Regenerative polypeptide and its use

Abstract

Described herein are polypeptides containing an FGF17, IGF2, or BMP™ amino acid sequence and an amino acid sequence from a heterologous polypeptide, which are useful for treating soft tissue and muscle diseases, disorders, and injuries. Also described herein are synergistic combinations of a fibroblast growth factor receptor agonist and a glycosaminoglycan, an insulin-like growth factor 1 receptor (IGF1R) agonist and a short-chain fatty acid, and a BMP receptor agonist and an mTOR activator and / or a glycosaminoglycan. Also described are methods for treating muscle and soft tissue diseases, including administering the polypeptides and / or synergistic compositions.

Description

[Technical Field]

[0001] References to sequence listings, tables, or computer programs A formal copy of the sequence listing will be submitted via EFS-Web as an ASCII-formatted text file (filename "JTI015_ST25.txt", created on December 17, 2020, size 208 kilobytes) along with the specification. The sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. [Background technology]

[0002] background As average life expectancy increases, "healthy aging" is becoming increasingly important. People desire to lead more active lifestyles as they age, and as a result, many age-related disorders can significantly impact the quality of life for aging individuals. Regenerative treatments are useful for treating age-related diseases. In addition, many treatments targeting age-related disorders can be applied to younger people who have diseases or injuries, or who have genetic or developmental disorders that lead to premature tissue loss, wasting, or debilitation. [Overview of the initiative] [Means for solving the problem]

[0003] overview Described herein are polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequence and an amino acid sequence from a heterologous polypeptide, which are useful for treating diseases, disorders, and injuries of soft tissues and muscles. The IGF2 amino acid sequence may include an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 89. The BMP7 amino acid sequence may include an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 89. The BMP7 amino acid sequence may include a fragment consisting of 15 to 30 amino acids that is at least about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 93. The FGF17 amino acid sequence may contain an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54. The FGF17 amino acid sequence may contain deletions of amino acids G181-T203, deletions of amino acids 197-T203, deletions of amino acids 204-216, deletions of amino acids 181-216, R204Q / K207Q, deletions of amino acids 197-216, K191A / K193A / S200A, and mutations selected from combinations thereof. FGF17, IGF2, and / or BMP7 may contain at least one N-, C-, or O-linked glycosylated amino acid.

[0004] Heterogeneous polypeptides may be immunoglobulin molecules or fragments thereof, albumin molecules, transferrin molecules, XTEN sequences, proline-alanine-serine polymers, homoamino acid polymers, glycine-rich sequences, gelatin-like polymers, elastin-like peptides, carboxy-terminal peptides, or combinations thereof. Immunoglobulin molecule fragments may include the hinge domain of IgG, the CH2 domain of IgG, the CH3 domain of IgG, or any combination thereof. Immunoglobulin molecules or fragments thereof may contain one or more mutations that reduce the effector function of the immunoglobulin molecule fragment. Further described herein are fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and combinations of BMP receptor agonists and mTOR activators and / or glycosaminoglycans.

[0005] This specification describes methods for treating diseases of muscle and soft tissue, comprising the step of administering a polypeptide, and / or a composition combining the polypeptide with a small fatty acid, an mTOR activator, and / or a glycosaminoglycan. Muscle diseases that can be treated include acute and chronic diseases or conditions of muscle wasting, such as sarcopenia, cachexia, muscular dystrophy, and muscle injury. Soft tissue regeneration may be useful for treating acute and chronic diseases or conditions of muscle wasting. [Brief explanation of the drawing]

[0006] [Figure 1A] This shows that purified IGF2-hFcm enhanced the differentiation of human myoblasts. [Figure 2A] This shows that sodium butyrate increased muscle fusion. [Figure 2B] This shows that sodium butyrate increased IGF2 activity. [Figure 2C]Shows that sodium butyrate increases IGF2 activity. [Figure 3A] Shows changes in the area proportion of eMyHC-positive cells after treatment with additional doses of vehicle, IGF2, sodium butyrate, or IGF2 in combination with sodium butyrate. [Figure 3B] Shows changes in the area proportion of eMyHC-positive cells after treatment with additional doses of vehicle, IGF2, sodium butyrate, or IGF2 in combination with sodium butyrate. [Figure 4A] Shows that IGF2 receptors are expressed on chondrocytes and osteocytes. [Figure 5A] Shows that BMP7 induces proliferation of myoblasts (measured by newly formed nuclei). [Figure 5B] Shows that BMP7 induces proliferation of myoblasts (measured by total number of nuclei). [Figure 6A] Shows that leucine increases the mitogenic activity of BMP7. [Figure 7A] Shows that hyaluronic acid (HA) increases the mitogenic activity of BMP7. [Figure 8A] Shows that BMP7 receptors are expressed in human myoblasts. [Figure 9A] Shows a treatment for chondrocyte proliferation in cartilage injury and osteoarthritis. [Figure 10A] Shows that FGF17-hFcm, either as part of the culture supernatant or in purified form, promotes proliferation of mouse myoblasts (Fig. 10B). [Figure 10B] Shows that FGF17-hFcm, either as part of the culture supernatant or in purified form, promotes proliferation of mouse myoblasts (Fig. 10B). [Figure 11A] Shows that mutations in the FGF17 sequence improve protein expression levels in CHO cells. [Figure 11B] Shows that mutated FGF17 sequence, either as part of the culture supernatant or in purified form, promotes proliferation of mouse myoblasts. [Figure 12A]Further FGF17 variants demonstrate improved protein expression levels in CHO cells. [Figure 12B] This study demonstrates that further mutations in the FGF17 sequence, either as part of the culture medium supernatant or purified, enhanced the proliferation of mouse myoblasts. [Figure 13A] This shows that the FGF17 receptor was expressed in human myoblasts. [Figure 14A] This shows that heparin increased the mitotic activity of FGF17. [Figure 15A] This shows that hyaluronic acid (HA) increased the mitotic activity of FGF17. [Figure 16A] This paper presents an overview of experiments demonstrating that intramuscular administration of FGF17 enhanced muscle regeneration in an aged mouse model with BaCl2 damage. [Figure 16B] Intramuscular administration of FGF17 enhanced muscle regeneration in an aged mouse model of BaCl2 damage (measured by the formation of new fibers). [Figure 16C] This study demonstrates that intramuscular administration of FGF17 enhanced muscle regeneration by reducing fibrosis in an aged mouse model of BaCl2 damage. [Figure 17A] This paper presents an overview of the experiment demonstrating that systemic administration of FGF17 protects against dexamethasone-induced muscle atrophy. [Figure 17B] Systemic administration of FGF17 protected against dexamethasone-induced muscle atrophy (measured by the percentage change in muscle mass (Figure 17B), by the specific force of the forelimbs (Figure 17C), and by the force of both limbs (Figure 17D)). [Figure 17C] Systemic administration of FGF17 protected against dexamethasone-induced muscle atrophy (measured by the percentage change in muscle mass (Figure 17B), by the specific force of the forelimbs (Figure 17C), and by the force of both limbs (Figure 17D)). [Figure 17D] Systemic administration of FGF17 protected against dexamethasone-induced muscle atrophy (measured by the percentage change in muscle mass (Figure 17B), by the specific force of the forelimbs (Figure 17C), and by the force of both limbs (Figure 17D)). [Modes for carrying out the invention]

[0007] Detailed explanation In certain embodiments, disclosed herein are therapeutically active proteins or polypeptide sequences, derivatives, or fragments thereof that increase the proliferation, regeneration, or function of progenitor cells via activation of cell surface receptors and secretory signals, multimerizing components, or stabilizing components. By modifying and combining specific polypeptide sequences, we have created secretory therapeutically active proteins applicable for the regeneration of muscle and soft tissues that are useful in treating acute and chronic muscle wasting diseases or conditions such as sarcopenia, cachexia, muscular dystrophy, and muscle injury. In certain embodiments, disclosed herein are methods for treating individuals having acute and chronic muscle wasting diseases or conditions such as sarcopenia, cachexia, muscular dystrophy, and muscle injury.

[0008] In certain embodiments, disclosed herein are polypeptides comprising an FGF8 subfamily amino acid sequence and a heterologous polypeptide amino acid sequence, wherein the heterologous polypeptide enhances the stability or biological function of the FGF8 subfamily amino acid sequence. In certain embodiments, disclosed herein are compositions comprising an FGFR agonist and a glycosaminoglycan.

[0009] In certain embodiments, disclosed herein is a polypeptide comprising an IGF2 amino acid sequence and a heterologous polypeptide amino acid sequence, wherein the heterologous polypeptide amino acid sequence enhances the stability or biological function of the IGF2 amino acid sequence. In certain embodiments, disclosed herein is a composition comprising an IGF1R agonist and a short fatty acid chain.

[0010] In certain embodiments, disclosed herein is a polypeptide comprising a BMP7 amino acid sequence and a heterologous polypeptide amino acid sequence, wherein the heterologous polypeptide enhances the stability or biological function of the BMP7 amino acid sequence. In certain embodiments, disclosed herein is a composition comprising a BMP7 receptor agonist and a glycosaminoglycan.

[0011] The secretory signal sequence may be naturally occurring with a therapeutically active protein or polypeptide sequence, or it may be differently selected, modified, or created to optimize expression levels via secretory efficiency, processing rate, or cell line-specific processing. Further examples and sequence numbers are shown in Table 1. In certain embodiments, the polypeptide may include a secretory signal peptide. In certain embodiments, the secretory signal peptide is one of sequence numbers 10-16. The production of the fusion polypeptide may be carried out in heterologous production systems (e.g., bacteria, yeast, mammals, insects, etc.).

[0012] Polypeptides can induce regenerative effects in target cell types via membrane receptors. Examples of stem cell secretomes selected for their ability to improve muscle and soft tissue regeneration are listed in Table 2, and these include, for example, FGF17, BMP7, IGF2, and their variants. Multimerizing components can link two or more protein components other than themselves. Multimerizing components can take the form of amino acid linker sequences that link components other than themselves in tandem to form a single continuous amino acid sequence. Alternatively, multimerizing components can take the form of dimerizing proteins or protein domains, in which case covalent disulfide bonds or non-covalent bonds can drive dimerization. Examples are shown in Table 3.

[0013] Stabilizing components reduce degradation, increase translation or post-translational folding, reduce unfolding rate, increase half-life, and / or other desirable pharmacokinetic parameters (e.g., serum half-life, C1).max AUC, T max Improvements such as those mentioned above may be achieved. Examples may include abundant circulating proteins or their fragments, such as crystallizable fragment (Fc) regions from albumin or human antibodies. Further examples are shown in Table 3.

[0014] Several definitions In the following description, several specific details are provided for the purpose of enabling a thorough understanding of the various embodiments. However, those skilled in the art will understand that the embodiments provided can be carried out without these details. Unless otherwise required by context, throughout the following specification and claims, the word “comprise” and its conjugations (“comprises” and “comprising”) shall be interpreted as an inclusive, open form meaning “including but not limited to.” Where used herein and in the claims, the singular forms “a,” “an,” and “the” shall have their plural meaning unless explicitly stated otherwise. Also, note that the words “or” and “or” shall generally be used to mean “and / or,” unless explicitly stated otherwise. Furthermore, the headings provided herein are provided for convenience only and shall not describe the scope or meaning of the claimed embodiments.

[0015] Where used herein, the term "about" refers to an amount in the vicinity of 10 percent of the stated amount.

[0016] Where used herein, the terms “individual,” “patient,” or “subject” are interchangeable to refer to an individual diagnosed with, suspected of having, or at risk of developing, at least one disease that can be treated with the compositions and methods described herein. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cattle, sheep, pig, goat, llama, alpaca, or yak. ​​In certain embodiments, the individual is a human.

[0017] Where used herein, the terms “treat” or “treating” refer to an intervention in an individual’s physiological or pathological condition that is designed or intended to alleviate at least one sign or symptom associated with that individual’s physiological or pathological condition. Since a variety of individuals may be affected by a particular disease, a person skilled in the art will recognize that not all individuals will respond to a given treatment in the same way, or that some may not respond at all.

[0018] Where used herein, the term “heterogeneous” refers to a nucleotide or amino acid sequence from a different source (e.g., a gene, polypeptide, or organism) than the amino acid or nucleotide sequence being compared to the heterogeneous sequence. Heterogeneous includes biological sequences derived from different organisms, or sequences derived from different sources (e.g., genes or proteins) belonging to the same organism. Heterogeneous sequences include recombinant DNA molecules containing nucleotide sequences from different sources, fusion proteins containing amino acid sequences from different sources, and naturally occurring or synthetically derived epitopes or purified tags.

[0019] As used herein, the term "muscle" refers to skeletal muscle and not to smooth muscle or cardiac muscle.

[0020] As used herein, the term “soft tissue” refers to connective tissue, including but not limited to tendons, ligaments, and cartilage.

[0021] As used herein, the term "proliferative activity" refers to activity that induces cell division or proliferation.

[0022] As used herein, the term "fusion-enhancing activity" refers to activity that enhances the formation of multinucleated cells through the fusion of multiple cells (such as the formation of multinucleated muscle fibers through the fusion of multiple muscle cells), or activity that promotes the differentiation of terminally differentiated stem cells or progenitor cells into committed cell lineages (such as the growth of myoblasts into muscle cells, or the increase in cell size of expanding muscle fibers).

[0023] The terms “polypeptide” and “protein” are used interchangeably to refer to polymers consisting of amino acid residues, not limited to the shortest length. Polypeptides, including the antibodies and antibody chains and other peptides (e.g., linkers and binding peptides) provided, may contain amino acid residues that are native and / or non-native. These terms also include post-expression modifications of polypeptides (e.g., glycosylation, sialylation, acetylation, and phosphorylation). In some embodiments, polypeptides may contain modifications to their native or natural sequence, insofar as the protein maintains the desired activity. Such modifications may be intentionally made by site-directed mutagenesis or accidental, such as mutations in the host producing the protein or errors in PCR amplification.

[0024] The percentage of sequence identity (%) relative to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after the sequence has been aligned and gaps introduced as necessary to maximize the percentage of sequence identity (conservative substitutions are not considered part of sequence identity). Alignment performed for the purpose of determining the percentage of amino acid sequence identity can be carried out in various well-known ways, such as using commonly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Optimal parameters for sequence alignment can be determined, which include algorithms necessary to maximize alignment over the entire length of the comparison sequence. However, for the purposes of this specification, the % value of amino acid sequence identity is obtained using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was developed by Genentech, and its source code, along with user documentation, has been filed with the United States Copyright Office (Washington, DC, 20559) and is registered under United States Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX® operating systems, including Digital UNIX® V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0025] When performing amino acid sequence comparison using ALIGN-2, the amino acid sequence identity % between a given amino acid sequence A and a given amino acid sequence B (alternatively, a given amino acid sequence A that has or contains a specific amino acid sequence identity % with respect to a given amino acid sequence B) is calculated as follows: multiply the fraction X / Y by 100 (where X is the number of amino acid residues recorded as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B). It is understood that if the lengths of amino acid sequence A and amino acid sequence B are not equal, then the amino acid sequence identity % of A with respect to B is not equal to the amino acid sequence identity % of B with respect to A. Unless otherwise specifically stated, all amino acid sequence identity % values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0026] Polypeptides described herein may be encoded by nucleic acids. Nucleic acids are polynucleotides of the type that contain two or more nucleotide bases. In certain embodiments, nucleic acids are components of vectors that can be used to transport polynucleotide-encoding polypeptides into cells. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is bound. One type of vector is a genomic integrated vector (or “integrated vector”) that can be incorporated into the chromosomal DNA of a host cell. Another type of vector is an “episome” vector, such as a nucleic acid that can replicate outside of a chromosome. A vector capable of directing the expression of a gene to which it is operably bound is referred herein to as an “expression vector.” Preferred vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, and viral vectors. Regulatory elements in expression vectors used in the control of transcription, such as promoters, enhancers, and polyadenylation signals, may be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in the host (usually conferred by the origin of replication) and select genes that facilitate recognition of the transformant may be further incorporated. Vectors derived from viruses such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses may be used. Plasmid vectors may be linearized and incorporated into locations on the chromosome. The vector may contain sequences that direct site-specific integration into a predetermined location or a limited number of sites in the genome (e.g., AttP-AttB recombination). Furthermore, the vector may contain sequences derived from transposition factors.

[0027] FGF17 polypeptide In certain embodiments, what is described herein is an FGF17 polypeptide comprising an FGF17 amino acid sequence. The FGF17 amino acid sequence may be human FGF17. The FGF17 amino acid sequence may have at least about 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 54. The FGF17 amino acid sequence may have 100% identity with SEQ ID NO: 54. The FGF17 amino acid sequence may have at least about 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 55. The FGF17 amino acid sequence may have 100% identity with SEQ ID NO: 55. The FGF17 amino acid sequence may have at least about 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 56. The FGF17 amino acid sequence may have 100% identity with SEQ ID NO: 56. The FGF17 amino acid sequence may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, and the sequence contains the R204Q and K207Q mutations. The FGF17 amino acid sequence may be 100% identical to SEQ ID NO: 57. The FGF17 amino acid sequence may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58. The FGF17 amino acid sequence may be 100% identical to SEQ ID NO: 58. The FGF17 amino acid sequence may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 59, and the sequence contains the K191A, K193A, and S200A mutations. The FGF17 amino acid sequence may be 100% identical to SEQ ID NO: 59.

[0028] The FGF17 polypeptides described herein may be fusion proteins or polypeptides that may contain further heterologous (non-FGF17) amino acid sequences that enhance the expression, stability, or function of the FGF17 polypeptide. Such heterologous amino acid sequences may increase the expression of the FGF17 fusion polypeptide from a cell line (e.g., CHO cells, or other cell lines suitable for mass production) by 10%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 200%, 400%, 500%, 1,000%, or more compared to a polypeptide that does not contain the heterologous amino acid sequence. The heterologous amino acid sequence may increase the bioavailability of the FGF17 polypeptide in vivo by 10%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 200%, 400%, 500%, 1,000%, or more compared to the polypeptide without the heterologous amino acid sequence (for example, it may increase Tl / 2). The heterologous amino acid sequence may increase the function of the FGF17 polypeptide in vivo by 10%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 200%, 400%, 500%, 1,000%, or more compared to the polypeptide without the heterologous amino acid sequence (for example, via signaling through the FGF receptor).

[0029] The FGF17 amino acid sequence of the FGF17-heteropolypeptide fusion protein may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54. The FGF17 amino acid sequence of the fusion protein may be 100% identical to SEQ ID NO: 54. The FGF17 amino acid sequence of the fusion protein may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55. The FGF17 amino acid sequence of the fusion protein may be 100% identical to SEQ ID NO: 55. The FGF17 amino acid sequence of the fusion protein may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56. The FGF17 amino acid sequence of the fusion protein may be 100% identical to SEQ ID NO: 56. The FGF17 amino acid sequence of the fusion protein may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, and the sequence may contain the R204Q and K207Q mutations. The FGF17 amino acid sequence of the fusion protein may be 100% identical to SEQ ID NO: 57. The FGF17 amino acid sequence of the fusion protein may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58. The FGF17 amino acid sequence of the fusion protein may be 100% identical to SEQ ID NO: 58. The FGF17 amino acid sequence of the fusion protein may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 59, and the sequence may contain the K191A, K193A, and S200A mutations. The FGF17 amino acid sequence of the fusion protein may be 100% identical to that of SEQ ID NO: 59.

