MXPA00003233A - Fibroblast growth factor with hepatocyte proliferation activity - Google Patents

Abstract

An additional member of the fibroblast growth factor (FGF) family of polypeptides has now been discovered and purified which has been found to exert a hepatocyte-specific mitogenic activity that distinguishes it from other FGF members. DNA molecules encoding this polypeptide can be used in gene therapy modes of administration to stimulate the proliferation of hepatocytes in vivo, or alternatively, they can be utilized in recombinant methods to produce the polypeptide for cell culture use or for therapeutic use to treat liver conditions requiring the generation and growth of hepatocytes.

Description

FACTOR OF GROWTH OF FIBROBLASTS WITH ACTIVITY OF PROLIFERATION OF HEPATOCYTES Field of the Invention This invention relates to a polypeptide (herein referred to as "FGF-16") which is a newly discovered element of the fibroblast growth factor family, as well as to homologs, analogs and derivatives thereof, and to nucleic acid molecules encoding these polypeptides, to methods for recombinant production thereof, to antibodies raised or raised against polypeptides, to methods for the use of polypeptides, and to pharmaceutical compositions of polypeptides for therapeutic use.

Background of the Invention The family of fibroblast growth factors (FGF) is now known to consist of at least fourteen elements, especially, FGF-1 to FGF-10 and homologous factors FHF-1 to FHF-4. The FGF-1 (aFGF) and the FGF-2 (bFGF), which were originally isolated from the mitogens for the REF.:119231 Fibroblasts of the brain and the pituitary gland are widely expressed in adult and developing tissues. These polypeptides exhibit multiple biological activities, including angiogenesis, mitogenesis, cell differentiation and wound healing; see Baird et al., Cancer Cells, Volume 3, pages 239-243 (1991) and Burgess et al., Annual Review of Biochemistry, Volume 58, pages 575-606 (1989). The FGF-3 (also known as "int-2"), the FGF-4 (also known as "hst / kFGF"), the FGF-5 and the FGF-6 were each originally identified as oncogenic products: see Dickson et al., Annual Review of the New York Academy of Sciences, volume 683, pages 18-26 (1991); Yoshiba et al., Annual Review of the New York Academy of Sciences, Volume 683, pages 27-37 (1991); Goldfarb et al. , Annual Review of the New York Academy of Sciences, Volume 683, pages 38-52 (1991); and Coulier et al., Annual Review of the New York Academy of Sciences, Volume 683, pages 53-61 (1991). FGF-7 (also referred to as "KGF") was isolated as a mitogenic factor for cultured keratinocytes; see Aaronson et al. , Annual Review of the New York Academy of Sciences, Volume 683, pages 62-77 (1991). FGF-8 and FGF-9 were isolated as an androgen-induced growth factor and a glial activation factor from mouse mammary carcinoma cells and human glioma cells, respectively; see Tanaka et al., Proceedings of the National Academy of Sciences USA, Volume 89, pages 8928-8932 (1991) and Miyamoto et al., Molecular Cell Biology, Volume 13, pages 4251-4259 (1993). FGF-10 was identified from the embryos of rats by the polymerase chain reaction (PCR) based on homology; see Yamasaki et al., Journal of Biological Chemistry, volume 271, pages 15918-15921 (1996). These FGFs are expressed predominantly during embryonic development and in some adult tissues. The four homologous factors (or "FHFs") were identified from the human retina by a combination of random cDNA sequencing, searches of existing sequence databases and polymerase chain reactions based on homology; see Smallwood et al., Proceedings of the National Academy of Sciences USA, Volume 93, pages 9850-9857 (1996). These FHFs are expressed predominantly in mature and developing nervous systems. It has been proposed that FHF-1, FHF-2, FHF-3, and FHF-4 should be designated as FGF-11, FGF-12, FGF-13 and FGF-14, respectively, in accordance with the recommendation of the Committee. of Nomenclature (Nomenclature Committee); see Coulier et al., Journal of Molecular Evolution, Volume 44, pages 43-56 (1997).

Brief Description of the Invention Briefly described, this invention encompasses the discovery and identification of an additional element of the family of fibroblast growth factors (FGF) of the polypeptides., designated herein as "FGF-16", as well as the fragments of the peptides and analogs thereof, as well as the nucleic acid molecules (including degenerate sequences) that encode such polypeptides. Also included within the scope of this invention are the chemical derivatives (ie, the polymer) of the polypeptides as well as the fragments and analogues composed of alternative sequences of the polypeptide, but having similar biological activity and retaining the same biological function. Additional aspects of the invention comprise vectors and transformed hosts useful for the expression of the nucleic acid molecules and the recombinant production of the polypeptides. As a further aspect, the invention involves the use of nucleic acid molecules or polypeptides to stimulate the proliferation and development of hepatocytes, both in vitro and in vivo, including the use of nucleic acids and encoded polypeptides to treat the disorders of the liver.

Brief Description of the Figures FIGURE 1. This Figure (comprised of Figures 1A and IB) shows the nucleic acid sequence of the coding region of the rat cDNA for FGF-16 from nucleotide 17 to nucleotide 637 (SEQ ID NO: 1) , and the predicted amino acid sequence of the encoded polypeptide (SEQ ID NO: 2). The start and stop codons in the nucleic acid sequence are underlined. The numbers above the individual nucleotides and below the individual amino acids refer to the nucleotide and amino acid sequences, respectively.

FIGURE 2. This Figure compares the amino acid sequence of FGF-16 of the rat with that of the FGF-9 of the rat (SEQ ID NO: 3). The numbers refer to the positions of the respective amino acid residues. The asterisks indicate those amino acid residues of FGF-16 and FGF-9 which are identical.

FIGURE 3. This Figure shows the detection of recombinant FGF-16 from the cell lysate of Sf9 cells which have been transfected with the recombinant baculovirus containing the cDNA for rat FGF-16. The culture medium and cell extracts of the Sf9 cells infected with the recombinant baculovirus were subjected to electrophoresis of the polyacrylate ida-SDS gel (12.5%). FGF-16 from the recombinant rat was detected by Western blot analysis using anti-E tag antibodies. Band 1, the cell lysate; band 2, the culture medium. The Wide Interval of the Pre-stained Protein Marker (New England Biolabs, Beverly, Massachusetts) was used as the standard for estimating the molecular mass of the polypeptides.

FIGURE 4. This Figure shows mRNA expression for rat FGF-16 in various adult tissues. The RNA aliquots (10 μg each) were subjected to electrophoresis on a denaturing agarose gel (1%) containing formaldehyde, and then transferred onto a nitrocellulose membrane. Hybridization was performed with a rat FGF-16 cDNA probe labeled with 32P. The positions of the 28S and 18S RNAs are indicated as "28S" and "18S", respectively. The bands or bands of "Br", "He", "Lu", "Li", "Ki", "BA" and "WA" indicate the mRNA of the brain of the adult, the heart, the lung, the liver, the kidney, brown adipose tissue and white adipose tissue, respectively.

FIGURE 5. This Figure, comprised of parts A, B and C, shows the location of the mRNA for FGF-16 in a sagittal section of a rat embryo (E19). The sagittal section was hybridized with a sense FGF-16 cRNA probe (Figure 5C) or antisense (Figure 5B) labeled with 35S. The section was also counterstained with hexaoxiline and eosin (Figure 5A). The "He" and "BA" marks indicate heart tissue and brown adipose tissue, respectively.

FIGURE 6. This Figure (comprised of Figures 6A and 6B) shows the nucleotide sequence of the coding region of the full-length human cDNA for FGF-16 starting at nucleotide 23 and ending at nucleotide 643 (SEC ID NO: 4) and the predicted amino acid sequence of the encoded polypeptide (SEQ ID NO: 5). The start and stop codons in the nucleic acid sequence are underlined.

FIGURE 7. This Figure, comprised of parts A, B, C and D, shows the sections of the liver labeled with BrdU from two mice injected with the control buffer (Figures 7A and 7B, respectively), side by side with the BrdU-labeled sections of the liver from the two mice injected with an N-terminal analogue with a des-34 truncation of the rat FGF-16 (des-N-34") at a dose of five milligrams per kilogram per day for seven days (Figures 7C and 7D, respectively) Comparisons show a significant increase in hepatocellular BrdU labeling in the livers of mice treated with des-N-34.

Detailed description of the invention In addition to the polypeptide of Figures 1A and IB (SEQ ID NO: 2) and the polypeptide of Figures 6A and 6B (SEQ ID NO: 5), also proposed as part of this invention are the fragments (i.e., "subsequences"). ), analogs and derivatives of such polypeptides which are substantially biologically equivalent or share one or more important biological properties, such as the growth of hepatocytes and the activity of proliferation. By "substantially biologically equivalent" is meant to have the same properties of the polypeptides as described herein, even if they are in different degrees. Preferably, such analogs will cross-react with raised or enhanced antibodies against the polypeptides of Figures 1A-1B and Figures 6A-6B. The term "analogue" is specifically proposed to mean molecules representing one or more substitutions, deletions and / or additions of amino acids, derived from the linear array of amino acids of the full length polypeptides of Figures 1A-1B and 6A- 6B, which are also substantially equivalent or share one or more biological properties. Analogs of the preferred polypeptides especially according to this invention are those which possess a degree of homology (ie, the identity of the amino acid residues) with the polypeptide of Figures 1A-1B (SEQ ID NO: 2) or the polypeptide of Figures 6A-6B (SEQ ID NO: 5) in excess of eighty percent (80%), and even more preferably, in excess of ninety percent (90%). The identity of the percentage sequence can be determined by the standard methods that are commonly used to compare the similarity in the position of the amino acids of two polypeptides to generate an optimal alignment of two respective sequences. By way of illustration, using a known computer program, such as BLAST or FASTA, two polypeptides are aligned for optimal comparison or matching of their respective amino acids (either along the entire length of one or both sequences, or at length of a predetermined portion of one or both sequences). The programs provide a "failure" opening penalty and a "failure" gap penalty, and a dialing matrix such as PAM 250. A standard dial matrix can be used in conjunction with the computer program; see Dayhoff et al., in Atlas of Protein Sequence and Structure, volume 5, Supplement 3 (1978). The percentage identity (or "homology" as used here) can then be calculated as follows: Total number of identical comparisons X 100 [No. of waste in the alignment region, not including non-identical waste at either or both ends and waste opposite a hole Analogous polypeptides according to this invention will typically have one or more substitutions, deletions and / or amino acid insertions, as mentioned. Usually, the substitutions will be conservative so that they have little effect or no effect on the total net charge, polarity or hydrophobicity of the protein. Examples of conservative substitutions are described below.

Substitutions of Conservative Amino Acids Basic: arginine lysine histidine Acids glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine The polypeptides of this invention may or may not have an amino terminal methionine, depending on the manner in which they are prepared. Typically, an amino terminal methionine residue will be present when the polypeptide is recombinantly produced in a non-secretory bacterial strain (e.g., E. coli) as the host. The polypeptide fragments (i.e., the subsequences) included within this invention will be those that have less than the full length sequence, but which possess substantially the same biological activity and are truncated at the amino terminus, the carboxy terminus, and / or internally. FGF-16 analogs comprising a truncated material of the amino acids will include those in which one or more amino acid residues are omitted from the full-length mature sequence (207 amino acids) starting from the N-terminus to approximately position 35 of the amino acids. amino acids (referred to herein as the "des-N" analogs). Also contemplated are truncated analogs in which one or more amino acid residues are omitted from the C-terminus to about position 19 of the amino acids (referred to herein as the "des-C" analogs), including the C-terminal truncations in those FGF-16 polypeptides in which amino acid residues have also been omitted from the N-terminal or extremity. When substitutions and omissions are made of the particular amino acid residues within the amino acid sequence that is naturally present (it is "natural or wild") of FGF-16, relatively conservative substitutions are preferred so that they do not adversely affect the desired biological properties to some substantial degree. Therefore, for example, residues or regions which are known or suspected to be involved in the specificity of the receptor or the binding of heparin should generally be avoided if alterations at these sites will impair these properties. A region that is believed to be important for the receptor binding specificity within FGF-16 extends from tyrosine 147 to tyrosine 161, in both the human and rat forms. Important sites for the binding of heparin (both in the rat and in humans) include arginine 68, threonine 69, asparagine 142, asparagine 166, lysine 167, arginine 172, arginine 176, glutamine 181, lysine 182 and phenylalanine 183. In general, the analogs, fragments and derivatives of the polypeptides according to this invention will be useful for the same purposes for which the polypeptides of SEQ ID NO: 2 and SEC ID NO: 5 are useful, and in particular as stimulating factors for the proliferation and growth of hepatocytes in vitro and in vivo.

