ENGINEERED FGF COMPOSITIONS AND METHODS OF USE THEREOF

Abstract

The present invention relates to the development of stable mutants of FGF-1 and FGF-2. In particular, it relates to novel engineered FGF-1 and FGF-2 polypeptides as well as polynucleotides, DNA constructs, and vectors encoding such polypeptides. In another aspect, pharmaceutical compositions and hydrogels including the disclosed polypeptides, polynucleotides, DNA constructs, and vectors are provided. In a still further aspect, methods of treating conditions using the compositions disclosed herein are provided.

Description

INTRODUCTION

The present invention relates to the development of stable mutants of FGF-1 and FGF-2. It relates in particular to polynucleotides, polypeptides, and pharmaceutical compositions including such mutant forms of FGF-1 and FGF-2 as well as methods of treating conditions using such compositions.

FGFs play an important role in various cellular processes like cell proliferation, migration, differentiation and induce processes such as regeneration, morphogenesis and angiogenesis. FGFs exert their effects upon binding to their specific receptors. These molecules are known to bind to heparin to increase the efficiency of mitogenic activity.

Growth factors like FGF-1 and FGF-2 also have a huge clinical significance. FGF-1 is known to play a crucial role in wound healing and other significant clinical conditions. For example, there are reports demonstrating the FGF-1 has significant nerve regeneration and potent angiogenic activity. These events are critical for proper healing after an injury. Hence administration of FGF-1 during an injury can quicken the process of healing and helps in safe recovery from trauma. Although FGF-1 and FGF-2 proteins are promising therapeutics, they are highly susceptible to proteolytic degradation especially by thrombin which is usually present in abundance at the site of a wound in the fibrin clots. Thus, there is a need in the art for new FGF-based treatments having increased stability against proteolytic degradation, higher biological activity, and a prolonged circulation half-life.

SUMMARY

The present invention relates to the development of stable mutants of FGF-1 and FGF-2. In one aspect, FGF polypeptides are provided. The FGF polypeptides may include SEQ ID NO: 4, SEQ ID NO: 6, a polypeptide having 80% sequence identity to amino acids 1-121; 123-140 of SEQ ID NO: 4, a polypeptide having 80% sequence identity to amino acids 1-121; 123-140 of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, a polypeptide having 80% sequence identity to amino acids 1-137; 139-155 of SEQ ID NO: 10, a polypeptide having 80% sequence identity to amino acids 1-137; 139-155 of SEQ ID NO: 12, fragments including amino acid residue 122 of SEQ ID NO: 4 or SEQ ID NO: 6, fragments including amino acid residue 138 of SEQ ID NO: 10 or SEQ ID NO: 12, a polypeptide having 80% sequence identity to at least one of SEQ ID NOs: 4, 6, 10 or 12, fragments comprising amino acid residue 122 of SEQ ID NO: 4 or SEQ ID NO: 6, fragments comprising amino acid residue 138 of SEQ ID NO: 10 or SEQ ID NO: 12, a fragment of SEQ ID NOs: 4 or 6 comprising amino acids 112-128 and a fragment of SEQ ID NOs: 10 or 12 comprising amino acids 128-144.

In another aspect, polynucleotides encoding any one of the FGF polypeptides described herein are provided.

In a further aspect, provided herein are pharmaceutical compositions including any one of the FGF polypeptides or polynucleotides described herein.

In another aspect, methods of treating a condition are provided. The methods may include administering any one of the compositions (FGF polypeptides, FGF polynucleotides, or pharmaceutical compositions) described herein to a subject in an amount effective to treat the condition.

In still further aspects, the compositions may be included in a hydrogel or may be encoded by a construct and/or produced as a fusion protein with a membrane permeable protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of the wild type (wt) FGF-1 molecule (SEQ ID NO: 1) with a codon for arginine at position 136 underlined. Also represented is the translated amino acid sequence of the wtFGF-1 (SEQ ID NO: 2) with the residue R136 underlined. As shown with SEQ ID NO: 2, the recombinant clone used in the Examples is devoid of the first 14 amino acids present at the N-terminus. Truncated FGF-1 showed no difference in biological activity compared to the full length form and is generally used and accepted in the field.

FIG. 2 shows the nucleotide sequence of FGF-1R136E molecule (SEQ ID NO: 3) with a codon for glutamic acid at position 136 underlined. Also represented is the translated amino acid sequence of the FGF-1R136E polypeptide (SEQ ID NO: 4) with the R136E substitution underlined. The R136E substitution is at position 122 in the sequence shown.

FIG. 3 shows the nucleotide sequence of FGF-1R136D molecule (SEQ ID NO: 5) with a codon for aspartic acid at position 136 of the full-length sequence underlined. Also represented is the translated amino acid sequence of the FGF-1R136D polypeptide (SEQ ID NO: 6) with the R136E substitution underlined. As noted above, the R136D substitution is at position 122 in the sequence shown.

FIG. 4 shows the 3D structure of the FGF-1 molecule using a ribbon diagram. Amino acid residue R136 that is very important in the function of the FGF-1 mitogenic activity is highlighted and represented in stick form (FIG. 4A). In the picture shown in FIG. 4B, the amino acid residues that play an important role in the heparin binding are highlighted. All these amino acid residues are shown in sticks and colored in orange.

FIG. 5 shows a graph of a mitogenic activity assay of the wild type and mutant forms FGF-1, where the mutant FGF-1 R136E shows approximately 2 fold increased mitogenic activity compared to the wtFGF-1 and other mutant forms of FGF-1. This signifies that charge reversal especially using glutamic acid in place of arginine at position 136 might actually be enhancing the overall stability of the FGF-1 molecule.

FIG. 6 shows graphs of the thrombin cleavage activity of wildtype (wtFGF-1), R136K and R136E mutants of FGF-1 in the absence and presence of SOS (sucrose octa sulfate an analog for heparin). When compared with wtFGF-1, R136K and R136E mutants show minimal degradation both in the absence (FIG. 6A) and presence (FIG. 6B) of heparin.

FIG. 7 shows graphs representing the limited trypsin digestion of wildtype (wtFGF-1) and mutant (R136K & R136E) FGF-1. The R136E mutant shows a very limited cleavage by trypsin compared to wtFGF-1 and the R136K mutant in the absence of SOS (sucrose octa sulfate an analog for heparin) (FIG. 7A). This effect might be due to a change in a confirmation that would give limited access for trypsin and also due to the reduction of one potential cleavage site either in the form of arginine or lysine. But in presence of SOS (FIG. 7B) no significant difference was observed between the three molecules under observation which signifies that upon binding to heparin there is a conformational change resulting in more compact structure decreasing the ability of trypsin to access the cleavage sites.