[0030] The amino acid sequence of the FGF17 fusion polypeptide may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 60, SEQ ID NO: 61, or SEQ ID NO: 62. The amino acid sequence of the fusion polypeptide may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65, and such sequence contains the R204Q and K207Q mutations. The amino acid sequence of the fusion polypeptide may be 100% identical to SEQ ID NO: 66, SEQ ID NO: 66, or SEQ ID NO: 71, and such sequence contains the K191A, K193A, and S200A mutations. The amino acid sequence of the fusion polypeptide may be 100% identical to SEQ ID NO: 72. The amino acid sequence of the fusion polypeptide may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 69, and the sequence may contain the R204Q and K207Q mutations.

[0031] The FGF17 amino acid sequence may be at least approximately 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to one of sequence numbers 54-70 or 74, with one, two, three, four, five, six, seven, eight, nine, or ten amino acids deleted from the N-terminus and / or C-terminus of the polypeptide.

[0032] IGF2 fusion protein This specification describes specific IGF2 polypeptides useful for therapeutic purposes, including IGF2 fusion polypeptides that enhance the in vivo stability and function of the IGF2-containing polypeptides.

[0033] In certain embodiments, what is described herein is an IGF receptor ligand polypeptide. The IGF2 polypeptide may comprise an IGF2 amino acid sequence. The IGF2 amino acid sequence may be that of a human IGF2 polypeptide. The human IGF2 polypeptide may comprise amino acids 25-91 of SEQ ID NO: 79 (i.e., SEQ ID NO: 76). The IGF2 amino acid sequence may have at least about 90%, 95%, 97%, 98%, 99%, or 100% identity with one of SEQ ID NOs: 76, 79, 81, or 86. The IGF2 amino acid sequence may have 100% identity with SEQ ID NO: 76. The IGF2 amino acid sequence may be at least approximately 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to one of sequence numbers 76, 79-81, 86, or 88, and may have one, two, three, four, five, six, seven, eight, nine, or ten amino acids deleted from the N-terminus and / or C-terminus of the polypeptide.

[0034] In certain cases, the IGF2 polypeptide described herein is a fusion protein or polypeptide that may contain further heterologous (non-IGF2) amino acid sequences that increase the expression, stability, or function of the IGF2 polypeptide compared to a polypeptide that does not contain heterologous amino acid sequences. Such heterologous amino acid sequences may increase the expression of the IGF2 fusion polypeptide from a cell line (e.g., CHO cells, or other cell lines suitable for mass production) by 10%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 200%, 400%, 500%, 1,000%, or more compared to a polypeptide that does not contain heterologous amino acid sequences. The heterologous amino acid sequence may increase the bioavailability or other pharmacokinetic factors of the IGF2 polypeptide in vivo by 10%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 200%, 400%, 500%, 1,000%, or more compared to polypeptides without the heterologous amino acid sequence (for example, T 1 / 2 AUC, C max , T max(This may increase the following). The heterologous amino acid sequence may improve and / or increase the pharmacodynamics and / or function of the IGF2 polypeptide in vivo by 10%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 200%, 400%, 500%, 1,000%, or more compared to the polypeptide without the heterologous amino acid sequence (e.g., via signaling through the IGF receptor).

[0035] Further described herein are IGF receptor ligand fusion polypeptides, or polypeptides containing a heterogeneous amino acid sequence from IGF2. IGF receptor ligand fusions contain heterogeneous amino acid sequences that enhance the stability or function of the IGF receptor ligand. The IGF2 amino acid sequence of an IGF2-heterogeneous polypeptide fusion protein may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76. The IGF2 amino acid sequence of a fusion protein may be 100% identical to SEQ ID NO: 76. The IGF2 amino acid sequence of a fusion protein may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 80. The IGF2 amino acid sequence of a fusion protein may be 100% identical to SEQ ID NO: 80. The IGF2 amino acid sequence of a fusion protein may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 88. The IGF2 amino acid sequence of the fusion protein may be 100% identical to that of Sequence ID No. 88. Further representative sequences can be found in Table 2.

[0036] BMP7 Fusion Protein This specification describes specific therapeutically useful BMP7 polypeptides, including BMP7 fusion polypeptides that enhance the in vivo stability and function of the BMP7-containing polypeptides.

[0037] In one embodiment, a BMP7 polypeptide comprising a BMP7 amino acid sequence is described herein. The BMP7 amino acid sequence may be a human BMP7 amino acid sequence. The BMP7 amino acid sequence may contain, or consist of, amino acids 293-431 of BMP7. The BMP7 amino acid sequence may contain the BMP7 knuckle domain (SEQ ID NO: 92). The BMP7 sequence may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 89. The BMP7 sequence may be 100% identical to SEQ ID NO: 89. The BMP7 sequence may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 92. The BMP7 polypeptide sequence may contain one, two, three, four, or more repeats of the BMP7 knuckle domain.

[0038] A fusion polypeptide can be created by further fusing the BMP7 amino acid sequence with a heterologous amino acid sequence, either directly or with a linker sequence between the BMP7 amino acid sequence and the heterologous polypeptide amino acid sequence. The fusion polypeptide may contain the human BMP7 amino acid sequence. The fusion polypeptide may contain, or consist of, amino acids 293-431 of BMP7. The fusion polypeptide may contain the BMP7 knuckle domain (SEQ ID NO: 92). The amino acid sequence of the fusion polypeptide may contain a BMP7 amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 89. The fusion polypeptide may contain a BMP7 amino acid sequence that is 100% identical to SEQ ID NO: 89. The amino acids of the fusion polypeptide may contain a BMP7 amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 92. The fusion polypeptide may contain a BMP7 amino acid sequence that is 100% identical to SEQ ID NO: 92. The amino acid sequence of the fusion polypeptide may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 90. The amino acid sequence of the fusion polypeptide may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 91. The amino acid sequence of the fusion polypeptide may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 91.

[0039] The BMP7 amino acid sequence may be at least approximately 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOs. 20, 21, 32, or 89, and may have one, two, three, four, five, six, seven, eight, nine, or ten amino acids deleted from the N-terminus and / or C-terminus of the polypeptide.

[0040] In some embodiments, the BMP7 sequence may be a fragment of the BMP7 sequence. The knuckle domain of BMP7 contains amino acids 98-129 of SEQ ID NO: 89 (SEQ ID NO: 32). A fragment consisting of 15-30 amino acids from the knuckle domain may activate BMP signaling. The BMP7 sequence may contain the knuckle domain of BMP. The BMP7 sequence may be a fragment consisting of 15-30 amino acids of SEQ ID NO: 32. The BMP7 sequence may consist of at least about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 29, 30, 31, 32, 33, 34, or 35 amino acids of SEQ ID NO: 32.

[0041] Secretion signal peptides In certain embodiments, the fusion polypeptide may include a secretory signaling peptide. The secretory signaling peptide may be SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. Production of the fusion polypeptides herein in heterologous production systems (e.g., bacteria, yeast, mammals, insects, etc.) may involve the use of a secretory signaling sequence suitable for the particular host cell.

[0042] A multimerizing component links two or more protein components other than itself. A multimerizing component may include an amino acid linker sequence that links identical or different components other than itself into a single continuous amino acid sequence. Suitable linkers include polypeptide linkers or spacers, such as the Gly-Ser linker described herein. A multimerizing component may take the form of a protein or protein domain that multimerizes or dimerizes, resulting in dimerization being driven by a covalent disulfide bond (e.g., by the addition of one or more novel cysteine ​​residues) or a non-covalent bond (e.g., a leucine zipper). A multimerizing component may link or multimerize multiple IGF2 amino acid sequences. A multimerizing component may link or multimerize two IGF2 amino acid sequences. These two IGF2 amino acid sequences may be the same or different and may be selected from any of the IGF2 sequences described herein. The multimerizing component may conjugate or multimerize two, three, four, five, or more IGF2 amino acid sequences. The multimerizing component may conjugate or multimerize the IGF2 amino acid sequence with another polypeptide that provides enhanced fusion, increased proliferation, increased plasma half-life, or other improvements in pharmacokinetic or pharmacodynamic parameters.

[0043] The FGF17, IGF2, or BMP7 amino acid sequences may include their functional fragments, mutated sequences, or modified polypeptides. Table 2 lists some examples of fragments, polypeptides, and modified polypeptides. The IGF2 or BMP7 sequence may be N-, C-, or O-linked glycosylated. The IGF2 sequence may have one amino acid glycosylated. The IGF2 sequence may have glycosylated sites corresponding to Thr96, Thr99, or Thr163 in SEQ ID NO: 31. The BMP7 sequence may contain at least one glycosylated amino acid. The BMP7 may have glycosylated residues Asn10, Asn29, or Asn90 in SEQ ID NO: 89.

[0044] The FGF17, IGF2, or BMP7 receptor ligand polypeptides and receptor ligand fusion polypeptides described herein are encoded by nucleic acids that can promote the production of the receptor ligand polypeptide or fusion polypeptide. These nucleic acids may be compatible with bacterial, yeast, insect, or mammalian expression systems. They may include promoters / enhancers (structural or induced), polyadenylation signals, selection markers (such as antibiotic resistance), origins of replication, or other associated nucleic acid sequences. FGF17, IGF2, or BMP7 sequences can be taken from many organisms. FGF17, IGF2, or BMP7 sequences may include human FGF17, IGF2, or BMP7 amino acid sequences. FGF17, IGF2, or BMP7 sequences may include cat, dog, or horse FGF17, IGF2, or BMP7 sequences. FGF17, IGF2, or BMP7 sequences may include sequences from mice, rats, rabbits, dogs, cats, horses, cattle, sheep, pigs, goats, llamas, alpacas, yaks, or monkeys.

[0045] FGF17 nucleic acid sequence In certain embodiments, the FGF17 nucleic acid sequence is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17. The FGF17 nucleic acid sequence may be 100% identical to SEQ ID NO: 17. The FGF17 nucleic acid sequence is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22. The FGF17 nucleic acid sequence may be 100% identical to SEQ ID NO: 22. The FGF17 nucleic acid sequence may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23. The FGF17 nucleic acid sequence may be 100% identical to SEQ ID NO: 23.

[0046] IGF2 nucleic acid sequence The IGF2 nucleic acid sequence may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to sequence number 39. The IGF2 nucleic acid sequence may be 100% identical to sequence number 39. The IGF2 nucleic acid sequence may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to sequence number 43. The IGF2 nucleic acid sequence may be 100% identical to sequence number 43. The IGF2 nucleic acid sequence may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to sequence number 46. The IGF2 nucleic acid sequence may be 100% identical to sequence number 46.

[0047] BMP7 nucleic acid sequence The BMP7 nucleic acid sequence may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to sequence number 51. The BMP7 nucleic acid sequence may be 100% identical to sequence number 51. The BMP7 nucleic acid sequence may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to sequence number 52. The BMP7 nucleic acid sequence may be 100% identical to sequence number 52. The BMP7 nucleic acid sequence may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to sequence number 53. The BMP7 nucleic acid sequence may be 100% identical to sequence number 53.

[0048] Heterogeneous peptides Heterogeneous polypeptides, which include a portion of the fusion proteins described herein, may include, consist of, or essentially consist of, fragments of immunoglobulin molecules, albumin molecules, transferrin molecules, XTEN sequences, proline-alanine-serine polymers, homoamino acid polymers, glycine-rich sequences, gelatin-like polymers, elastin-like peptides, carboxy-terminal peptides, or combinations thereof.

[0049] In one embodiment described herein, the therapeutic polypeptide is an FGF receptor ligand polypeptide or an FGF17 polypeptide. In one embodiment described herein, the therapeutic polypeptide is an IGF receptor ligand polypeptide or an IGF2 polypeptide. In one embodiment described herein, the therapeutic polypeptide is a BMP receptor ligand polypeptide or a BMP7 polypeptide.

[0050] In one embodiment described herein, a therapeutic polypeptide is fused directly or via a linker to a heterologous polypeptide amino acid sequence, the heterologous amino acid sequence enhancing the function or stability of the therapeutic polypeptide.

[0051] Heterogeneous peptides can improve the pharmacokinetics, pharmacodynamics, stability, or biological function of therapeutic amino acid sequences. Heterogeneous sequences can be fused to therapeutic amino acid sequences at the C-terminus or N-terminus. Therapeutic amino acid sequences can be fused to heterogeneous sequences at the N-terminus. Therapeutic amino acid sequences can be fused to heterogeneous sequences at the C-terminus. Flexible linkers can be used between the therapeutic amino acid sequence and the heterogeneous sequence at the N-terminus. Flexible linkers can be used between the therapeutic amino acid sequence and the heterogeneous sequence at the C-terminus. Spacers can be used between the therapeutic amino acid sequence and the heterogeneous sequence at the N-terminus. Spacers can be used between the therapeutic amino acid sequence and the heterogeneous sequence at the C-terminus.

[0052] Heterogeneous peptides can improve the pharmacokinetics, pharmacodynamics, stability, or biological function of the IGF2 amino acid sequence. Fusion proteins can be used to improve the pharmacokinetics of bioactive molecules, for example, by increasing their half-life, as described in Strohl, “Fusion Proteins for Half-Life Extension of Biologies as a Strategy to Make Biobetters,” BioDrugs (2015) 29:215-239. The half-life of a polypeptide is increased by fusing it to a molecule or fragment of a molecule with a long half-life, such as immunoglobulin, albumin, or transferrin. XTEN sequences are repeating amino acid polymers containing amino acid residues A, E, G, P, S, and T, which are normally inactive but can increase the half-life of a peptide when fused to it. Furthermore, the half-life of polypeptides can also be extended by fusing them with homoamino acid polymer sequences such as proline-alanine-serine polymers (repeat proline, alanine, and serine), glycine-rich sequences (GGGS), gelatin-like proteins, and small repeat sequences such as elastin-like sequences (VPGxG, where x is any amino acid other than proline). The half-life of polypeptides in serum can also be extended by fusing them with carboxy-terminal peptides (CTPs) due to the strong negative charge (change) of the CTPs. Heterogeneous polypeptides may include fragments of immunoglobulin molecules, albumin molecules, transferrin molecules, XTEN sequences, proline-alanine-serine polymers, homoamino acid polymers, glycine-rich sequences, gelatin-like polymers, elastin-like peptides, carboxy-terminal peptides, or combinations thereof.

[0053] Immunoglobulins are large effector molecules; for example, IgG immunoglobulin has a plasma half-life of approximately 21 days. Fusing an immunoglobulin fragment to a second polypeptide can increase the half-life of the second polypeptide. Immunoglobulin molecular fragments may include the hinge domain of IgG, the CH2 domain of IgG, the CH3 domain of IgG, or any combination thereof. Immunoglobulin molecular fragments may include the hinge domain of IgG1, the CH2 domain of IgG1, the CH3 domain of IgG1, or any combination thereof. Immunoglobulin molecular fragments may include the hinge domain of IgG4, the CH2 domain of IgG4, the CH3 domain of IgG4, or any combination thereof.

[0054] In some cases, mutations in an immunoglobulin molecule or fragment can increase the half-life or stability of the immunoglobulin molecule or fragment. An immunoglobulin molecule fragment may contain the hinge domain of IgG1, the CH2 domain of IgG1, the CH3 domain of IgG1, or any combination thereof, along with one or more of the following amino acid mutations in the immunoglobulin molecule: P329G, L234A, and L235A. The immunoglobulin molecule fragment may also contain an IgG4 molecule. An immunoglobulin molecule fragment may contain an IgG4 molecule having at least one of the following amino acid mutations in the immunoglobulin molecule: N434A, N434H, T307A / E380A / N434A, M252Y / S254T / T256E, 433K / 434F / 436H, T250Q, T250F, M428L, M428F, T250Q / M4, according to the EU numbering system. 28L, N434S, V308W, V308Y, V308F, M252Y / M428L, D259I / V308F, M428L / V308F, Q311V / N434S, T307Q / N434A, E258F / V427T, S228P, L235E, S228P / L235E / R409K, S228P / L235E, K370Q, K370E, deletion of G446, deletion of K447, and combinations thereof.

[0055] Secretory signal sequences are sequence motifs that target proteins to secretory pathways within a cell. Secretory sequences can be cleaved from proteins to produce mature secretory proteins. Polypeptides may contain secretory signal sequences. Polypeptides may contain human FGF17, IGF2, or BMP7 secretory sequences (SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12). Polypeptides may contain secretory signals such as SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0056] Linker and spacer A linker or spacer is a short amino acid sequence that separates different domains within a single protein, or a domain between two fusion proteins. As used herein, the terms “linker” and “spacer” are indistinguishable. Linkers may be rigid or flexible. Rigid linkers can prevent undesirable interactions between different domains. Proline-rich linkers tend to be relatively rigid, while glycine-rich linkers tend to be relatively flexible. Flexible linkers allow interactions between domains within a single protein. Another use of flexible linkers is to covalently link a protein complex to a binding partner to create a stable protein complex. Flexible linkers can also be used to enhance dimerization. Linkers and spacers are discussed in Chichili et al, Linkers in the Structural biology of protein-protein interactions, Protein Sci. Feb 2013. 22(2): 153-167.

[0057] The fusion polypeptides described herein may further include a linker or spacer amino acid sequence for separating the therapeutic polypeptide from a heterologous polypeptide. The linker or spacer may be a peptide linker or spacer. The linker or spacer may be a flexible linker or spacer. The linker may be three alanines (AAA). The peptide linker may be a glycine-serine linker. The linker may be GGGGS (4GS) (in a single-letter amino acid code), or a polymer of a 4GS linker (such as a repeat of two, three, four, or five 4GS linkers). The glycine-serine linker may include the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 95, or two, three, four, or five repetitions of SEQ ID NO: 94 or SEQ ID NO: 95. The linker may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids that do not originate from any of the polypeptide sequences in Table 2 or the heterologous polypeptide amino acid sequences in Table 3.

[0058] The linker or spacer may be one amino acid residue or longer. In certain embodiments, the length of the peptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids. The peptide linker may have at least one amino acid residue but does not exceed the length of 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues.

[0059] A combination of FGFR agonist and glycosaminoglycan In certain embodiments, compositions comprising FGFR agonists and glycosaminoglycans are disclosed herein. There are four types of FGF receptors: FGF1R, FGFR2, FGFR3, and FGFR4, which are expressed in various tissues throughout the body. Chemical agonists of FGFR include, but are not limited to, PF-05231023 and SUN11602. Other polypeptides, including dekafin and hexafin, can also activate FGFR signaling. Such compositions may contain unexpected synergistic effects and are useful in treating conditions or disorders of muscle and / or soft tissue. Furthermore, these synergistic effects may be enhanced by methods including the separate administration of FGFR agonists and glycosaminoglycans. The combinations described herein can impart further therapeutic utility and function to each component of the combination.

[0060] FGFR1 can be activated by FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8, FGF10, FGF17, FGF19, FGF20, FGF21, FGF22, and FGF23. FGFR1 agonists may be FGFR1 agonist antibodies, FGF polypeptides or their functional fragments, FGF17 or its functional fragments, PF-05231023, SUN11602, Decafin, Hexafin, or combinations thereof.

[0061] FGFR2 includes two isoforms that are alternatively spliced. FGFR2IIIb binds to FGF1, FGF3, FGF10, and FGF22, while FGFR2IIIc binds to FGF1, FGF2, FGF4, FGF6, FGF8, FGF9, FGF17, and FGF18. FGFR2 agonists may be FGFR2 agonist antibodies, FGF polypeptides or their functional fragments, FGF17 or its functional fragments, PF-05231023, SUN11602, Decafine, Hexaphine, or combinations thereof.