Nucleic acids This invention also encompasses nucleic acid molecules that encode any of the aforementioned polypeptides. Accordingly, in addition to nucleic acid molecules having the particular sequences shown in Figures 1A and IB (SEQ ID NO: 1) and Figures 6A and 6B (SEQ ID NO: 4), degenerate nucleic sequences are also included. of them (that is, they differ in one or more bases) that encode the same polypeptide. In addition, this invention encompasses nucleic acid molecules that encode biologically active precursors and derivatives of the polypeptides described herein. In addition, the invention encompasses the nucleic acid molecules encoding the complementary strands (ie, antisense), as well as the nucleic acid molecules which hybridize (or could hybridize but for the variability of the nucleotide sequence due to degeneracy of the codons) to the nucleic acid molecules of Figures 1A-1B or Figures 6A-6B, or to the fragments or degenerate sequences thereof or their complementary strands, preferably under relatively stringent conditions (e.g. such as those described later). The present invention also encompasses nucleic acid molecules that can encode additional amino acid residues that flank the 5 'or 3' portions of the region encoding the "mature" polypeptide (i.e., the processed product collected from the host), such as the sequences encoding the alternative pre / pro regions (i.e., the sequences responsible for the secretion of the polypeptide through the membranes of the cell) instead of the "natural" pre / pro regions. Additional sequences may also constitute sequences that are coding, including regulatory sequences such as transcription or translational promoters, depending on the host cell. The nucleic acid molecules can still include several internal non-coding sequences (introns) that are known to be present within the genes. In general, when the term "stringent conditions" is used herein, it refers to hybridization and washing under conditions that allow the binding of a nucleic acid molecule such as an oligonucleotide or a cDNA molecule to the highly homologous sequences. A severe or strict wash solution, suitable for use with DNA probes at a temperature of 55-65 ° C, is composed of 0.015 M sodium chloride, 0.0015 M sodium citrate (0.1 X SSC, where 1 X SSC = 0.15 M sodium chloride and 0.015 M sodium citrate) and 0.1 percent SDS. Another slightly less severe wash solution is composed of 0.2 X SSC and 0.1 percent SDS, and can be used at a temperature between 50 and 65 ° C. Where the oligonucleotide probes are used to select the cDNA or the genomic libraries, the following severe or stringent washing conditions can be used. One protocol uses 6 X SSC with 0.05 percent sodium pyrophosphate at a temperature of 35-62 ° C, depending on the length of the oligonucleotide probe. For example, probes of 14 base pairs are washed at 35-40 ° C, probes of 17 base pairs are washed at 45-50 ° C, probes of 20 base pairs are washed at 52-57 ° C, and Probes of 23 base pairs are washed at 57-63 ° C. The temperature can be increased 2-6 ° C where the non-specific binding of the bottom seems to be high. Another protocol uses tetramethyl onium chloride (TMAC) for the washing of oligonucleotide probes. A suitable severe wash solution is composed of TMAC 3 M, 50 mM Tris-HCl, pH 8.0, and 0.2 percent SDS. The wash temperature for use with this solution is a function of the length of the probe. For example, a probe of 17 base pairs is typically washed at about 45-50 ° C. RNA molecules that are homologous with any of the aforementioned DNA molecules are also included within the scope of this invention.

Recombinant Expression The polypeptides of the invention can be prepared using well known recombinant technology methods such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) and / or Ausubel et al., Editors, Current Protocols in Molecular Biology, Green Publishers Inc., and Wiley and Sons, New York (1994). A gene or cDNA encoding the polypeptide or the truncated version thereof can be obtained, for example, by selecting a genomic or cDNA library, or by PCR amplification. Alternatively, a DNA molecule encoding the polypeptide can be prepared by chemical synthesis using methods well known to the skilled artisan, such as those described by Engels et al. in Angew. Chem. Intl. D., Volume 28, pages 716-734 (1989). Typically, the DNA encoding the polypeptide will be several hundred nucleotides in length. Nucleic acids larger than about one hundred nucleotides can be synthesized as several fragments using these same methods, and the fragments can then be ligated together to form a nucleotide sequence of the desired length. The nucleic acid molecules of this invention (either genes or cDNAs) can be inserted into an appropriate expression vector for expression in a suitable host organism or cell using recombinant methods. The vector is selected to be functional in the particular host employed (ie, the vector is compatible with the machinery of the host cell, such that amplification and / or expression of the gene can occur). The polypeptide, fragment or analog can be amplified / expressed in prokaryotic, yeast, insect (baculovirus systems) and / or eukaryotic host cells, or transgenic non-human animal species such as the host. The selection of the host cell will depend at least in part on whether the polypeptide expression product is going to be glycosylated. If glycosylation is desired, then host cells of yeast, insects or mammals are preferred for use, because yeast cells will glycosylate the polypeptide, and insect and mammalian cells can glycosylate and / or phosphorylate the a manner similar to glycosylation and / or "natural" phosphorylation. Vectors used in any of the host cells to express the polypeptide may also contain a 5 'flanking sequence (also referred to as a "promoter") and other regulatory elements of expression operably linked to the nucleic acid (DNA) molecule that will be expressed, as well as to the enhancer (s), an origin of replication element, a transcriptional termination element, a complete sequence of introns containing a splice site of the donor and the acceptor, a sequence of the signal peptide, an element of the ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Each of these elements is described later. Optionally, the vector may also contain a "tag" sequence, i.e., an oligonucleotide sequence located at the 5 'or 3' end of the coding sequence for the polypeptide encoding polyHis (such as hexaHis) or another small immunogenic sequence. . This tag will be expressed in the carrier of the protein, and can serve as an affinity tag for the purification of the polypeptide from the host cell. Optionally, the tag can be subsequently removed from the purified polypeptide by various means, for example, with the use of a selective peptidase. The flanking sequence of 5 'may be the natural flanking sequence 5', or it may be homologous (i.e., from the same species and / or the strain as the host), heterologous (ie from a different species of the host cell or strain), hybrid (ie, a combination of the 5 'flanking sequences). from more than one source), or synthetic. The source of the 5 'flanking sequence can be any unicellular prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, so long as the flanking sequence 5' is functional in, and can be activated by the machinery of the cell Guest. Where the 5 'flanking sequence is not known, a DNA fragment containing a 5' flanking sequence can be isolated from a larger piece of the DNA it can contain, for example, a coding sequence or even another gene or genes. Isolation can be effected by digestion of the restriction endonuclease using one or more carefully selected enzymes to isolate the appropriate DNA fragment. After digestion, the desired fragment can be isolated by the purification of the agarose gel, or by other methods known to the skilled artisan. The selection of suitable enzymes to carry out this purpose will be readily apparent to a person skilled in the art. The origin of the replication element is typically a part of the commercially purchased prokaryotic expression vectors, which aid in the amplification of the vector in a host cell. The amplification of the vector up to a certain number of copies, in some cases, may be important for optimal expression of the polypeptide. If the vector of choice does not contain an origin of the replication site, one can be synthesized chemically based on a known sequence and then ligated into the vector. The transcription termination element is typically located 3 'to the end of the sequence encoding the polypeptide and serves to terminate transcription of the polypeptide. Usually, the transcription termination element in the prokaryotic cells is a fragment rich in G-C followed by a poly-T sequence. Although the element is easily cloned from a library or even commercially purchased as part of a vector, it can also be easily synthesized using methods for nucleic acid synthesis such as those referred to above. A selectable marker gene element encodes a protein necessary for the survival and growth of a host cell that has grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, eg, ampicillin, tetracycline or kanamycin for prokaryotic host cells, (b) complement the auxotrophic deficiencies of the cell, or ( c) they supply critical nutrients not available from the complex medium. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene and the tetracycline resistance gene. The ribosome binding element, commonly called the Shine-Delgarno sequence (for prokaryotes) or the Kozak sequence (for eukaryotes), is necessary for the initiation of translation for mRNA. The element is typically located 3 'with respect to the promoter and 5' with respect to the coding sequence of the polypeptide to be synthesized. The Shine-Dalgarno sequence is varied but is typically a polypurine (ie, having a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be easily synthesized using the methods described above and used in a prokaryotic vector. In those cases where it is desirable for the polypeptide to be secreted from the host cell, a signal sequence can be used to direct the polypeptide out of the host cell where it is synthesized. Typically, the sequence of the signal is placed in the coding region of the nucleic acid sequence, or directly at the 5 'end of the coding region. Many of the sequences of the signal have been identified, and any of them that are functional in the selected host cell can be used here. Accordingly, the signal sequence can be homologous or heterologous with respect to the polypeptide. Additionally, the signal sequence can be synthesized chemically using methods such as those referred to herein above. The host cells can be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as yeast, insect or vertebrate cells). The host cell, when cultured under suitable nutrient conditions, can synthesize the polypeptide, which can be subsequently collected by the isolation of the culture medium (if the host cell secretes it in the medium) or directly from the host cell that produces it. (if it is not secreted). After the collection, the polypeptide can be purified using methods such as molecular sieve chromatography, affinity chromatography, and the like. In general, if the polypeptide is expressed in E. coli it will contain a methionine residue in the N-terminus in its recovered form (ie, Met "1), unless it is expressed in an E. coli strain in the which methionine is enzymatically cleaved by the host.The cells or suitable cell lines can also be animal cells, and especially mammalian cells, such as Chinese hamster ovary (CHO) cells or 3T3 cells. Suitable mammalian hosts and methods for transformation, cultivation, amplification, selection and production and purification of the product are already known in the art.Other suitable mammalian cell lines are the COS-1 and COS- cell lines. 7 of the monkey, and the CV-1 cell line Additional exemplary mammalian host cells include primate cell lines and rodent cell lines, including cell lines is transformed Normal cells, cell strains derived from the in vitro culture of primary tissue, as well as primary explants, are also suitable. The candidate cells may be genotypically deficient in the selection gene, or they may contain a dominantly acting selection gene. Still other suitable mammalian cell lines include, but are not limited to, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss mice, Balb-c or NIH, hamster cell lines BHK or HaK. The insertion (also referred to as the "transformation" or "transfection") of the vector into the selected host cell can be effected using calcium chloride, electroporation, microinjection, lipofection or the DEAE-dextran method. The particular method selected will depend, in part, on the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan. See, for example, Sambrook et al., Supra. The host cells containing the vector can be cultured using standard means well known to the skilled artisan. The medium will usually contain all the nutrients necessary for the growth and survival of the transformed cells. Suitable media for the culture of E. coli cells are, for example, the Luria Broth (LB) and / or the Terrific Broth (TB). Suitable means for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all of which can be supplemented with serum and / or growth factors when required by the particular cell line that is cultured. A suitable medium for the cultivation of insect cells is Grace's medium supplemented with yeast hydrolyzate (yeastolate), lactalbumin hydrolyzate and / or fetal bovine serum, when necessary. Typically, an antibiotic or other compound useful for the selective growth of transformed cells is added as a supplement to the medium. The compound to be used will be chosen by the selectable marker element present on the plasmid with which the host cell was transformed. For example, where the selectable marker element is kanamycin resistance, the compound added to the culture medium will be kanamycin. The amount of the polypeptide produced in the host cell can be evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS polyacrylamide gel electrophoresis, electrophoresis of non-denaturing gel, HPLC separation, immunoprecipitation, and / or activity assays such as assays. of displacement of the DNA binding gel.

