Heparin-Associated Polypeptides and Uses Thereof

BACKGROUND

As the average life span increases, increasing emphasis is placed upon “healthy aging.” Individuals would like to live more active lifestyles as they age, and as a result, many aging disorders can have a significant impact on the quality of life of aging individuals. Treatments directed to regenerative ends have utility for treating aging diseases. Additionally, many treatments for aging disorders can be applicable to younger individuals who have suffered illness, injury, or who possess genetic or developmental defects leading to premature tissue loss, wasting, or weakening.

SUMMARY

As individuals age, tissue progenitor cells lose their regenerative potential. Described herein, in certain aspects, are heparin-associated polypeptides that can restore some or all of this regenerative potential, and are thus useful in the treatment of aging disorders that result in tissue loss or underperformance, and rehabilitation from injury. Described herein are therapeutic compositions comprising heparin-associated polypeptides and methods of treating disorders associated with aging, injury, or illness. The therapeutic compositions may comprise one or more heparin-associated polypeptides that possess mitogenic (i.e., regenerative) and/or fusion promoting activity to a somatic cell, such as a tissue progenitor cell. The therapeutic compositions may have activity towards muscle and soft tissue progenitor cells. These compositions may possess utility in treating sarcopenia, cachexia, muscular dystrophy, acute and chronic muscle wasting diseases, and muscle, ligament, or tendon injury, or any combination of these diseases or conditions.

In one aspect, described herein is a composition comprising: (a) a first therapeutic polypeptide comprising a first polypeptide of Table 2, and (b) a second therapeutic polypeptide comprising a second polypeptide of Table 2. The first polypeptide of Table 2 may be a first polypeptide of Table 1 and/or the second polypeptide of Table 2 is a second polypeptide of Table 1. The first polypeptide of Table 2 may comprise THBS1, THBS2, THBS4, FGF17, BMP7, VTN, POSTN, IGF2, or IL-15, or any combination thereof. The second polypeptide of Table 2 may comprise THBS1, THBS2, THBS4, FGF17, BMP7, VTN, POSTN, IGF2, or IL-15, or any combination thereof.

The first polypeptide of Table 2 may comprise BMP7 and/or a sequence comprising at least about 90% homology HAPs ID NO: 72; and the second polypeptide of Table 2 may comprise FGF17 and/or a sequence comprising at least about 90% homology to amino acids 23-216 of HAPs ID NO: 7.

The first polypeptide of Table 2 may comprise BMP7 and/or a sequence comprising at least about 90% homology to HAPs ID NO: 72; and the second polypeptide of Table 2 may comprise IGF2 and/or a sequence comprising at least about 90% homology to amino acids 25-91 of HAPs ID NO: 11.

The first polypeptide of Table 2 may comprise THBS1 and/or a sequence comprising at least about 90% homology to amino acids 19-1170 of HAPs ID NO: 9; and the second polypeptide of Table 2 may comprise THBS2 and/or a sequence comprising at least about 90% homology to amino acids 19-1172 of HAPs ID NO: 4.

The first polypeptide of Table 2 may comprise THBS1 and/or a sequence comprising at least about 90% homology to amino acids 19-1170 of HAPs ID NO: 9; and the second polypeptide of Table 2 may comprise THBS4 and/or a sequence comprising at least about 90% homology to amino acids 27-961 of HAPs ID NO: 8.

The first polypeptide of Table 2 may comprise THBS1 and/or a sequence comprising at least about 90% homology to amino acids 19-1170 of HAPs ID NO: 9; and the second polypeptide of Table 2 may comprise FGF17 and/or a sequence comprising at least about 90% homology to amino acids 23-216 of HAPs ID NO: 7.

The first polypeptide of Table 2 may comprise THBS1 and/or a sequence comprising at least about 90% homology to amino acids 19-1170 of HAPs ID NO: 9; and the second polypeptide of Table 2 may comprise VTN and/or a sequence comprising at least about 90% homology to amino acids 20-478 of HAPs ID NO: 1.

The first polypeptide of Table 2 may comprise THBS1 and/or a sequence comprising at least about 90% homology to amino acids 19-1170 of HAPs ID NO: 9; and the second polypeptide of Table 2 may comprise POSTN and/or a sequence comprising at least about 90% homology to amino acids 22-836 of HAPs ID NO: 6.

The first polypeptide of Table 2 may comprise THBS1 and/or a sequence comprising at least about 90% homology to amino acids 19-1170 of HAPs ID NO: 9; and the second polypeptide of Table 2 may comprise IGF2 and/or a sequence comprising at least about 90% homology to amino acids 25-91 of HAPs ID NO: 11.

The first polypeptide of Table 2 may comprise THBS1 and/or a sequence comprising at least about 90% homology to amino acids 19-1170 of HAPs ID NO: 9; and the second polypeptide of Table 2 may comprise IL-15 and/or a sequence comprising at least about 90% homology to amino acids 49-162 of HAPs ID NO: 10.

The first polypeptide of Table 2 may comprise THBS2 and/or a sequence comprising at least about 90% homology to amino acids 19-1172 of HAPs ID NO: 4; and the second polypeptide of Table 2 may comprise THBS4 and/or a sequence comprising at least about 90% homology to amino acids 27-961 of HAPs ID NO: 8. It may be, the composition further may comprise IL-15 and/or a polypeptide comprising at least about 90% homology to amino acids 49-162 of HAPs ID NO: 10. The composition may further comprise IGF2 and/or a polypeptide comprising at least about 90% homology to amino acids 25-91 of HAPs ID NO: 11.

The first polypeptide of Table 2 may comprise THBS4 and/or a sequence comprising at least about 90% homology to amino acids 27-961 of HAPs ID NO: 8; and the second polypeptide of Table 2 may comprise FGF17 and/or a sequence comprising at least about 90% homology to amino acids 23-216 of HAPs ID NO: 7.

The first polypeptide of Table 2 may comprise THBS4 and/or a sequence comprising at least about 90% homology to amino acids 27-961 of HAPs ID NO: 8; and the second polypeptide of Table 2 may comprise VTN and/or a sequence comprising at least about 90% homology to amino acids 20-478 of HAPs ID NO: 1.

The first polypeptide of Table 2 may comprise THBS4 and/or a sequence comprising at least about 90% homology to amino acids 27-961 of HAPs ID NO: 8; and the second polypeptide of Table 2 may comprise POSTN and/or a sequence comprising at least about 90% homology to amino acids 22-836 of HAPs ID NO: 6.

The first polypeptide of Table 2 may comprise THBS4 and/or a sequence comprising at least about 90% homology to amino acids 27-961 of HAPs ID NO: 8; and the second polypeptide of Table 2 may comprise IGF2 and/or a sequence comprising at least about 90% homology to amino acids 25-91 of HAPs ID NO: 11.

The first polypeptide of Table 2 may comprise THBS4 and/or a sequence comprising at least about 90% homology to amino acids 27-961 of HAPs ID NO: 8; and the second polypeptide of Table 2 may comprise IL-15 and/or a sequence comprising at least about 90% homology to amino acids 49-162 of HAPs ID NO: 10.

The first polypeptide of Table 2 may comprise FGF17 and/or a sequence comprising at least about 90% homology to amino acids 23-216 of HAPs ID NO: 7; and the second polypeptide of Table 2 may comprise VTN and/or a sequence comprising at least about 90% homology to amino acids 20-478 of HAPs ID NO: 1.

The first polypeptide of Table 2 may comprise FGF17 and/or a sequence comprising at least about 90% homology to amino acids 23-216 of HAPs ID NO: 7; and the second polypeptide of Table 2 may comprise POSTN and/or a sequence comprising at least about 90% homology to amino acids 22-836 of HAPs ID NO: 6.

The first polypeptide of Table 2 may comprise FGF17 and/or a sequence comprising at least about 90% homology to amino acids 23-216 of HAPs ID NO: 7; and the second polypeptide of Table 2 may comprise IGF2 and/or a sequence comprising at least about 90% homology to amino acids 25-91 of HAPs ID NO: 11.

The first polypeptide of Table 2 may comprise FGF17 and/or a sequence comprising at least about 90% homology to amino acids 23-216 of HAPs ID NO: 7; and the second polypeptide of Table 2 may comprise IL-15 and/or a sequence comprising at least about 90% homology to amino acids 49-162 of HAPs ID NO: 10.

The first polypeptide of Table 2 may comprise FGF17 and/or a sequence comprising at least about 90% homology to amino acids 23-216 of HAPs ID NO: 7; and the second polypeptide of Table 2 may comprise BMP-7 and/or a sequence comprising at least about 90% homology to amino acids 293-431 of HAPs ID NO: 70.

The first polypeptide of Table 2 may comprise VTN and/or a sequence comprising at least about 90% homology to amino acids 20-478 of HAPs ID NO: 1; and the second polypeptide of Table 2 may comprise POSTN and/or a sequence comprising at least about 90% homology to amino acids 22-836 of HAPs ID NO: 6.

The first polypeptide of Table 2 may comprise VTN and/or a sequence comprising at least about 90% homology to amino acids 20-478 of HAPs ID NO: 1; and the second polypeptide of Table 2 may comprise IGF2 and/or a sequence comprising at least about 90% homology to amino acids 25-91 of HAPs ID NO: 11.

The first polypeptide of Table 2 may comprise VTN and/or a sequence comprising at least about 90% homology to amino acids 20-478 of HAPs ID NO: 1; and the second polypeptide of Table 2 may comprise IL-15 and/or a sequence comprising at least about 90% homology to amino acids 49-162 of HAPs ID NO: 10.

The first polypeptide of Table 2 may comprise POSTN and/or a sequence comprising at least about 90% homology to amino acids 22-836 of HAPs ID NO: 6; and the second polypeptide of Table 2 may comprise IGF2 and/or a sequence comprising at least about 90% homology to amino acids 25-91 of HAPs ID NO: 11.

The first polypeptide of Table 2 may comprise POSTN and/or a sequence comprising at least about 90% homology to amino acids 22-836 of HAPs ID NO: 6; and the second polypeptide of Table 2 may comprise IL-15 and/or a sequence comprising at least about 90% homology to amino acids 49-162 of HAPs ID NO: 10.

The first polypeptide of Table 2 may comprise IGF2 and/or a sequence comprising at least about 90% homology to amino acids 25-91 of HAPs ID NO: 11; and the second polypeptide of Table 2 may comprise IL-15 and/or a sequence comprising at least about 90% homology to amino acids 49-162 of HAPs ID NO: 10.

The first polypeptide of Table 2 may comprise IGF2 and/or a sequence comprising at least about 90% homology to amino acids 25-91 of HAPs ID NO: 11; and the second polypeptide of Table 2 may comprise BMP-7 and/or a sequence comprising at least about 90% homology to amino acids 293-431 of HAPs ID NO: 70.

Also described is a method of producing a composition suitable for the treatment of an aging disorder, a muscle wasting disorder, a muscle injury, or an injury to a connective tissue, or a combination thereof, the method comprising admixing a pharmaceutically acceptable excipient, carrier, or diluent with the mitogenic and/or fusion promoting polypeptide or plurality of mitogenic and/or fusion promoting polypeptides. Also described is a method of producing a mitogenic and/or fusion promoting polypeptide comprising culturing a cell line comprising a nucleic acid encoding mitogenic and/or fusion promoting polypeptide or plurality of mitogenic and/or fusion promoting polypeptides under conditions sufficient to produce the mitogenic and/or fusion promoting polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the features described herein will be obtained by reference to the following detailed description that sets forth illustrative examples, in which the principles of the features described herein are utilized, and the accompanying drawings of which:

FIG. 1A shows experimental experiment overview of intramuscular administration of the entire pool of heparin-associated polypeptides from undifferentiated hESC cells (HAPs) promoted muscle regeneration and decreased fibrosis in aged mice (FIG. 1B) and genetically obese mice (FIG. 1C). Squares denote injury inducing intramuscular injection (IM) with Barium Chloride; circles denote administration of treatment or vehicle.

FIG. 2A Quantification of the regenerative index calculated as the number of newly regenerated fibers per mm{circumflex over ( )}2 of injury area in aged mice following IM treatment with HAPs. Regenerated fibers were identified as fibers with central nuclei, ****p<0.0001, **p=0.0011 (One-way Anova with multiple hypothesis correction)

FIG. 2B Histogram showing the fibrotic index calculated as the percentage of the fibrotic area decreased in aged mice following IM treatment with HAPs **p=0.009, ***p=0.0003. (One-way Anova with multiple hypothesis correction)

FIG. 3A Quantification of the regenerative index calculated as the number of newly regenerated fibers per mm{circumflex over ( )}2 of injury area in genetically obese mice following IM treatment with HAPs. Regenerated fibers were identified as fibers with central nuclei, ****p<0.0001, **p=0.0011 (One-way Anova with multiple hypothesis correction)

FIG. 3B Histogram showing the fibrotic index calculated as the percentage of the fibrotic area decreased in genetically obese mice following IM treatment with HAPs **p=0.009, ***p=0.0003. (One-way Anova with multiple hypothesis correction)

FIGS. 4A-4B illustrate representative results from an in vitro assay useful to validate the regenerative capacity of factors identified by mass spectroscopy. FIG. 4A shows proliferation rate changes expressed as % of nuclei stained with BrdU from cells treated with fusion media (neg. control), defined growth media (pos. control), Optimem, supernatant from differentiated HAPs, supernatant from undifferentiated HAPs, heparin binding proteins eluted from supernatant of undifferentiated HAPs under two different conditions, and supernatant of undifferentiated HAPs that has been depleted of heparin binding proteins. FIG. 4B shows data expressed as % imaged area stained for embryonic myosin heavy chain (eMyHC).

FIGS. 4C-4D show differentially enriched genes and pathways driven by heparin-associated proteins (HAPs) cocktail in aged human muscle cells. (C) RNA expression heatmap of top 50 differentially expressed (DE) genes in aged human muscle cells treated with cocktail of HAPs or vehicle. Cells were treated with indicated factor every 24 h for 96 h. (D) HAPs induce extracellular matrix and cell surface interactions to activate signal cascades promoting proliferative homeostasis. Reactome pathway analysis performed on all upregulated or downregulated DE genes (cutoff=p-value 0.01). Enriched pathways were obtained from GSEA Reactome_2016 gene sets. Enrichment is shown according to the p-value. Positive values are upregulated pathways and negative values are downregulated pathways

FIGS. 5A-5B show quantitation and representative images demonstrating the proliferation effect of IGFBP7 (330 ng/mL), POSTN (330 ng/mL), SPON1 (330 ng/mL), MST1 (330 ng/mL), and RARRES2 (330 ng/mL) (FIG. 5A); and VTN (10 μg/mL), FGF17 (500 ng/mL), IGF2 (2 μg/mL), FGF4 (500 ng/mL), FGF1 (500 ng/mL), and FGF6 (1 μg/mL) (FIG. 5B) in injury activated primary mouse myoblasts grown in vitro. FIG. 5C shows quantitation and representative images demonstrating the increased cellular fusion effect of THBS1 (330 ng/mL), THBS2 (330 ng/mL), and STC2 (875 ng/mL) in injury activated primary mouse myoblasts grown in vitro.

FIGS. 6A-6E show quantitation of immunofluorescent stained cell images demonstrating the proliferation effect of specific heparin-associated polypeptides. FIG. 6A shows the effect of IGFBP5 at 1 μg/mL, FIG. 6B shows the effect of THBS4 at 1 μg/mL, FIG. 6C shows the effect of VTN at 10 μg/mL, FIG. 6D shows the effect of FGF17 at 250 ng/mL, and FIG. 6E shows the effect of IGFBP7 at 500 ng/mL—all demonstrated notable effects in injury activated primary human myoblasts, young (18 years old) and aged (both 68 years old), grown in vitro. FIGS. 6F-6G, show quantitation and representative images demonstrating the increased cellular fusion effect of POSTN (1 μg/mL) FIG. 6F, PDGFRL (5 μg/mL) FIG. 6G in injury activated primary human myoblasts, young (18 years) and old (both 69 years old), grown in vitro.

FIG. 7A provides an exemplary proliferative dose response of mouse myoblasts cultured with hPSC-derived factor Thrombospondin 1 (THBS1) applied at 125 ng/mL, 250 ng/mL, and 500 ng/mL, 1000 ng/ml and 200 ng/ml.

FIG. 7B provides an exemplary proliferative dose response of mouse myoblasts cultured with hPSC-derived factor Platelet derived growth factor-like (PDGFRL) proteins were applied at 625 ng/mL, 1250 ng/mL, 2500 ng/mL, 5000 ng/mL, and 10000 ng/mL which demonstrated a dose-dependent increase in % fusion as expressed in % imaged area stained for embryonic myosin heavy chain (eMyHC).

FIG. 7C provides an exemplary proliferative dose response of mouse myoblasts cultured with hPSC-derived factor Fibroblast growth factor 17 (FGF17) applied at 12.5 ng/ml, 25 ng/ml, 50 ng/ml, 100 ng/ml, and 200 ng/ml which demonstrated a dose-dependent increase in proliferating cells as measured by % EdU positive nuclei.

FIG. 7D shows the shows that the combination of the THBS1 and FGF17 produced potentiation type synergy (CI<0.68, p<7.92E-7

FIG. 8 provides examples of synergistic combinations of heparin-associated polypeptides relative to the vehicle only control (FM) or to treatment with either of the individual heparin-associated polypeptides. Combination Index (CI) values and probability values (p-values) from statistical tests for the synergy models for these and other combinations are reported in Table 10.

FIG. 9A shows a bar graph quantitation of % EdU+ mouse myoblast in response to BMP7, demonstrating BMP7 drives proliferation. Error bars indicate standard deviation and asterisks indicate a p-value of less than 0.05 by Welch's One-Tailed T-test.

FIG. 9B shows a bar graph quantitation of cell count of human myoblast in response to BMP7, demonstrating BMP7 drives proliferation or improves cell survival. Myoblast were cultured 72 h in the presence of BMP7 at indicated dose. Fresh media and BMP7 was added every 24 h. Error bars indicate standard deviation and asterisks indicate a p-value of less than 0.05 by Welch's One-Tailed T-test

FIG. 10A shows a bar graph quantitation of % EdU+ mouse myoblast in response to BMP7, IGF2, or BMP7/IGF2 combined. Error bars indicate standard deviation and asterisks indicate a p-value of less than 0.05 by Welch's One-Tailed T-test, values reported in Table 18.

FIGS. 10B-10C show bar graph quantitation of the fold change from fusion media (vehicle control) in male or female human myoblast cell line in response to BMP7, IGF2, or BMP7/IGF2 combined. Error bars indicate standard deviation and asterisks indicate a p-value of less than 0.05 by Welch's One-Tailed T-test.

FIG. 11A shows a bar graph quantitation of % EdU+ mouse myoblast in response to FGF17, BMP7, or FGF17/BMP7 combined. Error bars indicate standard deviation and asterisks indicate a p-value of less than 0.05 by Welch's One-Tailed T-test.

FIGS. 11B-11C shows bar graph quantitation of the fold change from fusion media (vehicle control) in male or female human myoblast cell line in response to FGF17, BMP7, or BMP7/FGF17 combined. Error bars indicate standard deviation and asterisks indicate a p-value of less than 0.05 by Welch's One-Tailed T-test.

FIG. 12A illustrates a histogram plot of the CD36 receptor on the cell surface of mouse myoblasts.

FIG. 12B illustrates a histogram plot of the ITGA3 receptor on the cell surface of mouse myoblasts.

FIG. 12C illustrates a histogram plot of the ITGA6 receptor on the cell surface of mouse myoblasts.

FIG. 12D illustrates a histogram plot of the ITGB1 receptor on the cell surface of mouse myoblasts.

FIG. 12E shows a bar graph of RNA expression in young and aged human myoblast cell lines. Myoblast were cultured 96 h in fusion media. Fresh media was added every 24 h. Expression are expressed as FPKM.

FIG. 13A shows a bar graph of FGF17 receptor RNASEQ expression in young and aged human myoblast cell lines. Myoblast were cultured 96 h in fusion media. Fresh media was added every 24 h. Expression values are expressed as FPKM.

FIG. 13B shows a bar graph of BMP7 receptors RNASEQ expression in young and aged human myoblast cell lines. Myoblast were cultured 96 h in fusion media. Fresh media was added every 24 h. Expression values are expressed as FPKM.

FIG. 13C shows a bar graph of IGF2 and IGF2 receptor RNASEQ expression in young and aged human myoblast cell lines. Myoblast were cultured 96 h in fusion media. Fresh media was added every 24 h. Expression values are expressed as FPKM.

FIGS. 14A and 14B show expression levels of myogenic markers in myoblasts treated with FGF2, BMP7, THBS1, FGF17, THBS4 or IGF2 for 48 and 72 hours respectively.

FIG. 15A-E shows the Gene Ontology terms for transcripts upregulated in aged human myoblasts compared to vehicle as measured by RNA sequencing treated with a pool of all HAPs FIG. 15A, BMP7 FIG. 15B, FGF17 FIG. 15C, IGF2 FIG. 15D, or THBS1 FIG. 15E.

FIG. 16 shows the results of an experiment analysis using an acute injury model in aged mice of the effects of individual heparin-associated polypeptides with proliferative effects in vitro.

FIG. 16A. Administration of 20 ul of heparin-associated polypeptides (HAPs) FGF17 (500 ng/mL, p <2.23E-4), THBS1 (2 μg/mL, p<5.83E-5), THBS2 (2 μg/mL, p<2.67E-4), and VTN (10 μg/mL, p<1.13E-2) resulted in improved new fiber formation (regenerative index) in aged mice compared to vehicle-treated aged mice to levels similar or better than young mice. FIG. 16B. Administration of 20 ul of heparin-associated polypeptides PPDGFRL (5 μg/mL, p<3.85E-2) and IGFBP7 (1 μg/mL, p<6.63E-3) resulted in improved new fiber formation (regenerative index compared to vehicle-treated aged mice. Regenerative index was calculated as the number of newly regenerated fibers per mm{circumflex over ( )}2 of injury area. Stars indicate degree of significance from one-way ANOVA test

FIG. 17A and FIG. 17B shows the results of an experiment for in vivo injury and individual heparin-associated polypeptide administration followed by muscle excision, dissociation, ex vivo culturing of activated myoblasts and quantitation by chemical and immunofluorescent labelling. FIG. 17A provides resulting quantitation that demonstrates the regenerative effect of heparin-associated polypeptide administration (FGF17) of 20 ul at 500 ng/ml improved the regeneration of new myoblasts in aged mice above the vehicle-treated aged mice (p<7.57E-8) to a level similar to those seen in young mice. FIG. 17B provides resulting quantitation that demonstrates the regenerative effect of administration of heparin-associated polypeptides FGF17 (500 ng/ml) and THBS4 (2 μg/mL)—each improved the regeneration of new myoblasts in aged mice above the vehicle-treated aged mice (p<1.57E-2, 4.64E-2 respectively, one-sided test) compared to vehicle treated control.

FIG. 18A shows the experimental overview. Intramuscular injection of 1.2% of BaCl₂(7 ul/TA) was used to generate chemical injury in the TAs of 78 weeks old mice. Factors were administered via intramuscular injection after 2 h and 48 h of muscle injury.

FIG. 18B shows quantification of the regenerative index calculated as the number of newly regenerated fibers per mm²of injury area demonstrating the effect of individual HAPs at saturating doses compared to combination treatments at subsaturating doses had synergistic efficacy: FGF17 at 500 ng/ml, THBS1 at 2000 ng/ml, THBS2 at 2000 ng/l and VTN at 10000 ng/ml. Regenerated fibers were identified as fibers with central nuclei, significant p-values are indicated with a star.

FIG. 18C shows the number of regenerating fibers after treatment with BMP7 combined with IGF2. FIG. 18D shows the fibrotic index calculated as the percentage of the fibrotic area, demonstrating the effect of individual HAPs at maximal dose (FGF17 at 500 ng/ml and THBS1 at 2000 ng/ml) and demonstrating a combined effect when used in pairwise treatments at a below maximal dose (FGF17 and BMP7 at 12.5 ng/ml each; BMP7 and IGF2 at 25 ng/ml and 60 ng/ml). Significant p-values are indicated with a star. One-way Anova corrected for multiples comparison using Tukey method was used to compare the data.

FIG. 19A shows the experiment overview for intramuscular injection of 10 ug of Cardiotoxin (CTX) to generate injury in the TAs of 21M old mice to test muscle regeneration. BMP7 was administered via intramuscular injection after 1d, 3d and 5d of muscle injury. At day 8, mice were euthanized and TAs were collected.

FIG. 19B Representative images and quantification. H&E staining showing the regenerated fibers and the injury area from Vehicle and BMp7 groups.

FIG. 19C Quantification of the regenerative index calculated as the number of newly regenerated fibers per mm{circumflex over ( )}2 of injury area. Regenerated fibers were identified as fibers with central nuclei, **p=0.0059 (unpaired t-test)

FIG. 19D Histogram of the frequency of muscle fiber cross sectional area in histological analysis showing muscle fiber size distribution increased in the BMP7 treatment group. *p=0.0325, *p=0.0350 (2-way Anova multiple comparisons).

FIG. 20A shows the experimental overview for assessing muscle regeneration from intramuscular administration of IGF2 in a cardiotoxin injured old mice model. Intramuscular injection of 10 ug of Cardiotoxin/TA was used to generate focal injury in the TAs of 21M old mice. 20 ul of IGF2 (2 ug/ml) or Vehicle (PBS) were administered via intramuscular injection at day 2, 4 and 6. Injured muscles were collected at 7 dpi.

FIG. 20B Representative images and quantification. H&E staining showing the regenerated fibers and the injury area from Vehicle and IGF2 groups.

FIG. 20C The quantification of the regenerative index calculated as the number of newly regenerated fibers per mm{circumflex over ( )}2 of injury area is also shown. Regenerated fibers were identified as fibers with central nuclei. significant p-values are shown using unpaired t-test. ** p=0.0016 (Unpaired t-test).

FIG. 20D Histogram of the frequency of muscle fiber cross sectional area in histological analysis showing muscle fiber size distribution increased in the IGF2 treatment group. *p=0.0324, **p=0.0074. 2-way Anova multiple comparisons

FIG. 21A shows the experimental overview. Dexamethasone (25 mg/kg i.p.) was administered to 12 weeks old mice for 20 days simultaneously with a subcutaneous injection of FGF17 (0.5 mg/kg). Muscle weight was assessed on Day 21. Forelimb grip strength and both limb grip strength were measured on Day 7, 13 and 21

FIG. 21B shows the TAs muscle weight over initial body weight shown as the percentage change from vehicle. **p=0.0062. (Unpaired t-test).

FIG. 21C shows the forelimb force measured on Day 21. The bar plot shows the specific forelimb force calculated as the ratio of forelimb force in N over the weight in g, *p=0.0268. (Unpaired t-test).

FIG. 21D shows the specific forelimb force calculated as the ratio of forelimb force in N over the weight in g. **p=0.001 (Unpaired t-test).

FIG. 21E Both limb force measured on Day 21 calculated as the ratio of both limb force in N over the weight in g *p=0.0117. (Unpaired t-test).

FIG. 22A Experiment overview and groups were systemic administration of BMP7 protected against Dexamethasone induced muscle atrophy. Dexamethasone (25 mg/kg i.p.) was administered to 12 weeks old mice for 14 days simultaneously with a subcutaneous injection of BMP7 (0.03 mg/kg) or vehicle (saline). Mice were euthanized on Day 15.

FIG. 22B TAs and FIG. 22C GCs muscle weight were assessed on Day 15. *** p=0.0001, **p=0.0014 (unpaired t-test)

FIG. 22D Measures of muscle strength or function improved by BMP7 treatment. FIG. 22D forelimb maximum force in mN assessed at Day 13 ** p=0.021. (unpaired t-test)

FIG. 22E Specific forelimb maximum force calculated as the ratio of forelimb force in mN over the weight in g ***p=0.0007. (unpaired t-test)

FIG. 22F distance **p=0.0265, FIG. 22G time to exhaustion **p=0.0051, FIG. 22H max speed **p=0.007, FIG. 22I and work **p=0.0056 were measured on Day 14 using an induced treadmill running model set to progressively increase speed 2 m/min every subsequent 2 min. Unpaired t-test was used to compare data.

FIG. 23A shows experiment overview and groups were systemic administration of BMP7 protected against aging induced muscle dysfunction. Subcutaneous injection of BMP7 (30 ug/kg) or vehicle (PBS) were administered to 21-24M old mice for 14 days. Muscle function was assessed at days 13 and 14.

FIG. 23 B,C Treadmill performance measured at day 14 using an induced treadmill running model set to progressively increase speed 2 m/min every subsequent 2 min. (B) Distance ran shown. *p=0.0371 (C) Time to exhaustion *p=0.0298. Unpaired t-test was used to compare data.

FIG. 23 D-G forelimb Grip strength force was assessed at day 13. (D) forelimb Grip strength force was assessed at day 13. The graph shows forelimb grip strength force, **** p<0.0001. (E) the graph shows specific forelimb maximum force calculated as the ratio of forelimb force in mN over the weight in g ***p=0.0001. (F) Bothlimb Grip strength force was assessed at day 13. The graph shows bothlimb grip strength force, *** p=0.0003. (G) the graph shows specific bothlimb maximum force calculated as the ratio of forelimb force in mN over the weight in g **p=0.0011. Unpaired t-test was used to compare data.

FIG. 23H-K show 4 representative graphs out of 37 readouts measured which showed systemic treatment with BMP7 resulted in no adverse events. No changes were observed in (H) the white blood cell count p=0.6503, (I) Alkaline phosphatase activity p<0.9999, (J) Creatinine concentration p=0.5995 and (K) Amylase activity p=0.5468. Unpaired t-test was used to analyze this data.

FIG. 24A provides representative quantitation of immunofluorescence images demonstrating the proliferation enhancing effects of HAPs (hPSC factors) and specific HAPss at various does on primary human myoblasts derived from a patient with type 1 myotonic dystrophy.

FIG. 24B provides quantitation of immunofluorescence images demonstrating the hypertrophy enhancing effects of HAPs (hPSC factors) and specific heparin-associated polypeptides administration at various does on primary human myoblasts derived from a patient with type 1 myotonic dystrophy.

FIG. 25A-C IGF2 treatment promoted proliferation and fusion in DM1 human myoblast (32 year old caucasian female) cells. FIG. 25A Bar graph and table quantitation of % EdU+ human myoblast and FIG. 25B % area eMyHC in response to IGF2. Significant p-values (EdU: Vehicle˜IGF2: 6.8E-3, % eMyHC Area: Vehicle˜IGF2: 1.9E-4) (*p<0.05 by Students Two-Tailed T-test, n=3-6). FIG. 25C Bar graph of MYH3 and CKM expression fold change in DM1 human myoblast in response to indicated treatment compared to vehicle as measured by qPCR. Myoblasts were cultured 96 h in the presence of factors (IGF2 200 ng/mL, n=3). Fresh media and factors were added every 24 h. Mean∓S.D. Significant p-values (MYH3: Vehicle˜IGF2: 1.13E-03, CKM: Vehicle IGF2: 7.67E-03) FIG. 25D Bar graph of ATP1B1 expression fold change in DM1 human myoblast in response to indicated treatment compared to FM (vehicle) as measured by qPCR. Myoblasts were cultured 48 h in the presence of factors (IGF2 200 ng/mL, n=3) Fresh media and factors were added every 24 h. Mean∓S.D. Significant p-values (Vehicle˜IGF2: 3.11E-05) (*p<0.05 by Students Two-Tailed T-test, n=3)

DETAILED DESCRIPTION

In one aspect, described herein is a composition comprising a mitogenic and/or fusion promoting polypeptide, wherein the polypeptide is a heparin-associated polypeptide secreted from a stem cell or a transformed cell line, wherein the heparin-associated polypeptide possesses mitogenic and/or fusion promoting activity. The composition may be for use in a method of treating an aging disorder, a muscle wasting disorder, a muscle injury, or an injury to a connective tissue, or a combination thereof. The composition may be for use in a method of increasing proliferation of a muscle cell or connective tissue cell precursor in an individual. The individual may be afflicted with or suspected of being afflicted with an aging disorder, a muscle wasting disorder, a muscle injury, or an injury to a connective tissue, or a combination thereof. The aging disorder may be sarcopenia. The muscle wasting disorder is muscular dystrophy. The muscle wasting disorder may be cachexia, e.g., muscular cachexia. In another aspect, described herein, is a composition comprising a mitogenic and/or fusion promoting polypeptide, wherein the mitogenic and/or fusion promoting polypeptide may comprise FST, CTGF, THBS1, THBS2, THBS3, HGFAC, IGFBP3, IGFBP5, IGFBP7, IGFBP4, SFRP1, STC1, STC2, IGFBP2, SPON1, MST1, POSTN, PTN, RARRES2, DRAXIN, CLC, LTBP1, ADAMTS7, NOV, PDGFRL, AGRN, FGFBP3, ADAMTS19, THBS4, PLAT, GDF15, CHRDL1, FRZB, COL1A1, FN1, LAMA1, LAMAB1, LAMAC1, TNC, VTN, MDK, LEFTYA, MATN2, ANOS1, APOB, CLEC3A, COCH, CSF2, FBLN1, FGF-17, TGFBI, CXCL12, HDGF2, NTS, GDNF, VEGF-165, TIMP1, TCN2, PAMR1, WISP2, HGF, IGF-2, ANG, PDGFD, FSTL1, SFRP2, VASN, FGF-4, TIMP2, NDNF, FGF1, CKB, DAG1, PLAU, PTPRS, FLT1, OLFML3, BTC, IL-13, IL-15, APLN, IL-10, HB-EGF, FGF-6, FGF-13, CTNNB1, GLG1, CCL14, NAMPT, BMP-7, HDGF, FGF-19, GASP-1, BMP-2, TGFb1, FGF-18, ADAMTS1 a protein listed in Table 2, or a protein listed in Table 1, or any combination thereof. The mitogenic and/or fusion promoting polypeptide may comprise one or more of VTN, POSTN, FGF17, THBS2, THBS4, IGF2, IL-15, THBS1, and BMP7. The mitogenic and/or fusion promoting polypeptide may comprise VTN. The mitogenic and/or fusion promoting polypeptide may comprise THBS2. The mitogenic and/or fusion promoting polypeptide may comprise POSTN. The mitogenic and/or fusion promoting polypeptide may comprise FGF17. The mitogenic and/or fusion promoting polypeptide may comprise THBS4. The mitogenic and/or fusion promoting polypeptide may comprise IGF2. The mitogenic and/or fusion promoting polypeptide may comprise IL-15. The mitogenic and/or fusion promoting polypeptide may comprise THBS1. The mitogenic and/or fusion promoting polypeptide may comprise BMP7. The composition may comprise a mixture of a plurality of different mitogenic and/or fusion promoting polypeptides. The plurality of different mitogenic and/or fusion promoting polypeptides may comprise three, four, or five different mitogenic and/or fusion promoting polypeptides. The plurality of polypeptides may comprise one, two, three, four, five, six, seven, eight, nine, ten or more polypeptides of Table 2. The plurality of polypeptides may comprise one, two, three, four, five, six, seven, eight, nine, ten or more polypeptides of Table 1. The plurality of mitogenic and/or fusion polypeptides may comprise BMP7 and IGF2. The plurality of mitogenic and/or fusion polypeptides may comprise BMP7 and and FGF17. The plurality of mitogenic and/or fusion promoting polypeptides may comprise IGF2, THBS2, and THBS4. The plurality of mitogenic and/or fusion promoting polypeptides may comprise IL-15, THBS2, and THBS4. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS2 and THBS4. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS2, THBS4, and VTN. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS2, THBS4, and ANOS1. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS2, THBS4, and IL-15. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS2, THBS4, and IGF2. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS1 and FGF17. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS2 and VTN. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS1 and VTN. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS1 and THBS2. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS2 and FGF17. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS1 and THBS4. The plurality of mitogenic and/or fusion promoting polypeptides may comprise VTN and FGF17. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS4 and VTN. The plurality of mitogenic and/or fusion promoting polypeptides may comprise THBS4 and FGF17. The composition may be for use in a method of treating an aging disorder, a muscle wasting disorder, a muscle injury, or an injury to a connective tissue, or any combination thereof. The composition may be for use in a method of increasing proliferation of a muscle cell and/or connective tissue cell precursor in an individual. The individual may be afflicted with or suspected of being afflicted with an aging disorder, a muscle wasting disorder, a muscle injury, or an injury to a connective tissue, or a combination thereof. The aging disorder may be sarcopenia. The muscle wasting disorder may be muscular dystrophy. The muscle wasting disorder may be cachexia, e.g., muscular cachexia.

In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims that follow, the word “comprise” and variations thereof, such as, “may comprise” and “comprising” are to be construed in an open, inclusive sense, that is, as “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 employed in its sense including “and/or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

As used herein a composition that is “consisting essentially” of the recited components is a composition that only has the recited elements as active ingredients, but can comprise other non-active components that do not appreciably modify the function or activity of the recited components. Any list disclosed herein that is recited as “comprising” can be recited as “consisting essentially,” to exclude non-recited polypeptide or protein components.

As used herein “heparin-associated polypeptide” means any polypeptide that directly binds to heparin with a K_Dof less than 1 micromolar, or any polypeptide that associates with one or more polypeptides that bind directly to heparin with a K_Dof less than 1 micromolar. This K_Dcan be measured using a method such as surface plasmon resonance. See e.g., Nguyen et al., “Surface plasmon resonance: a versatile technique for biosensor applications.” Sensors (Basel). 2015 May 5; 15(5):10481-510. Alternatively, a heparin-associated polypeptide is one that is enriched by a factor of at least 5-fold, 10-fold, 100-fold, or 1,000 from a complex mixture of polypeptides (e.g., a cell supernatant) by the use of heparin bound to a bead or other matrix support, or co-purifies with such a polypeptide.

As used herein “heparin-binding polypeptide” (HAP) means any polypeptide that directly binds to heparin with a K_Dof less than 1 micromolar. Heparin-binding polypeptides can interact with heparin at steady-state under normal growth conditions, but in other instances heparin-binding polypeptides may interact with heparin transiently under normal growth conditions or only under certain conditions as a result of a signaling or environmental stimulus. Heparin binding-polypeptides may interact with heparin as a result of post-translational modifications such as phosphorylation, dephosphorylation, acetylation, deacetylation, lipidation, delipidation, glycosylation, or deglycosylation, or combinations thereof.

As used herein “pluripotent stem cell” or “pluripotent cell” (PSC) means a cell that has the ability to differentiate into several different cell types that are derivatives of all of the three germinal layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells are capable of forming teratomas. Examples of pluripotent stem cells are embryonic stem cells (ESCs), embryonic germ stem cells (EGCs), embryonic Carcinoma Cells (ECCs), and induced pluripotent stem cells (iPSCs). PSC may be from any organism of interest, including, primate, human (hPSCs); canine; feline; murine; equine; porcine; avian; camel; bovine; ovine, and so on.

As used herein “somatic cell” means any cell of an organism that, in the absence of experimental manipulation, does not ordinarily give rise to all types of cells in an organism. In other words, somatic cells are cells that have differentiated sufficiently that they will not naturally generate cells of all three germ layers of the body, i.e., ectoderm, mesoderm and endoderm. For example, somatic cells would include muscle cells and muscle progenitor cells, the latter of which may be able to self-renew and naturally give rise to all or some cell types of the skeletal, cardiac, or smooth muscle but cannot give rise to cells of the ectoderm or endoderm lineages.

As used herein the term “about” refers to an amount that is near the stated amount by 10% or less.

As used herein the terms “individual” “subject,” and “patient” are interchangeable. The individual can be mammal such as a horse, cow, pig, chicken, goat, rabbit, mouse, rat, dog, or cat. The individual may be a human person.

The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. Polypeptides, including the provided polypeptide chains and other peptides, e.g., linkers and binding peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-translational modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins, errors due to PCR amplification, or errors in protein translation.

