Engineered FGF1 / FGF2 fused heterodimer compositions and methods of use thereof

Abstract

The present invention provides fusion proteins comprising a fibroblast growth factor 1 (FGF1) polypeptide and a fibroblast growth factor 2 (FGF2) polypeptide. Pharmaceutical compositions and wound dressings comprising the fusion proteins, polynucleotides and constructs encoding the fusion proteins, and methods of using the fusion proteins to treat a condition in a subject are also provided.

Description

[0001] ENGINEERED FGF1 / FGF2 FUSED HETERODIMER COMPOSITIONS AND METHODS OF USE THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 519,501, filed August 14, 2023, the contents of which are incorporated by reference in their entireties.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant P20 GM139768 awarded by the National Institutes of Health, grant DE-FG02-01ER15161 awarded by the Department of Energy, and grant 1945465 awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007] This application includes a sequence listing in XML format titled “169879.00169_ST26. xml”, which is 9,437 bytes in size and was created on August 6, 2024. The sequence listing is electronically submitted with this application via Patent Center and is incorporated herein by reference in its entirety.

[0008] INTRODUCTION

[0009] Wound healing is a complex process that involves the coordinated efforts of several cell types, including keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets. It involves migration, infiltration, proliferation, and differentiation of cells; and it leads to an inflammatory response, the formation of new tissue, and ultimately wound closure. This complex process is executed and regulated by an equally complex signaling network involving numerous growth factors, cytokines, and chemokines. Of particular importance in this signaling network is the fibroblast growth factor (FGF) family (Behm, Babilas, Landthaler, & Schreml, 2012; Yamakawa & Hayashida, 2019).

[0010] FGF signaling regulates the growth and replication of new cells throughout the human body. It is mediated by fibroblast growth factor receptors (FGFR 1-4), and it regulates a broad spectrum of biological activities, including cellular proliferation, survival, migration, and differentiation (Hui, Jin, Li, Liu, & Wang, 2018; Ornitz & Itoh, 2015).

[0011] The FGF family consists of 23 proteins that are categorized into 7 subfamilies. The FGF1 subfamily is the most important and well-studied FGF subfamily and it consists of human fibroblast growth factor 1 (hFGFl) and human fibroblast growth factor 2 (hFGF2). hFGFl and hFGF2 are both mitotic proteins that play significant roles in cell development and survival, with hFGFl promoting cell proliferation and hFGF2 promoting angiogenesis. Both hFGFl and hFGF2 are critical for wound healing. Application of recombinant hFGFl and hFGF2 to a wound has been shown to induce cell development, migration, proliferation, and differentiation, allowing for controlled external modulation of the healing process (Hui et al., 2018; Nurkesh, Jaguparov, Jimi, & Saparov, 2020; Yamakawa & Hayashida, 2019).

[0012] Unfortunately, previous efforts to use these proteins to treat chronic wounds have not been successful, most likely due to the rapid degradation of the proteins at the wound site. While hFGFl and hFGF2 are both promising therapeutic agents for wound treatment, their use is limited by low thermodynamic stability under physiological temperatures, heparin dependency, and the presence of proteases at the wound site. Thus, there is a need in the art for stable, protease-resistant FGF -based therapeutics.

[0013] SUMMARY

[0014] In a first aspect, the present invention provides fusion proteins comprising: (a) a fibroblast growth factor 1 (FGF1) polypeptide; (b) a fibroblast growth factor 2 (FGF2) polypeptide; and (c) a linker peptide that links the FGF1 polypeptide to the FGF2 polypeptide. In some embodiments, the FGF1 polypeptide has at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 and the FGF2 polypeptide has at least 90% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0015] In a second aspect, the present invention provides polynucleotides encoding the fusion protein disclosed herein.

[0016] In a third aspect, the present invention provides constructs comprising the polynucleotide disclosed herein operably linked to a promoter.

[0017] In a fourth aspect, the present invention provides pharmaceutical compositions comprising the fusion protein disclosed herein and a pharmaceutically acceptable carrier. In a fifth aspect, the present invention provides wound dressings comprising the fusion protein disclosed herein.

[0018] In a sixth aspect, the present invention provides methods for treating a condition in a subject in need thereof using the compositions disclosed herein.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1A shows temperature induced unfolding of hFGFl, hFGF2, FGF1 / 2 heterodimer PM FGF1 / 2 heterodimer, and super_FGFl / 2 heterodimer in the absence of heparin.

[0021] FIG. IB shows temperature induced unfolding of hFGFl, hFGF2, FGF1 / 2 heterodimer PM FGF1 / 2 heterodimer, and super_FGFl / 2 heterodimer in the presence of heparin.

[0022] FIG. 2A shows urea induced unfolding of hFGFl, hFGF2, FGF1 / 2 heterodimer PM FGF1 / 2 heterodimer, and super_FGFl / 2 heterodimer in the absence of heparin.

[0023] FIG. 2B shows urea induced unfolding of hFGFl, hFGF2, FGF1 / 2 heterodimer PM FGF1 / 2 heterodimer, and super_FGFl / 2 heterodimer in the presence of heparin.

[0024] FIG. 3 shows densitometric data from the limited trypsin digestion of hFGFl (A), hFGF2 (B), FGF1 / 2 heterodimer (C), PM FGF1 / 2 heterodimer (D), and super_FGFl / 2 heterodimer (E) obtained from SDS-PAGE gels.

[0025] FIG. 4 shows densitometric data of the limited thrombin digestion of FGF1 (A), FGF2 (B), FGF1 / 2 heterodimer (C), PM FGF1 / 2 heterodimer (D), and super_FGFl / 2 heterodimer (E) obtained from SDS-PAGE gels.

[0026] FIG. 5 shows isothermograms representing titration of hFGFl, hFGF2, FGF1 / 2 heterodimer PM FGF 1 / 2 heterodimer, and super FGF 1 / 2 heterodimer with heparin.

[0027] FIG. 6 shows the results of a cell proliferation assay for hFGFl, hFGF2, FGF 1 / 2 heterodimer, PM_FGFl / 2 heterodimer, and super_FGFl / 2 heterodimer. Actively growing NIH- 3T3 cells were adapted in serum deprivation followed with addition of different concentrations of mitogen. Cell number was quantified using the Giosensor™ cAMP assay.

[0028] FIG. 7 shows cartoon representations of the FGF 1 / 2 heterodimer at the beginning (t = 0 ps) and end (t = 2.4 ps) of a simulation. FGF1 (blue), the heparin-binding region of FGF1 (green), FGF2 (red), the heparin-binding region of FGF2 (gold), and the linker (grey) are shown.

