Regenerated polypeptides and uses thereof

Abstract

Described herein is a polypeptide comprising an IGF2 amino acid sequence and an amino acid sequence derived from a heterologous polypeptide, useful for treating soft tissue and muscle diseases, disorders, and injuries. Mutations in the IGF2 amino acid sequence improved the stability of the molecule by reducing backbone cleavage. Also described herein is the synergistic effect of a combination of an Insulin-like Growth Factor 1 Receptor (IGF1R) agonist and a short chain fatty acid. Also described is a method of treating muscle and soft tissue diseases, comprising administering the polypeptide and / or the synergistic composition.

Description

[Technical field]

[0001] Reference to a sequence listing, table, or computer program An official copy of the Sequence Listing was submitted contemporaneously with the specification as an ASCII formatted text file via EFS-Web with a filename of "JTI021_ST25.txt", a creation date of June 7, 2022, and a size of 102 kilobytes. The Sequence Listing filed via EFS-Web is a part of this specification and is incorporated by reference in its entirety. [Background technology]

[0002] As life expectancy increases, there is a growing emphasis on "healthy aging". As individuals age, they will likely want to live a more active lifestyle, and as a result, many aging disorders can have a significant impact on the quality of life of aging individuals. Treatments that target regenerative end points have utility for treating aging diseases. In addition, many treatments for aging disorders may be applicable to young individuals who suffer from disease, injury, or have genetic or developmental defects that result in premature tissue loss, wasting, or debilitation. Summary of the Invention

[0003] As an individual ages, tissue progenitor cells lose their regenerative capacity.

[0004] Described herein is a polypeptide comprising an IGF2 amino acid sequence and an amino acid sequence derived from a heterologous polypeptide, which is useful for treating diseases, disorders and injuries of soft tissue and muscle.Also described herein is a mutation in the IGF2 amino acid sequence that improves the stability of the molecule by reducing backbone cleavage.Also described herein is a mutation in the IGF2 sequence that blocks the cleavage of the peptide backbone.Also described is a method for treating muscle and soft tissue diseases, which comprises administering the polypeptide and / or synergistic composition. [Brief description of the drawings]

[0005] [Figure 1A] 1 shows differentiation of human myoblasts promoted with purified IGF2-hFcm. [Figure 1B] 1 shows differentiation of human myoblasts promoted by purified IGF2-LhFc4. [Figure 2A] Purified HSA-L-IGF2R61A promoted the differentiation of human myoblasts. [Figure 2B] FIG. 1 shows that IGF2 and IGF2 receptor were expressed in human myoblasts. [Figure 2C] FIG. 1 shows that IGF2 and IGF2 receptor were expressed in human myoblasts. [Figure 3A] This indicates that sodium butyrate promoted muscle fusion. [Figure 3B] This shows that sodium butyrate enhanced the activity of IGF2. [Figure 3C] This shows that sodium butyrate enhanced the activity of IGF2. [Figure 4A] The change in percent area of ​​eMyHC positive cells treated with increasing doses of vehicle, IGF2, sodium butyrate, or IGF2 and sodium butyrate are presented. [Figure 4B] The change in percent area of ​​eMyHC positive cells treated with increasing doses of vehicle, IGF2, sodium butyrate, or IGF2 and sodium butyrate are presented. [Diagram 5] FIG. 1 shows that IGF2 enhances MYOG expression in DM1 human myoblasts. [Figure 6] It has been shown that the IGF2 receptor is expressed on chondrocytes and bone cells. [Figure 7A] FIG. 1 shows that IGF2 treatment promoted proliferation in DM1 human myoblasts (32 year old Caucasian female). [Figure 7B] FIG. 1 shows that IGF2 treatment promoted fusion in DM1 human myoblasts (32 year old Caucasian female). [Figure 8A] FIG. 1 shows that IGF2 enhanced the expression of MYH3 and CKM in DM1 human myoblasts (32 year old Caucasian female). [Figure 8B] 1 shows that IGF2 enhanced the expression of ATP1B1 in DM1 human myoblasts (32 year old Caucasian female). [Figure 9A] FIG. 1 depicts systemic administration of IGF2 / sodium butyrate prevents age-induced muscle dysfunction. [Figure 9B] 13 depicts an experimental outline of systemic administration of IGF2 / NaB to prevent age-induced muscle dysfunction as measured by grip strength. [Figure 9C] FIG. 1 depicts systemic administration of IGF2 / sodium butyrate prevents age-induced muscle dysfunction as measured by hindlimb grip strength. [Figure 9D] FIG. 1 depicts systemic administration of IGF2 / sodium butyrate prevents age-induced muscle dysfunction as measured by forelimb force. [Figure 9E] FIG. 1 depicts systemic administration of IGF2 / sodium butyrate prevents age-induced muscle dysfunction as measured by treadmill performance. [Figure 9F] FIG. 1 depicts systemic administration of IGF2 / sodium butyrate prevents age-induced muscle dysfunction as measured by running time to exhaustion. [Figure 9G] FIG. 1 depicts systemic administration of IGF2 / sodium butyrate prevents age-induced muscle dysfunction as measured by maximum running speed. [Figure 10A] FIG. 1 depicts an experimental outline to demonstrate that long-term systemic administration of IGF2 / sodium butyrate was safe for liver, kidney, and pancreatic function. [Figure 10B] This indicates that systemic administration of IGF2 / sodium butyrate had no adverse effects on white blood cells. [Figure 10C] This indicates that systemic administration of IGF2 / sodium butyrate had no adverse effect on albumin concentrations. [Figure 10D] This indicates that systemic administration of IGF2 / sodium butyrate had no adverse effect on creatinine concentrations. [Figure 10E] Systemic administration of IGF2 / sodium butyrate did not have an adverse effect on calcium concentrations (FIG. 10E). [Figure 11A]1 shows an experimental outline of systemic administration of IGF2 and sodium butyrate to prevent dexamethasone-induced muscle atrophy. [Figure 11B] FIG. 1 shows that systemic administration of IGF2 and sodium butyrate prevents dexamethasone-induced muscle atrophy as measured by forelimb force. [Figure 11C] FIG. 1 shows that systemic administration of IGF2 and sodium butyrate prevents dexamethasone-induced muscle atrophy as measured by specific fore and hind limb strength. [Figure 11D] FIG. 1 shows that systemic administration of IGF2 and sodium butyrate prevents dexamethasone-induced muscle atrophy as measured by forelimb force. [Figure 11E] FIG. 1 depicts systemic administration of IGF2 and sodium butyrate to prevent dexamethasone-induced muscle atrophy, as measured by forelimb specific force, calculated as the ratio of forelimb force (in mN) to body weight. [Figure 11F] FIG. 1 shows that systemic administration of IGF2 and sodium butyrate prevents dexamethasone-induced muscle atrophy as measured by muscle fiber cross-sectional area. [Figure 12A] FIG. 12A shows that systemic administration of IGF2 and sodium butyrate improves tibialis anterior muscle weight versus vehicle treatment, regenerating and promoting muscle health in the D2-mdx model of Duchenne muscular dystrophy. [Figure 12B] FIG. 12B shows that systemic administration of IGF2 and sodium butyrate improves forelimb grip strength versus vehicle treatment, regenerating and enhancing muscle function in the D2-mdx model of Duchenne muscular dystrophy. [Figure 12C] FIG. 12C shows that systemic administration of IGF2 and sodium butyrate improves forelimb grip strength versus vehicle treatment and regenerates and enhances forelimb muscle function in the D2-mdx model of Duchenne muscular dystrophy. [Figure 12D] FIG. 12D shows that systemic administration of IGF2 and sodium butyrate improves treadmill running distance versus vehicle treatment, regenerating and enhancing muscle function in the D2-mdx model of Duchenne muscular dystrophy. [Figure 12E]FIG. 12E shows that systemic administration of IGF2 and sodium butyrate improves treadmill running distance versus vehicle treatment, regenerating and enhancing muscle function in the D2-mdx model of Duchenne muscular dystrophy. [Figure 12F] FIG. 12F shows that systemic administration of IGF2 and sodium butyrate improves treadmill running distance versus vehicle treatment, regenerating and enhancing muscle function in the D2-mdx model of Duchenne muscular dystrophy. [Figure 13A] FIG. 13A shows that HSA-L-IGF2 is cleaved when expressed from CHO cells, as visualized on reducing SDS-PAGE. [Figure 13B] FIG. 13B shows that HSA-L-IGF2 is cleaved when expressed from CHO cells as visualized by reducing SDS-PAGE followed by Western blotting detecting the 6xHIS tag, and cleavage of IGF2 confirmed by dual-tagged IGF2 as visualized by reducing SDS-PAGE followed by Western blotting detecting the 6xHIS tag and the antibody constant region hFc4 tag. [Figure 14] FIG. 14 depicts an IGF2 truncation tagged with amino acid sequences at each end (HSA and hFc4) as visualized by reducing SDS-PAGE followed by Western blotting detecting the 6xHIS and hFc4 tags. [Figure 15A] FIG. 15A shows that cleavage of dual-tagged (HSA and hFc4) IGF2 was blocked by the mutations, as visualized by reducing SDS-PAGE. [Figure 15B] FIG. 15B shows that cleavage of dual-tagged (HSA and hFc4) IGF2 was blocked, as visualized by reducing SDS-PAGE followed by Western blotting detecting the 6xHIS and hFc4 tags. [Figure 16A]FIG. 16A depicts the results of an in vitro myogenesis assay demonstrating that the HSA-IGF2R61A mutant sequence retains equivalent activity towards IGF2 in human DM1 muscle precursors from a 32-year-old female. [Figure 16B] FIG. 16B depicts the results of an in vitro myogenesis assay demonstrating that IGF2 retains equivalent activity to HSA-IGF2R61A in human DM1 muscle precursors from a 32-year-old female. [Figure 17A] FIG. 17A depicts the results of an in vitro myogenesis assay demonstrating that the HSA-IGF2R61A variant sequence retains equivalent activity towards IGF2 in human muscle precursors from a healthy 32-year-old female. [Figure 17B] FIG. 17B depicts the results of an in vitro myogenesis assay demonstrating that IGF2 exhibits equivalent activity to equimolar amounts of HSA-IGF2R61A in human muscle precursors from a healthy 32-year-old female. [Figure 18A] FIG. 18A shows a noncompartmental analysis fit of the pharmacodynamic data for intravenous administration of HSA-IGF2R61 in mice, demonstrating significantly improved serum half-life compared to the native sequence of IGF2. [Figure 18B] FIG. 18B depicts noncompartmental analysis fits of the pharmacodynamic data for intravenous administration of IGF2 and HSA-IGF2R61 in mice. [Figure 19] FIG. 19 shows that HSA-IGF2R61A interacts with rhIGFBP3 as demonstrated by a mass shift upon size exclusion chromatography by HPLC. [Figure 20] FIG. 20 shows that the mutant sequence HSA-IGF2R61A retains equivalent or increased activity towards HSA-IGF2 at equimolar concentrations. [Figure 21A] Figures 21A-F show results from an acute injury mouse model: Figure 21A shows the fatigue index of mice administered 6HIS-HSA-IGF2R61A or control (vehicle). [Figure 21B]Figures 21A-F show results from an acute injury mouse model. Figure 21B shows force production as measured by specific force frequency in mice administered 6HIS-HSA-IGF2R61A or control (vehicle). [Figure 21C] Figures 21A-F show results from an acute injury mouse model. Figure 21C shows force production as measured by maximum contraction rate in mice administered 6HIS-HSA-IGF2R61A or control (vehicle). [Figure 21D] Figures 21A-F show results from an acute injury mouse model, and Figure 21D shows the association rate of mice administered 6HIS-HSA-IGF2R61A compared to mice administered control (vehicle). [Figure 21E] Figures 21A-F show results from an acute injury mouse model. Figure 21E shows the regeneration index, measured as the number of new muscle fibers per square millimeter, for mice administered 6HIS-HSA-IGF2R61A or control (vehicle). [Figure 21F] Figures 21A-F depict results from an acute injury mouse model, in which Figure 21F shows muscle mass in mice administered 6HIS-HSA-IGF2R61A compared to mice administered a control (vehicle). [Figure 22A] Figures 22A-E show results from a sarcopenia mouse model: Figure 22A shows blood glucose normalized to baseline levels in mice administered 6 mg / kg 6HIS-HSA-IGF2R61A and 0.3 g / kg sodium butyrate (NaB) or control (vehicle). [Figure 22B] Figures 22A-E show results from a sarcopenia mouse model. Figure 22B shows force generation as measured by limb grip strength (normalized to body weight) for both mice administered 6 mg / kg 6HIS-HSA-IGF2R61A and mice administered 0.3 g / kg sodium butyrate (NaB) or control (vehicle). [Figure 22C]Figures 22A-E show results from a sarcopenia mouse model. Figure 22C shows force generation as measured by forelimb grip strength (normalized to body weight) for mice administered 6 mg / kg 6HIS-HSA-IGF2R61A and 0.3 g / kg sodium butyrate (NaB) or control (vehicle). [Figure 22D] Figures 22A-E show results from a sarcopenic mouse model. Figure 22D shows recovery of muscle force development in mice administered 6 mg / kg 6HIS-HSA-IGF2R61A and 0.3 g / kg sodium butyrate (NaB) or control (vehicle). [Figure 22E] Figures 22A-E show results from a sarcopenia mouse model. Figure 22E shows force frequency normalized to body weight for mice administered 6 mg / kg 6HIS-HSA-IGF2R61A and 0.3 g / kg sodium butyrate (NaB) or control (vehicle). [Figure 23A] Figures 23A-B show results from a sarcopenia mouse model: Figure 23A shows fiber type distribution in mice administered 6 mg / kg 6HIS-HSA-IGF2R61A and 0.3 g / kg sodium butyrate (NaB) or control (vehicle). [Figure 23B] Figures 23A-B show results from a sarcopenia mouse model: Figure 23B shows cross-sectional area (CSA) of mice administered 6 mg / kg 6HIS-HSA-IGF2R61A and 0.3 g / kg sodium butyrate (NaB) or control (vehicle). [Figure 24A] Figures 24A-D show results from a mouse model of muscular dystrophy (DM1): Figure 24A shows muscle fiber distribution in mice administered 6 mg / kg 6HIS-HSA-IGF2R61A and 0.3 g / kg sodium butyrate (NaB) or control (vehicle). [Figure 24B]Figures 24A-D show results from a muscular dystrophy (DM1) mouse model. Figure 24B shows the cross-sectional area (CSA) of mice administered 6 mg / kg 6HIS-HSA-IGF2R61A and 0.3 g / kg sodium butyrate (NaB) or control (vehicle). [Figure 24C] Figures 24A-D show results from a muscular dystrophy (DM1) mouse model. Figure 24C shows the change in seizures from baseline in mice administered 6 mg / kg 6HIS-HSA-IGF2R61A and 0.3 g / kg sodium butyrate (NaB) or control (vehicle). [Figure 24D] Figures 24A-D show results from a muscular dystrophy (DM1) mouse model. Figure 24D shows changes in novel object recognition in mice administered 6 mg / kg 6HIS-HSA-IGF2R61A and 0.3 g / kg sodium butyrate (NaB) or control (vehicle). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] In one embodiment, disclosed herein is a therapeutically active protein or polypeptide sequence or a derivative or fragment thereof that enhances the growth or regeneration or function of progenitor cells through activation of cell surface receptors, and one or more of a secretion signal, a multimerization component, or a stabilization component. The inventors have modified and combined certain polypeptide sequences to create a secreted therapeutically active protein with application to muscle and soft tissue regeneration, useful for treating acute and chronic muscle wasting diseases or disorders, such as sarcopenia, cachexia, muscular dystrophy, and muscle injury. In one embodiment, disclosed herein is a method of treating individuals with acute and chronic muscle wasting diseases or disorders, such as sarcopenia, cachexia, muscular dystrophy, and muscle injury.