If the polypeptide has been designed to be secreted from the host cells, the majority of the polypeptide will likely be found in the cell culture medium. If, on the other hand, the polypeptide is not secreted, it is present in the cytoplasm (for eukaryotic cells, gram-positive bacteria and insect host cells) or in the periplasm (for host cells of gram bacteria -negative). For the intracellular polypeptide, the host cells are typically first mechanically or osmotically altered to release the cytoplasmic contents in a buffered solution. The polypeptide is then isolated from this solution. The purification of the polypeptide from the solution can then be effected using a variety of techniques. If the polypeptide has been synthesized to contain a label such as Hexahistidine or another small peptide at any of the carboxyl or amino termini, it can be purified in a one step process by passing the solution through an affinity column in the cell. which the column matrix has a high affinity for the label or for the polypeptide directly (ie, a monoclonal antibody). For example, polyhistidine binds with great affinity and specificity to nickel, therefore an affinity column of nickel (such as the nickel columns of Qiagen) can be used for purification. (See, for example, Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1994). Where, on the other hand, the polypeptide does not have a label and the antibodies are not available, other well known methods for purification can be used. Such methods include, without limitation, ion exchange chromatography, molecular sieve chromatography, hydrophobic interaction chromatography, reverse phase chromatography, electrophoresis of the natural gel in combination with gel elution, and preparative isoelectric focusing. These operations can be performed in low pressure or CLAR modes. In some cases, two or more of these techniques can be combined to achieve increased purity. If it is anticipated that the polypeptide will be found primarily in the periplasmic space of bacteria or the cytoplasm of eukaryotic cells, the contents of the periplasm or the cytoplasm, including inclusion bodies (eg, Gram-negative bacteria) if the polypeptide processed has formed such complexes, can be extracted from the host cell using any standard technique known to the skilled artisan. For example, host cells can be used to release the contents of the periplasm by the use of a French press, homogenization and / or sound application. The homogenate can then be centrifuged.

Other Modes of DNA Expression In addition to the typical methods of recombinant expression in heterologous hosts such as described, the polypeptides of this invention can be expressed by other known means, which may or may not involve the use of expression vectors. For example, techniques of homologous recombination can be employed to facilitate the expression of a polypeptide of this invention, without the use of an expression vector. By way of illustration, a suitable DNA construct, comprising a regulatory element, can be inserted into the genome of a cell by homologous recombination in such a way that it is in close proximity to a segment of the endogenous gene encoding the FGF -16 and stimulates the expression of it in situ. See U.S. Pat. No. 5, 212, 011 (Chappel). The product of polypeptide expression is then collected and purified and can be used in the same manner as polypeptides produced by recombinant expression in a heterologous host. Alternatively, the isolated nucleic acid molecules described herein can be used directly in cell or gene therapy applications. Suitable vectors for gene therapy, such as retroviral or adenoviral vectors, are known and can be modified to incorporate the nucleic acid encoding the polypeptide of the present invention for administration to the patient and expression at a desired location in vivo. . See, for example, Verma, Scientific American, pages 68-84 (November 1990). In these situations, the genomic DNA, the cDNA, and / or the synthetic DNA encoding the polypeptide or a fragment or variant thereof can be operably linked to a constitutive or inducible promoter which is active in the tissue in which it is desired. the expression, then inserted into a suitable vector. In a particular method of application, the cells are removed from the patient, transfected with the gene therapy vector using standard transfection procedures for eukaryotic cells, and tested for production and secretion of the polypeptide. Such cells can be liver cells, bone marrow cells, cells derived from the umbilical cord or blood progenitor cells, for example. Alternatively, the cells can be pretreated with a regulatory element of the DNA or segment to activate by means of homologous recombination the genomic expression of an endogenous DNA molecule encoding the polypeptide in the manner described above. These cells that express and secrete the polypeptide can then be reintroduced into the patient such that they are then viable and function as a localized source of the polypeptide in situ. The cellular supply can be regulated to last for a preselected period of time, such as days, weeks or months, at the end of which the recipient can receive another "dose" (i.e., a fresh transplant of the cells that secrete the polypeptide) . Other methods may involve the targeted delivery of DNA constructs directly to tissues or organs in the body for in situ expression of therapeutically effective amounts, such as those described in further detail below.

Polypeptide derivatives Chemically modified polypeptides, in which the polypeptide is linked to a polymer to modify the properties (referred to herein as the "derivatives"), are included within the scope of the present invention. The polymer is typically soluble in water so that the protein to which it is fixed is not precipitated in an aqueous environment, such as a physiological environment. The polymer can have a unique reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled. A preferred reactive aldehyde is polyethylene glycol propionaldehyde, which is stable in water, or C1-C10 mono alkoxy or aryl xi derivatives thereof (see U.S. Patent No. 5,252,714). The polymer can be branched or unbranched. Preferably, for therapeutic use of the preparation of the final product, the polymer will be pharmaceutically acceptable. By way of illustration, the water soluble polymer, or a mixture thereof if desired, can be selected from the group consisting of polyethylene glycol (PEG), monomethoxy polyethylene glycol, dextran, cellulose, or other polymers based on carbohydrates, polymers and polymers. (N-vinyl pyrrolidone) polyethylene glycol, homopolymers of propylene glycol, a copolymer of polypropylene oxide / ethylene oxide, polyoxyethylated polyols (for example, glycerol) and polyvinyl alcohol.

Pharmaceutical Compositions For therapeutic purposes, the polypeptides of this invention, or fragments, analogs or derivatives thereof, will typically be formulated into suitable pharmaceutical compositions adapted for therapeutic delivery, which constitutes a further aspect of this invention. Such pharmaceutical compositions will typically comprise an effective amount of the polypeptide (or fragment, analog or derivative), alone or together with one or more carriers, excipients or other standard ingredients for a pharmaceutical composition. By "effective amount" is meant an amount sufficient to produce a measurable biological effect on the cells or organ being treated, such as the proliferation or growth of hepatocytes or the regeneration of liver tissue (See later for additional details). The carrier material can be water for injection, preferably supplemented with other common materials in solutions for administration to mammals. Typically, the polypeptide will be administered in the form of a composition comprising a purified form of the polypeptide (which can be chemically modified) in conjunction with one or more carriers, excipients, or physiologically acceptable diluents. Neutral buffered brine or brine mixed with whey albumin are exemplary appropriate carriers. Other carriers, diluents, and standard excipients may be included when desired. The pharmaceutical compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, ed. ., Mack Publishing Company, 1990) in the form of a lyophilized cake or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the receptors and are preferably inert in the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate, succinate or other organic acid salts; antioxidants such as ascorbic acid; polypeptides of low molecular weight; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, sucrose, lactose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; the counterions that form salts such as sodium; and / or nonionic surfactants such as Tween or Pluronics. Any composition of this invention which is proposed to be used for in vivo administration must be sterile. Sterilization is easily effected by filtration through the sterile filtration membranes. Where the composition is lyophilized, sterilization using these methods can be conducted either prior to, or after, lyophilization and reconstitution. The composition for parenteral administration will commonly be stored in the lyophilized form or in solution.

Preferred Stabilizing Agents The stability of the polypeptides of this invention below, at or about room temperature in the buffered solution, can be improved, and any tendency toward aggregation of the reduced protein, by the addition of an organic or inorganic sulfate (e.g. , sodium sulfate or ammonium sulfate) and EDTA. Preferably, the sulfate salt is employed in an amount of about 10 mM or greater, and the EDTA in an amount of about 1 μM to about 10 mM.

Dosages and Administration Routes The amount of the polypeptide that will be effective in the treatment of a particular disorder or condition will depend on the nature of the polypeptide and the disorder or condition, as well as the age and general health of the recipient, and can be determined by standard clinical procedures. Wherever possible, it will be desirable to determine the dose response curve of the pharmaceutical composition primarily in vitro, as in bioassay systems, and then in animal model systems useful in vivo prior to testing in humans. In general, suitable amounts in vivo can be developed based on an understanding of the therapeutically known effective doses for the wild type protein on which the analogs are based. The expert doctor, considering the therapeutic context, the type of disorder under treatment, and other applicable factors, will be able to figure out the proper dosage without undue effort. For in vivo administration to humans, it is anticipated that amounts in the range of about thirty micrograms to about three hundred micrograms per kilograms of body weight per day will be adequate. Typically, a physician will administer the composition of the polypeptide until a dose is reached, which achieves the desired effect. The composition can be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the polypeptide) over time, or on a continuous basis. The route of administration of the therapeutically active polypeptides or the pharmaceutical compositions of the polypeptides can be according to any of the known methods, such as the oral route or by injection or infusion by the intravenous (or intraarterial), intraperitoneal, or intralesional routes. , or by the use of sustained release systems or implants. Where desired, the compositions can be administered continuously by infusion, bolus injection or by an implant device. Alternatively or additionally, the polypeptides of this invention may be administered locally by means of implantation in the affected area of a membrane, sponge, or other appropriate material upon which the polypeptide has been absorbed. Where an implant device is used, the device can be implanted in any suitable tissue or organ. The polypeptides of this invention can also be administered in a sustained release formulation or preparation. Suitable examples of the sustained release preparations include the semipermeable polymer matrices in the form of shaped articles, for example, films or microcapsules. Sustained-release matrices include polyesters, hydrogels, polylactides (US Patent No. 3,773,919), L-glutamic acid copolymers and gamma ethyl-L-glutamine (Sidman et al, Biopolymers, volume 22, pages 547-556, 1983), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed, Mater. Res., Volume 15, pages 167-277, 1981, and Langer, Chem. Tech., Volume 12, pages 98- 105, 1982), ethylene vinyl acetate (Langer et al., Supra) or poly-D (-) - 3-hydroxybutyric acid. Sustained release compositions can also include liposomes, which can be prepared by any of the various methods known in the art.; see, for example, Epstein et al., Proc. Natl. Acad. Sci. USA, volume 82, pages 3688-3692 (1985), and Hwang et al., Proc. Natl. Acad. Sci. USA, Volume 77, pages 4030-4034 (1980).

Applications of Gene and Cell Therapy In certain situations, it may be desirable to use the so-called therapy methods of the genes or cells for administration. Such a delivery form may be particularly effective for the in vivo stimulation of the proliferation of hepatocytes in the liver. In these situations, the genomic DNA, the cDNA and / or the synthetic DNA encoding the polypeptide or a fragment or variant thereof can be operably linked to a constitutive or inducible promoter which is active in the tissue within which the composition will be injected. This construct can then be inserted into a suitable vector such as an adenovirus vector or a retrovirus vector to create a gene therapy vector. The cells of the patient to be treated can be removed from the patient, transfected with the gene therapy vector using standard transfection procedures for eukaryotic cells, and tested for the production and secretion of the polypeptides. Those cells that express and secrete the polypeptide can then be reintroduced into the patient in such a way that they are viable and function as a localized source of the polypeptide in situ. Alternatively, the DNA construct can be injected directly into the tissue of the organ to be treated, where it can be absorbed in vivo and expressed locally in the cells, provided that the DNA is operably linked to a promoter that is active. in such tissue. The construction of the DNA may also additionally include the sequence of vectors of vectors such as an adenovirus vector, a retroviral vector and / or an Herpes Airus vector, to aid absorption in the cells. For the in vivo regeneration of hepatocytes in the liver, the use of Moloney retroviral vectors can be especially effective; see Bosch et al., Journal of Clinical Investigation, volume 98, Number 12, pages 2683-2687 (1966). The DNA / vector construct can be mixed with a pharmaceutically acceptable carrier or carriers for injection.

Antibody Formation and Diagnostic Applications The polypeptides of this invention can also be used according to standard procedures for generating antibodies which are useful for medically related purposes. In particular, such antibodies, preferably in the labeled form, will be useful for diagnostic purposes both in vivo and in vitro to detect the presence of the polypeptides in a fluid or tissue sample, for example. Various methods known in the art can be employed for the production of polyclonal antibodies that recognize the epitopes of the polypeptides. For the production of antibodies, several host animals can be immunized by injection with an analogous polypeptide, or fragment or derivative thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants can be used to increase the immune response, depending on the host species, including but not limited to Freund mineral gels such as aluminum hydroxide (alum), surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as Bacillus Calmette-Guerin and Corynebacterium parvum. For the preparation of monoclonal antibodies directed towards the polypeptides, any technique that provides for the production of the antibody molecules by the continuous cell lines in the culture can be used. For example, the hybridoma technique originally developed by Kohler and Milstein and described in Nature, volume 256, pages 495-497 (1975), as well as the trioma technique, the human B cell hybridoma technique described by Kozbor et al. in Immunology Today, Volume 4, page 72 (1983), and the Hybridoma-EBV technique to produce monoclonal antibodies described by Colé et al in "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, Inc., pages 77-96 (1985), are all useful for the preparation of monoclonal antibodies. In addition, a molecular clone of an antibody for an epitope or epitopes of the polypeptide can be prepared by known techniques. In particular, the recombinant DNA methodology can be used to construct the nucleic acid sequences encoding a monoclonal antibody molecule or antigen binding region thereof.; see, for example, Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982).