A recombinant protein may be a protein expressed in a system other than a human, e.g., the protein is expressed from bacteria, yeast, or mammalian cells in culture. In some cases, the protein is expressed from Chinese Hamster Ovary cells (CHO cells). In some cases, the protein is expressed from mouse myeloma cells, e.g., (NS0) cells. In some cases, the protein is expressed from E. coli.

Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. 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 a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or may comprise a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

“Exogenous” with respect to a nucleic acid or polynucleotide indicates that the nucleic acid is part of a recombinant nucleic acid construct, or is not in its natural environment. For example, an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct. An exogenous nucleic acid also can be a sequence that is native to an organism and that has been reintroduced into cells of that organism. An exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. The exogenous elements may be added to a construct, for example using genetic recombination. Genetic recombination is the breaking and rejoining of DNA strands to form new molecules of DNA encoding a novel set of genetic information. Often exogenous nucleic acids will include a translatable sequence lacking introns that has been cloned from a cDNA.

As described herein a “mitogenic polypeptide” is one that induces one or more stages of mitosis, including interphase, prophase, metaphase, anaphase, and telophase. A mitogenic polypeptide may be one that induces mitosis in any one or more of a soft-tissue cell, a soft-tissue precursor cell, a muscle cell, a muscle precursor cell, or a tenocyte.

As described herein a “fusion promoting” polypeptide is one that promotes fusion of muscle cells or muscle cell precursors. Fusion of muscle precursors like C2C12 cells is an experimental marker of differentiation and can be monitored by increases in eMyHC expression, cell size, or increased number of nuclei per eMyHC positive cell a by a statistically measurable change of at least 25% magnitude (p<0.05) relative to vehicle treated cells grown in otherwise identical conditions.

Reference to a fusion, fusion polypeptide, or fusion protein may refer to a synthetically and/or recombinantly produced molecule in which two or more amino acid sequences are connected, e.g., by a peptide bond and/or linker. In some cases, the two or more amino acid sequences are linked via a linker comprising one or more amino acids. In other cases, the two or more amino acid sequences are not linked via a linker, e.g., the two sequences are directly connected by a peptide bond. In some cases, at least one of the two or more amino acid sequences may comprise a polypeptide described herein. For example, the polypeptide described herein is a polypeptide comprising VTN, POSTN, PDGFRL, THBS2, THBS4, THBS1, IL-15, or IGF2, or a combination thereof.

Reference to a conjugate, polypeptide conjugate, or protein conjugate may refer to a synthetically and/or recombinantly produced molecule comprising a chemical entity covalently bound to one or more amino acids of an amino acid sequence. In some cases, the conjugation is selective such that the chemical entity is connected to a specific amino acid of the amino acid sequence. The amino acid sequence may comprise a polypeptide described herein. For example, the polypeptide described herein is a polypeptide comprising VTN, POSTN, FGF17, THBS2, THBS4, THBS1, IL-15, IGF2, or BMP7, or a combination thereof.

A polypeptide described herein may be a proteoform of a protein listed in Table 2. As used herein a proteoform may describe a molecular form of a protein product arising from a gene encoding a protein, such as a protein listed in Table 2. In some cases, a proteoform includes proteins that arise from the same gene as a result of genetic variation, alternatively spliced RNA transcripts, post-translational modifications, or polypeptide cleavage event.

Heparin-Associated Polypeptides

In one aspect, polypeptides described herein that are useful for treating an aging disease or injury comprise one or more polypeptides secreted from an induced pluripotent stem cell, an embryonic stem cell, a tissue progenitor cell, or a transformed cell line that bind to heparin. A plurality of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more HAPs are included in a composition may comprise a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the composition may comprise one, two, three, four, five, six, seven, eight, nine, ten or more polypeptides of Table 2. In some cases, the composition may comprise one, two, three, four, five, six, seven, eight, nine, ten or more polypeptides of Table 1.

In certain aspects, there are three biochemical features that are common across all potential therapeutic HAPs: 1) they are secreted by human pluripotent stem cells; 2) they can be purified by heparin agarose beads from a complex mixture, and 3) their molecular weight equals or exceeds 3.5 kDa.

In certain aspects, there are certain structure-function relationships that potentially link disparate therapeutic polypeptides into a genus of heparin-associated therapeutic polypeptides. Included among these are the ability to be secreted, which may require: 1) an N-terminal signal sequence (aprox. 15-30 amino acids in length); and/or 2) the presence of one or more post translational modifications added in the Endoplasmic Reticulum or the Golgi apparatus to promote stability, such as glycosylation or disulfide bonds. It is estimated that 2,000 to 3,000 genes encoded by the human genome produce a secreted polypeptide in one or more cell types. In addition to being secretory polypeptides the therapeutic polypeptides may comprise a heparin-binding domain, or, alternatively associate with heparin-binding domain comprising polypeptides. Heparin is a linear polymer of saccharides in 1-4 alpha linkages that form a spiraling chain, commonly associated with its role in binding plasma proteins to reduce clotting (See Capila and Lindhart, “Heparin-protein interactions” Angew Chem Int Ed Engl. 2002 Feb. 1; 41(3):391-412). Currently, predicting heparin-binding from protein sequence alone is a challenge for the field due to the structural heterogeneity of heparin polymers and the large and variable number of shallow binding pockets thought to be important for stabilizing the interaction. Several hundred HAPs have been empirically tested for heparin binding, using a few heparin chain configurations. Based on these studies many binding motifs have been proposed, but none have been proven necessary and sufficient. One common motif appears to be a sequence of repeating basic residues that orient onto a common surface of the secondary structure for interacting with the matching pattern of sulfate groups on heparin chains. Therefore, many heparin-binding therapeutic polypeptides may contain patterns of basic residues (arginine or lysine) clustered in some part of the protein, though agnostic to the exact sequence.

The heparin-associated therapeutic polypeptide is a secreted polypeptide. The heparin-associated therapeutic polypeptide may comprise a secreted polypeptide that may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more disulfide bonds. The heparin-associated therapeutic polypeptide may comprise a secreted polypeptide that may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or N-liked or 0-linked glycans. The heparin-associated therapeutic polypeptide may be greater than about 3.5 kilodaltons. The heparin-associated may be greater than about 5, 7.5, 10, 15, or 20 kilodaltons. The heparin-associated therapeutic polypeptide may be one that may comprise a region exhibiting enrichment for basic amino acids arginine or lysine. The region can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length, and comprise an amount of basic residues that is greater than would be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 150%, or 200% greater than expected given random chance. The heparin-associated therapeutic polypeptide does not comprise a basic DNA binding motif, such as those found in bZIP transcription factors. The HAP may comprise heparin binding polypeptide.

The HAPs, described herein, can comprise one or more amino acid modifications that promote stability and/or facilitate production. The polypeptide can comprise one or more covalent modifications that promote stability (e.g., PEGylation). Other modifications of the HAP(s) are contemplated herein. For example, the HAP(s) may be linked to one of a variety of non-proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP may comprise be fused or conjugated to another protein to increase stability and or bioavailability. The HAP may comprise be a fusion with an Fc region of an immunoglobulin or with serum albumin.

The HAPs described herein can be encapsulated in nanospheres or nanoparticles to increase stability. The nanospheres or nanoparticles may comprise biodegradable or bioabsorbable. Certain types of nanospheres can be deployed such as polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres or nanospheres. The HAP may be included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA).

The heparin-associated-polypeptide may be concatemerized to increase stability and or bioavailability. The HAP(s) may comprise concatemers of the same or of different heparin-associated binding polypeptides. Concatemers can be separated by polypeptide linkers, for example a Gly-Ser linker of any suitable length. The Gly-Ser linker may comprise a G₄S₁linker. The concatemers may comprise 1, 2, 3, 4, 5 or more of the same HAP as a single polypeptide separated by a Gly-Ser linker. The concatemers may comprise 1, 2, 3, 4, 5 or more different HAPs as a single polypeptide separated by a Gly-Ser linker. The concatemers may comprise 1, 2, 3, 4, 5 or more of the same HAP covalently linked through a non-peptide linkage, such as for example a disulfide bridge. The concatemers may comprise 1, 2, 3, 4, 5 or more different HAPs covalently linked through a non-peptide linkage, such as for example a disulfide bridge. The concatemers may comprise 1, 2, 3, 4, 5 or more of the same HAP non-covalently linked, such as for example, by a streptavidin-biotin interaction or protein-protein interaction. The concatemers may comprise 1, 2, 3, 4, 5 or more different HAPs non-covalently linked such as for example, by a streptavidin-biotin interaction or protein-protein interaction.

Additional modifications to HAP comprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 amino acids from the N-terminal or C-terminal ends of the HAP. The HAP may comprise the deletion of known inhibitory domains or deletion of domains not associated with the heparin-associated-polypeptides functions in inducing proliferation of muscle, connective, or soft-tissue cell precursors.

The HAPs herein can comprise cleavage products of a pro-protein. Cleavage of a pro-protein can result in activation or higher activity of said pro-protein. HAPs may be produced that correspond to a cleaved or active form of the pro-protein. The HAPs may comprise only the active domain of a heparin associated pro-protein (e.g., the minimal portion sufficient to create a biological effect).

The HAP may comprise one or more of the polypeptides listed in Table 1 and/or Table 2. The polypeptide may be at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a polypeptide listed in Table 1 and/or Table 2, or an isoform thereof. The polypeptide may be at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a sequence selected from POLYPEPTIDE ID NOS: 1-44, 55, 56, and 58-72.

The HAP may comprise one or more of the polypeptides listed in Table 2, Table 1, or a proteoform thereof. The HAP may be at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a polypeptide listed in Table 2, Table 1, or a proteoform thereof. The HAP may comprise a polypeptide at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to FST, CTGF, THBS1, THBS2, THBS3, HGFAC, IGFBP3, IGFBP5, IGFBP7, IGFBP4, SFRP1, STC1, STC2, IGFBP2, SPON1, MST1, POSTN, PTN, RARRES2, DRAXIN, CLC, LTBP1, ADAMTS7, NOV, PDGFRL, AGRN, FGFBP3, ADAMTS19, THBS4, PLAT, GDF15, CHRDL1, FRZB, COL1A1, FN1, LAMA1, LAMAB1, LAMAC1, TNC, VTN, MDK, LEFTYA, MATN2, ANOS1, APOB, CLEC3A, COCH, CSF2, FBLN1, FGF-17, TGFBI, CXCL12, HDGF2, NTS, GDNF, VEGF-165, TIMP1, TCN2, PAMR1, WISP2, HGF, IGF-2, ANG, PDGFD, FSTL1, SFRP2, VASN, FGF-4, TIMP2, NDNF, FGF1, CKB, DAG1, PLAU, PTPRS, FLT1, OLFML3, BTC, IL-13, IL-15, APLN, IL-10, HB-EGF, FGF-6, FGF-13, CTNNB1, GLG1, CCL14, NAMPT, BMP-7, HDGF, FGF-19, GASP-1, BMP-2, TGFb1, FGF-18, ADAMTS1 or any combination thereof. The HAP may comprise a polypeptide at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to VTN, POSTN, FGF17, THBS2, THBS4, THBS1, IL-15, IGF2, or BMP7, or any combination thereof. The HAP may comprise THBS1. The HAP may comprise THBS2. The HAP may comprise THBS4. The HAP may comprise FGF17. The HAP may comprise VTN. The HAP may comprise POSTN. The HAP may comprise IGF2. The HAP may comprise IL-15. The HAP may comprise BMP7. Described herein is a composition that may comprise comprising any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a polypeptide Table 2, Table 1, FST, CTGF, THBS1, THBS2, THBS3, HGFAC, IGFBP3, IGFBP5, IGFBP7, IGFBP4, SFRP1, STC1, STC2, IGFBP2, SPON1, MST1, POSTN, PTN, RARRES2, DRAXIN, CLC, LTBP1, ADAMTS7, NOV, PDGFRL, AGRN, FGFBP3, ADAMTS19, THBS4, PLAT, GDF15, CHRDL1, FRZB, COL1A1, FN1, LAMA1, LAMAB1, LAMAC1, TNC, VTN, MDK, LEFTYA, MATN2, ANOS1, APOB, CLEC3A, COCH, CSF2, FBLN1, FGF-17, TGFBI, CXCL12, HDGF2, NTS, GDNF, VEGF-165, TIMP1, TCN2, PAMR1, WISP2, HGF, IGF-2, ANG, PDGFD, FSTL1, SFRP2, VASN, FGF-4, TIMP2, NDNF, FGF1, CKB, DAG1, PLAU, PTPRS, FLT1, OLFML3, BTC, IL-13, IL-15, APLN, IL-10, HB-EGF, FGF-6, FGF-13, CTNNB1, GLG1, CCL14, NAMPT, BMP-7, HDGF, FGF-19, GASP-1, BMP-2, TGFb1, FGF-18, ADAMTS1 a proteoform thereof, or a combination thereof; and a pharmaceutically acceptable excipient, carrier, or diluent. The composition may comprise a plurality of peptides from Table 2; and optionally a pharmaceutically acceptable excipient, carrier, or diluent. In some cases the plurality may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides of Table 2. In some cases, one or more of the plurality of polypeptides is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a polypeptide of Table 2. The composition may comprise a plurality of peptides from Table 1; and optionally a pharmaceutically acceptable excipient, carrier, or diluent. In some cases the plurality may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides of Table 1. In some cases, one or more of the plurality of polypeptides is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a polypeptide of Table 1. The composition may comprise THBS1. The composition may comprise THBS2. The composition may comprise THBS4. The composition may comprise FGF17. The composition may comprise VTN. The composition may comprise POSTN. The composition may comprise IGF2. The composition may comprise IL-15. The composition may comprise BMP7. The composition may comprise IGF2, THBS2, and THBS4. The composition may comprise IL-15, THBS2, and THBS4. The composition may comprise THBS2 and THBS4. The composition may comprise THBS2, THBS4, and VTN. The composition may comprise THBS2, THBS4, and ANOS1. The composition may comprise THBS2, THBS4, and IL-15. The composition may comprise THBS2, THBS4, and IGF2. The composition may comprise THBS1 and FGF17. The composition may comprise THBS2 and VTN. The composition may comprise THBS1 and VTN. The composition may comprise THBS1 and THBS2. The composition may comprise THBS2 and FGF17. The composition may comprise THBS1 and THBS4. The composition may comprise VTN and FGF17. The composition may comprise THBS4 and VTN. The composition may comprise THBS4 and FGF17. The composition may comprise BMP7 and IGF2. The composition may comprise BMP7 and FGF17. A composition may comprise any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to CTGF, THBS1, THBS2, THBS3, HGFAC, IGFBP3, IGFBP5, IGFBP7, IGFBP4, SFRP1, STC1, STC2, IGFBP2; and a pharmaceutically acceptable excipient, carrier, or diluent. Described herein, is a composition that may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides, wherein one or more the polypeptides are at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, BMP7, or THBS4; and a pharmaceutically acceptable excipient, carrier, or diluent.

Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 1 or amino acids 20-478 of HAPs ID NO: 1, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 1. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 2, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 2. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 3, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 3. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 4 or amino acids 19-1172 of HAPs ID NO: 4, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 4. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 5, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 5. Described herein is a composition that may comprise polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 6 or amino acids 22-836 of HAPs ID NO: 6, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 6. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 7 or amino acids 23-216 of HAPs ID NO: 7, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 7. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 8 or amino acids 27-961 of HAPs ID NO: 8, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 8. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 9 or amino acids 19-1170 of HAPs ID NO: 9, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 9. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 10 or amino acids 49-162 of HAPs ID NO: 10, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 10. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 11 or amino acids 25-91 of HAPs ID NO: 11, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 11.

Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 12, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 12. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 13, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 13. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 14, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 14. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 15 and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 15. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 16, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 16. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 17, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 17. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 18, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 18. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 19, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 19.

Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 20, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 20. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 21, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 21. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 22, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 22. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 23, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 23. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 24, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 24. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 25, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 25. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 26, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 26. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 27, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 27. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 28, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 28. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 29, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 29.

Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 30, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 30. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 31, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 31. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 32, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 32. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 33, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 33. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 34, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 34. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 35, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 35. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 36, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 36. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%. 97%. 98%, 99%, or 100% homology or identity to HAPs ID NO: 37, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 37. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 38, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 38. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 39, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 39.

Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 40, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 40. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 41, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 41. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 42, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 42. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 43, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 43. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 44, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 44. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 58, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 58. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 59, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 59. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 60, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 60. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 61, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 61. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 62, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 62. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 63, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 63. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 64, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 64. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 65, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 65. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 66, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 66. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 67, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 67. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 68, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 68. Described herein is a composition that may comprise a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology or identity to HAPs ID NO: 72, and a pharmaceutically acceptable excipient, carrier, or diluent. In some cases, the polypeptide does not comprise a signal sequence of HAPs ID NO: 72.

Described herein is a composition that may comprise comprising 1, a plurality, or 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to ADAMTS12, INS-IGF2, AOC1, SOD3, CLU, ITIH1, APLP1, THBS1, COCH, ITIH2, APLP2, THBS3, COL11A1, LAMA1, APOB, TNXB, COL12A1, LAMA2, APOE, VEGFA, COL14A1, LAMA5, APOH, VTN, COL18A1, LAMB1, APP, ZNF207, COL1A1, LAMB2, CCDC80, COL1A2, LTF, CFH, COL2A1, MATN2, CLEC3B, COL3A1, MDK, COL25A1, COL5A1, MST1, COL5A3, COL5A2, NID1, CYR61, COL6A1, NPNT, F2, COL6A2, OLFML3, FGF2, COL6A3, PCOLCE, FGFBP3, CTGF, POSTN, FSTL1, DCD, PTN, HDGF, DRAXIN, RARRES2, KNG1, ECM1, RELN, NDNF, FBLN1, SFRP1, NRP1, FBN1, SLIT3, PAFAH1B1, FBN2, SPON1, PCOLCE2, FN1, STC1, PTPRF, FST, STC2, PTPRS, HGFAC, SVEP1, RPL22, IGFBP2, THBS2, RPL29, BMP7, and combinations thereof; and a pharmaceutically acceptable excipient, carrier, or diluent. Described herein is a composition that may comprise comprising a plurality of polypeptides at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to CTGF, THBS1, THBS2, THBS3, HGFAC, IGFBP3, IGFBP5, IGFBP7, IGFBP4, SFRP1, STC1, STC2, IGFBP2; and a pharmaceutically acceptable excipient, carrier, or diluent.

Described herein is a composition that may comprise consisting essentially of 1, a plurality, or 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to ADAMTS12, INS-IGF2, AOC1, SOD3, CLU, ITIH1, APLP1, THBS1, COCH, ITIH2, APLP2, THBS3, COL11A1, LAMA1, APOB, TNXB, COL12A1, LAMA2, APOE, VEGFA, COL14A1, LAMA5, APOH, VTN, COL18A1, LAMB1, APP, ZNF207, COL1A1, LAMB2, CCDC80, COL1A2, LTF, CFH, COL2A1, MATN2, CLEC3B, COL3A1, MDK, COL25A1, COL5A1, MST1, COL5A3, COL5A2, NID1, CYR61, COL6A1, NPNT, F2, COL6A2, OLFML3, FGF2, COL6A3, PCOLCE, FGFBP3, CTGF, POSTN, FSTL1, DCD, PTN, HDGF, DRAXIN, RARRES2, KNG1, ECM1, RELN, NDNF, FBLN1, SFRP1, NRP1, FBN1, SLIT3, PAFAH1B1, FBN2, SPON1, PCOLCE2, FN1, STC1, PTPRF, FST, STC2, PTPRS, HGFAC, SVEP1, RPL22, IGFBP2, THBS2, BMP7, and RPL29; and a pharmaceutically acceptable excipient, carrier, or diluent. Described herein is a composition that may comprise consisting essentially of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to CTGF, THBS1, THBS2, THBS3, HGFAC, IGFBP3, IGFBP5, IGFBP7, IGFBP4, SFRP1, STC1, STC2, IGFBP2; and a pharmaceutically acceptable excipient, carrier, or diluent.

In certain embodiments, compositions comprising HAPs do not comprise fibroblast growth factors (FGF). In certain embodiments, compositions comprising HAPs do not comprise fibroblast growth factor 2 (FGF2). In certain embodiments, compositions comprising HAPs do not comprise FGF19, Angiogenin, BTC, IL-13 R alpha 2, Siglec-5/CD170, IL-15, APJ, IGFBP-2, Chordin-Like 1, GASP-1/WFIKKNRP, MFRP, IL-10 R alpha, Chem R23, HB-EGF, FGF-6, HGF, IL-16, IL-7 R alpha, TRAIL R3/TNFRSF10C, BMP-6, IL-1 F9/IL-1 H1, IL-1 beta, Kremen-2, TRAIL R4/TNFRSF10D, CXCR1/IL-8 RA, Ck beta 8-1/CCL23, Beta-catenin, FGF-13 1B, TRAIL/TNFSF10, CCL14/HCC-1/HCC-3, or FGF-4, or a combination thereof.

In certain aspects, heparin-associated binding polypeptides and compositions of heparin-associated binding polypeptides herein comprise polypeptides that increase the proliferation of muscle cell precursors, and/or increase their differentiation into muscle cells. The HAPs increase proliferation of a muscle cell precursor by at least about 20%, 30%, 40%, 50%, or 100% compared to a muscle cell precursor not treated with the heparin-associated binding polypeptide. The HAPs increase proliferation of a myoblast by at least about 20%, 30%, 40%, 50%, 100%, 200%, or 500% compared to a myoblast not treated with the heparin-associated binding polypeptide. The myoblast is a human myoblast cell line. The myoblast is a mouse myoblast cell line (e.g., C2C12). Proliferation can be measured by BrdU or EdU incorporation, which can be quantified using suitable methods such as, by way of non-limiting embodiment, microscopy, flow cytometry, or ELISA.

The HAPs increase differentiation and/or fusion of a muscle cell precursor by at least about 50%, 75%, 100%, 200%, or 500% compared to a muscle cell precursor not treated with the heparin-associated binding polypeptide. The HAPs increase differentiation of a myoblast by at least about 50%, 75%, or 100% compared to a myoblast not treated with the heparin-associated binding polypeptide. The myoblast is a human myoblast cell line. The myoblast is a mouse myoblast cell line (e.g., C2C12). Differentiation can be measured and/or quantified by eMyHC staining, which detects fusion of a myoblast or muscle cell precursor. This staining can be quantified, for example, by microscopy or flow cytometry.

HAPs that increase muscle or connective tissue cell precursor proliferation and/or differentiation are useful in methods of treating muscle or connective tissue disorders. These disorders can arise from the normal aging process, injury related to trauma or physical exertion, genetic predispositions, or incident to other disease states.

Heparin-associated binding polypeptides that are useful for increasing muscle cell precursor differentiation or proliferation are described herein, and in certain embodiments comprise Vitronectin (VTN), Stanniocalcin-2 (STC2), Periostin (POSTN), Agrin (AGRN), Fibroblast growth factor (FGF17, also known as Fibroblast growth factor 13 or FGF13), Thrombospondin 2 (THBS2), follistatin (FST), Thrombospondin 4 (THBS4), Thrombospondin 1 (THBS1), Insulin-like growth factor 2 (IGF2), Bone morphogenic protein 7 (BMP7), or Interleukin 15 (IL-15), or any combination thereof. In certain embodiments, any one, two, three, four, or five of VTN, STC2, AGRN, THBS2, or FST are present in a composition useful for increasing muscle cell precursor proliferation or muscle cell differentiation. In certain embodiments, any one, two, three, four, five, six, seven, or eight of VTN, POSTN, FGF17, THBS2, THBS1, IL-15, IGF2, and THBS4 are present in a composition useful for increasing muscle cell precursor proliferation or muscle cell differentiation.

In certain embodiments, a heparin-associated binding polypeptide composition may comprise Vitronectin (VTN). VTN may be further included in the composition with any one, two, three, four, five, six, seven, eight, nine, or all polypeptides selected from STC2, AGRN, POSTN, FGF17, THBS2, FST, THBS1, IL-15, IGF2, and THBS4. The composition may comprise VTN and STC2. The composition may comprise VTN and AGRN. The composition may comprise VTN and THBS2. The composition may comprise VTN and FST. The composition may comprise VTN and POSTN. The composition may comprise VTN and FGF17. The composition may comprise VTN and THBS4. The composition may comprise VTN and THBS1. The composition may comprise VTN and IGF2. The composition may comprise VTN and IL-15. Human VTN is disclosed in HAPs ID NO: 1. The VTN of the heparin-associated binding polypeptide composition may comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to HAPs ID NO: 1 or amino acids 20-478 of HAPs ID NO: 1. The VTN polypeptide lacks a secretory leader sequence, e.g., amino acids 1-19 of HAPs ID NO: 1. The VTN polypeptide is modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids from the N-terminal or C-terminal end of the polypeptide, including increments therein. The VTN polypeptide may comprise one or more additional modifications to increase stability. The VTN polypeptide is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP is fused or conjugated to another protein to increase stability and or bioavailability. The VTN polypeptide is fused with an Fc region of an immunoglobulin or with serum albumin. The VTN polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from STC2, AGRN, THBS2, THBS1, THBS4, FGF17, POSTN, IGF2, IL-15 and FST. The VTN polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, and THBS4. The VTN polypeptide is present in a concatemer with one, two, three, four, or more distinct VTN polypeptides. The VTN polypeptide is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA). In some cases, the VTN polypeptide is prepared recombinantly in an expression system (e.g., bacteria, yeast, mammalian, insect). In some cases, the VTN polypeptide is prepared by chemical synthesis.

In certain embodiments, a heparin-associated binding polypeptide composition may comprise Periostin (POSTN). POSTN may be further included in the composition with any one, two, three, four, five, six, or all polypeptides selected from VTN, FGF17, THBS2, THBS1, IGF2, IL-15, and THBS4. The composition may comprise POSTN and VTN. The composition may comprise POSTN and FGF17. The composition may comprise POSTN and THBS2. The composition may comprise POSTN and THBS4. The composition may comprise POSTN and THBS1. The composition may comprise POSTN and IGF2. The composition may comprise POSTN and IL-15. Human POSTN is disclosed in HAPs ID NO: 6. The POSTN of the heparin-associated binding polypeptide composition may comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to HAPs ID NO: 6 or amino acids 22-836 of HAPs ID NO: 6. The POSTN polypeptide lacks a secretory leader sequence, e.g., amino acids 1-21 of HAPs ID NO: 6. The POSTN polypeptide is modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids from the N-terminal or C-terminal end of the polypeptide, including increments therein. The POSTN polypeptide may comprise one or more additional modifications to increase stability. The POSTN polypeptide is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP is fused or conjugated to another protein to increase stability and or bioavailability. The POSTN polypeptide is fused with an Fc region of an immunoglobulin or with serum albumin. The POSTN polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from VTN, FGF17, THBS2, THBS1, IGF2, IL-15, BMP7, and THBS4. The POSTN polypeptide is present in a concatemer with one, two, three, four, or more distinct POSTN polypeptides. The POSTN polypeptide is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA). In some cases, the POSTN polypeptide is prepared recombinantly in an expression system (e.g., bacteria, yeast, mammalian, insect). In some cases, the POSTN polypeptide is prepared by chemical synthesis.

In certain embodiments, a heparin-associated binding polypeptide composition may comprise Fibroblast growth factor (FGF17). FGF17 may be further included in the composition with any one, two, three, four, five, six, seven, or all polypeptides selected from VTN, POSTN, THBS2, THBS1, IL-15, IGF2, BMP7, and THBS4. The composition may comprise FGF17 and VTN. The composition may comprise FGF17 and POSTN. The composition may comprise FGF17 and THBS2. The composition may comprise FGF17 and THBS4. The composition may comprise FGF17 and THBS1. The composition may comprise FGF17 and IGF2. The composition may comprise FGF17 and IL-15. Human FGF17 is disclosed in HAPs ID NO: 7. The FGF17 of the heparin-associated binding polypeptide composition may comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to HAPs ID NO: 7, or amino acids 23-216 of HAPs ID NO: 7. The FGF17 polypeptide lacks a secretory leader sequence, e.g., amino acids 1-22 of HAPs ID NO: 7. The FGF17 polypeptide is modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids from the N-terminal or C-terminal end of the polypeptide, including increments therein. The FGF17 polypeptide may comprise one or more additional modifications to increase stability. The FGF17 polypeptide is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP is fused or conjugated to another protein to increase stability and or bioavailability. The FGF17 polypeptide is fused with an Fc region of an immunoglobulin or with serum albumin. The FGF17 polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from VTN, POSTN, THBS2, THBS1, IGF2, IL-15, BMP7, and THBS4. The FGF17 polypeptide is present in a concatemer with one, two, three, four, or more distinct FGF17 polypeptides. The FGF17 polypeptide is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA). In some cases, the FGF17 polypeptide is prepared recombinantly in an expression system (e.g., bacteria, yeast, mammalian, insect). In some cases, the FGF17 polypeptide is prepared by chemical synthesis.

In certain embodiments, a heparin-associated binding polypeptide composition may comprise Stanniocalcin-2 (STC2). STC-2 may be further included in the composition with any one, two, three, four or more polypeptides selected from VTN, AGRN, THBS2, THBS1, THBS4, POSTN, FGF17, IGF2, IL-15, BMP7, and FST. The composition may comprise STC2 and VTN. The composition may comprise STC2 and AGRN. The composition may comprise STC2 and THBS2. The composition may comprise STC2 and FST. Human STC2 is disclosed in HAPs ID NO: 2. The STC2 of the heparin-associated binding polypeptide composition may comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to HAPs ID NO: 2. The STC2 polypeptide lacks a secretory leader sequence. The STC2 polypeptide is modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids from the N-terminal or C-terminal end of the polypeptide, including increments therein. The STC2 polypeptide may comprise one or more additional modifications to increase stability. The STC2 polypeptide is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP is fused or conjugated to another protein to increase stability and or bioavailability. The STC2 polypeptide is fused with an Fc region of an immunoglobulin or with serum albumin. The STC2 polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from VTN, AGRN, THBS2, THBS1, THBS4, FGF17, POSTN, IGF2, BMP7, IL15, and FST. The STC2 polypeptide is present in a concatemer with one, two, three, four, or more distinct STC2 polypeptides. The STC2 polypeptide is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA). In some cases, the STC-2 polypeptide is prepared recombinantly in an expression system (e.g., bacteria, yeast, mammalian, insect). In some cases, the STC-2 polypeptide is prepared by chemical synthesis.

In certain embodiments, a heparin-associated binding polypeptide composition may comprise Agrin (AGRN). AGRN may be further included in the composition with any one, two, three, four or more polypeptides selected from VTN, STC2, THBS2, THBS1, THBS4, FGF17, POSTN, IGF2, IL-15, BMP7, and FST. The composition may comprise AGRN and VTN. The composition may comprise AGRN and STC2. The composition may comprise AGRN and THBS2. The composition may comprise AGRN and FST. Human AGRN is disclosed in HAPs ID NO: 3. The AGRN of the heparin-associated binding polypeptide composition may comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to HAPs ID NO: 3. The AGRN polypeptide lacks a secretory leader sequence. The AGRN polypeptide is modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids from the N-terminal or C-terminal end of the polypeptide, including increments therein. The AGRN polypeptide may comprise one or more additional modifications to increase stability. The AGRN polypeptide is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP is fused or conjugated to another protein to increase stability and or bioavailability. The AGRN polypeptide is fused with an Fc region of an immunoglobulin or with serum albumin. The AGRN polypeptide is present in a concatemer with one, two, three, or four other distinct polypeptides selected from VTN, STC2, THBS2, THBS1, THBS4, FGF17, POSTN, IGF2, IL-15, and FST. The AGRN polypeptide is present in a concatemer with one, two, three, four, or more distinct AGRN polypeptides. The AGRN polypeptide is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA). In some cases, the AGRN polypeptide is prepared recombinantly in an expression system (e.g., bacteria, yeast, mammalian, insect). In some cases, the AGRN polypeptide is prepared by chemical synthesis.

In certain embodiments, a heparin-associated binding polypeptide composition may comprise Thrombospondin 2 (THBS2). THBS2 may be further included in the composition with any one, two, three, four, five, six or all polypeptides selected from VTN, STC2, AGRN, THBS1, IL-15, IGF2, and FST. The composition may comprise THBS2 and VTN. The composition may comprise THBS2 and STC2. The composition may comprise THBS2 and AGRN. The composition may comprise THBS2 and FST. The composition may comprise AGRN and FST. The composition may comprise THBS2 and THBS1. The composition may comprise THBS2 and IGF2. The composition may comprise THBS2 and IL-15. THBS2 may be further included in the composition with any one, two, three, four, five, six, or all polypeptides selected from VTN, POSTN, FGF17, THBS1, IL-15, IGF2, and THBS4. The composition may comprise THBS2 and VTN. The composition may comprise THBS2 and POSTN. The composition may comprise THBS2 and FGF17. The composition may comprise THBS2 and THBS4. The composition may comprise FGF17 and THBS4. Human THBS2 is disclosed in HAPs ID NO: 4. The THBS2 of the heparin-associated binding polypeptide composition may comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to HAPs ID NO: 4 or amino acids 19-1,172 of HAPs ID NO: 4. The THBS2 polypeptide lacks a secretory leader sequence, e.g., amino acids 1-18 of HAPs ID NO: 4. The THBS2 polypeptide is modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids from the N-terminal or C-terminal end of the polypeptide, including increments therein. The THBS2 polypeptide may comprise one or more additional modifications to increase stability. The THBS2 polypeptide is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP is fused or conjugated to another protein to increase stability and or bioavailability. The THBS2 polypeptide is fused with an Fc region of an immunoglobulin or with serum albumin. The THBS2 polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from VTN, STC2, AGRN, THBS1, THBS4, FGF17, POSTN, IGF2, IL-15, and FST. The THBS2 polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from VTN, POSTN, THBS1, IGF2, IL-15, FGF17, and THBS4. The THBS2 polypeptide is present in a concatemer with one, two, three, four, or more distinct THBS2 polypeptides. The THBS2 polypeptide is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA). In some cases, the THBS2 polypeptide is prepared recombinantly in an expression system (e.g., bacteria, yeast, mammalian, insect). In some cases, the THBS2 polypeptide is prepared by chemical synthesis.

In certain embodiments, a heparin-associated binding polypeptide composition may comprise Thrombospondin 4 (THBS4). THBS4 may be further included in the composition with any one, two, three, four, five, six or all polypeptides selected from VTN, POSTN, FGF17, THBS1, IL-15, IGF2, and THBS2. The composition may comprise THBS4 and VTN. The composition may comprise THBS4 and POSTN. The composition may comprise THBS4 and FGF17. The composition may comprise THBS4 and THBS2. The composition may comprise THBS4 and THBS1. The composition may comprise THBS4 and IL-15. The composition may comprise THBS4 and IGF2. Human THBS4 is disclosed in HAPs ID NO: 8. The THBS4 of the heparin-associated binding polypeptide composition may comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to HAPs ID NO: 8 or amino acids 27-961 of HAPs ID NO: 8. The THBS4 polypeptide lacks a secretory leader sequence, e.g., amino acids 1-26 of HAPs ID NO: 8. The THBS4 polypeptide is modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids from the N-terminal or C-terminal end of the polypeptide, including increments therein. The THBS4 polypeptide may comprise one or more additional modifications to increase stability. The THBS4 polypeptide is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP is fused or conjugated to another protein to increase stability and or bioavailability. The THBS4 polypeptide is fused with an Fc region of an immunoglobulin or with serum albumin. The THBS4 polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from VTN, POSTN, FGF17, THBS1, IGF2, IL-15, and THBS2. The THBS4 polypeptide is present in a concatemer with one, two, three, four, or more distinct THBS4 polypeptides. The THBS4 polypeptide is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA). In some cases, the THBS4 polypeptide is prepared recombinantly in an expression system (e.g., bacteria, yeast, mammalian, insect). In some cases, the THBS4 polypeptide is prepared by chemical synthesis.

In certain embodiments, a heparin-associated binding polypeptide composition may comprise follistatin (FST). FST may be further included in the composition with any one, two, three, four or more polypeptides selected from VTN, STC2, AGRN, THBS1, THBS4, FGF17, POSTN, IGF2, IL-15, and THBS2. The composition may comprise FST and VTN. The composition may comprise FST and STC2. The composition may comprise FST and AGRN. The composition may comprise FST and THBS2. Human FST is disclosed in HAPs ID NO: 5. The FST of the heparin-associated binding polypeptide composition may comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to HAPs ID NO: 5. The FST polypeptide lacks a secretory leader sequence. The FST polypeptide is modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids from the N-terminal or C-terminal end of the polypeptide, including increments therein. The FST polypeptide may comprise one or more additional modifications to increase stability. The FST polypeptide is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP is fused or conjugated to another protein to increase stability and or bioavailability. The FST polypeptide is fused with an Fc region of an immunoglobulin or with serum albumin. The FST polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from VTN, STC2, AGRN, THBS1, THBS4, FGF17, POSTN, IGF2, IL-15, BMP7, and THBS2. The FST polypeptide is present in a concatemer with one, two, three, four, or more distinct FST polypeptides. The FST polypeptide is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA).

In certain embodiments, a heparin-associated binding polypeptide composition may comprise Thrombospondin 1 (THBS1). THSB1 may be further included in the composition with any one, two, three, four, five, six, seven, eight or all polypeptides selected from VTN, STC2, AGRN, THBS4, FGF17, POSTN, IGF2, IL-15, BMP7, and THBS2. The composition may comprise THSB1 and VTN. The composition may comprise THSB1 and STC2. The composition may comprise THSB1 and AGRN. The composition may comprise THSB1 and THBS2. The composition may comprise THSB1 and THBS4. The composition may comprise THSB1 and FGF17. The composition may comprise THSB1 and POSTN. The composition may comprise THSB1 and IGF2. The composition may comprise THSB1 and IL-15. Human THSB1 is disclosed in HAPs ID NO: 9. The THSB1 of the heparin-associated binding polypeptide composition may comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to HAPs ID NO: 9 or amino acids 19-1170 of HAPs ID NO: 9. The THSB1 polypeptide lacks a secretory leader sequence. The THSB1 polypeptide is modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids from the N-terminal or C-terminal end of the polypeptide, including increments therein. The THSB1 polypeptide may comprise one or more additional modifications to increase stability. The THSB1 polypeptide is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP is fused or conjugated to another protein to increase stability and or bioavailability. The THSB1 polypeptide is fused with an Fc region of an immunoglobulin or with serum albumin. The THSB1 polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from VTN, STC2, AGRN, THBS4, FGF17, POSTN, IGF2, IL-15, BMP7, and THBS2. The THSB1 polypeptide is present in a concatemer with one, two, three, four, or more distinct THSB1 polypeptides. The THSB1 polypeptide is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA). In some cases, the THBS1 polypeptide is prepared recombinantly in an expression system (e.g., bacteria, yeast, mammalian, insect). In some cases, the THBS1 polypeptide is prepared by chemical synthesis.