[0007] In one embodiment, 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 one embodiment, disclosed herein is a composition comprising an IGF1R agonist and a short chain fatty acid.

[0008] The secretory signal sequence may be either naturally occurring with the therapeutically active protein or polypeptide sequence, or another one selected, modified, or engineered to optimize expression yield through secretion efficiency, processing kinetics, or cell line specific processing. Further examples and SEQ ID NOs are found in the sequence listing at the end of this disclosure. In some embodiments, the polypeptide may include a secretory signal peptide. In some embodiments, the secretory signal peptide is SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16. Production of the fusion polypeptide herein in a heterologous production system (e.g., bacteria or yeast) may result in incorrect cleavage of the signal sequence of the fusion polypeptide or non-specific initial cleavage at the C-terminus of the fusion polypeptide. Processing of the secretory sequence or N- or C-terminal processing may result in loss of amino acids from either the N- or C-terminus of the polypeptide.

[0009] There are several polypeptide sequences that can induce regenerative effects through membrane receptors. Examples from stem cell secretomes selected for their ability to improve muscle and soft tissue regeneration are listed in the sequence table and include IGF2 and its variants. Multimerization components link two or more separate protein components. Multimerization components can take the form of a linker sequence of amino acids that tandemly link other components into a single continuous amino acid sequence. Or multimerization components can take the form of a protein or protein domain that dimerizes, resulting in a covalent disulfide bond or non-covalent association that drives dimerization. Examples are disclosed in the sequence table at the end of this disclosure.

[0010] The stabilizing component can reduce the rate of degradation, increase translational or post-translational folding, reduce the rate of unfolding, or increase the circulating half-life. Examples can include abundant circulating proteins or fragments thereof, such as albumin or the fragment crystallizable (Fc) region from a human antibody. Further examples are disclosed in the sequence listing at the end of this disclosure.

[0011] Specific Definitions In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the provided embodiments may be practiced without these details. Unless otherwise required by context, throughout the following specification and claims, the term "comprise" and variations thereof (such as "comprises" and "comprising") should be interpreted in an inclusive sense, i.e., "including, but not limited to." As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in the sense of including "and / or" unless the content clearly dictates otherwise. Moreover, the headings provided herein are merely for convenience and do not interpret the scope or meaning of the claimed embodiments.

[0012] As used herein, the term "about" refers to an amount closer to 10% than the stated amount.

[0013] As used herein, the terms "individual", "patient" or "subject" are used interchangeably and refer to an individual who has been diagnosed with, is suspected of having, or is at risk of developing at least one disease for which the compositions and methods described are useful for treating. In some embodiments, the individual is a mammal. In some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In some embodiments, the individual is a human.

[0014] As used herein, the term "treat" or "treating" refers to an intervention in an individual's physiological or disease state designed or intended to improve at least one sign or symptom associated with said physiological or disease state. One skilled in the art will recognize that given a heterogeneous population of individuals suffering from a disease, not all individuals will respond equally, or at all, to a given treatment.

[0015] As used herein, the term "heterologous" refers to a nucleotide or amino acid sequence that is derived from a different source (e.g., a gene, polypeptide, or organism) compared to the amino acid or nucleotide sequence that is said to be heterologous. Heterologous includes biological sequences derived from different organisms or sequences derived from different sources (e.g., genes or proteins) of the same organism. Heterologous sequences include recombinant DNA molecules that contain nucleotide sequences from different sources, fusion proteins that contain amino acid sequences from different sources, and epitopes or purification tags of natural or synthetic origin.

[0016] As used herein, the term "muscle" refers to skeletal muscle, and not smooth or cardiac muscle.

[0017] As used herein, the term "soft tissue" refers to connective tissue including, but not limited to, tendons, ligaments, and cartilage.

[0018] As used herein, the term "mitogenic activity" refers to activity that induces cell division or proliferation.

[0019] As used herein, the term "fusion-promoting activity" refers to activity that promotes the fusion of cells into multinucleated cells, e.g., the fusion of muscle cells into multinucleated muscle fibers, or activity that advances the differentiation of terminally differentiated stem cells or progenitor cells toward a committed cell lineage type, such as the progression of myoblasts into muscle cells or the increase in cell size of an expanding muscle fiber.

[0020] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including antibodies and antibody chains provided as well as other peptides, such as linkers and linking peptides, may contain amino acid residues, including natural and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, the polypeptide may contain modifications relative to the native or native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host that produces the protein or by errors due to PCR amplification.

[0021] Percent (%) sequence identity 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 aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Suitable parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the sequence comparison computer program ALIGN-2 is used to generate % amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been submitted with user documentation to the US Copyright Office, Washington, DC, 20559, and is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., (South San Francisco, Calif.) or may be compiled from the 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 do not vary.

[0022] In the context of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, a given amino acid sequence A having or containing a certain % amino acid sequence identity with a given amino acid sequence B) is calculated as the fraction X / Y times 100, where X is the number of amino acid residues scored 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 to be understood that the length of amino acid sequence A is not equal to the length of amino acid sequence B, and the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0023] The polypeptides described herein may be encoded by nucleic acids. Nucleic acids are a type of polynucleotide that contains two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to introduce a polynucleotide encoding a polypeptide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. One type of vector is a genomic integrated vector or "integration vector" that can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. A vector that can induce the expression of a gene to which it is operably linked is referred to herein as an "expression vector." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In expression vectors, regulatory elements such as promoters, enhancers, polyadenylation signals, etc. for use in controlling transcription can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host (usually conferred by an origin of replication), and a selection gene to facilitate recognition of transformants, can be further integrated. Vectors derived from viruses such as lentivirus, retrovirus, adenovirus, adeno-associated virus, etc. may be used. Plasmid vectors can be linearized for integration into chromosomal locations. Vectors can contain sequences that induce site-specific integration (e.g., AttP-AttB recombination) into a defined location or a limited set of sites in the genome. Additionally, vectors can contain sequences derived from transposable elements.

[0024] IGF2 fusion protein Insulin-like growth factor (IGF) ligands IGF1 and IGF2 are involved in many cell signaling and developmental processes. IGF2 is one of the major embryonic growth factors in humans, with minimal expression in adults, where transient bursts localize to skeletal muscle cells that transition through multiple cell states. Further complexity in its regulation arises from its genomic imprinting, with IGF2 being one of the few proteins expressed only from the paternal copy. Its effects are differentially mediated by the cell surface receptors to which it binds: insulin receptor, insulin-like growth factor receptor 1 (IGF1R), and insulin-like growth factor receptor 2 (IGF2R). IGF1R activates many signal transductions, including pathways involved in cell proliferation, cell differentiation, and cell survival. IGF2R is involved in attenuating the signaling response. Described herein are certain therapeutically useful IGF2 polypeptides, including IGF2 fusion polypeptides that enhance the in vivo stability and function of IGF2, including combinations of polypeptides or IGF2 or IGF2 fusion proteins with an IGF binding protein (IGFBP), such as IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, or IGFBP6.

[0025] In certain aspects, IGF receptor ligand polypeptides are described herein. In certain aspects, IGF2 polypeptides are described herein that include an IGF2 amino acid sequence. In certain embodiments, the IGF2 amino acid sequence is that of a human IGF2 polypeptide. In certain embodiments, the human IGF2 polypeptide includes amino acids 25-91 of SEQ ID NO:32 (i.e., SEQ ID NO:29). In certain embodiments, the IGF2 amino acid sequence is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:29. In certain embodiments, the IGF2 amino acid sequence is 100% identical to SEQ ID NO:29. In certain embodiments, the IGF2 amino acid sequence includes an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:29, but with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids deleted from the N-terminus or C-terminus.

[0026] In certain embodiments, the IGF2 amino acid sequence is at least about 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 29 and has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deleted from the N-terminus or C-terminus of the polypeptide. In certain embodiments, the IGF2 fusion polypeptide amino acid sequence is at least about 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32 and has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deleted from the N-terminus or C-terminus of the polypeptide. In certain embodiments, the IGF2 fusion polypeptide amino acid sequence is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34 and has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deleted from the N-terminus or C-terminus of the polypeptide. In certain embodiments, the IGF2 amino acid sequence is at least about 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39 and has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deleted from the N-terminus or C-terminus of the polypeptide.

[0027] In certain IGF2, the polypeptides described herein are fusion proteins or polypeptides that may contain additional heterologous (non-IGF2) amino acid sequences that enhance the expression, stability, or function of the IGF2 polypeptide compared to a polypeptide that does not contain the heterologous amino acid sequence. These heterologous amino acid sequences may increase the expression of the IGF2 fusion polypeptide from a cell line (e.g., CHO cells or other suitable cell line for bulk 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. These heterologous amino acid sequences may increase the bioavailability of the IGF2 polypeptide in vivo (e.g., increase T1 / 2) 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. These heterologous amino acid sequences may increase the function of the IGF2 polypeptide in vivo (e.g., signaling through the IGF receptor) 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.

[0028] Also described herein are IGF receptor ligand fusion polypeptides or polypeptides that include a heterologous amino acid sequence in IGF2. In an embodiment, the IGF receptor ligand fusion is to a heterologous amino acid sequence that promotes the stability or function of the IGF receptor ligand. In an embodiment, the IGF2 amino acid sequence of the IGF2-heterologous polypeptide fusion protein is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:29. In an embodiment, the IGF2 amino acid sequence of the fusion protein is 100% identical to SEQ ID NO:29. In an embodiment, the IGF2 amino acid sequence of the IGF2-fusion protein is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:33. In an embodiment, the IGF2 amino acid sequence of the fusion protein is 100% identical to SEQ ID NO:33. In certain embodiments, the IGF2 amino acid sequence of the IGF2-fusion protein is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41. In certain embodiments, the IGF2 amino acid sequence of the fusion protein is 100% identical to SEQ ID NO: 41. Further representative sequences are found in the sequence listing at the end of this disclosure.

[0029] Secretory signal peptide In some aspects, the fusion polypeptide may include a secretory signal peptide. In some embodiments, the secretory signal peptide is any known mammalian secretory signal peptide. The production of the fusion polypeptide herein in a heterologous production system (e.g., bacteria or yeast) may result in incorrect cleavage of the signal sequence of the fusion polypeptide or non-specific initial cleavage at the C-terminus of the fusion polypeptide. Processing of the secretory sequence or N- or C-terminal processing may result in loss of amino acids from either the N- or C-terminus of the polypeptide.

[0030] A multimerization component links two or more separate protein components. A multimerization component may include a linker sequence of amino acids that links the same or different other components into a single contiguous amino acid sequence. Suitable linkers include polypeptide linkers such as Gly-Ser linkers or spacers described herein. A multimerization component can take the form of a protein or protein domain that multimerizes or dimerizes, resulting in a covalent disulfide bond (e.g., by the addition of one or more de novo cysteine ​​residues) or a non-covalent bond (e.g., leucine zipper) that drives the dimerization. In an embodiment, a multimerization component can link or multimerize multiple IGF2 amino acid sequences. In an embodiment, a multimerization component can link or multimerize two IGF2 amino acid sequences. The two IGF2 amino acid sequences can be the same or different and can be selected from any of the IGF2 sequences described herein. In certain embodiments, the multimerization moiety may link or multimerize two, three, four, five, or more IGF2 amino acid sequences, hi certain embodiments, the multimerization moiety may link or multimerize an IGF2 amino acid sequence with another polypeptide that provides a fusion-promoting or growth-promoting function, or increased plasma half-life.

[0031] In some embodiments, the IGF2 amino acid sequence may include its functional fragment, mutant sequence, or modified polypeptide. The sequence table lists some exemplary fragments, polypeptides, and modified polypeptides. In some embodiments, the IGF2 sequence is glycosylated in N-linked, C-linked, or O-linked manner. In some embodiments, the IGF2 sequence is glycosylated at one amino acid. In some embodiments, the IGF2 sequence is glycosylated at a site corresponding to Thr96, Thr99, or Thr163.

[0032] IGF family proteins are substrates for several proteases for the process during maturation and degrade intracellularly and extracellularly. The M16A family zinc metalloprotease known as insulin degrading enzyme (IDE) has high affinity (about 100 nM) for IGF2 as a substrate and degrades it rapidly (Malito et al. Cell Mol Life Sci. 2008;65:2574-85). Herein, mutations in IGF2 that reduce protease-mediated cleavage of the IGF2 peptide backbone are described. In some embodiments, these mutations are in the C domain, SEQ ID NO:42. In some embodiments, the mutations are specific to changing a positively charged amino acid, such as arginine or lysine, to another amino acid. In some embodiments, the mutation is arginine 61 or arginine 64, such as SEQ ID NO:35 or SEQ ID NO:37. In some embodiments, the mutations change a positively charged amino acid to another amino acid with a lower molecular weight. In some embodiments, the mutation mutates the amino acid sequence to one or more alanines, such as SEQ ID NO:34 or SEQ ID NO:36.

[0033] The IGF2 receptor ligand polypeptides and receptor ligand fusion polypeptides described herein may be encoded by nucleic acids that facilitate 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 (either constitutive or inducible), polyadenylation signals, selection markers (such as antibiotic resistance), origins of replication, or other accessory nucleic acid sequences. IGF2 sequences can be used from many organisms. In some embodiments, the IGF2 sequence includes a human IGF2 amino acid sequence. In some embodiments, the IGF2 sequence includes a feline, dog, or horse IGF2 sequence. In some embodiments, the IGF2 sequence includes a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, yak, or monkey sequence.

[0034] IGF2 nucleic acid sequence In some embodiments, the IGF2 nucleic acid sequence is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17. In some embodiments, the IGF2 nucleic acid sequence is 100% identical to SEQ ID NO: 17. In some embodiments, the IGF2 nucleic acid sequence is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21. In some embodiments, the IGF2 nucleic acid sequence is 100% identical to SEQ ID NO: 21. In some embodiments, the IGF2 nucleic acid sequence is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23. In some embodiments, the IGF2 nucleic acid sequence is 100% identical to SEQ ID NO: 23. In some embodiments, the IGF2 amino acid sequence is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. In one embodiment, the IGF2 amino acid sequence is 100% identical to SEQ ID NO:32.

[0035] Heterologous peptides A heterologous polypeptide comprising a portion of a fusion protein described herein can comprise, consist of, or consist essentially of a fragment of an immunoglobulin molecule, an albumin molecule, a transferrin molecule, an XTEN sequence, a proline-alanine-serine polymer, a homoamino acid polymer, a glycine-rich sequence, a gelatin-like polymer, an elastin-like peptide, a carboxy-terminal peptide, or a combination thereof.

[0036] In one embodiment described herein, the therapeutic polypeptide is an IGF receptor ligand polypeptide or an IGF2 polypeptide.

[0037] In one aspect described herein, a therapeutic polypeptide is fused, either directly or via a linker, to a heterologous polypeptide amino acid sequence, where the heterologous amino acid sequence confers increased function or stability to the therapeutic polypeptide.