Description of the Specific Modalities The invention is described in further detail with respect to the following materials, methods and procedures, which are understood to be illustrative only and are not intended to be limiting.

I. Preparation of RNA from Adult Rat and Tissue Embryos RNA was prepared from Wistar rat tissues (male) using a commercially available RNA extraction set or set (Pharmacia Biotech, Uppsala, Sweden). Poly (A) + RNA was prepared using an oligo (dT) -cellulose (Type 2, Collaborative Bio edical Products, Bedford, Massachusetts).

II. Isolation and Analysis of the cDNAs of the Rat FGF Family Five micrograms of the rat (A) + RNA from the rat brain were incubated for sixty minutes at 37 ° C in twenty microliters (μl) of a reaction mixture containing three hundred units of reverse transcriptase of murine leukemia virus. Moloney (GIBCO-BRL, Gaithersburg, Maryland), fifteen units of the human placental RNase inhibitor (Wako Puré Chemicals, Osaka, Japan) and half a microgram (μg) of a random hexadeoxinucleotide primer. To amplify the cDNAs of the FGF family, the PCR was carried out for thirty cycles in twenty-five microliters of a reaction mixture containing an aliquot of the above cDNA solution, 0.05 units per microliter (unit / μl) of Taq DNA polymerase (Wako Puré Chemicals) and five picomoles per microliter (pmol / μl) of each of the sense and antisense degenerate primers representing all possible codons corresponding to the consensus amino acid sequences of the FGF-3 and FGF-7 of the mouse, YLAMNK and YNTYAS, respectively; see Moore et al., EMBO Journal, volume 5, pages 919-924 (1986) and Mason et al., Mech. Dev., Volume 45, pages 15-30 (1994). The amplified DNA of the expected size (approximately 110 base pairs) was cloned into the pGEM-T DNA vector (Promega, Madison, Wisconsin). The nucleotide sequence of the cloned DNA was determined by a DNA sequencer (Applied Biosystems, Foster, California). To determine the entire coding region, the cDNA synthesized from the poly (A) + rat heart RNA was analyzed by the Rapid Amplification method of the cDNA ends (RACE); Frohman et al., Proceedings of the National Academy of Sciences USA, Volume 78, pages 3824-3828 (1988). The cDNA covering the entire coding region was amplified by PCR (twenty-five cycles) in a reaction mixture containing an aliquot of the cDNA solution from the heart of the rat, 0.05 units / μl of the Ex Taq DNA polymerase (TaKaRa , Kyoto, Japan) and 0.4 pmoles / μl of each of the sense and antisense primers for their 5 'and 3' noncoding regions, and it was then cloned into the pGEM-T vector of the DNA.

III. Expression of rat FGF-16 cDNA in Sf9 Cells A cDNA of FGF-16 with a DNA fragment (75 base pairs) encoding an E tag (GAPVPYPDPLEPR) (SEQ ID NO: 6) and a 6XHis tag (HHHHHH) at the 3 'end of the coding region was constructed in the DNA of the transfer vector, pBacPAK9 (Clontech, Palo Alto, California). The recombinant baculovirs containing the FGF-16 cDNA with the tag sequences were obtained by cotransfection of the Sf9 cells with the recombinant pBacPAK9 and an I-digested expression vector Nsu36 (Promega) The Sf9 cells were infected with the resulting recombinant baculovirus to produce the FGF-16 with the tags.

IV. Detection of FGF-16 from the recombinant rat by Western blot analysis The culture supernatant and the cell lysate of the Sf9 cells infected with the recombinant baculovirus were subjected to polyacrylamide gel • sodium dodecyl sulfate (SDS) electrophoresis (12.5%), then transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham, Buckinghamshire, England). The membrane was then incubated with a salted solution buffered with phosphate (PBS) containing 0.1% Tween and 5% non-fat dry milk. The incubation was conducted for one hour at room temperature with anti-E tag antibodies (Pharmacia Biotech) in PBS containing 0.1% Tween 20 (PBS-T). After washing in PBS-T, the membrane was treated for one hour at room temperature with goat anti-mouse immunoglobulin conjugated with horseradish peroxidase (Cappel, Durham, North Carolina). The membrane was washed four times in PBS-T and reacted with the chemiluminescent horseradish peroxidase substrate (Amersham). The protein labeled E was visualized by exposure of the membrane to the X-ray film (RX Medical, Fuji Photo Film Co., Tokyo, Japan).

V. Analysis of Northern Spotting The aliquots of the RNA (10 μg each) of the tissues of the rat were dissolved on a denaturing agarose gel (1%) containing formaldehyde, and then transferred to a cellulose membrane in 20X SSC (IX SSC; 0.15M). NaCl / 0.015 M sodium citrate) overnight. The membrane was then baked at 80 ° C for two hours under vacuum, then prehybridized at 60 ° C for four hours in the hybridization solution (5X SSC / 0.1% sodium dodecyl sulfate (SDS) / 4X Denhardt's solution / 100 μg / ml of denatured salmon sperm DNA with heating / 5% dextran sulfate and sodium), and followed by hybridization at 60 ° C for eighteen hours in a hybridization solution containing a labeled FGF-16 cDNA probe with 32P, labeled by a random primer labeling kit (TaKaRa) with deoxycytidine 5 '- [a-32P] triphosphate (approximately 110 TBq / mmol) (ICN Biomedicals Inc., Costa Mesa, California). The membrane was then washed at room temperature three times, for twenty minutes each time, in IX SSC and 0.1% SDS, and twice at 60 ° C in 0.2X SSC and 0.1% SDS. The washed membrane was analyzed with a radio imaging analyzer (BAS 2000, Fuji Photo Film Co.).

SAW. In Situ Hybridization The embryos of Wistar rats (El9) were frozen in powdered dry ice. The sagittal sections were cut at sixteen micrometers (μm) by a cryostat, then mounted to liquefy on polylysine-coated slides, and stored at -85 ° C until hybridization. The rat FGF-16 sense and antisense cRNA probes, labeled with 35S, were transcribed using the SP6 RNA polymerase and the T7 RNA polymerase (TaKaRa) with 5'-a. { 35S] uridine thiotriphosphate (approximately 30 TBq / mmoles) (Amersham), respectively. Sections were examined by in situ hybridization with the probe labeled as described in Yamasaki et al., Supra. To determine the regional location of the rat FGF mRNA, the labeled sections were exposed to an X-ray film (Hyperfilm-ß max, Amersham). The sections were visualized by the counterstained with hematoxylin and eosin.

VII. Isolation of the cDNA encoding the rat FGF-16 The elements of the FGF family have a conserved core region, as noted above. Accordingly, for example, amino acid residues 96 to 101 (YLAMNK) and 126 to 131 (YNTYAS) of FGF-3 are identical to those of the corresponding regions of FGF-7: see also Moore et al. and Masón et al., previously. Accordingly, the degenerate oligonucleotide primers representing all possible codons corresponding to the consensus sequences of FGF-3 and FGF-7 were designed to isolate, by the polymerase chain reaction (PCR), the cDNA fragments which encode the novel elements of the FGF family. A cDNA fragment encoding a novel element of the FGF family, FGF-10, was previously isolated from the embryos of the rat by PCR using these same primers: see Yamasaki et al., Supra. The amino acid sequence of FGF-10 is more highly homologous (60%) with respect to the amino acid sequences of FGF-3 and FGF-7. FGFs are abundantly present in the brain as well as in embryos; see Baird et al. and Burgess et al., previously. Accordingly, an attempt has been made to isolate the cDNA fragments encoding the novel elements of the FGF family of the rat brain by PCR using the consensus primers described above. The cDNA synthesized from the poly (A) + RNA of the rat brain was amplified by PCR using the primers. DNA of the expected size (approximately 110 base pairs), which was a major amplified product, was cloned. Fifty-five clones were isolated and their nucleotide sequences were determined. Forty-seven clones were found to be cDNA clones related to FGF. Among them, thirty-three clones had sequences identical to that of the rat FGF-10 cDNA and nine clones had sequences identical to those of the FGF-7 cDNA. Four clones had a highly homologous sequence with that of the mouse FGF-3 cDNA, indicating that they were cDNAs of rat FGF-3. Only one clone had a sequence similar but not identical to that of the cDNAs encoding the known elements of the FGF family, suggesting that this cDNA encoded a novel element of the FGF family. The expression of the mRNA encoding this FGF found in the adult rat tissue was preliminarily examined by PCR with the mRNA-specific primers. The data indicated that the mRNA was expressed in the heart much more abundantly than in the brain. The cDNA covering the entire coding region of the FGF was isolated from the heart by Rapid Amplification of the cDNA End Method (RACE); see Frohman et al., cited above.

VIII. Structure of Rat FGF-16 The nucleotide sequence of the cDNA coding region allowed elucidation of the entire 207 amino acid long sequence of a rat FGF polypeptide (SEQ ID NO: 2), which contained a conserved amino acid core of approximately one hundred twenty amino acids (ie, amino acids 58-149 and 161-189). The full length sequence for this FGF of the rat is described in Figures 1A and IB. The cDNA fragment was originally amplified by PCR based on the homology with the primers for the consensus amino acid sequences of FGF-3 and FGF-7. Although a consensus sequence, YNTYAS, was found in amino acids 144 to 149 of the polypeptide, another consensus sequence, YLAMNK, was not found. However, a sequence similar to the final consensus sequence was found at amino acids 114 to 119: YLGMNE (see Figures 1A-1B). This result indicates that the cDNA fragment was amplified from the brain cDNA by PCR with a misaligned or mismatched primer. Two cysteine residues that are well conserved in other elements' of the FGF family are also conserved in this rat FGF polypeptide (specifically, amino acids 67 and 133). Because this polypeptide is the sixteenth document element of the FGF family, it is now tentatively designated here as "FGF-16". The amino acid sequence of FGF-16 was found to be more highly homologous (73%) than that of FGF-9 (see Figure 2). Analysis of hydropathy plots confirmed the low hydrophobicity of the amino-terminal region of FGF-16, similar to FGF-9, indicating that FGF-16 does not have a typical signal sequence; see Hopp and Woods, Proceedings of the National Academy of Sciences USA, volume 78, pages 3824-3828 (1991).

IX. Expression of Rat FGF-16 cDNA in Sf9 Cells FGF-3, FGF-4, FGF-5, FGF-6, FGF-7 and FGF-8, with their typical signal sequences at their amino termini, are known to be efficiently secreted from the cells; see Tanaka et al., Dickson et al., Yoshida et al., Goldfarb et al. and Coulier et al., previously. In contrast, FGF-1, FGF-2, FGF-9 and FHF-1 to FHF-4 do not have typical signal sequences at their amino terminals; see Baird et al., Burgess et al., Miyamoto et al. and Smallwood et al., supra. FGF-1, FGF-12 and FHF-1 to FHF-4 are not secreted. In contrast, however, FGF-9 is efficiently secreted despite the absence of a typical signal sequence. To examine whether FGF-16 is secreted, Sf9 cells were infected with the recombinant baculovirus containing the rat FGF-16 cDNA with the 3 'terminal extension encoding the E and 6XHis tags. To detect the recombinant FGF-16 with an extension of carboxy-terminal 25 amino acids of the labels, both the culture supernatant and the cell lysate were examined by Western blot analysis using anti-E-tag antibodies. A major band of approximately 26 kilodaltons was detected especially in the culture supernatant (see Figure 3). The observed molecular mass of the main band was consistent with the calculated molecular mass of the recombinant FGF-16 (ie, 26,462 daltons). The 26 kilodalton protein was purified by affinity chromatography using a chelating column, and then subjected to an amino acid sequence determination using a commercial protein sequencer. The amino-terminal sequence could not be determined, however, which indicates that the amino-terminal amino acid is probably blocked.

X. Expression of Rat FGF-16 mRNA in the Tissues of the Adult Rat The expression of FGF-16 mRNA in the tissues of the adult rat was investigated as follows: The RNA of the brain, heart, lung, liver, kidney, brown adipose tissue and white adipose tissue was examined by Northern blot analysis using a 32P labeled FGF-16 cDNA probe. The integrity of the RNA was confirmed by electrophoresis on a denaturing agarose gel containing formaldehyde. The labeled probe hybridized strongly to a 1.8 kilobase mRNA in the heart (see Figure 4). The labeled mRNA was also detected moderately in brown adipose tissue. However, the mRNA was not detected in the brain, lungs, liver, kidney and white adipose tissue. The expression of FGF-16 mRNA in other tissues was also examined, including the small intestine, muscles, thymus, stomach, pancreas, spleen and testes by PCR with specific primers for the FGF-16 mRNA. the rat. The mRNA of FGF-16 was detected in these tissues at much lower levels than in the heart. Accordingly, the FGF-16 mRNA is predominantly expressed in the heart and brown adipose tissue.