In certain embodiments, a heparin-associated binding polypeptide composition may comprise Interleukin-15 (IL-15). IL-15 may be further included in the composition with any one, two, three, four or more polypeptides selected from VTN, STC2, AGRN, THBS1, THBS4, IGF2, POSTN, FGF17, BMP7, and THBS2. The composition may comprise IL-15 and VTN. The composition may comprise IL-15 and STC2. The composition may comprise IL-15 and AGRN. The composition may comprise IL-15 and THBS2. The composition may comprise IL-15 and THBS1. The composition may comprise IL-15 and THBS4. The composition may comprise IL-15 and IGF2. The composition may comprise IL-15 and POSTN. The composition may comprise IL-15 and FGF17. The composition may comprise IL-15 and THBS1. Human IL-15 is disclosed in HAPs ID NO: 10. The IL-15 of the heparin-associated binding polypeptide composition may comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to HAPs ID NO: 10 or amino acids 49-162 of HAPs ID NO: 10. The IL-15 polypeptide lacks a secretory leader sequence. The IL-15 polypeptide is modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids from the N-terminal or C-terminal end of the polypeptide, including increments therein. The IL-15 polypeptide may comprise one or more additional modifications to increase stability. The IL-15 polypeptide is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP is fused or conjugated to another protein to increase stability and or bioavailability. The IL-15 polypeptide is fused with an Fc region of an immunoglobulin or with serum albumin. The IL-15 polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from VTN, STC2, AGRN, THBS1, THBS4, FGF17, POSTN, IGF2, BMP7, and THBS2. The IL-15 polypeptide is present in a concatemer with one, two, three, four, or more distinct IL-15 polypeptides. The IL-15 polypeptide is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA). In some cases, the IL-15 polypeptide is prepared recombinantly in an expression system (e.g., bacteria, yeast, mammalian, insect). In some cases, the IL-15 polypeptide is prepared by chemical synthesis.

In certain embodiments, a heparin-associated binding polypeptide composition may comprise Insulin-like growth factor 2 (IGF2). IGF2 may be further included in the composition with any one, two, three, four or more polypeptides selected from VTN, STC2, AGRN, THBS1, THBS4, POSTN, FGF17, IL-15, BMP7, and THBS2. The composition may comprise IGF2 and VTN. The composition may comprise IGF2 and STC2. The composition may comprise IGF2 and AGRN. The composition may comprise IGF2 and THBS2. The composition may comprise IGF2 and THBS1. The composition may comprise IGF2 and THBS4. The composition may comprise IGF2 and IL-15. The composition may comprise IGF2 and POSTN. The composition may comprise IGF2 and FGF17. Human IGF2 is disclosed in HAPs ID NO: 11. The IGF2 of the heparin-associated binding polypeptide composition may comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to HAPs ID NO: 11 or amino acids 25-91 of HAPs ID NO: 11. The IGF2 polypeptide lacks a secretory leader sequence. The IGF2 polypeptide is modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids from the N-terminal or C-terminal end of the polypeptide, including increments therein. The IGF2 polypeptide may comprise one or more additional modifications to increase stability. The IGF2 polypeptide is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP is fused or conjugated to another protein to increase stability and or bioavailability. The IGF2 polypeptide is fused with an Fc region of an immunoglobulin or with serum albumin. The IGF2 polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from VTN, STC2, AGRN, THBS1, THBS4, FGF17, POSTN, IL-15, BMP7, and THBS2. The IGF2 polypeptide is present in a concatemer with one, two, three, four, or more distinct IGF2 polypeptides. The IGF2 polypeptide is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA). In some cases, the IGF2 polypeptide is prepared recombinantly in an expression system (e.g., bacteria, yeast, mammalian, insect). In some cases, the IGF2 polypeptide is prepared by chemical synthesis.

In certain embodiments, a heparin-associated binding polypeptide composition may comprise Bone Morphogenic Protein 7 (BMP7). BMP7 may be further included in the composition with any one, two, three, four or more polypeptides selected from VTN, STC2, AGRN, THBS1, THBS4, POSTN, FGF17, IL-15, IGF2, and THBS2. The composition may comprise IGF2 or FGF-17. The composition may comprise IGF2 and STC2. The composition may comprise IGF2 and AGRN. The composition may comprise IGF2 and THBS2. The composition may comprise IGF2 and THBS1. The composition may comprise IGF2 and THBS4. The composition may comprise IGF2 and IL-15. The composition may comprise IGF2 and POSTN. The composition may comprise IGF2 and FGF17. Human BMP7 is disclosed in HAPs ID NO: 72. The BMP7 of the heparin-associated binding polypeptide composition may comprise an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to HAPs ID NO: 11 or amino acids 25-91 of HAPs ID NO: 11. The BMP7 polypeptide lacks a secretory leader sequence. The BMP7 polypeptide is modified by a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids from the N-terminal or C-terminal end of the polypeptide, including increments therein. The BMP7 polypeptide may comprise one or more additional modifications to increase stability. The BMP7 polypeptide is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. The HAP is fused or conjugated to another protein to increase stability and or bioavailability. The BMP7 polypeptide is fused with an Fc region of an immunoglobulin or with serum albumin. The BMP7 polypeptide is present in a concatemer with one, two, three, four or more distinct polypeptides selected from VTN, STC2, AGRN, THBS1, THBS4, FGF17, POSTN, IL-15, IGF2, and THBS2. The BMP7 polypeptide is present in a concatemer with one, two, three, four, or more distinct BMP7 polypeptides. The BMP7 polypeptide is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA). In some cases, the BMP7 polypeptide is prepared recombinantly in an expression system (e.g., bacteria, yeast, mammalian, insect). In some cases, the BMP7 polypeptide is prepared by chemical synthesis.

The heparin-associated binding polypeptide composition may comprise any two polypeptides selected from VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, BMP7, and THBS4. The composition may comprise THBS1. The composition may comprise THBS2. The composition may comprise THBS4. The composition may comprise FGF17. The composition may comprise VTN. The composition may comprise POSTN. The composition may comprise IGF2. The composition may comprise IL-15. The composition may comprise VTN and POSTN. The composition may comprise VTN and FGF17. The composition may comprise VTN and THBS2. The composition may comprise VTN and THBS1. The composition may comprise VTN and IGF2. The composition may comprise VTN and IL-15. The composition may comprise VTN and THBS4. The composition may comprise POSTN and FGF17. The composition may comprise POSTN and THBS2. The composition may comprise POSTN and THBS1. The composition may comprise POSTN and IGF2. The composition may comprise POSTN and IL-15. The composition may comprise POSTN and THBS4. The composition may comprise FGF17 and THBS2. The composition may comprise FGF17 and THBS1. The composition may comprise FGF17 and IGF2. The composition may comprise FGF17 and IL-15. The composition may comprise FGF17 and THBS4. The composition may comprise THBS2 and THBS1. The composition may comprise THBS2 and IGF2. The composition may comprise THBS2 and IL-15. The composition may comprise THBS2 and THBS4. The composition may comprise THBS1 and IGF2. The composition may comprise THBS1 and IL-15. The composition may comprise THBS1 and THBS4. The composition may comprise IGF2 and IL-15. The composition may comprise IGF2 and THBS4. The composition may comprise IL-15 and THBS4.

The heparin-associated binding polypeptide composition comprising any two polypeptides selected from VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, BMP7, and THBS4, may comprise one or more additional modifications to increase stability. In certain embodiments, one or more of the HAPs is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. In certain embodiments, one or more of the HAPs is fused or conjugated to another protein to increase stability and or bioavailability. In certain embodiments, one or more of the HAPs is fused with an Fc region of an immunoglobulin or with serum albumin. In certain embodiments, one or more of the HAPs is present in a concatemer with one, two, three, or four other distinct polypeptides selected from THBS2, VTN, POSTN, FGF17, THBS1, IGF2, IL-15, BMP7, and THBS4. In certain embodiments, one or more of the HAPs is present in a concatemer with one, two, three, four, or more distinct polypeptides. In certain embodiments, one or more of the HAPs is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA).

The heparin-associated binding polypeptide composition may comprise any three polypeptides selected from VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, BMP7, and THBS4. The composition may comprise THBS1. The composition may comprise THBS2. The composition may comprise THBS4. The composition may comprise FGF17. The composition may comprise VTN. The composition may comprise POSTN. The composition may comprise IGF2. The composition may comprise IL-15. The composition may comprise IGF2, THBS2, and THBS4. The composition may comprise IL-15, THBS2, and THBS4. The composition may comprise THBS2 and THBS4. The composition may comprise THBS2, THBS4, and VTN. The composition may comprise THBS2, THBS4, and ANOS1. The composition may comprise THBS2, THBS4, and IL-15. The composition may comprise THBS2, THBS4, and IGF2. The composition may comprise THBS1 and FGF17. The composition may comprise THBS2 and VTN. The composition may comprise THBS1 and VTN. The composition may comprise THBS1 and THBS2. The composition may comprise THBS2 and FGF17. The composition may comprise THBS1 and THBS4. The composition may comprise VTN and FGF17. The composition may comprise THBS4 and VTN. The composition may comprise THBS4 and FGF17.

The composition may comprise VTN, POSTN, and FGF17. The composition may comprise VTN, POSTN, and THBS2. The composition may comprise VTN, POSTN, and FGF17. The composition may comprise VTN, POSTN, THBS2. The composition may comprise VTN, POSTN, and THBS1. The composition may comprise VTN, POSTN, IGF2. The composition may comprise VTN, POSTN, and IL-15. The composition may comprise VTN, POSTN, and THBS4.

The composition may comprise VTN, FGF17, and THBS2. The composition may comprise VTN, FGF17, and POSTN. The composition may comprise VTN, FGF17, and THBS2. The composition may comprise VTN, FGF17, and THBS1. The composition may comprise VTN, FGF17, and IGF2. The composition may comprise VTN, FGF17, and IL-15. The composition may comprise VTN, FGF17, and THBS4.

The composition may comprise VTN, THBS2, and POSTN. The composition may comprise VTN, THBS2, and FGF17. The composition may comprise VTN, THBS2, and THBS1. The composition may comprise VTN, THBS2, and IGF2. The composition may comprise VTN, THBS2, and IL-15. The composition may comprise VTN, THBS2, and THBS4.

The composition may comprise VTN, THBS1, and POSTN. The composition may comprise VTN, THBS1, and FGF17. The composition may comprise VTN, THBS1, and THBS2. The composition may comprise VTN, THBS1, and IGF2. The composition may comprise VTN, THBS1, and IL-15. The composition may comprise VTN, THBS1, and THBS4.

The composition may comprise VTN, IGF2, and POSTN. The composition may comprise VTN, IGF2, and FGF17. The composition may comprise VTN, IGF2, and THBS2. The composition may comprise VTN, IGF2, and THBS1. The composition may comprise VTN, IGF2, and IL-15. The composition may comprise VTN, IGF2, and THBS4.

The composition may comprise VTN, IL-15, and POSTN. The composition may comprise VTN, IL-15, and FGF17. The composition may comprise VTN, IL-15, and THBS2. The composition may comprise VTN, IL-15, and THBS1. The composition may comprise VTN, IL-15, and IGF2. The composition may comprise VTN, IL-15, and THBS4.

The composition may comprise VTN, TBHS4, and POSTN. The composition may comprise VTN, TBHS4, and FGF17. The composition may comprise VTN, TBHS4, and THBS2. The composition may comprise VTN, TBHS4, and THBS1. The composition may comprise VTN, TBHS4, and IGF2. The composition may comprise VTN, TBHS4, and IL-15.

The composition may comprise POSTN, FGF17, and VTN. The composition may comprise POSTN, FGF17, and TBHS2. The composition may comprise POSTN, FGF17, and THBS1. The composition may comprise POSTN, FGF17, and IGF2. The composition may comprise POSTN, FGF17, and IL-15. The composition may comprise POSTN, FGF17, and THBS4.

The composition may comprise POSTN, THBS2, and VTN. The composition may comprise POSTN, THBS2, and FGF17. The composition may comprise POSTN, THBS2, and THBS1. The composition may comprise POSTN, THBS2, and IGF2. The composition may comprise POSTN, THBS2, and IL-15. The composition may comprise POSTN, THBS2, and THBS4.

The composition may comprise POSTN, THBS1, and VTN. The composition may comprise POSTN, THBS1, and FGF17. The composition may comprise POSTN, THBS1, and THBS2. The composition may comprise POSTN, THBS1, and IGF2. The composition may comprise POSTN, THBS1, and IL-15. The composition may comprise POSTN, THBS1, and THBS4.

The composition may comprise POSTN, IGF2, and VTN. The composition may comprise POSTN, IGF2, and FGF17. The composition may comprise POSTN, IGF2, and THBS2. The composition may comprise POSTN, IGF2, and THBS1. The composition may comprise POSTN, IGF2, and IL-15. The composition may comprise POSTN, IGF2, and THBS4.

The composition may comprise POSTN, IL-15, and VTN. The composition may comprise POSTN, IL-15, and FGF17. The composition may comprise POSTN, IL-15, and THBS2. The composition may comprise POSTN, IL-15, and THBS1. The composition may comprise POSTN, IL-15, and IGF2. The composition may comprise POSTN, IL-15, and THBS4.

The composition may comprise POSTN, THBS4, and VTN. The composition may comprise POSTN, THBS4, and FGF17. The composition may comprise POSTN, THBS4, and THBS2. The composition may comprise POSTN, THBS4, and THBS1. The composition may comprise POSTN, THBS4, and IGF2. The composition may comprise POSTN, THBS4, and IL-15.

The composition may comprise FGF17, THBS2, and VTN. The composition may comprise FGF17, THBS2, and POSTN. The composition may comprise FGF17, THBS2, and THBS1. The composition may comprise FGF17, THBS2, and IGF2. The composition may comprise FGF17, THBS2, and IL-15. The composition may comprise FGF17, THBS2, and THBS4.

The composition may comprise FGF17, THBS1, and VTN. The composition may comprise FGF17, THBS1, and POSTN. The composition may comprise FGF17, THBS1, and THBS2. The composition may comprise FGF17, THBS1, and IGF2. The composition may comprise FGF17, THBS1, and IL-15. The composition may comprise FGF17, THBS1, and THBS4.

The composition may comprise FGF17, IGF2, and VTN. The composition may comprise FGF17, IGF2, and POSTN. The composition may comprise FGF17, IGF2, and THBS2. The composition may comprise FGF17, IGF2, and THBS1. The composition may comprise FGF17, IGF2, and IL-15. The composition may comprise FGF17, IGF2, and THBS4.

The composition may comprise FGF17, IL-15, and VTN. The composition may comprise FGF17, IL-15, and POSTN. The composition may comprise FGF17, IL-15, and THBS2. The composition may comprise FGF17, IL-15, and THBS1. The composition may comprise FGF17, IL-15, and IGF2. The composition may comprise FGF17, IL-15, and THBS4.

The composition may comprise FGF17, THBS4, and VTN. The composition may comprise FGF17, THBS4, and POSTN. The composition may comprise FGF17, THBS4, and THBS2. The composition may comprise FGF17, THBS4, and THBS1. The composition may comprise FGF17, THBS4, and IGF2. The composition may comprise FGF17, THBS4, and IL-15.

The composition may comprise THBS2, THBS1, and VTN. The composition may comprise THBS2, THBS1, and POSTN. The composition may comprise THBS2, THBS1, and FGF17. The composition may comprise THBS2, THBS1, and IGF2. The composition may comprise THBS2, THBS1, and IL-15. The composition may comprise THBS2, THBS1, and THBS4.

The composition may comprise THBS2, IGF2, and VTN. The composition may comprise THBS2, IGF2, and POSTN. The composition may comprise THBS2, IGF2, and FGF17. The composition may comprise THBS2, IGF2, and THBS1. The composition may comprise THBS2, IGF2, and IL-15. The composition may comprise THBS2, IGF2, and THBS4.

The composition may comprise THBS2, IL-15, and VTN. The composition may comprise THBS2, IL-15, and POSTN. The composition may comprise THBS2, IL-15, and FGF17. The composition may comprise THBS2, IL-15, and THBS1. The composition may comprise THBS2, IL-15, and IGF2. The composition may comprise THBS2, IL-15, and THBS4.

The composition may comprise THBS2, THBS4, and VTN. The composition may comprise THBS2, THBS4, and POSTN. The composition may comprise THBS2, THBS4, and FGF17. The composition may comprise THBS2, THBS4, and THBS1. The composition may comprise THBS2, THBS4, and IGF2. The composition may comprise THBS2, THBS4, and IL-15.

The composition may comprise THBS1, IGF2, and VTN. The composition may comprise THBS1, IGF2, and POSTN. The composition may comprise THBS1, IGF2, and FGF17. The composition may comprise THBS1, IGF2, and THBS2. The composition may comprise THBS1, IGF2, and IL-15. The composition may comprise THBS1, IGF2, and THBS4.

The composition may comprise THBS1, IL-15, and VTN. The composition may comprise THBS1, IL-15, and POSTN. The composition may comprise THBS1, IL-15, and FGF17. The composition may comprise THBS1, IL-15, and THBS2. The composition may comprise THBS1, IL-15, and IGF2. The composition may comprise THBS1, IL-15, and THBS4.

The composition may comprise THBS1, and THBS4, and VTN. The composition may comprise THBS1, and THBS4, and POSTN. The composition may comprise THBS1, and THBS4, and FGF17. The composition may comprise THBS1, and THBS4, and THBS2. The composition may comprise THBS1, and THBS4, and IGF2. The composition may comprise THBS1, and THBS4, and IL-15.

The composition may comprise IGF2, IL-15, and VTN. The composition may comprise IGF2, IL-15, and POSTN. The composition may comprise IGF2, IL-15, and FGF17. The composition may comprise IGF2, IL-15, and THBS2. The composition may comprise IGF2, IL-15, and THBS1. The composition may comprise IGF2, IL-15, and THBS4.

The composition may comprise IGF2, THBS4, and VTN. The composition may comprise IGF2, THBS4, and POSTN. The composition may comprise IGF2, THBS4, and FGF17. The composition may comprise IGF2, THBS4, and THBS2. The composition may comprise IGF2, THBS4, and THBS1. The composition may comprise IGF2, THBS4, and IL-15.

The composition may comprise IL-15, and THBS4, and VTN. The composition may comprise IL-15, and THBS4, and POSTN. The composition may comprise IL-15, and THBS4, and FGF17. The composition may comprise IL-15, and THBS4, and THBS2. The composition may comprise IL-15, and THBS4, and THBS1. The composition may comprise IL-15, and THBS4, and IGF2.

The composition may comprise VTN, POSTN, and FGF17. The composition may comprise VTN, POSTN, and THBS2. The composition may comprise VTN, POSTN, and THBS4. The composition may comprise VTN, FGF17, and POSTN. The composition may comprise VTN, FGF17, and THBS2. The composition may comprise VTN, FGF17, and THBS4. The composition may comprise VTN, THBS2, and POSTN. The composition may comprise VTN, THBS2, and FGF17. The composition may comprise VTN, THBS2, and THBS4. The composition may comprise VTN, THBS4, and POSTN. The composition may comprise VTN, THBS4, and FGF17. The composition may comprise VTN, THBS4, and THBS2. The composition may comprise POSTN, VTN, and THBS4. The composition may comprise POSTN, VTN, and FGF17. The composition may comprise POSTN, VTN, and THBS2. The composition may comprise POSTN, FGF17, and THBS4. The composition may comprise POSTN, FGF17, and VTN. The composition may comprise POSTN, FGF17, and THBS2. The composition may comprise POSTN, THBS2, and THBS4. The composition may comprise POSTN, THBS2, and VTN. The composition may comprise POSTN, THBS2, and FGF17. The composition may comprise POSTN, THBS4, and THBS2. The composition may comprise POSTN, THBS4, and VTN. The composition may comprise POSTN, THBS4, and FGF17. The composition may comprise FGF17, VTN, and THBS2. The composition may comprise FGF17, VTN, and THBS4. The composition may comprise FGF17, VTN, and POSTN. The composition may comprise FGF17, POSTN, and THBS2. The composition may comprise FGF17, POSTN, and THBS4. The composition may comprise FGF17, POSTN, and VTN. The composition may comprise FGF17, THBS2, and POSTN. The composition may comprise FGF17, THBS2, and THBS4. The composition may comprise FGF17, THBS2, and VTN. The composition may comprise FGF17, THBS4, and POSTN. The composition may comprise FGF17, THBS4, and THBS2. The composition may comprise FGF17, THBS4, and VTN. The composition may comprise THBS2, VTN, and POSTN. The composition may comprise THBS2, VTN, and FGF17. The composition may comprise THBS2, VTN, and THBS4. The composition may comprise THBS2, POSTN, and VTN. The composition may comprise THBS2, POSTN, and FGF17. The composition may comprise THBS2, POSTN, and THBS4. The composition may comprise THBS2, FGF17, and VTN. The composition may comprise THBS2, FGF17, and POSTN. The composition may comprise THBS2, FGF17, and THBS4. The composition may comprise THBS2, THBS4, and VTN. The composition may comprise THBS2, THBS4, and POSTN. The composition may comprise THBS2, THBS4, and FGF17. The composition may comprise THBS4, VTN, and THBS2. The composition may comprise THBS4, VTN, and POSTN. The composition may comprise THBS4, VTN, and FGF17. The composition may comprise THBS4, POSTN, and THBS2. The composition may comprise THBS4, POSTN, and VTN. The composition may comprise THBS4, POSTN, and FGF17. The composition may comprise THBS4, FGF17, and THBS2. The composition may comprise THBS4, FGF17, and VTN. The composition may comprise THBS4, FGF17, and POSTN. The composition may comprise THBS4, THBS2, and FGF17. The composition may comprise THBS4, THBS2, and VTN. The composition may comprise THBS4, THBS2, and POSTN.

The heparin-associated binding polypeptide composition comprising any three polypeptides selected from VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, BMP7, and THBS4, may comprise one or more additional modifications to increase stability. In certain embodiments, one or more of the HAPs is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. In certain embodiments, one or more of the HAPs is fused or conjugated to another protein to increase stability and or bioavailability. In certain embodiments, one or more of the HAPs is fused with an Fc region of an immunoglobulin or with serum albumin. In certain embodiments, one or more of the HAPs is present in a concatemer with one, two, three, or four other distinct polypeptides selected from THBS2, VTN, POSTN, FGF17, THBS1, IGF2, IL-15, BMP7, and THBS4. In certain embodiments, one or more of the HAPs is present in a concatemer with one, two, three, four, or more distinct polypeptides. In certain embodiments, one or more of the HAPs is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA).

The heparin-associated binding polypeptide composition may comprise any four polypeptides selected from VTN, POSTN, FGF17, BMP7, THBS1, IGF2, IL-15, THBS2, and THBS4. The composition may comprise BMP7, VTN, POSTN, and FGF17. The composition may comprise BMP7, VTN, POSTN, and THBS2. The composition may comprise BMP7, VTN, POSTN, and THBS4. The composition may comprise BMP7, VTN, FGF17, and POSTN. The composition may comprise BMP7, VTN, FGF17, and THBS2. The composition may comprise BMP7, VTN, FGF17, and THBS4. The composition may comprise BMP7, VTN, THBS2, and POSTN. The composition may comprise BMP7, VTN, THBS2, and FGF17. The composition may comprise BMP7, VTN, THBS2, and THBS4. The composition may comprise BMP7, VTN, THBS4, and POSTN. The composition may comprise BMP7, VTN, THBS4, and FGF17. The composition may comprise BMP7, VTN, THBS4, and THBS2. The composition may comprise BMP7, POSTN, VTN, and THBS4. The composition may comprise BMP7, POSTN, VTN, and FGF17. The composition may comprise BMP7, POSTN, VTN, and THBS2. The composition may comprise BMP7, POSTN, FGF17, and THBS4. The composition may comprise BMP7, POSTN, FGF17, and VTN. The composition may comprise BMP7, POSTN, FGF17, and THBS2. The composition may comprise BMP7, POSTN, THBS2, and THBS4. The composition may comprise BMP7, POSTN, THBS2, and VTN. The composition may comprise BMP7, POSTN, THBS2, and FGF17. The composition may comprise BMP7, POSTN, THBS4, and THBS2. The composition may comprise BMP7, POSTN, THBS4, and VTN. The composition may comprise BMP7, POSTN, THBS4, and FGF17. The composition may comprise BMP7, FGF17, VTN, and THBS2. The composition may comprise BMP7, FGF17, VTN, and THBS4. The composition may comprise BMP7, FGF17, VTN, and POSTN. The composition may comprise BMP7, FGF17, POSTN, and THBS2. The composition may comprise BMP7, FGF17, POSTN, and THBS4. The composition may comprise BMP7, FGF17, POSTN, and VTN. The composition may comprise BMP7, FGF17, THBS2, and POSTN. The composition may comprise BMP7, FGF17, THBS2, and THBS4. The composition may comprise BMP7, FGF17, THBS2, and VTN. The composition may comprise BMP7, FGF17, THBS4, and POSTN. The composition may comprise BMP7, FGF17, THBS4, and THBS2. The composition may comprise BMP7, FGF17, THBS4, and VTN. The composition may comprise BMP7, THBS2, VTN, and POSTN. The composition may comprise BMP7, THBS2, VTN, and FGF17. The composition may comprise BMP7, THBS2, VTN, and THBS4. The composition may comprise BMP7, THBS2, POSTN, and VTN. The composition may comprise BMP7, THBS2, POSTN, and FGF17. The composition may comprise BMP7, THBS2, POSTN, and THBS4. The composition may comprise BMP7, THBS2, FGF17, and VTN. The composition may comprise BMP7, THBS2, FGF17, and POSTN. The composition may comprise BMP7, THBS2, FGF17, and THBS4. The composition may comprise BMP7, THBS2, THBS4, and VTN. The composition may comprise BMP7, THBS2, THBS4, and POSTN. The composition may comprise BMP7, THBS2, THBS4, and FGF17. The composition may comprise BMP7, THBS4, VTN, and THBS2. The composition may comprise BMP7, THBS4, VTN, and POSTN. The composition may comprise BMP7, THBS4, VTN, and FGF17. The composition may comprise BMP7, THBS4, POSTN, and THBS2. The composition may comprise BMP7, THBS4, POSTN, and VTN. The composition may comprise BMP7, THBS4, POSTN, and FGF17. The composition may comprise BMP7, THBS4, FGF17, and THBS2. The composition may comprise BMP7, THBS4, FGF17, and VTN. The composition may comprise BMP7, THBS4, FGF17, and POSTN. The composition may comprise BMP7, THBS4, THBS2, and FGF17. The composition may comprise BMP7, THBS4, THBS2, and VTN. The composition may comprise BMP7, THBS4, THBS2, and POSTN. The composition may comprise VTN, POSTN, FGF17, and THBS2. The composition may comprise VTN, POSTN, FGF17, and THBS4. The composition may comprise VTN, POSTN, THBS2, and FGF17. The composition may comprise VTN, POSTN, THBS2, and THBS4. The composition may comprise VTN, POSTN, THBS4, and FGF17. The composition may comprise VTN, POSTN, THBS4, and THBS2. The composition may comprise VTN, FGF17, POSTN, and THBS4. The composition may comprise VTN, FGF17, POSTN, and THBS2. The composition may comprise VTN, FGF17, THBS2, and THBS4. The composition may comprise VTN, FGF17, THBS2, and POSTN. The composition may comprise VTN, FGF17, THBS4, and THBS2. The composition may comprise VTN, FGF17, THBS4, and POSTN. The composition may comprise VTN, THBS2, POSTN, and FGF17. The composition may comprise VTN, THBS2, POSTN, and THBS4. The composition may comprise VTN, THBS2, FGF17, and POSTN. The composition may comprise VTN, THBS2, FGF17, and THBS4. The composition may comprise VTN, THBS2, THBS4, and POSTN. The composition may comprise VTN, THBS2, THBS4, and FGF17. The composition may comprise VTN, THBS4, POSTN, and THBS2. The composition may comprise VTN, THBS4, POSTN, and FGF17. The composition may comprise VTN, THBS4, FGF17, and THBS2. The composition may comprise VTN, THBS4, FGF17, and POSTN. The composition may comprise VTN, THBS4, THBS2, and FGF17. The composition may comprise VTN, THBS4, THBS2, and POSTN. The composition may comprise POSTN, VTN, THBS4, and FGF17. The composition may comprise POSTN, VTN, THBS4, and THBS2. The composition may comprise POSTN, VTN, FGF17, and THBS4. The composition may comprise POSTN, VTN, FGF17, and THBS2. The composition may comprise POSTN, VTN, THBS2, and THBS4. The composition may comprise POSTN, VTN, THBS2, and FGF17. The composition may comprise POSTN, FGF17, THBS4, and THBS2. The composition may comprise POSTN, FGF17, THBS4, and VTN. The composition may comprise POSTN, FGF17, VTN, and THBS2. The composition may comprise POSTN, FGF17, VTN, and THBS4. The composition may comprise POSTN, FGF17, THBS2, and VTN. The composition may comprise POSTN, FGF17, THBS2, and THBS4. The composition may comprise POSTN, THBS2, THBS4, and VTN. The composition may comprise POSTN, THBS2, THBS4, and FGF17. The composition may comprise POSTN, THBS2, VTN, and THBS4. The composition may comprise POSTN, THBS2, VTN, and FGF17. The composition may comprise POSTN, THBS2, FGF17, and THBS4. The composition may comprise POSTN, THBS2, FGF17, and VTN. The composition may comprise POSTN, THBS4, THBS2, and FGF17. The composition may comprise POSTN, THBS4, THBS2, and VTN. The composition may comprise POSTN, THBS4, VTN, and FGF17. The composition may comprise POSTN, THBS4, VTN, and THBS2. The composition may comprise POSTN, THBS4, FGF17, and VTN. The composition may comprise POSTN, THBS4, FGF17, and THBS2. The composition may comprise FGF17, VTN, THBS2, and THBS4. The composition may comprise FGF17, VTN, THBS2, and POSTN. The composition may comprise FGF17, VTN, THBS4, and THBS2. The composition may comprise FGF17, VTN, THBS4, and POSTN. The composition may comprise FGF17, VTN, POSTN, and THBS2. The composition may comprise FGF17, VTN, POSTN, and THBS4. The composition may comprise FGF17, POSTN, THBS2, and VTN. The composition may comprise FGF17, POSTN, THBS2, and THBS4. The composition may comprise FGF17, POSTN, THBS4, and VTN. The composition may comprise FGF17, POSTN, THBS4, and THBS2. The composition may comprise FGF17, POSTN, VTN, and THBS4. The composition may comprise FGF17, POSTN, VTN, and THBS2. The composition may comprise FGF17, THBS2, POSTN, and THBS4. The composition may comprise FGF17, THBS2, POSTN, and VTN. The composition may comprise FGF17, THBS2, THBS4, and POSTN. The composition may comprise FGF17, THBS2, THBS4, and VTN. The composition may comprise FGF17, THBS2, VTN, and POSTN. The composition may comprise FGF17, THBS2, VTN, and THBS4. The composition may comprise FGF17, THBS4, POSTN, and VTN. The composition may comprise FGF17, THBS4, POSTN, and THBS2. The composition may comprise FGF17, THBS4, THBS2, and VTN. The composition may comprise FGF17, THBS4, THBS2, and POSTN. The composition may comprise FGF17, THBS4, VTN, and THBS2. The composition may comprise FGF17, THBS4, VTN, and POSTN. The composition may comprise THBS2, VTN, POSTN, and FGF17. The composition may comprise THBS2, VTN, POSTN, and THBS4. The composition may comprise THBS2, VTN, FGF17, and POSTN. The composition may comprise THBS2, VTN, FGF17, and THBS4. The composition may comprise THBS2, VTN, THBS4, and POSTN. The composition may comprise THBS2, VTN, THBS4, and FGF17. The composition may comprise THBS2, POSTN, VTN, and THBS4. The composition may comprise THBS2, POSTN, VTN, and FGF17. The composition may comprise THBS2, POSTN, FGF17, and THBS4. The composition may comprise THBS2, POSTN, FGF17, and VTN. The composition may comprise THBS2, POSTN, THBS4, and FGF17. The composition may comprise THBS2, POSTN, THBS4, and VTN. The composition may comprise THBS2, FGF17, VTN, and POSTN. The composition may comprise THBS2, FGF17, VTN, and THBS4. The composition may comprise THBS2, FGF17, POSTN, and VTN. The composition may comprise THBS2, FGF17, POSTN, and THBS4. The composition may comprise THBS2, FGF17, THBS4, and VTN. The composition may comprise THBS2, FGF17, THBS4, and POSTN. The composition may comprise THBS2, THBS4, VTN, and FGF17. The composition may comprise THBS2, THBS4, VTN, and POSTN. The composition may comprise THBS2, THBS4, POSTN, and FGF17. The composition may comprise THBS2, THBS4, POSTN, and VTN. The composition may comprise THBS2, THBS4, FGF17, and POSTN. The composition may comprise THBS2, THBS4, FGF17, and VTN. The composition may comprise THBS4, VTN, THBS2, and POSTN. The composition may comprise THBS4, VTN, THBS2, and FGF17. The composition may comprise THBS4, VTN, POSTN, and THBS2. The composition may comprise THBS4, VTN, POSTN, and FGF17. The composition may comprise THBS4, VTN, FGF17, and THBS2. The composition may comprise THBS4, VTN, FGF17, and POSTN. The composition may comprise THBS4, POSTN, THBS2, and FGF17. The composition may comprise THBS4, POSTN, THBS2, and VTN. The composition may comprise THBS4, POSTN, VTN, and FGF17. The composition may comprise THBS4, POSTN, VTN, and THBS2. The composition may comprise THBS4, POSTN, FGF17, and VTN. The composition may comprise THBS4, POSTN, FGF17, and THBS2. The composition may comprise THBS4, FGF17, THBS2, and VTN. The composition may comprise THBS4, FGF17, THBS2, and POSTN. The composition may comprise THBS4, FGF17, VTN, and THBS2. The composition may comprise THBS4, FGF17, VTN, and POSTN. The composition may comprise THBS4, FGF17, POSTN, and THBS2. The composition may comprise THBS4, FGF17, POSTN, and VTN. The composition may comprise THBS4, THBS2, FGF17, and POSTN. The composition may comprise THBS4, THBS2, FGF17, and VTN. The composition may comprise THBS4, THBS2, VTN, and POSTN. The composition may comprise THBS4, THBS2, VTN, and FGF17. The composition may comprise THBS4, THBS2, POSTN, and VTN. The composition may comprise THBS4, THBS2, POSTN, and FGF17.

The composition may comprise VTN, POSTN, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, POSTN, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, POSTN, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, POSTN, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, POSTN, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, POSTN, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, POSTN, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, POSTN, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise VTN, FGF17, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, FGF17, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, FGF17, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, FGF17, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, FGF17, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, FGF17, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, FGF17, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise VTN, THBS2, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, THBS2, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, THBS2, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, THBS2, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, THBS2, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, THBS2, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise VTN, THBS1, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, THBS1, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, THBS1, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, THBS1, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, THBS1, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, THBS1, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise VTN, IGF2, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, IGF2, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, IGF2, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, IGF2, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, IGF2, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, IGF2, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise VTN, IL-15, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, IL-15, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, IL-15, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, IL-15, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, IL-15, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, IL-15, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise VTN, TBHS4, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, TBHS4, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, TBHS4, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, TBHS4, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, TBHS4, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise VTN, TBHS4, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise POSTN, FGF17, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, FGF17, TBHS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, FGF17, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, FGF17, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, FGF17, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, FGF17, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise POSTN, THBS2, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS2, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS2, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS2, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS2, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS2, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise POSTN, THBS1, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS1, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS1, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS1, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS1, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS1, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise POSTN, IGF2, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, IGF2, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, IGF2, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, IGF2, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, IGF2, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, IGF2, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise POSTN, IL-15, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, IL-15, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, IL-15, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, IL-15, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, IL-15, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, IL-15, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise POSTN, THBS4, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS4, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS4, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS4, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS4, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise POSTN, THBS4, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise FGF17, THBS2, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS2, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS2, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS2, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS2, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS2, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise FGF17, THBS1, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS1, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS1, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS1, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS1, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS1, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise FGF17, IGF2, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, IGF2, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, IGF2, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, IGF2, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, IGF2, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, IGF2, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise FGF17, IL-15, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, IL-15, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, IL-15, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, IL-15, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, IL-15, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, IL-15, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise FGF17, THBS4, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS4, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS4, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS4, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS4, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise FGF17, THBS4, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise THBS2, THBS1, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, THBS1, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, THBS1, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, THBS1, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, THBS1, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, THBS1, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise THBS2, IGF2, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, IGF2, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, IGF2, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, IGF2, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, IGF2, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, IGF2, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise THBS2, IL-15, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, IL-15, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, IL-15, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, IL-15, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, IL-15, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, IL-15, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise THBS2, THBS4, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, THBS4, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, THBS4, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, THBS4, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, THBS4, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS2, THBS4, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise THBS1, IGF2, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, IGF2, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, IGF2, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, IGF2, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, IGF2, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, IGF2, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise THBS1, IL-15, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, IL-15, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, IL-15, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, IL-15, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, IL-15, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, IL-15, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise THBS1, and THBS4, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, THBS4, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, THBS4, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, THBS4, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, THBS4, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise THBS1, THBS4, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise IGF2, IL-15, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IGF2, IL-15, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IGF2, IL-15, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IGF2, IL-15, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IGF2, IL-15, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IGF2, IL-15, THBS4, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise IGF2, THBS4, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IGF2, THBS4, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IGF2, THBS4, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IGF2, THBS4, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IGF2, THBS4, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IGF2, THBS4, IL-15, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The composition may comprise IL-15, THBS4, VTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IL-15, THBS4, POSTN, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IL-15, THBS4, FGF17, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IL-15, THBS4, THBS2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IL-15, THBS4, THBS1, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4. The composition may comprise IL-15, THBS4, IGF2, and a polypeptide comprising BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, or THBS4.

The heparin-associated binding polypeptide composition comprising any four polypeptides selected from BMP7, VTN, POSTN, FGF17, THBS2, THBS1, IGF2, IL-15, and THBS4, may comprise one or more additional modifications to increase stability. In certain embodiments, one or more of the HAPs is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. In certain embodiments, one or more of the HAPs is fused or conjugated to another protein to increase stability and or bioavailability. In certain embodiments, one or more of the HAPs is fused with an Fc region of an immunoglobulin or with serum albumin. In certain embodiments, one or more of the HAPs is present in a concatemer with one, two, three, four or more distinct polypeptides selected from BMP7, THBS2, VTN, POSTN, FGF17, THBS1, IL-15, IGF2, and THBS4. In certain embodiments, one or more of the HAPs is present in a concatemer with one, two, three, four, or more distinct polypeptides. In certain embodiments, one or more of the HAPs is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA).