FIG. 8 shows the DSC profiles of wildtype (wtFGF-1), R136K & R136E forms of FGF-1 in the absence (FIG. 8A) and presence of SOS (FIG. 8B). The R136E mutant shows a Tm of 56.1° C. compared with the wt (49.3° C.) and R136K (50.3° C.) mutants in the absence of SOS (FIG. 8A). This signifies the thermal stability of R136E independent of heparin. In presence of SOS, there is no significant difference in Tm observed between the wt and mutant forms of FGF-1 (FIG. 8B). Navy Blue=wtFGF-1, Magenta=R136K mutant, Cyan=R136E mutant.

FIG. 9 shows an overlay of 1H-15N HSQC profile of wt (wtFGF-1) and mutant R136K and R136E forms of FGF-1. Very few amino acid peaks were perturbed in the mutants when compared with the wtFGF-1

FIG. 10 shows the binding isotherms of the wt (wtFGF-1; FIG. 10A) and mutant R136K (FIG. 10B) and R136E (FIG. 10C) forms of FGF-1 titrated with SOS. Both wtFGF-1 and R136K show strong affinity to SOS when compared with R136E, which is evidence showing heparin independent mitogenic activity for R136E.

FIG. 11 shows the western blot analysis for the detection of phosphorylated forms of Akt (FIG. 11A) and ERK (FIG. 11B) proteins in the presence of wt (wtFGF-1) and R136E FGF-1 (MT). Compared between both Akt and ERK pathways, it appears that R136E was involved in upregulating the ERK pathway which is critical for wound healing processes. C=control, WT=wtFGF-1, MT=R136E mutant.

FIG. 12 shows monolayers of quiescent Swiss 3T3 cells which were wounded and incubated for 40 h with 5 ng of “wt” (wtFGF-1) or “mutant” FGF-1 (R136E), or without FGF-1 (“control”). FIG. 12A shows the migrating fronts of the wounds after 40 h. Bar—80 μm. FIG. 12B shows representative views of wound closing. Bar—200 μm. FIG. 12C shows cell migration to the central area of the wound, 40 h after wounding. Average cell numbers in the standard median areas (21.5×4 mm) of the wounds are presented.

FIG. 13 shows the nucleotide sequence of the wild type (wt) FGF-2 molecule (SEQ ID NO: 7) with a codon for lysine at position 138 underlined. Also represented is the translated amino acid sequence of the wtFGF-2 (SEQ ID NO: 8) with the residue K138 underlined.

FIG. 14 shows the nucleotide sequence of FGF-2K138E molecule (SEQ ID NO: 9) with a codon for glutamic acid at position 138 underlined. Also represented is the translated amino acid sequence of the FGF-2K138E polypeptide (SEQ ID NO: 10) with the K138E substitution underlined.

FIG. 15 shows the nucleotide sequence of FGF-2K138D molecule (SEQ ID NO: 11) with a codon for aspartic acid at position 136 underlined. Also represented is the translated amino acid sequence of the FGF-2K138D polypeptide (SEQ ID NO: 12) with the K138D substitution underlined.

DETAILED DESCRIPTION

Provided herein are FGF polynucleotides, polypeptides, and pharmaceutical compositions comprising novel substitutions conferring greater resistance to proteases and higher biological activity as compared to wild-type FGF polypeptides. In one aspect, the present inventors have discovered novel FGF polypeptides. The FGF polypeptides may include SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, or SEQ ID NO: 12.

As used herein, the terms “protein” or “polypeptide” or “peptide” may be used interchangeably to refer to a polymer of amino acids. A “polypeptide” as contemplated herein typically comprises a polymer of naturally occurring amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). The proteins contemplated herein may be further modified in vitro or in vivo to include non-amino acid moieties. These modifications may include but are not limited to acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation, lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, enzymatic addition such as polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine) are also contemplated.

As used herein, the term “FGF polypeptide” refers to FGF-1 and FGF-2 polypeptides. The FGF polypeptides provided herein are engineered polypeptides representing substitution mutants of the wild-type polypeptide. The FGF polypeptides disclosed herein may include “mutant” FGF polypeptides and variants thereof. As used herein the term “wild-type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms. As used herein, a “variant” refers to a polypeptide molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule. A variant may include a fragment of a reference molecule. For example, a FGF polypeptide variant molecule may have one or more insertions, deletions, or substitution of at least one amino acid residue relative to the mutant FGF polypeptides described herein.

The DNA sequence of the “wild-type” FGF-1 protein from humans is presented as SEQ ID NO:1 while the amino acid sequence of residues 15-155 is depicted as SEQ ID NO: 2. See FIG. 1. The DNA sequence of the “wild-type” FGF-2 protein from humans is presented as SEQ ID NO: 7 while the amino acid sequence of residues 1-155 is depicted as SEQ ID NO: 8. See FIG. 13.

Amino acid sequences for the engineered mutant FGF polypeptides disclosed herein are provided as SEQ ID NO: 4 (FGF-1R136E), SEQ ID NO: 6 (FGF-1R136D), SEQ ID NO: 10 (FGF-2K138E), and SEQ ID NO: 12 (FGF-2K138D). These sequences may be used as reference sequences.

The FGF polypeptides provided herein may be full-length polypeptides (i.e., SEQ ID NOS: 4, 6, 10, or 12) or may be fragments of the full-length FGF polypeptide including amino acid residue 122 of SEQ ID NO: 4 or SEQ ID NO: 6 or amino acid residue 138 of SEQ ID NO: 10 or SEQ ID NO: 12.

As used herein, a “fragment” is a portion of an amino acid sequence which is identical in sequence to but shorter in length than a reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one amino acid residue. For example, a fragment may comprise from 5 to 155 contiguous amino acid residues of a reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or 150 contiguous amino acid residues of a reference polypeptide. Fragments may be preferentially selected from certain regions of a molecule. The term “at least a fragment” encompasses the full length polypeptide. A fragment of a FGF-1 polypeptide may comprise or consist essentially of a contiguous portion of an amino acid sequence of the full-length FGF polypeptide (SEQ ID NOS: 4, 6, 10, or 12). A fragment may include an N-terminal truncation, a C-terminal truncation, or both truncations relative to the full-length FGF polypeptide.

Preferably, a fragment of a FGF-1 polypeptide includes amino acid residue 122 of SEQ ID NO: 4 or SEQ ID NO: 6. Preferably, a fragment of a FGF-2 polypeptide includes amino acid residue 138 SEQ ID NO: 10 or SEQ ID NO: 12. Suitable fragments include or consist of amino acid residues 112-128, 106-134, 100-140, 50-140, 25-140, 10-140, 2-140 or any range therein of SEQ ID NOs: 4 or 6, or amino acid residues 128-144, 120-150, 110-155, 100-155, 80-155, 60-155, 40-155, 20-155, 10-155, 2-155 or any range therein of SEQ ID NOs: 10 or 12.