[0038] In one aspect of the invention, the heterologous peptide increases the stability or biological function of the therapeutic amino acid sequence. In an embodiment, the heterologous sequence can be fused to the therapeutic amino acid sequence at the C-terminus or N-terminus of the therapeutic amino acid sequence. In an aspect, the therapeutic amino acid sequence is fused to the heterologous sequence at the N-terminus. In an embodiment, the therapeutic amino acid sequence is fused to the heterologous sequence at the C-terminus. In an embodiment, a flexible linker is used between the therapeutic amino acid sequence and the heterologous sequence at the N-terminus. In an embodiment, a flexible linker is used between the therapeutic amino acid sequence and the heterologous sequence at the C-terminus. In an embodiment, a spacer is used between the therapeutic amino acid sequence and the heterologous sequence at the N-terminus. In an embodiment, a spacer is used between the therapeutic amino acid sequence and the heterologous sequence at the C-terminus.

[0039] Heterologous peptides can be used to increase the stability or biological function of the IGF2 amino acid sequence. Fusion proteins can be used to improve the pharmacokinetics of biologically active molecules, such as by extending half-life, as discussed in Strohl, “Fusion Proteins for Half-Life Extension of Biologies as a Strategy to Make Biobetters,” BioDrugs (2015) 29:215-239. Fusing a polypeptide to a molecule or fragment of a molecule with a long half-life, such as immunoglobulin, albumin, or transferrin, increases the half-life of the polypeptide. XTEN sequences are repeating amino acid polymers containing amino acid residues A, E, G, P, S, and T, which, when fused to a peptide, can extend the half-life of the peptide that is otherwise inactive. Fusion of small repeating sequences, such as proline-alanine-serine polymers (proline, alanine, and serine repeats), homoamino acid polymer sequences, such as glycine-rich sequences (GGGS), gelatin-like proteins, and elastin-like sequences (VPGxG, where x is any amino acid except proline), can also extend the half-life of a polypeptide. Fusion of a polypeptide to a carboxy-terminal peptide (CTP) can increase the half-life of the polypeptide in serum due to the strong negative alteration of the CTP. In certain embodiments, the heterologous polypeptide comprises a fragment of an immunoglobulin molecule, an albumin molecule, a transferrin molecule, an XTEN sequence, a proline-alanine-serine polymer, a homoamino acid polymer, a glycine-rich sequence, a gelatin-like polymer, an elastin-like peptide, a carboxy-terminal peptide, or a combination thereof. In certain embodiments, a heterologous peptide that improves the pharmacokinetics of a biologically active molecule is genetically encoded to generate a fusion protein. In certain embodiments, the heterologous peptide that improves the pharmacokinetics of a biologically active molecule is post-translationally linked to the biologically active molecule, either covalently or non-covalently, and in certain embodiments, the non-covalent linkage can be driven by a genetically or covalently modified portion of the biologically active molecule.

[0040] Immunoglobulins are large effector molecules produced by the immune system. IgG immunoglobulins have a plasma half-life of about 21 days. When an immunoglobulin fragment is fused to a second polypeptide, it can increase the half-life of the second polypeptide. In some embodiments, the fragment of an immunoglobulin molecule comprises an IgG hinge domain, an IgG CH2 domain, an IgG CH3 domain, or any combination thereof. In some embodiments, the fragment of an immunoglobulin molecule comprises an IgG1 hinge domain, an IgG1 CH2 domain, an IgG1 CH3 domain, or any combination thereof. In some embodiments, the fragment of an immunoglobulin molecule comprises an IgG4 hinge domain, an IgG4 CH2 domain, an IgG4 CH3 domain, or any combination thereof.

[0041] In some circumstances, the mutation of the immunoglobulin molecule or fragment may increase the half-life or stability of the immunoglobulin molecule or fragment. In some embodiments, the fragment of the immunoglobulin molecule comprises the hinge domain of IgG, the CH2 domain of IgG1, the CH3 domain of IgG1, or any combination thereof with one or more of the following amino acid mutations in the immunoglobulin molecule: P329G, L234A, and L235A. In some embodiments, the fragment of the immunoglobulin molecule comprises an IgG4 molecule. In some embodiments, the fragment of an immunoglobulin molecule comprises in the immunoglobulin molecule the following amino acid mutations of IgG4 according to the EU numbering system: N434A, N434H, T307A / E380A / N434A, M252Y / S254T / T256E, 433K / 434F / 436H, T250Q, T250F, M428L, M428F, T250Q / M428L, N434S, V308W, V308 Y, 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.

[0042] A secretory signal sequence is a sequence motif that directs a protein into the secretory pathway within a cell. A secretory sequence can be cleaved from a protein to generate a mature secreted protein. In some embodiments, a polypeptide comprises a secretory signal sequence. In some embodiments, a polypeptide comprises a human IGF2 secretory sequence (SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12). In some aspects, a polypeptide comprises a secretory signal that is SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

[0043] Linkers and spacers A linker or spacer is a short amino acid sequence that separates different domains in a single protein or between fusion proteins. As used herein, the terms "linker" and "spacer" are interchangeable. A linker can be either rigid or flexible. Rigid linkers can prevent undesired interactions between different domains. Proline-rich linkers tend to be more rigid, while glycine-rich linkers tend to be more flexible. Flexible linkers can allow domains within a single protein to interact. Another use of flexible linkers is to covalently link protein complexes with binding partners to generate stable protein complexes. Flexible linkers can also be used to promote dimerization. Linkers and spacers are reviewed in Chichili et al, Linkers in the Structural biology of protein-protein interactions, Protein Sci. Feb 2013. 22(2):153-167.

[0044] The fusion polypeptide described herein may further comprise a linker or spacer amino acid sequence separating the therapeutic polypeptide and the heterologous polypeptide. In an embodiment, the linker or spacer is a peptide linker or spacer. In an embodiment, the linker or spacer is a flexible linker or spacer. In an embodiment, the linker is three alanines (AAA). In an embodiment, the peptide linker is a glycine-serine linker. In an embodiment, the linker is (in single letter amino acid code): GGGGS (4GS) or a multimer of 4GS linker, for example, 2, 3, 4, or 5 repeats of 4GS linker. In an embodiment, the glycine-serine linker comprises the amino acid sequence set forth in SEQ ID NO: 43 or 44, or 2, 3, 4, or 5 repeats of SEQ ID NO: 43 or 44. In certain embodiments, the linker comprises 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 are not derived from a polypeptide sequence in the Sequence Listing or from a heterologous polypeptide amino acid sequence in the Sequence Listing.

[0045] The linker or spacer may be a single amino acid residue or longer. In some embodiments, 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 long. In some embodiments, the peptide linker has at least one amino acid residue but is no longer than 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 long.

[0046] Combination of IGF1R agonist and short-chain fatty acids In certain aspects, compositions comprising an IGF1R agonist and a short chain fatty acid are disclosed herein. IGF1R signaling activates downstream pathways, including pathways involved in cell proliferation, cell differentiation, and cell survival. Two IGF ligands, IGF1 and IGF2, activate IGF1R signaling. An additional peptide that activates IGF1R signaling is INS. Other agonists of IGF1R include, but are not limited to, demethylasteriquinone B1, ginsenoside Rg5, and human antimicrobial peptide LL-37. In some embodiments, the IGF1R agonist comprises an IGF1R agonist antibody, an IGF polypeptide or a functional fragment thereof, IGF2 or a functional fragment thereof, insulin, demethylasteriquinone B1, ginsenoside Rg5, LL-37, or a combination thereof. These compositions include unexpected synergistic effects and are useful for treating muscle and / or soft tissue diseases or disorders. This synergistic effect may also be enhanced by methods involving separate administration of an IGF1R agonist and a short chain fatty acid.

[0047] In some embodiments, the IGF2R agonist is an IGF ligand. In some embodiments, the IGF1R agonist is IGF2. In some embodiments, the IGF2 polypeptide is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:29. In some embodiments, the IGF2 polypeptide is 100% identical to SEQ ID NO:29. In some embodiments, the IGF2 polypeptide is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:33. In some embodiments, the IGF2 polypeptide is 100% identical to SEQ ID NO:33. In some embodiments, the IGF2 polypeptide is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:34. In some embodiments, the IGF2 polypeptide is 100% identical to SEQ ID NO:34.

[0048] In some embodiments, the composition comprises an IGF1R agonist and a short-chain fatty acid. Short-chain fatty acids include, but are not limited to, butyrate, phenylbutyrate, valproic acid, propionic acid, methanoic acid, ethanoic acid, 2-methylpropanoic acid, 3-methylbutanoic acid, pentanoic acid, and their multimerized versions, such as tributyrin. Butyrate includes, but is not limited to, butyric acid, sodium butyrate, methylbutyrate, ethyl butyrate, butyl butyrate, pentyl butyrate, or sodium butyrate. In some embodiments, the short-chain fatty acid is butyrate. In some embodiments, the butyrate is butyric acid. In some embodiments, the butyrate is sodium butyrate. In some embodiments, the short-chain fatty acid is phenylbutyrate, valproic acid, propionic acid, methanoic acid, ethanoic acid, 2-methylpropanoic acid, 3-methylbutanoic acid, or pentanoic acid, or their multimerized versions, such as tributyrin.

[0049] The methods described herein also include administering an IGF1R agonist and a short chain fatty acid. The administration can be in the same composition, separate formulations. When separate formulations are administered, they can be administered simultaneously (e.g., during the same treatment) or separately, effectively separated by at least 1 hour, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more.

[0050] treatment index In certain aspects, the fusion polypeptides comprising an IGF ligand amino acid sequence and a heterologous polypeptide, compositions comprising an IGF1R agonist and short chain fatty acids, and methods described herein are useful for treating diseases and disorders involving soft tissue damage, degradation, or destruction, or for use in treating individuals with aging disorders, muscle wasting disorders, muscle damage, damage to connective tissue, or damage to non-muscle soft tissue, or any combination thereof.

[0051] Aging disorders that lead to the deterioration and loss of muscle tissue are such disorders. For example, sarcopenia is the degenerative loss of skeletal muscle mass, quality and strength, and can be associated with aging. Other muscle disorders that lead to acute muscle damage can be treated by the polypeptides, compositions and methods described herein. Disorders include muscle rupture, pulled muscle, and contusion. Rupture is the separation of muscle tissue. Pulled muscle is contraction-induced injury in which muscle fibers are torn by extensive mechanical stress, and can be classified into grade I, II, or III. Muscle contusion is muscle hematoma. Muscle damage can also be caused by non-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 (acrodynia), Crohn's disease, untreated / severe type 1 diabetes, anorexia nervosa, chemotherapy, muscular dystrophy, or other genetic diseases that cause immobility, and hormone deficiency.Certain disorders that are weakness of specific muscles (e.g., dysphagia or scapular-humeral muscular dystrophy) can also be treated by the polypeptides described herein.Further soft tissue disorders that can be treated using the polypeptides comprising the IGF ligand amino acid sequence described herein and compositions comprising IGF1R agonists and short-chain fatty acids are those that damage tendons, ligaments, or cartilage.

[0052] In some embodiments, the muscle wasting disease is muscular dystrophy.In some embodiments, the muscular dystrophy includes myotonic muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapular-humeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, or distal muscular dystrophy.In some embodiments, the muscular dystrophy is myotonic dystrophy.

[0053] In some embodiments, the aging disorder is sarcopenia. In some embodiments, the muscle wasting disorder is cachexia. In some embodiments, the cachexia is a result of cancer, AIDS, end stage renal disease, or cardiovascular disease. In some embodiments, the injury is muscle injury. In some embodiments, the muscle wasting is limb immobilization or disuse atrophy. In some embodiments, the muscle injury is a pulled or torn muscle. In some embodiments, the muscle injury is a grade III pulled muscle. In some embodiments, sarcopenia contributes to the development of muscle injury. In some embodiments, the injury is a ligament injury. In some embodiments, the ligament injury is a rupture or tear. In some embodiments, the injury is a tendon injury. In some embodiments, the tendon injury is a rupture or tear. In some embodiments, the injury is a cartilage injury.

[0054] In some embodiments, the compositions described herein are for use in a method for treating myositis, including dermatomyositis, polymyositis, necrotizing myopathy (also called necrotizing autoimmune myopathy or immune-mediated necrotizing myopathy), juvenile myositis, or sporadic inclusion body myositis.

[0055] In some embodiments, the compositions described herein are for use in a method for treating cartilage-related disorders.In some embodiments, the cartilage-related disorders can be caused by tearing, injury, or wear.In some embodiments, the cartilage-related disease can be osteoarthritis, osteochondritis dissecans, achondroplasia, or degenerative cartilage lesions.

[0056] In some embodiments, the compositions described herein are for use in the method of increasing proliferation or promoting survival of cells associated with soft tissue injury.In some embodiments, the compositions comprising the polypeptide comprising the IGF ligand amino acid sequence described herein and the IGF1R agonist and short chain fatty acid are useful in the method of increasing proliferation or promoting survival of any one or more of muscle cells, muscle progenitor cells, tenocytes, tenocyte progenitor cells, chondrocytes, chondrocyte progenitor cells, mesenchymal stem cells, or fibroblasts.

[0057] Muscle fibrosis is the excessive accumulation of extracellular matrix components, including collagen. Muscle fibrosis impairs muscle function, adversely affects muscle regeneration after injury, and increases muscle susceptibility to re-injury. In some embodiments, the compositions described herein are for use in methods for reducing muscle fibrosis. In some embodiments, fibrosis is associated with aging, muscular dystrophy, or injury. In some embodiments, the IGF ligand is IGF2.

[0058] In order to differentiate into mature muscle cells, myoblasts must fuse to form multinucleated cells. In some embodiments, the fusion polypeptides comprising IGF ligand amino acid sequences and heterologous polypeptides, compositions comprising IGF1R agonists and short chain fatty acids, and methods described herein are for use in methods to increase myoblast fusion. In some embodiments, the IGF ligand is IGF2.

[0059] In some embodiments, the fusion polypeptide comprising an IGF ligand amino acid sequence and a heterologous polypeptide, the composition comprising an IGF1R agonist and a short chain fatty acid, and the method described herein are for use in a method for increasing muscle mass. In some embodiments, the muscle mass is increased by at least about 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, or more than 50%. In some embodiments, the IGF ligand is IGF2.

[0060] In some embodiments, the fusion polypeptide comprising an IGF ligand amino acid sequence and a heterologous polypeptide, the composition comprising an IGF1R agonist and a short chain fatty acid, and the method described herein are for use in a method for increasing grip strength. In some embodiments, the grip strength is increased by at least about 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, or more than 50%. In some embodiments, the IGF ligand is IGF2.

[0061] In an embodiment, the fusion polypeptide comprising IGF ligand amino acid sequence and heterologous polypeptide, the composition comprising IGF1R agonist and short chain fatty acid, and the method described herein are for use in the method for increasing muscle endurance.In an embodiment, muscle endurance is increased by at least about 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, or more than 50%.In an embodiment, the IGF ligand is IGF2.

[0062] Treatment Method In certain aspects, disclosed herein is a method of treating an individual with a disorder, comprising administering to the individual an IGF1R agonist and a short chain fatty acid. In some embodiments, the IGF1R agonist and the short chain fatty acid are administered in separate formulations. In some embodiments, the IGF1R agonist and the short chain fatty acid are administered simultaneously. In some embodiments, the IGF1R agonist and the short chain fatty acid are administered at different times.