[0029] FIG. 8 shows a cartoon representation of the super_FGFl / 2 heterodimer with the mutated residues highlighted. FGF1 (blue), FGF2 (red), and the linker (grey) are shown. FIG. 9 shows unique salt-bridge interactions that facilitate the formation of FGF1 -FGF2 dimer interface. R281 (gray purple) of FGF2 (red), which is located in the heparin-binding pocket (gold), forms a salt-bridge with D68 (light blue) or D70 (hot pink) of FGF1 (blue). E82 (light green) of the FGF1 (blue) forms a salt-bridge with KI 87 (yellow) or K296 (orange) of FGF2 (red). D28 (light pink) in FGFl(blue) has a salt-bridge interaction with residue R205 (dark purple) of FGF2 (red). D68 (light blue) of FGFl(blue) forms a salt-bridge with K296 of FGF2 (red).

[0030] FIG. 10 shows a root mean square deviation (RMSD) time series for the FGF1 / 2 heterodimer. The RMSD for the initial conformation is shown on the left, and the RMSD for the final conformation is shown on the right.

[0031] FIG. 11 shows an internal RMSD time series for the individual hFGFl and hFGF2 subunits.

[0032] FIG. 12 shows an internal RMSD time series for the heparin-binding regions of hFGFl and hFGF2.

[0033] FIG. 13 shows root mean square fluctuation (RMSF) estimations for the individual hFGFl and hFGF2 subunits.

[0034] FIG. 14 shows the FGF1 / 2 heterodimer release profile for poly(ethylene glycol)-co- poly(acrylic acid) (PEG-co-PAA) hydrogel as a function of time based on fluorescence spectroscopy ( / .ex, = 280 nm; emv = 308 nm).

[0035] FIG. 15 shows the results of a cell proliferation assay for protein (FGF1 / 2 heterodimer and shFGFl) released from the PEG-co-PAA hydrogel.

[0036] DETAILED DESCRIPTION

[0037] The present invention provides fusion proteins comprising a fibroblast growth factor 1 (FGF1) polypeptide and a fibroblast growth factor 2 (FGF2) polypeptide. Pharmaceutical compositions and wound dressings comprising the fusion proteins, polynucleotides and constructs encoding the fusion proteins, and methods of using the fusion proteins to treat a condition in a subject are also provided.

[0038] Fusion proteins:

[0039] As is described in the Examples, the inventors have generated FGF1-FGF2 fusion proteins that have increased thermal stability, chemical stability (Cm), resistance to proteolytic cleavage, and mitogenic activity as compared to the individual proteins FGF1 and FGF2. The fusion proteins are heterodimers comprising an FGF1 protein linked to an FGF2 protein via a flexible linker peptide. Thus, in a first aspect, the present invention provides fusion proteins comprising: (a) a FGF1 polypeptide; (b) a FGF2 polypeptide; and (c) a linker peptide that links the FGF1 polypeptide to the FGF2 polypeptide.

[0040] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to refer to polymers of amino acids connected by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. Proteins may be modified to include non-amino acid moieties (e.g., via acetylation, glycosylation) and may include amino acid analogs.

[0041] A “fusion protein” is an engineered protein created by joining together two or more genes that originally encoded separate proteins, such that the two or more genes are transcribed and translated as a single unit to produce a single protein. The fusion proteins of the present invention are “heterodimers,” i.e., dimers comprising two similar but non-identical subunits.

[0042] Fibroblast growth factor 1 (FGF1) and fibroblast growth factor 2 (FGF2) are mitotic proteins that both play a critical role in wound healing. As used herein, the terms “hFGFl” and “hFGF2” refer to human forms of FGF1 and FGF2, respectively, and the terms “FGF1” and “FGF2” refer to all forms (i.e., both human and non-human) of FGF1 and FGF2, respectfully. The FGF1 and FGF2 polypeptides used in the fusion proteins of the present invention may be from any organism. However, in preferred embodiments, the FGF1 and FGF2 polypeptides are derived from hFGFl and hFGF2.

[0043] In some embodiments, the FGF1 polypeptide comprises the wild-type hFGFl protein of SEQ ID NO: 1. As used herein the term “wild-type” refers to the typical form of a protein that occurs in nature. In other embodiments, the FGF1 polypeptide comprises an hFGFl variant. As used herein, the term “variant” refers to a protein that has an amino acid sequence that differs from a reference protein, such as a wild-type protein. A variant may have one or more amino acid insertions, deletions, or substitutions relative to the reference protein or may be a fragment of the reference protein.

[0044] As used herein, a “fragment” is a portion of a polypeptide that is identical in sequence to, but shorter in length than, the full-length polypeptide. For example, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous amino acid residues of a full-length polypeptide. Fragments may be preferentially selected from certain regions of a polypeptide. A fragment may include an N-terminal truncation, a C-terminal truncation, or both an N-terminal and C-terminal truncation relative to the full-length polypeptide. Preferably, polypeptide fragments used with the present invention are functional fragments. As used herein, a “functional fragment” is a fragment that retains at least 20%, 40%, 60%, 80%, or 100% of the biological (e.g., mitogenic) activity of the corresponding full-length polypeptide.

[0045] The hFGFl variants used in the fusion protein may have at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to the wild-type hFGFl protein of SEQ ID NO: 1, and may comprise one or more substitution mutation relative to SEQ ID NO: 1. A “substitution mutation” or “substitution” is a replacement of one amino acid in a reference sequence with a different amino acid. A substitution may be conversative (i.e., a replacement with an amino acid that has similar properties) or radical (i.e., a replacement with an amino acid that has different properties). For example, an hFGFl variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions relative to SEQ ID NO: 1.

[0046] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window. The aligned sequences may comprise additions or deletions (i.e., gaps) relative to each other for optimal alignment. The percentage is calculated by determining the number of matched positions at which an identical nucleic acid base or amino acid residue occurs in both sequences, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100. Protein and nucleic acid sequence identities can be evaluated using the Basic Local Alignment Search Tool ("BLAST"), which is well known in the art (Karlin and Altschul, Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc. Natl. Acad. Sci. USA (1990) 87: 2267-2268; Altschul et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. (1997) 25: 3389-3402). The BLAST programs identify homologous sequences by identifying similar segments between a query amino acid or nucleic acid sequence and a test sequence, which is preferably obtained from a protein or nucleic acid sequence database. The BLAST programs can be used with the default parameters or with modified parameters provided by the user.