FGF polypeptides may be useful for a variety of reasons. For example, FGF polypeptides which contain the substitutions noted above can be used inter alia for raising antibodies. Such polypeptides are typically less than full-length proteins. Preferably such residues are at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 23, 25, 30, 40, 50 or more residues in length. As an example, if the polypeptide is 6 residues in length, than it can comprise residues including the substitution site (i.e. residue 136 of FGF-1 (residue 122 of SEQ ID NO: 4 or SEQ ID NO: 6), residue 138 of FGF-2). Sufficient residues are desired to form a good immunogen or blocking antigen for use in assays. It may be desirable to conjugate or genetically fuse additional sequences to the polypeptide, for example, to boost immunogenicity, to enhance purification, to facilitate production or expression, or to facilitate detection. Any sequences as are convenient may be used for these or other purposes. The size of these additional sequences may vary greatly, but typically will be at least 2, 4, 6, or 8 amino acid residues in length. Suitably the additional sequences will be less than 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 amino acids in length.

A “deletion” in a polypeptide refers to a change in the amino acid sequence which results in the absence of one or more amino acid residues. A deletion may remove at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, or more amino acids residues. A deletion may include an internal deletion and/or a terminal deletion (e.g., an N-terminal truncation, a C-terminal truncation or both of a reference polypeptide).

“Insertions” and “additions” in a polypeptide refer to changes in an amino acid sequence resulting in the addition of one or more amino acid residues. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more amino acid residues. A variant of a FGF-1 polypeptide may have N-terminal insertions, C-terminal insertions, internal insertions, or any combination of N-terminal insertions, C-terminal insertions, and internal insertions.

Regarding polypeptides, the phrases “percent identity,” “% identity,” and “% sequence identity” refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.

As described herein, variants of the engineered mutant FGF polypeptides disclosed herein may have 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, or 50% amino acid sequence identity relative to a reference molecule (e.g., relative to the FGF-1 or FGF-2 full-length mutant polypeptides (i.e., SEQ ID NOs: 4, 6, 10, and 12). In some embodiments, variants of the engineered mutant FGF polypeptides disclosed herein may have 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, or 50% amino acid sequence identity relative to a reference molecule (e.g., relative to the FGF-1 or FGF-2 full-length mutant polypeptides (i.e., SEQ ID NOS: 4, 6, 10, and 12) and include amino acid residue 122 of SEQ ID NO: 4 or SEQ ID NO: 6 or amino acid residue 138 of SEQ ID NO: 10 or SEQ ID NO: 12. Suitably the variants include the substitution mutations identified herein.

Polypeptide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

The amino acid sequences of the FGF polypeptide variants as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, or derivative FGF polypeptide may include conservative amino acid substitutions relative to a reference molecule. “Conservative amino acid substitutions” are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.

The amino acid sequences of the FGF polypeptide variants as contemplated herein may include may include modifications made apparent by a sequence alignment of the FGF polypeptides disclosed herein and other FGF polypeptides. A person of ordinary skill in the art, could easily align the FGF polypeptides disclosed herein with FGF polypeptides from, for example, other species to determine what additional variants (i.e. substitutions, insertions, deletions, etc.) could be made to the engineered FGF polypeptides. For example, a person of ordinary skill in the art would appreciate that modifications in a reference FGF polypeptide could be based on alternative amino acid residues that occur at the corresponding position in other homologous FGF polypeptides from other species.

The disclosed FGF polypeptides, mutants, or variants described herein may have one or more functional or biological activities exhibited by a reference polypeptide (e.g., one or more functional or biological activities exhibited by wild-type FGF-1 polypeptide (SEQ ID NO: 2) or wild-type FGF-2 polypeptide (SEQ ID NO: 8)). For example, the disclosed FGF polypeptides, mutants, variants, or derivatives thereof may have increased mitogenic activity or increased stability to protease degradation.

In another aspect of the present invention, fusion proteins are provided. The fusion proteins may include any one of the FGF polypeptides disclosed herein and a membrane permeable peptide. The membrane permeable peptide may be any polypeptide having less than 50, 40, 30, or 20 amino acids that can penetrate cell membranes and deliver conjugated FGF polypeptides into cells. Suitable membrane permeable peptide may include, without limitation, a cell-penetrating polypeptide (CPP), TAT, Pep-1, Penetratin, SynB1, SynB3, PTD-4, PTD-5, Transportan, MAP, SBP, FBP, Polyarginines, or Polylysines.

Polynucleotides encoding any of the FGF polypeptides disclosed herein are also provided. The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic, natural, or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).

The polynucleotides may encode FGF polypeptides including an R136D substitution of FGF-1, an R136E substitution of FGF-1, a K138E substitution of FGF-2, or a K138D substitution of FGF-2. Suitably the polynucleotides are at least 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500 nucleotides long and include the nucleotide coding for the substitution. Suitably the polynucleotide encodes the full-length polypeptide. Polynucleotides encoding partial polypeptides comprising the site of the substitutions indicated are also provided. Polynucleotide sequences of the polypeptides comprising the R136D and R136E substitutions and the wild-type FGF-1 are provided in FIGS. 2-3. The polynucleotide sequences may include the polynucleotide sequences of SEQ ID NO: 3 or SEQ ID NO: 5. Polynucleotide sequences of the polypeptides comprising the K138E and K136D substitutions and the wild-type FGF-2 are provided in FIGS. 14-15. The polynucleotide sequences may include the polynucleotide sequences of SEQ ID NO: 9 or SEQ ID NO: 11.

Isolated polynucleotides homologous to the polynucleotides described herein are also provided. Those of skill in the art also understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide. In some embodiments, the polynucleotides may be codon-optimized for expression in a particular cell such as, without limitation, a mammalian cell or a prokaryotic cell. While particular nucleotide sequences which are found in humans are disclosed herein any nucleotide sequences may be used which encode a desired form of the substituted polypeptides described herein. Thus non-naturally occurring sequences may be used. These may be desirable, for example, to enhance expression in heterologous expression systems of polypeptides or proteins. Computer programs for generating degenerate coding sequences are available and can be used for this purpose as well as other means.

The isolated polynucleotides or polypeptides provided herein may be prepared by methods available to those of skill in the art. Isolated indicates that the polynucleotides or proteins are not in their naturally occurring state. Such preparations may be cell-free preparations. The polynucleotide or polypeptides may be extracted from the cells by breaking the cell membrane and optionally removing non-desired components. The polypeptides may be made as secreted polypeptides and further isolated using means known to those of skill in the art. Alternatively, desired proteins or nucleic acids can be purified using sequence-specific reagents, including but not limited to oligonucleotide probes, primers, and antibodies. Techniques for isolating cell-free preparations are well known in the art, and any that are convenient can be used. The term “substantially isolated or purified” refers to polypeptides or polynucleotides that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.