[0063] In one aspect, disclosed herein is a method for treating an individual with a disorder, comprising administering to the individual with the disorder a polypeptide comprising an IGF ligand amino acid sequence and butyric acid. In some embodiments, the polypeptide comprising an IGF ligand amino acid sequence and butyric acid are administered in separate formulations. In some embodiments, the polypeptide comprising an IGF ligand amino acid sequence and butyric acid are administered simultaneously. In some embodiments, the polypeptide comprising an IGF ligand amino acid sequence and butyric acid are administered at different times.

[0064] In one aspect, disclosed herein is a method for treating an individual with a disorder, comprising administering to the individual with the disorder a polypeptide comprising an IGF2 amino acid sequence and a short chain fatty acid. In some embodiments, the IGF ligand amino acid sequence and the short chain fatty acid are administered in separate formulations. In some embodiments, the IGF2 amino acid sequence and the short chain fatty acid are administered simultaneously. In some embodiments, the IGF2 amino acid sequence and the short chain fatty acid are administered at different times.

[0065] In one aspect, disclosed herein is a method for treating an individual with a disorder, comprising administering to the individual with the disorder a polypeptide comprising an IGF2 amino acid sequence and butyrate.In some embodiments, the polypeptide comprising an IGF2 amino acid sequence and butyrate are administered in separate formulations.In some embodiments, the polypeptide comprising an IGF2 amino acid sequence and butyrate are administered simultaneously.In some embodiments, the polypeptide comprising an IGF2 amino acid sequence and butyrate are administered at different times.

[0066] In some embodiments, the treatment is administered by any suitable route, such as, for example, subcutaneously, intravenously, or intramuscularly. In some embodiments, the treatment is administered in a suitable dosing regimen, such as weekly, twice weekly, monthly, twice monthly, once every three weeks, or once every four weeks. The treatment is administered in any therapeutically effective amount. In some embodiments, the therapeutically effective amount is from about 0.001 mg / kg to about 1 mg / kg.In certain embodiments, the therapeutically effective amount is from about 0.001 mg / kg to about 0.002 mg / kg, from about 0.001 mg / kg to about 0.005 mg / kg, from about 0.001 mg / kg to about 0.01 mg / kg, from about 0.001 mg / kg to about 0.02 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.001 mg / kg to about 0.1 mg / kg, from about 0.001 mg / kg to about 0.2 mg / kg, from about 0.001 mg / kg to about 0.5 mg / kg, from about 0.001 mg / kg to about 1 mg / kg, from about 0.002 mg / kg to about 0.005 mg / kg, or from about 0. 002mg / 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 0.0 05mg / kg~about 0.2mg / kg, about 0.005mg / kg~about 0.5mg / kg, about 0.005mg / kg~about 1mg / kg, about 0.01mg / kg~about 0.02mg / kg, about 0.01mg / kg~about 0.05mg / kg, about 0.01mg / kg~about 0.1mg / k g, about 0.01mg / kg to about 0.2mg / kg, about 0.01mg / kg to about 0.5mg / kg, about 0.01mg / kg to about 1mg / kg, about 0.02mg / kg to about 0.05mg / kg, about 0.02mg / kg to about 0.1mg / kg, about 0.02mg / kg to about 0.2mg / kg, about 0.02 mg / kg to about 0.5 mg / kg, about 0.02 mg / kg to about 1 mg / kg, about 0.05 mg / kg to about 0.1 mg / kg, about 0.05 mg / kg to about 0.2 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.05 mg / kg to about 1 mg / kg, about 0.1 mg / kg to about 0.2 mg / kg, about 0.1 mg / kg to about 0.5 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.2 mg / kg to about 0.5 mg / kg, about 0.2 mg / kg to about 1 mg / kg, or about 0.5 mg / kg to about 1 mg / kg.In certain embodiments, the therapeutically effective amount is about 0.001 mg / kg, about 0.002 mg / kg, about 0.005 mg / kg, about 0.01 mg / kg, about 0.02 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.5 mg / kg, or about 1 mg / kg. In certain embodiments, the therapeutically effective amount is at least about 0.001 mg / kg, about 0.002 mg / kg, about 0.005 mg / kg, about 0.01 mg / kg, about 0.02 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, or about 0.5 mg / kg. In some embodiments, the therapeutically effective amount is up to about 0.002 mg / kg, about 0.005 mg / kg, about 0.01 mg / kg, about 0.02 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.5 mg / kg, or about 1 mg / kg. In some embodiments, the therapeutically effective amount is from about 0.1 mg / kg to about 50 mg / kg.In certain embodiments, the therapeutically effective amount is from about 0.1 mg / kg to about 0.2 mg / kg, from about 0.1 mg / kg to about 0.5 mg / kg, from about 0.1 mg / kg to about 1 mg / kg, from about 0.1 mg / kg to about 2 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 20 mg / kg, from about 0.1 mg / kg to about 50 mg / kg, Approx. 0.2 mg / kg ~ approx. 0.5 mg / kg, approx. 0.2 mg / kg ~ approx. 1 mg / kg, approx. 0.2 mg / kg ~ approx. 2 mg / kg, approx. 0.2 mg / kg ~ approx. 5 mg / kg, approx. 0.2 mg / kg ~ Approximately 10mg / kg, approximately 0.2mg / kg to approximately 20mg / kg, approximately 0.2mg / kg to approximately 50mg / kg, approximately 0.5mg / kg to approximately 1mg / kg, approximately 0.5mg / kg to approximately 2mg / kg, approximately 0.5mg / kg to about 5mg / kg, about 0.5mg / kg to about 10mg / kg, about 0.5mg / kg to about 20mg / kg, about 0.5mg / kg to about 50mg / kg, about 1mg / kg to about 2 mg / kg, 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 m g / kg, about 2 mg / kg to about 10 mg / kg, about 2 mg / kg to about 20 mg / kg, about 2 mg / kg to about 50 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 20 mg / kg, about 5 mg / kg to about 50 mg / kg, about 10 mg / kg to about 20 mg / kg, about 10 mg / kg to about 50 mg / kg, or about 20 mg / kg to about 50 mg / kg. In some embodiments, the therapeutically effective amount is about 0.1 mg / kg, about 0.2 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, or about 50 mg / kg. In some embodiments, the therapeutically effective amount is at least about 0.1 mg / kg, about 0.2 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, or about 20 mg / kg. In some embodiments, the therapeutically effective amount is up to about 0.2 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, or about 50 mg / kg.

[0067] In some embodiments, the individual being treated is a mammal. In some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In some embodiments, the individual is a dog, cat, or horse. In some embodiments, the individual being treated is a human.

[0068] Method of production A polypeptide comprising an IGF2 ligand amino acid sequence can be purified or synthesized in any suitable manner. The nucleic acid encoding the polypeptide can be cloned into a suitable vector and expressed in a suitable cell line. In some embodiments, the cell line is a prokaryotic cell line. In some embodiments, the cell line is a eukaryotic cell line. In some embodiments, the cell line is a mammalian cell line. In some embodiments, the polypeptide can be expressed from E. coli. In some embodiments, the polypeptide can be expressed from yeast cells, including but not limited to Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, or Yarrowia lipolytica. In some embodiments, the polypeptide can be expressed from mouse myeloma cells, including but not limited to NSO, Sp2 / 0, and FO. In some embodiments, the polypeptide can be expressed from Chinese hamster ovary (CHO) cells. In some embodiments, the polypeptides may be expressed by mammalian cells, including but not limited to COS cells, Vero cells, or BHK cells. In some embodiments, the polypeptides may be expressed from human cells, including but not limited to HeLa cells, HEK-293 cells, CAP cells, CAP-T cells, PER.C6® cells.

[0069] The supernatant from such an expression system may be subjected to one or more purification steps, including centrifugation, ultracentrifugation, filtration, diafiltration, tangential flow filtration, dialysis, chromatography (e.g., cation exchange, ion exchange, hydrophobic interaction, reverse phase, affinity, or size exclusion). The polypeptide may be purified to a degree suitable for human administration. Additionally, the polypeptide may be synthesized for inclusion in a formulation administered to a human individual. In certain embodiments, the polypeptide may be produced by a suitable peptide synthesis method, such as solid phase synthesis.

[0070] In one embodiment, the mammalian expression vector pmax Cloning is used to make C-terminal 6xHis-tagged, StrepII-tagged, and human IgGl Fc-tagged vectors. DNA fragments encoding secreted myogenic factors are amplified by PCR from human open reading frame (ORF) clones and then inserted into the tagged vectors by In-Fusion cloning technology (Takara Bio Inc.). Expression vectors carrying secreted myogenic factors are transfected with 6xl0 ng / ml of IgGl-tagged vectors using ExpiFectamine CHO transfection kit (Thermo Scientific). 6 The vector is transiently transfected into ExpiCHO-S cells at a density of 100 / ml.

[0071] The expressed myogenic factors with different tags in the culture supernatant are affinity purified by using different purification media. In some embodiments, the polypeptide comprises an Fc region. For these polypeptides, matrices or resins containing protein A, protein G, protein L or any combination thereof can be used. The matrices or resins can be appropriately loaded onto columns to facilitate batch purification.

[0072] Purification of immunoglobulin fusion proteins In certain embodiments, the heterologous sequence may comprise an immunoglobulin or a fragment thereof. When the polypeptide comprises an immunoglobulin or a fragment thereof, the polypeptide may be purified by protein A, G, or L affinity. Protein 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 coupled to a solid matrix such as cross-linked uncharged agarose (Sepharose, not including the charged fraction of native agarose), tris-acryl, cross-linked dextran, or silica-based materials. Such methods are well known in the art, for example, coupling to a CNBr-activated matrix via the primary amino functional groups of the protein. Protein A binds with high affinity and high specificity to the Fc portion of IgG, i.e., the Cγ2-Cγ3 interface region of IgG, as previously described by Langone et al., 1982. In particular, it binds strongly to human allotypes or subclasses IgG1, IgG2, and IgG3, and mouse allotypes or subclasses IgG2a, IgG2b, and IgG3.

[0073] After purification by Protein A, G or L, the bound fraction can be eluted and passed over or through additional resins or matrices, including one or more ion exchange columns. The first ion exchanger is usually an anion exchanger. The pH of the buffer used for loading and running the first ion exchanger is set to counter the total change of Fc-containing fusion polypeptide and Protein A to be separated by the ion exchanger in flow-through mode according to the invention, taking into account the pi of the Fc-containing fusion polypeptide and Protein A. The operation mode of the first anion exchanger according to the invention requires a buffer exchange between the acidic or neutralized eluate from the Protein A affinity chromatography step and the equilibration buffer of the first anion exchanger. After the first anion exchanger, the Fc-containing fusion polypeptide can be considered ready for use in the application or in need of further purification by customary purification methods. In a further preferred embodiment, the first ion exchange step is followed by a second ion exchange step in which the antibody is loaded as essentially monomeric, non-aggregated antibody, bound to a second ion exchange medium, and eluted by increasing salt and / or pH with a buffer other than the loading buffer.

[0074] In certain embodiments, the method according to the invention allows recovery of at least 70%, 80% or 90% of the Fc-containing fusion polypeptide loaded onto the first ion exchanger in the flow-through of the ion exchanger, in certain embodiments, only one type of species of Fc-containing fusion polypeptide is present in the mixture, disregarding glycoforms and the resulting processing variants of the same Fc-containing fusion polypeptide.

[0075] Master cell bank and transgenic cells In some embodiments, a master cell bank is described herein that includes cells that include a nucleic acid encoding one or more IGF ligands or IGF2 fusion polypeptides that are integrated into the genome to generate a transgenic cell line. In some embodiments, the master cell bank includes a plurality of cells that each include a nucleic acid encoding an IGF ligand or an IGF2 fusion polypeptide. In some embodiments, the nucleic acid is maintained extrachromosomally on a plasmid or yeast artificial chromosome. In some embodiments, the nucleic acid is integrated into a chromosomal location. In some embodiments, the cell is a yeast cell. In some embodiments, the yeast is Pichia pastoris or Saccharomyces cerevisiae. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a 293T cell or a derivative thereof (e.g., 293T-Rex). In some embodiments, the cell is a bacterial cell.

[0076] In some embodiments, the transgenic mammalian, yeast, or bacterial cells are a master cell bank that includes a cryopreservative suitable for freezing to at least about -80° C. or below. In some embodiments, the master cell bank includes about 10 to about 30% glycerol or DMSO and is suitable for long-term storage at about -80° C. or below. In some embodiments, the master cell bank is capable of storing the transgenic mammalian, yeast, or bacterial strains for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more.

[0077] Pharmaceutically acceptable excipients, carriers, and diluents The polypeptides comprising the IGF2 amino acid sequence and the amino acid sequence from a heterologous polypeptide described herein can be administered in a pharmaceutical composition comprising one or more pharma- ceutically acceptable excipients, carriers, or diluents. The exact components may vary based on the preferred route of administration. The excipients used in the pharmaceutical composition can provide additional functions to the polypeptide by making it suitable for a particular route of administration (e.g., intravenous, topical, subcutaneous, or intramuscular), increasing the stability of the polypeptide, increasing the penetration of the desired tissue (e.g., muscle or skin), increasing the residence time at a particular site, increasing solubility, enhancing the efficacy of the polypeptide, and / or reducing the inflammatory response that occurs upon administration.

[0078] In some embodiments, the composition is included in the pharmaceutical composition with a solubilizing emulsifier or dispersant. In some embodiments, the solubilizer can allow for a highly concentrated solution of the fusion polypeptide, at least about 2 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, or more than 20 mg / mL. The carbomer in the aqueous pharmaceutical composition acts as an emulsifier and a viscosity adjuster. In some embodiments, the pharma- ceutically acceptable excipient comprises or consists of a carbomer. In some embodiments, the carbomer comprises or consists of carbomer 910, carbomer 934, carbomer 934P, carbomer 940, carbomer 941, carbomer 1342, or combinations thereof. The cyclodextrin in the aqueous pharmaceutical composition serves as a solubilizer and stabilizer. In some embodiments, the pharma-ceutically acceptable excipient comprises or consists of a cyclodextrin. In some embodiments, the cyclodextrin comprises or consists of alpha cyclodextrin, beta cyclodextrin, gamma cyclodextrin, or a combination thereof. The lecithin in the pharmaceutical composition may serve as a solubilizer. In some embodiments, the solubilizer comprises or consists of lecithin. The poloxamer in the pharmaceutical composition serves as an emulsifier, solubilizer, and dispersant. In some embodiments, the pharmaceutically acceptable excipient comprises or consists of a poloxamer. In some embodiments, the poloxamer comprises or consists of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or a combination thereof. The polyoxyethylene sorbitan fatty acid ester in the pharmaceutical composition serves as an emulsifier, solubilizer, surfactant, and dispersant. In some embodiments, the pharmaceutically acceptable excipient comprises or consists of a polyoxyethylene sorbitan fatty acid ester.In one embodiment, the polyoxyethylene sorbitan fatty acid ester comprises or consists of polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, polysorbate 120, or combinations thereof. The polyoxyethylene stearate in the pharmaceutical composition acts as an emulsifier, solubilizer, surfactant, and dispersant. In one embodiment, the pharma- ceutically acceptable excipient comprises or consists of polyoxyethylene stearate. In an embodiment, the polyoxyethylene stearate comprises or consists of polyoxyl 2 stearate, polyoxyl 4 stearate, polyoxyl 6 stearate, polyoxyl 8 stearate, polyoxyl 12 stearate, polyoxyl 20 stearate, polyoxyl 30 stearate, polyoxyl 40 stearate, polyoxyl 50 stearate, polyoxyl 100 stearate, polyoxyl 150 stearate, polyoxyl 4 distearate, polyoxyl 8 distearate, polyoxyl 12 distearate, polyoxyl 32 distearate, polyoxyl 150 distearate, or combinations thereof. The sorbitan ester in the pharmaceutical composition acts as an emulsifier, solubilizer, and non-ionic surfactant, as well as a dispersant. In an embodiment, the pharma- ceutically acceptable excipient comprises or consists of a sorbitan ester. In some embodiments, the sorbitan ester comprises or consists of sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan stearate, sorbitan trioleate, sorbitan sesquioleate, or combinations thereof. In some embodiments, solubility can be achieved using a protein carrier. In some embodiments, the protein carrier comprises recombinant human albumin.