[0047] In some embodiments, the FGF1 polypeptide comprises an engineered hFGFl variant. In previous work described in U.S. Patent Publication No. US 2019 / 0284252, which is hereby incorporated by reference in its entirety, the inventors generated an engineered hFGFl variant with enhanced thermal stability, resistance to proteolytic activity, and heparin-independent bioactivity as compared to wild-type hFGFl. This hFGFl variant comprises the mutations Q41P, S48L, H94S, KI 13N, and R123E relative to SEQ ID NO: 1 and is referred to herein as “super human acidic fibroblast growth factor 1 (super hFGFl)”. The amino acid sequence of super hFGFl is provided as SEQ ID NO: 2.

[0048] In the Examples, the inventors demonstrate that substituting super hFGFl for wild-type hFGFl in their FGF 1 / 2 heterodimer further increases the stability and mitogenic activity of the heterodimer. Thus, in some embodiments, the FGF1 polypeptide comprises at least one substitution mutation at a residue that is mutated in super hFGFl, namely residues Q41, S48, H94, KI 13, and R123. In these embodiments, (a) the substitution at residue Q41 may be selected from Q41P, Q41F, Q41M, Q41Y, Q41W, Q41I, Q41L, Q41V, and Q41A; (b) the substitution at residue S48 may be selected from S48L, S48A, S48V, S48P, S48T, S48M, S48I, S48F, S48Y, and S48W; (c) the substitution at residue H94 may be selected from H94S, H94T, H94K, H94R, and H94Y; (d) the substitution at residue KI 13 may be selected from KI 13N, KI 13Q, K113S, KI 13T, KI 13R, and KI 13Y; and / or (e) the substitution at residue R123 may be selected from R123E and R123D. The hFGFl variant may comprise substitutions at 1, 2, 3, 4, or all 5 of these residues. In some embodiments, the hFGFl variant comprises one or more of the specific substitution mutations made in super hFGFl, namely Q41P, S48L, H94S, KI 13N, and R123E. The hFGFl variant may comprise 1, 2, 3, 4 or all 5 of the specific substitution mutations made in super hFGFl. In some embodiments, the hFGFl variant comprises (a) the super hFGFl protein of SEQ ID NO: 2 or (b) a polypeptide having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity SEQ ID NO: 2.

[0049] In some embodiments, the FGF2 polypeptide comprises the wild-type hFGF2 protein of SEQ ID NO: 3 or a variant thereof. The 11FGF2 variant may have at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity SEQ ID NO: 3. The hFGF2 variant may comprise one or more substitution mutation relative to SEQ ID NO: 3. For example, the hFGF2 variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions relative to SEQ ID NO: 3.

[0050] In some embodiments, the FGF2 polypeptide comprises an engineered hFGF2 variant. Based on a pairwise sequence alignment and a comparison of the 3D structures of hFGFl and hFGF2, the inventors have generated an hFGF2 variant with substitution mutations that mimic the mutations made in super hFGFl . This hFGF2 variant comprises the mutations Q65I, C78S, N111G, K128N, and K138E relative to SEQ ID NO: 3 and is referred to herein as “super human acidic fibroblast growth factor 2 (super hFGF2)”. The amino acid sequence of super hFGF2 is provided as SEQ ID NO: 4.

[0051] In the Examples, the inventors demonstrate that substituting super hFGF2 for wild-type hFGF2 in their FGF1 / 2 heterodimer further increases the stability and mitogenic activity of the heterodimer. Thus, in some embodiments, the FGF2 polypeptide comprises at least one substitution mutation at a residue that is mutated in super hFGF2, namely residues Q65, C78, N111, K128, and K138. In these embodiments, (a) the substitution at residue Q65 may be selected from Q65P, Q65F, Q65M, Q65Y, Q65W, Q65I, Q65L, Q65V, and Q65A; (b) the substitution at residue C78 may be selected from C78S and C78A; (c) the substitution at N111 may be selected from N111G, N11 IS, N11 IQ, N11 IT, and N111Y; (d) the substitution at residue K128 may be selected from K128N, K128Q, K128S, K128T, K128R, and K128Y; and / or (e) the substitution at residue K138 may be selected from K138E and K138D. The hFGF2 variant may comprise substitutions at 1, 2, 3, 4, or all 5 of these residues. In some embodiments, the hFGF2 variant comprises one or more of the specific substitution mutations made in super hFGF2, namely Q65I, C78S, N111G, K128N, and K138E. The hFGF2 variant may comprise 1, 2, 3, 4 or all 5 of the specific substitution mutations made in super hFGF2. In some embodiments, the hFGF2 variant comprises (a) the super hFGF2 protein of SEQ ID NO: 4 or (b) a polypeptide having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity SEQ ID NO: 4.

[0052] The FGF1 polypeptides and FGF2 polypeptides described herein may include additional modifications (i.e., substitutions, insertions, deletions) other than those explicitly described. Modifications based on the alternative amino acid residues that occur at a corresponding position in an FGF homolog from another species are expected to conserve protein function. Accordingly, the additional modifications may be selected using a sequence alignment of a particular FGF polypeptide with the corresponding FGF polypeptide from another species.

[0053] The fusion proteins of the present invention comprise a linker peptide that connects the FGF1 polypeptide to the FGF2 polypeptide. As used herein, the term “linker peptide” refers to a peptide that links two segments of a fusion protein together via peptide bonds, allowing the two segments to be translated as a single polypeptide. A linker peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues. A linker peptide may comprise any amino acid sequence that does not substantially hinder the function of the two polypeptide segments that it links. In some embodiments, the linker is flexible such that it has no required fixed structure in solution and the polypeptide segments that it links are free to move relative to one another. Preferred amino acid residues for flexible linker sequences include glycine, alanine, serine, threonine, lysine, arginine, glutamine, and glutamic acid. In the Examples, the inventors utilized the linker peptide of SEQ ID NO: 8, which comprises 12 glycine residues. Thus, in some embodiments, the linker peptide comprises SEQ ID NO: 8 or a polypeptide having at least 90%, 92%, 94%, 95%, 96%, 98%, or 99% identity to SEQ ID NO: 8.

[0054] In the Examples, the inventors identified interactions between the FGF1 and FGF2 segments of their fusion proteins that result in the formation of a dimer interface. Specifically, two salt bridge interactions are formed between negatively charged residues (ASP68 or ASP70) in the FGF1 segment and a positively charged residue (ARG281) located in the heparin binding region of the FGF2 segment. The inventors hypothesize that these interactions may be responsible for the increased stability of the FGF1-FGF2 fusion proteins as compared to the individual proteins FGF1 and FGF2. Thus, in preferred embodiments, the linker peptide allows for the formation of these interactions.