In another aspect of the present invention, DNA constructs are provided. As used herein, the term “DNA construct” refers to recombinant DNA polynucleotides which may be single-stranded or double-stranded and may represent the sense or the antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods that include polynucleotide sequences derived from at least two different natural sources or they may be synthetic. Constructs thus may include new modifications to endogenous genes introduced by, for example, genome editing technologies. Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies.

The DNA constructs provided herein may be prepared by methods available to those of skill in the art. Notably each of the DNA constructs claimed are recombinant molecules and as such do not occur in nature. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, and recombinant DNA techniques that are well known and commonly employed in the art. Standard techniques available to those skilled in the art may be used for cloning, DNA and RNA isolation, amplification and purification. Such techniques are thoroughly explained in the literature.

The DNA constructs provided herein may include a promoter operably linked to any one of the polynucleotides described herein. The promoter may be a heterologous promoter or an endogenous promoter associated with the FGF polypeptide.

As used herein, the terms “heterologous promoter,” “promoter,” “promoter region,” or “promoter sequence” refer generally to transcriptional regulatory regions of a gene, which may be found at the 5′ or 3′ side of the FGF polynucleotides described herein, or within the coding region of the FGF polynucleotides, or within introns in the FGF polynucleotides. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. The typical 5′ promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

In some embodiments, the disclosed FGF polynucleotides are operably connected to the promoter. As used herein, a polynucleotide is “operably connected” or “operably linked” when it is placed into a functional relationship with a second polynucleotide sequence. For instance, a promoter is operably linked to an FGF polynucleotide if the promoter is connected to the FGF polynucleotide such that it may effect transcription of the FGF polynucleotides. In various embodiments, the FGF polynucleotides may be operably linked to at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 promoters.

Heterologous promoters useful in the practice of the present invention include, but are not limited to, constitutive, inducible, temporally-regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters. The heterologous promoter may be an animal, plant, bacterial, or fungal promoter. In mammalian cells, typical promoters include, without limitation, promoters for Rous sarcoma virus (RSV), human immunodeficiency virus (HIV-1), cytomegalovirus (CMV), SV40 virus, and the like as well as the translational elongation factor EF-1α promoter or ubiquitin promoter. Other promoters include the T3, T7 and SP6 promoter sequences, which are often used for in vitro transcription of RNA. Those of skill in the art are familiar with a wide variety of additional promoters for use in various cell types. In some embodiments, the heterologous promoter includes a mammalian promoter, either endogenous to the animal host or heterologous.

Vectors, including any of the DNA constructs or polynucleotides described herein, are provided. The term “vector” is intended to refer to a polynucleotide capable of transporting another polynucleotide to which it has been linked. In some embodiments, the vector may be a “plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome, such as some viral vectors or transposons. Vectors may carry genetic elements, such as those that confer resistance to certain drugs or chemicals.

Pharmaceutical compositions including any of the FGF polypeptides, polynucleotides, DNA constructs, or vectors described herein are provided. The pharmaceutical compositions may include a pharmaceutical carrier, excipient, or diluent (i.e., agents), which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often a pharmaceutical agent is in an aqueous pH buffered solution. Examples of pharmaceutical carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; 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, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ brand surfactant, polyethylene glycol (PEG), and PLURONICS™ surfactant.

Hydrogels including any of the FGF polypeptides, polynucleotides, DNA constructs, or vectors described herein are provided. Suitably, the hydrogels include any one of the FGF polypeptides disclosed herein. As used herein, a “hydrogel” refers to a gel in which the liquid component is water.

Methods of treating a condition are also provided. The methods may include administering any of FGF polypeptides, polynucleotides, DNA constructs, vectors, pharmaceutical compositions, or hydrogels described herein to a subject in an amount effective to treat the condition. The condition may include a wound (chronic and acute), Type 1 diabetes, Type 2 diabetes, obesity, internal injuries, a cardiovascular disorder, a cosmetic condition (i.e. whitening, wrinkling), critical limb ischemia, a nerve injury, a burn, hair loss (whether genetic or not, i.e. alopecia), a retinal disorder (i.e., retinopathy disorders), a muscular disorder, an arterial disease, an age related disorder, organ or tissue damage (whether or not from chemotherapy or radiation therapy), osteoporosis, a digestive tract ulcer (i.e., gastric ulcer), ulcerative colitis, a scar, an energy homeostasis disorder such as obesity, dyslipidemia, diabetes, insulin resistance, hyperglycemia or the metabolic syndrome, osteoarthritis, and acute renal failure.

The subject may be any mammal, suitably a human, domesticated animal such as a dog, cat, horse, cow, pig, or a mouse or rat. Treating the condition or treatment includes but is not limited to ameliorating at least one symptom of the condition, reducing or slowing further progression of the condition, reducing or slowing the spread of the condition to unaffected areas. Treating a subject refers to any type of treatment that imparts a benefit to a subject afflicted with a disease or at risk of developing the disease, including improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disease, delay the onset of symptoms or slow the progression of symptoms, etc.

An effective amount or a therapeutically effective amount as used herein means the amount of a composition that, when administered to a subject for treating a state, disorder or condition is sufficient to effect a treatment (as defined above). The therapeutically effective amount will vary depending on the compound, formulation or composition, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated.

The compositions (i.e. FGF polypeptides, polynucleotides, DNA constructs, vectors, pharmaceutical compositions, or hydrogels) described herein may be administered by any means known to those skilled in the art, including, but not limited to, oral, topical, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transcutaneous, nasopharyngeal, intra-lesional, intra-tumoral, intradermal, or transmucosal absorption. Thus the compositions may be formulated as an ingestible, injectable, topical or suppository formulation. The compositions may also be delivered with in a liposomal or time-release vehicle. The methods may also include an electrical stimulation or electroporation step to aid entry of the polypeptides, polynucleotides, or pharmaceutical compositions into the intracellular space. Administration of the compositions to a subject in accordance with the invention may exhibit beneficial effects in a dose-dependent manner. Thus, within broad limits, administration of larger quantities of the compositions is expected to achieve increased beneficial biological effects than administration of a smaller amount. Moreover, efficacy is also contemplated at dosages below the level at which toxicity is seen.