XI. Expression of Rat FGF-16 mRNA in the Rat Embryo To examine the expression of mRNA in FGF-16 in embryos (E19), sagittal sections of embryos were analyzed by in situ hybridization with a sense or antisense FGF-16 cRNA probe tagged with 36S, followed by frameradioradiography . With the antisense probe, discrete labeling was also observed in the brown adipose tissue and the heart of the embryo (Figure 5B). However, no labeling was observed with the sense probe as a control (Figure 5C). These results indicate that the FGF-16 mRNA in the embryo is also expressed predominantly in the heart and brown adipose tissue. FGF-1 and FGF-2 are widely expressed in adult tissues; see Baird et al. and Burgess et al, previously. In contrast, many other FGFs and FHFs are expressed tightly in adult tissues; see the references cited above. Although the amino acid sequence of FGF-16 is highly homologous with respect to that of FGF-9, the expression profile of FGF-16 is very different from that of FGF-9, as well as other elements of the FGF family. Therefore, FGF-16 seems to be a novel FGF which has a unique physiological role.

XII. Cloning of Human FGF-16 Using the information resulting from the cloning and sequencing of the gene for rat FGF-16, the gene for human FGF-16 was cloned and expressed as follows. A partial cDNA sequence from the rat FGF-16 (SEQ ID NO: 7), which starts at nucleotide 92 and ends at nucleotide 637 of the nucleic acid sequence shown in Figure 1, was used for the design of PCR primers for the amplification of human FGF-16 DNA. Some of the primer sequences were partially degenerate to increase the probability of the first annealing despite the codon differences which might exist between the human and rat FGF-16 DNA sequences. The PCR products were cloned into either the PCRII vector or the PCR2.1 vector (Invitrogen, Carlsbad, California) before sequencing. PCR amplification of the genomic DNA of the rat verified that the position of the last intron in the coding region of FGF-16 corresponds to the position of the intron in the other elements of the FGF family. Human genomic DNA prepared from HeLa cells was amplified with combinations of the FGF-16 primers from the rat and the products were analyzed by polyacrylamide gel electrophoresis (PAGE). The products of the PCR reactions whose size was unpredictable because the fragment that was amplified expanded one or more introns was analyzed by PCR packaged with the pairs of the primers that are expected to rest within the single exons. One such clustered or clustered PCR, primed with a pair of oligonucleotides, specifically, 5 '-CGG GAA CAG TTT GAA GAA AAC TGG TA-3' (SEQ ID NO: 8), corresponding to nucleotides 422-447 in the nucleic acid sequence in Figure 1, and 5 '-GAA AGT GNG TGA AYT TCT GRT G-3' (SEQ ID NO: 9), where "N" designates inosine, and which is complementary to nucleotides 554- 575 of the nucleic acid sequence of Figures 1A-1B, produced a PCR product of the expected size (0.15 kilobases), based on the FGF-16 sequence of the full-length rat. In the nucleic acid sequence of SEQ ID NO: 9, "Y" represents a mixture of C and T and "R" represents a mixture of A and G, according to the convention of the IUB (International Union of Biochemistry) . Both the packaged or packaged PCR product and the origin, the PCR product extending over the intron [1.7 kilobases, primed with 5 '-CCG CAC GGG CTT CCA CCT TGA-3' (SEQ ID NO: 10), the which is homologous to nucleotides 217-237 of the nucleic acid sequence of the rat of Figures 1A-1B, and 5 '-GAA AGT GIG TGA AYT TCT GRT G-3'] were cloned and sequenced. The sequence of the predicted peptide to be encoded by the exon sequences of this human FGF-16 genomic DNA fragment was highly homologous with rat FGF-16. The first cDNA in the strand was synthesized from the polyA + RNA of the human heart using a random primer adapter, 5 '-GGC CGG ATA GGC CTC ACN NNN NNT-3' (SEQ ID NO: 11), with "N" representing a random mixture of bases A, C, G and T. PCR (thirty-five cycles) 'was carried out with the 5' -CGC GGC TCG CCC ACÁ GAC TTC-3 '(SEQ ID NO: 12) , corresponding to nucleotides 155-175 of the nucleic acid sequence of the rat of Figure 1, and a PCR primer of human FGF-16 of the unique sequence, 5 '-CTG TCT CTC TGA GTC CGA ATG TT- 3 '(SEQ ID NO: 13), complementary to nucleotides 480-502 of the nucleic acid sequence of Figures 6A-6B, was designed based on the sequence of the human genomic fragment. The product band of approximately 340 base pairs in size was purified by PAGE and cloned into the pCRII vector (Invitrogen) for DNA sequencing. The human FGF-16 oligonucleotides were designed based on these sequences and used in 3 '-RACE and 5'-RACE of the human fetal brain cDNA and the human heart cDNA to extend the cDNA sequence in both directions to get the complete coding sequence. The 3 '-RACE procedure was carried out as follows. The cDNA of the first strand was prepared from the polyA + RNA of the human heart (Clontech Laboratories, Inc., Palo Alto, California) by standard methods using the oligo-DT adapter-primer, 5'-TTCGGCCGGATAGGCCTTTTTTTTTTTTTT-3 '(SEC ID NO: 14), and Superscript reverse transcriptase (GIBCO-BRL). The cDNA of the first strand was used as a 'model or template in the 3' PCR-RACE (thirty cycles, annealing temperature 55 ° C) primed with 100 nM each of 5'-TTCGGCCGGATAGGCCTTTTTTTTTTTTTT-3 'and 5' -CGG GAA CAG TTT GAA GAA AAC TGG TA-3 '. To partially suppress the priming by the oligo-dT primer adapter without suppressing the priming by the FGF-16 primer specific for the gene, a non-primer homolog, especially the 5'-TTCGGCCGGATAGGCCTTTTTTTTTTTTTTP-3 ', where p represents a 3'-phosphate group was added to a final concentration of 200 nM to this and the 3'-RACE PCRs as a competitive annealing and priming inhibitor by 5'-TTCGGCCGGATAGGCCTTTTTTTTTTTTTT-3 '. The additional amplification and enrichment of the FGF-16 DNA was carried out by diluting 0.4 μl of the product of the first PCR RACE in 40 μl of the fresh PCR mixture containing the same primers, and then the PCR was continued for another eighteen cycles. An aliquot of 0.4 μl of the product of this PCR was used as a model or template in a 3 'CRP packaged (twenty five cycles), priming with 5' -TTCGGCCGGATAGGCCTTTTTTTTTTTTTT-3 ', and a new primer of human FGF-16, especially the 5 '-GTA CA CAC CTA TGC CTC AAC CT-3' (SEQ ID NO: 15), which corresponds to nucleotides 451-473 of the human nucleic acid sequence of Figures 6A-6B, located downstream of 5 '-CGG GAA CAG TTT GAA GAA AAC TGG TA-3'. The main band of the packaged or embedded PCR product was cloned and sequenced. The sequence of this DNA fragment included the carboxy-terminal coding region and at least part of the 3'-UTR of the apparent human homologue of rat FGF-16. Three successive cycles of DNA sequence determination and 5'-RACE were required to obtain the cDNA sequence encoding the amino-terminal portion of human FGF-16. 5 '-RACE was performed on the cDNA of the first strand without glue using a new set of semi-random primer adapters. Six partially random primers, each capable of priming with a large number of different cDNAs, but only in a small fraction of the sites within any cDNA such as the FGF-16 cDNA, were used as the upstream (5 ') primers in combination with the downstream (3') primers specific for the FGF-16 gene. Each partially randomized primer consisted of a unique adapter sequence of eighteen nucleotides, 5'-GCAGTCGCTCCTTCCGTG-3 '(SEQ ID NO: 16), followed by the nine nucleotide random sequence, NNNNNNNNN (SEQ ID NO: 17), followed by a unique sequence of 4 or 56 nucleotides such as 5'-CACA-3 '. First, the cDNA of the human heart was used as the template or model in the thirty cycles of the PCR with the mixture of five primers of the partially randomized sequence, especially 5'-GCAGTCGCTCCTTCCGTGNNNNNNNNNX-3 '(SEQ ID NO: 18), where "N" designates random base (A, C, T or G) and "X" = AATG, TCTC, TTGG, CACA or AACC, and a primer of FGF-16, 5 '-CTC CTC GCT CAT TCA TTC CTA-3 '(SEQ ID NO: 19), which was complementary to nucleotides 366-386 of the human nucleic acid sequence of Figure 6. The first cycle of PCR was carried out differently from the cycles remaining, to allow a low severity for the annealing of the random primers. In particular, the specific primer of FGF-16 was not added until the second cycle, and the annealing step in the first cycle was carried out at 25 ° C in the presence of both Taq and Klenow DNA polymerases, followed by slow heating (ten minutes) at the Taq elongation temperature of 72 ° C. When the temperature was increased to 94 ° C during the start of the second PCR cycle, the FGF-16 primer was added and the PCR was continued. An aliquot of the product of this PCR was then used as the model or template in a PCR (twenty cycles) with the adapter primer, 5 '-GCAGTCGCTCCTTCCGTG-3', and 5'-AGT CCA CTC CCC GGA TGC TGA T-3 '(SEQ ID NO: 20), the latter being complementary to nucleotides 332-353 of the human nucleic acid sequence of Figure 6. The most prominent bands in the product of this PCR were cloned and sequenced, the sequence of a clone revealing the sequence of the initiation of human FGF-16 at nucleotide 149. The sequences of several clones contained an apparent intron and a 3 'splice site preceding nucleotide 298. The sequence of a clone revealed that the semi-random primer which ends in the sequence of four bases CACA is annealed inside the intron. To eliminate this unwanted priming event, this primer was omitted from the semi-random primer mix in the subsequent round of 5'-RACE. For this 5'-RACE, a mixture of the four remaining semi-random primers was incubated with the cDNA of the first strand of the human heart in a standard PCR mixture with the Taq polymerase and the nucleoside triphosphates, but without a primer. FGF-16 downstream, for periods of 10-15 minutes each at 25 ° C, 37 ° C, and 50 ° C, then one minute at 72 ° C for elongation of the strand, before adding the FGF primer -16, 5 '-AGT CCA CCC GGA TGC TGA T-3', and the adapter primer, 5'-GCAGTCGCTCCTTCCGTG-3 ', and proceeding with the PCR for thirty cycles. An aliquot of the product was further amplified in a packaging PCR (eighteen cycles) with the primers 5 '-GCAGGTCGCTCCTTCCGTG-3' and 5'-CTC CAG GAT TCC GAA GCG GCT GTG GTC GTG-3 '(SEQ ID NO: 21) , the latter being complementary to nucleotides 278-307 of the human nucleic acid sequence of Figure 6, followed by a 10: 1 dilution in an identical PCR mixture lacking the competitor and another ten PCR cycles. Although the resulting products appeared as a spot on the electrophoresis of the gel, they were cloned into a pCRII vector and the clones were obtained which contained the sequence of FGF-16 starting at nucleotide 19. A PCR technique similar to the 5'-RACE methods described above was used to amplify the genomic DNA sequences including the amino-terminal coding sequence of human FGF-16. A semi-random adapter-adapter, the 5'-NNT ANN ACN CCA CNC AAN NNN NAT G-3 '(SEQ ID NO: 22), with "N" at positions 1, 2, 5, 6, 9, 14 and 18 which designate inosine, and "N" at positions 19, 20, 21, and 22, which designate the bases selected at random from among A, C, T, and G, at a concentration of 2 μM, was used in a DNA synthesis reaction catalyzed by the Klenow polymerase at 25 ° C with 100 ng denatured human genomic DNA with heating as the model or template. An aliquot of 4 μi of the product was used as a template or model in a PCR (thirty cycles; 40 μl) barley with the 5 'adapter primer -GGT AGG ACG CCA CGC AAG-3' (SEQ ID NO: 23) and the 3 'primer of FGF-16, 5'-TCC GAA GCG GCT GTG GTC GTG-3' (SEQ ID NO: 24), the latter representing nucleotides 278-298 of the nucleic acid sequence of Figures 1A-1B, in the presence of an equimolar amount of the competitive oligonucleotide 5 '-GGT AGG ACG CCA CGC AAGp- 3' . The product of this PCR was further amplified in a packed PCR (twenty cycles) with the primers 5 '-GGT AGG ACG CCA CGC AAG-3' and 5 '-AAG ATC TCC AGG TGG AAG CCG-3', the latter being complementary with nucleotides 229-249 of the human nucleic acid sequence of Figures 6A-6B, again in the presence of an equimolar amount of the competitor, the 5'-GGT AGG ACG CCA CGC AAGp-3 '. The products were cloned in the PCR2.1 vector (Invitrogen). Colonies were selected to verify the presence of FGF-16 sequences by PCR with the 5 '-GGA TCT ACÁ CGG CTT CTC CTC GTC T-3' (SEQ ID NO: 25), which represents nucleotides 61-85 of the nucleic acid sequence of Figures 6A-6B, and 5'-CTG GGG AGT CAG CTA AGG GCA-3 '(SEQ ID NO: 26), which represent nucleotides 96-116 of the nucleic acid sequence of the Figures 1A-1B, and by colonies' hybridizations with the oligonucleotide labeled with 32P 5 '-GGA TCT ACÁ CGG CTT CTC CTC GTC T-3'. The sequences of these clones contained the sequence of the amino terminus of human FGF-16, completing the sequence of the region encoding human FGF-16 (Figures 6A-6B, SEQ ID NO: 5).