[0055] In the FGF1 / 2 fusion proteins tested in the Examples, the linker peptide links the C- terminus of the FGF1 polypeptide to the N-terminus of the FGF2 polypeptide. Thus, in some embodiments, the components of the fusion protein are arranged in the following order from N- terminus to C-terminus: FGF1 polypeptide-linker peptide-FGF2 polypeptide. In other embodiments, the components of the fusion protein are arranged in the reverse order: FGF2 polypeptide-linker peptide-FGFl polypeptide.

[0056] In the Examples, the inventors generated three FGF1-FGF2 fusion proteins. The first fusion protein, which is referred to herein as simply the “FGF1 / 2 heterodimer” (SEQ ID NO: 5), comprises wild-type hFGFl (SEQ ID NO: 1) linked to wild-type hFGF2 (SEQ ID NO: 3). Thus, in some embodiments, the FGF1 polypeptide comprises SEQ ID NO: 1 and the FGF2 polypeptide comprises SEQ ID NO: 3. In some embodiments, the fusion protein comprises SEQ ID NO: 5. The second, penta-mutant fusion protein, which is referred to herein as the “PM FGF1 / 2 heterodimer” (SEQ ID NO: 6), comprises super hFGFl (SEQ ID NO: 2) linked to wild-type hFGF2 (SEQ ID NO: 3). Thus, in some embodiments, the FGF1 polypeptide comprises SEQ ID NO: 2 and the FGF2 polypeptide comprises SEQ ID NO: 3. In some embodiments, the fusion protein comprises SEQ ID NO: 6. The third, deca-mutant fusion protein, which is referred to herein as the “super FGF 1 / 2 heterodimer” (SEQ ID NO: 7), comprises super FGF1 (SEQ ID NO: 2) linked to super hFGF2 (SEQ ID NO: 4). Thus, in some embodiments, the FGF1 polypeptide comprises SEQ ID NO: 2 and the FGF2 polypeptide comprises SEQ ID NO: 4. In some embodiments, the fusion protein comprises SEQ ID NO: 7.

[0057] In the Examples, the inventors demonstrate that their FGF1-FGF2 fusion proteins have (a) increased thermal stability, (b) increased chemical stability, (c) increased resistance to cleavage by trypsin, (d) increased resistance to cleavage by thrombin, and (e) increased mitogenic activity as compared to the individual proteins FGF1 and FGF2. Thus, in preferred embodiments, the fusion proteins have one or more of these characteristics.

[0058] In some embodiments, the fusion proteins further comprise a protein tag. A “tag” is a peptide that is included in a fusion protein to serve a particular function. Suitable tags for use in the fusion proteins of the present invention include, without limitation, (a) affinity tags for protein purification (e.g., chitin binding protein (CBP), maltose binding protein (MBP), Strep, glutathione-S-transferase (GST), and poly(His) tags), (b) solubilization tags (e.g., thioredoxin (TRX), poly(NANP), MBP, GST), (c) epitope tags for antibody-based detection (e.g., ALFA-tag, V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag and NE-tag), (d) fluorescent tags (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP)) and enzymatic tags (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose-6-phosphatase, acetylcholinesterase) for visual detection, (e) targeting tags that target the fusion proteins to a particular site or tissue type within the body (e.g., fibrin-, thrombin-, fibronectin-, or collagen- binding peptide), and (f) cell penetrating peptides that deliver the fusion proteins into cells.

[0059] The tag may be included on the N-terminus of the fusion protein, on the C-terminus of the fusion protein, between the FGF1 polypeptide and the linker peptide, or between the FGF2 polypeptide and the linker peptide. Optionally, the tag may be separated from the other components of the fusion protein by a protease cleavage site to allow for removal of the tag after it has served its purpose.

[0060] Polynucleotides:

[0061] In a second aspect, the present invention provides polynucleotides encoding a fusion protein disclosed herein. The terms “polynucleotide,” “nucleic acid,” and “oligonucleotide” are used interchangeably to refer a polymer of DNA or RNA. A polynucleotide may be singlestranded or double-stranded and may represent the sense or the antisense strand. A polynucleotide may be synthesized or obtained from a natural source. A polynucleotide may contain natural, non-natural, or altered nucleotides, as well as natural, non-natural, or altered internucleotide linkages (e.g., phosphoroamidate linkages, phosphorothioate linkages).

[0062] The polynucleotides of the present invention may be codon-optimized for expression in a particular cell type. “Codon optimization” is a process used to increase expression of a polynucleotide in a particular host cell by altering the sequence of the polynucleotide to accommodate the codon bias of the host cell. Computer programs for generating codon- optimized sequences for use in a particular host cell are known in the art.

[0063] Constructs:

[0064] In a third aspect, the present invention provides constructs comprising a polynucleotide disclosed herein operably linked to a promoter. As used herein, the term “construct” refers a to recombinant polynucleotide, i.e., a polynucleotide that was formed by combining at least two polynucleotide components from different sources. For example, a construct may comprise the coding region of one gene operably linked to a promoter that is (1) associated with another gene found within the same genome, (2) from the genome of a different species, or (3) synthetic. Constructs can be generated using conventional recombinant DNA methods.

[0065] As used herein, the term “promoter” refers to a DNA sequence that defines where transcription of a polynucleotide begins. RNA polymerase and the necessary transcription factors bind to the promoter to initiate transcription. Promoters are typically located directly upstream (i.e., at the 5' end) of the transcription start site. However, a promoter may also be located at the 3’ end, within a coding region, or within an intron of a gene that it regulates. Promoters may be derived in their entirety from a native or heterologous gene, may be composed of elements derived from multiple regulatory sequences found in nature, or may comprise synthetic DNA. A promoter is “operably linked” to a polynucleotide if the promoter is positioned such that it can affect transcription of the polynucleotide. Suitable promoters for use with the present invention include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters. The promoter may be an animal, plant, bacterial, fungal, or synthetic promoter. In some embodiments, the construct is a vector. The term “vector” refers to a DNA molecule that is used to carry a particular DNA segment into a host cell. Some vectors are capable of autonomous replication in a host cell (e.g., bacterial vectors that include an origin of replication and episomal mammalian vectors). Other vectors can be integrated into the genome of a host cell such that they are replicated along with the host genome (e.g., viral vectors and transposons). Vectors may include heterologous genetic elements that are necessary for propagation of the vector or for expression of an encoded gene product. Vectors may also include a reporter gene or a selectable marker gene. Suitable vectors include plasmids (i.e., circular double-stranded DNA molecules) and mini-chromosomes.