It will be appreciated that the specific dosage administered in any given case will be adjusted in accordance with the composition or compositions being administered, the disease to be treated or inhibited, the condition of the subject, and other relevant medical factors that may modify the activity of the compositions or the response of the subject, as is well known by those skilled in the art. For example, the specific dose for a particular subject depends on age, body weight, general state of health, diet, the timing and mode of administration, the rate of excretion, medicaments used in combination and the severity of the particular disorder to which the therapy is applied. Dosages for a given patient can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the compositions described herein and of a known agent, such as by means of an appropriate conventional pharmacological protocol.

The maximal dosage for a subject is the highest dosage that does not cause undesirable or intolerable side effects. The number of variables in regard to an individual treatment regimen is large, and a considerable range of doses is expected. The route of administration will also impact the dosage requirements. It is anticipated that dosages of the compositions will improve wound healing or other condition being treated by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more as compared to no treatment.

The effective dosage amounts described herein refer to total amounts administered, that is, if more than one composition is administered, the effective dosage amounts correspond to the total amount administered. The compositions can be administered as a single dose or as divided doses. For example, the composition may be administered two or more times separated by 4 hours, 6 hours, 8 hours, 12 hours, a day, two days, three days, four days, one week, two weeks, or by three or more weeks.

The FGF polypeptides, polynucleotides, DNA constructs, vectors, pharmaceutical compositions, or hydrogels described herein may be administered one time or more than one time to the subject to effectively improve wound healing or other condition being treated. Suitable dosage ranges are of the order of several hundred micrograms effective ingredient with a range from about 0.01 to 10 mg/kg/day, preferably in the range from about 0.1 to 1 mg/kg/day. Precise amounts of effective ingredient required to be administered depend on the judgment of the practitioner and may be peculiar to each subject. It will be apparent to those of skill in the art that the therapeutically effective amount of the polypeptides, polynucleotides, and pharmaceutical compositions described herein will depend, inter alia, upon the administration schedule, the unit dose of antigen administered, whether the composition is administered in combination with other therapeutic agents, the status and health of the recipient, and the therapeutic activity of the particular composition.

Given that the FGF polypeptides of the present invention confer greater resistance to proteases and higher biological activity, it is envisioned that compositions including or encoding such polypeptides would be useful in several different FGF applications.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat diabetes (i.e., type 1 or type 2). Treatment of type1 and type2 diabetes may be achieved by injecting the FGF compositions (either individually or in combination). Without being limited by theory, systemic FGF polypeptides could potentially activate the process of angiogenesis. Also the skeletal muscle cell multiplication drastically increases that could result in large uptake of blood glucose eventually restore the pancreas to function normally.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat internal injuries. Because the disclosed FGF polypeptides demonstrated protease resistant activity, they may be used in treating visceral injuries such as intestinal wall ruptures (sites with excess protease action like trypsin etc.).

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used in cosmetic applications. For example, a medicinal composition including the FGF compositions described herein (either individually or in combination) and FGF2, FGF5, FGF7 and/or FGF10 could be used in various cosmetic applications including, without limitation, whitening, anti-crinkle and anti-aging related problems.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat cardiovascular disorders. It has been shown that injection of FGF-1 into the intramyocardial region resulted in improved collateral artery growth and capillary formation and proliferation. Thus, the FGF compositions (either individually or in combination) may be used to improve collateral artery growth and capillary formation and proliferation.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat critical limb ischemia. For example, gene therapy based delivery of FGF polypeptides (either individually or in combination) could be used in treating end stage limb ischemia which would potentially reduce the chances of amputation.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used in treating nerve injuries. The disclosed FGF compositions (either individually or in combination) could be used to regenerate damaged cells in the spinal cord.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat burns. Several factors are responsible for causing burns such as heat, electricity, UV-light and corrosive chemicals. The disclosed FGF compositions (either individually or in combination) might quicken the process of healing of burns along with other pharmacologically active ingredients.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used in hydrogels. Hydrogels including the disclosed FGF compositions (either individually or in combination) could be used in design and development of scaffolds in the fields of tissue engineering and regenerative medicine.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used in regenerating hair growth. Growth factors like FGF1, FGF2, and FGF10 are known to be involved in the regulation of hair morphogenesis and hair growth. Due to the enhanced bioactive properties of the disclosed FGF compositions, the disclosed FGF compositions may be used to alter (increase or decrease) hair growth.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat wounds. For example, compositions including Parathyroidharmone (PTH), Collagen binding protein (CBD), and the disclosed FGF compositions (either individually or in combination) could be used in the treating both chronic and acute wounds.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to repair an injured retina. The disclosed FGF compositions (either individually or in combination) may be used for rejuvenating damaged cells of the retina such as in retinopathy disorders.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used in skeletal muscle development. For example, the disclosed FGF compositions (either individually or in combination) could be fused to other FGF family members that could facilitate the faster growth of skeletal muscle.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used in cell cultures such as mammalian cell cultures.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat arterial diseases. For example, the disclosed FGF compositions (either individually or in combination) may be used in treatment of peripheral arterial disease with intermittent claudication.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat aging related disorders.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to regenerate internal organs.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat osteoporosis.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat gastric ulcers.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat colitis such as ulcerative colitis. For example, the disclosed FGF compositions (either individually or in combination) may be used in combination with keratinocyte growth factor-1 to treat ulcerative colitis.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat scars developed from various types of injuries.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used in combination with biopolymers such as carboxy methyl benzyamine dextran sulfonate to treat digestive tract ulcers.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to affect differentiation of osteoblast cells. For example, the disclosed FGF compositions (either individually or in combination) may be used, for example by gene therapy, to enhance the down regulation of Wnt signaling proteins which would result in decreased differentiation of osteoblast cells.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat damaged tissue due to radiation or chemotherapy. For example, the disclosed FGF compositions (either individually or in combination) may be used in combination with FGF-20 to treat damaged tissue due to radiation and also due to extensive use of chemotherapy.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat diabetes and obesity. For example, FGF-21 controls the glucose-uptake of adipocytes independent of insulin which would decrease the load of blood glucose, triglycerides and glucagon. Thus, compositions including the FGF compositions described herein (either individually or in combination) and FGF-21 would be used in treating diabetes and obesity.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat disorders related to energy homeostasis. For example, the disclosed FGF compositions (either individually or in combination) may be used in combination with FGF8 to treat disorders of energy homeostasis such as obesity, dyslipidemia, diabetes, insulin resistance, hyperglycemia or the metabolic syndrome.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to control differentiation of cord cells. For example, the disclosed FGF compositions (either individually or in combination) may be used in combination with FGF4 to induce human umbilical cord mesenchymal stem cells to differentiate into hepatocytes.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat alopecia. For example, the disclosed FGF compositions (either individually or in combination) may be used in combination with FGF-10 to treat alopecia.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat osteoarthritis. For example, the disclosed FGF compositions (either individually or in combination) may be used in combination with FGF-9 to promote cartilage repair in patients suffering from osteoarthritis by reducing the abnormal differentiation of articular cartilage cells at the site of inflammation.