The heparin-associated binding polypeptide composition may comprise any three polypeptides selected from VTN, STC2, AGRN, THBS2, and FST. The composition may comprise VTN, STC2, and AGRN. The composition may comprise VTN, STC2, and THBS2. The composition may comprise VTN, STC2, and FST. The composition may comprise VTN, AGRN, and STC2. The composition may comprise VTN, AGRN, and THBS2. The composition may comprise VTN, AGRN, and FST. The composition may comprise VTN, THBS2, and STC2. The composition may comprise VTN, THBS2, and AGRN. The composition may comprise VTN, THBS2, and FST. The composition may comprise VTN, FST, and STC2. The composition may comprise VTN, FST, and AGRN. The composition may comprise VTN, FST, and THBS2. The composition may comprise STC2, VTN, and FST. The composition may comprise STC2, VTN, and AGRN. The composition may comprise STC2, VTN, and THBS2. The composition may comprise STC2, AGRN, and FST. The composition may comprise STC2, AGRN, and VTN. The composition may comprise STC2, AGRN, and THBS2. The composition may comprise STC2, THBS2, and FST. The composition may comprise STC2, THBS2, and VTN. The composition may comprise STC2, THBS2, and AGRN. The composition may comprise STC2, FST, and THBS2. The composition may comprise STC2, FST, and VTN. The composition may comprise STC2, FST, and AGRN. The composition may comprise AGRN, VTN, and THBS2. The composition may comprise AGRN, VTN, and FST. The composition may comprise AGRN, VTN, and STC2. The composition may comprise AGRN, STC2, and THBS2. The composition may comprise AGRN, STC2, and FST. The composition may comprise AGRN, STC2, and VTN. The composition may comprise AGRN, THBS2, and STC2. The composition may comprise AGRN, THBS2, and FST. The composition may comprise AGRN, THBS2, and VTN. The composition may comprise AGRN, FST, and STC2. The composition may comprise AGRN, FST, and THBS2. The composition may comprise AGRN, FST, and VTN. The composition may comprise THBS2, VTN, and STC2. The composition may comprise THBS2, VTN, and AGRN. The composition may comprise THBS2, VTN, and FST. The composition may comprise THBS2, STC2, and VTN. The composition may comprise THBS2, STC2, and AGRN. The composition may comprise THBS2, STC2, and FST. The composition may comprise THBS2, AGRN, and VTN. The composition may comprise THBS2, AGRN, and STC2. The composition may comprise THBS2, AGRN, and FST. The composition may comprise THBS2, FST, and VTN. The composition may comprise THBS2, FST, and STC2. The composition may comprise THBS2, FST, and AGRN. The composition may comprise FST, VTN, and THBS2. The composition may comprise FST, VTN, and STC2. The composition may comprise FST, VTN, and AGRN. The composition may comprise FST, STC2, and THBS2. The composition may comprise FST, STC2, and VTN. The composition may comprise FST, STC2, and AGRN. The composition may comprise FST, AGRN, and THBS2. The composition may comprise FST, AGRN, and VTN. The composition may comprise FST, AGRN, and STC2. The composition may comprise FST, THBS2, and AGRN. The composition may comprise FST, THBS2, and VTN. The composition may comprise FST, THBS2, and STC2. In certain embodiments, one or more of the HAPs comprise one or more additional modifications to increase stability. In certain embodiments, one or more of the HAPs is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. In certain embodiments, one or more of the HAP is fused or conjugated to another protein to increase stability and or bioavailability. In certain embodiments, one or more of the HAPs is fused with an Fc region of an immunoglobulin or with serum albumin. In certain embodiments, one or more of the HAPs is present in a concatemer with one, two, three, four or more distinct polypeptides selected from THBS2, VTN, STC2, AGRN, or FST. In certain embodiments, one or more of the HAPs is present in a concatemer with one, two, three, four, or more distinct polypeptides. In certain embodiments, one or more of the HAPs is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA).

The heparin-associated binding polypeptide composition may comprise any four polypeptides selected from VTN, STC2, AGRN, THBS2, and FST. The composition may comprise VTN, STC2, AGRN, and THBS2. The composition may comprise VTN, STC2, AGRN, and FST. The composition may comprise VTN, STC2, THBS2, and AGRN. The composition may comprise VTN, STC2, THBS2, and FST. The composition may comprise VTN, STC2, FST, and AGRN. The composition may comprise VTN, STC2, FST, and THBS2. The composition may comprise VTN, AGRN, STC2, and FST. The composition may comprise VTN, AGRN, STC2, and THBS2. The composition may comprise VTN, AGRN, THBS2, and FST. The composition may comprise VTN, AGRN, THBS2, and STC2. The composition may comprise VTN, AGRN, FST, and THBS2. The composition may comprise VTN, AGRN, FST, and STC2. The composition may comprise VTN, THBS2, STC2, and AGRN. The composition may comprise VTN, THBS2, STC2, and FST. The composition may comprise VTN, THBS2, AGRN, and STC2. The composition may comprise VTN, THBS2, AGRN, and FST. The composition may comprise VTN, THBS2, FST, and STC2. The composition may comprise VTN, THBS2, FST, and AGRN. The composition may comprise VTN, FST, STC2, and THBS2. The composition may comprise VTN, FST, STC2, and AGRN. The composition may comprise VTN, FST, AGRN, and THBS2. The composition may comprise VTN, FST, AGRN, and STC2. The composition may comprise VTN, FST, THBS2, and AGRN. The composition may comprise VTN, FST, THBS2, and STC2. The composition may comprise STC2, VTN, FST, and AGRN. The composition may comprise STC2, VTN, FST, and THBS2. The composition may comprise STC2, VTN, AGRN, and FST. The composition may comprise STC2, VTN, AGRN, and THBS2. The composition may comprise STC2, VTN, THBS2, and FST. The composition may comprise STC2, VTN, THBS2, and AGRN. The composition may comprise STC2, AGRN, FST, and THBS2. The composition may comprise STC2, AGRN, FST, and VTN. The composition may comprise STC2, AGRN, VTN, and THBS2. The composition may comprise STC2, AGRN, VTN, and FST. The composition may comprise STC2, AGRN, THBS2, and VTN. The composition may comprise STC2, AGRN, THBS2, and FST. The composition may comprise STC2, THBS2, FST, and VTN. The composition may comprise STC2, THBS2, FST, and AGRN. The composition may comprise STC2, THBS2, VTN, and FST. The composition may comprise STC2, THBS2, VTN, and AGRN. The composition may comprise STC2, THBS2, AGRN, and FST. The composition may comprise STC2, THBS2, AGRN, and VTN. The composition may comprise STC2, FST, THBS2, and AGRN. The composition may comprise STC2, FST, THBS2, and VTN. The composition may comprise STC2, FST, VTN, and AGRN. The composition may comprise STC2, FST, VTN, and THBS2. The composition may comprise STC2, FST, AGRN, and VTN. The composition may comprise STC2, FST, AGRN, and THBS2. The composition may comprise AGRN, VTN, THBS2, and FST. The composition may comprise AGRN, VTN, THBS2, and STC2. The composition may comprise AGRN, VTN, FST, and THBS2. The composition may comprise AGRN, VTN, FST, and STC2. The composition may comprise AGRN, VTN, STC2, and THBS2. The composition may comprise AGRN, VTN, STC2, and FST. The composition may comprise AGRN, STC2, THBS2, and VTN. The composition may comprise AGRN, STC2, THBS2, and FST. The composition may comprise AGRN, STC2, FST, and VTN. The composition may comprise AGRN, STC2, FST, and THBS2. The composition may comprise AGRN, STC2, VTN, and FST. The composition may comprise AGRN, STC2, VTN, and THBS2. The composition may comprise AGRN, THBS2, STC2, and FST. The composition may comprise AGRN, THBS2, STC2, and VTN. The composition may comprise AGRN, THBS2, FST, and STC2. The composition may comprise AGRN, THBS2, FST, and VTN. The composition may comprise AGRN, THBS2, VTN, and STC2. The composition may comprise AGRN, THBS2, VTN, and FST. The composition may comprise AGRN, FST, STC2, and VTN. The composition may comprise AGRN, FST, STC2, and THBS2. The composition may comprise AGRN, FST, THBS2, and VTN. The composition may comprise AGRN, FST, THBS2, and STC2. The composition may comprise AGRN, FST, VTN, and THBS2. The composition may comprise AGRN, FST, VTN, and STC2. The composition may comprise THBS2, VTN, STC2, and AGRN. The composition may comprise THBS2, VTN, STC2, and FST. The composition may comprise THBS2, VTN, AGRN, and STC2. The composition may comprise THBS2, VTN, AGRN, and FST. The composition may comprise THBS2, VTN, FST, and STC2. The composition may comprise THBS2, VTN, FST, and AGRN. The composition may comprise THBS2, STC2, VTN, and FST. The composition may comprise THBS2, STC2, VTN, and AGRN. The composition may comprise THBS2, STC2, AGRN, and FST. The composition may comprise THBS2, STC2, AGRN, and VTN. The composition may comprise THBS2, STC2, FST, and AGRN. The composition may comprise THBS2, STC2, FST, and VTN. The composition may comprise THBS2, AGRN, VTN, and STC2. The composition may comprise THBS2, AGRN, VTN, and FST. The composition may comprise THBS2, AGRN, STC2, and VTN. The composition may comprise THBS2, AGRN, STC2, and FST. The composition may comprise THBS2, AGRN, FST, and VTN. The composition may comprise THBS2, AGRN, FST, and STC2. The composition may comprise THBS2, FST, VTN, and AGRN. The composition may comprise THBS2, FST, VTN, and STC2. The composition may comprise THBS2, FST, STC2, and AGRN. The composition may comprise THBS2, FST, STC2, and VTN. The composition may comprise THBS2, FST, AGRN, and STC2. The composition may comprise THBS2, FST, AGRN, and VTN. The composition may comprise FST, VTN, THBS2, and STC2. The composition may comprise FST, VTN, THBS2, and AGRN. The composition may comprise FST, VTN, STC2, and THBS2. The composition may comprise FST, VTN, STC2, and AGRN. The composition may comprise FST, VTN, AGRN, and THBS2. The composition may comprise FST, VTN, AGRN, and STC2. The composition may comprise FST, STC2, THBS2, and AGRN. The composition may comprise FST, STC2, THBS2, and VTN. The composition may comprise FST, STC2, VTN, and AGRN. The composition may comprise FST, STC2, VTN, and THBS2. The composition may comprise FST, STC2, AGRN, and VTN. The composition may comprise FST, STC2, AGRN, and THBS2. The composition may comprise FST, AGRN, THBS2, and VTN. The composition may comprise FST, AGRN, THBS2, and STC2. The composition may comprise FST, AGRN, VTN, and THBS2. The composition may comprise FST, AGRN, VTN, and STC2. The composition may comprise FST, AGRN, STC2, and THBS2. The composition may comprise FST, AGRN, STC2, and VTN. The composition may comprise FST, THBS2, AGRN, and STC2. The composition may comprise FST, THBS2, AGRN, and VTN. The composition may comprise FST, THBS2, VTN, and STC2. The composition may comprise FST, THBS2, VTN, and AGRN. The composition may comprise FST, THBS2, STC2, and VTN. The composition may comprise FST, THBS2, STC2, and AGRN. In certain embodiments, one or more of the HAPs comprise one or more additional modifications to increase stability. In certain embodiments, one or more of the HAPs is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. In certain embodiments, one or more of the HAPs is fused or conjugated to another protein to increase stability and or bioavailability. In certain embodiments, one or more of the HAPs is fused with an Fc region of an immunoglobulin or with serum albumin. In certain embodiments, one or more of the HAPs is present in a concatemer with one, two, three, or four other distinct polypeptides selected from THBS2, VTN, STC2, AGRN, or FST. In certain embodiments, one or more of the HAPs is present in a concatemer with one, two, three, four, or more distinct polypeptides. In certain embodiments, one or more of the HAPs is included in a composition comprising a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA).

In some embodiments, a composition herein may comprise polypeptide 1 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 2 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 3 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 4 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 5 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 6 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 7 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 8 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 9 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 10 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 11 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 12 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 13 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 14 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 15 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 16 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 17 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 18 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 19 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 21 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 22 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 23 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 24 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 25 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 26 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 27 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 28 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 29 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 30 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 31 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 32 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 33 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 34 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 35 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 36 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 37 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 38 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 39 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 40 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 41 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 42 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 43 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 44 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 45 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 46 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 47 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 48 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 49 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 50 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 51 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 52 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 53 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 54 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 55 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 56 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 57 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 58 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 59 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 60 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 61 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 62 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 63 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 64 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 65 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 66 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 67 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 68 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 69 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 70 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 71 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 72 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 73 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 74 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 75 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 76 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 77 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 78 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 79 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 80 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 81 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 82 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 83 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 84 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 85 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 86 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 87 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 88 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 89 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 90 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 91 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 92 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 93 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 94 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 95 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 96 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 97 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 98 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 99 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 100 and one or more polypeptides from Table 2.

In some embodiments, a composition herein may comprise polypeptide 101 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 102 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 103 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 104 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 105 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 106 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 107 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 108 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 109 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 110 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 111 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 112 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 113 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 114 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 115 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 116 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 117 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 118 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 119 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 121 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 122 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 123 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 124 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 125 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 126 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 127 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 128 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 129 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 130 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 131 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 132 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 133 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 134 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 135 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 136 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 137 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 138 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 139 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 140 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 141 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 142 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 143 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 144 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 145 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 146 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 147 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 148 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 149 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 150 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 151 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 152 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 153 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 154 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 155 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 156 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 157 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 158 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 159 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 160 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 161 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 162 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 163 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 164 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 165 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 166 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 167 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 168 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 169 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 170 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 171 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 172 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 173 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 174 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 175 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 176 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 177 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 178 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 179 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 180 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 181 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 182 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 183 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 184 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 185 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 186 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 187 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 188 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 189 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 190 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 191 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 192 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 193 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 194 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 195 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 196 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 197 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 198 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 199 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 200 and one or more polypeptides from Table 2.

In some embodiments, a composition herein may comprise polypeptide 201 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 202 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 203 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 204 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 205 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 206 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 207 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 208 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 209 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 210 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 211 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 212 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 213 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 214 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 215 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 216 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 217 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 218 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 219 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 221 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 222 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 223 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 224 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 225 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 226 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 227 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 228 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 229 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 230 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 231 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 232 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 233 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 234 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 235 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 236 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 237 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 238 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 239 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 240 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 241 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 242 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 243 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 244 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 245 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 246 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 247 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 248 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 249 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 250 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 251 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 252 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 253 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 254 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 255 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 256 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 257 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 258 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 259 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 260 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 261 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 262 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 263 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 264 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 265 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 266 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 267 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 268 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 269 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 270 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 271 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 272 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 273 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 274 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 275 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 276 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 277 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 278 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 279 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 280 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 281 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 282 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 283 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 284 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 285 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 286 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 287 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 288 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 289 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 290 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 291 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 292 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 293 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 294 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 295 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 296 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 297 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 298 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 299 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 300 and one or more polypeptides from Table 2.

In some embodiments, a composition herein may comprise polypeptide 401 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 402 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 403 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 404 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 405 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 406 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 407 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 408 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 409 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 410 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 411 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 412 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 413 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 414 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 415 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 416 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 417 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 418 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 419 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 421 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 422 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 423 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 424 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 425 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 426 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 427 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 428 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 429 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 430 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 431 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 432 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 433 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 434 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 435 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 436 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 437 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 438 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 439 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 440 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 441 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 442 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 443 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 444 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 445 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 446 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 447 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 448 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 449 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 450 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 451 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 452 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 453 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 454 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 455 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 456 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 457 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 458 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 459 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 460 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 461 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 462 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 463 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 464 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 465 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 466 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 467 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 468 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 469 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 470 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 471 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 472 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 473 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 474 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 475 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 476 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 477 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 478 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 479 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 480 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 481 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 482 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 483 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 484 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 485 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 486 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 487 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 488 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 489 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 490 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 491 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 492 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 493 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 494 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 495 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 496 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 497 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 498 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 499 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 500 and one or more polypeptides from Table 2.

In some embodiments, a composition herein may comprise polypeptide 501 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 502 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 503 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 504 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 505 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 506 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 507 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 508 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 509 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 510 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 511 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 512 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 513 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 514 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 515 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 516 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 517 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 518 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 519 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 521 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 522 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 523 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 524 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 525 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 526 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 527 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 528 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 529 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 530 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 531 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 532 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 533 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 534 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 535 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 536 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 537 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 538 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 539 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 540 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 541 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 542 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 543 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 544 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 545 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 546 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 547 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 548 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 549 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 550 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 551 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 552 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 553 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 554 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 555 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 556 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 557 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 558 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 559 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 560 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 561 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 562 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 563 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 564 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 565 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 566 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 567 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 568 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 569 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 570 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 571 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 572 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 573 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 574 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 575 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 576 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 577 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 578 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 579 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 580 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 581 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 582 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 583 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 584 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 585 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 586 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 587 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 588 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 589 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 590 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 591 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 592 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 593 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 594 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 595 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 596 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 597 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 598 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 599 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 600 and one or more polypeptides from Table 2.

In some embodiments, a composition herein may comprise polypeptide 701 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 702 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 703 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 704 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 705 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 706 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 707 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 708 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 709 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 710 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 711 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 712 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 713 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 714 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 715 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 716 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 717 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 718 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 719 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 721 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 722 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 723 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 724 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 725 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 726 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 727 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 728 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 729 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 730 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 731 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 732 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 733 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 734 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 735 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 736 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 737 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 738 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 739 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 740 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 741 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 742 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 743 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 744 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 745 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 746 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 747 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 748 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 749 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 750 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 751 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 752 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 753 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 754 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 755 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 756 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 757 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 758 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 759 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 760 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 761 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 762 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 763 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 764 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 765 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 766 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 767 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 768 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 769 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 770 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 771 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 772 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 773 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 774 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 775 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 776 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 777 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 778 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 779 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 780 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 781 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 782 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 783 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 784 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 785 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 786 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 787 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 788 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 789 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 790 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 791 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 792 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 793 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 794 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 795 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 796 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 797 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 798 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 799 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 800 and one or more polypeptides from Table 2.

In some embodiments, a composition herein may comprise polypeptide 801 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 802 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 803 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 804 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 805 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 806 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 807 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 808 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 809 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 810 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 811 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 812 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 813 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 814 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 815 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 816 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 817 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 818 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 819 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 821 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 822 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 823 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 824 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 825 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 826 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 827 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 828 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 829 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 830 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 831 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 832 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 833 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 834 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 835 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 836 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 837 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 838 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 839 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 840 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 841 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 842 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 843 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 844 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 845 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 846 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 847 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 848 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 849 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 850 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 851 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 852 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 853 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 854 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 855 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 856 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 857 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 858 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 859 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 860 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 861 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 862 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 863 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 864 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 865 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 866 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 867 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 868 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 869 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 870 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 871 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 872 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 873 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 874 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 875 and one or more polypeptides from Table 2. In some embodiments, a composition herein may comprise polypeptide 876 and one or more polypeptides from Table 2.

In some cases, the one or more polypeptides from Table 2 may comprise IL-15. In some cases, the one of more polypeptides from Table 2 may comprise BMP7. In some cases, the one or more polypeptides from Table 2 may comprise THBS4. In some cases, the one or more polypeptides from Table 2 may comprise POSTN. In some cases, the one or more polypeptides from Table 2 may comprise THBS1. In some cases, the one or more polypeptides from Table 2 may comprise THBS2. In some cases, the one or more polypeptides from Table 2 may comprise VTN. In some cases, the one or more polypeptides from Table 2 may comprise FGF17. In some cases, the one or more polypeptides from Table 2 may comprise IGF2. In some cases, the one or more polypeptides from Table 2 may comprise polypeptide 1, 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, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, or 876. In some cases, one or a plurality of the polypeptides of the composition are HAPs. In some cases, one or a plurality of the polypeptides of the composition are mitogenic and/or fusion promoting polypeptides. In certain embodiments, one or more of the polypeptides of the composition comprise one or more additional modifications to increase stability. In certain embodiments, one or more of the polypeptides is covalently conjugated to polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polysialic acid, glycolic acid, or polypropylene glycol. In certain embodiments, one or more of the polypeptides is fused or conjugated to another protein to increase stability and or bioavailability. In certain embodiments, one or more of the polypeptides is fused with an Fc region of an immunoglobulin or with serum albumin. In certain embodiments, one or more of the polypeptides is present in a concatemer with one, two, three, four or more distinct polypeptides selected from Table 2 and/or Table 1. In certain embodiments, one or more of the polypeptides is present in a concatemer with one, two, three, four, or more distinct polypeptides. In certain embodiments, one or more of the polypeptides is included in the composition with a biodegradable or bioabsorbable carrier that promotes polypeptide stability. The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA), polyglycolic acid (PGA), or Poly(D,L-lactic-coglycolic-acid) (PLGA). The biodegradable or bioabsorbable carrier may comprise polylactic acid (PLA). The biodegradable or bioabsorbable carrier may comprise polyglycolic acid (PGA). The biodegradable or bioabsorbable carrier may comprise Poly(D,L-lactic-coglycolic-acid) (PLGA).

The HAPs increase the mitogenic (e.g., proliferative capacity) of a somatic cell that is a tissue cell or a tissue precursor, such as: a muscle cell, a muscle precursor cell, a tenocyte, a tenocyte precursor cell, a chondrocyte, a chondrocyte precursor, a mesenchymal stem cell, or a fibroblast. The cell can be a precursor cell derived from any mammal, such as, monkeys, apes, dogs, cats, horses, rats, mice, or humans. The precursor cell is a human precursor cell. The HAPs increase the proliferative capacity of a mouse myoblast by at least about 1.5-fold, about 2-fold, about 3-fold, or about 4-fold as measured by BrdU or EdU incorporation.

Therapeutic Indications

In certain aspects, HAPs and compositions comprising HAPs, described herein, are useful for treating diseases and disorders that involve soft-tissue injury, degradation, or destruction. Aging disorders that result in the deterioration and loss of muscle tissue are such soft-tissue disorders. Sarcopenia, for example, is the degenerative loss of skeletal muscle mass quality, and strength associated with aging. Injuries that result in acute muscle damage are other such disorders. The disorders include muscle ruptures, strains, and contusions. A rupture is a separating of the muscle tissues. Muscle strains are contraction-induced injuries in which muscle fibers tear due to extensive mechanical stress, and can be classified as a grade I, II, or III. Muscle contusions are muscle hematomas. Muscle injury can also be caused by non-mechanical stresses such as cachexia. Cachexia may be caused by malnutrition, cancer, AIDS, coeliac 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 mellitus, anorexia nervosa, chemotherapy, muscular dystrophy or other genetic diseases which cause immobility, and hormonal deficiencies. Certain disorders that are weaknesses of specific muscles such as dysphagia or facioscapulohumeral muscular dystrophy may also be treated by the polypeptides described herein. Additional soft-tissues disorders that may be treated using the HAPs described herein are those that inflict injury to the tendons, ligaments or cartilage. The muscle wasting disease is a muscular dystrophy. The muscular dystrophy may comprise myotonic muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, Limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital, muscular dystrophy, oculopharyngeal muscular dystrophy, or distal muscular dystrophy. The muscular dystrophy is Becker muscular dystrophy. The HAPs useful for treating a soft-tissue disorder comprise any one, two, three, four, or five HAPs selected from Vitronectin (VTN), Periostin (POSTN), Fibroblast growth factor (FGF17), Thrombospondin 2 (THBS2), Thrombospondin 4 (THBS4), Thrombospondin 1 (THBS1), Insulin-like growth factor 2 (IGF2), Bone morphogenic protein 7 (BMP7), and Interleukin 15 (IL-15). The HAPs useful for treating a soft-tissue disorder comprise any one, two, three, four, or five HAPs selected from Vitronectin (VTN), Stanniocalcin-2 (STC2), Agrin (AGRN), Thrombospondin 2 (THBS2), follistatin (FST), Periostin (POSTN), Fibroblast growth factor (FGF17), Thrombospondin 4 (THBS4), Thrombospondin 1 (THBS1), Insulin-like growth factor 2 (IGF2), and Interleukin 15 (IL-15).

The HAPs and compositions comprising HAPs, described herein, are for use in treating an individual with an aging disorder, a muscle wasting disorder, a muscle injury, an injury to a connective tissue, or an injury to a non-muscle soft-tissue, or any combination thereof. The aging disorder is sarcopenia. The muscle wasting disorder is cachexia. The cachexia is a result of a cancer. The cachexia is a result of AIDS. The injury is a muscle injury. The muscle wasting is atrophy do to limb immobilization or disuse. The muscle injury is a strain or a tear. The muscle injury is a Grade III strain. In certain embodiments, sarcopenia contributes to the incidence of the muscle injury. The injury is ligament damage. The ligament damage is a rupture or a tear. The injury is tendon damage. The tendon damage is a rupture or a tear. The injury is cartilage damage. The HAPs comprise any one, two, three, four, five or more HAPs selected from Vitronectin (VTN), Periostin (POSTN), Fibroblast growth factor (FGF17), Thrombospondin 2 (THBS2), Thrombospondin 4 (THBS4), Thrombospondin 1 (THBS1), Insulin-like growth factor 2 (IGF2), Bone morphogenic protein 7 (BMP7), and Interleukin 15 (IL-15). The HAPs comprise any one, two, three, four, five or more HAPs selected from Vitronectin (VTN), Stanniocalcin-2 (STC2), Agrin (AGRN), Thrombospondin 2 (THBS2), and follistatin (FST).

The HAPs and compositions comprising HAPs, described herein, are for use in a method of treating myositis. The myositis may comprise dermatomyositis, polymyositis, necrotizing myopathy (also called necrotizing autoimmune myopathy or immune-mediated necrotizing myopathy), juvenile myositis, or sporadic inclusion-body myositis. The HAPs comprise any one, two, three, four, five or more HAPs selected from Vitronectin (VTN), Periostin (POSTN), Fibroblast growth factor (FGF17), Thrombospondin 2 (THBS2), Thrombospondin 4 (THBS4), Thrombospondin 1 (THBS1), Insulin-like growth factor 2 (IGF2), Bone morphogenic protein 7 (BMP7), and Interleukin 15 (IL-15). The HAPs comprise any one, two, three, four, or five HAPs selected from Vitronectin (VTN), Stanniocalcin-2 (STC2), Agrin (AGRN), Thrombospondin 2 (THBS2), and follistatin (FST).

The HAPs and compositions comprising HAPs, described herein, are for use in a method of treating cartilage related-disorders. The cartilage related disorder may be due to tears, injuries, or wear. The cartilage-associated disease may be osteoarthritis, osteochondritis dissecans, achondroplasia, or degenerative cartilage lesions. The HAPs comprise any one, two, three, four, five, six, or more HAPs selected from Vitronectin (VTN), Periostin (POSTN), Fibroblast growth factor (FGF17), Thrombospondin 2 (THBS2), Thrombospondin 4 (THBS4), Thrombospondin 1 (THBS1), Insulin-like growth factor 2 (IGF2), Bone Morphogenic Protein 7 (BMP7), and Interleukin 15 (IL-15). The HAPs comprise any one, two, three, four, or five HAPs selected from Vitronectin (VTN), Stanniocalcin-2 (STC2), Agrin (AGRN), Thrombospondin 2 (THBS2), and follistatin (FST).

The HAPs and compositions comprising HAPs, described herein, are for use in a method of increasing proliferation or promoting survival of a cell associated with soft-tissue damage. The HAPs described herein are useful in a method of increasing proliferation or promoting survival of any one or more of a muscle cell, a muscle precursor cell, a tenocyte, a tenocyte precursor cell, a chondrocyte, a chondrocyte precursor cell, a mesenchymal stem cell, or a fibroblast. The HAPs comprise any one, two, three, four, five or more HAPs selected from Vitronectin (VTN), Periostin (POSTN), Fibroblast growth factor (FGF17), Thrombospondin 2 (THBS2), Thrombospondin 4 (THBS4), Thrombospondin 1 (THBS1), Insulin-like growth factor 2 (IGF2), Bone morphogenic protein 7 (BMP7), and Interleukin 15 (IL-15). The HAPs comprise any one, two, three, four, or five HAPs selected from Vitronectin (VTN), Stanniocalcin-2 (STC2), Agrin (AGRN), Thrombospondin 2 (THBS2), and follistatin (FST).

The HAPs compositions described herein can be administered separately or as a mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more heparin-binding or HAPs for the treatment of any disorder associated with muscle or soft-tissue.

In certain aspects, a method of treating a disease or condition, such as those described herein, in a subject in need thereof may comprise administering to the subject a composition comprising a polypeptide of Table 2. In some embodiments, the polypeptide of Table 2 is a polypeptide of Table 1. In some embodiments, the composition may comprise a pharmaceutically acceptable excipient, such as described herein. In some embodiments, the disease or condition may comprise an aging disorder, muscle wasting disorder, muscle injury, or injury to connective tissue, or a combination thereof. In some embodiments, the aging disorder may comprise sarcopenia. In some embodiments, the muscle wasting disorder may comprise muscular dystrophy. In some embodiments, the muscle wasting is a result of obesity. The muscle wasting is the result of a metabolic disorder. In some cases the metabolic disorder is diabetes. In some cases the diabetes is Type 2 Diabetes. In some embodiments, muscle wasting is a result of disease progression. In some embodiments, muscle wasting is a result of therapeutic treatment. In some embodiments, the muscle wasting is cachexia. In some embodiments, the therapeutic polypeptide promotes fusion of myocytes.

In some embodiments, the polypeptide is a heparin-associated binding polypeptide as described herein. In some embodiments, the polypeptide is a mitogenic and/or fusion promoting polypeptide as described herein. In some embodiments, a composition comprising a plurality of heparin-associated binding polypeptides as described herein is administered. In some embodiments, a composition comprising a plurality of mitogenic and/or fusion promoting polypeptides as described herein is administered.

In some embodiments, the polypeptide has been recombinantly produced. In some embodiments, the polypeptide has been produced in a mammalian cell culture. The polypeptide has been produced in a mammalian cell and the mammalian cell is a human cell. In some cases the human cell is a human embryonic kidney-derived epithelial cell (e.g., HEK293 cells). In some embodiments, the mammalian cell culture is a mouse myeloma cell culture. In some embodiments, the mammalian cell culture is a Chinese Hamster Ovary (CHO) cell culture. In some embodiments, the polypeptide has been produced in a non-mammalian cell culture, e.g., in bacteria, yeast, or insect cells. The polypeptide has been purified from a human biological sample. In some cases, the human biological sample is human plasma. In some embodiments, the composition is formulated for administration by injection to the subject. In some embodiments, the composition may comprise one or more polypeptides having at least about 90% homology to a sequence selected from HAPs ID NOS: 1-44, 55, 56, and 58-72. In some embodiments, the composition may comprise polypeptide 1, 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, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, or 876, or any combination thereof.

In some embodiments, the polypeptide may comprise VTN. In some cases, the VTN may comprise a polypeptide comprising at least about 90% homology or identity to HAPs ID NO: 1. In some cases, the VTN may comprise a polypeptide comprising at least about 90% homology or identity to amino acids 20-478 of HAPs ID NO: 1. In some cases, the VTN is purified from human plasma.

In some embodiments, the polypeptide may comprise POSTN. In some cases, the POSTN may comprise a polypeptide comprising at least about 90% homology or identity to HAPs ID NO: 6. In some cases, the POSTN may comprise a polypeptide comprising at least about 90% homology or identity to amino acids 22-836 of HAPs ID NO: 6. In some cases, the POSTN is expressed in a mouse myeloma cell line.

In some embodiments, the polypeptide may comprise FGF17. In some cases, the FGF17 may comprise a polypeptide comprising at least about 90% homology or identity to HAPs ID NO: 7. In some cases, the FGF17 may comprise a polypeptide comprising at least about 90% homology or identity to amino acids 23-216 of HAPs ID NO: 7. In some cases, the FGF17 is expressed in a bacterial cell. In some embodiments, the bacterial cell is E. coli.

In some embodiments, the polypeptide may comprise THBS2. In some cases, the THBS2 may comprise a polypeptide comprising at least about 90% homology or identity to HAPs ID NO: 4. In some cases, the THBS2 may comprise a polypeptide comprising at least about 90% homology or identity to amino acids 19-1172 of HAPs ID NO: 4. In some cases, the THBS2 is expressed in a mouse myeloma cell line.

In some embodiments, the polypeptide may comprise THBS4. In some cases, the THBS4 may comprise a polypeptide comprising at least about 90% homology or identity to HAPs ID NO: 8. In some cases, the THBS4 may comprise a polypeptide comprising at least about 90% homology or identity to amino acids 27-961 of HAPs ID NO: 8. In some cases, the THBS4 is expressed in Chinese hamster ovary cell.

In some embodiments, the polypeptide may comprise IGF2. In some cases, the IGF2 may comprise a polypeptide comprising at least about 90% homology or identity to HAPs ID NO: 11. In some cases, the IGF2 may comprise a polypeptide comprising at least about 90% homology or identity to amino acids 25-91 of HAPs ID NO: 11. In some cases, the IGF2 is expressed in a bacterial cell. In some embodiments, the bacterial cell is E. coli.

In some embodiments, the polypeptide may comprise IL-15. In some cases, the IL-15 may comprise a polypeptide comprising at least about 90% homology or identity to HAPs ID NO: 10. In some cases, the IL-15 may comprise a polypeptide comprising at least about 90% homology or identity to amino acids 49-162 of HAPs ID NO: 10. In some cases, the IL-15 is expressed in a bacterial cell. In some embodiments, the bacterial cell is E. coli.

In some embodiments, the polypeptide may comprise THBS1. In some cases, the THBS1 may comprise a polypeptide comprising at least about 90% homology or identity to HAPs ID NO: 9. In some cases, the THBS1 may comprise a polypeptide comprising at least about 90% homology or identity to amino acids 19-1170 of HAPs ID NO: 9. In some cases, the THBS1 is expressed in a mouse myeloma cell line.

In some embodiments, the polypeptide may comprise BMP7. In some cases, the BMP7 may comprise a polypeptide comprising at least about 90% homology or identity to HAPs ID NO: 72. In some cases, the BMP7 may comprise a polypeptide comprising at least about 90% homology or identity to HAPs ID NO: 72. In some cases, the BMP7 is expressed in a mouse myeloma cell line.

In some embodiments, the polypeptide may comprise IL-15, and the composition further may comprise THBS2. In some embodiments, the polypeptide may comprise IL-15, and the composition further may comprise THBS4. In some embodiments, the polypeptide may comprise THBS4, and the composition further may comprise THBS2. In some embodiments, the polypeptide may comprise IL-15, and the composition further may comprise THBS2 and THBS4.

In some embodiments, the polypeptide may comprise IGF2, and the composition further may comprise THBS2. In some embodiments, the polypeptide may comprise IGF2, and the composition further may comprise THBS4. In some embodiments, the polypeptide may comprise THBS4, and the composition further may comprise THBS2. In some embodiments, the polypeptide may comprise IGF2, and the composition further may comprise THBS2 and THBS4.

Schedules Routes of Administration and Amounts

The HAPs can be administered by any suitable route such as, for example, subcutaneous, intravenous, or intramuscular. The HAPs are administered on a suitable dosage schedule, for example, weekly, twice weekly, monthly, twice monthly, once every three weeks, or once every four weeks. The HAPs can be administered in any therapeutically effective amount. The therapeutically acceptable amount is about 0.001 mg/kg to about 1 mg/kg. The therapeutically acceptable amount is about 0.001 mg/kg to about 0.002 mg/kg, about 0.001 mg/kg to about 0.005 mg/kg, about 0.001 mg/kg to about 0.01 mg/kg, about 0.001 mg/kg to about 0.02 mg/kg, about 0.001 mg/kg to about 0.05 mg/kg, about 0.001 mg/kg to about 0.1 mg/kg, about 0.001 mg/kg to about 0.2 mg/kg, about 0.001 mg/kg to about 0.5 mg/kg, about 0.001 mg/kg to about 1 mg/kg, about 0.002 mg/kg to about 0.005 mg/kg, about 0.002 mg/kg to about 0.01 mg/kg, about 0.002 mg/kg to about 0.02 mg/kg, about 0.002 mg/kg to about 0.05 mg/kg, about 0.002 mg/kg to about 0.1 mg/kg, about 0.002 mg/kg to about 0.2 mg/kg, about 0.002 mg/kg to about 0.5 mg/kg, about 0.002 mg/kg to about 1 mg/kg, about 0.005 mg/kg to about 0.01 mg/kg, about 0.005 mg/kg to about 0.02 mg/kg, about 0.005 mg/kg to about 0.05 mg/kg, about 0.005 mg/kg to about 0.1 mg/kg, about 0.005 mg/kg to about 0.2 mg/kg, about 0.005 mg/kg to about 0.5 mg/kg, about 0.005 mg/kg to about 1 mg/kg, about 0.01 mg/kg to about 0.02 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.01 mg/kg to about 0.1 mg/kg, about 0.01 mg/kg to about 0.2 mg/kg, about 0.01 mg/kg to about 0.5 mg/kg, about 0.01 mg/kg to about 1 mg/kg, about 0.02 mg/kg to about 0.05 mg/kg, about 0.02 mg/kg to about 0.1 mg/kg, about 0.02 mg/kg to about 0.2 mg/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. The therapeutically acceptable 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. The therapeutically acceptable 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. The therapeutically acceptable amount is at most 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. The therapeutically acceptable amount is about 0.1 mg/kg to about 50 mg/kg. The therapeutically acceptable amount is 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 0.1 mg/kg to about 2 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 20 mg/kg, about 0.1 mg/kg to about 50 mg/kg, about 0.2 mg/kg to about 0.5 mg/kg, about 0.2 mg/kg to about 1 mg/kg, about 0.2 mg/kg to about 2 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 20 mg/kg, about 0.2 mg/kg to about 50 mg/kg, about 0.5 mg/kg to about 1 mg/kg, about 0.5 mg/kg to about 2 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 20 mg/kg, about 0.5 mg/kg to about 50 mg/kg, about 1 mg/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 mg/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. The therapeutically acceptable 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. The therapeutically acceptable 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. The therapeutically acceptable amount is at most 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.

Nucleic Acids

TABLE 3

Nucleic acid sequences.

Protein
Size

Protein
mRNA

Name
(AA)
UniProtKB
accession#
accession#
Gene ID
SEQ ID

FGF17
216
O60258.1
NP_003858.1
NM_003867.4
8822
45

THBS1
1170
P07996.2
NP_003237.2
NM_003246.4
7057
46

THBS2
1172
P35442.2
NP_003238.2
NM_003247.3
7058
47

THBS4
961
P35443.2
NP_003239.2
NM_003248.6
7060
48

IGF2
180
P01344.1
NP_000603.1
NM_000612.6
3481
49

IL15
162
P40933.1
NP_000576.1
NM_000585.5
3600
50

IGFBP7
282
Q16270.1
NP_001544.1
NM_001553.3
3490
51

VTN
478
P04004
NP_000629.3
NM_000638.4
7448
52

POSTN
836
Q15063.2
NP_006466.2
NM_006475.3
10631
53

PDGFRL
375
Q15198.1
NP_006198.1
NM_006207.2
5157
54

ANOS1
3730
P23352
NP_000207.2
NM_000216.4
3730
57

BMP7
431
P18075
NP_001710.1
NM_001719.3
655
73

Described herein is a composition that may comprise nucleic acids that encode the HAPs described herein. The nucleic acids are exogenous. The nucleic acid is a plasmid. The nucleic acid is a viral vector. The viral vector is an adenovirus, lentivirus, retrovirus, adeno-associated virus, or vaccinia virus. The nucleic acid may comprise RNA. The nucleic acid encodes any of the polypeptides listed in Table 1 or Table 2, or VTN, STC2, AGRN, POSTN, FGF17, THBS2, FST, THBS1, IL-15, IGF2, THBS4, or BMP7. The nucleic acid encodes any one or more polypeptides described herein. Nucleic acids according to this description can comprise additional nucleic acid sequences sufficient to propagate the vector or express a polypeptide encoded by the vector. The nucleic acid may comprise a universal promoter, such as the CMV promoter, or an inducible promoter system such as a TET_ON, TET_OFFor GAL4. The nucleic acid is expressed via a tissue specific promoter or one compatible with a eukaryotic or prokaryotic cellular expression system. The nucleic acid can further comprise a sequence encoding a suitable purification tag (e.g., HIS-tag, V5, FLAG, MYC).

Production of Heparin-Associated or Heparin-Binding Polypeptides

Once a polypeptide is determined as a heparin-associated or heparin-binding polypeptide it can be purified or synthesized in any suitable manner. A nucleic acid encoding the polypeptide can be cloned into a suitable vector and expressed in a suitable cellular system. The cellular system is a prokaryotic cell system. The cellular system is a eukaryotic cell system. The cellular system is a mammalian cell system. The supernatants from such an expression system can be subjected to one or more purification steps involving centrifugation, ultracentrifugation, filtration, diafiltration, tangential-flow filtration, dialysis, chromatography (e.g., cation exchange, ion exchange, hydrophobic interaction, reverse phase, affinity, or size exclusion). The polypeptides can be purified to an extent suitable for human administration. Additionally, polypeptides can be synthesized for inclusion in a formulation to be administered to a human subject. The polypeptides can be produced by a suitable peptide synthesis method, such as solid-phase synthesis.