[0062] FGFR3 can be activated by at least FGF1, FGF2, FGF4, FGF5, FGF6, FGF8, FGF9, FGF16, FGF17, FGF18, FGF20, FGF9, FGF19, FGF21, and FGF23, and FGF17. Mutations in FGFR3 are associated with defects in chondrocyte proliferation and calcification, as well as achondroplasia. FGFR3 agonists may be FGFR3 agonist antibodies, FGF polypeptides or their functional fragments, FGF17 or its functional fragments, PF-05231023, SUN11602, decafin, hexaphine, or combinations thereof.

[0063] FGFR4 can be activated by at least FGF1, FGF2, FGF4, FGF6, FGF7, FGF8, FGF9, FGF16, FGF17, and FGF18. FGFR4 agonists may be FGFR4 agonist antibodies, FGF polypeptides or their functional fragments, FGF17 or its functional fragments, PF-05231023, SUN11602, Decafine, Hexaphine, or combinations thereof.

[0064] FGFR agonists may be members of the FGF8 subfamily. FGFR agonists may be FGFR1 agonists. FGFR agonists may be FGF17. FGF17 polypeptides may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54. FGF17 may be 100% identical to SEQ ID NO: 54. FGF17 polypeptides may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55. FGF17 polypeptides may be 100% identical to SEQ ID NO: 55. FGF17 polypeptides may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56. FGF17 may be 100% identical to SEQ ID NO: 56. The FGF17 polypeptide may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, and the sequence contains the R204Q and K207Q mutations. In certain embodiments, the FGF17 polypeptide is 100% identical to SEQ ID NO: 57. In certain embodiments, the FGF17 polypeptide is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58. The FGF17 polypeptide may be 100% identical to SEQ ID NO: 58. The FGF17 polypeptide may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 59, and the sequence contains the K191A, K193A, and S200A mutations. The FGF17 polypeptide may be 100% identical to SEQ ID NO: 59.

[0065] Glycosaminoglycans are linear polysaccharides containing repeating disaccharide units. There are four classes of glycosaminoglycans: heparin / heparin sulfate, chondroitin sulfate / dermatan sulfate, keratin sulfate, and hyaluronic acid. A glycosaminoglycan may be heparin / heparin sulfate, chondroitin sulfate / dermatan sulfate, keratin sulfate, or hyaluronic acid. In some embodiments, the glycosaminoglycan contains heparin. The glycosaminoglycan may also contain hyaluronic acid.

[0066] Heparin can be derived from natural products and is often referred to as unfractionated heparin. Heparin can also be defined based on its molecular weight. Low molecular weight heparins include dalteparin, enoxaparin, sertoparin, aldepalin, parnaparin, reviparin, nadropalin, and danaparoid. Heparin can be administered as a mixture with other compounds, such as danaparoid, which is a mixture of heparin sulfate, dermatan sulfate, and chondroitin sulfate. Compositions may include low molecular weight heparin, heparin sulfate, unfractionated heparin, heparin tetrasaccharide, dalteparin, tinzaparin, enoxaparin, sertoparin, aldepalin, parnaparin, reviparin, nadropalin, heparin flush, danaparoid, fondaparinux, or combinations thereof.

[0067] Hyaluronic acid (HA) is a polymer molecule and can exhibit a wide range of molecular weights. Hyaluronic acid can be used in almost any average modal molecular weight formulation. Molecules include, for example, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, HA may be 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000 kDa, or higher, or any range that can be derived therefrom. HA may include low molecular weight HA (about 500-700 kilodaltons kDa), medium molecular weight HA (700-1000 kDa), and high molecular weight HA (1.0-4.0 million daltons (MDa)). HA may include natural preparations, synthetic preparations, or combinations thereof. In some embodiments, HA is low molecular weight, medium molecular weight, low molecular weight, or a combination thereof. In some embodiments, HA is natural HA, synthetic HA, or a combination thereof.

[0068] HA may be a hyaluronic acid derivative. Examples of possible chemical modifications to HA include any reaction between the four reactive groups of HA—namely, the acetamide terminus, carboxyl terminus, hydroxyl terminus, and reducing terminus—and a drug. HA derivatives include, but are not limited to, hydrophobized hyaluronan, maleimide-modified HA, methacrylated hyaluronic acid, or sulfated hyaluronic acid. In some embodiments, HA is modified at the acetamide group, carboxyl group, hydroxyl group, reducing terminus, or a combination thereof. HA may include hydrophobized hyaluronan, maleimide-modified HA, methacrylated hyaluronic acid, sulfated hyaluronic acid, or a combination thereof. HA may be covalently crosslinked via proteins or organic molecules to form higher molecular weight sites.

[0069] Furthermore, methods comprising administering FGFR agonists and glycosaminoglycans are described herein. Administration may be carried out in the same composition or in separate preparations. If separate preparations are administered, they may be effectively administered simultaneously (for example, during the same procedure) or separately with intervals of at least 1 hour, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or longer. If separate preparations are administered, they may be administered via the same or different routes selected from intravenous, intradermal, and subcutaneous. Whether separate or single-dose, the preparations may be administered directly to the site of muscle or soft tissue injury.

[0070] IGF1R agonist and short fatty acid chain combination In certain embodiments, disclosed herein are compositions comprising IGF1R agonists and short fatty acid chains. IGF1R signaling activates downstream pathways, including those involved in cell proliferation, cell differentiation, and cell survival. Two IGF ligands, IGF1 and IGF2, activate IGF1R signaling. A further peptide that activates IGF1R signaling is INS. Other IGF1R agonists include, but are not limited to, demethylasteriquinone B1, ginsenoside Rg5, and human antimicrobial peptide LL-37. IGF1R agonists may include IGF1R agonist antibodies, IGF polypeptides or their functional fragments, IGF2 or its functional fragments, insulin, demethylasteriquinone B1, ginsenoside Rg5, LL-37, or combinations thereof. Such compositions may exhibit unexpected synergistic effects and may be useful in treating muscle and / or soft tissue conditions or disorders. Furthermore, this synergistic effect may be enhanced by methods including administering the IGF1R agonist and the short fatty acid chain separately.

[0071] An IGF2R agonist may be an IGF ligand. An IGF1R agonist may be IGF2. An IGF2 polypeptide may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76. An IGF2 polypeptide may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 80. An IGF2 polypeptide may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 81. An IGF2 polypeptide may be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 81. An IGF2 polypeptide may be at least 100% identical to SEQ ID NO: 81.

[0072] The composition may contain an IGF1R agonist and a short fatty acid chain. The short fatty acid chain includes, but is not limited to, buttyrates, phenylbutyric acid, valproic acid, propionic acid, methaneic acid, ethaneic acid, 2-methylpropanoic acid, 3-methylbutanoic acid, pentanoic acid, and their polymers (such as triptyline). Butyrates include, but are not limited to, butyric acid, sodium butyrate, methyl butyrate, ethyl butyrate, butyl butyrate, pentyl butyrate, or sodium butyrate. The short-chain fatty acid may be butyrate. The butyrate may be butyric acid. The butyrate may be sodium butyrate. The short-chain fatty acid may be phenylbutyric acid, valproic acid, propionic acid, methaneic acid, ethaneic acid, 2-methylpropanoic acid, 3-methylbutanoic acid, pentanoic acid, or their polymers (such as triptyline).

[0073] Furthermore, methods comprising administering an IGF1R agonist and a short fatty acid chain are described herein. The administration may be carried out in the same composition or in separate preparations. If separate preparations are administered, they may be effectively administered simultaneously (for example, during the same procedure) or separately with intervals of at least 1 hour, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or longer.

[0074] BMP receptor agonist and mTOR activator combination In certain embodiments, disclosed herein are compositions comprising BMP receptor agonists and mTOR activators. BMP receptors activate downstream signaling via the TGF-beta pathway and are involved in many cellular functions, including differentiation, proliferation, and migration. There are two classes of BMP receptors: type I BMPs (ACVR1, BMPR1A, and BMPR1B) and type II BMPs (BMP2R, ACVR2A, and ACVR2B). Type I BMP receptors bind exclusively to BMP ligands, while type II BMP receptors bind to BMP and related proteins, including activin, Gdf9, and GDf11. BMP receptor agonists may include ACVR1 agonists, BMPR1A agonists, BMPR1B agonists, BMP2R agonists, ACVR2A agonists, ACVR2B agonists, or combinations thereof. BMP receptor agonists may include ACVR1 agonists, ACVR2A agonists, ACVR2B agonists, BMPR1A agonists, or combinations thereof. BMP receptors may include ACVR1 agonists. BMP receptor agonists may include ACVR2A agonists. BMP receptors may include ACVR2B agonists. BMP receptors may include BMPR1A agonists. BMP receptor agonists may include ACVR1 agonist antibodies, BMPR1A agonist antibodies, BMPR1B agonist antibodies, BMP2R agonist antibodies, ACVR2A agonist antibody antibodies, ACVR2B agonist antibody antibodies, BMP polypeptides or their functional fragments, BMP7 or its functional fragments, ventromorphin, SB4, tacrolimus, isoliquitigenin, allantolactone, PD407824, or combinations thereof. These compositions exhibit unexpected synergistic effects and are useful in treating muscle and / or soft tissue conditions or disorders. Furthermore, these synergistic effects can be enhanced by methods including the separate administration of BMP receptor agonist antibodies and leucine.

[0075] The BMP7 polypeptide may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 89. The BMP7 polypeptide may be 100% identical to SEQ ID NO: 89. The BMP7 polypeptide may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 90. The BMP7 polypeptide may be 100% identical to SEQ ID NO: 90. The BMP7 polypeptide may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 91. The BMP7 polypeptide may be 100% identical to SEQ ID NO: 91. The BMP7 polypeptide may be at least approximately 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 93. The BMP7 polypeptide may be 100% identical to SEQ ID NO: 93.

[0076] The mammalian target of rapamycin (mTOR) pathway is a crucial regulator of skeletal muscle mass and proliferation. mTOR is a serine / threonine kinase involved in various cellular processes, including cell proliferation, differentiation, autophagy, survival, and metabolism. mTOR activation via the mTORC1 complex is required for both myofibril muscle protein synthesis and skeletal muscle hypertrophy. Inactivation of the mTORC1 complex has been found to be associated with decreased muscle mass and muscle struct in aging muscle wasting, cachexia, and atrophy due to physical activity.

[0077] The mTOR signaling pathway can be activated by many different signals, including amino acids, polypeptides, and small molecules. mTOR activators can be amino acids. These may include leucine, valine, isoleucine, or combinations thereof. Another example is leucine. MTOR activators can be polypeptides. These polypeptides may include brain-rich Ras homolog (Rheb), tuberous sclerosis complex (TSC), protein kinase B (PKB), extracellular signal-regulated kinase 1 / 2 (ERK1 / 2), p90 ribosomal s6 kinase 1 (RSK1), Wnt ligand, or combinations thereof. MTOR activators can also be small molecules. These may include MHY1485, NV-5138, 3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO), or combinations thereof. mTOR activators may include leucine, valine, isoleucine, brain-rich Ras homolog (Rheb), tuberous sclerosis (TSC), protein kinase B (PKB), extracellular signal-regulated kinase 1 / 2 (ERK1 / 2), p90 ribosomal s6 kinase 1 (RSK1), Wnt ligand, MHY1485, NV-5138, 3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO), or combinations thereof.

[0078] mTOR activators may contain leucine. The amino acid leucine is an important part of mTOR signaling in skeletal muscle. Leucine is an essential branched-chain amino acid in human food and beverages. It is the most common amino acid in human proteins, appearing at a frequency of nearly one in ten amino acids (UniProtKB / Swiss-Prot release 2013_04-2013). The circulating and intracellular concentrations of this amino acid are monitored and strictly controlled as part of feedback mechanisms that control major anabolic and catabolic processes such as cell division, protein synthesis, and autophagy. The mTORC1 complex is involved in some of the regulatory effects of leucine, and the driving of protein synthesis and cell cycle progression by the activation of the latter is partly driven by intracellular leucine concentration. Combinations of BMP receptor agonist antibodies with leucine or other branched-chain amino acids exhibit synergistic pro-mitotic activity.

[0079] Synthetic leucine derivatives may be used. Leucine may include l-leucine, glycyl-l-leucine, acetyl-l-leucine, l-leucine ethyl ester, and l-leucine methyl ester, caproic acid, phthaloyl-l-leucine, benzoyl-dl-leucine, or combinations thereof. Leucine may be a salt. Leucine may include l-leucenium hydrogen maleate, leucine hydrochloride, or combinations thereof.

[0080] Furthermore, a method comprising administering a BMP receptor agonist antibody and an mTOR activator is described herein. The administration may be carried out in the same composition or in separate preparations. If separate preparations are administered, they may be effectively administered simultaneously (for example, during the same procedure) or separately with intervals of at least 1 hour, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or longer.

[0081] Treatment indications In certain embodiments, compositions and methods comprising fusion polypeptides, FGFR agonists and glycosaminoglycans, FGF8 subfamily amino acid sequences and heterologous polypeptides, as described herein, are useful for treating diseases and disorders involving damage, degradation, or destruction of soft tissues, or for use in treating individuals having aging disorders, muscle wasting disorders, muscle injuries, connective tissue injuries, or non-muscle soft tissue injuries, or any combination thereof.

[0082] In certain embodiments, compositions and methods described herein, comprising a fusion polypeptide containing an IGF ligand amino acid sequence and a heterologous polypeptide, an IGF1R agonist and a short fatty acid chain, are useful for treating diseases and disorders involving damage, degradation, or destruction of soft tissue, or for use in treating individuals having aging disorders, muscle wasting disorders, muscle injuries, connective tissue injuries, or non-muscle soft tissue injuries, or any combination thereof.

[0083] In certain embodiments, compositions comprising a fusion polypeptide containing a BMP7 amino acid sequence, a BMP receptor agonist and a glycosaminoglycan, a composition comprising a BMP receptor agonist and an mTOR activator, and methods described herein are useful for treating diseases and disorders involving damage, degradation, or destruction of soft tissues, or for use in treating individuals having aging disorders, muscle wasting disorders, muscle injuries, connective tissue injuries, or non-muscle soft tissue injuries, or any combination thereof.

[0084] Aging disorders that lead to deterioration and reduction of muscle tissue include the following: For example, sarcopenia is a progressive decrease in the quantity, quality and strength of skeletal muscle and may be associated with aging. Other muscle disorders that lead to acute muscle injury are those that can be treated by polypeptides, compositions and methods described herein. These disorders include muscle tears, strains, and contusions. A tear is a separation of muscle tissue. A muscle strain is an injury caused by contraction in which muscle fibers tear due to great mechanical stress and may be classified as grade I, II, or III. A muscle contusion is a hematoma of the muscle. Muscle injury can also be caused by stress other than mechanical stress, such as cachexia. Cachexia can be caused by malnutrition, cancer, AIDS, celiac disease, chronic obstructive pulmonary disease, multiple sclerosis, rheumatoid arthritis, congestive heart failure, tuberculosis, familial amyloid polyneuropathy, mercury poisoning (acromiomyopathy), Crohn's disease, untreated / severe type 1 diabetes mellitus, anorexia nervosa, chemotherapy, muscular dystrophy or other genetic disorders causing immobility, and hormone deficiencies. Certain disorders, such as dysphagia or facioscapulohumeral muscular dystrophy, which involve weakness of specific muscles, can also be treated with the polypeptides described herein. Further soft tissue disorders that can be treated with polypeptides comprising the FGF8 subfamily amino acid sequence described herein and / or compositions comprising FGFR agonists and glycosaminoglycans are those causing damage to tendons, ligaments, or cartilage. Further soft tissue injuries that can be treated using polypeptides comprising the IGF ligand amino acid sequence described herein, as well as compositions comprising an IGF1R agonist and a short fatty acid chain, are injuries to tendons, ligaments, or cartilage. Further soft tissue injuries that can be treated using fusion polypeptides comprising the BMP7 amino acid sequence, a BMP receptor agonist, and a glycosaminoglycan, as described herein, as well as methods, are injuries to tendons, ligaments, or cartilage.

[0085] Muscle wasting diseases can be muscular dystrophy. Muscular dystrophy may include myotonic muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, or distal muscular dystrophy. Muscular dystrophy can be myotonic dystrophy.

[0086] Aging disorders can be sarcopenia. Muscle wasting disorders can be cachexia. Cachexia can result from cancer, AIDS, end-stage renal disease, or cardiovascular disease. Injuries can be muscle injuries. Muscle wasting can be atrophy due to immobilization or disuse of limbs. Muscle injuries can be strains or lacerations. Muscle injuries can be grade III strains. Sarcopenia can contribute to the development of muscle injuries. Injuries can be ligament injuries. Ligament injuries can be ruptures or lacerations. Injuries can be tendon injuries. Tendon injuries can be ruptures or lacerations. Injuries can be cartilage injuries.

[0087] The compositions described herein are for use in methods for treating myositis. Myositis may include dermatomyositis, polymyositis, necrotizing myopathy (also known as necrotizing autoimmune myopathy or immune-mediated necrotizing myopathy), juvenile myositis, or sporadic inclusion body myositis.

[0088] The compositions described herein may be used in methods for treating cartilage-related disorders. Cartilage-related disorders may be caused by lacerations, injuries, or abrasion. Cartilage-related diseases may include osteoarthritis, osteochondritis dissecans, achondroplasia, or degenerative cartilage damage.

[0089] The compositions described herein may be used in methods for increasing the proliferation or promoting the survival of cells associated with soft tissue injury. The polypeptides comprising IGF ligand amino acid sequences, as well as compositions comprising IGF1R agonists and short fatty acid chains, described herein may be useful in methods for increasing the proliferation or promoting the survival of any one or more of the following: muscle cells, muscle progenitor cells, tendinocytes, tendinocyte progenitor cells, chondrocytes, chondrocyte progenitor cells, mesenchymal stem cells, or fibroblasts.

[0090] Myofibrosis is an excessive accumulation of extracellular matrix components, including collagen. Myofibrosis impairs muscle function, negatively affects muscle regeneration after injury, and makes muscles more susceptible to re-injury. The compositions described herein may be used in methods to alleviate myofibrosis. Fibrosis may be associated with aging, muscular dystrophy, or injury. The IGF ligand may be IGF2.

[0091] Myoblasts must fuse to form multinucleated cells in order to differentiate into mature muscle cells. In certain embodiments, compositions and methods described herein, comprising an IGF ligand amino acid sequence and a fusion polypeptide containing a heterologous polypeptide, an IGF1R agonist, and a short fatty acid chain, are for use in methods to increase myoblast fusion. The IGF ligand may be IGF2.

[0092] Compositions and methods described herein, including fusion polypeptides comprising IGF ligand amino acid sequences and heterologous polypeptides, IGF1R agonists and short fatty acid chains, may be used in methods for increasing muscle mass. Muscle mass may increase by at least about 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, or more than 50%. The IGF ligand may be IGF2.

[0093] Compositions and methods described herein, including fusion polypeptides comprising IGF ligand amino acid sequences and heterologous polypeptides, IGF1R agonists and short fatty acid chains, may be used in methods for increasing grip strength. Grip strength may increase by at least about 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, or more than 50%. The IGF ligand may be IGF2.

[0094] Compositions and methods described herein, including fusion polypeptides comprising IGF ligand amino acid sequences and heterologous polypeptides, IGF1R agonists and short fatty acid chains, may be used in methods for increasing muscle endurance. Muscle endurance may be increased by at least about 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, or more than 50%. The IGF ligand may be IGF2.