In some embodiments, the FGF compositions described herein (either individually or in combination) may be used to treat acute renal failure. For example, the disclosed FGF compositions (either individually or in combination) may be used in combination with inhibin beta and FGF-2 to treat acute renal failure.

The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of” and “consisting of” those certain elements.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.

Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a protein” or “an RNA” should be interpreted to mean “one or more proteins” or “one or more RNAs,” respectively.

The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

Examples

Materials and Methods

Polymerase Chain Reaction

Polymerase chain reaction (PCR) for amplification and generation of FGF1 mutant plasmid DNA was performed using a QuikChange Site-Directed Mutagenesis Kit. The reaction mixture consisted of 1 pmol each of forward & reverse primers, 5 uL of 10× Pfu reaction buffer, 1 uL dNTP, 10-15 ng of wild-type plasmid DNA, 1 uL Pfu DNA polymerase and autoclaved deionized water to give a total volume of 50 uL. Cycling parameters were as per the manufacturer's instructions.

After PCR, the reaction mixture was subjected to Dpn1 digestion to eliminate the methylated parental DNA. Mutant pDNA was used to transformed cXL1-Blue supercompetent cells. Plasmid extraction was performed using a QIAprep Spin Miniprep Kit (250), and the correctness of DNA was verified by DNA sequencing.

FGF-1 Expression

The expression of wt and mutant FGF-1 proteins was achieved by transforming B121DE3 PlysS Escherichia coli with the wt and mutant FGF1 plasmid DNA and the transformed expression host cells were grown in LB medium. ¹⁵N-labeled protein was prepared by growing the cells in M9-minimal medium containing ¹⁵N-labeled NH₄Cl as the sole source of nitrogen. Induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) was performed when the optical density (O.D.) of the inoculated medium was between 0.5-0.6. Cells were harvested by centrifugation and resuspended in buffer containing 10 mM phosphate, 1 mM EDTA, 0.1% beta-mercaptoethanol, and 1 mM PMSF at a pH of 7.2.

FGF-1 Purification

The purification of FGF-1(wt) and the FGF-1 mutant proteins was performed on heparin-sepharose affinity column. Pure protein was obtained using 10 mM phosphate buffer, pH 7.2, with an increasing sodium chloride concentration in a step-gradient manner. FGF-1(wt) and mutant proteins eluted at different salt concentrations based on their affinity toward the immobilized heparin moiety. Purity of the protein was confirmed by resolving the samples on 15% SDS-PAGE. All purified protein samples were subjected to dialysis against a buffer of 10 mM phosphate, 100 mM NaCl, 50 mM ammonium sulfate, pH 6.5.

Mitogenic Activity

The mitogenic activities of WT FGF-1 and all mutants were determined through cell proliferation assays using NIH-3T3 mouse embryonic fibroblast cells maintained in tissue culture under specific conditions (37° C. and 7% CO₂). Dulbecco's Modified Essential Medium (DMEM) containing 10% newborn calf serum (NCS), L-Glutamine, and penicillin were used to grow the fibroblast cells. After the cells reached a confluency rate of 80%, they were diluted and an appropriate amount of the cell culture was transferred to a new flask to continue their growth. In a 96-well plate, approximately 4000 cells/80 μL of media were added. The well plate was then incubated for 24 hours in absence and presence of heparin and also with the fixed concentrations of wt and mutant FGF1 proteins in order for the cells to adhere to the bottom of the plate. After the 24-hour time period, the medium was removed, the cells were rinsed with PBS, and the medium was replaced with DMEM with 0.5% NCS starvation medium and incubated for another 18 to 24 hours. A Biotek EL808 microplate reader was used to determine the absorbance of the wells in the well plate at 450 nm.

Limited Trypsin Digestion and Thrombin Cleavage

Limited proteolytic digestion experiments were performed at 37° C. using trypsin (Sigma) as the proteolytic enzyme in the absence and presence of sucrose octasulfate. Proteolytic digestions were carried out by adding 100 units of enzyme to 1 mL of 37.504 FGF-1. The reaction was stopped at 5-minute time intervals by the addition of saturated trichloroacetic acid (TCA). The degree of proteolytic cleavage was estimated by densitometry of the intensity of the 16 kDa band corresponding to the undigested FGF-1. The intensity of the FGF-1 band prior to the addition of trypsin was used as 100% undigested protein.

Differential Scanning Calorimetry

Differential Scanning calorimetry was performed on an N-DSC III Differential Scanning calorimeter. The concentration of protein was 75 uM in 10 mM phosphate, 100 mM NaCl, 50 mM ammonium sulfate, pH 6.5. Scans were performed from 10-90° C. with 1° C./min ramping temperature. Data was plotted using CpCalc Version 2.2.0.10 software supplied by the manufacturer.

2D ¹H-¹⁵N HSQC NMR Spectroscopy

NMR experiments were performed on a Bruker Avance DMX-700 MHz spectrometer equipped with a 5 mm inverse cryoprobe at 25° C. ¹⁵N labeled protein samples were prepared in 10 mM phosphate, 100 mM sodium chloride, 50 mM ammonium sulfate, pH 6.5 added with 10% D20. The data was analyzed using XWINNMR 3.5 software supplied by Bruker.

Isothermal Titration Calorimetry of the FGF-1 Mutants with SOS

The binding of FGF-1(wt) and the mutants with SOS was monitored by measuring the heat changes during the titration of SOS into the cell containing FGF-1 using a Microcal VP-ITC MicroCalorimeter. For WT hFGF-1, and mutant proteins were in the cell and at a concentration of 0.05 mM in a buffer of 10 mM phosphate, 100 mM NaCl, 50 mM ammonium sulfate, pH 6.5. The SOS in the syringe was at a concentration of 0.5 mM in a buffer of 10 mM phosphate buffer (pH 6.5) containing 100 mM NaCl and 50 mM ammonium sulfate. The volume of SOS injections was 6 uL and injections were continued every 5 minutes for a total of 49 injections at 25° C. The data was analyzed using Origin scientific plotting software.

Western Blot Analysis of Phosphaorylated Forms of Akt and ERK Proteins

NIH 3T3 cells grown in DMEM and after starvation the cells were added with fixed concentration of buffer blank, WT and mutant FGF1 proteins. Cells were harvested by centrifugation at 1500×g and lysed. Samples were resolved on SDS-PAGE and the proteins were transferred on to a nitrocellulose membrane. Phosphorylated Akt and ERK proteins were detected by using specific polyclonal antibodies. Densitometry of the bands was plotted as the relative difference in expression of the phosphorylated protein.