[0066] Pharmaceutical compositions:

[0067] In a fourth aspect, the present invention provides pharmaceutical compositions comprising a fusion protein disclosed herein and a pharmaceutically acceptable carrier. As used herein, the term “carrier” refers to a substance used to deliver an agent to a targeted site within the body. A “pharmaceutically acceptable carrier” is a carrier that is compatible with the other ingredients of the composition and that is not deleterious to the subject to which the composition is to be administered. Pharmaceutically acceptable carriers include, but are not limited to, excipients, diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., thimerosal, benzyl alcohol, parabens), solubilizing agents (e.g., glycerol, polyethylene glycerol), emulsifiers, liposomes, nanoparticles, and adjuvants. Pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, or emulsions.

[0068] In the Examples, the inventors demonstrate that their FGF1 / 2 heterodimer is gradually released from a hydrogel made of the copolymer polyethylene glycol)-co-poly(acrylic acid) (PEG-co-PAA). Thus, in some embodiments, the carrier is a hydrogel. As used herein, the term “hydrogel” refers to a gel in which the liquid component is water. In specific embodiments, the carrier is PEG-co-PAA or a derivative thereof. In some embodiments, the carrier allows for time dependent release of the agent.

[0069] The pharmaceutical compositions of the present invention may further include additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), antioxidants (e.g., ascorbic acid, sodium metabisulfite), bulking substances or tonicity modifiers (e.g., lactose, mannitol).

[0070] Wound dressings: In a fifth aspect, the present invention provides wound dressings comprising a fusion protein disclosed herein. As used here, the term “wound dressing” refers to a product that is applied directly to a wound. The wound dressing may be embedded or implanted with the fusion protein so that the fusion protein is transferred to the wound when the wound dressing is applied. Examples of suitable wound dressings include, but are not limited to, gauze pads, films, bandages, artificial skin, ointments, creams, sprays, and intradermal injections. The wound dressing may include granules, beads, gels, hydrogels, liquids, and / or pastes that comprise the fusion protein.

[0071] Methods of use:

[0072] In a sixth aspect, the present invention provides methods for treating a condition in a subject in need thereof using the compositions disclosed herein. The methods comprise administering a fusion protein, pharmaceutical composition, or wound dressing disclosed herein to the subject.

[0073] As used herein, “treating” describes something that is done to a subject to combat a condition. Treating may involve controlling the condition, lessening its symptoms or complications, slowing its progression, and / or eliminating it. In preferred embodiments, a therapeutically effective amount of the composition is administered to the subject. The term “therapeutically effective amount” refers to an amount sufficient to achieve one or more desirable treatment outcome. For any active agent, a therapeutically effective amount can be estimated initially in cell culture assays or in an animal model.

[0074] The methods of the present invention can be used to treat any condition in which treatment with the fusion proteins described herein is beneficial, e.g., a condition in which it is useful to stimulate cell proliferation and / or angiogenesis. Examples of such conditions include, without limitation, wounds (e.g., internal, external, chronic, acute, wounds related to diabetes), tissue or organ damage, a fractured bone, a gastric ulcer, ulcerative colitis, a scar, a nerve injury, a bum, hair loss (e.g., alopecia), osteoporosis, a cardiovascular disorder, a cosmetic condition (i.e. whitening, wrinkling), ischemia (e.g., critical limb ischemia), a retinal disorder, a muscular disorder, an arterial disease, an age related disorder, a metabolic disorder (e.g., obesity, dyslipidemia, diabetes, insulin resistance, hyperglycemia), osteoarthritis, and acute renal failure.

[0075] As used herein, the term “administering” refers to the introduction of a substance into or onto a subject's body. Methods of administration are well known in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraoral administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, intradermal administration, intrathecal administration, and subcutaneous administration. Administration can be continuous or intermittent. Administration may be performed in a single dose or in multiple doses.

[0076] The “subject” to which the methods are applied may any mammal. Suitable mammals include, but are not limited to, humans, cows, horses, sheep, pigs, goats, rabbits, dogs, cats, bats, mice, and rats. In certain embodiments, the methods may be performed on lab animals (e.g., mice and rats) for research purposes. In other embodiments, the methods are used to treat commercially important farm animals (e.g., cows, horses, pigs, rabbits, goats, sheep, and chickens) or companion animals (e.g., cats and dogs). In a preferred embodiment, the subject is a human.

[0077] 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.

[0078] 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.

[0079] 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 descriptions found in the cited references.

[0080] 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.

[0081] EXAMPLES

[0082] The following example demonstrates the therapeutic potential of their FGF1-FGF2 (FGF1 / 2) heterodimers.

[0083] Development of the FGF1 / 2 heterodimers We have developed and tested three different FGF1 / 2 heterodimers. The first, wild-type heterodimer, which is referred to herein as the “FGF1 / 2 heterodimer” (SEQ ID NO: 5), comprises wild-type hFGFl (SEQ ID NO: 1) linked to wild-type hFGF2 (SEQ ID NO: 3) via a 12-glycine linker. The second, penta-mutant heterodimer, which is referred to herein as the “PM FGF1 / 2 heterodimer” (SEQ ID NO: 6), comprises super hFGFl (SEQ ID NO: 2) linked to wild-type hFGF2 (SEQ ID NO: 3) via a 12-glycine linker. And the third, deca-mutant heterodimer, which is referred to herein as the “super_FGFl / 2 heterodimer” (SEQ ID NO: 7), comprises super FGF1 (SEQ ID NO: 2) linked to super hFGF2 (SEQ ID NO: 4) via a 12-glycine linker.

[0084] Purification of the FGF1 / 2 heterodimers, as well as the individual proteins hFGFl and hFGF2, was performed using heparin Sepharose column chromatography. The proteins were eluted at a higher salt concentration (>1200 mM NaCl). Protein stability

[0085] Protein stability against temperature and chemical denaturants is directly related to protein half-life. Proteins with higher melting temperature (Tm) and chemical denaturation (Cm) values are expected to have increased biological activity for an extended period of time at physiological temperatures. Thus, the several methods were used to compare the stability of the FGF1 / 2 heterodimer, PM FGF1 / 2 heterodimer, and super_FGFl / 2 heterodimer to that of hFGFl and hFGF2.