[0095] Treatment method In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder an FGFR agonist and a glycosaminoglycan. The FGFR agonist and the glycosaminoglycan may be administered in separate preparations. The FGFR agonist and the glycosaminoglycan may be administered simultaneously. The FGFR agonist and the glycosaminoglycan may be administered at different times. The glycosaminoglycan may be heparin. The glycosaminoglycan may be hyaluronic acid.

[0096] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder an FGFR1 agonist and a glycosaminoglycan. The FGFR1 agonist and the glycosaminoglycan (e.g., heparin) may be administered in separate preparations. The FGFR1 agonist and the glycosaminoglycan (e.g., heparin) may be administered simultaneously. The FGFR1 agonist and the glycosaminoglycan (e.g., heparin) may be administered at different times. The glycosaminoglycan may be heparin. The glycosaminoglycan may be hyaluronic acid.

[0097] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder a polypeptide and glycosaminoglycan (e.g., heparin) or a compound or mixture containing a glycosaminoglycan. The polypeptide and glycosaminoglycan (e.g., heparin) containing the FGF8 subfamily amino acid sequence may be administered in separate preparations. The polypeptide and glycosaminoglycan (e.g., heparin) containing the FGF8 subfamily amino acid sequence may be administered simultaneously. The polypeptide and glycosaminoglycan (e.g., heparin) containing the FGF8 subfamily amino acid sequence may be administered at different times. The glycosaminoglycan may be hyaluronic acid.

[0098] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder a polypeptide containing the FGF17 amino acid sequence and a glycosaminoglycan (e.g., heparin) or a compound or mixture containing a glycosaminoglycan. The polypeptide containing the FGF17 amino acid sequence and the glycosaminoglycan (e.g., heparin) may be administered in separate preparations. The polypeptide containing the FGF17 amino acid sequence and the glycosaminoglycan (e.g., heparin) may be administered simultaneously. The polypeptide containing the FGF17 amino acid sequence and the glycosaminoglycan may be administered at different times. The glycosaminoglycan may be hyaluronic acid.

[0099] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering a polypeptide comprising an FGF8 subfamily amino acid sequence. The polypeptide may comprise an FGF17 amino acid sequence. The polypeptide may comprise an FGF17 fusion protein.

[0100] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual an IGF1R agonist and a short fatty acid chain (e.g., butyrate). The IGF1R agonist and the short fatty acid chain (e.g., butyrate) may be administered in separate preparations. The IGF1R agonist and the short fatty acid chain (e.g., butyrate) may be administered simultaneously. The IGF1R agonist and the short fatty acid chain (e.g., butyrate) may be administered at different times.

[0101] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder a polypeptide and a butyrate containing an IGF ligand amino acid sequence. The polypeptide and the butyrate containing the IGF ligand amino acid sequence may be administered in separate preparations. The polypeptide and the butyrate containing the IGF ligand amino acid sequence may be administered simultaneously. The polypeptide and the butyrate containing the IGF ligand amino acid sequence may be administered at different times.

[0102] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder a polypeptide containing an IGF2 amino acid sequence and a short fatty acid chain (e.g., butyrate). The polypeptide containing the IGF ligand amino acid sequence and the short fatty acid chain (e.g., butyrate) may be administered in separate preparations. The polypeptide containing the IGF2 amino acid sequence and the short fatty acid chain (e.g., butyrate) may be administered simultaneously. The polypeptide containing the IGF2 amino acid sequence and the short fatty acid chain (e.g., butyrate) may be administered at different times.

[0103] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder a BMP receptor agonist and a glycosaminoglycan. The BMP receptor agonist and the glycosaminoglycan may be administered in separate preparations. The BMP receptor agonist and the glycosaminoglycan may be administered simultaneously. The BMP receptor agonist and the glycosaminoglycan may be administered at different times. The glycosaminoglycan may be heparin. The glycosaminoglycan may be hyaluronic acid.

[0104] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder a polypeptide containing the BMP7 amino acid sequence and hyaluronic acid or a compound or mixture containing hyaluronic acid. The polypeptide containing the BMP7 amino acid sequence and hyaluronic acid are administered in separate preparations. In some embodiments, the polypeptide containing the BMP7 amino acid sequence and hyaluronic acid are administered simultaneously. In some embodiments, the polypeptide containing the BMP7 amino acid sequence and hyaluronic acid are administered at different times.

[0105] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder a polypeptide containing a BMP7 amino acid sequence and heparin or a compound or mixture containing heparin. In some embodiments, the polypeptide containing the BMP7 amino acid sequence and heparin are administered in separate preparations. In some embodiments, the polypeptide containing the BMP7 amino acid sequence and heparin are administered simultaneously. In some embodiments, the polypeptide containing the BMP7 amino acid sequence and heparin are administered at different times.

[0106] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder a BMP receptor agonist and an mTOR activator. In some embodiments, the BMP receptor agonist and the mTOR activator are administered in separate preparations. In some embodiments, the BMP receptor agonist and the mTOR activator are administered simultaneously. In some embodiments, the BMP receptor agonist and the mTOR activator are administered at different times. In certain embodiments, the mTOR activator is leucine.

[0107] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder a polypeptide comprising a BMP7 amino acid sequence and leucine or a compound or mixture comprising leucine. In some embodiments, the polypeptide comprising the BMP7 amino acid sequence and leucine are administered in separate preparations. In some embodiments, the polypeptide comprising the BMP7 amino acid sequence and leucine are administered simultaneously. In some embodiments, the polypeptide comprising the BMP7 amino acid sequence and leucine are administered at different times.

[0108] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder a BMP receptor agonist and a glycosaminoglycan. In some embodiments, the BMP receptor agonist and the glycosaminoglycan are administered in separate preparations. In some embodiments, the BMP receptor agonist and the glycosaminoglycan are administered simultaneously. In some embodiments, the BMP receptor agonist and the glycosaminoglycan are administered at different times. In certain embodiments, the glycosaminoglycan is heparin. In certain embodiments, the glycosaminoglycan is hyaluronic acid.

[0109] In certain embodiments, disclosed herein are methods for treating an individual with a disorder, comprising administering to the individual with the disorder a polypeptide containing a BMP7 amino acid sequence and a glycosaminoglycan (e.g., heparin or hyaluronic acid) or a compound or mixture containing a glycosaminoglycan (e.g., heparin or hyaluronic acid). In some embodiments, the polypeptide containing the BMP7 amino acid sequence and the glycosaminoglycan (e.g., heparin or hyaluronic acid) are administered in separate preparations. In some embodiments, the polypeptide containing the BMP7 amino acid sequence and the glycosaminoglycan (e.g., heparin or hyaluronic acid) are administered simultaneously. In some embodiments, the polypeptide containing the BMP7 amino acid sequence and the glycosaminoglycan (e.g., heparin or hyaluronic acid) are administered at different times.

[0110] The treatment can be administered via any preferred route, such as subcutaneously, intravenously, or intramuscularly. In certain embodiments, the treatment is administered according to a preferred dosing schedule, such as once a week, twice a week, once a month, twice a month, once every three weeks, or once every four weeks. The treatment can be administered in any therapeutically effective dose, which may range from approximately 0.001 mg / kg to approximately 1 mg / kg.The therapeutically effective dose is approximately 0.001 mg / kg to 0.002 mg / kg, approximately 0.001 mg / kg to 0.005 mg / kg, approximately 0.001 mg / kg to 0.01 mg / kg, approximately 0.001 mg / kg to 0.02 mg / kg, approximately 0.001 mg / kg to 0.05 mg / kg, approximately 0.001 mg / kg to 0.1 mg / kg, approximately 0.001 mg / kg to 0.2 mg / kg, approximately 0.001 mg / kg to 0.5 mg / kg, approximately 0.001 mg / kg to 1 mg / kg, approximately 0.002 mg / kg to 0.005 mg / kg, approximately 0.002 mg / kg kg~about 0.01mg / kg, about 0.002mg / kg~about 0.02mg / kg, about 0.002mg / kg~about 0.05mg / kg, about 0.002mg / kg~about 0.1mg / kg, about 0.002mg / kg~about 0.2mg / kg, about 0.002mg / kg~about 0.5mg / kg, about 0.002mg / kg to about 1mg / kg, about 0.005mg / kg to about 0.01mg / kg, about 0.005mg / kg to about 0.02mg / kg, about 0.005mg / kg to about 0.05mg / kg, about 0.005mg / kg to about 0.1mg / kg, about 0.005mg / kg ~ approximately 0.2 mg / kg, approximately 0.005 mg / kg ~ approximately 0.5 mg / kg, approximately 0.005 mg / kg ~ approximately 1 mg / kg, approximately 0.01 mg / kg ~ approximately 0.02 mg / kg, approximately 0.01 mg / kg ~ approximately 0.05 mg / kg, approximately 0.01 mg / kg ~ approximately 0.1 mg / kg, approximately 0.01 mg / kg ~ approximately 0.2 mg / kg, approximately 0.01 mg / kg ~ approximately 0.5 mg / kg, approximately 0.01 mg / kg ~ approximately 1 mg / kg, approximately 0.02 mg / kg ~ approximately 0.05 mg / kg, approximately 0.02 mg / kg ~ approximately 0.1 mg / kg, approximately 0.02 mg / kg ~ approximately 0.2 mg / kg It could be approximately 0.02 mg / kg to approximately 0.5 mg / kg, approximately 0.02 mg / kg to approximately 1 mg / kg, approximately 0.05 mg / kg to approximately 0.1 mg / kg, approximately 0.05 mg / kg to approximately 0.2 mg / kg, approximately 0.05 mg / kg to approximately 0.5 mg / kg, approximately 0.05 mg / kg to approximately 1 mg / kg, approximately 0.1 mg / kg to approximately 0.2 mg / kg, approximately 0.1 mg / kg to approximately 0.5 mg / kg, approximately 0.2 mg / kg to approximately 0.5 mg / kg, or approximately 0.5 mg / kg to approximately 1 mg / kg.The therapeutically effective dose may be approximately 0.001 mg / kg, approximately 0.002 mg / kg, approximately 0.005 mg / kg, approximately 0.01 mg / kg, approximately 0.02 mg / kg, approximately 0.05 mg / kg, approximately 0.1 mg / kg, approximately 0.2 mg / kg, approximately 0.5 mg / kg, or approximately 1 mg / kg. The therapeutically effective dose may be at least approximately 0.001 mg / kg, approximately 0.002 mg / kg, approximately 0.005 mg / kg, approximately 0.01 mg / kg, approximately 0.02 mg / kg, approximately 0.05 mg / kg, approximately 0.1 mg / kg, approximately 0.2 mg / kg, or approximately 0.5 mg / kg. The therapeutically effective dose may be up to approximately 0.002 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, or 1 mg / kg. The therapeutically effective dose may range from approximately 0.1 mg / kg to approximately 50 mg / kg.The therapeutically effective doses are approximately 0.1 mg / kg to 0.2 mg / kg, 0.1 mg / kg to 0.5 mg / kg, 0.1 mg / kg to 1 mg / kg, 0.1 mg / kg to 2 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 20 mg / kg, 0.1 mg / kg to 50 mg / kg, and 0.2 mg / kg. kg~about 0.5mg / kg, about 0.2mg / kg~about 1mg / kg, about 0.2mg / kg~about 2mg / kg, about 0.2mg / kg~about 5mg / kg, about 0.2mg / kg~about 10mg / kg kg, about 0.2 mg / kg to about 20 mg / kg, about 0.2 mg / kg to about 50 mg / kg, about 0.5 mg / kg to about 1 mg / kg, about 0.5 mg / kg to about 2 mg / kg, about 0.5 mg / kg~about 5mg / kg, about 0.5mg / kg~about 10mg / kg, about 0.5mg / kg~about 20mg / kg, about 0.5mg / kg~about 50mg / kg, about 1mg / kg~about 2mg / kg g, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 50 mg / kg, about 2 mg / kg to about 5 mg / k The therapeutically effective dose may be approximately 0.1 mg / kg, approximately 0.2 mg / kg, approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 10 mg / kg, approximately 20 mg / kg, or approximately 20 mg / kg. The therapeutically effective dose may be at least approximately 0.1 mg / kg, approximately 0.2 mg / kg, approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, or approximately 20 mg / kg. The therapeutically effective dose may be at most approximately 0.2 mg / kg, approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 20 mg / kg, or approximately 50 mg / kg.

[0111] The individual being treated may be a mammal. Mammals may include mice, rats, rabbits, dogs, cats, horses, cattle, sheep, pigs, goats, llamas, alpacas, or yaks. The individual may be a dog, cat, or horse. The individual being treated may be a human.

[0112] Manufacturing method Polypeptides containing FGF17, IGF, or BMP7 ligand amino acid sequences can be purified or synthesized by any preferred method. The nucleic acids encoding the polypeptides can be cloned into a preferred vector and expressed in a preferred cell line. The cell line may be a prokaryotic cell line. The cell line may be a eukaryotic cell line. The cell line may be a mammalian cell line. Polypeptides can be expressed from Eschericia coli. Polypeptides can be expressed from yeast cells, including but not limited to Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, or Yarrowia lipolytica. Polypeptides can be expressed from mouse myeloma cells, including but not limited to NS0, Sp2 / 0, and FO. Polypeptides can be expressed from Chinese hamster ovary (CHO) cells. Polypeptides can be expressed from mammalian cells, including but not limited to COS cells, Vero cells, or BHK cells. Polypeptides can be expressed from human cells, including but not limited to HeLa cells, HEK-293 cells, CAP cells, CAP-T cells, and PER.C6® cells.

[0113] The supernatant from such an expression system can be subjected to one or more purification steps involving centrifugation, ultracentrifugation, filtration, dialysfiltration, tangential flow filtration, dialysis, or chromatography (e.g., cation exchange, ion exchange, hydrophobic interaction, reversed phase, affinity, or size exclusion). The polypeptide can be purified to a degree suitable for administration to humans. Furthermore, the polypeptide can be synthesized for encapsulation in preparations administered to human subjects. The polypeptide can be produced by preferred peptide synthesis methods such as solid-phase synthesis.

[0114] Using the mammalian expression vector pmax cloning, vectors can be constructed with 6xHis tags, StrepII tags, and human IgG1 Fc tags at the C-terminus. DNA fragments encoding secretory myogenic factors are amplified from human open reading frame (ORF) clones by PCR and then inserted into the tagged vector using in-fusion cloning technology (Takara Bio). The expression vector containing the secretory myogenic factor is then transfected into ExpiCHO-S cells using the ExpiFectamine CHO transfection kit (Thermo Scientific) at a rate of 6 × 10⁶ per ml. 6 Transient transfection occurs at individual density.

[0115] Myogenic factors expressed with different tags in the culture supernatant are affinity-purified using different purification media. Polypeptides may contain Fc regions. A matrix or resin containing protein A, protein G, protein L, or any combination thereof can be used for these polypeptides. Batch purification can be facilitated by properly loading this matrix or resin onto a column.

[0116] Purification of immunoglobulin fusion proteins Heterogeneous sequences may contain immunoglobulins or their fragments. If a polypeptide contains immunoglobulins or their fragments, the polypeptide can be purified using the affinity of protein A, G, or L. Proteins A and G are cell surface proteins found in Staphylococcus aureus. They have the property of binding to the Fc region of mammalian antibodies, particularly IgG class antibodies. For use in protein A or G affinity chromatography, protein A or G is conjugated to a solid matrix such as cross-linked uncharged agarose (Sepharose; natural agarose with the charged fraction removed), trisacryl, cross-linked dextran, or silica-based materials. Methods for this are widely known in the art (e.g., binding to a CNBr-activated matrix via the primary amino function of the protein). As previously described by Langone et al. (1982), protein A binds to the Fc portion of IgG (i.e., the Cγ2-Cγ3 interface region of IgG) with high affinity and high specificity. This strongly binds to human allotypes or subclasses IgG1, IgG2, and IgG3, as well as mouse allotypes or subclasses IgG2a, IgG2b, and IgG3.

[0117] After purification with protein A, G, or L, the bound fraction can be eluted and passed through one or more ion exchange columns containing further resin or matrix. The first ion exchanger is typically an anion exchange resin. The pH of the buffer used to load and flow through the first ion exchanger is set to impose opposing total charge changes on the Fc-containing fusion polypeptide and protein A, taking into account the pI of the Fc-containing fusion polypeptide and protein A, as separated using the ion exchanger in flow-through mode according to the present invention. The operating mode of the first anion exchanger according to the present invention requires buffer exchange of the acidic or neutral eluate from the protein A affinity chromatography step with the equilibrium buffer of the first anion exchanger. After being subjected to the first anion exchanger, the Fc-containing fusion polypeptide can be applied, or further purification by conventional purification methods may be deemed necessary. In a more preferred embodiment, a second ion exchange step is performed following the first ion exchange step, in which the antibody is loaded and bound by a second ion exchange medium, and eluted as an essentially monomeric and non-aggregated antibody by increasing the salt and / or pH using a buffer other than the loading buffer.

[0118] In the methods disclosed herein, at least 70%, 80%, or 90% of the Fc-containing fusion polypeptide loaded onto the first ion exchanger can be recovered during the flow-through of the ion exchanger.

[0119] Master cell bank and genetically modified cells Described herein is a master cell bank that may contain cells having nucleic acids encoding one or more IGF ligands or IGF2 fusion polypeptides incorporated into their genomes for the creation of genetically modified cell lines. A master cell bank may contain a plurality of cells, each containing nucleic acids encoding IGF ligands or IGF2 fusion polypeptides. The nucleic acids may be maintained extrachromosomally on plasmids or yeast artificial chromosomes. The nucleic acids may be incorporated into chromosomal locations. The cells may be yeast cells. The yeast may be Pichia pastrix or Saccharomyces cerevisiae. The cells may be mammalian cells. The mammalian cells may be 293T cells or their derivatives (e.g., 293T-Rex). The cells may be bacterial cells.

[0120] Genetically modified mammalian, yeast, or bacterial cells may be stored in a master cell bank containing a cryopreservative suitable for freezing at or below approximately -80°C. The master cell bank may contain approximately 10-30% glycerin or DMSO and may be suitable for long-term storage at or below approximately -80°C. The master cell bank can store genetically modified mammalian, yeast, or bacterial strains for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years, or longer.

[0121] Medicinally acceptable excipients, carriers, and diluents Polypeptides comprising FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans described herein, can be administered in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, or diluents. The exact components may vary based on the preferred route of administration. Excipients used in the pharmaceutical composition may provide further functionality to the polypeptide by optimizing the polypeptide for a specific route of administration (e.g., intravenous, topical, subcutaneous, or intramuscular), increasing polypeptide stability, increasing penetration into the target tissue (e.g., muscle or skin), increasing residence time at a specific site, increasing solubility, increasing polypeptide efficacy, and / or reducing inflammatory responses occurring concurrently with administration.