Endothelial Migration/Wound Healing Assay

A line was drawn at the bottom of 6 well plates for indication of where to make the scratches and for orientation during microscopy. Murine 2H11 endothelial cells were cultured to near confluency (˜70-90%) in 6 well dishes over night, then rinsed and put in starvation media (0.5%-0.1% serum) for 24 hrs. Using a sterile 200 μl or 1000 μl pipette tip, three scratches were made through the cells perpendicular to the previously drawn line. The cells were rinsed gently with serum free medium and then 1.5 ml medium with 2, 20, or 40 μg/ml of wild type or mutant FGFs was added. Pictures were taken at 0, 6, 12, 24 and 48 hrs (phase contrast-10×) of the scratches and the width of the scratch was measured (at 6 and 12 hrs fresh medium containing FGFs was also re-added). For data analysis, each scratch diameter was subtracted from the baseline diameter and the percent that the diameter of the scratch decreased was calculated.

Results

The 3D structure of FGF-1 shows residues important for binding to the fibroblast growth factor receptor molecule (FGFR1) and residues that binds to heparin. See, e.g., FIG. 4. It is known that FGF-1 shows a stretch of positive amino acids at the C-terminus side of the molecule that is important for binding to heparin. One of those residues R136 is critical and also sensitive to secondary proteolytic degradation by thrombin. Therefore, using site-directed mutagenesis protocol, wtFGF-1 was subjected to mutation at position 136 whereby residue arginine was replaced to glutamic acid. This new recombinant vector containing nucleic acid sequence of mutated FGF-1 codes for mutated form of FGF-1 with glutamic acid instead of arginine at position 136. The translated protein product with the above substitution showed an increased resistance to degradation by thrombin and an enhanced biological activity compared to the wtFGF-1. Wound healing assay using Swiss 3T3 cells showed a striking difference between wtFGF-1 and R136E. Cells treated with wtFGF-1 exhibited epithelioid cobblestone-like morphology even at the migrating front of monolayer wounds, while mutant FGF-1R136E induced spindle-like or triangular cell shapes, and cells migrated individually, not as a defined front. Because of different characters of wound closing, we assessed cell migration by number of cells, which reached median areas of the wounds. We found that by 40 hours after wounding the presence of cells in the central wound areas was at least twice higher with mutant FGF-1R136E than with wtFGF-1.

To generate the mutation, following bases were changed in the wtFGF-1

1.
(SEQ ID NO: 13)
5′TGCAAACGCGGTCCTCGGACTCACTATGGCCAG 3′-wtFGF
2.
(SEQ ID NO: 14)
5′TGCAAACGCGGTCCTGAGACTCACTATGGCCAG 3′-R136E
3.
(SEQ ID NO: 15)
5′TGCAAACGCGGTCCTAAGACTCACTATGGCCAG 3′-R136K

Effects of Mutations on Mitogenic Activity of FGF-1

Mitogenic assay was performed to determine the cell proliferation activity of wild-type FGF-1 and mutant proteins. FIG. 5 shows the activity of WT FGF-1 and each mutant as a measure of number of cells multiplied. Although some mutants were less active, all showed some degree of activity. Interestingly, R136E showed higher activity compared to WT FGF-1 or any of the other mutants tested. This could be a result of the increased stability R136E mutation conferred to the FGF1 molecule. R136E mutant showed an increased thermal stability, resistance to proteolytic digestion. Despite the decrease in heparin-binding affinity, R136E is most active, suggesting heparin-binding may not be a requirement for FGF-1 signaling.

Effects of Mutations on Proteolytic Digestion of FGF1

To determine the effects of the charge-reversal mutations on solvent exposure and stability against enzymatic cleavage, resistance to trypsin and thrombin proteases was performed both in the absence and presence of SOS at 37° C. FIG. 6 and FIG. 7 shows the percentage of undigested hFGF-1 versus time for wt hFGF-1, R136K, R136E in the absence and presence of SOS. The degree of proteolytic cleavage was determined by densitometric analysis of the 16 kDa band corresponding to the undigested hFGF-1; the intensity of the hFGF-1 band prior to the addition of trypsin corresponds to 100% undigested protein. In the absence of SOS, ˜40% of the WT hFGF-1 remains intact after incubation with trypsin for 60 minutes. FGF1-R136E mutant was significantly more stable in the presence of trypsin and thrombin than WT: after 1 hour, ˜80% of R.136E remained uncleaved compared to ˜40% of WT showed an increased protease resistance compared to wt and R136K mutant. Also R136E mutant showed no significant difference in the absence and presence of heparin this supports the mitogenic data where the R136E showed higher activity in the absence of heparin.

Effects of Mutations on Thermal Stability of FGF1

Differential Scanning calorimetry (DSC) directly measures the thermal stability of a protein. DSC was performed to determine the melting temperature (T_m) of WT hFGF-1 and mutation alone and in the presence of sucrose octasulfate (SOS). The changes in T_m values are reflective of the effect the mutant has on the thermal stability of the protein as well as the effect the mutation has on the SOS binding affinity. The T_m value of WT hFGF-1 was 49.3° C. in the absence of SOS; this value increased to 61.12° C. when 0.5 mM SOS was added, giving a ΔT_m, of 11.9° C. (FIG. 8). Compared to R136K, R136E mutant showed an increase in thermal stability of over 6° C. compared to wtFGF1 in the absence of heparin. However, the T_m values were similar in the presence of SOS resulting in a lower ΔT_m, value of R136E (5.2° C.), reflective of some loss of SOS/heaprin affinity observed in ITC FIG. 8).

Although it was originally expected that inserting a negatively charged amino acid among several positively charged residues would result in increased stability (due to the possible formation of salt bridges), the DSC results have shown the opposite, with the exception of R136E. The reason for this observation may be due to the distances between the inserted negative charge and the surrounding positive residues. Although the formation of a salt bridge increases stability, the oppositely charged amino acids must be within 5 Å of each other, otherwise long-range electrostatic interactions can occur, which are often destabilizing. (Kumar and Nussinov 2002). For instance, in a study of 22 ion pairs in 14 NMR conformer ensembles for 11 nonhomologous proteins, 92.1% of salt bridges, 68% of N—O bridges, and only 33% of longer-range ion pairs were stabilizing, likely because close-range electrostatic interactions have a more favorable geometrical orientation and electrostatic strength (Kumar and Nussinov 2001; Kumar and Nussinov 2002). Mutation of R136 to glutamic acid would result in the formation of salt bridge which might contribute to the increased stability.