Master Cell Bank and Transgenic Cells

Described herein is a master cell bank comprising a cell that may comprise a nucleic acid encoding one or more HAPs integrated into its genome creating a transgenic cell-line. The master cell bank may comprise a plurality of cells that each comprise a nucleic acid encoding a HAP. The nucleic acid is maintained extrachromosomally on a plasmid or yeast artificial chromosome. The nucleic acid is integrated into a chromosomal location. The cell is a yeast cell. The yeast is Pichia pastoris or Saccharomyces cerevisiae. The cell is a mammalian cell. The mammalian cell is a 293T cell or derivative thereof (e.g., 293T-Rex). The cell is a bacterial cell.

The transgenic mammalian, yeast, or bacterial cell is a master cell bank that may comprise a cryopreservative suitable for freezing to at least about −80° or below. The master cell bank may comprise glycerol at between about 10 and about 30%, and is suitable for long-term storage at about −80° or below. The master cell bank can preserve a transgenic mammalian, yeast, or bacterial strain for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.

Pharmaceutically Acceptable Excipients, Carriers, and Diluents

The HAP(s) described herein can be administered in a pharmaceutical composition that may comprise one or more pharmaceutically acceptable excipients, carriers, or diluents. The exact components can differ based upon the preferred route of administration. The excipients used in a pharmaceutical composition can provide additional function to the polypeptide by making the polypeptide suitable for a particular route of administration (e.g., intravenous, topical, subcutaneous, or intramuscular), increasing polypeptide stability, increasing penetration of a desired tissue (e.g., muscle or skin), increasing residence time at particular site, increasing solubility, enhancing the efficacy of the polypeptide, and/or reducing inflammatory reactions coincident with administration.

The HAP(s) described herein are included in a pharmaceutical composition with a solubilizing emulsifying, or dispersing agent. The solubilizing agent can allow high-concentration solutions of HAPs that exceed at least about 2 mg/mL, 5 mg/mL, 10 mg/mL, 15 mg/mL, or 20 mg/mL. Carbomers in an aqueous pharmaceutical composition serve as emulsifying agents and viscosity modifying agents. The pharmaceutically acceptable excipient may comprise or consists of a carbomer. The carbomer may comprise or consists of carbomer 910, carbomer 934, carbomer 934P, carbomer 940, carbomer 941, carbomer 1342, or combinations thereof. Cyclodextrins in an aqueous pharmaceutical composition serve as solubilizing and stabilizing agents. The pharmaceutically acceptable excipient may comprise or consists of a cyclodextrin. The cyclodextrin may comprise or consists of alpha cyclodextrin, beta cyclodextrin, gamma cyclodextrin, or combinations thereof. Lecithin in a pharmaceutical composition may serve as a solubilizing agent. The solubilizing agent may comprise or consists of lecithin. Poloxamers in a pharmaceutical composition serve as emulsifying agents, solubilizing agents, and dispersing agents. The pharmaceutically acceptable excipient may comprise or consists of a poloxamer. The poloxamer may comprise or consists of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or combinations thereof. Polyoxyethylene sorbitan fatty acid esters in a pharmaceutical composition serve as emulsifying agents, solubilizing agents, surfactants, and dispersing agents. The pharmaceutically acceptable excipient may comprise or consists of a polyoxyethylene sorbitan fatty acid ester. The polyoxyethylene sorbitan fatty acid ester may comprise 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. Polyoxyethylene stearates in a pharmaceutical composition serve as emulsifying agents, solubilizing agents, surfactants, and dispersing agents. The pharmaceutically acceptable excipient may comprise or consists of a polyoxyethylene stearate. The polyoxyethylene stearate may comprise 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. Sorbitan esters in a pharmaceutical composition serve as emulsifying agents, solubilizing agents, and non-ionic surfactants, and dispersing agents. The pharmaceutically acceptable excipient may comprise or consists of a sorbitan ester. The sorbitan ester may comprise or consists of sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan stearate, sorbitan trioleate, sorbitan sesquioleate, or combinations thereof. Solubility may be achieved with a protein carrier. The protein carrier may comprise recombinant human albumin.

The HAP(s) of the current disclosure are formulated to increase stability. Polypeptides in aqueous formulations may require stabilization to prevent degradation. The stabilizer may comprise pH buffers, salts, amino acids, polyols/disaccharides/polysaccharides, liposomes, surfactants, antioxidants, reducing agents, or chelating agents. The stabilizer may comprise or consists of a polyol/non-reducing sugar. The non-reducing sugar may comprise or consists of sucrose, mannitol, trehalose, raffinose, stachyose, xylitol, starch, verbascose, or combinations thereof. Polypeptides can be encapsulated in liposomes to increase stability. The stabilizer may comprise or consists of liposomes. The liposomes comprise or consists of ipalmitoylphosphatidylcholine (DPPC) liposomes, phosphatidylcholine:cholesterol (PC:Chol) (70:30) liposomes, or dipalmitoylphosphatidylcholine: dipalmitoylphosphatidylserine (DPPC:DPPS) liposomes (70:30). Non-ionic surfactants can increase the stability of a polypeptide. The stabilizer may comprise or consists of a non-ionic surfactant. The non-ionic surfactant may comprise or consists of polysorbates (e.g., poly sorbate 80, poly sorbate 20), alkylsaccharides alkyl ethers and alkyl glyceryl ethers, polyoxyethelene (4) lauryl ether; polyoxyethylene cetyl ethers, polyoxyethylene stearyl ethers, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, or combinations thereof 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. The stabilizer may comprise or consists of an antioxidant or reducing agent. The reducing agent may comprise or consists of dithiothreitol, ethylenediaminetetraacetic acid, 2-Mercaptoethanol, Tris(2-carboxyethyl)phosphine hydrochloride, Tris(hydroxypropyl)phosphine, or combinations thereof. The antioxidant may comprise or consists of methionine, ascorbic acid, citric acid, alpha tocopherol, sodium bisulfite, ascorbyl palmitate, erythorbic acid, or combinations thereof. Chelating agents can stabilize polypeptides by reducing the activity of proteases. The stabilizer may comprise or consists of a chelating agent. The chelating agent may comprise or consists of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(O-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), metal complexes (e.g. Zn-protein complexes), or combinations thereof. Buffer agents can stabilize polypeptides by reducing the acid hydrolysis of polypeptides. The stabilizer may comprise or consists of a buffer agent. The buffer agent may comprise or consists sucrose octa-sulfate, ammonium carbonate, ammonium phosphate, boric acid, sodium citrate, potassium citrate, lactic acid, 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES), hydroxymethylaminomethane (Tris), calcium carbonate, calcium phosphate or combinations thereof.

The HAP(s) also may be entrapped in or associated with microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

The HAP(s) of the current disclosure may be formulated or delivered with an anti-inflammatory agent. The anti-inflammatory agent may comprise or consists of a corticosteroid. The corticosteroid may comprise 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 certain emboidments, the anti-inflammatory may comprise or consists of a non-steroidal anti-inflammatory (NSAID). The NSAID may comprise or consists of aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac, or tolmetin.

The HAP(s) of the current disclosure are included in a pharmaceutical composition suitable for intravenous administration comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. The polypeptides of the current disclosure are administered suspended in a sterile solution. The solution is one commonly used for administration of biological formulations, and may comprise, for example, about 0.9% NaCl or about 5% dextrose. The solution further may comprise one or more of buffers, for example, acetate, citrate, histidine, succinate, phosphate, potassium phosphate, bicarbonate and hydroxymethylaminomethane (Tris); surfactants, for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; polyol/disaccharide/polysaccharides, for example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, for example, glycine, histidine, leucine, or arginine; antioxidants, for example, ascorbic acid, methionine; or chelating agents, for example, EDTA, or EGTA.

The HAP(s) of the current disclosure are included in a pharmaceutical composition suitable for intramuscular or subcutaneous administration comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. Formulations suitable for intramuscular or subcutaneous injection can include 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 (inositol, propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like) 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 the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for subcutaneous injection also contain optional additives such as preserving, wetting, emulsifying, and dispensing agents.

The HAP(s) of the current disclosure are formulated for topical administration as a cream, gel, paste, ointment, or emulsion. Excipients in a cream, gel, paste, ointment, or emulsion can comprise gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidol silicon dioxide, phosphates, sodium dodecyl sulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethycellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, sugars, and starches.

The excipient used with the HAP(s) described herein will allow for storage, formulation, or administration of highly concentrated formulations. A highly concentrated HAP(s) may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 20, 25, 40, 45, 50 or more milligrams per milliliter.

The polypeptides of the current disclosure are shipped/stored lyophilized and reconstituted before administration. Lyophilized HAP formulations may comprise a bulking agent such as, mannitol, sorbitol, sucrose, trehalose, and dextran 40. The lyophilized formulation can be contained in a vial comprised of glass. The HAPs when formulated, whether reconstituted or not, can be buffered at a certain pH, generally less than 7.0. The pH can be between 4.5 and 6.5, 4.5 and 6.0, 4.5 and 5.5, 4.5 and 5.0, or 5.0 and 6.0.

EXAMPLES

The following illustrative examples are representative of embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

Example 1—Isolation of Heparin-Associated Polypeptides

hESC secretome collection (differentiated vs undifferentiated): Human embryonic or induced pluripotent stem cells (H1, H9, H7 lines, and 2 iPSC lines derived from 1 healthy young adult female (18-25 years) and 1 aged female (greater than 65 year) donor), were cultured in triplicate on 10 cm plates on diluted Matrigel (1:30), in mTeSR-1 (Stem Cell Technologies), for a total media volume of 10 mL per plate. Another triplicate set of hPSCs/iPSCs were cultured on 10 cm plates and differentiated after plating in mTeSR-1 by changing the medium to DMEM/F12 with 10% Bovine Growth Serum (Hyclone), and culturing for an additional 7 days. hPSCs/iPSCs and differentiated hPSCs/iPSCs (6 plates in total) were washed twice with Opti-MEM (Gibco) and then cultured in Opti-MEM for 16 hours. 10 ml media was then collected per plate as hPSCs/iPSCs—secretome or differentiated hPSCs/iPSCs—secretome containing media. Media was spun for 5 min at 1000 g and transferred to new tubes to remove cell debris, aliquoted and flash frozen at 2 mL per plate as 0.5 mL aliquots and stored at −80 C; remaining 8 mL/plate was used immediately for heparin-associated protein purification.

Heparin-Associated Protein Purification

10 mL of Heparin-Agarose Type I Beads (H 6508, Sigma Aldrich) was washed with molecular grade water and preconditioned in 1 mL OptiMEM as recommended by manufacturer. 8-9 ml secretome containing media was incubated with 1 ml Heparin-Agarose Beads for 2 hours shaking at 4° C. to allow binding. Remaining heparin-depleted hPSCs/iPSCs-conditioned medium or differentiated hPSCs/iPSCs-conditioned medium was aliquoted in 15 mL tubes, flash frozen and stored at −80 C to serve as negative controls for efficacy testing. Protein bound heparin beads were washed twice via a 10-minute incubation at 4° C. in 1 mL sterile PBS+0.05% Tween. Proteins were eluted twice for 15 minutes at 4° C. in 400 μl of elution buffer A (Eluted-A) (0.01M HEPES pH 7.5+1.5M NaCl+0.1% BSA) per 10 cm plate for the first two plates, or elution buffer B (Eluted-B) lacking BSA (0.01M HEPES pH 7.5+1.5M NaCl) for the 3rd 10 cm plate, to collect proteins in a total of 800 μl of elute per original plate. The proteins were desalted by diffusion dialysis (3500 MWCO) (by a 2-hour dialysis shaking at 4 C in 500 ml McCoy's 5A Medium or similar tissue culture medium (Gibco) followed by overnight (not more than 16 hours) dialysis shaking at 4 C in 200 ml OptiMEM (Gibco). The collected eluate was aliquoted in appropriately capped tubes, flash frozen and stored at −80 C.

Secretome Heparin-Associated Fraction Validation Assays:

BCA assay (Pierce) was performed for total protein yield in the eluate using 2 ul per sample in triplicate according to manufacturer's instructions from each sample.

SDS-PAGE Silver Stain/SDS-PAGE Coomassie was performed for protein integrity and rough MW analysis (loading <5-10 μg per lane for each sample).

Mouse Myoblast Proliferation Assay

Reduced regeneration from an individual's tissue progenitor cells is a hallmark of aging, therefore assays that measure mitogenic capacity in tissue progenitor cells serve as a read-out for potential success of any given heparin-associated polypeptide (HAP) as a regenerative factor. Measuring the increased proliferation rate of treated mouse or human muscle progenitor cells will provide good basis for potentially therapeutic regenerative factors for treating individuals who have suffered illness, injury, or who possess genetic or developmental defects leading to premature tissue loss, wasting, or weakening. As a control, the assay will also be performed on proteins purified from differentiated cells, which result in no in myoblast proliferation, cultured in medium conditioned by differentiated cells, or purified heparin-associated fractions.

Mouse muscle progenitor cells (early passage myoblasts) were cultured and expanded 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), at 37° C. and 5% CO2. For experimental conditions, cells were plated at 40,000 cells/well on Matrigel coated 8-well chamber slides in 250-500 μL medium per well (1:100 matrigel: PBS) in mouse fusion medium: DMEM (Gibco)+2% horse serum (Hyclone). One hour after plating, mouse myoblasts were treated with 50% respective medias:

TABLE 4

8-well Chamber Slide A: Eluted HAPs from H9/H7 hPSCs and 2

iPSC lines - 4 slides total, 1 for each cell line tested.

Fusion
50% FM/
50% FM/50%
50% FM/50%

Media (FM)
50% Eluted-A
Differentiated
Differentiated

(250 μL)
Heparin-
hPSC-
hPSC-

associated
conditioned
conditioned

proteins
OptiMem
OptiMem

50% FM
50% FM/50%
50% FM/50%
50% FM/

(125 μL)/
hPSC-
Heparin-
50% Eluted-B

50% Growth
conditioned
depleted
Heparin-

Media
OptiMEM
hESC-
associated

(125 μL)

conditioned
proteins

OptiMEM
(no BSA)

TABLE 5

Assay for eluted heparin-associated proteins

(Control). 50%
50% FM/
50% FM/50%
50% FM/

FM/
50% Eluted-A
Differentiated
50% Eluted-B

50% OptiMEM
Heparin-
hPSC-conditioned
Heparin-

associated
OptiMem
associated

proteins

proteins (no BSA)

TABLE 6

8-well Chamber Slide B: Eluted Heparin-

associated Protein Serial Dilution

Fusion Media
50% FM/
75% FM/25%
75% FM/12.5%

(FM)
50% Eluted-A
Eluted-A
Eluted-A

(250 μL)
Heparin-
Heparin-
Heparin-

associated
associated
associated

protein
protein
protein

81.25% FM/
84.375% FM/
98.44% FM/
75% FM/

6.25% Eluted-
3.125% Eluted-
1.56% Eluted-
25% hPSC-

A Heparin-
A Heparin-
A Heparin-
conditioned

associated
associated
associated
OptiMEM

protein
protein
protein

Mouse myoblasts were cultured for 24 hours in the above conditions, at 37° C. in 10% CO₂incubator. BrdU (300 μM) in DMSO was added for 2 hours prior to fixation with cold 70% ethanol and stored at 4° C. until staining.

Quantifying Regenerative Index

Following permeabilization in PBS+0.25% Triton X-100, antigen retrieval was performed via a 10-minute 4 N HCl treatment followed by PBS washes. Primary staining was performed overnight at 4° C. in PBS+2% FBS. Primary antibodies include: a species-specific monoclonal antibody for mouse anti-embryonic Myosin Heavy Chain (eMyHC, hybridoma clone 1.652, Developmental Studies Hybridoma Bank) and Rat-anti-BrdU (Abcam Inc. ab6326). Secondary staining with fluorophore-conjugated, species-specific antibodies (Donkey anti-Rat-488, #712-485-150; Donkey anti-Mouse-488, #715-485-150; all secondary antibodies from Jackson ImmunoResearch) was performed for 1 hour at room temperature at a 1:500 dilution in PBS+2% FBS. Nuclei are visualized by Hoechst staining.

In one experiment, old mice were injured with BaCl₂and injected with the HAPs. After 5 days, mice were euthanized, and the muscles were analyzed for regeneration. As depicted in FIG. 1, the muscles of old mice that had received an injection of the heparin-binding proteins had a higher regenerative index than the muscles of the untreated old mice.

For cell quantification, 5 images per well were collected at 20× in each of the channels as well as DIC to achieve at least 2000 imaged cells per condition. Using the Hoechst stain to tally cell number, the percent of cells positive for BrdU and eMyHC were tabulated and reported.

Human muscle progenitor cells (myoblasts) were similarly activated to proliferate when conditioned with hPSC-secreted heparin-associated proteins. Proliferation assays were performed on human myoblasts to test protein candidate factors for enhanced precursor cell activity in an in vitro screening assay. Conditions for culturing human muscle cells were optimized to reflect the slower rate of growth and differentiation of human muscle cells, where early passage human myoblasts were cultured for 72 hours with daily medium changes rather than 24 hours, and pulsed for 4 hours with BrdU instead of 2 hours.

Example 2—Characterization of the Protein Components of the Heparin Bead Binding hPSC Secretomes
Protein Quantification

The protein concentration in the eluted sample was determined using the bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Waltham, MA). The protocol was performed as follows: A volume containing 100 μg protein was extracted and disulfide bonds were reduced with 5 mM tris-(2-carboxyethyl)-phosphine (TCEP), at room temperature for 25 min, and alkylated with 10 mM iodoacetamide at room temperature for 30 min in the dark). Excess iodoacetamide was quenched with 15 mM dithiothreitol (room temperature, 15 min in the dark). At this point the samples were split, with 20 μg analyzed immediately via SDS-PAGE Silver Stain, 20 μg saved for SDS-PAGE Coomassie stained gel band analysis, and 60 μg proceeded to in-solution mass spectrometry sample preparation.

Quantify the Size Distribution of Proteins

Silver staining provides a sensitive, rapid, low cost way to survey the complexity and general molecular weight distribution of the proteins in a complex mixture. By running a matched sample treated to remove glycans, the presence of this PTM common secreted proteins can be determined by the resulting shift in apparent molecular weight. Additional rounds of selective glycosylation reactions can then be run to gain insight into the identity and structure of glycan modifications on proteins of interest. Five micrograms of sample can be removed and treated with Protein Deglycosylation Mix II (NEB) to remove all N-linked and simple O-linked glycans as well as some complex O-linked glycans, which can be visualized by molecular weight shifts relative to an untreated control on a silver stained SDS-PAGE gel.

A 4-12% acrylamide gel (BioRad) in 1×MOPS buffer was loaded gel with samples (>0.20 ug/lane) and ladder (as per manufacturer's instructions), run at 200V for 45 minutes or until sample front neared the bottom of the gel, and incubated in 50% methanol/50% LC grade water >1 hour. Stain solution was prepared adding a solution of 0.8 g AgNO3 in 4 mL LC grade H₂O dropwise into a solution of 1 mL 0.36% NaOH+1.4 mL 14.8M ammonium hydroxide under constant stirring followed by the addition of LC grade water to a final volume of 100 mL. Gel staining proceeded by incubating gel in stain solution for 15 minutes, before washing twice with LC grade water, allowing 5-8 minutes of incubation per wash step. The silver stain was developed by incubation in a solution of 0.25 mL 1% citric acid+25 uL 37% formaldehyde in 50 mL LC grade water for 10-15 minutes in the dark (or until desired density was achieved). Developer solution was removed, and the gel washed with LC grade water to slow development for an imaging series, or development was stopped by incubation in a solution of 45% methanol, 10% acetic acid.

In-Solution Mass Spectrometry Sample Preparation

Methanol-chloroform precipitation was performed prior to protease digestion (a standard trichloroacetic acid-based precipitation protocol would be substituted here if protein yield from the heparin bead eluates are below 25 μg total). In brief, four parts neat methanol was added to each sample and vortexed, one part chloroform was added to the sample and vortexed, and three parts water was added to the sample and vortexed. The sample was centrifuged at 4,000 RPM for 15 min at room temperature and subsequently washed twice with 100% methanol, prior to air-drying. Samples were resuspended in 50 mM HEPES pH 8.5 and digested at room temperature for 12 hrs with LysC protease at a 100:1 protein-to-protease ratio. Trypsin was then added at a 100:1 protein-to-protease ratio and the reaction was incubated 6 hours at 37° C. Peptide concentrations in the digests were measured using the Quantitative Colorimetric Peptide assay kit (Pierce). From each sample 10 μg of peptide digestion solution was taken and enzymatic activity quenched with formic acid to a final pH of <2 before de-salting via C-18 Stagetips, using a standard formic acid/acetonitrile buffer system. Stagetips were eluted directly into autosampler vials in a buffer of 70% acetonitrile and 1% formic acid, dried in a vacuum concentrator, and stored at −80 C until being resuspended to ˜1 ug/μl of Buffer A (typically ˜0.2% formic acid, 5% acetonitrile) for mass spectrometry analysis.

SDS-PAGE Coomassie and In-Gel Band Mass Spectrometry Sample Preparation

A gel-based sample preparation pipeline may be employed if the abundance distribution of the sample is heavily skewed, or where only a few species of proteins account for a substantial majority of the molecules in the sample. This size-based separation method has been shown to effectively improve depth of proteomic coverage in biochemically purified protein mixtures.

Briefly, the protocol begins by running >20 μg per lane of sample out on an SDS-PAGE as in the Silver Stain method above, staining and destaining by Coomassie as per manufacturer's instructions, excising sections of the gel containing potentially interesting proteins, and cutting excised gel sections into 1 mm×1 mm squares. Ensure gel pieces are at neutral pH by adding 50-100 μl 100 mM Ammonium bicarbonate, let sit for 10 minutes and discard. Wash gel pieces with 100-150 μl 50 mM Ammonium bicarbonate/50% acetonitrile for 10 minutes, vortexing every 5 minutes to dehydrate. Depending on intensity of stain, repeat step 9 until the gel pieces are clear. Discard solution phase and dry samples in speed vac for 5-10 minutes. To digest proteins add 5 pmol sequencing grade trypsin (Promega Corp.) in 50 mM Ammonium bicarbonate and 0.02% Protease Max to each sample and incubate overnight in 37° C. on a shaking heatblock. Spin down samples at 1000G for 2 minutes, pull off all liquid, and transfer to a glass autosampler vial. Add 40-50 μl 1% formic acid, 66% acetonitrile 33% 100 mM Ammonium bicarbonate and incubate for 10 minutes at 37° C. to increase peptide release from gel. Spin at 10,000G for 2 minutes to pellet insoluble protein or detergent degradation production. Extract all solution being sure to avoid pellet areas and combine into autosampler vial. Speed vac total combined extracts to dryness and store at −80 C until being resuspended to ˜1 μg/μl of Buffer A (typically ˜0.2% formic acid, 5% acetonitrile) for mass spectrometry analysis.

nHPLC-MS2 Instrumentation and Analysis

Two, 3-hr gradients were collected per sample using an Orbitrap Fusion instrument coupled to a Waters liquid chromatography (LC) pump (Thermo Fisher Scientific). Peptides are fractionated on a 100 μm inner diameter microcapillary column packed with ˜25 cm of Accucore 150 resin (1.2 μm, 150 Å, ThermoFisher Scientific). For each analysis, 1 μg per sample was loaded onto the column. Peptides were separated using a 3 hr gradient of 6 to 46% acetonitrile in 0.2% formic acid at a flow rate of ˜400 nL/min. Instrument settings for the Orbitrap fusion were as follows: FTMS1 resolution (120,000), ITMS2 isolation window (0.4 m/z), ITMS2 max ion time (120 ms), ITMS2 AGC (2E4), ITMS2 CID energy (35%), dynamic exclusion window (90 sec). A TOP10 method was used where each FTMS1 scan was used to select up to 10 FTMS2 precursors for interrogation by HCD-MS2 with readout in the orbitrap.

Data Analysis

Resulting mass spectra were searched using commercially available analysis software (e.g., Byonic) against a human database publicly available from Uniprot which was concatenated with common contaminants and reversed sequences of the human and contaminant proteins as decoys for FDR determination. Searches restricted the precursor ion tolerance to 20 ppm, and product ion tolerance window was set to 0.5 m/z. Searches allowed up to two missed cleavages, including static carbamidomethylation of cysteine residues (+57.021 Da) and variable oxidation of methionine residues (+15.995 Da). Additional variable modifications may be included, particularly glycosylations, based on the results of the gel shift assay following de-glycosylation treatment or the preview search PTM scan. Results were filtered to a 1% FDR at the protein level per sample.

Example 3—In Vitro Screening of Stem Cell Secreted Factors

A deeper understanding of a given protein factor's contribution to the regenerative effects of the pool of heparin-associated hPSC secretome will be gained by screening against an established panel of assays for cellular age. Assays include measurements of reactive oxygen species (ROS) production or tolerance, cytoplasmically and in the mitochondria, telomerase activity, measurements of proteostasis capacity via lysosomal, autophagy, and proteasomal routes, epigenetic re-patterning, and cellular energy balance (e.g., ATP/ADP and NAD/NADH ratios). Many of these assays leverage the high-throughput automated microscopy described above to make these measurements in a variety of cell types, including fibroblast, endothelial cells, mesenchymal stem cells, and chondrocytes. Collectively these metrics can inform both the pathway and the mechanisms by which the heparin-associated hPSC secretome or its individual components enact their regenerative effects.

To begin screening and validating potential regenerative protein factors protein coding sequences will be collected from a publicly available source, such as used for the proteomics analysis (e.g., UniProt). The sequence for each of the proteins will be used to construct a DNA sequence encoding the proteins. The sequences are then each cloned into a plasmid vector system tailored for inducible or constitutive high-copy expression (in mammalian or prokaryotic settings). Alternatively, such a plasmid vector system may be designed in silico. Such a plasmid can be transformed into a pool of cells where the encoded protein was transiently expressed from the plasmid. Alternatively, the gene of interest could be incorporated in the genomes of a pool of cells (e.g. lentiviral transduction for mammalian cells or homologous recombination for S. cerevisiae) to create stable cell lines for recombinant protein production.

To de-bulk the target factor list and validate recombinant expression for factor production, a viable route would be to express the construct in a human cell line (like 293T-REx), which exploits: 1) that proteins of interest naturally purify themselves during the secretion process; and 2) will be processed in a natural context, potentially preserving important post translational processing steps. T-REx cells will be grown on 10 cm plates to ˜50% confluence in DMEM with 10% Bovine Growth Serum (Hyclone), 2 mM L-glutamine, and 1% Pen-Strep before initiating translation of a target protein of interest for 48 hr. The media would be collected, spun at 2,000 g to purify, and the supernatant used for heparin-associated enrichment of target factors in mouse myoblast regeneration assays.

Machine Learning Classifier

By combining and statistically comparing the information from the Regenerative index assay, the Panel of Cellular Age Makers, the Proteomics we can create deep feature vectors for each protein factor, the pool of all factors (from each repeat of the assays), and the negative control pool (from each repeat of the assays). Treating the pool of all factors (or known factors such as FGF-2) as True Positives, and the negative control pool (or known non-functional proteins such as BSA) as True Negatives a supervised clustering algorithm can be trained to classify protein factors. Using a standard 10-fold cross validation scheme to assess the relative accuracy, recall, and confusion matrix graphs of the output of various algorithms' outputs (eg, Naive Bayes, Support Vector Machine, Linear Regression, or Random Forest) trained classifier most likely to successfully distinguish proteins with regenerative potential from the set of target factors can be selected. Target factors (or tested combinations) can then be rank ordered by the probability they derive from the regenerative set compared to the null set. A number of the top scoring target factors (or tested combinations) will then be selected for GMP-grade production for in vivo and in vitro validation.

Based on the complexity of the original heparin-associated fraction of the hPSC secretome and the limits to which individual proteins can recapitulate the activity of the whole pool, we will test combinations of factors as well. In the simplest approach, we would combine the 293T-REx secretome containing media from two or more cell lines each producing a given factor, and test their combined regenerative efficacy across a range of concentrations in an isobologram analysis using the regenerative index from the Myoblast Regeneration assay.

Example 4—In Vivo Testing of Stem Cell Secreted Factors

There are two main aspects of muscle degeneration with aging, acute loss following trauma and chronic wasting (sarcopenia), and both of them will be tested. As the therapeutic approach to each case is expected to be different two arms for the in vivo validation is envisioned to specifically test each use-case for the factors as therapeutics in humans. The following Acute Injury Model and Sarcopenia/Chronic Administration Model for the most promising proteins emerging from the machine learning classifier can be carried out.

Acute Injury Model

Animals were kept under standard animal husbandry condition. Animals were fed standard chow, and have ad libitum access to food and water. Temperature were kept at 22° C. and 12 h light/12 h dark cycles. Animals were acclimated prior to study initiation. The experimental groups were: C57BL/6J male mice, N=18; Young: 12-13 weeks old (3-month-old) mice, n=6; Aged: 77-78 weeks old (18-month-old) mice, n=12. This design was used to test any single factor identified and validated in in vitro assays or complex mixtures of 2 or more factors, as shown in FIG. 1.

TABLE 7

Experimental design of acute injury model

Dose
Adminis-
Blood
Tissue

Group

Test
(mg/kg)
tration
collection
collection

Young,
6
vehicle
n/a
i.m, q.d; into
terminal
TA/GA

Vehicle

injured

muscles into

Aged,
6
vehicle
n/a
muscle

Tissue-TEK

Vehicle

site on day

OCT; Brain,

Aged,
6
Test
0.1
0, and 2

liver, heart,

Test

factor

and lung into

factor

4% PFA

On Day 0, mice were weighed and underwent muscle injury with focal injection of barium chloride (BaCl₂, 14 μL, 1.2% w/v in saline, Sigma-Aldrich) in the Tibialis anterior (TA; Day 0) of both the right and left hindlegs. Injections of vehicle or factor A (0.1 mg/kg) were administered intramuscularly (i.m) 2 h following the BaCl₂into the TA injured hindleg sites, and again 48 hours later on day 2 (i.m.) into the TA injured hindleg sites. Also on day 2, BaCl₂(BaCl₂, 14 μL, 1.2% w/v in saline, Sigma-Aldrich) was injected into the Gastrocnemius (GA, Day 2, i.m.) muscles of both right and left hind legs. Injections of vehicle or a factor were sequentially administered (i.m.) following the BaCl₂into the TA hindleg sites post-injury, and again 48 hours later on day 4 (i.m.) into the GA injured hind leg sites. Bromodeoxyuridine (BrdU) was be administered (100 mg/kg, i.p.) once daily for 3 days, day 2-4, before sacrifice to label proliferating cells.

On day 5, animals were sacrificed, and animal weight recorded followed by collecting 0.5 ml of terminal blood via cardiac puncture which was processed to plasma and stored at 80° C. We then dissected the skin from the GA/TA muscles of each hind leg and took photos (prior to excision). After excision of exclusively the GA or TA muscle, excised tissue was photographed, weighed, then immersed in Tissue-TEK OCT and rapidly frozen in chilled isopentane bath precooled in liquid nitrogen and stored at −80 C. Cryosectioning and H&E were performed to ensure muscle injury site was appropriately visualized. A diagram of the time points for these experiments can be seen in FIG. 2A. Muscle tissue composition from new skeletal muscle fibers, fibrotic tissue, and adipose (fat), were measured. Muscle regeneration, as defined as the number of number of new myofibers with centrally located nuclei per millimeter, fibrosis as defined as the area of fibrotic scarring, size of the fibers, as defined as the width and area, adipose tissue, as defined by the amount of fat surrounding the muscle, were measured to assess level of regeneration.

Young muscle regeneration after acute focal injury had the highest regenerative index (measured as the number of new myofibers with centrally located nuclei per millimeter squared). Aged muscle regeneration following acute injuries performed on average 30-50% less than young muscle regeneration, in part due to the reduction of mitogenic muscle precursor/progenitor cell activity (FIG. 2B). It was predicted that systemic treatment of aged animals, injured animals, animals with genetic diseases causing muscle wasting, or animals injured by radiation or other tissue damaging treatment, the HAPs, individually or in various combinations, will improve skeletal muscle regeneration by 20-50%, approaching comparable levels to young healthy animals. It was also predicted that the composition of fibrous scar tissue and fat tissue will be reduced to levels comparable to younger animals by treatment with the HAPs. And indeed, treatment with the HAPs improved the skeletal muscle regeneration of sarcopenic mice to levels indistinguishable from the young by both the number of new myoblasts and the reduction in fibrous scar tissue (FIG. 2B).

Sarcopenia/Chronic Administration Model

After arrival, animals will be kept under standard animal husbandry condition. Animals will be fed standard chow, and have ad libitum access to food and water. Temperature will be kept at 22° C. and 12 h light/12 h dark cycles. Animals will be acclimated prior to study initiation, including any in vivo assay acclimation, if necessary. The experimental design was C57BL/6J male mice, N=18; Young: 12-13 weeks old (3-month-old) mice, n=6; Aged: 77-78 weeks old (18-month-old) mice, n=12. This design can be used to test any single factor identified and validated in in vitro assays or complex mixtures of 2 or more factors.

TABLE 8

Design of sarcopenia/chronic administration model

Adminis-

Blood
Tissue

Group
n
Test
(g/kg)
tration
In vivo assay
collection
collection

Young,
6
vehicle
n/a
i.p, q.d; on
2 sets of: Animal
terminal
TA/GA

Vehicle

day −8 to +5
weight, grip strength,

muscles into

Aged
6
vehicle
n/a

running wheel

Tissue-TEK

Vehicle

performance;

OCT; Brain,

Aged,
6
Test
0.1

horizontal

liver, heart,

Test

factor

bar; 1 set of:

and lung into

factor

In capacitance

4% PFA

On Day 0, mice will have the following in vivo healthspan measurements will be performed over 1 day as a baseline for age-based parameters: Weight, running wheel performance, grip strength, and horizontal bar. Each assay should be run for 4 trials per assay per animal. These healthspan assays will be repeated on day −1. After one day of rest on day −9, mice will begin 1× daily injections (0.1 mg/kg) of vehicle or factor A for the remainder of the experiment until sacrifice (days −8 to +5, 13 days of dosing). On day −4, 6 days after dosing begins, mice will undergo a repeat of the healthspan assays. On day 0, 5 days prior to sacrifice, mice will undergo muscle injury with focal injection of BaCl₂(BaCl₂, 14 μL, 1.2% w/v in saline, Sigma-Aldrich) in the Tibialis anterior (TA; Day 0) of the right hindleg only. On day 2, the Gastrocnemius (GA; Day 2) muscle of the right hind leg will then receive BaCl₂(BaCl₂, 14 μL, 1.2% w/v in saline, Sigma-Aldrich). BrdU will be administered (100 mg/kg, i.p.) once daily for 3 days, day 2-4, before sacrifice. On day +5, prior to take-down, the animals will have an in vivo in capacitance assay run. On day +5, animals will be sacrificed, and animal weight recorded. Collect 0.5 ml of blood via cardiac puncture, process to plasma and store plasma samples at 80° C. will then dissect the skin from the GA/TA muscles of each hind leg and take photos (prior to excision). After excision of exclusively the GA or TA muscle, weigh the muscles, then place the muscles in chilled isopentane bath precooled in liquid nitrogen and stored at −80 C. Perform cryosectioning and H&E, ensuring muscle injury site is appropriately visualized. Carefully excise the inguinal white adipose tissue (WAT) and weigh tissue. Discard WAT post-weighing.

Collected brain, liver, heart and lung can be post-fixed in 4% PFA for 72 hours, after 72 hours, transferred into 30% sucrose in 1×PBS and stored at −4° C. (brain, liver, heart, lung).

Muscle tissue composition, from new skeletal muscle fibers, fibrotic tissue, and adipose (fat), will be measured. Muscle regeneration, as defined as the number of number of new myofibers with centrally located nuclei per millimeter, fibrosis, as defined as the area of fibrotic scarring, size of the fibers, as defined as the width and area, adipose tissue, as defined by the amount of fat surrounding the muscle, will be measured to assess level of regeneration. Weights of the animals during the duration of treatment with HAP(s), as well as healthspan assays including performance on a running wheel (speed, distance, duration), grip strength, and performance on a horizontal bar will take into account the phenotypic outcomes of treatment of the aged animals systemically with the HAPs for sarcopenia.

The horizontal bar test is performed as described previously (Malinowska et al. 2010) at 8 months (n=6 WT, n=7 MPS IIIB) and 10 months (n=3 WT, n=4 MPS IIIB) of age. In brief, a 300-mm metal wire, 2 mm in diameter, was secured between two posts 320 mm above a padded surface. The mouse was allowed to grip the center of the wire and the time to fall or reach the side was recorded, and after 2 min the test was stopped. Crossing the bar in x seconds was scored as 240-x, remaining on the bar was scored as 120, and falling off the bar after y seconds was recorded as the value of y. The test was repeated three times as a practice run followed by a 10-min rest prior to three tests where the score was recorded.

Young muscle regeneration after acute focal injury has the highest regenerative index (measured as the number of new myofibers with centrally located nuclei per millimeter squared). Aged muscle regeneration following acute injuries performs on average 30-50% less than young muscle regeneration, in part due to the reduction of mitogenic muscle precursor/progenitor cell activity. We predict systemic treatment of aged animals, injured animals, animals with genetic diseases causing muscle wasting, or animals injured by radiation or other tissue damaging treatment, the HAPs, individually or in various combinations, will improve skeletal muscle regeneration by 20-50%, approaching comparable levels to young healthy animals. We also predict the composition of fibrous scar tissue and fat tissue will be reduced to levels comparable to younger animals by treatment with the HAPs.

Animals will also have better healthspan outcomes: reduced weight, fat composition, scar tissue around muscles, increased running speed, duration, and distance, increased grip strength, and enhanced performance on the horizontal bar test.

Genetically Obese Muscle Dystrophy Model

Genetically obese (ob/ob) mice were injected with BaCl₂on day 0 in the TA muscle. 3 mice were treated with vehicle only, 3 mice were injected with the hPSC factors and 3 mice were treated with FGF19 (positive control) on day 0 and day 2. On day 5, the mice were euthanized, the TA muscles perfused with PBS, and dissected, as depicted in FIG. 3A. Muscles were then analyzed for regenerative index and fibrotic index, as described in Example 1. Mice that had been treated with either the hPSC factors or FGF19 had a significant increase in the regenerative index and a significant decrease in the fibrotic index when compared to untreated mice, as depicted in FIG. 3B.

Methods of Testing Muscle Strength, Endurance and Function

Forelimb and Both limb grip strength test: After 30 min acclimation, the mice are introduced to the grip strength meter. For forelimb grip strength, the mice held by the tail are allowed to grasp the grip bar with only its forelimbs. For both limb measurements the mice are placed on the grid and allowed to grasp the grid with both limbs. The force generated by each mouse is calculated as the average of 5-6 measurements.

Limb endurance test: Mice are allowed to discover and acclimate the rodent treadmill environment through 2 training sessions of 10 min each at 10 m/min on separate days prior to the endurance test. For the endurance test, mice are placed in the individual lanes of the rodent treadmill. The speed is gradually increased at 2 m/min until exhaustion is reached. Exhaustion is defined as a mouse staying on a grill electrified to deliver a shock of 2 Hz, intensity 5 for 3-5 seconds.

In vivo tetanic force measurement: Mice will be under anesthesia using regulated delivery of isoflurane during the whole process. Following anesthetization, the animal is placed onto a heated chamber with the foot is secured on the foot pedal of an Aurora force transducer. The 2 electrodes are placed specifically to stimulate the sciatic nerve. The force generated by the ankle torsion of the animal's hind limb, as opposed to direct force is measured in response to a series of stimulation that includes 20, 50, 70, 100, 150 and 200 Hz.

In situ tetanic force measurement: This experiment is performed using Aurora force measurement. Mice are under anesthesia during the whole process. A small incision in the skin around the Anterior Tibialis exposes the Achilles tendon which is connected via surgical suture to the Aurora force transducer through a hook. The force generated by the muscle in response to a series of stimulation that includes 20, 50, 70, 100, 150 and 200 Hz by 2 electrodes placed on the anterior tibialis is recorded.