[0122] The composition may be included in the pharmaceutical composition together with a solubilizer, emulsifier, or dispersant. The solubilizer can enable high-concentration fusion polypeptide solutions of at least about 2 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, or more than 20 mg / mL. In aqueous pharmaceutical compositions, carbomers act as emulsifiers and viscosity modifiers. pharmaceutically acceptable excipients may include or consist of carbomers. Carbomers may include or consist of carbomer 910, carbomer 934, carbomer 934P, carbomer 940, carbomer 941, carbomer 1342, or combinations thereof. In aqueous pharmaceutical compositions, cyclodextrins act as solubilizers and stabilizers. pharmaceutically acceptable excipients may include or consist of cyclodextrins. Cyclodextrins may include or consist of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, or combinations thereof. Lecithin in pharmaceutical compositions may act as a solubilizer. Solubilizers may include or consist of lecithin. Poloxamers in pharmaceutical compositions may act as emulsifiers, solubilizers, and dispersants. Medicinally acceptable excipients may include or consist of poloxamers. Poloxamers may include or consist of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or combinations thereof. Polyoxyethylene sorbitan fatty acid esters in pharmaceutical compositions may act as emulsifiers, solubilizers, surfactants, and dispersants. Medicinally acceptable excipients may include or consist of polyoxyethylene sorbitan fatty acid esters. Polyoxyethylene sorbitan fatty acid esters may include or consist of polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, polysorbate 120, or combinations thereof.Polyoxyethylene stearate in pharmaceutical compositions serves as an emulsifier, solubilizer, surfactant, and dispersant. pharmaceutically acceptable excipients may include or consist of polyoxyl stearate. Polyoxyethylene stearate may include or consist of polyoxyl 2, polyoxyl 4, polyoxyl 6, polyoxyl 8, polyoxyl 12, polyoxyl 20, polyoxyl 30, polyoxyl 40, polyoxyl 50, polyoxyl 100, polyoxyl 150, polyoxyl 4, polyoxyl 8, polyoxyl 12, polyoxyl 32, polyoxyl 150, or combinations thereof. In pharmaceutical compositions, sorbitan esters serve as emulsifiers, solubilizers, nonionic surfactants, and dispersants. Medicinally acceptable excipients may include or consist of sorbitan esters. Sorbitan esters may include or consist of sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan stearate, sorbitan trioleate, sorbitan sesquioleate, or combinations thereof. Solubility may be achieved by protein carriers. Protein carriers may include recombinant human albumin.

[0123] Polypeptides containing FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans described herein, may be formulated to increase stability. Polypeptides may need to be stabilized in aqueous preparations to prevent degradation. Stabilizers may include pH buffers, salts, amino acids, polyols / disaccharides / polysaccharides, liposomes, surfactants, antioxidants, reducing agents, or chelating agents. Stabilizers may include or consist of polyols / non-reducing sugars. Non-reducing sugars may include or consist of sucrose, mannitol, trehalose, raffinose, stachyose, xylitol, starch, velvascose, or combinations thereof. Polypeptides can be encapsulated in liposomes to increase their stability. Stabilizers may include or be derived from liposomes. Liposomes may include or be derived from dipalmitoylphosphatidylcholine (DPPC) liposomes, phosphatidylcholine:cholesterol (PC:Chol) (70:30) liposomes, or dipalmitoylphosphatidylcholine:dipalmitoylphosphatidylserine (DPPC:DPPS) liposomes (70:30). Nonionic surfactants can increase the stability of polypeptides. Stabilizers may include or be derived from nonionic surfactants. Nonionic surfactants may include or consist of polysorbates (e.g., polysorbate 80, polysorbate 20), alkyl sugar alkyl ethers and alkyl glyceryl ethers, polyoxyethylene(4) lauryl ether; polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, or combinations thereof. Polypeptides can be formulated with protein surfactants such as recombinant human serum albumin as a stabilizer.Antioxidants or reducing agents can increase the stability of polypeptides. Stabilizers may include or consist of antioxidants or reducing agents. Reducing agents may include or consist of dithiothreitol, ethylenediaminetetraacetic acid, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride, tris(hydroxypropyl)phosphine, or combinations thereof. Antioxidants may include or consist of methionine, ascorbic acid, citric acid, alpha-tocopherol, sodium bisulfite, ascorbyl palmitate, erythorbic acid, or combinations thereof. Chelating agents can stabilize polypeptides by reducing protease activity. Stabilizers may include or consist of chelating agents. Chelating agents may include or consist of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), metal complexes (e.g., Zn-protein complexes), or combinations thereof. Buffering agents can stabilize polypeptides by reducing their acid hydrolysis. Stabilizers may include or consist of buffering agents. Buffering agents may include or consist of sucrose octa-sulfate, ammonium carbonate, ammonium phosphate, boric acid, sodium citrate, potassium citrate, lactic acid, 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES), hydroxymethylaminomethane (Tris), calcium carbonate, calcium phosphate, or combinations thereof.

[0124] Furthermore, polypeptides comprising FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans, as described herein, can be encapsulated or conjugated in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively) prepared by coacervation technology or interfacial polymerization, or in colloidal transport systems (e.g., liposomes, albumin spheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, edited by Oslo, A. (1980).

[0125] Polypeptides comprising FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans described herein, may be formulated or carried together with anti-inflammatory agents. The anti-inflammatory agents may include or consist of corticosteroids. Corticosteroids may include, or consist of, hydrocortisone, cortisone, betamethasone (Celestone), prednisone (Prednisone Intensol), prednisolone (Orapred, Prelone), triamcinolone (Aristospan Intra-Articular, Aristospan Intralesional, Kenalog), methylprednisolone (Medrol, Depo-Medrol, Solu-Medrol), or dexamethasone (Dexamethasone Intensol). Anti-inflammatory agents may include, or consist of, nonsteroidal anti-inflammatory drugs (NSAIDs). NSAIDs may include, or consist of, aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketrolac, nabumetone, naproxen, oxaprozin, piroxicam, sarsalat, sulindac, or tolmetine.

[0126] Polypeptides comprising FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans described herein, may be incorporated into pharmaceutical compositions suitable for intravenous administration, comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. Polypeptides relating to this disclosure can be administered suspended in a sterile solution. The solution may be one commonly used for the administration of biological preparations and may, for example, contain about 0.9% NaCl or about 5% dextrose. Furthermore, the solution may contain one or more of the following: buffers such as acetate, citrate, histidine, succinate, phosphate, potassium phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); surfactants such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; polyols / disaccharides / polysaccharides such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids such as glycine, histidine, leucine, or arginine; antioxidants such as ascorbic acid or methionine; or chelating agents such as EDTA or EGTA.

[0127] Polypeptides comprising FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans described herein, may be included in pharmaceutical compositions suitable for intramuscular or subcutaneous administration, comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. Preparations suitable for intramuscular or subcutaneous injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders that can be reconstituted in sterile injection solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include ethanol, polyols (such as inositol, propylene glycol, polyethylene glycol, glycerin, and cremophor) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Appropriate fluidity is maintained, for example, by using coating agents such as lecithin, and in the case of dispersions, by maintaining the required particle size, and by using surfactants. Preparations suitable for subcutaneous injection may also contain optional additives such as preservatives, humectants, emulsifiers, and suspending agents.

[0128] Polypeptides comprising FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, as described herein, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans, may be formulated as creams, gels, pastes, ointments, or emulsions for topical administration. Excipients in creams, gels, pastes, ointments, or emulsions may include gelatin, casein, lecithin, gum arabic, cholesterol, tragacanth gum, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycol, polyoxyethylene stearate, colloidal silicon dioxide, phosphates, sodium dodecyl sulfate, calcium carboxymethylcellulose, sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, sugars, and starch.

[0129] Polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequence and an amino acid sequence from a heterologous polypeptide, as described herein, or excipients used with combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans, may enable the storage, formulation, or administration of highly concentrated preparations. In certain embodiments, the highly concentrated fusion polypeptide is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 20, 25, 40, 45, or 50 milligrams or more per milliliter.

[0130] Polypeptides and / or compositions relating to this disclosure can be transported / stored, lyophilized, and reconstituted before administration. Lyophilized ligand-fusion polypeptide preparations may contain fillers such as mannitol, sorbitol, sucrose, trehalose, and dextran 40. Lyophilized preparations can be placed in glass vials. Fusion polypeptides, whether reconstituted or not, can be buffered to a specific pH (generally less than 7.0) at formulation. In certain embodiments, the pH may be 4.5–6.5, 4.5–6.0, 4.5–5.5, 4.5–5.0, or 5.0–6.0.

[0131] kit Further described herein are kits comprising, in a suitable container, one or more polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequence and an amino acid sequence from a heterologous polypeptide, or one or more combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans, and further comprising one or more additional components selected from instructions for use, diluents, excipients, carriers, and administration devices.

[0132] In one embodiment, a method for preparing a treatment for a disease or disorder of soft tissue or muscle is described herein, comprising the step of mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with a polypeptide comprising an FGF17, IGF2, or BMP7 amino acid sequence and an amino acid sequence from a heterologous polypeptide, or a combination of a fibroblast growth factor receptor agonist and glycosaminoglycan, an insulin-like growth factor 1 receptor (IGF1R) agonist and short-chain fatty acid, and a BMP receptor agonist and an mTOR activator and / or glycosaminoglycan. In one embodiment, a method for preparing a treatment for a disease or disorder of soft tissue or muscle for storage or transport is described herein, comprising the step of lyophilizing one or more antibodies relating to the present disclosure.

[0133] The invention disclosed herein may be better understood from the details of the experiments described below. However, those skilled in the art will readily understand that the specific methods and results described herein only illustrate the invention as further fully described in the appended claims below. Unless otherwise noted, this disclosure is not limited to specific procedures or materials, and these may vary. It will also be understood that the technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them. [Examples]

[0134] Example 1 Recombinant protein expression and purification Mammalian expression plasmids containing genes with different tags were transiently transfected into CHO cells. The genes were expressed, producing proteins, which were subsequently secreted into the culture medium. The proteins in the medium were visualized on a polyacrylamide gel, and their activity was measured by an in vitro functional assay. The recombinant proteins in the medium were then affinity-purified. The purified proteins were visualized on a polyacrylamide gel to assess their purity, and their biological activity was investigated by an in vitro functional assay.

[0135] Expression vector construction: Using the mammalian expression vector pmax cloning, vectors were constructed with 6×His tags, StrepII tags, and human IgG1 and IgG4 Fc tags at the C-terminus. DNA fragments encoding secretory myogenic factors were amplified from human open reading frame (ORF) clones by PCR and subsequently inserted into the tagged vectors using in-fusion cloning technology (Takara Bio).

[0136] Expression of secretory myogenic polypeptides: Expression vectors containing secretory myogenic factors were transfected into ExpiCHO-S cells using the ExpiFectamine CHO transfection kit (ThermoScientific) at a rate of 6 × 10⁶ per ml. 6 Transient transfection was performed at a density of [number] cells. After 18–22 hours, CHO feed and enhancer were added to the transfected cultures. Subsequently, expressed proteins were monitored every 24 hours by SDS-PAGE until the maximum expression level was achieved. In most cases, the cell cultures were harvested on day 4 and the cells were centrifuged. The supernatant was centrifuged again to remove cell debris. The clarified culture supernatant containing secretory myogenic factors was stored at -80°C or processed promptly for use.

[0137] Measurement of expression levels of secretory myogenic polypeptides: To measure the improved expression levels of secretory myogenic factors, the following three protein analysis techniques were applied: sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Western blotting was performed to identify myogenic factors. ELISA was used to measure the absolute amount of myogenic factors in the culture supernatant.

[0138] Isolation of the prepared myogenic polypeptides: Myogenic factors expressed with different tags in the culture supernatant were affinity-purified using different purification media. For Fc fusion factors, they were specifically bound using either protein A magnetic beads (GenScript) or protein A membrane columns (Takara Bio). For 6×His-tagged factors, the factors were isolated using NTA-magnetic beads (NEB).

[0139] Example 2: Purified IGF2-hFcm enhanced the differentiation of human myoblasts. Figure 1A: Suspended CHO cells were transiently transfected with a plasmid encoding IGF2-hFcm. IGF2-hFcm was affinity-purified using a protein A membrane column. The purified IGF2-hFcm was added to human myoblast cultures for 96 hours. Myosin heavy chains (MyHCs) were immunostained and imaged by fluorescence microscopy. The percentage of MyHC area in human myoblasts treated with purified IGF2-hFcm was significantly higher than that in human myoblasts treated with vehicle control (One-Way ANOVA Tukey Honest Significant Difference, n=2~6).

[0140] [Table 1]

[0141] Example 3: IGF2-LhFc4 enhanced the differentiation of human myoblasts. I Suspended CHO cells were transiently transfected with a plasmid encoding GF2-LhFc4. IGF2-LhFc4 was affinity-purified using a protein A membrane column. The purified IGF2-LhFc4 was added to human myoblast cultures for 96 hours, with daily medium changes. Myosin heavy chains (MyHC) were immunostained and imaged by fluorescence microscopy. The percentage of MyHC area in human myoblasts treated with purified IGF2-LhFc4 was significantly higher than that in human myoblasts treated with vehicle control (One-Way ANOVA Tukey Honest Significant Difference, n=2~6).

[0142] [Table 2]

[0143] Example 4 Differentiation of purified HSA-L-IGF2R61A human myoblasts H Suspended CHO cells were transiently transfected with a plasmid encoding SA-L-IGF2R61A. HSA-L-IGF2R61A was affinity-purified using a protein A membrane column. The purified HSA-L-IGF2R61A was added to human myoblast cultures for 96 hours, with daily medium changes. Myosin heavy chains (MyHC) were immunostained and imaged by fluorescence microscopy. The percentage of MyHC area in human myoblasts treated with purified HSA-L-IGF2R61A was significantly higher than that in human myoblasts treated with vehicle control (One-Way ANOVA Tukey Honest Significant Difference, n=2~6).

[0144] [Table 3]

[0145] Example 5: IGF2 and IGF2 receptor are expressed in human myoblasts. IGF in young (17-21 year old Caucasian male) and aged (68-69 year old Caucasian male) human myoblast cell lines 2 and IGF2 receptor body R Bar graphs and quantitative tables for NASeq expression. Myoblasts were cultured for 96 hours in growth medium (GM) or fusion medium (FM). Fresh medium was added every 24 hours. Mean ± SEM. n=6. Expression is expressed as FPKM. Significant p-value (young GM to aged GM: 3.54E-04).

[0147] [Table 4]

[0149] [Table 5]

[0150] Example 6: Sodium butyrate increases muscle fusion. Mouse myoblasts were treated with PBS or sodium butyrate at concentrations of 0.1 nM, 1 nM, and 10 nM. The myoblasts were cultured for 48 hours, with fresh medium added every 24 hours. Cells were treated with EdU (30 μM) for 2–5 hours, fixed with ethanol, stained with Hoechst 3342, and immunostained to examine proliferation (measured by the percentage of EdU-positive stained cells (%EdU)) and differentiation (measured by the increase in area of ​​cells positively stained with embryonic myosin heavy chain (%eMyHC) relative to a negative control containing only medium and vehicle). Compared to untreated myoblasts, cells treated with 1 nM sodium butyrate showed significant differences (see figure). 2 As shown in A, the fusion rate increased. Significance was determined by a p-value of less than 0.05 using the one-way ANOVA Tukey Honest Significant Difference test.

[0151] figure 2 A: Bar graph showing fusion indices in response to sodium butyrate (NaBut) compared to the vehicle. Myoblasts were cultured for 48 hours in the presence of the indicated dose of NaBut. Fresh medium and NaBut were added every 24 hours. Mean ± SD. Tables also show quantification of fusion indices and p-values. (Student's two-tailed t-test) * (p<0.05, n=3~5)

[0152] [Table 6]

[0153] Example 7: Sodium butyrate increases IGF2 activity. Human myoblasts were treated with either PBS (vehicle), IGF2 (15 ng / mL), sodium butyrate, or IGF2 and sodium butyrate. Fresh medium was added every 24 hours. After 96 hours, cells were treated with EdU (30 μM) for 2–5 hours, fixed with ethanol, stained with Hoechst 3342, and immunostained to examine proliferation (measured by the percentage of EdU-positive stained cells (%EdU)) and differentiation (measured by the increase in area of ​​cells positively stained with embryonic myosin heavy chain (%eMyHC) relative to a negative control treated with only medium and vehicle). The total area of ​​eMyHC-positive cells was analyzed, and treated cells were compared to cells treated with vehicle alone. Cells treated with IGF alone and cells treated with IGF2 and sodium butyrate showed a significant increase in differentiation. Compared to cells treated with IGF2 alone, the total area of ​​eMyHC cells was significantly increased in cells treated with 1 nM and 100 nM sodium butyrate and IGF2. Significance was determined by a one-way ANOVA Tukey Honest Significant Difference test, with a p-value of less than 0.05.

[0154] figure 2B: Bar graph of the fusion index of mouse myoblasts in response to sodium butyrate (NaBut) compared to vehicle. Mouse myoblasts were cultured for 48 hours in the presence of the indicated doses of NaBut. Fresh medium and NaBut were added every 24 hours. Mean ± S.D. Table quantification of fusion index and p-values is shown. (by Student's two-tailed t-test * p<0.05, n=3-5). Significant p-values (vehicle vs IGF2: 0.015 ug / mL: 6.33E-06, vehicle vs NaBut: 1 nM IGF2: 0.015 ug / mL: 1.79E-11, vehicle vs NaBut: 100 nM IGF2: 0.015 ug / mL: 1.79E-11) Figure 2 Table of data for B

[0155] [Table 7]

[0156] Figure 2 C Quantification by bar graph of the area percentage of eMyHC-positive human myoblasts in response to the indicated treatment compared to IGF2 (15 ng / mL). Myoblasts were cultured for 96 hours in the presence of the indicated dose of BMP7. Fresh medium and BMP7 were added every 24 hours. Mean ± S.D. (by One-Way ANOVA Tukey Honest Significant Difference * p<0.05, n=2-12) Figure 2 Table of data for C

[0157] [Table 8]

[0158] Example 8 Sodium butyrate increases IGF2 activity Human myoblasts were treated with either PBS (vehicle), IGF2 (15 ng / mL), sodium butyrate, or IGF2 and sodium butyrate. Fresh medium was added every 24 hours. After 48 hours, cells were treated with EdU (30 μM) for 2–5 hours, fixed with ethanol, stained with Hoechst 3342, and immunostained to examine proliferation (measured by the percentage of EdU-positive stained cells (%EdU)) and differentiation (measured by the increase in area of ​​cells positively stained with embryonic myosin heavy chain (%eMyHC) relative to a negative control containing only medium and vehicle). Figure 3 As shown in A, the total area of ​​eMyHC-positive cells was analyzed, and treated cells were compared to cells treated with the vehicle alone. Myoblasts treated with 0.03 ug / mL IGF2 or a combination of IGF2 and sodium butyrate showed a significant increase in eMyHC-positive area compared to cells cultured with the vehicle alone.

[0159] figure 3 A: Bar graph showing area % of eMyHC-positive senescent human myoblasts (68-year-old Caucasian male) in response to the indicated treatment, compared to the vehicle. Myoblasts were cultured for 96 hours in the presence of the indicated doses of the factor. Fresh medium and factor were added every 24 hours. Mean ± SD. Significant p-values ​​(vehicle ~ IGF2: 0.03 ug / mL: 1.42E-08, vehicle ~ NaBut: 1 nM IGF2: 0.03 ug / mL: 1.79E-11, vehicle ~ NaBut: 10 nM IGF2: 0.03 ug / mL: 1.80E-11, vehicle ~ NaBut: 100 nM IGF2: 0.03 ug / mL: 1.79E-11). Figure. 3Bar graph quantification of area % of eMyHC-positive human myoblasts (68-year-old Caucasian male) in response to the indicated treatment, compared to IGF2 (15 ng / mL). Myoblasts were cultured for 96 hours in the presence of the indicated dose of BMP7. Mean ± SD. Significant p-values ​​(IGF2~NaBut: 1 nM IGF2: 0.03 ug / mL: 1.88E-3, Vehicle~NaBut: 10 nM IGF2: 0.03 ug / mL: 4.80E-3, Vehicle~NaBut: 100 nM IGF2: 0.03 ug / mL: 1.87E-3) (One-Way ANOVA Tukey Honest Significant Difference) * (p<0.05, n=2~12) figure 3 Table of data about A

[0160] [Table 9]

[0161] Myoblasts treated with a combination of IGF2 and sodium butyrate were compared to cells treated with IGF2 alone. (Figure) 3 As shown in B and Table 12, all cells treated with the combination of IGF2 showed a significant increase compared to cells treated with IGF2 alone. Significance was determined by a one-way ANOVA Tukey Honest Significant Difference test, with a p-value of less than 0.05.