Effect of Muattions on the Amino Acid Perturbrations in FGF1

NMR spectroscopy is useful for the determination of residues involved in ligand binding as well as the conformational changes of a protein caused by a mutation. 2D ¹H-¹⁵N HSQC NMR spectra were acquired for WT hFGF-1 and all mutants in the absence and presence of SOS. An overlay of WT hFGF-1 with each mutant reveals the residues shifted due to the mutation (FIG. 9), while the overlay of the spectra for each mutant in the presence of SOS shows the residues that shift upon binding to SOS. R136E (data not shown) have a perturbation plot similar to that of WT overlaid with WT in the presence of SOS. This indicates that the binding between R133E and R136E to SOS is not greatly affected by the mutation.

Effects of Mutations on Heparin Binding Affinity of FGF1

The method used to study the affinities of WT hFGF-1 and each mutant for heparin and the heparin analog, sucrose octasulfate (SOS), was Isothermal Titration calorimetry (ITC). ITC is a method used to directly evaluate the affinity and enthalpy of binding reactions. The dissociation constant (K_d) obtained from the isotherm represents the affinity of the protein to SOS or heparin; the binding event for both found to be exothermic. The K_d value for wt hFGF-1 was 2.6 μM for SOS (FIG. 10A). R136E showed a significant decrease in binding with K_d values of 72.4 μM (FIG. 10C) for SOS and 16.72 μM for heparin (data not shown) confirms the charge reversal effect on heparin binding affinity. R136K showed a K_d for SOS similar to that of WT (FIG. 10B) as the basic amino acid arginine replaced with another basic amino acid lysine. This indicates that, although each mutant falls within the heparin binding region of hFGF-1, they each contribute differently to the binding.

Effect of Mutations on the Phosphorylation of Akt and ERK Proteins

FGF1 is known to activate various cellular pathways upon binding to FGF receptor. Two important pathways among them Akt and ERK1/2 are critical for apoptosis and cell differentiation. FGF1-R136 mutant showed a marginal difference in phosphorylation of Akt and four fold increase in the ERK protein phosphorylation compared to that of WT. See FIG. 11. This confirms that the increased stability of the protein has a drastic effect on the cell proliferation property of this mutant protein.

Effect of Mutations on Wound Healing Property of FGF1

The wound healing potency of the wild-type FGF-1 was then compared to R136E. See FIG. 12. Results from this assay indicate that R136E has a pronounced (˜two fold) effect compared to that of WT in inducing endothelial cell proliferation and migration to the site of wound at a much larger extent.

CONCLUSIONS

Heparin is proposed to function in two ways: it binds to FGFs and FGFRs, which is a requirement for the FGF/FGFR signaling complex formation, or it functions to stabilize the complex, thereby acting only to facilitate the formation of the FGF/FGFR complex. In 1991, Yayon et al. showed that Chinese hamster ovary cells expressing FGFR1, but lacking in the expression of cell-surface heparin-like glycosaminoglycans (HLGAGs), could not bind to FGF-2. However, upon addition of heparin or heparin sulfate, binding did occur. This led to the conclusion that heparin is a requirement for FGF/FGFR complex formation. However, the question remained unanswered as to whether the removal of heparin diminished binding because FGF or FGFR was destabilized, or because, without heparin, it is impossible for FGF and FGFR to come together to form the signaling complex. A more recent study involving the mutant K118E (K132E) of FGF-1 showed that this mutant had much lower activity than WT FGF-1. The study, however, went on to show that when this mutant was stabilized by additional mutations, mitogenic activity was restored. In the current study, several mutations were constructed in order to evaluate how stability versus heparin-binding affinity affects mitogenic activity of FGF-1. Despite each charge reversal mutation falling within the heparin-binding region of hFGF-1, the effects of each mutation on stability and heparin affinity differed greatly. Although arginine has been shown to bind heparin more tenaciously, the results of trypsin digestion, DSC, ITC and 2D ¹H-¹⁵N HSQC NMR indicate that the loss in heparin binding for R136E is less significant than that of WT. Based on all the above listed biophysical, biochemical and cell based activity assays, we found that R136E mutant of FGF1 exhibited an increased thermal, protease stability with an enhanced bioactivity.

Claims

1. A FGF polypeptide comprising a polypeptide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, a polypeptide having 80% sequence identity to at least one of SEQ ID NOs: 4, 6, 10 or 12 and comprising amino acid residue of 122 of SEQ ID NO: 4 or SEQ ID NO: 6 or amino acid residue 138 of SEQ ID NO: 10 or SEQ ID NO: 12, fragments comprising amino acid residue 122 of SEQ ID NO: 4 or SEQ ID NO: 6, fragments comprising amino acid residue 138 of SEQ ID NO: 10 or SEQ ID NO: 12, a fragment of SEQ ID NOs: 4 or 6 comprising amino acids 112-128 and a fragment of SEQ ID NOs: 10 or 12 comprising amino acids 128-144.

2. The FGF polypeptide of claim 1, wherein the FGF polypeptide comprises SEQ ID NO: 4 or a polypeptide having 80% sequence identity to SEQ ID NO: 4 and comprising amino acid residue of 122 of SEQ ID NO: 4.

3. The FGF polypeptide of claim 2, wherein the FGF polypeptide comprises SEQ ID NO: 4.

4. A fusion protein comprising the composition of claim 1 and a membrane permeable peptide.

5. A polynucleotide encoding the polypeptide of claim 1.

6. The polynucleotide of claim 5, wherein the polynucleotide is codon-optimized for expression in a cell.

7. The polynucleotide of claim 6, wherein the cell is a prokaryotic cell.

8. The polynucleotide of claim 6, wherein the cell is mammalian cell.

9. The polynucleotide of claim 5, wherein the polynucleotide is DNA.

10. The polynucleotide of claim 5, wherein the polynucleotide comprises SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 11.

11. A DNA construct comprising a promoter operably linked to the polynucleotides of claim 5.

12. The DNA construct of claim 11, wherein the promoter is a heterologous promoter.

13. The DNA construct of claim 11, wherein the promoter is a mammalian promoter.

14. A vector comprising the polynucleotides of claim 5.

15. The vector of claim 14, wherein the vector comprises a plasmid.

16. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutical carrier.

17. A hydrogel comprising the composition of claim 1.

18. A method of treating a condition comprising administering the composition of claim 1 to a subject in an amount effective to treat the condition, wherein the condition is selected from the group consisting of a wound, Type 1 diabetes, Type 2 diabetes, obesity, internal injuries, a cardiovascular disorder, a cosmetic condition, critical limb ischemia, a nerve injury, a burn, hair loss, a retinal disorder, a muscular disorder, an arterial disease, an age related disorder, organ or tissue damage, osteoporosis, a digestive tract ulcer, ulcerative colitis, a scar, an energy homeostasis disorder, osteoarthritis, and acute renal failure.

19. The method of claim 18, wherein the condition is a wound.

20. (canceled)

21. The method of claim 18, wherein the mammal is a human.