[0086] Differential scanning calorimetry (DSC) is an analytical technique that measures the amount of heat required to increase the temperature of a sample. DSC can measure several thermodynamic parameters, including heat capacity (Cp; the amount of heat required to raise the temperature of a unit mass of a protein by one degree) and melting temperature (Tm; the midpoint of the unfolding transition of a protein in solution, which corresponds to 50% folded and 50% unfolded). Protein samples were subjected to DSC. The ratios of relative fluorescence intensity at 308 nm and 350 nm were used to calculate the fraction of unfolded protein, and a plot of temperature versus fraction of unfolded protein was used to determine the melting temperature. The results of this experiment demonstrate that the FGF1 / 2 heterodimer (Tm: 52°C) and its variants have much higher thermal stabilities than the individual proteins hFGFl (Tm: 42°C) and hFGF2 (Tm: 38°C). The super_FGFl / 2 heterodimer had the greatest stability, followed by the PM FGF1 / 2 heterodimer, and then the FGF1 / 2 heterodimer. This trend was observed in both the absence of heparin (FIG. 1A) and in the presence of heparin (FIG. IB).

[0087] Urea is a chaotropic agent that disrupts non-covalent interactions within proteins, leading to denaturation. Chemical denaturation (Cm; the midpoint concentration of a denaturant at which a protein is half denatured) can be calculated by monitoring the unfolding of proteins in the presence of increasing concentrations of urea via the ratio of relative fluorescence intensity at 308 nm and 350 nm. The FGF1 / 2 heterodimer and its variants were more resistant to chemical- induced denaturation with urea than both FGF1 alone (Cm: 2. IM) and FGF2 alone (Cm: 2.8M). The super_FGFl / 2 heterodimer (Cm: 3.8M) found to be more resistant than the PM FGF1 / 2 heterodimer (Cm: 3.7M) and the FGF1 / 2 heterodimer (Cm: 2.9M). This trend was observed in both the absence of heparin (FIG. 2A) and in the presence of heparin (FIG. 2B).

[0088] Trypsin is a serine protease that cleaves peptide bonds at the C-terminus of lysine and arginine residues. The results of a trypsin digestion experiment demonstrate that the FGF1 / 2 heterodimer variants were more resistant to trypsin digestion than the individual FGF proteins. The super_FGFl / 2 heterodimer was the most resistant, followed by the PM_FGFl / 2 heterodimer (FIG. 3)

[0089] Thrombin is a serine protease that cleaves between arginine and glycine residues in its specific cleavage site. The results of a thrombin digestion experiment demonstrate that the FGF 1 / 2 heterodimer variants were more resistant to thrombin cleavage than the individual FGF proteins. The super_FGFl / 2 heterodimer was the most resistant, followed by the PM_FGFl / 2 heterodimer (FIG. 4).

[0090] Together, these data suggest that the FGF 1 / 2 heterodimer and variants thereof described herein will have an improved in vivo half-life as compared to the individual proteins hFGFl and hFGF2.

[0091] Heparin binding

[0092] Heparin binding assays using isothermal titration calorimetry (ITC) are a powerful method for studying the interaction between heparin and proteins. ITC was used to directly evaluate the heparin-binding affinity of the FGF 1 / 2 heterodimer and its variants. The resulting isothermograms are shown in FIG. 5 and the dissociation constant (Kd) values are provided in Table 1. A lower Kd value signifies a stronger binding affinity, while a higher Kd value indicates a weaker binding affinity. Table 1. Heparin binding affinity

[0093] Cell proliferation activity

[0094] FGF proteins are known to stimulate the proliferation of a variety of cell types, including fibroblasts, endothelial cells, and epithelial cells. Thus, the cell proliferation that occurs in the presence of an FGF protein can be quantified and used an indicator of that protein’s FGF activity. Cell proliferation activity is significantly higher in the presence of the FGF 1 / 2 heterodimers as compared to in the presence of the individual proteins hFGFl and hFGF2 (FIG. 6), suggesting that the heterodimers have enhanced bioactivity. The increased activity may result from the combination of hFGFl’s induction of cell proliferation and hFGF2’s induction of angiogenesis. (Note: Induction of angiogenesis can result in increased cell proliferation by increasing the supply of oxygen and essential nutrients delivered to tissues.) The super_FGFl / 2 heterodimer induced the greatest proliferation, followed by the PM FGF1 / 2 heterodimer, and then the FGF 1 / 2 heterodimer. Thus, the increased activity of these proteins is correlated with increased stability.

[0095] Computational validation of stability

[0096] Modeller (Eswar et al., 2006) was used to model the FGF 1 / 2 heterodimer using X-ray crystal structures of the hFGFl monomer (PDB: 1RG8, resolution: 1.1 A), hFGF2 monomer (PDB:4OEE, resolution: 1.5 A), and a 12-glycine linker that connects the C-terminus of hFGFl to the N-terminus of hFGF2. VMD (Visual Molecular Dynamics) (Humphrey, Dalke, & Schulten, 1996) was used to visualize the 3-Dimensional structure of the protein. The modeled system was simulated using an all-atom unbiased molecular dynamics (MD) simulation for 2.4 microseconds. In this simulation, the hFGFl and hFGF2 subunits approached each other and formed a dimer interface at the end of the simulation. This interface remained stable for approximately 600 ns. Additionally, the heparin-binding region of both hFGFl (residues 112-128) and hFGF2 (residues 112-128) remained stable and did not relocate from the proteins’ cores during the 2.4 ps simulation (FIG. 7). The positions of the super FGF mutations are highlighted on this model in FIG. 8.

[0097] The formation of FGF1 / 2 heterodimer interface is facilitated by electrostatic interactions between positively charged residues in hFGF2 and negatively charged residues in hFGFl. Two salt bridge interactions are formed between negatively charged residues (ASP68 or ASP70) in FGF1 and a positively charged residue (ARG281) located in the heparin binding region of FGF2. The salt bridges (FIG. 9) occur almost simultaneously after 1.7 ps of simulation time and remain stable for almost 500-700 ns. Hence, these salt bridge interactions appear to be crucial for stabilizing the dimer's new conformation.

[0098] A root-mean-square deviation (RMSD) time series with respect to the initial and final confirmations of the FGF 1 / 2 heterodimer model (FIG. 10) demonstrates the stability of this fusion protein at the last one-third of the simulation time. The model initially has an RMSD value of approximately 25 A from their initial and final conformations, indicating high flexibility at least within the first 1.6 ps of simulation. The RMSD with respect to the final configuration changes from approximately 1 to 3 A within 600 ns (0.6 ps) at the end of simulation and indicates that this new conformation remains stable during this time frame.