[0162] figure 3 Table of data for B

[0163] [Table 10]

[0164] Example 9: IGF2 increases MYOG expression in DM1 human myoblasts. FBar graphs showing the multiplicative changes in myogenic gene expression in DM1 human myoblasts in response to the indicated treatments, compared to the M (vehicle). Myoblasts were cultured for 48 hours in the presence of factors (BMP7 50 ng / mL, butyrate 100 nM, IGF2 200 ng / mL). Mean ± SD. Significant p-values ​​(FM~IGF2: 4.94E-04, FM~IGF2_NaBut: 6.53E-03) * Table of mean and p-values ​​for MYF5, MYOD1, and MYOG (n=3) (p<0.01).

[0165] De Data table

[0166] [Table 11]

[0167] Example 10: IGF2 receptor is expressed on chondrocytes and osteocytes. figure 4 A: Bar graph showing IGF2 receptor expression on cartilage-associated cells. Data obtained from Ramilowsky et al, Nature 2015. figure 4 Table of RNA expression data (TPM) for A

[0168] [Table 12]

[0169] Example 11: IGF2 treatment enhances proliferation and fusion in DM1 human myoblasts (32-year-old Caucasian female). E Bar graph showing the percentage of dU-positive human myoblasts (32-year-old Caucasian woman), IPercentage of MyHC area in response to GF2. Myoblasts were cultured for 72 hours for proliferation and 96 hours for fusion (in the presence of the indicated factors). Mean ± SD. Mean ± SD. Significant p-values ​​(EdU: vehicle ~ IGF2: 6.8E-3, %eMyHC area: vehicle ~ IGF2: 1.9E-4) (by Student's two-tailed t-test) * p<0.05, n=3~6).

[0170] De Data table

[0171] [Table 13]

[0172] De Data table

[0173] [Table 14]

[0174] Example 12: IGF2 increases the expression of MYH3, CKM, and ATP1B1 in DM1 human myoblasts (32-year-old Caucasian woman). Bi Bar graphs showing the fold change in MYH3 and CKM expression in DM1 human myoblasts (32-year-old Caucasian female) in response to the indicated treatment, compared to the vehicle. Myoblasts were cultured for 96 hours in the presence of the factor (IGF2 200 ng / mL). Mean ± SD. Significant p-values ​​(MYH3: vehicle ~ IGF2: 1.13E-03, CKM: vehicle ~ IGF2: 7.67E-03) F Bar graph showing the multiplicative change in ATP1B1 expression in DM1 human myoblasts (32-year-old Caucasian female) in response to the indicated treatment, compared to the vehicle (M). Myoblasts were cultured for 48 hours in the presence of the factor (IGF2 200 ng / mL). Mean ± SD. Significant p-value (vehicle ~ IGF2: 3.11E-05) (Student's two-tailed t-test). * p<0.05, n=3).

[0175] De Data table

[0176] [Table 15]

[0177] De Data table

[0178] [Table 16]

[0179] Example 13: Systemic administration of IGF2 / NaB protects against age-induced muscle dysfunction. I Mice aged 21–24 months were administered subcutaneously with GF2 (50 ug / kg) or NaB (1.2 g / kg), IGF2 / NaB (150 ug / kg, 1.2 g / kg), or vehicle (PBS) for 14 days. Muscle function was evaluated on days 13 and 14. 。1 Grip strength evaluated on the third day. First grade Fu is This shows the grip strength of both limbs. **** p<0.0001, ** p=0.0043, ** p=0.001 (One-way ANOVA, multiple comparisons) . before Limb strength, **** p<0.0001, * p=0.0368, * p=0.0187 (One-way ANOVA, multiple comparisons) 。2 Treadmill performance was measured on day 14 using an induced treadmill running model, where the speed was set to gradually increase by 2 m / min every minute. The distance covered is shown. *** p=0.0005, * p=0.0459, **** p<0.0001 (One-way ANOVA, multiple comparisons ) Fatigue Time until deterioration *** p=0.0002, **p=0.0024 (One-way ANOVA, multiple comparisons ) most large speed *** p=0.0004, ** p=0.001 3. thing (kj) ** p=0.0026, ** p=0.0035 (One-way ANOVA, multiple comparisons).

[0180] Example 14: Systemic administration of IGF2 / NaB is safe. Bi Subcutaneous injections of either Hickle or IGF2 / NaB were administered to 21-month-old mice for 14 days. Blood and serum were collected, and complete blood counts and metabolic panels for liver, kidney, and pancreatic function were evaluated. . measurement These are four representative graphs from the 37 defined readouts, showing white blood cell count (independent t-test, p=0.8020), albumin concentration (independent t-test, p>0.9999), creatinine concentration (independent t-test, p=0.5490), and calcium concentration (independent t-test, p=0.811).

[0181] Example 15: Systemic administration of IGF2 / But protects against dexamethasone-induced muscle atrophy. De Xamethasone (25 mg / kg intraperitoneally) was administered to 12-week-old mice for 14 days simultaneously with subcutaneous injection of IGF2 / NaB (150 ug / kg, 1.2 g / kg) or vehicle (PBS). Muscle function was evaluated on days 13-14. Grip strength was evaluated on day 13; the graph shows the grip strength of both limbs measured on day 13. Power and ratio of both limbs Power and This shows that the specific force of each limb was calculated as the ratio of the force (mN) of each limb to the weight (g). *** p=0.0003, *** p=0.0004 (independent t-test) 。1 The graph shows the grip strength measured on day 3, and the forelimb grip strength measured on day 13. Power and Ratio of forelimbs Power and This shows that the specific force of the forelimb was calculated as the ratio of the force of the forelimb (mN) to its weight (g). ** p=0.0012, ***p=0.0005 (independent t-test) 。1 On day 5, the mice were euthanized and TA was collected for histological analysis. The graph shows the muscle fiber size distribution as evaluated using SMASH software. ** p=0.054, * p=0.037, and **** p<0.0001 (2-way ANOVA, multiple comparisons).

[0182] Example 16: BMP7 induces myoblast proliferation. figure 5 A) Bar graph quantification of EdU-positive mouse myoblasts % in response to BMP7. Myoblasts were cultured for 48 hours in the presence of the indicated doses of BMP7. Fresh medium and BMP7 were added every 24 hours, followed by 2 hours of EdU application and fixation. Mean ± SD. Significant p-values ​​(vehicle-BMP7 0.025 ug / mL: 1.21E-07, vehicle-BMP7 0.075 ug / mL: 8.05E-07, vehicle-BMP7 0.2255 ug / mL: 3.37E-03, vehicle-BMP7 0.9 ug / mL: 4.99E-02). Figure. 5 B) Bar graph quantification of % of EdU-positive human myoblasts (68-year-old Caucasian male) that responded to BMP7. Myoblasts were cultured for 72 hours in the presence of the indicated dose of BMP7. EdU was applied to the cells for 4 hours, after which they were fixed. Mean ± SD. Significant p-value (vehicle ~ BMP7 1.56 ng / mL: 0.04). (Welch's one-sided t-test) * (p<0.05, n=2) figure 5 Table of data for A: BMP7 mouse myoblast proliferation

[0183] [Table 17]

[0184] figure 5 Table of data for B: BMP7 Human Myoblast Proliferation

[0185] [Table 18]

[0186] Example 17: Leucine increases the mitotic activity of BMP7. figure 6 A) Bar graph of EdU-positive mouse myoblast percentage compared to vehicle. Mouse myoblasts were cultured for 48 hours in the presence of the indicated factors, followed by 2 hours of EdU application and then fixation. Mean ± SD. Significant p-values ​​(FM~BMP7: 0.008 ug / mL: 3.17E-02, FM~Leucine: 300 uM BMP7: 0.008 ug / mL: 2.77E-03, FM~Leucine: 100 uM BMP7: 0.008 ug / mL: 3.72E-05, FM~Leucine: 900 uM BMP7: 0.008 ug / mL: 2.52E-03). (One-Way ANOVA Tukey Honest Significant Difference) * (p<0.05, n=2~6) Example 18: Hyaluronic acid (HA) increases the mitotic activity of BMP7.

[0187] figure 7 A: Bar graph of EdU-positive mouse myoblast percentage compared to vehicle. Mouse myoblasts were cultured for 48 hours in the presence of the indicated factor. EdU was applied and fixed. Mean ± SD. (Student's one-sided t-test for increased activity) * (p<0.05, n=2~6) Example 19: BMP7 receptor is expressed in human myoblasts.

[0188] figure 8 A: Bar graph showing BMP7 receptor RNASeq expression in young and aged human myoblast cell lines (Caucasian males aged 68-69 years). Myoblasts were cultured in fusion medium for 96 hours. Fresh medium was added every 24 hours, followed by RNA extraction and sequencing. Mean ± SEM. n=3. Expression is expressed as FPKM.

[0189] Example 20 Treatment for chondrocyte proliferation in cartilage damage and osteoarthritis figure 9 A: Bar graph showing the expression of the BMP7 receptor on cartilage-associated cells. Data is from Ramilowsky et al., Nature, 2015.

[0190] Example 21: FGF17-hFcm promotes the proliferation of mouse myoblasts. Figure 1 0 A) Suspended CHO cells were transiently transfected with either an empty control plasmid or a plasmid encoding FGF17-hFcm. After 4 days, the culture supernatant was collected and added to mouse myoblast cultures for 48 hours, followed by 2 hours of EdU application and then fixation. The proportion of EdU-positive mouse myoblasts treated with the culture supernatant of CHO cells expressing FGF17-hFcm was significantly higher than the proportion of EdU-positive mouse myoblasts treated with either the vehicle control or the culture supernatant of CHO cells expressing an empty control vector (One-Way ANOVA Tukey Honest Significant Difference, n=2~6). Figure 1 0 B) Suspended CHO cells were transiently transfected with a plasmid encoding FGF17-hFcm. After 4 days, the culture supernatant was collected, and FGF17-hFcm was affinity-purified using a protein A membrane column. The purified FGF17-hFcm was added to mouse myoblast cultures for 48 hours, followed by 2 hours of EdU application and fixation. The proportion of EdU-positive mouse myoblasts treated with purified FGF17-hFcm was significantly higher than the proportion of EdU-positive mouse myoblasts treated with the culture supernatant of CHO cells expressing an empty control vector (One-Way ANOVA Tukey Honest Significant Difference, n=2~6).

[0191] Figure 1 0 Table of data about A

[0192] [Table 19]

[0193] Figure 1 0 Table of data for B

[0194]

Table 20

[0195] Example 22: FGF17 Mutants Improved Protein Expression Levels in CHO Cells Figure 1 1 A: SDS-PAGE of culture supernatant from CHO cells transiently transfected with plasmids encoding FGF17-hFcm or mutants thereof (FGF17d181-216-hFcm, FGF17d204-216-hFcm, FGF17R204QK207Q-hFcm). The expressed FGF17-hFcm protein and mutant proteins are indicated by white arrows. Figure 1 1 B) Culture supernatant from CHO cells transiently transfected with plasmids encoding different FGF17 variants was added to mouse myoblast cultures for 48 hours, followed by 2 hours of EdU application and fixation. The percentage of EdU-positive mouse myoblasts treated with culture supernatant from CHO cells expressing wild-type FGF17-hFcm or mutant FGF17-hFcm (AA204-216 deletion mutant and R204Q K207Q point mutant) was significantly higher than that of EdU-positive mouse myoblasts treated with culture supernatant from CHO cells expressing an empty control vector (One-Way ANOVA Tukey Honest Significant Difference, n=2~6).

[0196] Figure 1 1 B

[0197]

Table 21

[0198] Example 23: FGF17 mutant improved protein expression levels in CHO cells. Figure 1 2 A) SDS-PAGE of culture supernatant from CHO cells transiently transfected with plasmids encoding FGF17-hFcm or its variants (FGF17d197-216-hFcm, FGF17K191AK193AS200A-hFcm). Expressed FGF17-hFcm protein and mutant proteins are indicated by white arrows. Figure 1 2 B) Culture supernatants from CHO cells transiently transfected with plasmids encoding different FGF17 were added to mouse myoblast cultures for 48 hours, followed by 2 hours of EdU application and fixation. The proportion of EdU-positive mouse myoblasts treated with culture supernatants from CHO cells expressing wild-type FGF17-hFcm or mutant FGF17-hFcm (AA197-216 deletion mutant and K191AK193AS200A point mutant) was significantly higher than that of mouse myoblasts treated with culture supernatants from CHO cells expressing an empty control vector (One-Way ANOVA Tukey Honest Significant Difference, n=2~6).

[0199] Figure 1 2 B

[0200] [Table 22]

[0201] Example 24: Human serum albumin (HSA)-fused FGF17 is more stable in culture medium than FGF17 without an HSA-fused tag. HSA-FGF17 and FGF17 were incubated in a medium in a CO₂ incubator at 37°C. Aliquots were taken at different time points (days 0, 1, 3, 5, and 7) and stored at -80°C. The activity of each sample was evaluated by an in vitro proliferation assay using mouse myoblasts. The number of nuclei at each time point from the proliferation assay was normalized to the number of nuclei on day 0.

[0202] Example 25 Differential induction of myogenic gene expression by FGF17 in mouse myoblasts RNA expression (fold change relative to FM) of myogenic genes in response to vehicle (FM) in a mouse myoblast cell line, monitored by real-time qPCR. Myoblasts were cultured in fusion medium for 48 hours. Mean ± SD. n=3. (Y, Z) Differential induction of myogenic gene expression by FGF17 in human myoblasts. Quantitative table (determined by real-time qPCR) of RNA expression (fold change relative to FM (fusion medium (DMEM + 2% horse serum))) of myogenic genes in response to FM or rh-FGF17 in an aged human myoblast cell line. Myoblasts were cultured in FM for (B) 48 hours or (C) 72 hours. Mean ± SD. n=3.

[0203]

Table 23

[0204] Example 26 FGF17 receptor is expressed in human myoblasts Figure 13 A: Bar graph of FGF17 receptor RNASeq expression in young and aged human myoblast cell lines. Myoblasts were cultured in fusion medium for 96 hours, with fresh medium added every 24 hours, followed by RNA extraction and sequencing. Mean ± SEM. n=3. Expression values are reported as FPKM.

[0205]

Table 24

[0206] Example 27: Heparin increases the mitotic activity of FGF17. figure 14 A: Bar graph of % EdU-positive mouse myoblasts compared to vehicle. Myoblasts were cultured for 48 hours in the presence of the indicated factors. Fresh medium and factors were added every 24 hours, followed by 2 hours of EdU application and fixation. Mean ± SD. Table quantification of %EdU and p-values ​​(One-Way ANOVA Tukey Honest Significant Difference) * (p<0.05, n=2~6)

[0207] [Table 25]

[0208] Example 28: Hyaluronic acid (HA) increases the mitotic activity of FGF17. figure 15A Bar graph showing % EdU-positive mouse myoblasts compared to the vehicle. Myoblasts were cultured for 48 hours in the presence of the factors shown. Fresh medium and factors were added every 24 hours, followed by 2 hours of EdU application and fixation. Mean ± SD. Tables show quantitative data for %EdU and p-values. (Increased activity by Student's one-sided t-test) * (p<0.05, n=2~6)

[0209] [Table 26]

[0210] Example 29: Dextran sulfate (DS) increases the fission-promoting activity of FGF17. eachBar graphs comparing total EdU-positive mouse myoblasts under different conditions. Myoblasts were cultured for 48 hours in the presence of the indicated factors. Fresh medium and factors were added every 24 hours, followed by 2 hours of EdU application and fixation. Mean ± standard deviation (SD). EdU count per field of view and p-values ​​for mouse assays (Student's one-sided t-test for increased activity). * Table quantification of evidence for the effect (p<0.05, n=3~6, synergistic value less than 1). each Bar graphs for total EdU-positive human myoblasts under different conditions. Myoblasts were cultured for 72 hours in the presence of the indicated factors, followed by 4 hours of EdU application and fixation. Fresh medium and factors were added every 24 hours. Mean ± standard deviation (SD). EdU-positive count and p-values ​​for human assays (by Student's one-sided t-test for increased activity). * Table quantification of evidence for the effect (p<0.05, n=3~6, synergistic value less than 1).

[0211] De Data table

[0212] [Table 27]

[0213] De Data table

[0214] [Table 28]

[0215] Example 30: Intramuscular administration of FGF17 enhanced muscle regeneration in an aged mouse model with BaCl2 damage. figure 16 A) Overall experiment. Chemical damage was induced in the TA of 78-week-old mice using intramuscular injection of 1.2% BaCl2 (7 ul / TA). FGF17 (500 ng / mL) was administered intramuscularly 2 and 48 hours after muscle injury. Figure 16B) Quantification of the regeneration index, calculated as the number of newly regenerated fibers per 1 mm² of damaged area. Regenerated fibers were identified as fibers with a central nucleus. **** p<0.0001 (independent t-test). figure 16C A histogram showing the fibrosis index, calculated as the percentage of fibrotic area. * p=0.0186 (independent t-test).

[0216] Example 31: Systemic administration of FGF17 protects against dexamethasone-induced muscle atrophy. figure 17 A) Overall experimental setup and group. Dexamethasone (25 mg / kg intraperitoneally) was administered to 12-week-old mice for 20 days simultaneously with subcutaneous injection of FGF17 (0.5 mg / kg). Muscle weight was assessed on day 21. Grip strength of the forelimbs and both limbs was measured on days 7, 13, and 21. (Figure) 17 B) TA muscle weight as a percentage change from vehicle body weight. *** p=0.0005, * p=0.0499 。2 The histogram of the forelimb force measured on day 1 shows the relative force of the forelimb, calculated as the ratio of forelimb force (N) to weight (g). * p=0.0458, ** p=0.0014 Figure 17C The force of both limbs measured on day 21 was calculated as the ratio of force (N) to weight (g). *** p=0.001 * p=0.0102. Data were compared using Tukey's method, which corrects for one-way Anova for multiple comparisons.

[0217] Example 32 Treatment for chondrocyte proliferation in cartilage damage and osteoarthritis, and induction of chondrocyte proliferation by FGF17 RNA expression indicates that the FGF17 receptor was expressed on cartilage-associated cells. 。FBar graph quantification of EdU-positive human chondrocyte percentages in response to GF17. Chondrocytes were cultured for 48 hours in the presence of the indicated dose of FGF17. 0.1 ug / mL of FGF18 was added as a positive control. Fresh medium and FGF17 were added every 24 hours. A table of EdU-positive chondrocyte percentages and p-values ​​is shown. Mean ± SD. (Tukey Honest Significant Difference T-test) * (p<0.05, n=2~3) Table: RNA expression (TPM) of FGFR1

[0218] [Table 29]

[0219] De Data table

[0220] [Table 30]

[0221] Example 33: In vitro assay for measuring myogenic activity Mouse myoblast proliferation assay Since reduced regeneration from tissue progenitor cells in an individual is a prominent feature of age- or disease-related dysfunction, assays measuring the pro-mitotic capacity of tissue progenitor cells serve as a leadout for the likelihood of treatment success. By measuring the increased proliferation rate, degree of differentiation, and cell viability of treated mouse or human muscle progenitor cells, a good benchmark can be provided for potential regenerative factors that may be used therapeutically in the treatment of individuals with disease, injury, or genetic or developmental disorders that lead to premature tissue loss, wasting, or debilitation.