XIII. Recombinant Expression of Rat FGF-16 To provide sufficient amounts of material for biological characterization, the rat FGF-16 polypeptides were recombinantly expressed in the bacterial cells as follows: A) Construction of Expression Vectors. Vectors for recombinant expression in bacterial cells were constructed in the following manner. 1) Preparation of pAMG21rFGF-16. The full-length DNA of the rat FGF-16 described above (Figures 1A-1B, SEQ ID NO: 1) was cloned into the plasmid vector pGEM-T (Promega, Madison, Wisconsin). Using standard molecular biology techniques and protocols suggested by the manufacturer, the rat FGF-16 insert (rFGF-16) was cloned into the plasmid vector pAMG21 (American Type Culture Collection, Rockville, Maryland, Access No. 98113 ). The oligonucleotide amplifiers for PCR were designated to be partially homologous with the sequence of rFGF-16 at the 5 'and 3' ends of the rFGF-16 insert in pGEM-T rFGF-16 and to contain the sites of recognition of the restriction endonuclease Ndel and Kpnl, respectively. PCR was performed using the following primers: AAA CAA CAT ATG GCT GAA GTT GGT GGT GTC TTT GCC TCC TTG GA (SEQ ID NO: 27) and AAA CAG GGT ACC TTT ACC TAT AGC GGA AGA GGT (SEQ ID NO: 28) (with the regions homologous to rFGF-16 underlined) and pGEM-T rFGF-16 (ampliTaq DNA Polymerase, Perkin-Elmer, Foster City, California) as the template or model. This PCR product was purified (set or QIAquick® PCR purification set, QIAGEN, Santa Clarita, California) and then digested with the restriction endonucleases Ndel and Kpnl (Boehringer Mannheim, Indianapolis, Indiana). The resultant 631 base pair DNA fragment was purified by extraction with an agarose gel (QIAquick® Gel Extraction Kit or set, QIAGEN) and ligated with a similarly purified 6,067 kilobase pAMG21 vector fragment (ATCC # 98113) . Transformation of the appropriate E. coli host strain (GM120, ATCC # 55764) with this binding reaction (Gene Pulser®, BioRad, Richmond, California) was plated on Luria agar plates with 40 μg / ml of kanamycin to select recombinant bacteria. No colony was observed. Selection in the culture of the liquid with kanamycin and subsequent plating on the Luria agar plates with 40 μg / ml kanamycin failed to recover any viable colonies. However, PCR of the binding reaction using a primer at the 3 'end of the rFGF-16 insert and a 5' primer of the vector to the insert of rFGF-16, CGT ACA GGT TTA CGC AAG AAA ATG G (SEQ. NO: 29), revealed that the correct construction of the plasmid was present in the junction. This PCR product was purified (set or QIAquick® PCR purification set) and then cut with the restriction endonuclease Xbal and Kpnl (Boehringer Mannheim). The DNA fragment of 667 base pairs resulting was purified by extraction with an agarose gel (set or QIAquick® Gel extraction set) and ligated or ligated with the 631 kilobase Kpnl-Pstl DNA fragment of similarly purified pAMG21. Transformation of the appropriate E. coli host strain (GM120; ATCC # 55764) with this binding or ligation (Gene Pulser® BioRad) produced the kanamycin-resistant colonies. The plasmid DNA was purified from one of these colonies (Game or Set of QIAGEN® Plasmids, QIAGEN) and the DNA sequence was confirmed.

) Preparation of pAMG21_EN34rFGF-16. Using standard molecular biology techniques and protocols suggested by the manufacturer, pAMG21_EN34rFGF-16 was cloned by ligation of the oligonucleotide linkers of Ndel-Pstl TAT GAA CGA GCG CCT GGG CCA GAT CGA GGG GAA GCT GCA (SEQ ID NO. : 30) and GCT TCC CCT CGA TCT GGC CCA GGC GCT CGT TCA (SEQ ID NO: 31) with a fragment of pAMG21_EN34rFGF-16, above, purified by extraction with an agarose gel (set or extraction set of QIAquick® Gel , QIAGEN). The linkers were treated with kinase and annealed (Polynucleotide kinase, Boehringer Mannheim) prior to binding or ligation (T4 DNA ligase, Boehringer Mannheim). The pAMG21 vector plasmid (ATCC # 98113) contains a kanamycin resistance gene, consequently the transformation of the appropriate E. coli host strain (GM120; ATCC # 55764) with this binding or binding reaction (Gene Pulser ®, BioRad) produced the kanamycin-resistant colonies on Luria agar plates using 40 μg / ml kanamycin in the growth medium. Plasmid DNA was purified from one of these colonies (set or set of QIAGEN® Plasmids, QIAGEN) and the DNA sequence was confirmed.

B) Expression of the FGF-16 of the Full Length Rat in E. Coli.

FGF-16 from the full-length rat (Figures 1A-1B, SEQ ID NO: 1) was expressed in E. coli using pAMG21rFGF-16. The cells were grown in a ten-liter fermentor at pH 7 and a temperature of 30 ° C. Dissolved oxygen levels were maintained greater than or equal to fifty percent. When the fermentation medium reached an optical density of 10, the cells were induced using ten milliliters of the storage solution composed of 500 nanograms per milliliter (ng / ml) of N- (beta-ketocapropyl) -dl-homoserine lactone (Sigma Chemical Company, St. Louis, Missouri). After an induction period of twelve hours, the fermentation medium was cooled and the cells were mechanically used in water and centrifuged at 10,000 revolutions per minute (rpm) for two hours. The supernatant was subjected to ion exchange column chromatography of SP-Sepharose in 50 mM Tris HCL, pH 7.5. The bound proteins were eluted with a linear NaCl gradient. Fractions that elute about 0.3-0.6 M NaCl contained many bands, which vary in molecular weight between 15,000 and 28,000. The analysis of the sequence of these protein bands after electrospinning showed both N-terminally and intact truncated forms. One of the bands had an N-terminal sequence of NRE (ie, asparagine-arginine-glutamic acid) and appeared to be smaller in size before suffering extensive proteolytic digestions. In addition to the FGF-16 of the full length rat (SEQ ID NO: 1), the resulting cell paste included a truncation product in which segmentation between leucine 34 and asparagine 35 ("des-N-34") has occurred. ") (SEQ ID NO: 32), as well as a truncation product in which segmentation between alanine 9 and serine 10 (" des-N-9") has occurred (SEQ ID NO: 33). FGF-16 has a sequence homology with FGF-9, with seventy-three percent of the amino acid residues that are identical between full-length human FGF-9 and FGF-16 polypeptides. Both of these FGFs lack signal sequence, as is the case for the acid FGF and the basic FGF. However, human FGF-9 has been observed to be efficiently secreted in the conditioned medium of the COS cells transfected with the hFGF-9 cDNA. The analysis of the sequence of the purified samples showed an intact N-term and a truncation between leucine 33 and serine 34. This des-N-33 form of FGF-9 has been found to be as active as the form of full-length FGF-9 in a preliminary in vitro bioassay. When FGF-9 and FGF-16 are aligned based on sequence identity, the N-terminal cleavage sites observed for FGF-19 and FGF-16 are located close at the same position. Accordingly, it was decided to evaluate the des-N-34 form of the rat FGF-16, rather than the full length, for biological activity.

C. Expression of the FGF-16 of the Rat of Des-N-34 in E. coli. E. coli cells transformed with pAMG21ΔN34rFGF-16, which contains the cDNA encoding the des-N-34 form of rat FGF-16, were grown under the conditions mentioned above and used mechanically in 1M ammonium sulphate, 50 mM Tris HCl, pH 7.5 at 100 g / 1. The used suspension is centrifuged at 4_C and 10,000 rpm for two hours. The supernatant is batch-bound to the phenyl-Sepharose in 1 M ammonium sulfate, 50 mM Tris HCl, pH 7.5. The resin is extensively washed with the same buffer in a glass filter and transferred to a column. The bound proteins were further washed with the same buffer in the column and then eluted with a gradient of linear descending ammonium sulfate from 1 to 0 M. The fractions were analyzed by SDS-PAGE, and those fractions containing FGF-16 of the des-N-34 rat were grouped. The resulting set was mixed with 3.5 volumes of cold water and batch-bound to SP-Sepharose equilibrated in 50 mM Tris HCl, pH 7.0. The SP-Sepharose was packed in a column and the bound proteins were eluted with a linear NaCl gradient from 0 to 1 M. Based on the SDS-PAGE of the eluted material, the fractions containing the des-N-34 form of FGF-16 from the rat were pooled, then dialyzed against 1 M ammonium sulfate, 50 mM Tris HCl, pH 7.0. The dialyzed material was loaded onto the phenyl-Sepharose equilibrated in 1 M ammonium sulfate, 50 mM Tris HCl, pH 7.0. After extensive washing with the same buffer, the bound proteins were eluted with a gradient of linear ammonium sulfate from 1 to 0 M. The fractions containing the FGF-16 of the de-N-34 rat in amounts greater than Ninety percent were pooled and dialysed against the buffer solution of PBS as a final product. The inclusion of 0.1-10 mM EDTA during the purification increased the recovery of the final product. This procedure, including the use of 0.1 mM EDTA, also led to the efficient purification of the full-length form of rat FGF-16 when applied to lysates of E. coli cells that have been transformed with the Full-length cDNA for the rat FGF-16.

XIV. In vivo Biological Test of the FGF-16 of the Rat Derived from the E. Coli The in vivo biological effects of the des-N-34 form of FGF-16 from the recombinant rat derived from E. coli were evaluated in the normal mice as follows.

A. In vivo Administration to Mice. Five female BDF1 mice were administered with FGF-16 from the recombinant des-N-34 rat (SEQ ID NO: 32) by intraperitoneal injection (IP) at a dose of 5 mg / kg / day for seven days. Separately, a group of five female BDF1 mice received a control buffer under the same conditions. All mice were injected with 50 mg / kg bromodeoxyuridine (BrdU) (Aldrich Chemical Company, Milwaukee, Wisconsin) one hour prior to collection, radiographed, and then sacrificed. Body weights and selected organs were measured, blood was drawn for hematology and serum chemistry analysis, and organs were collected for histological analysis and BrdU labeling. Blood samples were analyzed to check the clinical chemistry on a Hitachi 717 System (Boehringer Mannheim) or the whole blood count on a Technicon H1E Analyzer (Miles Technicon Instrument Corp., Tarrytown, New York). Immunohistochemical staining of BrdU was done on 4 millimeter thick paraffin-soaked sections using an automatic TechMate Immunotherapist (BioTek Solutions, Santa Barbara, California). The sections were first digested with 0.1% protease (Sigma Chemical, St. Louis, Missouri), followed by 2N HCl. Brd was detected with a rat monoclonal antibody (MAb) for BrdU (Accurate Chemical, Westbury, New York) followed by a biotinylated anti-mouse / anti-rabbit secondary cocktail (BioTek) and a tertiary ABC bound to a phosphatase alkaline (BioTek). The dyeing reaction was visualized with the Red chromogen from BioTek. The hepatocytes labeled with BrdU were quantified by a protected pathologist for the treatment groups by the count of the hepatocytes labeled with BrdU in ten random high power fields (HPF-40X objective) by section of the liver and determination of the average number of the positive hepatocytes of BrdU by HPF.