[0099] An internal RMSD time series (FIG. 11) can be used to examine the relative stability of each subunit within the heterodimer. Comparing the internal RMSD of the FGF1 and FGF2 subunits reveals that FGF1 is more stable than FGF2 during the 2.4 ps simulation. The internal RMSD value of FGF1 varies between 1 and 2 A, while the value for FGF2 varies between about 1 and 5 A.

[0100] A comparison of the internal RMSD of the heparin-binding regions of the FGF1 and FGF2 subunits to their initial conformation indicates that this region is stable. The values do not change much, and they are less than 1.2 A during the simulation, which indicates that the heparin binding regions are stable and available for heparin binding (FIG. 12).

[0101] These results are also supported by root mean square fluctuation (RMSF) data for each subunit, which was calculated for the Ca atoms of each protein residue. RMSF is a numerical calculation that measures how much a protein residue moves or fluctuates from a reference position over time during a simulation. Based on RMSF plots (FIG. 13), FGF2 is more flexible than FGF1 . Moreover, the heparin-binding region of both FGF1 and FGF2 exhibit lower fluctuation compared to the rest of each protein.

[0102] These computational predictions agree with the experimental data and support the conclusion that the FGF1 / 2 heterodimer has greater stability than hFGFl or hFGF2 alone. Delivery by hydrogels

[0103] A hydrogel made of the copolymer polyethylene glycol)-co-poly(acrylic acid) (PEG-co- PAA) was used for delivery of hFGFl in our previous study (Wang et al., 2019). The release of hFGFl from this hydrogel can be controlled by the salt concentration. We tested the ability of the PEG-co-PAA hydrogel to bind and release the FGF1 / 2 heterodimer. The preliminary results showed that 10% of FGF1 / 2 heterodimer was released from the hydrogel over the first 3 days (FIG. 14), and that the released heterodimer is bioactive (i.e., stimulates cell proliferation) (FIG. 15).

[0104] In a new formulation of the hydrogel, gelatin will be used to increase the spacing of the PAA component in the copolymer, which is expected to improve the release rate of the FGF1 / 2 heterodimer. Gelatin is generally recognized as safe by the FDA and is used as a food ingredient. References

[0105] Agrawal, S., Kumar, V. G., Gundampati, R. K., Moradi, M., & Kumar, T. K. S. (2021).

[0106] Characterization of the structural forces governing the reversibility of the thermal unfolding of the human acidic fibroblast growth factor. Scientific Reports, 11(1). doi: 10.1038 / s41598-021-95050-2

[0107] Kumar, T. K. S., Agrawal, S., Gundampati, R. K., Jayanthi, S., Wang, T. J., Chen, J. Y. (2029) Engineered FGF1 and FGF2 compositions and methods of use thereof (US 2019 / 0284252A1). https: / / patents.google.com / patent / US20190284252Al / en

[0108] Behm, B., Babilas, P., Landthaler, M., & Schreml, S. (2012). Cytokines, chemokines and growth factors in wound healing. Journal of the European Academy of Dermatology and Venereology, 26(7), 812-820. doi: 10.1111 / j .1468-3083.2011.04415.x

[0109] Erzurum, V. Z., Bian, J. F., Husak, V. A., Ellinger, J., Xue, L., Burgess, W. H., & Greisler, H. P. (2003). R136K fibroblast growth factor- 1 mutant induces heparin-independent migration of endothelial cells through fibrin glue. Journal of Vascular Surgery, 37(5), 1075-1081. doi: 10.1067 / mva.2003.177 Eswar, N., Webb, B., Marti-Renom, M. A., Madhusudhan, M. S., Eramian, D , Shen, M.-Y., . Sali, A. (2006). Comparative protein structure modeling using Modeller. Current protocols in bioinformatics, Chapter 5, Unit-5.6. doi: 10.1002 / 0471250953.bi0506sl5

[0110] Guo, Y., Xu, B., Wang, Y., Li, Y., Si, H., Zheng, X., Fan, D. (2019). Dramatic promotion of wound healing using a recombinant human-like collagen and bFGF cross-linked hydrogel by transglutaminase. Journal of Biomaterials Science-Polymer Edition, 30(17), 1591- 1603. doi: 10.1080 / 09205063.2019.1652416

[0111] Hui, Q., Jin, Z., Li, X. K., Liu, C. X., & Wang, X. J. (2018). FGF Family: From Drug Development to Clinical Application. International Journal of Molecular Sciences, 19(7). doi:10.3390 / ijmsl9071875

[0112] Humphrey, W., Dalke, A., & Schulten, K. (1996). VMD: Visual molecular dynamics. Journal of Molecular Graphics & Modelling, 14(1), 33-38. doi: 10.1016 / 0263-7855(96)00018-5

[0113] Kawaguchi, H., Oka, H., Jingushi, S., Izumi, T., Fukunaga, M., Sato, K., Grp, T. (2010). A Local Application of Recombinant Human Fibroblast Growth Factor 2 for Tibial Shaft Fractures: A Randomized, Placebo-Controlled Trial. Journal of Bone and Mineral Research, 25(12), 2459-2467. doi: 10.1002 / jbmr,146

[0114] Kerr, R., Agrawal, S., Maity, S., Koppolu, B., Jayanthi, S., Kumar, G. S., . . . Kumar, T. K. S. (2019). Design of a thrombin resistant human acidic fibroblast growth factor (hFGFl) variant that exhibits enhanced cell proliferation activity. Biochemical and Biophysical Research Communications, 518(2), 191-196. doi: 10.1016 / j.bbrc.2019.08.029

[0115] Liu, T., Dan, W., Dan, N., Liu, X., Liu, X., & Peng, X. (2017). A novel grapheme oxidemodified collagen-chitosan bio-film for controlled growth factor release in wound healing applications. Materials Science & Engineering C-Materials for Biological Applications, 77, 202-211. doi : 10.1016 / j . msec.2017.03.256

[0116] Matsumine, H. (2015). Treatment of skin avulsion injuries with basic fibroblast growth factor. Plastic and reconstructive surgery. Global open, 3(4), e371-e371. doi : 10.1097 / gox.0000000000000341

