In another specific embodiment, the pharmaceutical composition of the present invention contains HFGF protein comprising the 40.sup.th to the 208.sup.th amino acids of SEQ ID NO:1 or a gene encoding said analogue. In further specific embodiment, the pharmaceutical composition contains another HFGF protein having the 69.sup.th to the 208.sup.th amino acids of SEQ ID NO:1. In additional embodiments, the pharmaceutical composition of the present invention contains HFGF protein comprising the 2.sup.nd to the 208.sup.th amino acids of SEQ ID NO:1, wherein Asp 87 is replaced by Glu 87, the 40.sup.th to the 208.sup.th amino acids of SEQ ID NO:1, whereinAsp 87 is replaced by Glu 87, 69.sup.th to the 208.sup.th amino acids of SEQ ID NO:1, whereinAsp 87 is replaced by Glu 87 or genes encoding said proteins.

The pharmaceutical compositions of the present invention are useful for preventing or treating alopecia and for promoting or accelerating hair growth and hair follicle repair. In a specific embodiment, the present invention provides methods of using HFGF or HFGF gene to prevent or treat or ameliorate alopecia, comprising administering a pharmaceutical composition containing HFGF or HFGF gene as an effective component to a patient in need thereof

Another aspect of the present invention is a HFGF gene encoding HFGF protein. A HFGF gene may be constituted of all possible degenerate sequences encoding said amino acid sequence. Furthermore, a HFGF gene may be in the form of cDNA or gDNA (genomic DNA), and it may comprise non-coding regions such as introns, promoters and/or enhancers. In one preferred embodiment of the invention, mutant HFGF genes encode the 2.sup.nd to the 208.sup.th amino acids, the 40.sup.th to the 208.sup.th amino acids or the 69.sup.th to the 208.sup.th of SEQ ID NO:1, wherein the amino acid residue at position 87 is glutamic acid or aspartic acid, and may further comprise an initiation codon at the 5'-end the nucleotide sequences.

In another preferred embodiment, mutant HFGF genes of the present invention comprise the 4.sup.th to the 627.sup.th or the 118.sup.th to the 627.sup.th nucleotides of SEQ ID NO: 2, wherein the codon corresponding to the amino acid at position 87 of the amino acid sequence encodes glutamic acid or aspartic acid, and may further comprise an initiation codon at the 5'-end of the nucleotide sequences. Of course, it would be routine for those skilled in the art to generate variants of the above nucleotide sequences by virtue of the degeneracy of the genetic code. Degenerate variants of the disclosed nucleic acid sequences are an aspect of the present invention.

In a specific embodiment to obtain a HFGF gene of the present invention, total RNA was extracted from hair follicle cells. As generally known to those of skill in the art, total RNA derived from a cell can be converted to cDNA by PCR or RT-PCR using oligonucleotide primers corresponding to specific nucleotide sequences of the gene or nucleic acid sequence intended to be amplified.

In this regard, oligonucleotide primers shown as SEQ ID NO: 3 and SEQ ID NO: 4 were utilized to amplify a HFGF gene comprising the 1.sup.st to the 627.sup.th nucleotides of SEQ ID NO: 2, and oligonucleotide primers shown as SEQ ID NO: 9 and SEQ ID NO: 10 were used to amplify a HFGF gene comprising the 118.sup.th to the 627.sup.th nucleotides of SEQ ID NO: 2.

In one embodiment, total RNA extracted from hair follicle cells was used as template to perform PCR with oligonucleotide primers shown as SEQ ID NO: 3 and SEQ ID NO: 4. The nucleotide sequence of a PCR product was identified, which provides a new cDNA sequence in which Glu is substituted for Lys at the 87.sup.th codon of that of human kgf-2 gene (see FIG. 1 and SEQ ID NO:2 (see Original Patent)).

The gene, newly isolated by the above process and referred to as HFGF gene, was inserted into the pGEM-T vector to construct a recombinant plasmid which can express HFGF of the present invention in a host cell. The construct was designated pGEM-T-KFG-2A and deposited in Korean Collection for Type Cultures which is an international depository authority under the regulations of Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedures, on Mar. 19, 2001 as Accession No. KCTC-1012BP.