Example 5-Additional Tests for Pro-Regenerative Factors

Mechanistic insight into a given HAP factor's pathway of action will be gained by establishing and screening against a panel of assays for cellular age. Assays include measurements of reactive oxygen species (ROS) production or tolerance cytoplasmically and in the mitochondria, telomerase activity, measurements of proteostasis capacity via lysosomal, autophagy, and proteasomal routes, epigenetic re-patterning, and cellular energy balance (e.g., ATP/ADP and NAD/NADH ratios). Many of these assay leverage high-throughput automated microscopy to make these measurements in a variety of cell types, including fibroblast, endothelial cells, mesenchymal stem cells, and chondrocytes. Collectively these metrics can inform both the pathway and the mechanisms by which the heparin-associated hPSC secretome or its individual components enact their regenerative effects. These deep profile vectors can be crucial for approaching combinations of factors rationally, and for machine learning predictions.

To test the cellular effects of secretomes toward reversing the hallmarks of aging, high-throughput automated imaging and quantification of single cells to achieve deep population level statistical power can be employed. Cellular component state profiles of Young, Aged, and Aged+Treatment in human fibroblasts and epithelial cells, myoblasts, mesenchymal stem cells, chondrocytes, and neural progenitor cells will be compared. Some examples of tests and methods include:

Epigenetic reprogramming: repressive mark H3K9me3, the heterochromatin-associated protein HP1γ, nuclear lamina support protein LAP2α

Nuclear membrane Folding/Blebbing: immunofluorescence of the nuclear membrane protein Lamin A/C

Proteolytic Activity: Cleavage of fluorescent-tagged chymotrypsin like substrate corresponds to proteasome 20S core particle activity. Wells are first stained with PrestoBlue Cell Viability dye (Life Technologies) for 10 minutes. Well signals are read using a TECAN fluorescence plate reader as a measure of cell count. Then cells are washed with HBSS/Ca/Mg before switching to original media containing the chymotrypsin like fluorogenic substrate LLVY-R110 (Sigma) which is cleaved by the proteasome 20S core particle. Cells are then incubated at 37° C. in 5% CO2 for 2 hours before signals are again read on the TECAN fluorescence plate reader. Readings are then normalized by PrestoBlue cell count.

Formation of autophagosomes: Autophagosome number and volume are measured by staining with CellTracker Deep Red (Sigma). The cells are then incubated at 37° C. in 5% CO2 for 20 minutes, washed 2 times using HBSS/Ca/Mg, and stained for 15 minutes using CellTracker Deep Red cell labeling dye. Cells are then switched to HBSS/Ca/Mg for single cell imaging using the Operetta High Content Imaging System (Perkin Elmer).

Energy Metabolism: ATP in the cells is measured using colorimetric assay using an ATP assay kit (ab83355; Abcam, Cambridge, MA) following manufacturer's instructions. Cells are washed in cold phosphate buffered saline and homogenized and centrifuged to collect the supernatant. The samples are loaded with assay buffer in triplicate. ATP reaction mix and background control (50 μL) is added to the wells of a 96 well plate and incubated for 30 min in dark. The average change in absorbance at 570 nm is used to estimate of the intracellular ATP concentration relative to the standard curve.

Mitochondrial Activity: To measure Mitochondria Membrane Potential, cells are washed twice with Ham's F10 (no serum or pen/strep). Subsequently, MuSCs are stained with MitoTracker Green FM (ThermoFisher, M7514) and DAPI for 30 minutes at 37° C., washed three times with Ham's F10, and analyzed using a BD FACSAria III flow cytometer. To measure Mitochondrial ROS Measurement. Cells are washed with HBSS/Ca/Mg and then switched to HBSS/Ca/Mg containing MitoSOX (Thermo), a live cell permeant flurogenic dye that is selectively targeted to mitochondria and fluoresces when oxidized by superoxide. Cells are incubated for 10 minutes at 37° C. in 5% C02. Cells are then washed twice with HBSS/Ca/Mg, and stained for 15 minutes using CellTracker Deep Red. Finally, cells are imaged in fresh HBSS/Ca/Mg using the Operetta High Content Imaging System (Perkin Elmer).

Deregulated Nutrient Sensing: levels of SIRT1 are measured.

Senescence: Senescence-associated beta-galactosidase staining is measured in cells washed twice with PBS then fixed with 15% Paraformaldehyde in PBS for 6 minutes. Cells are rinsed 3 times with PBS before staining with X-gal chromogenic substrate, which is cleaved by endogenous Beta galactosidase. Plates are kept in the staining solution, Parafilmed, to prevent from drying out, and incubated overnight at 37° C. with ambient CO2. The next day, cells are washed again with PBS before switching to a 70% glycerol solution for imaging under a Leica brightfield microscope.

Secretome of the cells: Mass-Spec or O-Link for inflammatory cytokines profiles.

Soft Tissue Deposition: Immunofluorescence for SOX9, MMP3, MMP13, and COL2A1 expression, the decrease of which is characterized by cartilage loss, pain, cleft-lip, and joint destruction.

Example 6—Identification of Pro-Regenerative Factors by Mass Spectroscopy

Factors enriched in the secretome of undifferentiated hPSCs can be determined by Mass spectroscopy. A schematic of a type of mass spectroscopy experiment employed herein is shown in FIG. 3A.

Five confluent, 15 cm plates of cells per biological replicate were washed with OptiMEM—a basal, synthetic medium-, and then incubated in OptiMEM for 16 hours, yielding roughly 100 ml of media. The media, now containing secreted factors, was collected, cells and cell debris removed by centrifugation, and flash frozen for storage at −80 C until processing. The target factors were enriched via affinity purification for heparin binding using heparin-agarose bead columns. Heparin-agarose beads (Sigma) were washed with water twice, and once with OptiMEM (minus phenol red), before incubating with factor containing culture media for 2 h at 4° C. shaking at 100 rpm. The ratio of bead slurry (˜50% beads) to media can be effective at 1:10, 1:20, 1:30, 1:40, and 1:50. Heparin-agarose beads were then collected into a column by centrifugation in an Amicon Pro Purification System column set in a 50 ml conical tube at 1000 g for 5 min, washed with 10× column volumes of PBS+0.05% tween at 4° C. twice. Factors were eluted via two repeats of the following: addition of a high salt solution (1.5M NaCl, 0.01M HEPES, pH 7.2, at ratio of 0.4 ml elution buffer per milliliter of bead slurry), incubated at 4° C. for 10 minutes at 100 rpm, and centrifugation at 1000 g for 5 min into a fresh collection tube.

Protein concentration in the eluted fraction was assayed by silver stain densitometry, and a BCA assay against standard curves for bovine serum albumin. Protein disulfide bonds were reduced by incubation in 5 mM tris-(2-carboxyethyl)-phosphine (TCEP) for 25 min, and the free cysteines alkylated with 10 mM iodoacetamide at room temperature for 30 min in the dark. Excess iodoacetamide was quenched with 15 mM dithiotreitol during a 15 min incubation. The eluates from all samples were then further purified by protein precipitation using trichloroacetic acid, prior to resuspending in digest buffer and 16 hr of digestion using a mixture of modified Trypsin and LysC to yield peptides predominantly with terminal arginine or lysine residues. The resulting peptide concentration were measured using a quantitative colorimetric peptide assay (Promega), and equimolar amounts of peptides from each biological replicate labeled at their free amines with tandem mass tags (TMT) using manufacturer recommended conditions before mixing the peptides. The mixed sample was desalted via reverse phase separation on a C18 StageTip prior to analysis via nHPLC-SPSMS3 on a Fusion Lumos (Thermo Fisher). A TOP10 method was used to select up to 10 MS2 precursors for identification by CID-MS2 analyzed in the ion trap. For synchronous precursor selection of up to 10 ion windows, the FTMS3 isolation window was 0.4 m/z, max ion time 150 ms, automatic gain control 1.5E5, and FTMS3 resolution was 50,000. Resulting spectra were searched using commercial MS analysis software against the Uniprot human database (2018) protein sequences (Swiss-Prot and TrEMBL) concatenated with their reversed sequences as decoys for FDR determination, appended to common contaminant sequences. Searches restricted the precursor ion tolerance to 50 ppm and the product ion tolerance window to 0.9 m/z (or 50 ppm), allowed no more than two missed cleavages, included static modification of lysine residues, arginine residues and peptide N-termini with TMT tags (+229.163 Da), static carbamidomethylation of cysteine residues (+57.021 Da), and variable oxidation of methionine residues (+15.995 Da).

Results were filtered to a 1% FDR at the peptide and then protein level using the target-decoy strategy. Peptides were assigned to protein groups, and individual proteins by the parsimony principle. Proteins were quantified by summing reporter ion intensities across all PSMs with greater than 70% of their spectral intensity deriving from matched ions and a summed signal to noise intensity greater than 200, normalizing channel level intensities, and computing the percent contribution of a given channel to the total signal. These values were then used for additional statistical modelling of differential abundance.

Heparin-associated proteins from undifferentiated and differentiated supernatants generated distinct sets of secreted factors. Combined results from such experiments are summarized in Table 2 shown previously herein, by the gene name, UniProt ID, Entrez Gene ID, and Ensembl ID.

Example 7—Validation of Pro-Regenerative Factors In Vitro Using High-Throughput Imaging

Mass spectroscopy can define candidate pro-regenerative factors, however, as shown in example 6, these experiments can generate large amounts of data that need to be further validated in relevant in vitro and in vivo models. The use of high-throughput imaging can help define individual factors and mixtures of factors that possess regenerative potential. Mouse muscle progenitor cells can be cultured with BrdU or Edu, in the presence or absence of specific potential pro-regenerative factors, and the degree of proliferation determined using high-throughput microscopy. BrdU or Edu staining indicates proliferation, while embryonic Myosin Heavy-Chain (eMyHC) staining indicates terminal differentiation of the progenitor cells. FIGS. 4A-4B show an example of data generated using high-throughput imaging.

Many of the factors detailed in Table 2 were tested individually for their ability to promote proliferation and/or fusion of mouse muscle progenitor cells in vitro. FIGS. 5A-5B show that there was a significant increase in the percent of proliferating cells of injury-activated primary mouse myoblasts grown in vitro for the hPSC factors, IGFBP7, POSTN, SPON1, MST1, RARES2, VTN, FGF1, IGF2, FGF4, FGF6, and FGF7, when compared to untreated cells. FIG. 5C shows that treatment with THBS1, THBS2, and STC2 resulted in a significant increase in the percent of fusion in injury activated primary mouse myoblasts, compared to untreated cells.

The factors were also able to affect proliferation and/or fusion in both young (18 years) and old (69 years) injury-activated primary human myoblasts. FIG. 6A shows that treatment with IGFBP5 at 1 μg/mL resulted in an increase in the percent of proliferating cells in both young and aged human myoblasts, compared to untreated myoblasts. FIG. 6B shows that treatment with THBS4 at 1 μg/mL resulted in an increase in the percent of proliferating cells in both young and aged human myoblasts, compared to untreated myoblasts. FIG. 6C shows that treatment with VTN at 10 μg/mL resulted in an increase in the percent of proliferating cells in both young and aged human myoblasts, compared to untreated myoblasts. FIG. 6D shows that treatment with FGF17 at 250 ng/mL resulted in an increase in the percent of proliferating cells in both young and aged human myoblasts, compared to untreated myoblasts. FIG. 6E shows that treatment with IGFBP7 T 500 NG/Ml resulted in an increase in the percent of fusion in both young and aged human myoblasts, compared to untreated myoblasts. FIG. 6F shows that treatment with 1 μg/mL of POSTN resulted in an increase in the percent of fusion in both young and aged human myoblasts, compared to untreated myoblasts. FIG. 6G shows that treatment with 5 μg/mL of PDGFRL resulted in an increase in the percent of fusion in both young and aged human myoblasts, compared to untreated myoblasts.

FIG. 7 shows an example of the dose dependent increasing cellular fusion of mouse myoblasts cultured with a HAP. In this case Platelet derived growth factor-like (PDGFRL) proteins were applied at 625 ng/mL, 1250 ng/mL, 2500 ng/mL, 5000 ng/mL, and 10000 ng/mL.

The effect of the candidate factors on myogenic activity was assayed in biological triplicate across a range of concentrations centered around expected physiological levels by adding each factor to mouse myoblasts for 48 hours or human myoblasts for 72 hours with daily media changes (DMEM +2% horse serum) and a second pulse of factors. After 24 hours, cells were pulsed for 2-5 hours with EdU (30 uM), ethanol fixed, stained with Hoescht 3342, immunostained for proliferation—as measured by the percent of cells staining positive for EdU (% EdU)−, and immunostained for differentiation—as measured by the increase in cellular area staining positive for embryonic myosin heavy chain (% eMyHC) relative to the negative controls, which received media and vehicle only. Wells were imaged on a Keyence BZ-100 at 4×, the images quantified in Cell Profiler, and the statistics were computed in R. FIGS. 8A-8B show an example of the proliferation dose response for two of the factors tested. Results for additional factors are summarized below in Table 9.

TABLE 9

Effect of individual factors on mouse myoblast growth and fusion

Proliferation
Fusion

(¹= % EdU or ²= nuclei counts)
(% eMyHC)

Effect size
Effect
Effect size
Effect

Concentration
(fold-change
significance
(fold-change
significance

Factor
Effect
(ug/mL)
to control)
(p-value)
to control)
(p-value)

AGRN
Proliferation²
2.5
1.14
0.01008
0.46
n.s.

APOB
Proliferation¹
0.1
1.28
0.008202
1.01
n.s.

BMP7
Proliferation¹
0.025
1.71
0.001448
0.17
n.s.

BTC
Proliferation¹/
0.5
1.4
0.03395
1.7
n.s.

Fusion

CHRDL1
Proliferation¹
2.5
1.62
0.004249
0.85
n.s.

CLEC3A
Proliferation¹
0.04
1.35
0.005224
1.09
n.s.

FGF1
Proliferation¹
0.5
2.89
0.005553
0.84
n.s.

FGF17
Proliferation¹
0.5
4.50
0.01809
0.09
n.s.

FGF4
Proliferation¹
0.5
4.01
0.01363
0.25
n.s.

FGF6
Proliferation¹/
1
3.8
0.007639
1.85
0.02367

Fusion

HGF
Proliferation¹
0.5
2.04
1.56E−05
0.39
n.s.

IGF2
Proliferation¹
2
1.66
0.0239
1.46
n.s.

IGF2
Proliferation²
0.03
2.28
0.0004336
—
—

IGFBP2
Proliferation¹
1
1.97
0.04376
1.03
n.s.

IGFBP7
Proliferation¹/
1
1.23
0.01132
1.05
0.04213

Fusion

IL15
Proliferation¹/
0.5
1.85
0.004579
2.03
0.02273

Fusion

MST1
Proliferation¹
1
1.36
0.03868
1.25
n.s.

NOV
Proliferation²
5
1.26
0.03359
0.65
n.s.

NTS
Proliferation¹
0.2
1.29
0.006777
1.1
n.s.

PAMR1
Fusion
0.4
0.88
n.s.
1.27
0.002882

PDGFD
Proliferation¹
0.1
1.47
3.94E−02
0.97
n.s.

PLAT
Proliferation²
2.2
1.27
0.02239
0.68
n.s.

POSTN
Fusion
0.25
0.65
n.s.
1.5
0.002679

THBS1
Proliferation¹/
1
1.26
0.003634
1.39
0.0008755

Fusion

THBS2
Proliferation¹/
1
1.54
n.s.
1.34
3.94E−05

Fusion

THBS4
Proliferation¹/
2
1.87
0.0163
1.23
0.0004685

Fusion

VTN
Proliferation¹
5
1.56
0.01231
1.01
n.s.

Proliferation
Fusion

(¹= % EdU or ²= nuclei counts)
(% eMyHC)

Effect

Effect

Effect size
significance
Effect size
significance

Concentration
(fold-change
(p-value,
(fold-change
(p-value,

Factor
Effect
(ug/mL)
to control)
t-test)
to control)
t-test)

AGRN
Proliferation²
2.5
1.14
0.01008
0.46
n.s.

APOB
Proliferation¹
0.1
1.28
0.008202
1.01
n.s.

BMP7
Proliferation¹
0.025
1.71
0.001448
0.17
n.s.

BTC
Proliferation¹/
0.5
1.4
0.03395
1.7
n.s.

Fusion

CHRDL1
Proliferation¹
2.5
1.62
0.004249
0.85
n.s.

CLEC3A
Proliferation¹
0.04
1.35
0.005224
1.09
n.s.

FGF1
Proliferation¹
0.5
2.89
0.005553
0.84
n.s.

FGF17
Proliferation¹
0.5
4.50
0.01809
0.09
n.s.

FGF4
Proliferation¹
0.5
4.01
0.01363
0.25
n.s.

FGF6
Proliferation¹/
1
3.8
0.007639
1.85
0.02367

Fusion

HGF
Proliferation¹
0.5
2.04
1.56E−05
0.39
n.s.

IGF2
Proliferation¹
2
1.66
0.0239
1.46
n.s.

IGF2
Proliferation²
0.03
2.28
0.0004336
—
—

IGFBP2
Proliferation¹
1
1.97
0.04376
1.03
n.s.

IGFBP7
Proliferation¹/
1
1.23
0.01132
1.05
0.04213

Fusion

IL15
Proliferation¹/
0.5
1.85
0.004579
2.03
0.02273

Fusion

MST1
Proliferation¹
1
1.36
0.03868
1.25
n.s.

NOV
Proliferation²
5
1.26
0.03359
0.65
n.s.

NTS
Proliferation¹
0.2
1.29
0.006777
1.1
n.s.

PAMR1
Fusion
0.4
0.88
n.s.
1.27
0.002882

PDGFD
Proliferation¹
0.1
1.47
3.94E−02
0.97
n.s.

PLAT
Proliferation²
2.2
1.27
0.02239
0.68
n.s.

POSTN
Fusion
0.25
0.65
n.s.
1.5
0.002679

THBS1
Proliferation¹/
1
1.26
0.003634
1.39
0.0008755

Fusion

THBS2
Proliferation¹/
1
1.54
n.s.
1.34
3.94E−05

Fusion

THBS4
Proliferation¹/
2
1.87
0.0163
1.23
0.0004685

Fusion

VTN
Proliferation¹
5
1.56
0.01231
1.01
n.s.

The effect of the combination of candidate factors on myogenic activity was assayed in biological triplicate across a range of concentrations centered around expected physiological levels by adding each factor to mouse myoblasts for 48 hours or human myoblasts for 72 hours with daily media changes (DMEM +200 horse serum) and a second pulse of factors. After 24 hours, cells were pulsed for 2-5 hours with EdU (30 uM), ethanol fixed, stained with Hoescht 3342, immunostained for proliferation—as measured by the percent of cells staining positive for EdU (% EdU)−, and immunostained for differentiation—as measured by the increase in cellular area staining positive for embryonic myosin heavy chain (% eMyHC) relative to the negative controls, which received media, individual factors, or vehicle only. Wells were imaged on a Keyence BZ-100 at 4×, the images quantified in Cell Profiler, and the statistics were computed in R. FIGS. 8C-SM show examples of the proliferation dose response for two or more of the factors tested individually and as a combination to test for synergy. Statistical metrics for increased myogenetic activity from the pair of factors relative to the controls are summarized below in Tables 10-14. Each polypeptide was produced using the method listed in Table 1. The magnitude of the combinations' effects relative to control (FM—negative control, HAPs—positive control) is shown. The Combination Index (CI) for synergy was calculated using the Highest Single Agent (HAS) model due to the linear dose responses for the individual factor, e.g. FIG. 8B. Tables 10-15 show additional data regarding the synergistic effects of the HAPs in both mice and human myoblasts.

TABLE 10

Synergistic combinations of HAPs

Factor 1
Factor 2
Factor 3

CI

Name
ug/mL
Name
ug/mL
Name
ug/mL
p-value
(HSA)

THBS2
0.125
THBS4
0.25
VTN
1
<0.05
0.886

THBS2
0.125
THBS4
0.25
ANOS1
1
<0.005
0.532

THBS2
0.125
THBS4
0.25

<0.005
0.633

THBS1
0.0625
FGF17
0.025

<0.005
0.667

THBS1
0.125
FGF17
0.1

<0.005
0.679

THBS2
0.125
THBS4
0.25
IL-15
1
<0.005
0.680

THBS2
0.125
THBS4
0.25
IGF2
0.05
<0.05
0.733

THBS2
0.0625
VTN
1.25

<0.005
0.763

THBS2
0.125
VTN
1.25

<0.005
0.763

THBS2
0.125
VTN
0.3125

<0.005
0.771

THBS2
0.0625
THBS4
0.125

<0.005
0.772

THBS2
0.125
THBS4
0.0625

<0.005
0.779

THBS1
0.0625
FGF17
0.1

<0.005
0.796

THBS1
0.0625
VTN
0.315

<0.005
0.840

THBS2
0.0625
THBS4
0.25

<0.005
0.850

THBS1
0.25
THBS2
0.25

<0.05
0.859

THBS2
0.0625
FGF17
0.1

<0.005
0.871

THBS2
0.0625
VTN
0.625

<0.005
0.876

THBS2
0.125
THBS4
0.125

<0.005
0.878

THBS1
0.0625
VTN
1.25

<0.05
0.880

THBS1
0.25
THBS4
0.0625

<0.005
0.888

THBS2
0.0625
FGF17
0.025

<0.005
0.890

THBS1
0.0625
THBS2
0.0625

<0.05
0.900

THBS1
0.25
VTN
1.25

<0.005
0.907

THBS2
0.125
FGF17
0.025

<0.005
0.913

VTN
0.3125
FGF17
0.025

<0.05
0.915

THBS4
0.25
VTN
0.3125

<0.05
0.922

THBS2
0.125
FGF17
0.1

<0.005
0.923

THBS1
0.25
VTN
0.625

<0.005
0.930

THBS2
0.0625
THBS4
0.0625

<0.05
0.942

THBS4
0.25
FGF17
0.1

<0.005
0.945

THBS4
0.0625
VTN
0.625

<0.05
0.950

VTN
0.625
FGF17
0.1

<0.005
0.952

THBS4
0.25
VTN
1.25

<0.05
0.954

TABLE 11

Additional data regarding synergistic combinations of HAPs (mouse myoblasts)

Single

Combo

Dose

Dose

Single
Single
% EdU

% EdU

Dose @
Dose
Fold
Single
Combo
Combo
Fold
Combo

Factor
saturation
% of
Change
Dose
Dose
Dose %
Change
Dose

Name
(ug/mL)
EB
to FM
% EdU
(ug/mL)
of EB
to FM
% EdU
CI_type
CI_Value

THBS1
2
50%
1.70
15%
0.0625
64%
1.16
14%
HSA
0.679

FGF17
0.5
239%
4.50
69%
0.1

148%
2.89
33%

combo

163%
3.63
29%
BI
0.194

THBS2
2
50%
1.25
13%
0.125
46%
0.88
10%
HSA
0.633

THBS4
2
40%
1.87
10%
0.25
53%
1.11
12%

Combo

71%
1.39
17%
BI
0.729

THBS1
2
50%
1.70
15%
0.0625
44.08%
0.75
10.63%
HSA
0.852

VTN
10
50%
1.50
16%
10
59.18%
1.01
14.27%

Combo

69.47%
1.19
16.75%
BI
0.845

THBS2
1
50%
1.54
13%
0.125
31.08%
0.85
7.86%
HSA
0.680

THBS4
2
40%
1.87
10%
0.25
29.33%
0.80
7.41%

IL15
0.5
52%
1.85

1
47.25%
1.28
11.90%
BI
1.239

Combo

57.91%
1.66
16.21%

THBS2
1
50%
1.54
13%
0.125
45.08%
0.79
9.64%
HSA
0.886

THBS4
2
40%
1.87
10%
0.25
61.15%
1.08
13.08%

VTN
10
50%
1.50
16%
10
54.46%
0.94
12.67%
BI
1.650

combo

69.02%
1.22
14.76%

THBS2
1
50%
1.54
13%
0.125
58.62%
0.86
15.23%
HSA
0.733

THBS4
2
40%
1.87
10%
0.25
62.99%
0.93
16.37%

IGF2
0.03
89%
2.28

0.5
85.78%
1.27
22.29%
BI
1.184

combo

116.95%
1.72
30.39%

FGF17
0.5
50%
1.70
15%
0.0125
49.74%
1.05
12.10
HSA
0.725

BMP7
0.025
117%
1.71
18%
0.0125
90.39%
1.91
21.98

combo

124.73%
2.63
30.33
BI
0.363

IGF2
0.03
89%
2.28
23%
0.012
41.54
1.69
8.04
HSA
0.73

BMP7
0.025
117%
1.71
18%
0.0125
112.33
4.39
21.73

combo

153.16
6.22
29.63
BI
−0.23

TABLE 12

Additional data regarding synergistic combinations of HAPs (mouse myoblasts)

Combo Dose

Single Dose
Single Dose
Single Dose
Combo Dose
Combo Dose
% EdU Fold
Combo Dose

Factor
(ug/mL)
% of Eb
% EdU
(ug/mL)
% of EB
Change to FM
% EdU

THBS1
2
50%
15%
0.125
64%
1.16
14%

FGF17
0.5
239%
69%
0.1

148%
2.89
33%

Combo

163%
3.63
29%

THBS2
2
50%
13%
0.125
46%
0.88
10%

THBS4
2
40%
10%
0.25
53%
1.11
12%

Combo

71%
1.39
17%

THBS1
2
50%
15%
0.0625
44.08%
0.75
10.63%

VTN
10
50%
16%
10
59.18%
1.01
14.27%

Combo

69.47%
1.19
16.75%

THBS2
2
50%
13%
0.125
31.08%
0.85
7.86%

THBS4
2
40%
10%
0.25
29.33%
0.8
7.41%

IL15
0.5
52%
12.50%
1
47.25%
1.28
11.90%

Combo

57.91%
1.66
16.21%

THBS2
2
50%
13%
0.125
45.08%
0.79
9.64%

THBS4
2
40%
10%
0.25
61.15%
1.08
13.08%

VTN
10
50%
16%
1
54.46%
0.94
12.67%

Combo

69.02%
1.22
14.76%

TABLE 13

Additional data regarding combinations of proteins with synergistic regenerative

effects in human myoblasts as measured by nuclei counts per well

Single dose

Combo dose

Nuclei

Nuclei

Counts

Counts

@

Fold

% of EB
Fold

Factor
saturation
% of
Change
Nuclei

Dose
(8x EB
Change
Nuclei
HSA

Name
(ug/mL)
EB
to FM
Counts

(ug/mL)
@ 856)
to FM
Counts
CI

THBS2
2
82.21
1.16
5000
THBS2/
0.5/0.5
187.85
1.13
1608

THBS4

THBS4
1
58.90
0.83
3582
IGF2
0.1
219.45
1.32
1879

IGF2
1
120.84
1.07
5532
THBS2/

239.08
1.44
2047
0.92

THBS4/

IGF2

THBS2
2
82.21
1.16
5000
THBS2/
0.5/0.5
187.85
1.13
1608

THBS4

THBS4
1
58.90
0.83
3582
IGF2
0.25
229.09
1.38
1961

IGF2
1
120.84
1.07
5532
THBS2/

278.39
1.67
2383
0.82

THBS4/

IGF2

THBS2
2
82.21
1.16
5000
THBS2/
0.5/0.5
187.85
1.13
1608

THBS4

THBS4
1
58.90
0.83
3582
IGF2
0.5
236.57
1.42
2025

IGF2
1
120.84
1.07
5532
THBS2/

291.41
1.75
2495
0.81

THBS4/

IGF2

THBS2
2
82.21
1.16
5000
THBS2/
0.125/0.125
192.52
1.16
1648

THBS4

THBS4
1
58.90
0.83
3582
IL15
0.125
202.22
1.22
1731

IL15
0.5
117.58
1.04
5383
THBS2/

234.40
1.41
2007
0.86

THBS4/

IL15

TABLE 14

Additional data regarding synergistic combinations of HAPs (human myoblasts)

Single

Combo

Dose

Dose

Single
Single
% EdU

Combo
% EdU

Dose @
Dose
Fold
Single
Combo
Dose
Fold
Combo

Factor
saturation
% of
Change
Dose
Dose
% of
Change
Dose
HSA

Name
(ug/mL)
EB
to FM
% EdU
(ug/mL)
EB
to FM
% EdU
CI

THBS1
7.5
98.93
1.61
10.99
0.5
78.22
0.85
8.95

FGF17
0.25
222.09
3.62
24.67
0.05
121.74
1.33
13.93

Combo

157.29
1.72
18
0.77

THBS2
7.5
112.8469
1.2
12.53448
0.125
57.09
0.95
7.82

THBS4
0.5
126.7002
2.07
14.07323
0.25
62.67
1.04
8.59

IL15
0.1
67.81
1.13
9.29
0.1
67.81
1.13
9.29

Combo

69
1.15
9.46
0.98

TABLE 15

Additional data regarding synergistic combinations of HAPs (human myoblasts)

Single

Combo

Dose

Dose

Single
Single
% EdU

Combo
% EdU
Combo

Dose @
Dose
Fold
Single

Combo
Dose
Fold
Dose

Factor
saturation
% of
Change
Dose

Dose
% of
Change
Dose
HSA

Name
(ug/mL)
EB
to FM
% EdU

(ug/mL)
EB
to FM
% EdU
CI

THBS1
7.5
98.93
1.61
10.99
THBS1
0.5
78.22
0.85
8.95

FGF17
0.25
222.09
3.62
24.67
FGF17
0.05
121.74
1.33
13.93

THBS1/
0.5/0.05
157.29
1.72
18.00
0.77

FGF17

THBS1
7.5
—
1.30
12.46
THBS1
0.125

0.989
11.80

FGF17
1
—
1.50
14.80
FGF17
0.025

1.09
12.80

THBS1/
0.125/0.025

1.281
14.30
0.85

FGF17

Example 8—BMP7 Induces Myoblast Proliferation

Mouse myoblast cells were cultured for 48 hours in the presence of BMP 7 at either 0.025 μg/mL, 0.075 μg/mL, 0.22 μg/mL, 0.45 μg/mL, 0.45 μg/mL, 0.9 μg/mL, 1.8 μg/mL, or vehicle only. Fresh media and BMP7 was added every 24 hours. After 48 hours, the cells were stained for EdU, Hoescht and eMyHC using methods similar to those in Example 1. Representative images of the images are seen in FIG. 9A. BMP7 treatment resulted in increased proliferation in the mouse myoblasts, as seen by the increase in EdU positive cells. The number of EdU positive nuclei was quantified, and this information is displayed in FIG. 9B and Table 16. Most doses of BMP7 resulted in an increase in the percent of EdU positive nuclei when compared to myoblasts treated with the vehicle alone. In particular, treatment with 0.025 μg/mL, 0.075 μg/ml, 0.2255 μg/mL, and 0.9 μg/ul resulted in a significant change in the percentage of EdU positive nuclei, indicating an increase in myoblast proliferation. Significance was determined by a Welch's one-tailed t-test with a p value <0.05.

TABLE 16

BMP7-induced proliferation in mouse myoblasts

BMP7
% EdU
p-value

Vehicle
10.56
—

0.025
ug/mL
18.08
1.21E−07

0.075
μg/mL
17.53
8.05E−07

0.2255
μg/mL
14.93
3.37E−03

0.45
μg/mL
12.93
n.s.

0.9
μg/mL
13.91
4.99E−02

1.8
μg/mL
10.37
n.s.

BMP7 treatment also induced proliferation of human myoblasts. Human myoblasts were cultured with BMP7 at a dose of either 0.78 ng/ml, 1.56 ng/ml, 3.12 ng/ml, 6.25 ng/mL. 12.5 ng/ml, or 25 ng/mL for 72 hours. Media and BMP7 was changed every 24 hours. After 72 hours, cells were stained and imaged for EdU as described in Example 1. The number of EdU positive nuclei was quantified and this information is displayed in FIG. 9C and Table 17 However, only the 1.56 ng/mL dose of BMP7 resulted in a significant increase in myoblast proliferation compared to cells treated with vehicle alone.

TABLE 17

BMP7-induced proliferation in human myoblasts

Nuclei

Human Myoblast
Counts
p-value

Vehicle
2083.5
—

0.78
ng/mL
2144.5
n.s.

1.56
ng/mL
2552.5
0.04

3.12
ng/mL
2408
n.s.

6.25
ng/mL
2422.5
n.s.

12.5
ng/mL
2706
n.s.

25
ng/mL
2509
n.s.

Example 9—IGF2 and BMP7 Combination Treatment is More Potent than the Single Factors Individually

Mouse myoblasts were cultured in media and with either PBS(vehicle), hPSC, IGF2, BMP7, or IGF2 and BMP7 combined. Fresh media and factors were added every 24 hours. After 48 hours, cells were stained and imaged for EdU as described in Example 1. The percent of EdU positive nuclei were quantified, as shown in FIG. 10A and Table 18. Compared to untreated cells, treatment with hPSC factors, BMP7, and BMP7/IGF2 resulted in a significant fold-change in proliferation. However, the combination treatment of BMP7/IGF2 resulted in the greatest fold-change of all treatment conditions, with a fold change of 6.22.

TABLE 18

BMP7-and IGF2-induced proliferation in mouse myoblasts

Factor

% EdU Fold

Name
(ug/mL)
Change
P-value

IGF2
0.012
1.69
n.s.

BMP7
0.006
4.56
1.39E−03

Combined

6.22
3.82E−06

Female and male myoblasts were cultured in media and with added factors. The factors consisted of either PBS (vehicle only), hPSC factors, IGF2, BMP7, or a combination of IGF2 and BMP7. After 72 hours, cells were stained and imaged for EdU as described in Example 1. The number of EdU positive nuclei was quantified in female myoblasts, application of either the hPSC factors or the combined IGF2 and BMP7 resulted in a significant increase in the number of EdU positive nuclei, while treatment with either factor alone did not result in a significant increase in proliferation, when compared to cells cultured with vehicle alone, as depicted in FIG. 10B and Table 19.

TABLE 19

BMP7- and IGF2-induced proliferation in human female myoblasts

Factor

Nuclei

Name
(ug/mL)
Counts
P-value

IGF2
0.03
1331
n.s.

BMP7
0.04
1041
n.s.

Combined

1546
8.32E−08

In human male myoblasts, there was a significant increase in nuclei count fold change when comparing either the hPSC factors or the combined IGF2/BMP7 condition to the fusion media, as depicted in FIG. 10C and Table 20. Treatment with either factor alone did not result in a significant increase compared to untreated cells.

TABLE 20

BMP7 and IGF2 induced proliferation in human male myoblasts

Factor

Nuclei

Name
(ug/mL)
Counts
P-value

IGF2
0.06
2723
n.s.

BMP7
0.003
2408
n.s.

Combined

2875
3.12E−02

Example 10—FGF17 and BMP7 Combination Treatment Induced Proliferation in Myoblasts

Mouse myoblasts were cultured in media and with either PBS (vehicle), hPSC, IGF2, BMP7, or IGF2 and BMP7 combined. Fresh media and factors were added every 24 hours. After 48 hours, cells were stained and imaged for EdU as described in Example 1. Representative images of the mouse myoblasts treated with the vehicle, hPSC factors, FGF17, BMP7, and the combination FGF17/BMP7 are shown in FIG. 11A. The number of EdU positive nuclei were quantified for each treatment condition, as depicted in FIG. 11B and Table 21. The cells treated with the hPSC factors, BMP7, and the combination treatment had significant increases in the percent fold change. Furthermore, the combination treatment of BMP7 and FGF17 produced the greatest fold-change increase in proliferation, with a fold-change of 2.63 when compared to untreated cells.

TABLE 21

Mouse myoblast proliferation

Factor
Dose
Fold

Name
(ug/mL)
Change
P-value

FGF17
0.012
1.05
n.s.

BMP7
0.012
1.91
3.47E−04

Combined

2.63
3.75E−05

Human myoblasts were also cultured with hPSC factors, FGF17, BMP7, and a combination FGF17/BMP7 treatment. The number of EdU positive nuclei were quantified for each treatment condition to assess myoblast proliferation. Culturing female human myoblast cells with either the hPSC factors or the combination FGF17/BMP7 produced a significant difference in the fold change of proliferating myoblasts, as depicted in FIG. 11C and Table 22. The combination factors resulted in a similar fold-change increase as culturing the cells with the hPSC factors. However, culturing the cells with either FGF17 or BMP7 alone did not produce a significant increase in proliferation.

TABLE 22

Human female myoblast proliferation

Dose
Nuclei

Factor Name
(ug/mL)
Counts
P-value

FGF17
0.0125
786
n.s.

BMP7
0.04
1041
n.s.

Combined

1470
3.90E−03

Culturing male human myoblast wells with the hPSC factors produced the greatest fold change increase in proliferation, compared to cells cultured with PBS, as depicted in FIG. 11D. Culturing male myoblast cells with either FGF17 or BMP7 alone did not produce a significant fold change in proliferation. However, cells that were cultured with a combination of FGF17 and BMP7 had a significant increase in proliferation, as depicted in FIG. 11D and Table 23.

TABLE 23

Male human myoblast proliferation

Dose
Nuclei

Factor Name
(ug/mL)
Counts
P-value

FGF17
0.0125
1985
n.s.

BMP7
0.00625
2422.5
n.s.

Combo

2872.5
7.25E−04

Example 11—THBS1 Receptor Expression in Mouse and Human Myoblasts

Mouse myoblast cells were cultured in growth media, as described in example 1. After 48 hours, the cells were analyzed via flow cytometry for the presence of THBS1 receptors on the cell surface. As depicted in FIG. 12A and Table 24, 46.9% of the cells were positive for the THBS1 receptor CD36. As depicted in FIG. 12B and Table 24, 97.4% of the cells were positive for the THBS1 receptor ITGA3. As depicted in FIG. 12C and Table 24, 82.4% of the cells were positive for the THBS1 receptor ITGA6. As depicted in FIG. 12D and Table 24, 99.9% of the cells were positive for the THBS1 receptor ITGB1.

TABLE 24

THBS1 receptors on the cell surface of mouse myoblasts

Receptor
Positive Cells (%)

CD36
46.9

ITGA3
97.4

ITGA6
82.4

ITGB1
99.9

Young and aged human myoblast cells were cultured in fusion media, with fresh media added every 24 hours. After 96 hours, 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).

As depicted in FIG. 12E and Table 25, both young and aged myoblasts showed expression of THBS1 receptors. CD36 expression was low in old myoblasts, with a FPKM of 0.871 compared to a FPKM of 5.032 in young myoblasts. ITGA2 expression was higher in old myoblasts than young myoblasts, with a FPKM of 99.259 compared to a FPKM of 44.177. ITGA4 expression was higher in young myoblasts than in old myoblasts, with a FPLKM of 24.348 compared to a FPKM of 15.409. ITGA6 expression was higher in old myoblasts than young myoblasts, with a FPKM of 49.069 compared to a FPKM of 43.355. ITGB1 expression was higher in young myoblasts than in old myoblasts, with a FPKM of 511.270 compared to a FPKM of 454.480.

TABLE 25

Human myoblast cells for receptors

GeneName
Young
Old
Young_SEM
Old_SEM

CD36
5.032
0.871
2.096
0.085

ITGA3
44.177
99.259
7.600
16.691

ITGA4
24.346
15.409
1.706
0.940

ITGA6
43.355
49.069
7.058
15.548

ITGB1
511.270
454.48
20.174
49.053

Example 12—Receptors for hPSC Factors are Expressed in Human Myoblasts

FGF17 was one of the factors present in the hPSC. FGF17 activates the receptor FGFR1. As depicted in Table 26 and FIG. 13A, both young and aged myoblasts expressed FGFR1.