[0079] In some embodiments, the polypeptide comprising the IGF2 amino acid sequence described herein and an amino acid sequence from a heterologous polypeptide is formulated to increase stability. The polypeptide in the aqueous formulation may require stabilization to prevent degradation. In some embodiments, the stabilizer comprises a pH buffer, a salt, an amino acid, a polyol / disaccharide / polysaccharide, a liposome, a surfactant, an antioxidant, a reducing agent, or a chelating agent. In some embodiments, the stabilizer comprises or consists of a polyol / non-reducing sugar. In some embodiments, the non-reducing sugar comprises or consists of sucrose, mannitol, trehalose, raffinose, stachyose, xylitol, starch, verbascose, or a combination thereof. To increase stability, the polypeptide may be encapsulated in a liposome. In some embodiments, the stabilizer comprises or consists of a liposome. In some embodiments, the liposome comprises or consists of ipalmitoyl phosphatidylcholine (DPPC) liposomes, phosphatidylcholine:cholesterol (PC:Chol) (70:30) liposomes, or dipalmitoyl phosphatidylcholine:dipalmitoyl phosphatidylserine (DPPC:DPPS) (70:30) liposomes. Non-ionic surfactants can enhance the stability of the polypeptide. In some embodiments, the stabilizer comprises or consists of a non-ionic surfactant. In some embodiments, the non-ionic surfactant comprises or consists of a polysorbate (e.g., polysorbate 80, polysorbate 20), alkyl saccharide 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. In some embodiments, the polypeptide is formulated with a protein surfactant, such as recombinant human serum albumin, as a stabilizer. Antioxidants or reducing agents can increase the stability of a polypeptide, hi certain embodiments, the stabilizer comprises or consists of an antioxidant or reducing agent.In certain embodiments, the reducing agent comprises or consists of dithiothreitol, ethylenediaminetetraacetic acid, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride, tris(hydroxypropyl)phosphine, or a combination thereof. In certain embodiments, the antioxidant comprises or consists of methionine, ascorbic acid, citric acid, alpha-tocopherol, sodium bisulfite, ascorbyl palmitate, erythorbic acid, or a combination thereof. The chelating agent can stabilize the polypeptide by reducing the activity of the protease. In certain embodiments, the stabilizing agent comprises or consists of a chelating agent. In certain embodiments, the chelating agent comprises or consists of ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), metal complexes (e.g., Zn-protein complexes), or a combination thereof. The buffer can stabilize the polypeptide by reducing acid hydrolysis of the polypeptide. In some embodiments, the stabilizer comprises or consists of a buffer, which in some embodiments consists of sucrose octa-sulfate, ammonium carbonate, diammonium 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.

[0080] The polypeptides comprising the IGF2 amino acid sequence and the amino acid sequence from a heterologous polypeptide described herein can also be entrapped in or associated with colloidal delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, for example, microcapsules prepared by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively). Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

[0081] A polypeptide comprising an IGF2 amino acid sequence as described herein and an amino acid sequence from a heterologous polypeptide can be formulated or delivered with an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent comprises or consists of a corticosteroid. In some embodiments, the corticosteroid comprises or consists of hydrocortisone, cortisone, ethamethasoneb (Celestone), prednisone (Prednisone Intensol), prednisolone (Orapred, Prelone), triamcinolone (Aristospan Intra-Articular, Aristospan Intralesional, Kenalog), methylprednisolone (Medrol, Depo-Medrol, Solu-Medrol) or dexamethasone (Dexamethasone Intensol). In some embodiments, the anti-inflammatory agent comprises or consists of a nonsteroidal anti-inflammatory drug (NSAID). In certain embodiments, the NSAID comprises or consists of aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac, or tolmetin.

[0082] In some embodiments, the polypeptide comprising the IGF2 amino acid sequence described herein and the amino acid sequence from a heterologous polypeptide is contained in a pharmaceutical composition suitable for intravenous administration, and the pharmaceutical composition comprises one or more pharma- ceutical acceptable excipients, carriers, and diluents.In some embodiments, the polypeptide of the present disclosure is administered suspended in a sterile solution.In some embodiments, the solution is one that is commonly used for the administration of biological agents, for example, contains about 0.9% NaCl or about 5% dextrose. In certain embodiments, the solution further comprises one or more of the following: a buffering agent, such as acetate, citrate, histidine, succinate, phosphate, potassium phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, such as glycine, histidine, leucine, or arginine; an antioxidant, such as ascorbic acid, methionine, or a chelating agent, such as EDTA, or EGTA.

[0083] In an embodiment, the polypeptide comprising the IGF2 amino acid sequence and the amino acid sequence from a heterologous polypeptide described herein is contained in a pharmaceutical composition suitable for intramuscular or subcutaneous administration, the pharmaceutical composition comprising one or more pharma- ceutical acceptable excipients, carriers, and diluents. Formulations suitable for intramuscular or subcutaneous injection can comprise physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include ethanol, polyols (such as inositol, propylene glycol, polyethylene-glycol, glycerol, cremophor, etc.), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity is maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. Formulations suitable for subcutaneous injection also contain optional additives such as preservatives, wetting agents, emulsifying agents, and dispersing agents.

[0084] In some embodiments, the polypeptide comprising the IGF2 amino acid sequence described herein and the amino acid sequence from heterologous polypeptide is formulated as cream, gel, paste, ointment or emulsion for topical administration.The excipient in cream, gel, paste, ointment or emulsion can include gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan ester, polyoxyethylene alkyl ether, polyoxyethylene castor oil derivative, polyoxyethylene sorbitan fatty acid ester, polyethylene glycol, polyoxyethylene stearate, silicon dioxide, phosphate, sodium dodecyl sulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, sugar and starch.

[0085] Excipients used with polypeptides comprising an IGF2 amino acid sequence and an amino acid sequence from a heterologous polypeptide as described herein allow for the storage, formulation, or administration of highly concentrated formulations. In certain embodiments, the high concentration fusion polypeptide(s) comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 20, 25, 40, 45, 50 milligrams per milliliter, or more.

[0086] In some embodiments, the polypeptides and / or compositions of the present disclosure are shipped / stored lyophilized and reconstituted prior to administration. In some embodiments, the lyophilized ligand fusion polypeptide formulation includes bulking agents such as mannitol, sorbitol, sucrose, trehalose, and dextran 40. The lyophilized formulation can be placed in a vial made of glass. The fusion polypeptide, when formulated, whether or not it is reconstituted, can be buffered at a specific pH, generally below 7.0. In some embodiments, the pH can be 4.5-6.5, 4.5-6.0, 4.5-5.5, 4.5-5.0, or 5.0-6.0.

[0087] kit Also described herein are kits comprising one or more polypeptides comprising an IGF2 amino acid sequence described herein and an amino acid sequence from a heterologous polypeptide in a suitable container, and one or more additional components selected from instructions for use, a diluent, an excipient, a carrier, and a device for administration.

[0088] In an embodiment, a method for preparing a treatment for a soft tissue or muscle disease or disorder is described herein, the method comprising combining one or more pharma- ceutically acceptable excipients, carriers, or diluents with a polypeptide comprising an IGF2 amino acid sequence and an amino acid sequence from a heterologous polypeptide as described herein.In an embodiment, a method for preparing a treatment for a soft tissue or muscle disease or disorder for storage or transport is described herein, the method comprises lyophilizing one or more antibodies of the present disclosure. EXAMPLES

[0089] Example 1 Recombinant Protein Expression and Purification Mammalian expression plasmids carrying genes with different tags were transiently transfected into CHO cells. The genes were expressed to produce proteins, which were then secreted into the culture medium. The proteins in the culture medium were visualized on polyacrylamide gels and their activities were measured by in vitro functional assays. The recombinant proteins in the culture medium were then affinity purified. The purified proteins were visualized on polyacrylamide gels to assess purity and assayed by in vitro functional assays to determine their biological activity.

[0090] Expression vector engineering Mammalian expression vector pmax Cloning was used to generate C-terminal 6xHis-tagged vectors, StrepII-tagged vectors, and human IgG1 and IgG4 Fc-tagged vectors. DNA fragments encoding secreted myogenic factors were amplified by PCR from human open reading frame (ORF) clones and then inserted into the tagged vectors by In-Fusion cloning technology (Takara Bio).

[0091] Expression of secreted myogenic polypeptide Expression vectors carrying secreted myogenic factors were transfected into 6x10 cells using the ExpiFectamine CHO transfection kit (Thermo Scientific). 6 The myogenic factors were transiently transfected into ExpiCHO-S cells at a density of 1000 / ml. After 18-22 h, CHO feed and enhancer were added to the transfected cultures. The expressed protein was then monitored by SDS-PAGE every 24 h to achieve maximum expression levels. 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 the secreted myogenic factors was stored at -80°C or immediately processed for use.

[0092] Measurement of expression levels of secreted myogenic polypeptides To measure the improved expression levels of secreted myogenic factors, three protein analysis techniques were applied: sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Western blots were performed to identify the myogenic factors. ELISA was used to measure the absolute amounts of myogenic factors in the culture supernatants.

[0093] Isolation of engineered myogenic polypeptides The expressed myogenic factors with different tags in the culture supernatant were affinity purified by using different purification media. For Fc-fusion factors, either Protein A magnetic beads (GenScript) or Protein A membrane columns (Takara Bio Inc.) were used to specifically bind to the Fc-fusion factors. For 6xHis-tagged factors, NTA magnetic beads (NEB) were used to isolate the factors.

[0094] Example 2 Purified IGF2-hFcm promoted the differentiation of human myoblasts. Figure 1A: Suspension CHO cells were transiently transfected with a plasmid encoding IGF2-hFcm. IGF2-hFcm was affinity purified by protein A membrane column. Purified IGF2-hFcm was added to cultures of human myoblasts for 96 hours. Myosin heavy chain (MyHC) was immunostained and imaged by fluorescence microscopy. The percentage of MyHC area in human myoblasts treated with purified IGF2-hFcm is significantly higher than the percentage of MyHC area in human myoblasts treated with vehicle control (One-Way ANOVA Tukey Honest Significant Difference, n=2-6).

[0095] [Table 1]

[0096] Example 3 IGF2-LhFc4 promoted the differentiation of human myoblasts. Figure 1b: Suspension CHO cells were transiently transfected with IGF2-LhFc4-encoding plasmid. IGF2-LhFc4 was affinity purified by protein A membrane column. Purified IGF2-LhFc4 was added to cultures of human myoblasts for 96 h with daily medium changes. Myosin heavy chain (MyHC) was immunostained and imaged by fluorescence microscopy. The percentage of area of ​​MyHC in human myoblasts treated with purified IGF2-LhFc4 is significantly higher than the percentage of area of ​​MyHC in human myoblasts treated with vehicle control (One-Way ANOVA Tukey Honest Significant Difference, n=2-6).

[0097] [Table 2]

[0098] Example 4 Differentiation of purified HSA-L-IGF2R61A human myoblasts. Figure 2A: A plasmid encoding HSA-L-IGF2R61A was transiently transfected into suspension CHO cells. HSA-L-IGF2R61A was affinity purified by protein A membrane column. Purified HSA-L-IGF2R61A was added to cultures of human myoblasts for 96 hours, and the medium was changed daily. Myosin heavy chain (MyHC) was immunostained and imaged by fluorescence microscopy. The percentage of area of ​​MyHC in human myoblasts treated with purified HSA-L-IGF2R61A is significantly higher than the percentage of area of ​​MyHC in human myoblasts treated with vehicle control (One-Way ANOVA Tukey Honest Significant Difference, n=2-6).

[0099] [Table 3]

[0100] Example 5 IGF2 and IGF2 receptor are expressed in human myoblasts. Bar graphs and quantification tables of RNASeq expression of IGF2 (Figure 2B) and IGF2 receptor (Figure 2C) in young (17-21 year old Caucasian male) and old (68-69 year old Caucasian male) human myoblast cell lines. Myoblasts were cultured in growth medium (GM) or fusion medium (FM) for 96 h. Fresh medium was added every 24 h. Mean ± SEM. n=6. Expression is expressed as FPKM. Significant p-value (young GM-old GM: 3.54E-04).

[0101] [Table 4]

[0102] Example 6 Sodium butyrate enhances muscle fusion. Mouse myoblasts were treated with PBS or sodium butyrate at 0.1 nM, 1 nM, and 10 nM concentrations. Myoblasts were cultured for 48 h, and fresh medium was added every 24 h. Cells were pulsed with EdU (30 uM) for 2–5 h, ethanol fixed, stained with Hoescht 3342, and immunostained for proliferation (measured by the percent of cells positive for EdU (%EdU)), and differentiation (measured by the increase in cell area staining positive for embryonic myosin heavy chain (%eMyHC) compared to negative controls that received only medium and vehicle). When compared to untreated myoblasts, cells treated with 1 nM sodium butyrate had increased fusion rates as shown in Figure 3A. Significance was determined by a p-value of less than 0.05 in a one-way ANOVA Tukey Honest Significant Difference test.

[0103] Figure 3A: Bar graph of fusion index in response to sodium butyrate (NaBut) compared to vehicle. Myoblasts were cultured for 48 h in the presence of the indicated doses of NaBut. Fresh medium and NaBut were added every 24 h. Tables of mean ± SD, quantification of fusion index and p-values ​​are also shown. (by two-tailed Student's T-test) * p<0.05, n=3-5)

[0104] [Table 5]

[0105] Example 7 Sodium butyrate enhances the activity of IGF2. 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 pulsed with EdU (30 uM) for 2-5 hours, ethanol fixed, stained with Hoescht 3342, and immunostained for proliferation (measured by the percent of cells positive for EdU (%EdU)), and differentiation (measured by the increase in cell area staining positive for embryonic myosin heavy chain (%eMyHC) compared to negative controls that received only medium and vehicle). The total area of ​​eMyHc positive cells was analyzed and treated cells were compared to cells treated with vehicle only. Cells treated with IGF alone, and the two conditions in which cells were treated with IGF2 and sodium butyrate, caused a significant increase in the amount of differentiation. There was a significant increase in the total area of ​​eMyHC cells in cells treated with 1 nM butyrate and 100 nM sodium butyrate plus IGF2 compared to cells treated with IGF2 alone, with significance determined by a p-value of less than 0.05 by one-way ANOVA Tukey's Honest Significant Difference test.