[0222] Mouse muscle progenitor cells (early passaged myoblasts) were cultured and grown in mouse growth medium (Ham's F-10 (Gibco), 20% bovine growth serum (Hyclone), 5 ng / mL FGF2, and 1% penicillin-streptomycin on a Matrigel-coated plate (Matrigel:PBS 1:300), at 37°C and 5% CO2). For the experimental conditions, cells were seeded at a rate of 40,000 cells per well on an 8-well Matrigel-coated chamber slide (Matrigel:PBS 1:100) containing 250-500 μL of mouse fusion medium (DMEM (Gibco) + 2% horse serum (Hyclone)) per well. One hour after seeding, the mouse myoblasts were treated with 50% of each medium, and then cultured for 24 hours at 37°C in a 10% CO2 incubator under the above conditions. After adding BrdU (300 μM) in DMSO for 2 hours, the samples were fixed with cold 70% ethanol and stored at 4°C until staining.

[0223] Quantitative analysis of the regeneration index Cells were permeabilized in PBS + 0.25% Triton X-100, followed by antigen retrieval. Primary staining was performed using primary antibodies containing species-specific monoclonal antibodies against mouse anti-embryonic myosin heavy chain (eMyHC, hybridoma clone 1.652, Developmental Studies Hybridoma Bank) and rat anti-BrdU (Abcam ab6326). Secondary staining was performed using fluorophore-conjugated species-specific antibodies (donkey anti-rat 488, #712-485-150; donkey anti-mouse 488, #715-485-150). Nuclei were visualized by Hoechst staining. Cell numbers were counted using Hoechst staining, and the percentage of cells positive for BrdU and eMyHC was reported in a table.

[0224] FGF17-hFcm promotes the proliferation of mouse myoblasts. Suspended CHO cells were transiently transfected with either an empty control plasmid or a plasmid encoding FGF17-hFcm. After 4 days, the culture supernatant was collected and added to mouse myoblast cultures for 48 hours, followed by 2 hours of EdU application and fixation. The proportion of EdU-positive mouse myoblasts treated with the culture supernatant of CHO cells expressing FGF17-hFcm was significantly higher than the proportion of EdU-positive mouse myoblasts treated with either the vehicle control or the culture supernatant of CHO cells expressing an empty control vector (One-Way ANOVA Tukey Honest Significant Difference, n=2-6).

[0225] Suspended CHO cells were transiently transfected with a plasmid encoding FGF17-hFcm. After 4 days, the culture supernatant was collected, and FGF17-hFcm was affinity-purified using a protein A membrane column. The purified FGF17-hFcm was added to mouse myoblast cultures for 48 hours, followed by 2 hours of EdU application and fixation. The proportion of EdU-positive mouse myoblasts treated with purified FGF17-hFcm was significantly higher than the proportion of EdU-positive mouse myoblasts treated with the culture supernatant of CHO cells expressing an empty control vector (One-Way ANOVA Tukey Honest Significant Difference, n=2~6).

[0226] Example 34 Myogenic gene profiling for regeneration-promoting factors The expression of myogenic factors Pax7, Myf5, Myod1, and Myog is an important indicator of the functional state of muscle progenitor cells. Factors upregulating Pax7 and Myf5 indicate activation of proliferative progenitor cells, while upregulation of Myod1 and Myog suggests muscle fiber regeneration. Readouts of the expression of these genes may provide the potential for success of any given polypeptide, including FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, as described herein, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans. Measuring myogenic genes in factor-treated mouse or human muscle progenitor cells may provide a good characterization of therapeutic effects for treating individuals with genetic disorders or developmental disorders that result in damage or premature tissue loss, wasting, or debilitation. As a control, this assay can also be performed on purified proteins from differentiated cells cultured in differentiated cell-conditioned media (resulting in no myoblast proliferation) or on purified heparin-binding fractions.

[0227] RNA was isolated from each well (RNeasy Mini Kit, Qiagen), and cDNA was obtained by reverse transcription (High Capacity Reverse Transcription Kit, Thermo Fisher Scientific). Real-time quantitative PCR was performed using QuantStudio3 (Thermo Fisher).

[0228] Senescent human myoblasts were cultured in well plates. Differentiated induction of myogenic gene expression occurred when cells were cultured in different media. As shown in Table 4, all factors resulted in changes in at least one myogenic receptor gene at 48 and 72 hours compared to cells cultured in fusion medium. Cells cultured with IGF2 showed increased MYOG levels at 48 hours and increased MYOD levels at 72 hours.

[0229] Table 4: Increased magnification of myogenic transcription factors in myoblasts cultured with IGF2

[0230] [Table 31]

[0231] Profiling myogenic genes in human or mouse progenitor cells For myogenic activity testing, human or mouse muscle progenitor cells may be seeded and cultured as described above. One hour after seeding, myoblasts may be treated with each factor. By analyzing the myoblasts to examine the expression of Pax7, Myf5, Myod1, and Myog, the regenerative effects of treatment with polypeptides containing FGF17, IGF2, or BMP7 amino acid sequences can be characterized. Furthermore, by performing tests, the effects of amino acid sequences from heterologous polypeptides, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans can be characterized.

[0232] Example 35: In vivo study of stem cell secretion factors Multiple in vivo muscle degeneration models can be tested. Polypeptides containing FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans described herein, have regenerative properties in in vitro models. Therefore, these in vivo models may exhibit similar regenerative and proliferative effects in the context of complete organ systems.

[0233] Acute injury model The experimental group may consist of C57BL / 6J male mice (N=18), young mice aged 12-13 weeks (3 months) (n=6), and aged mice aged 77-78 weeks (18 months) (n=12). This design can be used to test any single factor identified and demonstrated in in vitro assays, or polypeptides containing FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans.

[0234] On day 0, mice were weighed, and muscle damage could be induced by local injection of barium chloride (BaCl2, 10 μL, 1.2% w / v in physiological saline, Sigma-Aldrich) into the anterior tibia (Tibialis anterior, TA, day 0) of both the right and left hind limbs. Following BaCl2 injection into the TA-injured hind limb site, a vehicle or factor A (0.1 mg / kg) could be administered intramuscularly simultaneously, and again 48 hours later, on day 2, it could be administered intramuscularly to the TA-injured hind limb site. Also on day 2, BaCl2 (Ctx, 10 μL, 1.2% w / v in physiological saline, Sigma-Aldrich) was injected intramuscularly into the gastrocnemius (Gastrocnemius, GA, day 2, intramuscular) of both the right and left hind limbs. After BaCl2 is administered to the TA hindlimb site, vehicles or factors may be sequentially administered (intramuscularly) after injury, and again (intramuscularly) to the GA injured hindlimb site 48 hours later, on day 4. To label proliferating cells, bromodeoxyuridine (BrdU) was administered once daily for 3 days (days 2-4) (100 mg / kg, intraperitoneal), followed by sacrificial death.

[0235] On day 5, the animals were euthanized, their weight was recorded, and then 0.5 ml of terminal blood was collected by cardiac puncture, processed as plasma, and stored at 80°C. The animals were then perfused with 1×PBS, and the skin was carefully incised from the GA / TA muscle of each hind limb and photographed (before stimulation). After selective stimulation of the GA or TA muscle, the excised tissue was photographed and weighed, then placed in 25% sucrose in PBS at 4°C for 4 hours, rinsed in 1×PBS, immersed in Tissue-TEK OCT for rapid freezing, and the muscle tissue was stored frozen at 80°C. Frozen sectioning and H&E were performed to confirm that the muscle injury site was adequately visualized. Muscle tissue composition, fibrous tissue, and adipose (fat) from new skeletal muscle fibers could be measured. The level of regeneration can be assessed by measuring muscle regeneration (defined as the number of new muscle fibers with a central nucleus per millimeter), fibrosis (defined as the area of ​​fibrotic scarring), fiber size (defined as width and area), and adipose tissue (defined by the amount of fat surrounding the muscle).

[0236] Sarcopenia / Chronic Therapy Model As shown in Table 4, the experimental design uses C57BL / 6J male mice (N=18), young mice aged 12-13 weeks (3 months) (n=6), and aged mice aged 77-78 weeks (18 months) (n=12). This design can be used to test any single factor or complex mixture of two or more factors or synergistic small molecules identified and demonstrated in in vitro assays.

[0237] On day 0, mice may undergo the following in vivo healthspan measurements over 1 day as baseline for age-based parameters: weight, swivel wheel performance, grip strength, and horizontal bar. Each assay should be performed four times per animal for each assay. These healthspan assays may be repeated starting from day -1. After a 1-day rest on day -9, mice may be administered daily injections of vehicle or factor A (0.1 mg / kg) for the remainder of the experiment until sacrificial death (days -8 to +5, 13 days of administration). Six days after the start of administration, on day -4, mice may undergo a repeat of the healthspan assay. Five days before sacrificial death, on day 0, muscle damage may be induced by local injection of cardiotoxicity (Ctx, 10 μg, Sigma-Aldrich) in the anterior tibia (TA, day 0) of only the right hind limb of the mouse. Next, on day 2, cardiotoxicity (Ctx, 10 μg, Sigma-Aldrich) may be administered to the gastrocnemius muscle (GA, day 2) of the right hind limb. From days 2 to 4, BrdU (100 mg / kg, intraperitoneal) may be administered once daily for 3 days, after which sacrificial death may be performed. On day 5, an in vivo incapacitance assay may be performed on the animal before withdrawal. On day 5, the animal may be sacrificial death and its weight may be recorded. 0.5 ml of blood may be collected by cardiac puncture, processed as plasma, and the plasma sample may be stored at 80°C. The animal may then be perfused with 1×PBS. Carefully incise the skin from the GA / TA muscle of each hind limb and take photographs (before stimulation). After selectively stimulating the GA or TA muscles, the muscles can be weighed, then immersed in 25% sucrose in PBS at 4°C for 4 hours, followed by rinsing in 1×PBS, and Tissue-TEK OCT can be added to preserve the muscle tissue frozen at 80°C. Frozen sections can be prepared and H&E can be performed to confirm that the muscle injury site is adequately visualized. White adipose tissue (WAT) from the groin can be carefully excised and weighed.

[0238] Muscle tissue composition, fibrous tissue, and adipose (fat) from new skeletal muscle fibers can be measured. The level of regeneration can be assessed by measuring muscle regeneration (defined as the number of new muscle fibers with a central nucleus per millimeter), fibrosis (defined as the area of ​​fibrous scarring), fiber size (defined as width and area), and adipose tissue (defined by the amount of fat surrounding the muscle). A healthy lifespan assay, including the weight of the animal during treatment and performance on a swivel wheel (speed, distance, duration), grip strength, and horizontal bar, may take into account the phenotypic results of aging animals treated systemically with polypeptides containing FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and combinations of BMP receptor agonists and mTOR activators and / or glycosaminoglycans.

[0239] At 8 months of age (n=6WT, n=7 MPS IIIB) and 10 months of age (n=3WT, n=4 MPS IIIB), the horizontal bar test could be performed as previously described (Malinowska et al. 2010). Briefly, a 300 mm metal wire (2 mm in diameter) was fixed between two supports located 320 mm above a padded surface. The mouse was given the wire to grasp in the middle, and the time it took to fall or reach the end was recorded, with the test stopped after 2 minutes. If the mouse crossed the bar in x seconds, it could be recorded as 240-x; if it remained on the bar, it could be recorded as 120; and if it fell from the bar after y seconds, it could be recorded as the value y. The test was repeated three times as practice, followed by three more times after a 10-minute break, and the scores were recorded.

[0240] Furthermore, the animals may exhibit the following outcomes: reduced weight, fat composition, scar tissue around muscles, increased running speed, duration, and distance, increased grip strength, and improved performance on the horizontal bar test.

[0241] Hereditary obesity muscular dystrophy model BaCl2 can be injected into the TA muscle of hereditary obese (ob / ob) mice on day 0. On days 0 and 2, three mice can be treated with vehicle alone, three mice can be injected with hPSC factor, and three mice can be treated with FGF19 (positive control). On day 5, the mice can be euthanized, and the TA muscle can be perfused with PBS and dissected. The muscle can then be analyzed to determine the regeneration index and fibrosis index.

[0242] Atrophy model For the experimental design, C57BL / 6J male mice are administered daily for 20 days either a vehicle (N=7), dexamethasone (25 mg / kg intraperitoneal) (N=6), or dexamethasone plus treatment (N=6). This design can be used to test any single factor or a complex mixture of two or more factors identified and demonstrated in an in vitro assay.

[0243] On day 21, the animals are euthanized and their weight is recorded. 0.5 ml of blood is collected by cardiac puncture, processed to obtain plasma, and the plasma sample is stored at -80°C. The animals are perfused with 1×PBS. After carefully incising the skin from the GA / TA muscle of each hind limb, photographs are taken, followed by limited stimulation of the GA or TA muscle, weighing of the muscle, and then rapidly freezing in isopentane at -80°C.

[0244] Methods for testing muscle strength, endurance, and function Forelimb and bilimb grip strength tests: After acclimatization for 30 minutes, introduce the mouse to the dynamometer. For forelimb grip strength, the mouse is suspended by its tail and made to grip the grip bar with only its forelimbs. For bilimb measurement, the mouse is placed on a grid and made to grip the grid with both limbs. The force produced by each mouse is calculated as the average of 5-6 measurements.

[0245] Limb endurance test: Prior to the endurance test, mice are made accustomed to the rodent treadmill environment by being exposed to it and given two 10-minute training sessions at 10 m / min on different days. For the endurance test, mice are placed in individual lanes of the rodent treadmill. The speed is gradually increased by 2 m / min until exhaustion is reached. Exhaustion is defined as the mouse remaining on an electrostatic grill that is subjected to a 2 Hz shock at intensity 5 for 3-5 seconds.

[0246] In vivo tetanic force measurement: Mice are anesthetized throughout the entire process using controlled delivery of isoflurane. After anesthesia, the animals are placed in a heated chamber and their legs are fixed to the foot pedal of an Aurora force transducer. Two electrodes are attached, specifically to stimulate the sciatic nerve. The force (not direct force) generated by the twisting of the ankle joint of the animal's hind limbs is measured in response to a series of stimuli including 50, 100, 150, and 200 Hz.

[0247] In situ tetanic force measurement: This experiment is performed using Aurora force measurement. Mice are anesthetized throughout the entire procedure. The Achilles tendon is exposed by making a small incision in the skin around the anterior tibia and connected to the Aurora force transducer via a hook by surgical suturing. The force generated by the muscle in response to a series of stimuli, including 50, 100, 150, and 200 Hz, is recorded using two electrodes on the anterior tibia.

[0248] Example 36: In vivo stability of mitotic polypeptides assimilated by bioavailability and pharmacokinetics. Bioavailability in Organizations The bioavailability of therapeutic polypeptides can be evaluated in target tissues of young mice (10-12 weeks old) and aged mice (78 weeks old). For this experiment, one subcutaneous (SC) injection of the therapeutic composition may be administered to one cohort of young mice (10-12 weeks old, N=24) and one cohort of aged mice (78 weeks old, N=24). Four young mice (10-12 weeks old, N=6) and four aged mice (78 weeks old, N=6) may be administered a subcutaneous injection of the vehicle and used as controls. Four mice from each cohort may be euthanized at 30 minutes, 1 hour, 1.5 hours, 2 hours, or 4 hours. At each time point, blood may be collected by cardiac puncture, followed by collection of selected tissues such as the anterior tibia, gastrocnemius muscle, quadriceps muscle, heart, and diaphragm. The administered therapeutic polypeptide can be detected and quantified by enzyme-linked immunosorbent assay (ELISA). Tissue-level bioavailability can be determined by comparing the levels of the therapeutic polypeptide with samples taken from mice injected with the vehicle.

[0249] Pharmacokinetics of the synthesized mitotic polypeptide Pharmacokinetics (PK) in mice refers to the absorption, distribution, metabolism, and elimination of a drug from the body. The pharmacokinetic profiles of therapeutic polypeptides can be determined in young mice (10-12 weeks old) and older mice (78 weeks old). For this experiment, two administration routes, including subcutaneous (SC) and intravenous (IV) injection, can be investigated in both young (10-12 weeks old) and older (78 weeks old) mice. For each group, evaluation can be performed at six time points: 5, 15, 30, 60, 90, and 120 minutes, using endpoint sampling or serial sampling. At least four animals may be used for each time point, group, and route. The concentration of the prepared mitotic polypeptide in the sample can be measured by LC-MS / MS or ELISA. Various pharmacokinetics and absorption / elimination dynamics after administration via different routes can be calculated.

[0250] Example 37 Further testing of regenerative factors Mechanistic insights into the combined action pathways of polypeptides containing FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans can be obtained by establishing and screening a panel of assays for cell age. These assays include the production or resistance of reactive oxygen species (ROS) in the cytoplasm and mitochondria, measurement of telomerase activity, measurement of proteostatic capacity via the lysosomal, autophagy, and proteasome pathways, epigenetic repatterning, and cellular energy balance (e.g., ATP / ADP ratio and NAD / NADH ratio). Many of these assays utilize high-throughput automated microscopy to perform these measurements on a variety of cell types, including fibroblasts, endothelial cells, mesenchymal stem cells, and chondrocytes. Overall, these measurements can provide information about both the pathways and mechanisms by which the heparin-bound hPSC secretome or its individual components exert regenerative effects. These deep profile vectors may be important for a rational approach to factor combinations and for machine learning predictions.

[0251] To test the effects of secretomes on cells with the aim of improving prominent features of aging, the power of deep population-level statistics can be achieved through high-throughput automated imaging and quantification of single cells. Cellular component state profiles of young, aged, and aging+ treated human fibroblasts and epithelial cells, myoblasts, mesenchymal stem cells, chondrocytes, and neural progenitor cells can be compared. Some examples of tests and methods include:

[0252] Epigenetic reprogramming: repressive marker H3K9me3, heterochromatin-related protein HRIγ, nuclear lamina-supporting protein LAP2α.

[0253] Nuclear membrane folding / vesicle formation: Immunofluorescence of nuclear membrane proteins lamin A / C.

[0254] Proteolytic activity: The cleavage of the fluorescently tagged chymotrypsin-like substrate corresponds to proteasome 20S core particle activity. First, wells can be stained with PrestoBlue Cell Viability dye (Life Technologies) for 10 minutes. The signal in the wells can be read as a measure of cell number using a TECAN fluorescence plate reader. Next, the cells can be washed with HBSS / Ca / Mg and then replaced with the original medium containing the chymotrypsin-like fluorescence-generating substrate LLVY-R110 (Sigma) (which is cleaved by proteasome 20S core particles). The cells can then be incubated in 5% CO2 at 37°C for 2 hours, after which the signal can be read again with a TECAN fluorescence plate reader. The reading can then be standardized by PrestoBlue cell count.

[0255] Autophagosome formation: The number and volume of autophagosomes can be measured by staining with CellTracker Deep Red (Sigma). Cells can then be incubated in 5% CO2 at 37°C for 20 minutes, washed twice with HBSS / Ca / Mg, and stained with CellTracker Deep Red cell labeling dye for 15 minutes. Cells can then be switched to HBSS / Ca / Mg for single-cell imaging using the Operetta High Content Imaging System (Perkin Elmer).