B. Total Results of the Pathology. The livers and spleens of the mice injected with the FGF-16 of the des-N-34 rat were significantly larger than those of the mice injected with the control of the buffer. These results are summarized in Table 1, below.

C. Results of Clinical Pathology. Mice injected with FGF-16 from the de-N-34 rat had significant increases in serum triglycerides, lactate dehydrogenase (LDH) and total proteins, and a significant decrease in serum alkaline phosphatase. These results are also summarized in Table 1.

D. Results of Histopathology. The sections stained with hexatoxylin and eosin (H &E) and BrdU from the liver, spleen, lungs, brain, heart, kidney, adrenal organs, stomach, small intestine, pancreas, caecum, the colon, mesenteric lymph node, skin, mammary gland, trachea, esophagus, thyroid, parathyroid, salivary glands, urinary bladder, ovaries, bones and bone marrow were examined from the five mice injected with 34 FGF-16 and five mice injected with the control of the buffer solution. The only significant histological discovery was in increment 'significant in the 1 BrdU positive hepatocytes by the high power microscopic field in the mice treated with (des-34) FGF-16 compared with the mice injected with the control buffer (See Table 1 and Figure 7).

TABLE 1 WEIGHTS OF SELECTED ORGANS, CHEMICALS OF SERUM AND LABELING WITH HEPATOCELLULAR BRDU IN THE MICE TREATED WITH FGF-16 E. Conclusions. Mice injected with the de-N-34 form of rat FGF-16 exhibited an increase in hepatocellular BrdU labeling and a moderate but significant increase in liver weight, serum triglycerides, serum LDH. and total serum proteins, along with a decrease in serum alkaline phosphatase. Accordingly, FGF-16 induces hepatocellular proliferation and increased hepatic production of triglycerides and serum proteins, such as albumin. These effects in vivo are similar to, but of a magnitude slightly less than, the hepatic effects induced by FGF-7 (KGF); see Housley et al., Journal of Clinical Investigation, Volume 94, pages 1764-1777 (1994). The present findings indicate the potential effectiveness of FGF-16 in applications in which an increase in hepatocellular stimulation, proliferation and / or differentiation is required. Such applications include increasing the function of the liver to treat or prevent liver cirrhosis, fulminant liver failure, damage caused by acute viral hepatitis and / or toxic attacks to the liver. Particular methods of therapy for this purpose may include transfection of the cells of endogenous hepatocytes into the host organism or subject being treated with a vector comprising regulatory elements, such as those which have been described, operatively linked to a DNA molecule encoding FGF-16 or an analog thereof to effect in situ expression. The invention described above is now defined in the appended claims.

LIST OF THE SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Amgen Inc. (ii) TITLE OF THE INVENTION: A FACTOR OF GROWTH OF FIBROBLASTS (iii) NUMBER OF SEQUENCES: 33 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Amgen Inc. (B) STREET: 1840 DeHavilland Drive (C) CITY: Thousand Oaks (D) STATE: California (E) COUNTRY: USA (F) POSTAL AREA: 91320-1789 (v) READABLE FOR THE COMPUTER: (A) TYPE OF MEDIUM: Flexible magnetic disk (B) COMPUTER: compatible with IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0 , Version # 1.30 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (viii) INFORMATION OF THE MANDATE HOLDER: (A) NAME: Mazza, Richard J. (B) REGISTRATION NUMBER: 27,657 (C) REGISTRATION NUMBER / REFERENCE: A-469 (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: 805.447.4112 (B) TELEFAX: 805.447.1090 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 621 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: ATGGCGGAGG TCGGGGGCGT CTTTCCCTCC TTGGACTGGG ACCTGCAAGG CTTCTCCTCC 60 TCTCTGGGGA ACGTGCCCTT AGCTGACTCC CCGGGTTTCC TGAACGAGCG CCTGGGCCAG 120 ATCGAGGGGA AGCTGCAGCG CGGCTCGCCC ACAC? CTTCG CCCACCTGAA GGGGATCCTG 180 CGGCGCCGCC AGCTCTACTG CCGCACGGGC TTCCACCTTG AAATCTTCCC CAATGGCACG 2 0 GTÜCATGGCA CCCGCCACGA CCACAGCCGC TTCGGAATTC TGGAATTTAT CAGCTTGGCT 300 GTGGGGCTGA TCAGCATCCG GGG GTAGAC TCTGGCCTAT ACCTAGGA? T GAATGAGCGA 360 GGAGACCTGT TTGGATCG? A GAA? CTCACA CGAGAATGTG TTTTCCGGGA ACACTTTGAA ~ 420 GAAA? CTGGT ACAACACCTA TGCATCCACC TTGTACAAAC ACTCGGACTC GGAGAGACAG 48C T? 1 ATGTGG CCCTGAATAA AGACGGCTCA CCCCGGGAGG GATACAGGAC TAAACGACAC 540 CAG ?? ATT A CTCACTTTT? ACCCAGGCCA GTAGATCCTT C7? AGTTGCC CTCCATGTCC 600 AGAGACCTCT TCCGCTATAG G 621 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 207 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Mßc Wing Glu Val Gly Gly Val Phe Wing Sor Leu Asp Trp Asp Leu Gln 1 5 10 15 Gly Phß Ser Ser Leu Gly Asn Val Pro Leu Wing Asp Ser Pro Gly 20 25 30 Phe Leu Asn Glu Arg Leu Gly Gln He Glu Gly Ly_ Leu Gln Aro Gly 3S 40 45 Ser Pro Thr Asp Phe Wing His Leu Lys Gly He Leu Arg Arg? Rg Gln 50 55 60 Leu Tyr Cys Arg Thr Gly Phe His Leu Glu He Phe Pro Asn Gly Thr 65 70 75 80 Val His Gly Thr Arg His Asp His Ser Arg Phe Gly He Leu Glu Phe 85 90 95 He Ser Leu Ala Val Gly Leu He Ser He? Rg Gly Val Asp Ser Gly 100 105 110 Leu Tyr Leu Gly Met Asn Glu Arg Gly Glu Leu Phe Gly Ser Lys Lys 115 120 125 Leu Thr Arg Gl? Cys Val Phe Arg Glu Gln Phe Glu Glu? Sn Trp Tyr 130 13S 140 Asn Thr Tyr Ala Ser Thr Leu Tyr Lys His Ser Asp Ser Gl? Arg Gln 1.5 150 155 160 Tyr Tyr V l Wing Leu Asn Lys Asp Gly Ser Pro Arg Glu Gly Tyr Arg 165 170 175 Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro? Rg Pro Val Asp 180 185 190 Pro Ser Lys Leu Pro Ser Het Ser Arg Asp Leu Phe Arg Tyr Arg 195 200 205 INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 208 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: UNIQUE (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRITION: SEQ ID NO: 3: Mee Wing Pro Leu Gly Glu Val Gly Ser Tyr Phe Gly Val Gln Asp Wing 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu 20 25 30 Leu Ser? S His Leu Gly Gln Ser Glu Ala Gly Gly Leu Pro Arg Gly 35 40 45 Pro Wing Val Thr? Sp Leu Asp His Leu Lys Gly He Leu Arg Arg Arg 50 55 60 Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Glu He Phe Pro Asn Gly 65 70 75 80 Thr He Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly He Leu Glu 85 90 95. Phe He Ser He Wing Val Gly Leu Val Ser He Arg Gly Val Asp Ser 100 105 110 Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115 120 125 Lye Leu Thr Gln Glu Cya Val Phe Arg Glu Gln Phe Glu Glu Asn Trp 130 135 140 Tyr? Sn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp He Leu Ser Gln Ser 195 200 205 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 621 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 ATGGCAÜ? GG TGGGGGGCGT CTTCGCCTCC TTGGACTGGG ATCTACACGG CTTCTCCTCG 60 TCTCTGGGGA? CGTGCCCTT AGCTGACTCC CCACGTTTCC TGAACGAGCG CCTCGGCCAA 120 ? TCGACGGGA AGCTGCAGCG TGGCTCACCC ACAGACTTCG CCC? CCTGAA GGGGATCCTG 180 CGGCGCCGCC AGCTCTACTG CCGCACCGCC TTCCACCTGG AGATCTTCCC CAACGGCACG 240 GTGCACGGGA CCCGCCACG? CCAC? GCCGC TTCGGAATCC TGGAGTTTAT C? GCCTGGCf 300 GTGGGGCTGA TCAGC? TCCG GGGAGTGG? C TCTGGCCTGT ACCTAGGAAT GAATGAGCC? 360 GGAGA? CTCT ATGGGTCGAA GAAACTCACA CGTGA? TGTG TTTTCCGGGA ACAGTTTGAA 420 GA ?? CTGGT? CAACACCTA TGCCTCAACC TTGT? CAAAC ATTCGGACTC AGAGAGACAG 480 TATTACGTGG CCCTGAACAA AGATGGCTCA CCCCGGGAGG GATACAGGAC TAA? CGACAC 540 CAGA? ATTCA CTCACTTTTT ACCCAGGCCT GTAGATCCTT CTAAGTTGCC CTCCATGTCC 600 AGAGACCTCT TTCACTATAG G 621 '2) INFORMATION FOR SEQ ID NO: 5; (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 207 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Met Ala Glu Val Gly Gly Val Ph? Wing Ser Leu Asp Trp Asp Leu His 1 5? O? 5 Gly Phe Ser Ser Leu Gly Asn Val Pro Leu Wing Asp SQY Pro Glv 20 25 30 Phe Leu Asn Glu Arg Leu Gly Gln He Glu Gly Lys Leu G n Arg Gly 35 40 45 Ser Pro Thr Asp Phe Ala His Leu Lys Gly He Leu Arg Arg Arg Gln 50 55 60 Leu Tyr Cys Arg Thr Gly Pho His Leu Glu He Phe Pro Asn Gly Thr 65 70 75 80 Val His Gly Thr Arg His Asp His Be Arg Phe Gly He Leu Glu Phe 85 90 95 Be Ser Leu Ala Val Cly Leu Be Ser Arg Gly Val Asp Ser Gly 100 105 110 Leu Tyr Leu Gly Met Asn Glu Arg Gly Glu Leu Tyr Gly Ser Lys Lys 115 120 125 Leu Thr? Rg Glu Cys Val Ph? Arg Glu Gln Phe Glu Glu Asn Trp Tyr 130 135 140? Sn Thr Tyr Ala Ser Thr Leu Tyr Lys His Ser Asp be Glu Arg Gln 145 ISO 155 160 Tyr Tyr Val Wing Leu Asn Lys Asp Gly Ser Pro Arg Glu Gly Tyr Arg 165 170 175 Thr Lyo? Rg His Gln Lys Phe Thr HiS Phe Leu Pro Arg Pro Val? Sp 180 IBS 190 Pro Ser Lys Leu Pro Ser Met Ser Arg Asp Leu Phe Kis Tyr? Rg 195 aOO 205 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Gly Ala Pro Val Pro Tyr Pro Asp Phe Leu Glu Pro Arg 1 5 10 INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 536 base pairs (B) TYPE: nucleic acid (C) TI PO OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) SEQUENCE DESCRITION: SEQ ID NO: 7: CCCTTAGCTG ACTCCCCGGG TTTCCTGAAC GAGCGCCT G CCCAC? TCGA GGGGAAGCTG 60 CAGCGCCGCT CGCCCACAGA CTTCGCCCAC CTGAAGGGGA TCCTGCGGCG CCGCC? GCTC 120 TACTGCCGCA CGGGCTTCCA CCTTGAAATC TTCCCCAATG CCACGGTGCA TGGCACCCGC 180 C? CGACCACA GCCGCTTCGG AATTCTGGAA TTT? TCAGCT TGGCTGTGGG GCTGATCAGC 240 ATCCGGGGAG TAGACTCTGG CCTATACCT? GGAATGAATG AGCGAGC? GA GCTGTTTGGA 300 TCGAAGAAAC TCACACGAGA ATsTsTTTTC CGGGAAC? GT TTGAAGAAAA CTGGTACAAC 360 ACCTATGCAT CCACCTTGTA CAAACACTCG GACTCGGACA GACAGTATTA TGTGGCCCTG 420 AATA? AGACG GCTCACCCCG GGAGGGAT? C AGGACTAAAC CACACCAGAA ATTCACTCAC 480 TTTTTTACCC? GGCCAGTAGA TCCTTCTAAG TTCCCCTCCA TGTCC? GAGA CCTCTT 536 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: CGGGAAC? GT TTGAAGAAAA CTGGTA 26 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from nucleic acid (A) ) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 8 (D) OTHER INFORMATION: / note = "The" N "denotes Inosina" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: GA? AGTGNGT G? ACTTTCTG AGTG ^ 4 INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: CCGCACGGGC TTCCACCTTG A 21 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (ix) FEATURE: (A) NAME / KEY: misc_feature (B) LOCATION: 18..23 (D) OTHER INFORMATION: / note = "The" N "in positions 18-23 denotes a random base (A, C, T, or G) " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: GGCCGGATAG GCCTCACNNN NNNT 24 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12 CGCGGCTCGC CCACAGACTT C '(2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from nucleic acid (A) ) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: 23 CTGTCTCTCT GAGTCCGAAT GTT (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: TTCGGCCGGA TAGGCCTTTT TTTTTTTTTT 30 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs 5 (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from acid or nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: : 15 23 l J GTACAACACC TATGCCTCAA CCT (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: 0 (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid ( A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: GCAGTCGCTC CTTCCGTG 18 (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (ix) CHARACTERISTICS: (A) NAME / KEY: misc_fe_? t.ure (B) LOCATION: 1..9 (D) OTHER INFORMATION: / note = "The" N "denotes base at random" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: NNNNNNNNN '9 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (ix) FEATURE: (A) NAME / KEY: misc_feature (B) LOCATION: 18..28 (D) OTHER INFORMATION: / note = "The" N "in positions 18-28 denote a random base (A, C, T, or G) " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18 GCAGTCGCTC CTTCCGTGNN NNNNNNN (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: CTCCTCGCTC ATTCATTCCT A 21 (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from nucleic acid (A) ) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 0: AGTCCACTCC CCGGATGCTG AT 22 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: CTCCAGGATT CCGAAGCGGC TGTGGTCGTG 30 (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: one of (1, 2, 5, 6, 9, 14, 18, 19, 20, '21, 22) (D) OTHER INFORMATION: / note = "The" N "in positions 1, 2, 5, 6, 9, 14 and 18 denotes" Inosina "and in positions 19-22" bases at random " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: 25 NNTANN - CNC CACNCAANNN NNATG (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: G.T? GGACGC CACGCAAG 18 (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from nucleic acid (A) ) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: TCCGAAGCGG CTGTGGTCGT G 21 (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO". [xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: GGATCTACAC GGCTTCTCCT CGTCT 25 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: CTCGGGAGTC AGCTAAGGGC A 21 (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from nucleic acid (A) ) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: AAACAACATA TGGCTGAAGT TGGTGCTGTC TTTGCCTGGT TGG? ^ 4 (2) INFORMATION FOR SEQ ID NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: A? ACAAGGTA CCTTTACCTA TAGCGGAAGA GCT 33 (2) INFORMATION FOR SEQ ID NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: CGTACAGGTT TACGCA? GAA AATGG 25 (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from nucleic acid (A) ) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 0: TATG ?? CGAG CGCCTGGGCC? GATCGAGGG GAAGCTGCA 39 (2) INFORMATION FOR SEQ ID NO: 31: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: different from the nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: GCTTCCCCTC GATCTGGCCC AGGCGCTCGT TCA 33 (2) INFORMATION FOR SEQ ID NO: 32: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 173 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Aen Glu? Rg Leu Gly Gln He Glu Gly Lys Leu Gln? Rg Gly Ser Pro 1 5 10 15 Thr Asp Phe Wing His Leu Lys Gly He Leu Arg Arg Arg Gln Leu Tyr 20 25 30 Cyp? Rg Thr Gly Phe His Leu Glu He Phe Pro Asn Gly Thr Val His 35 40 45 Gly Thr Arg His Asp His Ser Arg Phe Gly He Leu Glu Phe He Ser 50 55 60 Leu Ala Val Gly Leu He Ser He Arg Gly Val Asp Ser Gly Leu Tyr 65 70 75 80 Leu Gly Met Asn Glu Arg Gly Glu Leu Phe Gly Ser Lys Lys Leu Thr 85 90 95 Arg Glu Cys Val Phß Arg Glu Gln Phß Glu Glu Asn Trp Tyr Asn Thr 100 105 110 Tyr Ala Ser Thr Leu Tyr Lys His Ser Asp Ser Glu Arg Gln Tyr Tyr 115 120 125 Val Ala Leu Asn Lys Asp Gly Ser Pro? Rg Glu Gly Tyr Arg Thr Lys 130 135 140? Rg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val Asp Pro Ser 145 150 155 160 Lys Leu Pro Ser Met Ser Arg Asp Leu Phe Arg Tyr Arg 165 170 (2) INFORMATION FOR SEQ ID NO: 33: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 198 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 33: Ser Leu Asp Trp Asp Leu Gln Gly Phe Being Ser Leu Gly Aen Val 1 5 10 15 Pro Leu Wing Asp Being Pro Gly Phe Leu Asn Glu Arg Leu Gly Gln He 20 25- 30 Gl? Gly Lys I.cu sm Arg Gly Ser Pro Thr Asp Phe Wing His Leu Lys 35 40 45 Gly He Leu Arg Arg Gln Leu Tyr Cys Arg Thr Gly Phe His Leu 50 55 60 Glu He Phe Pro Asn Gly Thr Val His Gly Thr Arg His Asp His Ser 65 70 75 80? Rg Phe Gly He Leu Glu Phe He Ser Leu Ala Val Gly Leu He Ser 85 90 95. He Arg Gly Val Asp Ser Gly Leu Tyr Leu Gly Met Asn Glu Arg Gly 100 105 110 Glu Leu Phe Gly Ser Lys Lys Leu Thr Arg Glu Cys Val Phe Arg Glu 115 120 125 Gln Phe Glu Glu Asn Trp Tyr Asn Thr Tyr Wing Ser Thr Leu Tyr Lys 130 135 140 His Sor Asp Ser Glu Arg Gln Tyr Tyr Val Wing Leu Asn Lys Asp Gly 145 150 155 160 Ser Pro Arg Glu Gly Tyr Arg Thr Lys Arg His Gln Lye Phe Thr His 165 170 175 Pho Leu Pro Arg Pro Val Asp Pro Ser Lye Leu Pro Ser Met Ser Arg 180 185 190 Asp Leu Phe Arg Tyr Arg 195 It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (39)