In a further aspect, the present invention provides a method for producing HFGF. In one embodiment, HFGF may be produced by direct synthetic processes to yield a protein corresponding to the amino acid sequence of SEQ ID NO:1. In another embodiment, HFGF may be isolated from hair follicles of human scalp skin. In still another embodiment, HFGF may be prepared by recombinant expression using a HFGF gene.

In a specific embodiment of the invention, HFGF is produced by subcloning a HFGF gene into a vector, transforming a host cell with said vector and culturing said transformant, wherein said HFGF gene encodes the 2.sup.nd to the 208.sup.th amino acids or the 40.sup.th to the 208.sup.th amino acids of SEQ ID NO:1, and may further comprise an initiation codon at the 5'-end of the nucleic acid sequences. A HFGF gene of the present invention may be a gene fusion in which additional nucleotide sequences are joined to a HFGF gene.

Examples of additional nucleotide sequences that may be fused to a HFGF gene sequence include sequences encoding signals (such as secretion signal sequences) for protein transport following protein expression, membrane anchor sequences, immunogenic determinants, tags, such as Histidine tags for aiding in the isolation or purification of protein; glutathione-S-transferase, and enzyme-specific restriction sequences etc. The additional sequences may be cut or removed after expression or purification of protein.

In one embodiment, a recombinant plasmid may be constructed by subcloning a HFGF gene into a commercial expression vector such as pET9c or pGEX-2T. The pET9c vector contains a T7 promoter for inducing high level expression of a target gene. The pGEX-2T vector contains a GST (Glutathione S Transferase)-encoding sequence upstream of the insertion site for a target gene, which results in expression of GST-fusion protein.

In one embodiment, a pGEX-2T-HFGF recombinant plasmid was constructed by subcloning a HFGF gene into the pGEX-2T vector, wherein said HFGF gene comprised the 118.sup.th to the 627.sup.th nucleotides of SEQ ID NO:2 wherein the sequence lacked the 1.sup.st to the 117.sup.th nucleotides encoding a signal sequence.

A transformant of the present invention may be prepared by transforming prokaryotic or eukaryotic host cells such as E. coli or yeast with a recombinant plasmid containing HFGF gene using well-known methodology, e.g. calcium chloride mediated transformation, calcium-phosphate precipitation, liposome mediation, microinjection, transfection by electroporation, etc.

In a preferred embodiment of the invention, E. coli strain BL21(DE3) is utilized as a host cell, that is, E. coli BL21(DE3) is transformed with a pGEX-2T-HFGF vector.

Generally, to isolate and purify protein from a transformant, the transformant is cultured for an appropriate time and then lysed. Subsequently, selective precipitation, chromatography, dialysis and/or filtration may be performed to purify the desired protein.

In a preferred embodiment of the invention, the above E. coli BL21(DE3) containing pGEX-2T-HFGF vector was cultured for 48 hours or more and lysed. Said cell lysate was then applied to a heparin-Sepharose column, hHFGF was eluted with a concentration-gradient using NaCl solutions and purified.

If a host cell transformed with pGEX-2T-BFGF vector is cultured, a GST-HFGF fusion protein is expressed. In this case, said fusion protein may be specifically purified using glutathione column chromatography. The GST moiety may be removed from the target protein by thrombin treatment. The selection and application of a suitable column for isolation and purification of protein will be appreciated by those skilled in the art.

In the case of subcloning a fused gene composed of a HFGF gene fused with additional nucleotide sequences into an expression vector, amino acids encoded by the additional nucleotides may be cut or removed from HFGF by using enzymes such as trypsin, or any other endopeptidase or endoprotease, after expression of the fusion protein.

The apparent molecular mass of HFGF determined by polyacrylaminde gel electrophoresis (PAGE) was approximately 20 kDa and that of a GST-HFGF fusion protein was approximately 45 kDa, consistent with the predicted molecular weights (see FIG. 2 (see Original Patent)).