TABLE 26

RNA Expression (in FPKM) of factor

receptors in human myoblasts

Young
Old

GeneName
(n = 6)
(n = 6)
Young_SEM
Old_SEM

FGFR1
18.069
23.122
0.843
1.836

ACVR1
28.705
31.643
0.746
2.070

ACVR2A
3.508
2.190
0.088
0.191

ACVR2B
0.083
0.129
0.016
0.018

BMPR1A
12.270
12.240
0.299
0.393

IGF2
37.589
26.511
5.690
3.341

IGF2R
37.033
41.306
0.761
2.503

BMP7 was one of the factors present in the hPSC. BMP7 is capable of interacting with several receptors, including ACVR1, ACVR2A, ACVR2B and BMPR1A. Both young and old myoblasts showed expression of multiple BMP7 receptors, as depicted in FIG. 13B and Table 26. ACVR1 was expressed at the highest levels in myoblasts, with a FPKM of 28.705 in young myoblasts and 31.643 in old myoblasts. BMPR1A had expression levels of 12.270 FPKMs and 12.240 FPKMs in young and old myoblasts, respectively. ACVR2A had expression levels of 3.508 and 2.190 FPKMs in young and old myoblasts respectively. ACVR2B has expression levels of 0.083 and 0.129 FPKMs, respectively.

Another hPSC factor was IGF2. Both IGF2 and IGF2R expression was present in human myoblasts, as depicted in FIG. 13C and Table 26. IGF2 levels were higher in young myoblasts than old myoblasts, with FPKMs of 37.589 and 26.511. respectively. IGF2R was expressed in both young and old myoblasts, with expression levels of 37,033 FPKMs and 41.306 FPKMs, respectively.

Example 13—Transcriptional Profiling
Myogenic Gene Profiling for Pro-Regenerative Factors

Expression of myogenic factors Pax7, Myf5, Myod1, and Myog are key indicators of the functional status of muscle progenitor cells. Factors upregulating of Pax7 and Myf5 indicate rejuvenation of proliferative progenitor cells whereas upregulation of Myod1 and Myog are indicative of muscle myofiber regeneration. A read-out of these gene expressions will provide potential success for any given HAP as a regenerative factor. Measuring myogenic genes in mouse or human muscle progenitor cells treated with factors will provide a good characterization of the therapeutic effect for treating individuals who have suffered injury, or who possess genetic or developmental defects leading to premature tissue loss, wasting, or weakening. As a control, the assay will also be performed on proteins purified from differentiated cells, which result in no in myoblast proliferation, cultured in medium conditioned by differentiated cells, or purified heparin-associated fractions.

Aged human myoblasts were cultured in well plates. Culturing the cells with the different medias resulted in differential induction of myogenic gene expression. All factors resulted in changes in at least one myogenic receptor gene at 48 hours and 72 hours when compared to cells cultured in fusion media, as depicted in Table 27 and FIGS. 14A-14B. 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).

Cells that had been cultured with FGF2 had increases in levels of both MYF and MYOG, but not MYOD. Cells that had been cultured with BMP7 had increases in levels of MYF5 and MYOG at 48 hours and all 3 receptors at 72 hours. Cells that had been cultured with THBS1 had increases in levels of MYF5 and MYOG at 48 hours and in levels of MYOD and MYOG at 72 hours. Cells that had been cultured with FGF17 had increases in levels of MYF5 and MYOG at 48 hours, and increases in levels of MYOD and MYOG at 72 hours. Cells that had been cultured with THBS4 had increases in levels of MYF5 and MYOG at 48 hours and increases in levels of all 3 factors at 72 hours. Cells that had been cultured with IGF2 had increases in levels of MYOG at 48 hours and levels of MYOD at 72 hours. Values were expressed as fold change compared to vehicle treated controls.

TABLE 27

Fold change increase in myogenic transcription factor expression

in aged human myoblasts cultured with different factors

MYF5-
MYOD1-
MYOG-
MYF5-
MYOD1-
MYOG-

Condition
48 h
48 h
48 h
72 h
72 h
72 h

FM
1.04
1.001
1.013
1.023
1.055
1.092

FGF2
1.918
0.448
4.851
0.784
4.339
5.075

BMP7
4.804
1.06
2.677
7.038
11.934
1.562

THBS1
1.354
0.968
3.735
0.605
1.174
19.906

FGF17
1.922
0.604
2.353
0.499
1.627
13.124

THBS4
1.307
0.91
3.773
1.014
4.299
17.925

IGF2
0.409
0.519
5.756
0.708
5.723
0.018

Myogenic Gene Profiling in Mouse Progenitor Cells

Mouse muscle progenitor cells plated at 10,000 cells/well on Matrigel coated 96-well plates in 100 μL medium per well (1:100 matrigel: PBS) in mouse fusion medium: DMEM (Gibco)+2% horse serum (Hyclone). One hour after plating, mouse myoblasts are treated with respective factors. Mouse myoblasts are analyzed for expression of Pax7, Myf5, Myod1, and Myog to characterize the regenerative effect of treatment with the therapeutic factor. Values were expressed as fold change compared to vehicle treated controls.

TABLE 28

Fold change increase in myogenic transcription factor expression

in mouse muscle progenitor cells cultured with different factors

Dose

Condition
(ug/mL)
Mean
SD
p-value

Gene:
Pax7

FM
2
1.011
0.185
—

THBS1
2
1.045
0.105
n.s.

IGF2
0.2
1.459
0.207
0.049

THBS4
0.5
1.511
0.567
n.s

FGF17
0.5
6.118
0.920
7.05E−04

BMP7
0.025
8.752
1.528
9.56E−04

Gene:
Myf5

FM
2
1.002
0.071
—

THBS1
2
0.921
0.094
n.s.

THBS4
0.2
1.365
0.319
n.s.

FGF17
0.5
1.376
0.201
1.69E−02

IGF2
0.5
1.383
0.051
1.66E−03

BMP7
0.025
40.738
5.627
6.65E−05

Gene:
Myod1

FM
2
1.002
0.086
—

THBS1
2
0.964
0.023
n.s.

THBS4
0.2
1.073
0.062
n.s.

FGF17
0.5
1.230
0.158
n.s.

IGF2
0.5
1.328
0.036
6.41E−03

BMP7
0.025
2.004
0.075
1.09E−04

Transcriptome Profiling in Human Progenitor Cells

Aged human myoblasts were cultured in well plates. Culturing the cells with the different medias resulted in differential induction of myogenic gene expression (FIG. 15A-E). Differentially enriched genes and pathways driven by heparin-associated proteins (HAPs) cocktail or individual factors including FGF17, IGF2, or BMP7 in aged human muscle cells. Cells were treated with indicated factor every 24 h for 96 h. Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and was further purified via polyA selection. cDNA was then generated and illumina adaptor indexes were ligated to generate RNAseq libraries (NEBNext Ultra). Libraries were sequenced using an Illumina HiSeq 4000 sequencer (Illumina). RNA abundance was obtained by STAR and RSEM software. Expression is normalized to Z-score. n=4 biological replicates. (FIG. 15A) HAPs induce extracellular matrix and cell surface interactions to activate signal cascades promoting proliferative homeostasis. Reactome pathway analysis performed on all upregulated or downregulated DE genes (cutoff=p-value 0.01) discovered upregulation of several categories (Cell Cycle, M Phase, Separation of Sister Chromatids, WNT signaling, and Mitotic Anaphase). Enriched pathways were obtained from GSEA Reactome_2016 gene sets. Enrichment is shown according to the p-value. Positive values are upregulated pathways and negative values are downregulated pathways.

Example 14—Plasma Membrane Receptor Profiling by Fractionating and Label-Free Mass Spectrometry

Plasma membrane receptor profiling was performed by fractionation and label-free mass spectrometry of aged, primary human myoblasts cultured in well plates in growth media. Cells were harvested by EDTA cold buffer and cell scraping. Fractionation followed the manufacturer's instructions for the Mem-Per Plus kit (ThermoFisher). Cellular fractions were prepared for mass spectrometry analysis as described in example 6 but omitting the TMT labelling aspects of the methods. Samples were analyzed using 2 hr gradients at 4-45% acetonitrile in 1% formic acid over a 20 cm C18 reverse phase columns (Ion Optiks) electrospraying into timsTOF PRO running PASEF, dynamic resampling, Top10 method. Resulting data were analyzed in commercially available software to identify proteins present in the various cellular fractions. Receptors for the HAPs FGF17, THBS1, VTN, and THBS4 were identified on 68-year-old primary human myoblasts, as listed in Table 29

TABLE 29

Receptors detected by label-free mass

spectrometry of fractionating myoblasts

Gene
Spectral
Matching

Name
Counts
Ligands

EGFR
5
FGF-17

FLT1
4
FGF-17

LRP1
4
THBS1

ITGB1
11
THBS1

ITGA6
8
THBS1, VTN, THBS4

ITGA7
10
THBS4

Example 15—In Vivo Testing of hPSC Factors Increases Regenerative Index and Reduces Fibrotic Index in an Acute Injury Model in Aged Mice

FIGS. 15A-15F show that aged mice (18 months) administered isolated, heparin-agarose bead purified hPSC showed an improved regenerative index and reduced fibrotic index. FIG. 15A shows a schematic of the experiment in this example. This experiment showed that hPSC-derived factors improved histological metrics of muscle health and function. As shown in FIG. 15B HAPs isolated from human pluripotent stem cells increased regenerative potential and reduce muscle fibrosis in aged mice subjected to a model of acute muscle injury. As shown in FIG. 15C, there was increased muscle regeneration for injured, aged mouse muscle treated with THBS1 (2 μg/mL) compared to young and vehicle-treated, aged mouse muscle.

FIG. 15D shows the experimental schematic of time-points for dosing and analysis using an acute injury model in aged mice to measure the effects of individual HAPs with fusion enhancing effects in vitro. Squares denote injury inducing intramuscular injection (IM) with Barium Chloride while circles denote administration of treatment or vehicle. FIG. 15E shows the results of the experiment outlined in FIG. 15D. Administration of 20 μl of HAPs PDGFRL (5 μg/mL, p<3.85E-2) and IGFBP7 (1 μg/mL, p<6.63E-3) resulted in improved new fiber formation (regenerative index compared to vehicle treated aged mice. Stars indicate degree of significance from one-way ANOVA tests. FIG. 15F provides representative images of immunofluorescence staining of sectioned mouse muscle (tibialis anterior) demonstrating increased muscle regeneration for injured, aged mouse muscle treated with POSTN (1 μg/mL) or IGFBP7 (1 μg/mL) compared to vehicle-treated, aged mouse muscle.

Other models for in vivo testing of hPSC factors include:

Disuse-Reload Injury Model

This mouse model is a way to observe muscle atrophy in a non-invasive way by contracting the hind limbs of a mouse and preventing extension and flexion, thus reducing the size and strength. The model will serve as an important measurement of muscle regeneration with biologic candidates.

The hind limb will be immobilized with Cast Tape extended position using sports tape to prevent flexion of the limb. Once the sports tape is in place, a strip of casting tape will be wrapped over the sports tape from the ankle upward, and air dried. The extension of the hind limb should stay rigid in its position for the duration of the study parameters.

The study begins after mice are acclimated and on Day −3, in which mice from all groups will be weighed. Assigned animals will be given daily i.p. injections of Vehicle control or Candidate Biologic for 3 days before undergoing hind limb immobilization on Day 0 for 7 days with continuous daily i.p. injections. Hind limbs will be observed for any adverse effects due to immobilization. On Day 7 of the study, all animals will be sacrificed, and muscle tissue weighed and harvested for further analysis.

Force Measurement

This study will be used to measure the force of pull in the hind limbs that the animal exerts upon skeletal muscle injury of the tibialis anterior (TA) and gastrocnemius (GA) muscles after injury induction with Barium chloride (BaCl₂). This model will serve to determine which of our biological candidates are efficacious in muscle regeneration.

Skeletal Muscle Injury Induction: Under anesthesia, BaCl₂will be administered in two sites on the TA and four sites on the GA (as previously described). Hair will be shaved on the left and right hind limbs prior to injection with small animal hair clippers. On Day 0 of the study, the TA muscle will undergo BaCl₂induced injury on two sites (previously described). On Day 4 of the study, the GA muscle will follow with BaCl₂induced injury on four sites. Candidate biologic will be administered on Days 0 and 2 in the injury sites of the TA and GA muscles. BrdU will be injected via IP (QD) on days 4-7 to label proliferating muscle precursor and fibrotic cells in order to measure their regenerative potential.

On terminal day 7, animals will be deeply anesthetized, and a force transducer will be used to measure twitch reactions in the hind limbs of each mouse being tested in the study, via a small incision in the TA to a small metal hook. This will be a terminal procedure. Grip strength measurements: the mice will rest on an angled mesh, facing away from the force meter and with its hind limbs at least one-half of the way down the length of the bar. The mouse's tail is pulled directly toward the meter and parallel to the bar. During this procedure, the mouse resists by grasping the mesh with all four limbs. Pulling is continued toward the meter until the hind limbs release.

Ex Vivo Regenerative Measurement

To confirm these data with age matched, primary muscle stem cells, injury-activated satellite cells associated with myofibers will be isolated from young and old muscle by dissecting the muscle groups of interest and dissociating the tissue to single cell suspensions by incubating in digestion medium (250 U/mL Collagenase type II in DMEM medium, buffered with 30 mM HEPES, pH 7.4) at 37 C for 1 hr., triturating the cell suspension, the myofibers were collected by centrifugation and myofibers further digested with 1 U/mL Dispase and 40 U/mL Collagenase type II in 30 mM HEPES at 37 C for 1 hr to free muscle stem cells, as depicted in FIG. 16A. Muscle stem cells can then be plated and cultured growth media containing serum (2-5%) from the same mouse. The regenerative and fusion potential of the cells will then be assayed as described above in in Example 7 and as demonstrated in FIGS. 16B, 16C, 17B, and 17C. This has the advantage of testing the effect of treatment while maintaining the exogenous, often inhibitory extracellular environmental cues contributed by the age appropriate serum.

Example 16—: Intramuscular Administration of FGF17, THBS1 and IGF2/BMP7 Combo Promote Regeneration of Muscle in a BaCl₂Injured Old Mouse Model

An old mouse model was used to assess the combination factors regenerative capabilities. On Day 0, 70-week-old mice were weighed and underwent muscle injury with focal injection of barium chloride (BaCl₂, 14 μL, 1.2% w/v in saline, Sigma-Aldrich) in the Tibialis anterior (TA; Day 0) of both the right and left hindlegs. Vehicle or factor A (0.1 mg/kg) were administered intramuscularly (i.m) 2 h following the BaCl₂into the TA injured hindleg sites, and again 48 hours later on day 3 (i.m.) into the TA injured hindleg sites. Bromodeoxyuridine (BrdU) was administered (100 mg/kg, i.p.) once daily for 3 days, day 2-4, before sacrifice to label proliferating cells. Treatments were administered as listed in Table 30.

TABLE 30

Treatment groups

Group
Factors
Concentration

A, n = 5
FGF17/THBS1
100 ng/ml/125 ng/ml

B, n = 5
FGF17/BMP7
12.5
ng/ml

C, n = 4
BMP7/IGF2
25 ng/ml/60 ng/ml

D. n = 4
FGF17
100
ng/ml

E, n = 4
THBS1
125
ng/ml

F, n = 3
BMP7
25
ng/ml

G. n = 4
IGF2
60
ng/ml

H, n = 3
FGF17
500
ng/ml

I, n = 4
THBS1
2000
ng/ml

J, n = 4
Vehicle
Saline solution

On day 6, animals were sacrificed, and animal weight recorded followed by collecting 0.5 ml of terminal blood via cardiac puncture which was processed to plasma and stored at 80° C. We then dissected the skin from the TA muscles of each hind leg and took photos (prior to excision). After excision of exclusively the TA muscle, excised tissue was photographed, weighed, then placed in chilled isopentane bath precooled in liquid nitrogen and stored at −80 C. Cryosectioning and H&E were performed to ensure the muscle injury site was appropriately visualized. A diagram of the time points for these experiments can be seen in FIG. 18A. Muscle tissue composition from new skeletal muscle fibers, fibrotic tissue, and adipose (fat) were measured. Muscle regeneration, as defined as the number of number of new myofibers with centrally located nuclei per millimeter, fibrosis as defined as the area of fibrotic scarring, size of the fibers, as defined as the width and area, adipose tissue, as defined by the amount of fat surrounding the muscle, were measured to assess level of regeneration.

FIGS. 18B-18C depicts the regenerative index of the treated and untreated muscles. When compared to the untreated (vehicle) group, mice that were treated with FGF17 (group H), THBS1 (group I), and the combination BMP7/IGF7 (group C) showed a significant increase in the regenerative index. When the muscle fibrosis was analyzed, mice that were treated with FGF17 (group H), FGF17/BMP7 (group B), and BMP7/IGF2 (group C) showed a significant decrease in the fibrotic index when compared to untreated mice, as depicted in FIG. 18D. Notably, a combination treatment of BMP7/IGF2 resulted in improved recovery from muscle injury as seen by both an increase in proliferation and a decrease in fibrosis. In this example, combining factors resulted in an improvement in muscle injury compared to untreated muscles.

Example 17—Systemic Administration of FGF17 Protects Against Dexamethasone Induced Muscle Atrophy

12-week-old mice were injected daily with either vehicle only (n=7), dexamethasone (n=6), or dexamethasone and FGF17 (n=6), as depicted in FIG. 19A. Dexamethasone was injected intraperitoneally at a concentration of 25 mg/kg to induce muscle atrophy. FGF17 was injected subcutaneously at a concentration of 0.5 mg/kg. Forelimb grip strength and both limb grip strength was assessed on days 7, 13, and 19, as described in previous Example 4. At 21 days, mice were euthanized, and the weight of the TA muscle was assessed.

Mice that had been administered dexamethasone had a significant decrease in TA weight when compared to mice that had not received dexamethasone, as depicted in FIG. 19B. Administration of FGF17 resulted in a significant increase of TA weight compared to untreated mice that had received dexamethasone. Both the forelimb specific force and the both limb specific force was significantly reduced in mice with dexamethasone-induced muscle atrophy compared to untreated mice. However, mice that had received both dexamethasone and FGF17 had a significant increase in forelimb specific forms and both limb specific force when compared to mice that received dexamethasone alone, as depicted in FIGS. 19C-19D.

Example 18—Modelling Treatment of a Muscular Dystrophy with Pro-Regenerative Factors In Vitro Using High-Throughput Imaging

Muscular dystrophies (MD) encompass a variety of muscular degeneration diseases typically due to genetic mutations in genes encoding proteins responsible for forming and stabilizing skeletal muscle. The phenotypic consequence of these genetic mutations is the progressive loss of muscle mass and strength over time, similar to sarcopenia but with different underlying causes. As HAPs provided phenotypic improvements on sarcopenic muscle, we tested for similar improvements in a model for MD.

Many of the factors detailed in Table 2 were tested individually for their ability to promote proliferation and/or fusion of human muscle progenitor cells from a patient with myotonic dystrophy type 1 (hMD)—a muscular dystrophy caused by mutations in the DMPK1 gene.

The effect of the candidate factors on myogenic activity was assayed in biological triplicate across a range of concentrations centered around expected physiological levels by adding each factor to hMD myoblasts for 72 hours with daily media changes (DMEM +2% horse serum) and a second pulse of factors at the first media change. After 72 hours, cells were pulsed for 2-5 hours with EdU (30 uM), ethanol fixed, stained with Hoechst 3342, immunostained for proliferation—as measured by the percent of cells staining positive for EdU (% EdU)−, and immunostained for differentiation—as measured by the increase in cellular area staining positive for embryonic myosin heavy chain (% eMyHC) relative to the negative controls, which received media and vehicle only. Wells were imaged on a Keyence BZ-100 at 4×, the images quantified in Cell Profiler, and the statistics were computed in R. FIGS. 24A, 25A and 24B, 25B show examples of the quantitation of the proliferation response and fusion response for several of the factors tested, respectively. Results of those and additional factors are summarized below in Table 31. Transcriptional profiling of these treated cells found IGF2 enhances MYH3, CKM, and ATP1B1 expression in DM1 human myoblast (32 year old caucasian female) cells FIGS. 25C and 25D.

TABLE 31

Effect of individual factors on dystrophic human myoblast growth and fusion

Proliferation (% EdU)
Fusion (% eMyHC)

Effect Size
Statistical
Effect Size
Statistical

Factor
Concentration
(% relative
Significance
(% relative
Significance

Name
(ug/mL)
to −control)
(p-value)
to −control)
(p-value)

FGF17
0.2
208%

<9E−14

FGF4
0.0125
269%
9.52E−13

FGF4
0.025
244%
6.83E−12

FGF4
0.05
235%
7.22E−10

FGF4
0.1
213%
8.11E−07

FGF4
0.2
184%
1.46E−02

IGF2
0.2
53%
2.57E−02

FGF1
0.05
157%
1.65E−06

FGF1
0.1
217%
2.87E−06

FGF1
0.2
213%
1.10E−10

FGF6
0.0125
277%
1.34E−09

FGF6
0.025
272%
4.54E−09

FGF6
0.05
261%
4.75E−08

FGF6
0.1
243%
1.03E−07

FGF6
0.2
237%
8.49E−06

PDGFRL
0.03125

2580%
1.47E−06

PDGFRL
0.0625

2240%
6.34E−07

PDGFRL
0.125

1410%
4.53E−03

PDGFRL
0.25

2570%
1.86E−08

PDGFRL
0.5

3440%
5.75E−11

IGF2
0.2
218
6.8E−3
1200%
1.9E−4

Example 19—Systemic Administration of Therapeutic Polypeptides Reverses Sarcopenia and Protects from Muscle Injury

A daily subcutaneous injection of therapeutic polypeptides or vehicle only is administered to 78-week-old mice for 14 days, as depicted in FIG. 21. The therapeutic polypeptides include FGF17 at a concentration of 500 ug/kg, IGF2 at a concentration of 100-1000 ug/kg, and BMP7 at a concentration of 10-100 μg/ug. In some experiments, treatment groups receive a single therapeutic factor while in other experiments, treatment groups receive a combination of factors. At 7 days, muscle function is assessed using forelimb grip strength and both grip strength. On day 12, 13 and 14, groups 1 and 2 are injected with BrdU intraperitoneally. On days 13-15, all mice are assessed for grip strength and an endurance test to determine max distance and max speed and tetanic force, as described in example 4.

At 15 days, mice in groups 1 and 2 are euthanized and the muscles are analyzed for markers of proliferation and fibrosis. At 15 days, an intramuscular injection of 1.2% of BaCl₂(7 ul/TA) is used to generate chemical injury in the TAs of group 3 and group 4. Mice from groups 3 and 4 continue to receive a therapeutic polypeptide injected subcutaneously on days 15-21. They also receive BrdU injections intraperitoneally on days 19, 20 and 21. On day 21, the TA muscles are tested for in situ tetanic force, using methods described in Example 4. The TA muscles are dissected and assessed for signs of proliferation and fibrosis.

Example 20—Systemic Administration of Therapeutic Polypeptides Predicted to Reverse Dexamethasone Induced Muscle Atrophy

12-week-old mice are divided into 3 treatment groups: group 1 which receives injections only of the vehicle, group 2 which receives injections of dexamethasone, and group 3 which receives injections of dexamethasone and therapeutic peptide. Dexamethasone (25 mg/kg i.p.) is administered for 14 days simultaneously with a subcutaneous injection of therapeutic polypeptides, as depicted in FIG. 22.

At 7 days, mice are assessed for forelimb and both limb grip strength, using the methods described in example 4. At days 13-15, mice are assessed for grip strength, in vivo and in situ tetanic force, and undergo a treadmill endurance test to determine max speed and max distance.

The therapeutic peptides predicted to be effective at reversing muscle atrophy are IGF2 at a concentration of 100-1000 ug/kg, BMP7 at a concentration of 10-100 μg/kg, POSTN at a concentration of 10-1000 μg/kg, IGFBP7 at a concentration of 100-1000 μg/kg, and PDGFRL at a concentration of 10-1000 μg/kg.

Example 21—Systemic Administration of Therapeutic Polypeptides Predicted to Improve Muscle Atrophy in Genetically Obese Mice

Thirteen-week-old genetically obese mice (ob ob) are injected subcutaneously with a therapeutic peptide for 14 days At day 7, forelimb and both grip strength are measured as described in Example 4. BrdU is injected on days 12, 13 and 14. On days 13, 14 and 15, forelimb and both limb grip strength, in vivo and in situ tetanic force are tested, and an endurance test to determine max distance and max speed is performed using methods described in Example 4. At 14 days, the mice are euthanized, and the TA muscles are dissected. Muscle weight and proliferation is analyzed.

The therapeutic peptides predicted to be effective at reversing muscle atrophy are IGF2 at a concentration of 100-1000 μg/kg, BMP7 at a concentration of 10-100 μg/kg, POSTN at a concentration of 10-1000 μg/kg, IGFBP7 at a concentration of 100-1000 μg/kg, and PDGFRL at a concentration of 10-1000 ug/kg.

Example 22—Systemic Administration of Therapeutic Polypeptides Predicted to Reverse of Slow Down Dystrophic Features in 70 Weeks Old Mdx Mice

Another class of human myopathies in need of treatment are the genetic abnormality induced muscular dystrophies, among which Duchenne muscular dystrophy is a rare but fatal case. Old genetically dystrophic (mdx) mice (>15 month old) show similar features to the human Duchenne muscular dystrophy (DMD), notably, a decrease in muscle regeneration leading to muscle wasting. Treatment with therapeutic polypeptide described herein can reverse the dystrophic features of old mdx mice. Mice will be fed ad libitum and housed under controlled conditions of lighting (12-hour light/12-hour dark) and temperature (22-24° C.). Mice will be allowed to acclimate for 3 days prior to the initiation of the experiment. During the acclimation period, the weight, Forelimb and both limb grip strength as well as in vivo tetanic force will be assessed to determine the baseline strength of each mouse. 70-week dystrophic mice (mdx) are injected with the therapeutic peptide subcutaneously for 14 days. At day 7, forelimb and both grip strength are measured as described in Example 4. BrdU is injected on days 12, 13 and 14. On days 13, 14 and 15, forelimb and both limb grip strength and tetanic force are tested, and an endurance test to determine max distance and max speed is performed using methods described in Example 4. A The right tibialis anterior and gastrocnemius will be collected, immersed in Tissue-TEK OCT and then flash frozen in chilled isopentane bath precooled in liquid nitrogen and stored at −80 C. Tissue will be sectioned and stained for Laminin to determine the cross-sectional area (CSA) of muscle fibers, for eMyHC to measure new fiber formation and for BrdU to assess the proliferation rate. The left anterior tibialis and gastrocnemius will be collected and flash frozen in liquid nitrogen for molecular analysis that include qPCR and western blot.

The therapeutic peptides predicted to be effective in this model are IGF2 at a concentration of 100-1000 ug/kg, FGF17 at a concentration of 10-100 ug/kg, FGF4 at a concentration of 100-1000 μg/kg, and FGF6 at a concentration of 100-1000 ug/kg.

Example 23—Systemic Administration of Therapeutic Polypeptides Predicted to Improve the Dystrophic Features in 6-Week-Old Mice

Between 3-6 weeks old, the skeletal muscle of mdx mice undergoes severe necrosis followed by an increase in the activation of satellite cells to promote muscle regeneration. Treatment with therapeutic polypeptide described herein can improve the regeneration process and therefore muscle health. Mice will be fed ad libitum and housed under controlled conditions of lighting (12-hour light/12-hour dark) and temperature (22-24° C.). Mice will be allowed to acclimate for 3 days prior to the initiation of the experiment. During the acclimation period, the weight, Forelimb and both limb grip strength as well as in vivo tetanic force will be assessed to determine the baseline strength of each mouse. 6-week-old dystrophic mice (mdx) are injected with the therapeutic peptide subcutaneously for 14 days. At day 7, forelimb and both grip strength are measured as described in Example 4. BrdU is injected on days 12, 13 and 14. On days 13, 14 and 15, forelimb and both limb grip strength and tetanic force are tested, and an endurance test to determine max distance and max speed is performed using methods described in Example 4.

Mice will be euthanized. The right tibialis anterior and gastrocnemius will be collected, immersed in Tissue-TEK OCT and then flash frozen in chilled isopentane bath precooled in liquid nitrogen and stored at −80 C. Tissue will be sectioned and stained for Laminin to determine the cross-sectional area (CSA) of muscle fibers, for eMyHC to measure new fiber formation and for BrdU to assess the proliferation rate. The left anterior tibialis and gastrocnemius will be collected and flash frozen in liquid nitrogen for molecular analysis that include qPCR and western blot.

The therapeutic peptides predicted to be effective in this model are IGF2 at a concentration of 100-1000 μg/kg, FGF17 at a concentration of 10-100 ug/kg, FGF4 at a concentration of 100-1000 μg/kg, and FGF6 at a concentration of 100-1000 ug/kg.

Example 24—Treatment for Chondrocyte Proliferation in Cartilage Injury and Osteoarthritis

Cartilage can become damaged as a result of a sudden injury or due to gradual wear and tear (osteoarthritis). Chondrocytes secrete the cartilage matrix and preadipocytes, osteocytes and tenocytes are all cell types associated with cartilage.

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

These cartilage-associated cells expressed receptors for many of hPSC factors Specifically, expression of FGFR1, ACVR1, ITGB1 and IGF2R was detected in cartilage associated cells. Subcutaneous preadipocytes, chondrocytes, osteocytes and tenocytes all expressed these receptors, as depicted in Table 31, indicating that these hEPSC factors may be able to affect cartilage proliferation.

TABLE 31

RNA expression in cartilage associated cells (TPM)

Cell Type
FGFR1
ACVR1
ITGB1
IGF2R

Preadipocyte
54.92
27.11
827.15
27.083

(Subcutaneous)

Chondrocyte
152.59
34.44
971.35
47.63

Osteocyte
202.57
39.79
290.03
83.96

Tenocyte
167.01
33.00
994.43
23.12

In Vitro Model Screening for Chondrocyte Proliferation in Cartilage Injury and Osteoarthritis

HAPs collectively and FGF17 specifically induced chondrocyte proliferation in healthy adult human chondrocytes as shown in Table 32. Chondrocytes were cultured for 48 h in the presence of HAPs at indicated dose. Fresh media and HAP was added every 24 h. Mean∓S.D. Table 32 of % EdU+ chondrocyte values and p-values by Tukey Honest Significant Difference T-test, n=2-3.

TABLE 32

Chondrocyte proliferation driven by HAPs

Condition
n
% EdU
sd
adj-p-val

Vehicle
3
0.744
0.247

FGF18
3
6.039
1.6370
1.29E−03

1x_HAPs
3
14.542
6.092
1.95E−02

2x_HAPs
3
31.037
6.097
1.28E−04

4x_HAPs
3
32.009
1.197
1.01E−04

HAPs collectively and FGF17 specifically induced chondrocyte proliferation in healthy adult human chondrocytes as shown in Table 32. Chondrocytes were cultured for 48 h in the presence of HAPs at indicated dose. Fresh media and FGF17 (Table 33) TGFB1 (Table 34), FGF1 (Table 34), or PDGFD (Table 34) was added every 24 h. Mean∓S.D. of % EdU+ chondrocyte values and p-values by Tukey Honest Significant Difference T-test, n=2-3.

TABLE 33

Chondrocyte proliferation driven by FGF17

Condition
n
% EdU
sd
adj-p-val

Vehicle
3
0.744
0.247

FGF18: 0.1 ug/mL
3
6.039
1.637
1.29E−03

FGF17: 0.1 ug/mL
2
4.675
0.198
1.92E−04

FGF17: 0.20 ug/mL
2
9.085
0.094
7.85E−06

FGF17: 0.40 ug/mL
2
16.773
0.621
5.42E−07

TABLE 34

Chondrocyte proliferation driven by

HAPs and pro-regenerative factors

Condition
N
Percent_EdU
SD
adj-pval

Vehicle
9
4.793
2.359

HAPs
6
28.794
4.159
3.07E−10

TGFB1
3
17.206
1.703
4.37E−04

FGF1
3
15.632
0.796
2.01E−03

PDGFD
3
44.225
2.697
2.18E−12

FGF17
3
26.342
7.521
1.39E−07

Example 25—Clinical Testing of Pro-Regenerative Factors

The purpose of this study is to determine the safety and tolerability of repeat dosing with multiple dose levels of heparin-associated polypeptide in healthy individuals or individuals diagnosed with sarcopenia, a muscular dystrophy, or recovery from surgery. The muscular dystrophy may be myotonic dystrophy. In addition, this study will generate data on the physical function, skeletal muscle mass and strength resulting from treatment with compositions comprising heparin-associated polypeptides. In addition, this study will generate data on the safety, tolerability, and pharmacokinetics of heparin-associated proteins in older adults with sarcopenia. Individuals will be administered placebo or heparin-associated binding proteins and monitored for 25 weeks of study. The following primary and secondary outcome measures will be assessed:

- Primary Outcome Measures: Safety and tolerability as assessed by various measures such as percent of adverse events per study arm.
- Secondary Outcome Measures: Plasma Pharmacokinetics (Cmax, Tmax, AUC) [Plasma at 0.5, 1, 1.5, 2, 4, 6, 8, 12 and 24 hrs after dosing.]
- Short Physical Performance Battery (SPPB). Change from baseline to week 25.
- 10-meter walk test. Change from baseline to week 25.
- Change in total lean body mass and appendicular skeletal muscle index measured by Dual-energy X-ray Absorptiometry (DEXA) from baseline to week 25.
- Inclusion Criteria: Diagnosis of sarcopenia, a muscular dystrophy, or recovery from surgery; Low muscle mass as confirmed by DXA; Low gait speed; SPPB score less than or equal to 9; Weigh at least 35 kg; with adequate dietary intake as determined by patient interview. Independently ambulatory to 10 meters.
- Protocol: Patients will be i.v.—administered placebo (5% dextrose solution) or treatment article (in 5% dextrose). Starting on day 1, week 1 and repeated every week (day one of weeks 1 through 25). At the end of week 13 and 25 patients will be assessed by the above methods for improvement. Doses will be selected from a traditional 3+3 design, and selected as the top two-doses that lack dose-limiting toxicity.

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

All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference 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 that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

TABLE 1

Exemplary Therapeutic Polypeptides

HAPs
Uniprot

Polypeptide
ID NO
ID
Modified Amino Acid Sequences Tested

Vitronectin (VTN)
1
P04004
Asp20 to Leu478, purified from Human

plasma-derived

STC2
2
O76061

AGRN
3
O00468

Thrombospondin 2
4
P35442
Gly19 to Ile1172, with a C-terminal 10-His

(THBS2)

tag (SEQ ID NO: 70), purified from Mouse

myeloma cell line, NS0

FST
5
P19883

Periostin (POSTN)
6
Q15063
Asn22 to Gln836, with a C-terminal 6-His tag

(SEQ ID NO: 69), purified from Mouse

myeloma cell line, NS0

Fibroblast growth
7
O60258
Thr23 to Thr216, purified from E. coli

factor 17

(FGF17)

Thrombospondin 4
8
P35443
Ala22 to Asn961, with a C-terminal 10-His

(THBS4)

tag (SEQ ID NO: 70), purified from Chinese

Hamster Ovary cell line

Thrombospondin 1
9
P07996
Asn19-Pro1170, with Thr523 Ala substitution

(THBS1)

plus 10His tag, purified from mouse myeloma

cell line, NS0

Interleukin-15
10
P40933
Asn49 to Ser162, purified from E. coli

Insulin-like growth
11
P01344
Ala25 to Glu91, purified from E. coli

factor 2 (IGF2)

Fibroblast growth
12
P09038
Pro143 to Ser288, purified E. coli

factor 2 (FGF 2)

Fibroblast growth
13
O95750
Leu25 to Lys216, purified from E. coli

factor 19 (FGF 19)

Angiogenin (ANG)
14
P03950
Gln25 to Pro147, purified from E. coli

Probetacellulin (BTC)
15
P35070
Asp32 to Tyr111, purified from E. coli

Interleukin -13
16
P35225
Arg21 to Ala228, purified from mouse

receptor alpha 2

myeloma cell line, NS0

Siglec-5/CD170
17
O15389
Glu17 to Thr434, purified from mouse

myeloma cell line, NS0

Apelin receptor (APJ)
18
P35414

Insulin-like growth
19
P18065
Glu40 to Gln328, purified from mouse

factor-binding protein

myeloma cell line, NS0

2 (IGFBP-2)

Chordin-Like 1
20
Q9BU40
Glu22-Cys450, purified from mouse myeloma

(CHRDL1)

cell line, NS0

WAP, Kazal,
21
Q8TEU8
Leu35 to His 576, purified from mouse

immunoglobulin,

myeloma cell line, NS0

Kunitz and NTR

domain-containing

protein 2

Membrane frizzled-
22
Q9BY79
Ser101 to Pro579, purified from mouse

related protein

myeloma cell line, NS0

(MFRP)

Interleukin -10
23
P22301
His22 to Asn235, purified from human cell

receptor alpha

line HEK293

Chemokine like
24
Q99788

receptor 1, Chemerin

Receptor 23 (Chem

R23)

HB-EGF
25
Q99075
Asp63 to Leu148, purified from insect cells

fibroblast growth
26
P10767
Gly67 to Ile208, purified from E. coli

factor 6

Hepatocyte Growth
27
P14210
Gln32 to Ser728, purified from insect cells

Factor

Interleukin-16
28
Q14005
Pro2 to Ser130, purified from E. coli

Interleukin-7 receptor
29
P16871
Glu21 to Lys261, purified from mouse

alpha

myeloma cell line, NS0

Tumor necrosis factor
30
O14798
Ala26 to Ala221, purified from mouse

receptor superfamily

myeloma cell line, NS0

member 10C

Bone morphogenetic
31
P22004
Gln382 to His51, 3 purified from E. coli

protein 6

Interleukin-36 gamma
32
Q9NZH8
Ser18 to Asp169, purified from E. coli

interleukin-1 receptor
33
P18510
Val2 to Asp155, purified from E. coli

antagonist

(IL-1RA)

Kremen protein 2
34
Q8NCW0
Gln19 to Ala364, purified from mouse

myeloma cell line, NS0

Tumor necrosis factor
35
Q9UBN6

receptor superfamily

member 10D

C-X-C chemokine
36
P25024

receptor type 1

C-C motif chemokine
37
P55773

23

Catenin, Beta
38
P35222

Fibroblast growth
39
Q92913

factor 13, 1B

Tumor necrosis factor
40
P50591
Glu108 to Leu261, purified from E. coli

ligand superfamily

member 10

C-C motif chemokine
41
Q16627
Ser35 to Glu111, purified from E. coli

14

Insulin-like growth
42
Q16270
Asp30 to Leu282 with a K95R mutation with

factor binding protein

an N-terminal 10-His tag (SEQ ID NO: 70),

7

purified from Mouse myeloma cell line, NS0

Fibroblast growth
43
P08620
Ser54 to Leu206, purified from E. coli

factor 4

Fibroblast growth
44
P55075
Gln23 to Arg204, purified from E. coli

factor 8

PDGFRL
55
Q15198
Gln22 to Ser375, purified from HEK293 cells

ANOS1
56
P23352
Ala25 to Tyr680, purified from CHO cells

THBS1 isoform 2
58
P07996-2

FGF17 isoform 2
59
O60258-2

POSTN isoform 2
60
Q15063-2

POSTN isoform 3
61
Q15063-3

POSTN isoform 4
62
Q15063-4

POSTN isoform 5
63
Q15063-5

POSTN isoform 6
64
Q15063-6

POSTN isoform 7
65
Q15063-7

IGF2 isoform 2
66
P01344-2

IGF2 isoform 3
67
P01344-3

IL15 isoform 2
68
P40933

Bone morphogenetic
72
P18075
Ser293 to His 431, purified from CHO cells

protein 7 (BMP7)

TABLE 2

Factors enriched in the supernatants of undifferentiated

human pluripotent stem cells.