1. A polypeptide having hepatocyte proliferation activity and growth activity, characterized in that it comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5, or a fragment, analog or derivative thereof.

2. A polypeptide according to claim 1, characterized in that 1 to 34 amino acids are deleted from the N-terminal region.

3. A polypeptide according to claim 2, characterized in that it comprises the amino acid sequence of SEQ ID NO: 334.

A polypeptide according to claim 2, characterized in that it comprises the amino acid sequence of SEQ ID NO: 34.

5. A polypeptide according to claim 1, characterized in that it has eighty percent or more of homology with SEQ ID NO: 2 or SEQ ID NO: 5 and which is also characterized by the proliferation of hepatocytes and growth activity .

6. A polypeptide according to claim 1, characterized in that it is human and has the amino acid sequence of SEQ ID NO: 5.

7. A polypeptide according to claim 1, characterized in that it has been produced by recombinant means.

8. A polypeptide derivative according to claim 1, characterized in that the polypeptide has been bound or conjugated to a polymer.

9. A polypeptide derivative according to claim 8, characterized in that the polymer is polyethylene glycol.

10. An isolated nucleic acid molecule encoding a polypeptide, fragment or analogue thereof according to claim 1.

11. A nucleic acid molecule according to claim 10, characterized in that it encodes the polypeptide of SEQ ID NO: 2 or the fragment or analog thereof.

12. A nucleic acid molecule according to claim 10, characterized in that it has the nucleotide sequence of SEQ ID NO: 1.

13. A nucleic acid molecule according to claim 10, characterized in that it encodes the polypeptide of SEQ ID NO: 5 or the fragment or analog thereof.

14. A nucleic acid molecule according to claim 13, characterized in that it has the nucleotide sequence of SEQ ID NO: 4.

15. An expression vector, characterized in that it comprises the regulatory elements of expression operably linked with a nucleic acid molecule according to claim 10.

16. An expression vector according to claim 15, characterized in that the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 1.

17. An expression vector according to claim 15, characterized in that the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 4.

18. A host cell, characterized in that it is transformed or transfected with a nucleic acid molecule according to claim 10.

19. A host cell, characterized in that it is transformed or transfected with an expression vector according to claim 15.

20. A transformed or transfected host cell according to claims 18 or 19, characterized in that it is prokaryotic or eukaryotic.

21. A transformed or transfected host cell according to claim 20, characterized in that it is an animal cell.

22. A transformed or transfected host cell according to claim 20, characterized in that it is a bacterial cell.

23. A transformed or transfected host cell according to claim 22, characterized in that it is an E. coli cell.

24. A pharmaceutical composition, characterized in that it comprises an effective amount of a polypeptide or fragment, analog or derivative thereof according to claim 1 and a pharmaceutically acceptable carrier.

25. An antibody to the polypeptide according to claim 1.

26. An antibody according to claim 25, characterized in that it is polyclonal.

27. An antibody according to claim 25, characterized in that it is monoclonal.

28. A method for expressing a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5, or a fragment or analog thereof, characterized in that it comprises: culturing host cells containing a DNA molecule encoding the polypeptide or fragment or analog operably linked to the regulatory elements to stimulate the expression thereof, under conditions such that the DNA molecule is expressed, and Isolate the polypeptide, fragment or analogue expressed from the host cell culture.

29. A method according to claim 28, characterized in that the molecule of DNA is a gene or cDNA.

30. A method according to claim 28, characterized in that the host cells have been transformed or transfected with an expression vector according to claim 15.

31. A method according to claim 30, characterized in that the host cells are E. coli cells.

32. A method according to claim 28, characterized in that the molecule of the DNA is an endogenous gene that is part of the genome of host cells.

33. A method according to claim 32, characterized in that the host cells are animal cells.

34. A method for cell therapy, characterized in that it comprises transforming or transfecting the cells within an organism with a vector comprising the regulatory elements of expression operably linked to a nucleic acid molecule according to claim 10 which is a molecule of DNA, whereby the expression of the DNA molecule occurs in situ in the host organism.

35. A method for stimulating the proliferation of hepatocyte cells, characterized in that it comprises contacting said cells with an effective amount of a polypeptide or fragment, analog or derivative thereof according to claim 1.

36. A method according to claim 35, characterized in that it is carried out in vitro.

37. A method according to claim 35, characterized in that it is carried out in vivo.

38. A method for stimulating the growth of hepatocytes in vivo by gene therapy, characterized in that it comprises transforming or transfecting the endogenous hepatocytes present within a host organism with an expression vector operably linked to the nucleic acid molecules encoding a polypeptide or fragment or analog thereof according to claim 1, in such a way that the nucleic acid molecules are capable of expressing the polypeptide, analog or fragment in situ.

39. A method according to claim 37 or 38, characterized in that it is used to treat a disease or disorder of the liver in the host organism. FACTOR OF GROWTH OF FIBROBLASTS WITH ACTIVITY OF PROLIFERATION OF HEPATOCYTES SUMMARY OF THE INVENTION The present invention relates to an additional element of the family of fibroblast growth factor (FGF) of the polypeptides that has now been discovered and purified, which has been found to exert a specific mitogenic activity for the hepatocytes that distinguishes it. of the other elements of FGF. The DNA molecules encoding this polypeptide can be used in the modes of therapy administration of the genes to stimulate the proliferation of the hepatocytes in vivo, or alternatively, they can be used in the recombinant methods to produce the polypeptide for the use of cell culture or for therapeutic use to treat liver conditions that require the generation and growth of hepatocytes.