[0106] FIG. 3B: Bar graph of fusion index of mouse myoblasts in response to sodium butyrate (NaBut) compared to vehicle. Mouse myoblasts were cultured for 48 h in the presence of the indicated doses of NaBut. Fresh medium and NaBut were added every 24 h. Tables of mean ± SD, quantification of fusion index, and p-values ​​are shown ( *Student's two-tailed T-test showed p values ​​< 0.05, n = 3-5). Significant p values ​​(Vehicle-IGF2: 0.015ug / mL: 6.33E-06, Vehicle-NaBut: 1 nM IGF2: 0.015ug / mL: 1.79E-11, Vehicle-NaBut: 100 nM IGF2: 0.015ug / mL: 1.79E-11)

[0107] [Table 6]

[0108] FIG. 3C. Bar graph quantification of % area of ​​eMyHC+ human myoblasts in response to the indicated treatments compared to IGF2 (15 ng / mL). Myoblasts were cultured in the presence of the indicated doses of IGF2 for 96 h. Fresh medium and IGF2 were added every 24 h. Mean ± SD ( * One-Way Anova Tukey Honest Significant Difference p<0.05, n=2-12).

[0109] [Table 7]

[0110] Example 8 Sodium butyrate enhances the activity of IGF2. 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 pulsed with EdU (30 uM) for 2-5 hours, ethanol fixed, stained with Hoescht3342, and immunostained for proliferation (measured by the percent of cells positive for EdU (%EdU)), and differentiation (measured by the increase in cell area staining positive for embryonic myosin heavy chain (%eMyHC) compared to negative controls that received only medium and vehicle). As seen in Figure 4A, the total area of ​​eMyHC positive cells was analyzed and treated cells were compared to cells treated with vehicle only. Myoblasts treated with either 0.03 μg / mL IGF2 or IGF2 combined with sodium butyrate showed a significant increase in eMyHC+ area compared to cells cultured with vehicle only.

[0111] [Table 8]

[0112] Myoblasts treated with a combination of IGF2 and sodium butyrate were compared to cells treated with IGF2 alone. As shown in Figure 4B and in the table, there was a significant increase in all cells treated with the combination compared to cells treated with IGF2 alone. Significance was determined by a p-value of less than 0.05 in a one-way ANOVA Tukey Honest Significant Difference test.

[0113] [Table 9]

[0114] Example 9 IGF2 enhances MYOG expression in DM1 human myoblasts. FIG. 5A is a bar graph of fold change in myogenic gene expression in DM1 human myoblasts in response to the indicated treatments compared to FM (vehicle). Myoblasts were cultured for 48 h in the presence of factors (BMP7 50 ng / mL, butyrate 10 nM, IGF2 200 ng / mL). Mean ± SD, significant p-values ​​(FM~IGF2: 4.94E-04, FM~IGF2_NaBut: 6.53E-03) ( * p<0.01) Table of means and p-values ​​for MYF5, MYOD1, and MYOG (n=3).

[0115] [Table 10]

[0116] Example 10 IGF2 receptor is expressed on chondrocytes and bone cells Figure 6A: Bar graph showing that IGF2 receptor is expressed on cartilage-related cells. Data from Ramilowsky et al., Nature 2015.

[0117] [Table 11]

[0118] Example 11 IGF2 treatment promoted proliferation and fusion in DM1 human myoblasts (32 year old Caucasian female). Figure 7A is a bar graph of % EdU+ human myoblasts (32 year old Caucasian female) and Figure 7B is % MyHC area in response to IGF2. 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 to IGF2: 6.8E-3, % eMyHC area: Vehicle to IGF2: 1.9E-4) by two-tailed Student's T-test * p<0.05, n=3–6).

[0119] [Table 12]

[0120] Example 12 IGF2 enhanced the expression of M17 / R, CKM, and ATP1B1 in DM1 human myoblasts (32 year old Caucasian female). FIG. 8A is a bar graph of fold change in MYH3 and CKM expression in DM1 human myoblasts (32 year old Caucasian female) in response to the indicated treatments compared to vehicle. Myoblasts were cultured in the presence of factors (IGF2 200 ng / mL) for 96 hours. Mean ± SD Significant p-values ​​(MYH3:vehicle to IGF2: 1.13E-03, CKM:vehicle to IGF2: 7.67E-03). FIG. 8B is a bar graph of ATP1B1 expression fold change in DM1 human myoblasts (32 year old Caucasian female) in response to the indicated treatments compared to FM (vehicle). Myoblasts were cultured in the presence of factors (IGF2 200 ng / mL) for 48 hours. Mean ± SD Significant p-values ​​(vehicle to IGF2: 3.11E-05) (by two-tailed Student's T-test) * p<0.05, n=3).

[0121] [Table 13]

[0122] Example 13 Systemic administration of IGF2 / NaB prevents age-induced muscle dysfunction. Figure 9A: Subcutaneous injections of IGF2 (50ug / kg) or NaB (1.2g / kg), IGF2 / NaB (150ug / kg; 1.2g / kg) or vehicle (PBS) were administered to 21-24 month old mice for 14 days. Muscle function was assessed on days 13 and 14. (Figure 9B) Grip strength was assessed on day 13. The first graph, Figure 9B, shows forelimb grip strength, **** p<0.0001, ** p=0.0043, ** p=0.001 (One-way ANOVA, multiple comparisons). Fig. 9C) Forelimb strength. **** p<0.0001, * p=0.0368, *p=0.0187 (One-way ANOVA, multiple comparisons). Figure 9D shows treadmill performance measured on day 14 using a guided treadmill running model set to progressively increase the speed by 2 m / min after every 2 min. Distance traveled is shown. *** p=0.0005, * p=0.0459, **** p<0.0001 (One-way ANOVA, multiple comparisons). Figure 9E) Time to exhaustion *** p=0.0002, ** p=0.0024 (One-way ANOVA, multiple comparisons). Figure 9F) Maximum speed *** p=0.0004, ** p=0.0013. Figure 9G) Work in kj units ** p=0.0026, ** p=0.0035 (One-way ANOVA, multiple comparisons).

[0123] Example 14 Systemic administration of IGF2 / NaB is safe. (Figure 10A) Subcutaneous injections of vehicle or IGF2 / NaB were administered to 21-month-old mice for 14 days, and blood and serum were collected to assess complete blood counts and metabolic panels of liver, kidney, and pancreatic function. Figures 10B-E are representative graphs of 4 of the 37 readouts measured, showing white blood cell counts (unpaired t-test, p=0.8020), albumin concentration (unpaired t-test, p>0.9999), creatinine concentration (unpaired t-test, p=0.5490), and calcium concentration (unpaired t-test, p=0.811).

[0124] Example 15 Systemic administration of IGF2 / But prevents dexamethasone-induced muscle atrophy. (Figure 11A) Twelve week old mice were administered dexamethasone (25mg / kg ip) simultaneously with subcutaneous injections of IGF2 / NaB (150ug / kg; 1.2g / kg) or vehicle (PBS) for 14 days (Figure 11B). Muscle function was assessed on days 13-14. Grip strength was assessed on day 13, and the forelimb forces measured on day 13 (Figure 11B), and forelimb specific forces (Figure 11C) are shown in the graphs. Forelimb specific forces were calculated as mN forelimb force per g body weight (Figure 11C). *** p=0.0003, *** p=0.0004) (unpaired t-test). Figure 11D) Grip strength was assessed on day 13, and graphs show forelimb force (Figure 11D), and specific forelimb force (Figure 11E) measured on day 13. Specific forelimb force was calculated as mN forelimb force per g body weight ( ** p=0.0012, *** p=0.0005) (unpaired t-test). FIG. 11F) On day 15, mice were euthanized and TA was collected for histological analysis. Myofiber distribution assessed using SMASH software is shown in the graph. ** p=0.054, * p=0.037, and **** p<0.0001 (2-way ANOVA, multiple comparisons).

[0125] Example 16 Myogenic Activity Assay, In Vitro Myoblast Proliferation Assay Reduced regeneration from tissue progenitor cells in an individual is a hallmark of age- or disease-related dysfunction, and therefore assays measuring mitogenic capacity in tissue progenitor cells serve as a readout for the potential success of treatment. Measuring the proliferation rate, degree of differentiation, and increased cell survival of treated mouse or human muscle progenitor cells provides a good basis for potential therapeutic regenerative agents to treat individuals suffering from disease, injury, or who have genetic or developmental defects that result in premature tissue loss, wasting, or debilitation.

[0126] Mouse muscle precursor cells (early passage 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 Matrigel-coated plates (1:300 Matrigel:PBS), 37°C, and 5% CO2). For experimental conditions, cells were plated at 40,000 cells / well in mouse fusion medium DMEM (Gibco) + 2% horse serum (Hyclone) on Matrigel-coated 8-well chamber slides with 250-500 μL medium per well. One hour after plating, mouse myoblasts were treated with 50% of each medium: Mouse myoblasts were cultured for 24 hours in the above conditions in a 37°C, 10% CO2 incubator. BrdU (300 μM) in DMSO was added for 2 h, followed by fixation with cold 70% ethanol and storage at 4°C until staining.

[0127] Human muscle precursor cells (early passage myoblasts) were cultured and grown in growth medium (Ham's F-10 (Gibco), 20% bovine growth serum (Hyclone), 5 ng / mL FGF2, and 1% penicillin-streptomycin) on Matrigel-coated plates (1:300 Matrigel:PBS) at 37°C and 5% CO2. For experimental conditions, cells were plated at 40,000 cells / well in human fusion medium DMEM (Gibco) + 2% horse serum (Hyclone) on Matrigel-coated 8-well chamber slides with 250-500 μL medium per well. One hour after plating, human myoblasts were treated with 50% of each medium: mouse myoblasts were cultured in the above conditions for 24 hours in a 37°C, 10% CO2 incubator. BrdU (300 μM) in DMSO was added for 2 h, followed by fixation with cold 70% ethanol and storage at 4°C until staining.

[0128] Testing in the in vitro myogenesis assay described above using healthy human muscle progenitors from a 32-year-old female demonstrated that the HSA-IGF2R61A (Figure 17A) variant retained equal activity to IGF2 (Figure 17B) at equimolar concentrations across a range of test article concentrations.

[0129] Testing in the in vitro myogenesis assay described above using healthy human muscle precursors from a 32-year-old female demonstrated that the mutant sequence HSA-IGF2R61A retained equal or increased activity relative to HSA-IGF2 at equimolar concentrations, before or after size-exclusion purification by HPLC (Figure 20).

[0130] Quantification of regeneration index After permeabilization in PBS + 0.25% Triton X-100, antigen retrieval was performed. Primary staining was performed using primary antibodies including species-specific monoclonal antibody for mouse anti-embryonic myosin heavy chain (eMyHC, hybridoma clone 1.652, Developmental Studies Hybridoma Bank), and Rat-anti-BrdU (Abeam Inc. ab6326). Secondary staining was performed with fluorochrome-conjugated species-specific antibodies (Donkey anti-Rat-488, #712-485-150; Donkey anti-Mouse-488, #715-485-150). Nuclei were visualized by Hoechst staining. Hoechst staining was used to calculate cell counts and reported as the percentage of cells positive for BrdU and eMyHC.

[0131] Testing in myotonic dystrophy muscle precursor cells Testing in the in vitro myogenesis assay described above using human DM1 muscle precursors from a 32-year-old female demonstrated that the HSA-IGF2R61A (FIG. 16A) variant retained equal activity towards IGF2 (FIG. 16B) at equimolar concentrations and across a range of test article concentrations.

[0132] Example 17 Myogenic gene profiling of regeneration-promoting factors Expression of myogenic factors Pax7, Myf5, Myod1, and Myog are important indicators of the functional state of muscle precursor cells. Upregulation of Pax7 and Myf5 factors indicates rejuvenation of proliferative precursor cells, while upregulation of Myod1 and Myog indicates muscle fiber regeneration. Readouts of these gene expression will provide potential success for any given polypeptide, including IGF2 amino acid sequences and amino acid sequences from heterologous polypeptides, or combinations of insulin-like growth factor 1 receptor (IGF1R) agonists and short chain fatty acids, as described herein. Measurement of myogenic genes in factor-treated mouse or human muscle precursor cells will provide good characterization of therapeutic efficacy for treating injured individuals, or individuals with genetic or developmental defects that result in premature tissue loss, wasting, or weakness. As a control, assays will also be performed on proteins purified from differentiated cells that do not produce myoblast proliferation, proteins cultured in media conditioned by differentiated cells, or purified heparin-related fractions.

[0133] 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).

[0134] Senescent human myoblasts were cultured in well plates. Culturing the cells in different media resulted in differential induction of myogenic gene expression. All factors led to changes in at least one myogenic receptor gene at 48 and 72 hours when compared to cells cultured in fusion medium, as shown in the table below. Cells cultured with IGF2 showed increased levels of MYOG at 48 hours and MYOD at 72 hours.

[0135] [Table 14]

[0136] Myogenic gene profiling in human or mouse progenitor cells Human or mouse muscle precursor cells are plated and cultured as described above for myogenic activity testing. One hour after plating, myoblasts will be treated with the respective factors. Myoblasts will be analyzed for the expression of Pax7, Myf5, Myod1, and Myog to characterize the regenerative effect of treatment with polypeptides containing IGF2 amino acid sequences, and test to characterize the effect of amino acid sequences derived from heterologous polypeptides or combinations of insulin-like growth factor 1 receptor (IGF1R) agonists and short-chain fatty acids.

[0137] Example 18 In vivo testing of stem cell secreted factors Several in vivo models of muscle degeneration are tested. Given that the polypeptide comprising the IGF2 amino acid sequence described herein and the amino acid sequence from a heterologous polypeptide, or the combination of an insulin-like growth factor 1 receptor (IGF1R) agonist and a short chain fatty acid, has regenerative properties in in vitro models, these in vivo models will show similar regenerative and proliferative effects in the context of intact organ systems.

[0138] Acute injury model The experimental groups will be: C57BL / 6J male mice, N = 18; young: 12-13 week old (3 month old) mice, n = 6; old: 77-78 week old (18 month old) mice, n = 12. This design will be used to test any single factor identified and validated in vitro assays of polypeptides containing IGF2 amino acid sequences and amino acid sequences from a heterologous polypeptide or combinations of insulin-like growth factor 1 receptor (IGF1R) agonists and short chain fatty acids.

[0139] On day 0, mice are weighed and subjected to muscle injury by local injection of barium chloride (BaCl2, 10 μL, 1.2% w / v in saline, Sigma-Aldrich) into the tibialis anterior (TA; day 0) muscles of both right and left hind limbs. BaCl2 is followed by co-injection of vehicle or factor A (0.1 mg / kg) intramuscularly (im) into the TA-injured hind limb site and again (im) 48 h later on day 2 into the TA-injured hind limb site. Also on day 2, BaCl2 (Ctx; 10 μL, 1.2% w / v in saline, Sigma-Aldrich) was injected (im) into the gastrocnemius muscles of both the right and left hind legs (GA, day 2). Following BaCl2, vehicle or factor injections were administered sequentially (im) into the TA hind limb injury site and again (im) 48 h later on day 4 into the GA injury hind limb site. Prior to sacrifice to label proliferating cells, bromodeoxyuridine (BrdU) was administered (100 mg / kg, ip) once daily for 3 days on days 2–4.

[0140] On day 5, the animals are sacrificed and the animal's weight is recorded, followed by collection of 0.5 ml of peripheral blood via cardiac puncture, which is processed into plasma and stored at -80°C. The animals are then perfused with 1xPBS, and the skin is carefully dissected from the GA / TA muscles of each hind leg and photographed (before resection). After excision of exclusively GA or TA muscles, the excised tissue is photographed, weighed, and then placed in 25% sucrose in PBS at 4°C for 4 hours, rinsed with 1xPBS, immersed in Tissue-TEK OCT, and flash frozen, after which the frozen muscle tissue is stored at -80°C. Cryosectioning and H&E will be performed to ensure that muscle injury sites are properly visualized. Muscle tissue composition from new skeletal muscle fibers, fibrous tissue, and adipose tissue (fat) will be measured. The level of regeneration is assessed by measuring muscle regeneration, defined as the number of new muscle fibers with centrally located nuclei per millimeter; fibrosis, defined as the area of ​​fibrotic scar; fiber size, defined as width and area; and adipose tissue, defined as the amount of fat surrounding the muscle.