[0256] Energy metabolism: ATP in cells is measured by colorimetric analysis using an ATP assay kit (ab83355, Abcam, Cambridge, MA) according to the manufacturer's instructions. Cells may be washed with cold phosphate-buffered saline, homogenized, and centrifuged to collect the supernatant. Samples may be loaded in three ways using assay buffer. The ATP reaction mixture and background control (50 μL) are added to the wells and incubated in the dark for 30 minutes. The plate is read using a SpectraMax M2e (Molecular Devices, SunnyVeil, CA) at OD 570 nm. Intracellular ATP concentration is estimated using the mean optical density and a calibration curve.

[0257] Mitochondrial activity: To measure mitochondrial membrane potential, cells may be washed twice with Ham's F10 (without serum, without penicillin / streptomycin). Subsequently, MuSCs may be stained with MitoTracker Green FM (Thermo Fisher, M7514) and DAPI at 37°C for 30 minutes, washed three times with Ham's F10, and analyzed using a BD FACSAria III flow cytometer.

[0258] Mitochondrial ROS measurement. Cells can be washed with HBSS / Ca / Mg, then switched to HBSS / Ca / Mg containing MitoSOX(Thermo) (a living cell-transmitting fluorescent dye selectively targeted to mitochondria that fluoresces when oxidized by superoxide). Cells can be incubated in 5% CO2 at 37°C for 10 minutes. Cells can then be washed twice with HBSS / Ca / Mg and stained with CellTracker Deep Red for 15 minutes. Finally, cells can be imaged in the fresh HBSS / Ca / Mg using the Operetta High Content Imaging System (Perkin Elmer).

[0259] Deregulated nutrient sensing: The level of SIRT1 can be measured.

[0260] Aging: Aging-related β-galactosidase staining is measured in cells, washed twice with PBS, and then fixed with 15% paraformaldehyde in PBS for 6 minutes. Cells may be rinsed three times with PBS and then stained with X-gal chromogenic substrate (cleaved by endogenous β-galactosidase). To prevent drying, plates may be kept in the staining solution and parafilmed, and incubated overnight at 37°C in ambient CO2. The following day, cells may be washed again with PBS and then switched to a 70% glycerin solution for imaging with a Leica bright-field microscope.

[0261] Cellular secretome: Mass spectrometry or O-binding for inflammatory cytokine profiling.

[0262] Soft tissue deposition: Immunofluorescence for SOX9, MMP3, MMP13, and COL2A1 expression (decreased expression is characterized by chondrotenopathy, pain, cleft lip, and joint destruction).

[0263] Example 38: Purified IGF2-hFcm enhanced myoblast differentiation. Suspended CHO cells were transiently transfected with a plasmid encoding IGF2-hFcm. After 4 days, the culture supernatant was collected, and IGF2-hFcm was affinity-purified using a protein A membrane column. The purified IGF2-hFcm was added to human myoblast cultures. Myosin heavy chain (MyHC) was immunostained and imaged by fluorescence microscopy. After quantifying the stained MyHC, the percentage of MyHC area was calculated as the percentage of pixels irradiated beyond the background within the stained channel in the field of view. The percentage of EdU in mouse myoblasts treated with purified IGF2-hFcm was significantly higher than that in mouse myoblasts treated with the culture supernatant of CHO cells expressing an empty control vector. Significance was determined by a p-value of less than 0.05 using the one-way ANOVA Tukey Honest Significant Difference test.

[0264] Table 5: IGF2 enhanced myoblast differentiation.

[0265] [Table 32]

[0266] In this example, it was found that the IGF2 fusion protein could induce cell proliferation. The IGF2 fusion protein and HAP share in vitro properties, which suggests that they may also share in vivo properties.

[0267] Example 39: Modeling of the treatment of muscular dystrophy with an IGF2 composition in vitro. Muscular dystrophy (MD) encompasses a range of muscle degenerative diseases typically resulting from genetic mutations in genes encoding proteins responsible for the formation and stabilization of skeletal muscle. The phenotypic consequences of these gene mutations are a time-dependent and progressive decline in muscle mass and strength, similar to that of sarcopenia, but with different underlying causes. Since HAP results in phenotypic improvement in sarcopenic muscles, it was tested to investigate similar improvements in models of MD.

[0268] IGF2 was tested for its ability to enhance the proliferation and / or fusion of human muscle progenitor cells from individuals with type 1 myotonic dystrophy (hMD) (a muscular dystrophy caused by mutations in the DMPK1 gene). The effect of IGF2 on myogenic activity was biologically assayed in three ways over a wide range of concentrations around the expected physiological level. This was done by adding each factor to hMD myoblasts for 72 hours, changing the medium daily (DMEM + 2% horse serum), and applying the factor a second time during the first medium change. After 72 or 96 hours, cells were treated with EdU (30 μM) for 2–5 hours, fixed with ethanol, stained with Hoechst 3342, and immunostained to examine proliferation (measured by the percentage of EdU-positively stained cells (%EdU)) and differentiation (measured by the increase in area of ​​cells positively stained with embryonic myosin heavy chain (%eMyHC) relative to a negative control with only culture medium and vehicle added). Wells were imaged at 4x magnification using Keyence BZ-100, images were quantified using Cell Profiler, and statistics were calculated in R. Furthermore, RNA was extracted from myoblasts, and the abundance of transcripts quantified by qPCR was selected. 。I This shows that GF2 treatment promoted the proliferation and differentiation of DM1 human myoblasts (from a 32-year-old Caucasian woman), respectively. 。I This study demonstrates that GF2 increased the expression of MYH3, CKM, and ATP1B1 in DM1 human myoblasts (from a 32-year-old Caucasian woman).

[0269] Example 40: Systemic administration of therapeutic polypeptide improves sarcopenia and protects against muscle damage. 78-week-old mice were administered daily subcutaneously with either a therapeutic polypeptide or a vehicle alone for 14 days. IGF2 was injected at a concentration of 100–1000 μg / kg. In some experiments, the treatment group received one therapeutic factor, while in others, the treatment group received a combination of factors. On day 7, muscle function was assessed by grip strength of the forelimbs and both limbs. On days 12, 13, and 14, BrdU was administered intraperitoneally to groups 1 and 2. On days 13–15, grip strength was assessed in all mice, and maximum distance, maximum speed, and tetanic force were examined by endurance tests.

[0270] On day 15, mice from groups 1 and 2 were euthanized, and their muscles were analyzed for markers of proliferation and fibrosis. On day 15, chemical damage was induced in the TA of mice from groups 3 and 4 using intramuscular injection of 1.2% BaCl2 (7 ul / TA). Mice from groups 3 and 4 continued subcutaneous injection of therapeutic polypeptide from days 15 to 21. They also received intraperitoneal BrdU injections on days 19, 20, and 21. On day 21, the TA muscle was tested for in situ tetanic force. The TA muscle was dissected and evaluated for signs of proliferation and fibrosis.

[0271] Example 41 Systemic administration of fusion polypeptide improves induced muscle atrophy. 12-week-old mice were divided into three treatment groups: Group 1 received vehicle injections only, Group 2 received dexamethasone injections, and Group 3 received dexamethasone and IGF2 fusion polypeptide injections. Dexamethasone (25 mg / kg intraperitoneal) was administered for 14 days simultaneously with subcutaneous injection of IGF2 fusion polypeptide.

[0272] On day 7, the grip strength of the forelimbs and both limbs of the mice will be evaluated. From days 13 to 15, the grip strength and in vivo tetanic force will be evaluated in the mice, and a treadmill endurance test will be performed to determine the maximum speed and maximum distance.

[0273] Example 42: Systemic administration of IGF2 fusion polypeptide is predicted to improve muscle atrophy in hereditary obese mice. Hereditary obese mice (ob / ob) at 13 weeks of age may be subcutaneously injected with IGF2 fusion polypeptide for 14 days. On day 7, grip strength of the forelimbs and both limbs may be measured. On days 12, 13, and 14, BrdU may be injected. On days 13, 14, and 15, grip strength of the forelimbs and both limbs, as well as tetanic force in vivo, may be tested, and endurance tests may be performed to determine maximum distance and maximum speed. On day 14, the mice may be euthanized, and the TA muscle may be dissected. Muscle weight and proliferation may be analyzed.

[0274] Example 43: Systemic administration of IGF2 fusion polypeptide is predicted to improve or slow down dystrophy characteristics in 70-week-old mdx mice. Another class of human myopathy requiring treatment is muscular dystrophy induced by genetic abnormalities, among which Duchenne muscular dystrophy, though rare, can be fatal. Aged hereditary dystrophy (mdx) mice (over 15 months of age) exhibit characteristics similar to human Duchenne muscular dystrophy (DMD), particularly reduced muscle regeneration leading to muscle wasting. Treatment with IGF2 fusion polypeptide can improve the dystrophic characteristics of aged mdx mice. During the acclimatization period, baseline strength can be determined for each mouse by evaluating weight, forelimb and biliary grip strength, and in vivo tetanic force. 70-week-old dystrophic mice (mdx) are injected subcutaneously with IGF2 fusion polypeptide for 14 days. On day 7, forelimb and biliary grip strength is measured. BrdU is injected on days 12, 13, and 14. On days 13, 14, and 15, grip strength of the forelimbs and both limbs, as well as tetanic force in vivo, will be tested, and endurance tests will be performed to determine maximum distance and maximum speed. The right anterior tibia and gastrocnemius muscle may be collected, immersed in Tissue-TEK OCT, and then rapidly frozen in a cold isopentane bath pre-cooled in liquid nitrogen, and stored at -80°C. The tissue may be sectioned, stained with laminin to examine the cross-sectional area (CSA) of muscle fibers, stained with eMyHC to measure new fiber formation, and stained with BrdU to assess proliferation rate. The left anterior tibia and gastrocnemius muscle may be collected, rapidly frozen in liquid nitrogen, and subjected to molecular analysis including qPCR and Western blotting.

[0275] IGF2 is expected to be effective at concentrations of 10–200 ug / kg.

[0276] Example 44: Systemic administration of IGF2 fusion polypeptide is predicted to improve dystrophy characteristics in 6-week-old mice. At 3–6 weeks of age, severe necrosis of the skeletal muscle of mdx mice is followed by increased activation of satellite cells, thereby enhancing muscle regeneration. Treatment with IGF2 fusion polypeptides described herein can improve the regeneration process and thus the health of the muscles. During the acclimatization period, baseline strength can be determined for each mouse by evaluating weight, forelimb and biliary grip strength, and in vivo tetanic force. Six-week-old dystrophy mice (mdx) are injected subcutaneously with IGF2 fusion polypeptide for 14 days. On day 7, forelimb and biliary grip strength is measured. On days 12, 13, and 14, BrdU is injected. On days 13, 14, and 15, forelimb and biliary grip strength and in vivo tetanic force are tested, and endurance tests are performed to determine maximum distance and maximum speed.

[0277] Mice can be euthanized. The right anterior tibia and gastrocnemius muscle can be collected, immersed in Tissue-TEK OCT, and then rapidly frozen in a cold isopentane bath pre-cooled in liquid nitrogen, and stored at -80°C. The tissue can be sectioned, laminin staining can be used to examine the cross-sectional area (CSA) of muscle fibers, eMyHC staining can be used to measure new fiber formation, and BrdU staining can be used to evaluate proliferation. The left anterior tibia and gastrocnemius muscle can be collected, rapidly frozen in liquid nitrogen, and subjected to molecular analysis including qPCR and Western blotting.

[0278] IGF2 is expected to be effective at concentrations of 10–200 μg / kg.

[0279] Example 45 Treatment for chondrocyte proliferation in cartilage damage and osteoarthritis Cartilage can be damaged as a result of sudden injury or by gradual wear and tear or inflammation, which can lead to disease conditions (e.g., osteoarthritis). Chondrocytes secrete the cartilage matrix, and the cell types associated with cartilage are all preadipocytes, osteocytes, and tendonocytes.

[0280] Preadipocytes, chondrocytes, osteocytes, and tendinocytes were cultured in well plates. RNA was isolated from each well (RNeasy Mini Kit, Qiagen), and cDNA was obtained by reverse transcription (High Capacity Reverse Transcription Kit, Thermo Fisher Scientific). Real-time quantitative PCR was performed using QuantStudio3 (Thermo Fisher).

[0281] These cartilage-associated cells expressed receptors for polypeptides containing FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or for combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans. Subcutaneous preadipocytes, chondrocytes, osteocytes, and tendinocytes all express one or more receptors, which suggests that polypeptides containing the aforementioned FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans may influence cartilage reduction, exacerbation of joint-related damage, or recovery from disease.

[0282] Example 46 Clinical trial of therapeutic composition The objective of this study is to investigate the safety, tolerability, and pharmacokinetics of repeated administration at multiple dose levels of polypeptides containing FGF17, IGF2, or BMP7 amino acid sequences and amino acid sequences from heterologous polypeptides, or combinations of fibroblast growth factor receptor agonists and glycosaminoglycans, insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids, and BMP receptor agonists and mTOR activators and / or glycosaminoglycans, in healthy individuals diagnosed with sarcopenia, muscular dystrophy, or individuals recovering from surgery. In certain embodiments, muscular dystrophy is myotonic dystrophy. Furthermore, this study may also yield data on physical function, skeletal muscle mass, and strength resulting from treatment with IGF2 fusion polypeptides in these individuals. Individuals may be administered either placebo or the IGF2 fusion polypeptide composition and monitored over a 25-week study period. The primary and secondary outcome measures described below may be evaluated.

[0283] Primary evaluation criteria: Safety and tolerability: Evaluated by various factors, including the rate of adverse events per research arm.

[0284] Secondary evaluation items: Plasma pharmacokinetics (Cmax, Tmax, AUC) [Plasma at 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 hours post-administration.] Short Physical Performance Battery (SPPB). Changes from baseline to week 25.

[0285] 10-meter walk test. Changes from baseline to week 25.

[0286] Changes in total lean body mass and limb skeletal muscle index, measured by dual-energy X-ray absorptiometry (DEXA), from baseline to week 25.

[0287] Inclusion Criteria: Diagnosis of sarcopenia or muscular dystrophy, or recovery from surgery; muscle loss confirmed by DXA; decreased walking speed; SPPB score of 9 or less; body weight of at least 35 kg; adequate dietary intake as determined by interview with the patient. Able to walk 10 meters independently.

[0288] Protocol Patients may be administered intravenously either a placebo (5% dextrose solution) or the treatment product (in 5% dextrose). Treatment should begin on day 1 of week 1 and be repeated weekly (day 1 of week 1 through week 25). Patient improvement may be assessed at the end of week 13 and week 25 using the method described above. Dosage may be selected from a conventional 3+3 design, and the top two doses that do not exhibit dose-limiting toxicity may be selected.

[0289] While preferred embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. Those skilled in the art will be able to conceive of a great many variations, modifications, and substitutions without departing from the present invention. It will be understood that in carrying out the present invention, various alternatives may be used to the embodiments of the invention described herein.

[0290] All publications, patent applications, issued patents, and other documents referenced herein are incorporated herein by reference to the same extent as each of those publications, patent applications, issued patents, or other documents is incorporated by specific and individual reference in its entirety. Where any definitions contained in a text incorporated by reference conflict with those in this disclosure, the former shall be excluded.

[0291] [Table 33]

[0292] Table 34

[0293] Table 35

[0294] Table 36

[0295] Table 37

[0296] Table 38

[0297] Table 39

[0298] Table 40

[0299] Table 41

[0300] Table 42

[0301] Table 43

[0302] Table 44

[0303] Table 45

[0304] Table 46

[0305] Table 47

[0306] Table 48

[0307] Table 49

[0308] Table 50

[0309] Table 51

[0310] Table 52

[0311] Table 53

[0312] Table 54

[0313] Table 55

[0314] Table 56

[0315] Table 57

[0316] Table 58

[0317] Table 59

[0318] Table 60

[0319] Table 61

[0320] Table 62

[0321] Table 63

[0322] Table 64

[0323] Table 65

[0324] Table 66

[0325] Table 67

[0326] Table 68

Claims

1. Use of a polypeptide comprising an insulin-like growth factor-2 (IGF2) amino acid sequence and a heterologous polypeptide amino acid sequence for the manufacture of a pharmaceutical composition for treating acute or chronic muscle-wasting diseases or conditions, including sarcopenia, cachexia, or muscular dystrophy, wherein the heterologous polypeptide amino acid sequence enhances the stability or biological function of the IGF2 amino acid sequence, wherein the heterologous polypeptide amino acid sequence comprises an N-terminal human serum albumin (HSA) heterologous polypeptide amino acid sequence, and the N-terminal HSA heterologous polypeptide amino acid sequence and the IGF2 amino acid sequence are separated by a flexible linker, the flexible linker comprising the amino acid sequence of Sequence ID No.

104.

2. The use according to claim 1, wherein the subject requiring treatment by use is systemically administered polypeptide by subcutaneous injection.

3. The use according to claim 2, wherein the IGF2 amino acid sequence includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

76.

4. The use according to claim 2, wherein the IGF2 amino acid sequence includes an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:

76.

5. The use according to claim 2, wherein the amino acid sequence of IGF2 includes a human IGF2 amino acid sequence.

6. The use according to any one of claims 1 to 5, wherein a disease or condition of acute or chronic muscle wasting is muscular dystrophy, wherein muscular dystrophy includes myotonic muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, or distal muscular dystrophy.

7. The use according to any one of claims 1 to 5, wherein the acute or chronic disease or condition of muscle wasting is myotonic muscular dystrophy.

8. The use according to any one of claims 1 to 7, wherein the proliferation and fusion of fibroblasts are enhanced.

9. The use according to any one of claims 1 to 8, wherein the differentiation of human fibroblasts is enhanced.

10. A composition comprising a therapeutically effective amount of a polypeptide containing an insulin-like growth factor-2 (IGF2) amino acid sequence and a heterologous polypeptide amino acid sequence, wherein the heterologous polypeptide amino acid sequence enhances the stability or biological function of the IGF2 amino acid sequence, and wherein the heterologous polypeptide amino acid sequence comprises an N-terminal human serum albumin (HSA) heterologous polypeptide amino acid sequence, and the N-terminal HSA heterologous polypeptide amino acid sequence and the IGF2 amino acid sequence are separated by a flexible linker, the flexible linker comprising the amino acid sequence of Sequence ID No.

104.

11. The composition according to claim 10, wherein the polypeptide is an IGF2-heteropolypeptide fusion protein.

12. The composition according to claim 11, wherein the IGF2 amino acid sequence of the IGF2-heterogeneous polypeptide fusion protein is at least 90% identical to that of SEQ ID NO:

76.

13. The composition according to claim 11, wherein the IGF2 amino acid sequence of the IGF2-heterogeneous polypeptide fusion protein is at least 98% identical to that of SEQ ID NO:

76.

14. The composition according to any one of claims 10 to 13, wherein the N-terminal HSA heterogeneous polypeptide amino acid sequence comprises the amino acid sequence of SEQ ID NO:

109.

15. The use according to any one of claims 1 to 9, wherein the acute or chronic muscle wasting disease or condition is cachexia.

16. The use according to any one of claims 1 to 9, wherein the acute or chronic disease or condition of muscle wasting is sarcopenia.

17. The composition according to any one of claims 11 to 14, wherein the heterogeneous polypeptide amino acid sequence essentially consists of a human serum albumin molecule.

Images