[0117] Nurkesh, A., Jaguparov, A., Jimi, S., & Saparov, A. (2020). Recent Advances in the Controlled Release of Growth Factors and Cytokines for Improving Cutaneous Wound Healing. Frontiers in Cell and Developmental Biology, 8. doi: 10.3389 / fcell.2020.00638 Ornitz, D. M., & Itoh, N. (2015). The Fibroblast Growth Factor signaling pathway. Wiley Interdisciplinary Reviews-Developmental Biology, 4(3), 215-266. doi: 10.1002 / wdev, 176

[0118] Prudovsky, I. (2021). Cellular Mechanisms of FGF-Stimulated Tissue Repair. Cells, 10(7). doi:10.3390 / cellsl0071830 Wang, T. J., Jones, J. D., Niyonshuti, II, Agrawal, S., Gundampati, R. K., Kumar, T. K. S., Chen, J. Y. (2019). Biocompatible, Injectable Anionic Hydrogels Based on Poly(Oligo Ethylene Glycol Monoacrylate-co- Acrylic Acid) for Protein Delivery. Advanced Therapeutics, 2(9). doi: 10.1002 / adtp.201900092

[0119] Wu, J., Zhu, J., He, C., Xiao, Z., Ye, J., Li, Y., . . . Xiao, J. (2016). Comparative Study of Heparin-Poloxamer Hydrogel Modified bFGF and aFGF for in Vivo Wound Healing

[0120] Efficiency. Acs Applied Materials & Interfaces, 8(29), 18710-18721. doi: 10.1021 / acsami.6b06047

[0121] Yamakawa, S., & Hayashida, K. (2019). Advances in surgical applications of growth factors for wound healing. Burns & Trauma, 7. doi : 10.1186 / s41038-019-0148- 1

Claims

CLAIMSWe claim:

1. A fusion protein comprising: a) a fibroblast growth factor 1 (FGF1) polypeptide comprising at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; b) a fibroblast growth factor 2 (FGF2) polypeptide comprising at least 90% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and c) a linker peptide that links the FGF1 polypeptide to the FGF2 polypeptide.

2. The fusion protein of claim 1, wherein the FGF1 polypeptide comprises SEQ ID NO: 1.

3. The fusion protein of claim 1, wherein the FGF1 polypeptide comprises at least one substitution mutation relative to SEQ ID NO: 1, and wherein the at least one substitution is at a residue selected from residues Q41, S48, H94, KI 13, and R123 of SEQ ID NO: 1.

4. The fusion protein of claim 3, wherein: a) the substitution at residue Q41 is selected from Q41P, Q41F, Q41M, Q41 Y, Q41W, Q41I, Q41L, Q41V, and Q41A; b) the substitution at residue S48 is selected from S48L, S48A, S48V, S48P, S48T, S48M, S48I, S48F, S48Y, and S48W; c) the substitution at residue H94 is selected from H94S, H94T, H94K, H94R, and H94Y; d) the substitution at residue KI 13 is selected from KI 13N, KI 13Q, KI 13S, KI 13T, K113R, and K113Y; e) the substitution at residue R123 is selected from R123E and R123D; or f) any combination of (a)-(e).

5. The fusion protein of claim 4, wherein the FGF1 polypeptide comprises the substitutions Q41P, S48L, H94S, K113N, and R123E.

6. The fusion protein of claim 5, wherein the FGF1 polypeptide comprises SEQ ID NO: 2.

7. The fusion protein of any one of claims 1 -6, wherein the FGF2 polypeptide comprises SEQ ID NO: 3.

8. The fusion protein of any one of claims 1-6, wherein the FGF2 polypeptide comprises at least one substitution mutation relative to SEQ ID NO: 3, and wherein the at least one substitution is at a residue selected from residues Q65, C78, N111, K128, and K138 of SEQ ID NO: 3.

9. The fusion protein of claim 8, wherein: a) the substitution at residue Q65 is selected from Q65P, Q65F, Q65M, Q65Y, Q65W, Q65I, Q65L, Q65V, and Q65A; b) the substitution at residue C78 is selected from C78S and C78A; c) the substitution at N111 is selected from N111G, N11 IS, N11 IQ, N11 IT, and N111Y; d) the substitution at residue K128 is selected from K128N, K128Q, K128S, K128T, K128R, and K128Y; and e) the substitution at residue K138 is selected from K138E and K138D; or f) any combination of (a)-(e).

10. The fusion protein of claim 9, wherein the FGF2 polypeptide comprises the substitutions Q65I, C78S, N111G, K128N, and K138E.

11. The fusion protein of claim 10, wherein the FGF2 polypeptide comprises SEQ ID NO: 4.

12. The fusion protein of any one of claims 1-11, wherein the linker peptide comprises at least 10 amino acids.

13. The fusion protein of claim 12, wherein the linker peptide comprises SEQ ID NO: 8.

14. The fusion protein of claim 1, wherein: a) the FGF1 polypeptide comprises SEQ ID NO: 1 and the FGF2 polypeptide comprises SEQ ID NO: 3;b) the FGF1 polypeptide comprises SEQ ID NO: 2 and the FGF2 polypeptide comprises SEQ ID NO: 3; c) the FGF1 polypeptide comprises SEQ ID NO: 2 and the FGF2 polypeptide comprises SEQ ID NO: 4; or d) the FGF1 polypeptide comprises SEQ ID NO: 1 and the FGF2 polypeptide comprises SEQ ID NO: 4.

15. The fusion protein of claim 14, wherein the fusion protein comprises: a) SEQ ID NO: 5; b) SEQ ID NO: 6; or c) SEQ ID NO: 7.

16. The fusion protein of any one of claims 1-1 , wherein the linker peptide links the C-terminus of the FGF1 polypeptide to the N-terminus of the FGF2 polypeptide.

17. A pharmaceutical composition comprising the fusion protein of any one of claims 1-16 and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition of claim 17, wherein the pharmaceutically acceptable carrier is a hydrogel.

19. A wound dressing comprising the fusion protein of any one of claims 1-16 or the pharmaceutical composition of any one of claims 17-18.

20. A polynucleotide encoding the fusion protein of any one of claims 1-16.

21. A construct comprising the polynucleotide of claim 20 operably linked to a promoter.

22. A method of treating a condition in a subject in need thereof, the method comprising administering the fusion protein of any one of claims 1-16, the pharmaceutical composition of claim 17 or 18, or the wound dressing of claim 19 to the subject.

23. The method of claim 22, wherein the condition is selected from a wound, organ damage, connective tissue damage, a metabolic disorder, and a nerve disorder.

24. The method of claim 22 or 23, wherein the administration is topical.

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