[0141] Sarcopenia / chronic administration model The experimental design is: C57BL / 6J male mice, N = 18; young: 12-13 week old (3 month old) mice, n = 6; old: 77-78 week old (18 month old) mice, n = 12. This design can be used to test any single factor or complex mixture of two or more factors or synergistic small molecules that have been identified and validated in in vitro assays.

[0142] On day 0, mice have the following in vivo health measurements performed over a 1-day period as a baseline for age-based parameters: weight, running wheel performance, grip strength, and horizontal bar. Each assay should be performed with 4 trials per assay per animal. These health assays are repeated on day -1. After 1 day of rest on day -9, mice begin daily injections of vehicle or factor A 1X (0.1 mg / kg) for the remainder of the experiment until sacrifice (days -8 to +5, day 13 of dosing). On day -4, 6 days after dosing begins, mice undergo one repeat of the health assays. On day 0, 5 days prior to sacrifice, mice are injected locally with cardiotoxin (Ctx; 10 μg, Sigma-Aldrich) in only the tibialis anterior muscle (TA; day 0) of the right hind limb to cause muscle injury. On day 2, cardiotoxin (Ctx; 10 μg, Sigma-Aldrich) is administered into the gastrocnemius muscle (GA; day 2) of the right hind leg. BrdU is administered (100 mg / kg, ip) once a day on days 2-4 for 3 days before sacrifice. On day +5, before feeding, animals undergo an in vivo incapacitance assay run. On day +5, animals are sacrificed and their weights are recorded. We collect 0.5 ml of blood via cardiac puncture, process it into plasma, and store the plasma samples at -80 °C. The animals are then perfused with 1x PBS. The skin is carefully incised from the GA / TA muscles of each hind leg and photographed (before excision). After excising exclusively GA or TA muscles, the muscles are weighed, then the muscles are placed in 25% sucrose in PBS at 4 °C for 4 h, then the muscles are rinsed in 1x PBS, Tissue-TEK OCT is added, and the frozen muscle tissue is stored at -80 °C. Cryosectioning and H&E are performed to ensure that the muscle injury site is properly visualized. Inguinal white adipose tissue (WAT) is carefully excised and weighed.

[0143] Muscle tissue composition from new skeletal muscle fibers, fibrous tissue, and adipose tissue (fat) is measured. Muscle regeneration, defined as the number of new muscle fibers with centrally located nuclei per millimeter, fibrosis, defined as the area of ​​fibrous scar, fiber size, defined as width and area, and adipose tissue, defined by the amount of fat surrounding the muscle, are measured to assess the level of regeneration. Animal body weight during the treatment period, as well as health status assays including performance on running wheels (speed, distance, duration), grip strength, and horizontal bar performance, will be considered as phenotypic outcomes of systemic treatment of aged animals with a polypeptide comprising an IGF2 amino acid sequence and an amino acid sequence from a heterologous polypeptide or a combination of an insulin-like growth factor 1 receptor (IGF1R) agonist and short chain fatty acids.

[0144] The horizontal bar test is performed at 8 months (n=6 WT, n=7 MPS IIIB) and 10 months (n=3 WT, n=4 MPS IIIB) of age as previously described (Malinowska et al. 2010). Briefly, a 300 mm metal wire with a diameter of 2 mm was fixed between two posts 320 mm above the padded surface. Mice were asked to grasp the center of the wire and the time to fall or reach to the side was recorded, and the test was stopped after 2 min. Crossing the bar in x seconds is scored as 240-x, remaining on the bar is scored as 120, and falling off the bar after y seconds is recorded as a value of y. The test is repeated three times as practice, followed by a 10 min rest before three trials in which scores are recorded.

[0145] The animals will also have better health outcomes: reduced body weight, fat composition, scar tissue around the muscles, increased running speed, duration, and distance, increased grip strength, and improved performance on the horizontal bar test.

[0146] Genetic obesity muscular dystrophy model Genetically obese (ob / ob) mice are injected with BaCl2 in the TA muscle on day 0. On days 0 and 2, three mice are treated with vehicle only, three mice are injected with hPSC factors, and three mice are treated with FGF19 (positive control). On day 5, mice are euthanized and the TA muscle is perfused with PBS and dissected. The muscle is then analyzed for regeneration and fibrosis index.

[0147] Methods for testing muscle endurance and function Forelimb and bilateral limb grip strength tests: After 30 min of acclimation, mice are introduced into the grip strength meter. For forelimb grip strength, mice held by the tail are made to grip the grip bar with only their forelimbs. For forelimb measurements, mice are placed on a grid and made to grip the grid with their forelimbs. The force generated by each mouse is calculated as the average of 5-6 measurements.

[0148] Limb endurance testing: Mice are allowed to discover and acclimate to the rodent treadmill environment through two training sessions of 10 min each at 10 m / min on separate days prior to endurance testing. For endurance testing, mice are placed into individual lanes of the rodent treadmill. Speed ​​is gradually increased by 2 m / min until exhaustion is reached. Exhaustion is defined as the mouse remaining for 3-5 s on the grill that is electrified to deliver a 2 Hz, intensity 5 shock.

[0149] In vivo tetanic force measurements: Mice are kept under anesthesia with regulated delivery of isoflurane during the entire process. After anesthesia, the animal is placed on a heated chamber and the feet are secured to the foot pedals of an Aurora force transducer. Two electrodes are specifically positioned to stimulate the sciatic nerve. In contrast to direct force, the force generated by ankle twisting of the animal's hind limb is measured in response to a series of stimuli including 50, 100, 150, and 200 Hz.

[0150] In situ tetanic force measurement: This experiment is performed using Aurora force measurement. Mice are kept under anesthesia during the entire process. A small incision in the skin around the tibialis anterior muscle exposes the Achilles tendon, which is connected to an Aurora force transducer via a surgical suture threaded through a hook. The force generated by the muscle in response to a series of stimuli including 50, 100, 150, and 200 Hz is recorded by two electrodes placed on the tibialis anterior.

[0151] Example 19. Stability of Mitogenic Polypeptides In Vivo Assayed by Bioavailability and Pharmacokinetics Tissue bioavailability The bioavailability of the therapeutic polypeptide is evaluated in target tissues in young (10-12 weeks old) and old (78 weeks old) mice. In this experiment, one cohort of young mice (10-12 weeks old; N=24) and one cohort of old mice (78 weeks old; N=24) receive one subcutaneous (SC) injection of the therapeutic composition. Four young mice (10-12 weeks old; N=6) and four old mice (78 weeks old; N=6) receive one SC injection of vehicle and serve as controls. Four mice from each cohort are euthanized after 30 min, 1 h, 1.5 h, 2 h, or 4 h. At each time point, blood is collected by cardiac puncture followed by collection of selected tissues such as tibialis anterior, gastrocnemius, quadriceps, heart, and diaphragm. Detection and quantification of the administered therapeutic polypeptide is detected by enzyme-linked immunosorbent assay (ELISA). Therapeutic polypeptide levels are compared to samples collected from vehicle-injected mice to determine tissue-level bioavailability.

[0152] Pharmacokinetics of engineered mitogenic polypeptides. Mouse pharmacokinetics (PK) describes the absorption, distribution, metabolism, and excretion of a drug from the body. The pharmacokinetic profile of a therapeutic polypeptide was determined in mice (10-12 weeks old). Mice were fed ad libitum and housed under controlled conditions of lighting (12 hours light / 12 hours dark) and temperature (22-24 °C). Mice were allowed to acclimate for 3 days before the start of the experiment. Intravenous (IV) injection in 10-12 week old mice. The engineered mitogenic polypeptide concentration in the samples was measured by ELISA. The various pharmacokinetics, as well as the absorption / release kinetics after different routes of administration, are calculated. Noncompartmental analysis fits of the pharmacodynamic data for intravenous administration of HSA-IGF2R6 (SEQ ID NO: 81) in mice (Figure 18A) show significantly improved serum half-life compared to the native sequence of IGF2 (SEQ ID NO: 76) (Figure 18B).

[0153] [Table 15]

[0154] Example 20 Purified IGF2-hFcm promoted myoblast differentiation Suspension 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 by protein A membrane column. The purified IGF2-hFcm was added into cultures of human myoblasts. Myosin heavy chain (MyHC) was immunostained and imaged by fluorescence microscopy. After quantification of stained MyHC, the percentage of area of ​​MyHC was calculated as the percentage of pixels in the field of view that were illuminated above background in the stained channel. The percentage of EdU in mouse myoblasts treated with purified IGF2-hFcm is significantly higher than that in mouse myoblasts treated with culture supernatant of CHO cells expressing an empty control vector. Significance was determined by a p-value of less than 0.05 in a one-way ANOVA Tukey Honest Significant Difference test.

[0155] [Table 16]

[0156] This example found that IGF2-fusion proteins can induce cell proliferation and share in vitro properties with HAP, suggesting shared in vivo properties.

[0157] Example 21 Modeling treatment of muscular dystrophy with IGF2 compositions in vitro Muscular dystrophies (MD) encompass a variety of myodegenerative diseases that typically result from genetic mutations in genes that code for proteins involved in the formation and stabilization of skeletal muscle. The phenotypic result of these genetic mutations is a progressive loss of muscle mass and strength over time, which is similar to sarcopenia, but the underlying causes are different. Because HAP has produced phenotypic improvements in sarcopenic muscle, we tested for similar improvements in a model of MD.

[0158] IGF2 was individually tested for its ability to promote proliferation and / or fusion of human muscle precursor cells derived from individuals with myotonic dystrophy type 1 (hMD), a muscular dystrophy caused by mutations in the DMPK1 gene. By adding each factor to hMD myoblasts for 72 hours, with daily medium changes (DMEM + 2% horse serum) and a second pulse of factor at the first medium change, the effect of IGF2 on myogenic activity was assayed over a range centered around expected physiological levels, in vivo in triplicate. After 72 or 96 hours, cells were pulsed with EdU (30uM) for 2-5 hours, ethanol fixed, stained with Hoescht3342, and immunostained for proliferation (measured by the percent of cells positive for EdU (%EdU)), and differentiation (measured by the increase in cell area staining positive for embryonic myosin heavy chain (%eMyHC) compared to negative controls that received only medium and vehicle). Wells were imaged on a Keyence BZ-100, images were quantified in Cell Profiler, and statistics were calculated in R. Additionally, RNA was extracted from myoblasts and selected transcript abundance was quantified by qPCR. Figures 7A and 7B show that IGF2 treatment promoted proliferation and differentiation, respectively, in DM1 human myoblasts (32-year-old Caucasian female). Figures 8A and 8B show that IGF2 enhanced expression of MYH3, CKM, and ATP1B1 in DM1 human myoblasts (32-year-old Caucasian female).

[0159] Example 22 Systemic Administration of Therapeutic Polypeptides Reverses Sarcopenia and Prevents Muscle Damage Daily subcutaneous injections of therapeutic polypeptide or vehicle alone are administered to 78-week-old mice for 14 days. IGF2 is injected at concentrations of 100-1000 pg / kg. In some experiments, treatment groups receive a single therapeutic factor, while in others, treatment groups receive a combination of factors. On day 7, muscle function is assessed using forelimb grip strength and grip strength of both limbs. On days 12, 13, and 14, groups 1 and 2 are injected intraperitoneally with BrdU. On days 13-15, all mice are assessed for grip strength and endurance tests to determine maximum distance, maximum velocity, and tetanic force.

[0160] On day 15, mice from groups 1 and 2 are euthanized and the muscles are analyzed for markers of proliferation and fibrosis. On day 15, chemical injury is created in the TA of groups 3 and 4 using an intramuscular injection of 1.2% BaCl2 (7ul / TA). Mice from groups 3 and 4 continue to receive subcutaneous injections of therapeutic polypeptides on days 15-21. They also receive intraperitoneal BrdU injections on days 19, 20, and 21. On day 21, TA muscles are tested for in situ tetanic strength. TA muscles are dissected and assessed for signs of proliferation and fibrosis.

[0161] Example 23 Systemic administration of the fusion polypeptide reversed the induced muscle atrophy. Mice at 12 weeks of age are divided into three treatment groups: Group 1 receives injections of vehicle only, Group 2 receives injections of dexamethasone, and Group 3 receives injections of the dexamethasone and IGF2 fusion polypeptide. Dexamethasone (25 mg / kg ip) is administered simultaneously with the subcutaneous injections of the IGF2 fusion polypeptide for 14 days.

[0162] On day 7, mice are assessed for forelimb and bilimb grip strength. On days 13-15, mice are assessed for grip strength, in vivo tetanic force and subjected to a treadmill endurance test to determine maximum speed and maximum distance.

[0163] Example 24 Systemic administration of an IGF2 fusion polypeptide predicted to ameliorate muscle atrophy in genetically obese mice. 13-week-old genetically obese mice (ob / ob) are subcutaneously injected with IGF2 fusion polypeptide for 14 days. On day 7, forelimb and bilimb grip strength is measured. BrdU is injected on days 12, 13, and 14. On days 13, 14, and 15, forelimb and bilimb grip strength and in vivo tetanic force are tested, and endurance tests are performed to determine maximum distance and maximum velocity. On day 14, mice are euthanized and TA muscles are dissected. Muscle weight and growth are analyzed.

[0164] Example 25 Systemic administration of an IGF2 fusion polypeptide was predicted to reverse or slow dystrophic features in 70 week old mdx mice. Another class of human myopathies requiring treatment are genetically induced muscular dystrophies, among which Duchenne muscular dystrophy is a rare but fatal case. Aged genetically dystrophic (mdx) mice (>15 months old) show similar characteristics to human Duchenne muscular dystrophy (DMD), especially reduced muscle regeneration resulting in muscle wasting. Treatment with IGF2 fusion polypeptides can reverse the dystrophic features of aged mdx mice. During the acclimation period, body weight, forelimb and both limb grip strength and in vivo tetanic force are assessed to determine the baseline strength of each mouse. 70-week-old dystrophic mice (mdx) are subcutaneously injected with IGF2 fusion polypeptides for 14 days. On day 7, forelimb and both limb grip strength is measured. BrdU is injected on days 12, 13, and 14. On days 13, 14, and 15, forelimb and bilimb grip strength and in vivo tetanic force are tested, and endurance testing is performed to determine maximum distance and maximum speed. Right tibialis anterior and gastrocnemius muscles are harvested, immersed in Tissue-TEK OCT, then flash frozen in a chilled isopentane bath pre-cooled in liquid nitrogen, and stored at -80°C. Tissues are sectioned and stained for laminin to determine myofiber cross-sectional area (CSA), eMyHC to measure new fiber formation, and BrdU to assess proliferation rate. Left tibialis anterior and gastrocnemius muscles are harvested and flash frozen in liquid nitrogen for molecular analysis including qPCR and Western blot.

[0165] IGF2 is predicted to be effective at concentrations between 10 and 200 μg / kg.