In another aspect, the present invention is also directed to pharmaceutical compositions containing HFGF, or a gene encoding a HFGF, as an effective component, wherein said HFGF comprises the 2.sup.nd to the 208.sup.th amino acids or the 40.sup.th to the 208.sup.th amino acids of SEQ ID NO:1, and may further comprise a Met residue at the N-terminus of said amino acid sequences, wherein said HFGF may be mature protein.

Furthermore, a HFGF gene of the present invention encodes the 2.sup.nd to the 208.sup.th amino acids or the 40.sup.th to the 208.sup.th amino acids of SEQ ID NO:1, and may further comprise an initiation codon at the 5'-end of the nucleic acid sequence, and said HFGF gene may be a fused gene bound to additional nucleotide sequences.

Mitogenic activity of HFGF on human hair follicles was measured to investigate the biological activities of HFGF. Particularly, HFGF was used to treat hair follicle cells from human scalp skin, and after a period of about 48 hours, cell proliferation was measured by colorimetric MTS assays.

Accordingly, as seen in FIG. 5 (see Original Patent), the addition of HFGF resulted in a dose-dependent stimulation of proliferation of human hair follicle cells with a maximum stimulatory effect observed at a HFGF concentration of 30 ng/ml; with an increased effect of 140% compared to negative control of 100%.

To exclude any effect caused by endogenous KGF-2, HFGF was used to treat human hair follicles in which dermal papilla (DP) were removed surgically. Cell proliferation was measured by colorimetric MTS assay.

Interestingly, HFGF stimulated the proliferation of DP-deleted hair follicle cells with a surprisingly increased level compared to DP-containing hair follicle cells (see FIG. 6A and 6B (see Original Patent)).

KGF-1 and KGF-2 are closely related proteins in the FGF family. Thus, it is appropriate to compare the stimulatory effect of HFGF to that of KGF-1 and KGF-2.

Accordingly, human hair follicle cells derived from scalp skin were treated with KGF-1, KGF-2 and HFGF, respectively, and, after a lapse of about 48 hours, proliferation rates were measured by colorimetric MTS assay. Further, human hair follicles, in which dermal papilla were removed surgically to exclude any effect by endogenous KGF-2, were treated with KGF-1, KGF-2 and HFGF, respectively.

The results showed that HFGF significantly stimulated the proliferation of human hair follicle cells compared to KGF-2 and KGF-1, this was independent of removal of dermal papilla (see FIG. 7A and 7B (see Original Patent)).

The HFGF of the present invention exhibit a stimulatory effect on the proliferation of hair follicle cells. HFGF of the present invention may be used as an effective component of a pharmaceutical composition to prevent or treat alopecia and to promote or accelerate hair growth and hair follicle repair.

In this regard, a HFGF gene encoding HFGF may also be used in a gene therapy regimen to prevent or treat alopecia and for promotion or acceleration of hair growth and hair follicle repair.

Pharmaceutical compositions of the present invention may be prepared by mixing a HFGF protein or a HFGF gene with a pharmaceutically acceptable excipient or adjuvant using traditional formulating methods. Said formulating methods may comprise inserting a HFGF gene into a vector for gene therapy.

In one embodiment, the present invention includes methods for preventing or treating alopecia with a HFGF protein or a gene encoding a HFGF protein. Said methods may comprise administering a pharmaceutical composition containing a HFGF protein or a gene encoding a HFGF protein as an effective component on a patient's scalp skin in a formulation comprising a cream, lotion, gel, ointment, salve, balm, or transdermal patch.

In a further embodiment, a pharmaceutical composition containing a HFGF protein or a gene encoding a HFGF protein may be administered parenterally, i.e. intravenously, subcutaneously, intramuscularly, percutaneously or transdermally, for example, by directly applying to scalp skin.
 

Claim 1 of 12 Claims

1. A method for stimulating hair follicle cell proliferation in a subject, said method comprising administering to the subject a composition comprising a polypeptide, wherein the polypeptide comprises the amino acid sequence of amino acids 69 to 208 of SEQ ID NO:1, and wherein Glu 87 is optionally replaced by Asp 87.
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.