[0166] Example 26 Systemic administration of IGF2 fusion polypeptide improved dystrophic features in 10-week-old mice Between 3 and 6 weeks of age, skeletal muscles of mdx mice undergo severe necrosis, followed by increased satellite cell activation to promote muscle regeneration. Treatment with the IGF2 fusion polypeptides described herein can improve the regeneration process and therefore muscle health. Mice were fed ad libitum and housed under controlled conditions of lighting (12 hours light / 12 hours dark) and temperature (22-24°C). Mice were allowed to acclimate for 3 days before the start of the experiment. During the acclimatization period, body weight, forelimb and both limb grip strength and in vivo tetanic force were assessed to determine the baseline strength of each mouse. 10-week-old dystrophic mice (mdx) were subcutaneously injected with IGF2 fusion polypeptides for 14 days. On day 7, forelimb and both limb grip strength is measured as described in Example 4. BrdU was injected on days 12, 13, and 14. On days 13, 14 and 15, grip strength and in vivo tetanic force of the forelimbs and both limbs were tested, and endurance tests were performed to determine maximum distance and maximum speed using the methods described in Example 4.

[0167] Mice were euthanized. Right tibialis anterior and gastrocnemius muscles were harvested, immersed in Tissue-TEK OCT, then flash frozen in a chilled isopentane bath pre-cooled in liquid nitrogen and stored at -80°C. Tissues were sectioned and stained for laminin to determine myofiber cross-sectional area (CSA), eMyHC to measure new fiber formation, and BrdU to assess proliferation rate. Left tibialis anterior and gastrocnemius muscles were collected and flash frozen in liquid nitrogen for molecular analysis including qPCR and Western blot.

[0168] IGF2 administered subcutaneously at a concentration of 150 μg / kg along with 1.2 g / kg sodium butyrate every 24 hours was effective in improving muscle weight (unpaired t-test, p=0.0055) (FIG. 12A), forelimb grip strength (unpaired t-test, p=0.0011) (FIG. 12B), forelimb grip strength (unpaired t-test, p<0.001) (FIG. 12C), treadmill distance (unpaired t-test, p=0.0035) (FIG. 12D), and treadmill time to exhaustion (unpaired t-test, p=0.0023) (FIG. 12E), as well as total work (unpaired t-test, p=0.0022) (FIG. 12F).

[0169] Example 27 Engineering the sequence of IGF2 to reduce backbone truncations The amino acid backbone of IGF2 is cleaved when expressed from Chinese Hamster Ovary (CHO) cells (FIG. 13A), as visualized by reducing SDS-PAGE and Western blot (FIG. 13B). CHO cells were grown to reach 4E6 cells / mL in a 37° C. incubator with a humidified atmosphere of 8% CO2 on an orbital shaker platform with a shaking speed of 125 rpm, and then a calculated volume of cells was transferred to fresh pre-warmed ExpiCHO™ Expression Medium in a shake flask. When the culture reached 4×10 6 ~6x10 6 Flasks were incubated in a 37 °C incubator with a humidified atmosphere of 8% CO2 on an orbital shaker platform until a density of 1000 viable cells / mL was reached. After mixing the plasmid in the microtube with 100ul of SFM, OptiPro medium (92μl x 10.5 = 966μl) and ExpiFectamine (8μl x 10.5 = 84μl) in the microtube, respectively, 100ul was added to the plasmid tube and mixed by pipetting, after 5 minutes, 200μl was added to each well and mixed by swirling, enhancer (15x10.2 = 153μl) was mixed with feed (600x10.2 = 6.12ml) and 615μl was added to each well. Culture supernatants collected at intervals were spun down at low and high speeds and then frozen at -80°C. Aliquots were analyzed for protein concentration by nanodrop and then equal amounts were loaded onto 4-12% SDS-PAGE gels for molecular weight distribution analysis. Cleavage of IGF2 was confirmed by expression of a construct of dual-tagged IGF2, n-terminus tagged with human serum albumin (HSA) and c-terminus tagged with human immunoglobulin heavy chain 4 (Figure 14). The sequence of IGF2 was engineered to prevent cleavage by mutation of one or more arginines, as confirmed by visualization on reducing SDS-PAGE followed by Western blotting to detect the 6xHIS tag (Figure 15B), and subsequent purification with a mini protein A column as visualized on reducing SDS-PAGE (Figure 15A).

[0170] Example 28 Clinical Trials of IGF2 Fusion Polypeptides In physiological conditions, IGF2 binds to IGF-binding proteins (IGFBPs). The aim of this study was to determine whether mutant sequences of IGF2 interact with IGFBPs. Retention time across a size-exclusion chromatography column is proportional to the size of the molecule. Thus, molecular interactions can be confirmed by a corresponding change in retention time on a size-exclusion chromatography column. Measuring the retention of HSA-IGF2R61A and rhIGFBP3 separately and as a 1:1 stoichiometric mixture demonstrated evidence of conserved affinity, as demonstrated by mass shifts in size-exclusion chromatography by HPLC (Figure 19).

[0171] Example 29 Clinical Trials of IGF2 Fusion Polypeptides The purpose of this study is to determine the safety, tolerability and pharmacokinetics of repeated administration of a polypeptide comprising an IGF2 amino acid sequence and an amino acid sequence from a heterologous polypeptide at multiple dose levels in healthy individuals or individuals diagnosed with sarcopenia, muscular dystrophy or recovery from surgery.In one embodiment, the muscular dystrophy is myotonic dystrophy.In addition, this study will generate data on the physical function, skeletal muscle mass and strength resulting from treatment with an IGF2 fusion polypeptide in such individuals.Individuals will be administered a placebo or an IGF2 fusion polypeptide composition and monitored for 25 weeks of the study.The following primary and secondary outcome measures will be evaluated:

[0172] Primary endpoint: Safety and tolerability assessed by various measures such as percent of adverse events per study arm.

[0173] Secondary outcome measures: Plasma pharmacokinetics (Cmax, Tmax, AUC) [plasma 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 hours after administration].

[0174] Short Physical Performance Battery (SPPB). Change from baseline to week 25.

[0175] 10 Meter Walk Test. Change from Baseline to Week 25.

[0176] Changes in total lean body mass and appendicular skeletal muscle index measured by dual-energy x-ray absorptiometry (DEXA) or MRI from baseline to week 25.

[0177] Inclusion criteria: A diagnosis of sarcopenia, muscular dystrophy, or recovery from surgery; low muscle mass confirmed by DXA; low walking speed; SPPB score ≤ 9; weight of at least 35 kg; with adequate dietary intake as determined by patient interview; independent walking up to 10 meters.

[0178] protocol Patients received placebo (5% dextrose solution) or treatment (in 5% dextrose) intravenously starting on week 1 day 1 and repeated weekly (weeks 1-25 days 1). At the end of weeks 13 and 25, patients were evaluated for improvement by the methods described above. Doses were selected from a traditional 3+3 design and chosen as the top 2 doses lacking dose-limiting toxicity.

[0179] Example 30 Administration of 6HIS-HSA-L-IGF2R61A enhances myogenic activation, fusion and maturation in an acute injury model The acute injury model of Example 18 was used. Brown female C57 / BL6 (NIA) mice (20 months old) were injected intramuscularly (IM) in the tibial anterior with cardiotoxin (10ug / 20uL) on day 1, and starting on day 2, mice were injected IM every other day with 0.4ug / 20uL of 6HIS-HSA-IGF2R61A (SEQ ID NO: 34) or vehicle (0.4ug / 20uL of HSA).

[0180] Mice administered 6HIS-HSA-IGF2R61A showed reduced muscle fatigue compared to control treatment (vehicle) as measured by specific fatigue index. These results are shown in FIG. 21A. Mice administered 6HIS-HSA-IGF2R61A showed increased force production compared to mice receiving control treatment (vehicle) as measured by specific force-frequency. These results are shown in FIG. 21B. Mice administered 6HIS-HSA-IGF2R61A showed increased force production comparable to control treatment (vehicle) as measured by maximum contraction velocity index. These results are shown in FIG. 21C. Mice administered 6HIS-HSA-IGF2R61A showed improved relationship ratio to control treatment (vehicle) index. These results are shown in FIG. 21D. Mice administered 6HIS-HSA-IGF2R61A showed an increased regeneration index as measured by the number of new fibers per square millimeter compared to control-treated (vehicle) mice. These results are shown in FIG. 21E. Mice administered 6HIS-HSA-IGF2R61A showed increased muscle mass compared to control-treated (vehicle) mice. These results are shown in FIG. 21F.

[0181] Example 31 Administration of 6HIS-HSA-L-IGF2R61A and Sodium Butyrate Preserves Muscle Function and Fiber Size in a Sarcopenia Model The sarcopenia model of Example 18 was used. Brown male C57 / BL6 (NIA) mice (83-86 weeks old) were injected with 6 mg / kg 6HIS-HSA-IGF2R61A (SEQ ID NO: 34) and 0.3 g / kg sodium butyrate (NaB) or vehicle every other day for 4 weeks to measure muscle regeneration and functional changes.

[0182] Blood glucose was measured in both groups and normalized to baseline levels within each mouse. Treatment with 6HIS-HSA-IGF2R61A and NaB reduced blood glucose compared to control (vehicle) treated mice. These results are shown in Figure 22A.

[0183] Limb grip strength was measured in both groups and normalized by body weight within each mouse to determine specific force production. Treatment with 6HIS-HSA-IGF2R61A and NaB increased specific force production compared to control (vehicle) treated mice. These results are shown in Figure 22B. Forelimb grip strength was measured in both groups and normalized by body weight within each mouse to determine specific force production. Treatment with 6HIS-HSA-IGF2R61A and NaB increased specific force production compared to control (vehicle) treated mice. These results are shown in Figure 22C. Restoration of muscle force production was measured in both groups. Treatment with 6HIS-HSA-IGF2R61A and NaB increased force production compared to control (vehicle) treated mice, as measured by twitch force. These results are shown in Figure 22D. Force-frequency was measured in both groups and normalized by body weight within each mouse to obtain non-force frequency. Treatment with 6HIS-HSA-IGF2R61A and NaB increased specific force production compared to control (vehicle) treated mice. These results are shown in Figure 22E.

[0184] Example 32 Administration of 6HIS-HSA-L-IGF2R61A and sodium butyrate in a sarcopenia model and preservation of muscle function and fiber size The sarcopenia model of Example 18 was used. Brown male C57 / BL6 (NIA) mice (23 months old) were injected daily with 6 mg / kg HSA-IGF2R61A (SEQ ID NO: 34) and 0.3 g / kg sodium butyrate (NaB) or vehicle for two weeks to measure muscle regeneration and functional changes.

[0185] Fiber type distribution was measured in both groups. Fiber type 2A was lower and type 2B was higher in mice treated with 6HIS-HSA-IGF2R61A and NaB compared to control (vehicle) treated mice. The results are shown in Figure 23A. Cross-sectional area (CSA) was measured and plotted by group. Mice treated with 6HIS-HSA-IGF2R61A and NaB showed increased muscle tissue with greater CSA values ​​compared to control (vehicle) treated mice. The results are shown in Figure 23B.

[0186] Example 33 Administration of 6HIS-HSA-L-IGF2R61A and sodium butyrate restores muscle function, fiber size, and fiber composition in a myotonic dystrophy (DM1) mouse model Brown male TRED960I(+ / +) / M2rtTA(+ / -) mice were fed a doxycycline chow diet from birth and observed for a muscle wasting phenotype by weeks 12-14. Mice were injected daily for 2 weeks with 6mg / kg 6HIS-HSA-IGF2R61A (SEQ ID NO:34) and 0.3g / kg sodium butyrate (NaB) or vehicle to measure muscle regeneration and functional changes, as well as post-mortem histological analysis.

[0187] The distribution of fiber types was measured in both groups. Fiber types 2A and 2B were significantly different in the two groups (P value < 0.05), with 2A decreased and 2B increased in mice treated with 6HIS-HSA-IGF2R61A and NaB compared to mice treated with control (vehicle). These results are shown in Figure 24A. Cross-sectional area (CSA) was measured and plotted by group. Mice treated with 6HIS-HSA-IGF2R61A and NaB showed increased muscle tissue with greater CSA values ​​compared to mice treated with control (vehicle). These results are shown in Figure 24B.

[0188] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0189] All publications, patent applications, issued patents, and other documents mentioned herein are incorporated by reference herein as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in the texts incorporated by reference are excluded to the extent that they conflict with definitions in this disclosure.

[0190] [Table 17-1]

[0191] [Table 17-2]

[0192] [Table 17-3]

[0193] [Table 17-4]

[0194] [Table 17-5]

[0195] [Table 17-6]

[0196] [Table 17-7]

[0197] [Table 17-8]

[0198]

Table 17-9

[0199]

Table 17-10

[0200]

Table 17-11

[0201]

Table 17-12

[0202]

Table 17-13

[0203]

Table 17-14

[0204]

Table 17-15

[0205]

Table 17-16

[0206]

Table 17-17

[0207]

Table 17-18

[0208]

Table 17-19

[0209]

Table 17-20

Claims

**Claim 1**: A fusion protein comprising an insulin-like growth factor 2 (IGF2) polypeptide fused to an N-terminal human serum albumin (HSA) polypeptide, wherein the IGF2 polypeptide comprises the amino acid sequence of SEQ ID NO: 29 and R61A in the amino acid sequence of SEQ ID NO: 32, the R61A mutation is present within the IGF2 C domain, and the fusion protein exhibits a decrease in cleavage within the IGF2 C domain. **Claim 2**: The fusion protein according to claim 1, wherein the HSA polypeptide of the fusion protein comprises the amino acid sequence of SEQ ID NO:

58. **Claim 3**: The fusion protein according to claim 1, further comprising a glycine-rich linker that binds the N-terminal HSA polypeptide to the IGF2 polypeptide. **Claim 4**: The fusion protein according to claim 3, wherein the glycine-rich linker is a glycine-serine linker comprising the amino acid sequence of SEQ ID NO:

63. **Claim 5**: The fusion protein according to claim 4, wherein the glycine-serine linker comprises a multimer of the 4GS linker of SEQ ID NO: 63 and has a repeat of 2, 3, 4, or 5 4GS linkers. **Claim 6**: The fusion protein according to claim 4, wherein the glycine-serine linker comprises the amino acid sequence of SEQ ID NO:

53. **Claim 7**: The fusion protein according to claim 4, comprising an amino acid sequence that is at least 98% identical to SEQ ID NO:

59. **Claim 8**: The fusion protein according to claim 4, comprising the amino acid sequence of SEQ ID NO:

59. **Claim 9**: The fusion protein according to claim 4, consisting essentially of the amino acid sequence of SEQ ID NO:

59. **Claim 10**: A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 9, and a pharmaceutically acceptable excipient, carrier, or diluent. **Claim 11**: Use of the fusion protein according to any one of claims 1 to 9 in the manufacture of a medicament for use in treating a muscle wasting disorder or disease in a subject in need of treatment of a muscle wasting disorder or disease.

12. The use according to claim 11, wherein the muscle wasting disorder or the muscle wasting disease is muscular dystrophy.

13. The use according to claim 12, wherein the muscular dystrophy is myotonic muscular dystrophy.

14. The use according to claim 11, wherein the muscle wasting disorder or the muscle wasting disease is sarcopenia.

15. The use according to claim 11, wherein the muscle wasting disorder or the muscle wasting disease is cachexia.

16. The use according to claim 11, wherein the muscle wasting disorder or the muscle wasting disease is a result of obesity, disease progression, metabolic disorder, therapeutic treatment, or a combination thereof.

17. The use according to claim 11, wherein the muscle wasting disease is caused by muscle injury or limb immobilization.

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