Uniprot
Entrez Gene

Gene Name
ID
ID
Ensembl ID
Polypeptide No.

A1BG
P04217
1
ENSG00000121410
1

A2M
P01023
2
ENSG00000175899
2

ABCF1
Q8NE71
23
ENSG00000204574
3

ACADVL
P49748
37
ENSG00000072778
4

ACLY
P53396
47
ENSG00000131473
5

ACP1
P24666
52
ENSG00000143727
6

ACP5
P13686
54
ENSG00000102575
7

ACTG1
P63261
71
ENSG00000184009
8

ACTN1
P12814
87
ENSG00000072110
9

ACTR3
P61158
10096
ENSG00000115091
10

ADAMTS1
Q9UHI8
9510
ENSG00000154734
11

ADAMTS12
P58397
81792
ENSG00000151388
12

ADAMTS19
Q8TE59
171019
ENSG00000145808
13

ADAMTS7
Q9UKP4
11173
ENSG00000136378
14

ADAMTS8
Q9UP79
11095
ENSG00000134917
15

ADRM1
Q16186
11047
ENSG00000130706
16

AEBP1
Q8IUX7
165
ENSG00000106624
17

AFM
P43652
173
ENSG00000079557
18

AFP
P02771
174
ENSG00000081051
19

AGPS
O00116
8540
ENSG00000018510
20

AGRN
O00468
375790
ENSG00000188157
21

AGT
P01019
183
ENSG00000135744
22

AHCYL2
Q96HN2
23382
ENSG00000158467
23

AHSG
P02765
197
ENSG00000145192
24

AIMP1
Q12904
9255
ENSG00000164022
25

ALB
P02768
213
ENSG00000163631
26

ALCAM
Q13740
214
ENSG00000170017
27

ALDH9A1
P49189
223
ENSG00000143149
28

ALDOA
P04075
226
ENSG00000149925
29

ALPL
P05186
249
ENSG00000162551
30

AMBP
P02760
259
ENSG00000106927
31

ANG
P03950
283
ENSG00000214274
32

ANGPTL4
Q9BY76
51129
ENSG00000167772
33

ANOS1
P23352
3730
ENSG00000011201
34

ANXA1
P04083
301
ENSG00000135046
35

ANXA2
P07355
302
ENSG00000182718
36

ANXA2P2
A6NMY6

37

AOC1
P19801
26
ENSG00000002726
38

AP2A1
O95782
160
ENSG00000196961
39

AP2A2
O94973
161
ENSG00000183020
40

AP3D1
O14617
8943
ENSG00000065000
41

APLP2
Q06481
334
ENSG00000084234
42

APOA1
P02647
335
ENSG00000118137
43

APOA2
P02652
336
ENSG00000158874
44

APOB
P04114
338
ENSG00000084674
45

APOC3
P02656
345
ENSG00000110245
46

APOD
P05090
347
ENSG00000189058
47

APOE
P02649
348
ENSG00000130203
48

APOH
P02749
350
ENSG00000091583
49

APOM
O95445
55937
ENSG00000204444
50

ARCN1
P48444
372
ENSG00000095139
51

ARHGEF1
Q92888
9138
ENSG00000076928
52

ARHGEF28
Q8N1W1
64283
ENSG00000214944
53

ARPC1B
O15143
10095
ENSG00000130429
54

ARRB1
P49407
408
ENSG00000137486
55

ARSK
Q6UWY0
153642
ENSG00000164291
56

ART4
Q93070
420
ENSG00000111339
57

ASNA1
O43681
439
ENSG00000198356
58

ASNS
P08243
440
ENSG00000070669
59

ATP6AP2
O75787
10159
ENSG00000182220
60

ATRN
O75882
8455
ENSG00000088812
61

AZGP1
P25311
563
ENSG00000160862
62

B3GALT6
Q96L58
126792
ENSG00000176022
63

B3GNT7
Q8NFL0
93010
ENSG00000156966
64

B4GALT1
P15291
2683
ENSG00000086062
65

B4GALT4
O60513
8702
ENSG00000121578
66

B4GAT1
O43505
11041
ENSG00000174684
67

BCAM
P50895
4059
ENSG00000187244
68

BGN
P21810
633
ENSG00000182492
69

BLVRB
P30043
645
ENSG00000090013
70

BMP1
P13497
649
ENSG00000168487
71

BMP7
P18075
655
ENSG00000101144
72

BOC
Q9BWV1
91653
ENSG00000144857
73

BRD3
Q15059
8019
ENSG00000169925
74

BSG
P35613
682
ENSG00000172270
75

BTBD17
A6NE02
388419
ENSG00000204347
76

BTD
P43251
686
ENSG00000169814
77

BZW2
Q9Y6E2
28969
ENSG00000136261
78

C11orf24
Q96F05
53838
ENSG00000171067
79

C1QA
P02745
712
ENSG00000173372
80

C1QBP
Q07021
708
ENSG00000108561
81

C1QC
P02747
714
ENSG00000159189
82

C1QTNF3
Q9BXJ4
114899
ENSG00000082196
83

C1QTNF3-
E9PGA6

ENSG00000273294
84

AMACR

C1QTNF4
Q9BXJ3
114900
ENSG00000172247
85

C1RL
Q9NZP8
51279
ENSG00000139178
86

C1S
P09871
716
ENSG00000182326
87

C20orf27
Q9GZN8
54976
ENSG00000101220
88

C3
P01024
718
ENSG00000125730
89

C4A
POCOL4
720
ENSG00000206340
90

C4B
A0A140TA29

ENSG00000236625
91

C4BPA
P04003
722
ENSG00000123838
92

C5
P01031
727
ENSG00000106804
93

C7
P10643
730
ENSG00000112936
94

C8B
P07358
732
ENSG00000021852
95

C9
P02748
735
ENSG00000113600
96

CA11
O75493
770
ENSG00000063180
97

CALM2
PODP24
801
ENSG00000143933
98

CALR
P27797
811
ENSG00000179218
99

CALU
O43852
813
ENSG00000128595
100

CAND1
Q86VP6
55832
ENSG00000111530
101

CANT1
Q8WVQ1
124583
ENSG00000171302
102

CANX
P27824
821
ENSG00000127022
103

CAPG
P40121
822
ENSG00000042493
104

CAPN1
P07384
823
ENSG00000014216
105

CAPZA2
P47755
830
ENSG00000198898
106

CARM1
Q86X55
10498
ENSG00000142453
107

CARS
P49589
833
ENSG00000110619
108

CBL
P22681
867
ENSG00000110395
109

CBX3
Q13185
11335
ENSG00000122565
110

CCAR2
Q8N163
57805
ENSG00000158941
111

CCBE1
Q6UXH8
147372
ENSG00000183287
112

CCDC80
Q76M96
151887
ENSG00000091986
113

CCK
P06307
885
ENSG00000187094
114

CCT2
P78371
10576
ENSG00000166226
115

CCT4
P50991
10575
ENSG00000115484
116

CCT7
Q99832
10574
ENSG00000135624
117

CD5
P06127
921
ENSG00000110448
118

CDC40
O60508
51362
ENSG00000168438
119

CDH1
P12830
999
ENSG00000039068
120

CDH9
Q9ULB4
1007
ENSG00000113100
121

CDON
Q4KMG0
50937
ENSG00000064309
122

CDSN
Q15517
1041
ENSG00000137197
123

CENPV
Q7Z7K6
201161
ENSG00000166582
124

CFB
P00751
629
ENSG00000241253
125

CFC1
P0CG37
55997
ENSG00000136698
126

CFD
P00746
1675
ENSG00000197766
127

CFH
P08603
3075
ENSG00000000971
128

CFI
P05156
3426
ENSG00000205403
129

CHAD
O15335
1101
ENSG00000136457
130

CHD4
Q14839
1108
ENSG00000111642
131

CHD8
Q9HCK8
57680
ENSG00000100888
132

CHGA
P10645
1113
ENSG00000100604
133

CHID1
Q9BWS9
66005
ENSG00000177830
134

CHRDL1
Q9BU40
91851
ENSG00000101938
135

CHST11
Q9NPF2
50515
ENSG00000171310
136

CHST6
Q9GZX3
4166
ENSG00000183196
137

CHSY1
Q86X52
22856
ENSG00000131873
138

CHSY3
Q70JA7
337876
ENSG00000198108
139

CILP2
Q8IUL8
148113
ENSG00000160161
140

CKAP5
Q14008
9793
ENSG00000175216
141

CKMT1A
C9J8F6

ENSG00000223572
142

CKMT2
P17540
1160
ENSG00000131730
143

CLDN6
P56747
9074
ENSG00000184697
144

CLEC3B
P05452
7123
ENSG00000163815
145

CLPX
O76031
10845
ENSG00000166855
146

CLSTN3
Q9BQT9
9746
ENSG00000139182
147

CLTC
Q00610
1213
ENSG00000141367
148

CLU
P10909
1191
ENSG00000120885
149

CNOT1
A5YKK6
23019
ENSG00000125107
150

COCH
O43405
1690
ENSG00000100473
151

COL11A1
P12107
1301
ENSG00000060718
152

COL11A2
P13942
1302
ENSG00000227801
153

COL12A1
Q99715
1303
ENSG00000111799
154

COL14A1
Q05707
7373
ENSG00000187955
155

COL16A1
Q07092
1307
ENSG00000084636
156

COL18A1
P39060
80781
ENSG00000182871
157

COL1A1
P02452
1277
ENSG00000108821
158

COL1A2
P08123
1278
ENSG00000164692
159

COL22A1
Q8NFW1
169044
ENSG00000169436
160

COL25A1
Q9BXS0
84570
ENSG00000188517
161

COL26A1
Q96A83
136227
ENSG00000160963
162

COL2A1
P02458
1280
ENSG00000139219
163

COL3A1
P02461
1281
ENSG00000168542
164

COL4A1
P02462
1282
ENSG00000187498
165

COL4A2
P08572
1284
ENSG00000134871
166

COL4A3
Q01955
1285
ENSG00000169031
167

COL4A6
Q14031
1288
ENSG00000197565
168

COL5A1
P20908
1289
ENSG00000130635
169

COL5A2
P05997
1290
ENSG00000204262
170

COL5A3
P25940
50509
ENSG00000080573
171

COL6A1
P12109
1291
ENSG00000142156
172

COL6A2
P12110
1292
ENSG00000142173
173

COL6A3
P12111
1293
ENSG00000163359
174

COL9A2
Q14055
1298
ENSG00000049089
175

COLEC10
Q9Y6Z7
10584
ENSG00000184374
176

COMP
P49747
1311
ENSG00000105664
177

COPA
P53621
1314
ENSG00000122218
178

COTL1
Q14019
23406
ENSG00000103187
179

CP
P00450
1356
ENSG00000047457
180

CPA4
Q9UI42
51200
ENSG00000128510
181

CPE
P16870
1363
ENSG00000109472
182

CPN1
P15169
1369
ENSG00000120054
183

CPNE1
Q99829
8904
ENSG00000214078
184

CPVL
Q9H3G5
54504
ENSG00000106066
185

CPXM1
Q96SM3
56265
ENSG00000088882
186

CPXM2
Q8N436
119587
ENSG00000121898
187

CPZ
Q66K79
8532
ENSG00000109625
188

CRIM1
Q9NZV1
51232
ENSG00000150938
189

CRISPLD1
Q9H336
83690
ENSG00000121005
190

CRLF1
O75462
9244
ENSG00000006016
191

CRYL1
Q9Y2S2
51084
ENSG00000165475
192

CS
O75390
1431
ENSG00000062485
193

CSDE1
O75534
7812
ENSG00000009307
194

CSF2RA
P15509
1438
ENSG00000198223
195

CST1
P01037
1469
ENSG00000170373
196

CST3
P01034
1471
ENSG00000101439
197

CST4
P01036
1472
ENSG00000101441
198

CTGF
Q5M8T4
1490

199

CTNNA1
P35221
1495
ENSG00000044115
200

CTSD
P07339
1509
ENSG00000117984
201

CTSV
O60911
1515
ENSG00000136943
202

CUL2
Q13617
8453
ENSG00000108094
203

CUL3
Q13618
8452
ENSG00000036257
204

CUL4B
Q13620
8450
ENSG00000158290
205

CUTA
O60888
51596
ENSG00000112514
206

CXADR
P78310
1525
ENSG00000154639
207

CXCL12
P48061
6387
ENSG00000107562
208

CYR61
Q6FI18
3491

209

DAG1
Q14118
1605
ENSG00000173402
210

DARS
P14868
1615
ENSG00000115866
211

DBNL
Q9UJU6
28988
ENSG00000136279
212

DCD
P81605
117159
ENSG00000161634
213

DDOST
P39656
1650
ENSG00000244038
214

DDR1
Q08345
780
ENSG00000137332
215

DDX17
Q92841
10521
ENSG00000100201
216

DDX39B
Q13838
7919
ENSG00000215425
217

DENND5A
Q6IQ26
23258
ENSG00000184014
218

DHFR
P00374
1719
ENSG00000228716
219

DHX29
Q7Z478
54505
ENSG00000067248
220

DKK1
O94907
22943
ENSG00000107984
221

DKK3
Q9UBP4
27122
ENSG00000050165
222

DKK4
Q9UBT3
27121
ENSG00000104371
223

DLG3
Q92796
1741
ENSG00000082458
224

DMBT1
Q9UGM3
1755
ENSG00000187908
225

DNAAF5
Q86Y56
54919
ENSG00000164818
226

DNAJB11
Q9UBS4
51726
ENSG00000090520
227

DNAJC3
Q13217
5611
ENSG00000102580
228

DNMT1
P26358
1786
ENSG00000130816
229

DRAXIN
Q8NBI3
374946
ENSG00000162490
230

DRG1
Q9Y295
4733
ENSG00000185721
231

DSG2
Q14126
1829
ENSG00000046604
232

ECM1
Q16610
1893
ENSG00000143369
233

EDA
Q92838
1896
ENSG00000158813
234

EDIL3
O43854
10085
ENSG00000164176
235

EEF1G
P26641
1937
ENSG00000254772
236

EFEMP1
Q12805
2202
ENSG00000115380
237

EFTUD2
Q15029
9343
ENSG00000108883
238

EGFLAM
Q63HQ2
133584
ENSG00000164318
239

EIF1AY
O14602
9086
ENSG00000198692
240

EIF2B4
Q9UI10
8890
ENSG00000115211
241

EIF2S1
P05198
1965
ENSG00000134001
242

EIF2S2
P20042
8894
ENSG00000125977
243

EIF3A
Q14152
8661
ENSG00000107581
244

EIF3C
Q99613
8663
ENSG00000184110
245

EIF3F
O00303
8665
ENSG00000175390
246

EIF3H
O15372
8667
ENSG00000147677
247

EIF3M
Q7L2H7
10480
ENSG00000149100
248

EIF5
P55010
1983
ENSG00000100664
249

EIF5B
O60841
9669
ENSG00000158417
250

ELAC2
Q9BQ52
60528
ENSG00000006744
251

ELP3
Q9H9T3
55140
ENSG00000134014
252

EMILIN2
Q9BXX0
84034
ENSG00000132205
253

EPHA4
P54764
2043
ENSG00000116106
254

EPHB2
P29323
2048
ENSG00000133216
255

EPHB4
P54760
2050
ENSG00000196411
256

EPRS
P07814
2058
ENSG00000136628
257

ERBB3
P21860
2065
ENSG00000065361
258

ERLIN1
O75477
10613
ENSG00000107566
259

ERVMER34-1
Q9H9K5
100288413
ENSG00000226887
260

EXTL2
Q9UBQ6
2135
ENSG00000162694
261

EZR
P15311
7430
ENSG00000092820
262

F10
P00742
2159
ENSG00000126218
263

F13A1
P00488
2162
ENSG00000124491
264

F2
P00734
2147
ENSG00000180210
265

F5
P12259
2153
ENSG00000198734
266

FAM129B
Q96TA1
64855
ENSG00000136830
267

FAP
Q12884
2191
ENSG00000078098
268

FAT1
Q14517
2195
ENSG00000083857
269

FBLN1
P23142
2192
ENSG00000077942
270

FBLN2
P98095
2199
ENSG00000163520
271

FBN1
P35555
2200

272

FBN2
P35556
2201
ENSG00000138829
273

FERMT2
Q96AC1
10979
ENSG00000073712
274

FGB
P02675
2244
ENSG00000171564
275

FGF17
O60258
8822
ENSG00000158815
276

FGF2
P09038
2247
ENSG00000138685
277

FGF8
P55075
2253
ENSG00000107831
278

FGF4
P08620
2249
ENSG00000075388
279

FGF6
P10767
2251
ENSG00000111241
280

FGFBP3
Q8TAT2
143282
ENSG00000174721
281

FGFR1
P11362
2260
ENSG00000077782
282

FGFR2
P21802
2263
ENSG00000066468
283

FGFR4
P22455
2264
ENSG00000160867
284

FGFRL1
Q8N441
53834
ENSG00000127418
285

FH
P07954
2271
ENSG00000091483
286

FLT1
P17948
2321
ENSG00000102755
287

FN1
P02751
2335
ENSG00000115414
288

FRAS1
Q86XX4
80144
ENSG00000138759
289

FRZB
Q92765
2487
ENSG00000162998
290

FST
P19883
10468
ENSG00000134363
291

FSTL1
Q12841
11167
ENSG00000163430
292

FUCA2
Q9BTY2
2519
ENSG00000001036
293

FXR1
P51114
8087
ENSG00000114416
294

GALNT1
Q10472
2589
ENSG00000141429
295

GALNT16
Q8N428
57452
ENSG00000100626
296

GALNT2
Q10471
2590
ENSG00000143641
297

GALNT7
Q86SF2
51809
ENSG00000109586
298

GANAB
Q14697
23193
ENSG00000089597
299

GARS
P41250
2617
ENSG00000106105
300

GBA
P04062
2629
ENSG00000177628
301

GC
P02774
2638
ENSG00000145321
302

GCNT1
Q02742
2650
ENSG00000187210
303

GDF11
O95390
10220
ENSG00000135414
304

GDF15
Q99988
9518
ENSG00000130513
305

GDF6
Q6KF10
392255
ENSG00000156466
306

GEMIN5
Q8TEQ6
25929
ENSG00000082516
307

GFAP
P14136
2670
ENSG00000131095
308

GGH
Q92820
8836
ENSG00000137563
309

GLB1
P16278
2720
ENSG00000170266
310

GLG1
Q92896
2734
ENSG00000090863
311

GM2A
P17900
2760
ENSG00000196743
312

GNAS
O95467
2778
ENSG00000087460
313

GOLM1
Q8NBJ4
51280
ENSG00000135052
314

GOT2
P00505
2806
ENSG00000125166
315

GPC1
P35052
2817
ENSG00000063660
316

GPC3
P51654
2719
ENSG00000147257
317

GPC4
O75487
2239
ENSG00000076716
318

GPI
P06744
2821
ENSG00000105220
319

GPRC5B
Q9NZH0
51704
ENSG00000167191
320

GPX4
P36969
2879
ENSG00000167468
321

GREM1
O60565
26585
ENSG00000166923
322

GRN
P28799
2896
ENSG00000030582
323

GRSF1
Q12849
2926
ENSG00000132463
324

GSN
P06396
2934
ENSG00000148180
325

GSPT1
P15170
2935
ENSG00000103342
326

GTF3C3
Q9Y5Q9
9330
ENSG00000119041
327

HABP2
Q14520
3026
ENSG00000148702
328

HADHB
P55084
3032
ENSG00000138029
329

HAPLN1
P10915
1404
ENSG00000145681
330

HAPLN3
Q96S86
145864

331

HAPLN4
Q86UW8
404037

332

HARS
P12081
3035
ENSG00000170445
333

HBB
P68871
3043
ENSG00000244734
334

HBS1L
Q9Y450
10767
ENSG00000112339
335

HDGF
P51858
3068
ENSG00000143321
336

HDGFL2
Q7Z4V5
84717
ENSG00000167674
337

HDLBP
Q00341
3069
ENSG00000115677
338

HGF
P14210
3082
ENSG00000019991
339

HGFAC
Q04756
3083
ENSG00000109758
340

HIST1H1C
P16403
3006
ENSG00000187837
341

HIST1H1E
P10412
3008
ENSG00000168298
342

HLA-C
P04222

ENSG00000225691
343

HMCN1
Q96RW7
83872
ENSG00000143341
344

HMCN2
Q8NDA2

ENSG00000148357
345

HMGB1
P09429
3146
ENSG00000189403
346

HMGB2
P26583
3148
ENSG00000164104
347

HMGB3
O15347
3149
ENSG00000029993
348

HMGN1
P05114
3150
ENSG00000205581
349

HMGN5
P82970
79366
ENSG00000198157
350

HNRNPA2B1
P22626
3181
ENSG00000122566
351

HNRNPDL
O14979
9987
ENSG00000152795
352

HP
P00738
3240
ENSG00000257017
353

HP1BP3
Q5SSJ5
50809
ENSG00000127483
354

HPR
P00739
3250
ENSG00000261701
355

HPX
P02790
3263
ENSG00000110169
356

HS3ST3A1
Q9Y663
9955
ENSG00000153976
357

HS6ST1
O60243
9394
ENSG00000136720
358

HS6ST2
Q96MM7
90161
ENSG00000171004
359

HSD17B10
Q99714
3028
ENSG00000072506
360

HSD17B4
P51659
3295
ENSG00000133835
361

HSP90AA1
P07900
3320
ENSG00000080824
362

HSP90AB1
P08238
3326
ENSG00000096384
363

HSP90B1
P14625
7184
ENSG00000166598
364

HSPA5
P11021
3309
ENSG00000044574
365

HSPG2
P98160
3339
ENSG00000142798
366

HTRA1
Q92743
5654
ENSG00000166033
367

HYOU1
Q9Y4L1
10525
ENSG00000149428
368

IARS
P41252
3376
ENSG00000196305
369

ICAM2
P13598
3384
ENSG00000108622
370

IDE
P14735
3416
ENSG00000119912
371

IDH1
O75874
3417
ENSG00000138413
372

IDH2
P48735
3418
ENSG00000182054
373

IGF1
P05019
3479
ENSG00000017427
374

IGF2
P01344
3481
ENSG00000167244
375

IGFBP2
P18065
3485
ENSG00000115457
376

IGFBP3
P17936
3486
ENSG00000146674
377

IGFBP4
P22692
3487
ENSG00000141753
378

IGFBP5
P24593
3488
ENSG00000115461
379

IGFBP6
P24592
3489
ENSG00000167779
380

IGFBP7
Q16270
3490
ENSG00000163453
381

IGFBPL1
Q8WX77
347252
ENSG00000137142
382

IGHA1
P01876

ENSG00000211895
383

IGHA2
P01877

ENSG00000211890
384

IGHG1
P01857

ENSG00000211896
385

IGHG2
P01859

ENSG00000211893
386

IGHG4
P01861

ENSG00000211892
387

IGHM
P01871

ENSG00000211899
388

IGKC
P01834

389

IGKV2-28
A0A075B6P5

ENSG00000244116
390

IGKV2D-40
P01614

ENSG00000251039
391

IGKV3D-20
A0A0C4DH25

ENSG00000211625
392

IGLC2
P0DOY2

ENSG00000211677
393

IGLC3
P0DOY3

ENSG00000211679
394

IGLV2-11
P01706

ENSG00000211668
395

IGSF1
Q8N6C5
3547
ENSG00000147255
396

IGSF10
Q6WRI0
285313
ENSG00000152580
397

ILF2
Q12905
3608
ENSG00000143621
398

INHBA
P08476
3624
ENSG00000122641
399

INS
P01308
3630
ENSG00000254647
400

INS-IGF2
F8WCM5
723961
ENSG00000129965
401

IPO11
Q9UI26
51194
ENSG00000086200
402

IPO5
O00410
3843
ENSG00000065150
403

IPO8
O15397
10526
ENSG00000133704
404

IQGAP1
P46940
8826
ENSG00000140575
405

ISOC1
Q96CN7
51015
ENSG00000066583
406

ITGAL
P20701
3683
ENSG00000005844
407

ITIH1
P19827
3697
ENSG00000055957
408

ITIH2
P19823
3698
ENSG00000151655
409

ITIH3
Q06033
3699
ENSG00000162267
410

ITIH4
Q14624
3700
ENSG00000055955
411

ITIH5
Q86UX2
80760
ENSG00000123243
412

ITLN2
Q8WWU7
142683
ENSG00000158764
413

JCHAIN
P01591
3512
ENSG00000132465
414

KARS
Q15046
3735
ENSG00000065427
415

KDM1A
O60341
23028
ENSG00000004487
416

KMT2A
Q03164
4297
ENSG00000118058
417

KNG1
P01042
3827
ENSG00000113889
418

KRT10
P13645
3858
ENSG00000186395
419

KRT14
P02533
3861
ENSG00000186847
420

KRT17
Q04695
3872
ENSG00000128422
421

KRT18
P05783
3875
ENSG00000111057
422

KRT8
P05787
3856
ENSG00000170421
423

LACRT
Q9GZZ8
90070
ENSG00000135413
424

LAG3
P18627
3902
ENSG00000089692
425

LAMA1
P25391
284217
ENSG00000101680
426

LAMA2
P24043
3908
ENSG00000196569
427

LAMA5
O15230
3911
ENSG00000130702
428

LAMB1
P07942
3912
ENSG00000091136
429

LAMB2
P55268
3913
ENSG00000172037
430

LAMC1
P11047
3915
ENSG00000135862
431

LARS
Q9P2J5
51520
ENSG00000133706
432

LCAT
P04180
3931
ENSG00000213398
433

LCN1
P31025
3933
ENSG00000160349
434

LDHA
P00338
3939
ENSG00000134333
435

LECT2
O14960
3950
ENSG00000145826
436

LEFTY1
O75610
10637
ENSG00000243709
437

LEFTY2
O00292
7044
ENSG00000143768
438

LEFTYA

439

LFNG
Q8NES3
3955
ENSG00000106003
440

LGALS3BP
Q08380
3959
ENSG00000108679
441

LGALS7
M0R281

ENSG00000205076
442

LIG3
P49916
3980
ENSG00000005156
443

LINGO1
Q96FE5
84894
ENSG00000169783
444

LIPG
Q9Y5X9
9388
ENSG00000101670
445

LMAN2
Q12907
10960
ENSG00000169223
446

LMNA
P02545
4000
ENSG00000160789
447

LOXL1
Q08397
4016
ENSG00000129038
448

LOXL2
Q9Y4K0
4017
ENSG00000134013
449

LOXL3
P58215
84695
ENSG00000115318
450

LPL
P06858
4023
ENSG00000175445
451

LRG1
P02750
116844
ENSG00000171236
452

LRP1
Q07954
4035
ENSG00000123384
453

LRPAP1
P30533
4043
ENSG00000163956
454

LRRC59
Q96AG4
55379
ENSG00000108829
455

LRRTM4
Q86VH4
80059
ENSG00000176204
456

LSR
Q86X29
51599
ENSG00000105699
457

LTBP1
Q14766
4052
ENSG00000049323
458

LTBP4
Q8N2S1
8425
ENSG00000090006
459

LTF
P02788
4057
ENSG00000012223
460

LUM
P51884
4060
ENSG00000139329
461

LYAR
Q9NX58
55646
ENSG00000145220
462

LYZ
P61626
4069
ENSG00000090382
463

MANF
P55145
7873
ENSG00000145050
464

MAP2K2
P36507
5605
ENSG00000126934
465

MAP4
P27816
4134
ENSG00000047849
466

MAPK1
P28482
5594
ENSG00000100030
467

MASP1
P48740
5648
ENSG00000127241
468

MATN2
O00339
4147
ENSG00000132561
469

MATN3
O15232
4148
ENSG00000132031
470

MATR3
P43243
9782
ENSG00000015479
471

MAZ
P56270
4150
ENSG00000103495
472

MBNL1
Q9NR56
4154
ENSG00000152601
473

MCM5
P33992
4174
ENSG00000100297
474

MDH2
P40926
4191
ENSG00000146701
475

MDK
P21741
4192
ENSG00000110492
476

MEGF10
Q96KG7
84466
ENSG00000145794
477

MEGF6
O75095
1953
ENSG00000162591
478

METAP2
P50579
10988
ENSG00000111142
479

METTL14
Q9HCE5
57721
ENSG00000145388
480

MFAP2
P55001
4237
ENSG00000117122
481

MFGE8
Q08431
4240
ENSG00000140545
482

MGAT1
P26572
4245
ENSG00000131446
483

MIF
P14174
4282
ENSG00000240972
484

MINPP1
Q9UNW1
9562
ENSG00000107789
485

MMP2
P08253
4313
ENSG00000087245
486

MMP9
P14780
4318
ENSG00000100985
487

MSMB
P08118
4477
ENSG00000263639
488

MSN
P26038
4478
ENSG00000147065
489

MST1
P26927
4485
ENSG00000173531
490

MST1L
Q2TV78
11223

491

MTDH
Q86UE4
92140
ENSG00000147649
492

MTHFD1
P11586
4522
ENSG00000100714
493

MTHFD2
P13995
10797
ENSG00000065911
494

MXRA5
Q9NR99
25878
ENSG00000101825
495

MYBBP1A
Q9BQG0
10514
ENSG00000132382
496

MYL3
P08590
4634
ENSG00000160808
497

MYL4
P12829
4635
ENSG00000198336
498

NAA15
Q9BXJ9
80155
ENSG00000164134
499

NAMPT
P43490
10135
ENSG00000105835
500

NASP
P49321
4678
ENSG00000132780
501

NCAM1
P13591
4684
ENSG00000149294
502

NCAN
O14594
1463
ENSG00000130287
503

NDNF
Q8TB73
79625
ENSG00000173376
504

NDST1
P52848
3340
ENSG00000070614
505

NECTIN1
Q15223
5818
ENSG00000110400
506

NECTIN3
Q9NQS3
25945
ENSG00000177707
507

NELL2
Q99435
4753
ENSG00000184613
508

NID1
P14543
4811
ENSG00000116962
509

NID2
Q14112
22795
ENSG00000087303
510

NIPBL
Q6KC79
25836
ENSG00000164190
511

NLGN3
Q9NZ94
54413
ENSG00000196338
512

NLGN4Y
Q8NFZ3
22829
ENSG00000165246
513

NME1-NME2
J3KPD9

ENSG00000011052
514

NMT1
P30419
4836
ENSG00000136448
515

NOLC1
Q14978
9221
ENSG00000166197
516

NOV
A0A024R9J4
4856

517

NPC2
P61916
10577
ENSG00000119655
518

NPM3
O75607
10360
ENSG00000107833
519

NPTX1
Q15818
4884

520

NPTX2
P47972
4885
ENSG00000106236
521

NPTXR
O95502
23467
ENSG00000221890
522

NRG1
Q02297
3084
ENSG00000157168
523

NRG2
O14511
9542
ENSG00000158458
524

NRP1
O14786
8829
ENSG00000099250
525

NRP2
O60462
8828
ENSG00000118257
526

NSUN5
Q96P11
55695
ENSG00000130305
527

NTS
P30990
4922
ENSG00000133636
528

NUBP2
Q9Y5Y2
10101
ENSG00000095906
529

NUCB1
Q02818
4924
ENSG00000104805
530

NUMA1
Q14980
4926
ENSG00000137497
531

NUP155
O75694
9631
ENSG00000113569
532

OAF
Q86UD1
220323
ENSG00000184232
533

OLA1
Q9NTK5
29789
ENSG00000138430
534

OLFM2
O95897
93145

535

OLFML2A
Q68BL7
169611
ENSG00000185585
536

OLFML3
Q9NRN5
56944
ENSG00000116774
537

ORM1
P02763
5004
ENSG00000229314
538

ORM2
P19652
5005
ENSG00000228278
539

P4HB
P07237
5034
ENSG00000185624
540

PACSIN2
Q9UNF0
11252
ENSG00000100266
541

PAFAH1B1
P43034
5048
ENSG00000007168
542

PAIP1
Q9H074
10605
ENSG00000172239
543

PAM
P19021
5066
ENSG00000145730
544

PAMR1
Q6UXH9
25891
ENSG00000149090
545

PAPLN
O95428
89932
ENSG00000100767
546

PAPPA
Q13219
5069
ENSG00000182752
547

PARP1
P09874
142
ENSG00000143799
548

PC
P11498
5091
ENSG00000173599
549

PCDH1
Q08174
5097
ENSG00000156453
550

PCLO
Q9Y6V0
27445
ENSG00000186472
551

PCOLCE
Q15113
5118
ENSG00000106333
552

PCOLCE2
Q9UKZ9
26577
ENSG00000163710
553

PCSK5
Q92824
5125
ENSG00000099139
554

PCSK9
Q8NBP7
255738
ENSG00000169174
555

PDCD6IP
Q8WUM4
10015
ENSG00000170248
556

PDGFD
Q9GZP0
80310
ENSG00000170962
557

PDGFRL
Q15198
5157
ENSG00000104213
558

PDIA3
P30101
2923
ENSG00000167004
559

PDIA4
P13667
9601
ENSG00000155660
560

PDIA5
Q14554
10954
ENSG00000065485
561

PDIA6
Q15084
10130
ENSG00000143870
562

PFAS
O15067
5198
ENSG00000178921
563

PFKP
Q01813
5214
ENSG00000067057
564

PFN1
P07737
5216
ENSG00000108518
565

PGD
P52209
5226
ENSG00000142657
566

PGLYRP2
Q96PD5
114770
ENSG00000161031
567

PHGDH
O43175
26227
ENSG00000092621
568

PI16
Q6UXB8
221476
ENSG00000164530
569

PIGR
P01833
5284
ENSG00000162896
570

PIP
P12273
5304
ENSG00000159763
571

PKDCC
Q504Y2
91461
ENSG00000162878
572

PKM
P14618
5315
ENSG00000067225
573

PLAT
P00750
5327
ENSG00000104368
574

PLAU
P00749
5328
ENSG00000122861
575

PLCB3
Q01970
5331
ENSG00000149782
576

PLEC
Q15149
5339
ENSG00000178209
577

PLG
P00747
5340
ENSG00000122194
578

PLIN4
Q96Q06
729359
ENSG00000167676
579

PLOD1
Q02809
5351
ENSG00000083444
580

PLOD2
O00469
5352
ENSG00000152952
581

PLOD3
O60568
8985
ENSG00000106397
582

PLTP
P55058
5360
ENSG00000100979
583

POLL
Q9UGP5
27343
ENSG00000166169
584

POMC
P01189
5443
ENSG00000115138
585

POSTN
Q15063
10631
ENSG00000133110
586

PPIA
P62937
5478
ENSG00000196262
587

PPIB
P23284
5479
ENSG00000166794
588

PPP1CA
P62136
5499
ENSG00000172531
589

PPP1CC
P36873
5501
ENSG00000186298
590

PPP2R1A
P30153
5518
ENSG00000105568
591

PPT1
P50897
5538
ENSG00000131238
592

PRB3
Q04118

ENSG00000197870
593

PRB4
P10163

594

PRCP
P42785
5547
ENSG00000137509
595

PRDX2
P32119
7001
ENSG00000167815
596

PRDX4
Q13162
10549
ENSG00000123131
597

PRDX5
P30044
25824
ENSG00000126432
598

PRG4
Q92954
10216
ENSG00000116690
599

PRKDC
P78527
5591
ENSG00000253729
600

PRMT1
Q99873
3276
ENSG00000126457
601

PRMT5
O14744
10419
ENSG00000100462
602

PROM1
O43490
8842
ENSG00000007062
603

PRPF19
Q9UMS4
27339
ENSG00000110107
604

PRPF40A
O75400
55660
ENSG00000196504
605

PRPF4B
Q13523
8899
ENSG00000112739
606

PRPF6
O94906
24148
ENSG00000101161
607

PRPF8
Q6P2Q9
10594
ENSG00000174231
608

PRPSAP2
O60256
5636
ENSG00000141127
609

PRR4
Q16378
11272
ENSG00000111215
610

PRSS2
P07478
5645
ENSG00000275896
611

PRSS23
O95084
11098
ENSG00000150687
612

PRSS3
P35030
5646
ENSG00000010438
613

PRTG
Q2VWP7
283659
ENSG00000166450
614

PSIP1
O75475
11168
ENSG00000164985
615

PSMB6
P28072
5694
ENSG00000142507
616

PSMD1
Q99460
5707
ENSG00000173692
617

PSMD2
Q13200
5708
ENSG00000175166
618

PSMD5
Q16401
5711
ENSG00000095261
619

PSMD6
Q15008
9861
ENSG00000163636
620

PSMD8
P48556
5714
ENSG00000099341
621

PSME3
P61289
10197
ENSG00000131467
622

PTK2
Q05397
5747
ENSG00000169398
623

PTK7
Q13308
5754
ENSG00000112655
624

PTN
P21246
5764
ENSG00000105894
625

PTPRC
P08575
5788
ENSG00000081237
626

PTPRD
P23468
5789
ENSG00000153707
627

PTPRF
P10586
5792
ENSG00000142949
628

PTPRS
Q13332
5802
ENSG00000105426
629

PTPRZ1
P23471
5803
ENSG00000106278
630

PUF60
Q9UHX1
22827
ENSG00000179950
631

PXDN
Q92626
7837
ENSG00000130508
632

PZP
P20742

ENSG00000126838
633

QPRT
Q15274
23475
ENSG00000103485
634

QSOX1
O00391
5768
ENSG00000116260
635

RAB7A
P51149
7879
ENSG00000075785
636

RACK1
P63244
10399
ENSG00000204628
637

RARRES2
Q99969
5919
ENSG00000106538
638

RBMX
P38159
27316
ENSG00000147274
639

RBP4
P02753
5950
ENSG00000138207
640

RCC1
P18754
1104
ENSG00000180198
641

RCOR1
Q9UKL0
23186
ENSG00000089902
642

RECQL
P46063
5965
ENSG00000004700
643

RELN
P78509
5649
ENSG00000189056
644

RNASE1
P07998
6035
ENSG00000129538
645

RNASE4
P34096
6038
ENSG00000258818
646

ROBO1
Q9Y6N7
6091
ENSG00000169855
647

RPL14
P50914
9045
ENSG00000188846
648

RPL18
Q07020
6141
ENSG00000063177
649

RPL19
P84098
6143
ENSG00000108298
650

RPL23A
P62750
6147
ENSG00000198242
651

RPL26
P61254
6154
ENSG00000161970
652

RPL29
P47914
6159
ENSG00000162244
653

RPL3
P39023
6122
ENSG00000100316
654

RPL35
P42766
11224
ENSG00000136942
655

RPL4
P36578
6124
ENSG00000174444
656

RPL9
A0A2R8Y5Y7

ENSG00000163682
657

RPLP0
P05388
6175
ENSG00000089157
658

RPLP1
P05386
6176
ENSG00000137818
659

RPLP2
P05387
6181
ENSG00000177600
660

RPN2
P04844
6185
ENSG00000118705
661

RPS13
P62277
6207
ENSG00000110700
662

RPS20
P60866
6224
ENSG00000008988
663

RPS23
P62266
6228
ENSG00000186468
664

RPS27A
P62979
6233
ENSG00000143947
665

RPS27L
Q71UM5
51065
ENSG00000185088
666

RPS3
P23396
6188
ENSG00000149273
667

RRBP1
Q9P2E9
6238
ENSG00000125844
668

RSF1
Q96T23
51773
ENSG00000048649
669

RSL1D1
O76021
26156
ENSG00000171490
670

RTF1
Q92541
23168
ENSG00000137815
671

RTN4
Q9NQC3
57142
ENSG00000115310
672

RTN4RL2
Q86UN3
349667
ENSG00000186907
673

RUVBL2
Q9Y230
10856
ENSG00000183207
674

S100A12
P80511
6283
ENSG00000163221
675

S100A13
Q99584
6284
ENSG00000189171
676

S100A7
P31151
6278
ENSG00000143556
677

S100A8
P05109
6279
ENSG00000143546
678

S100A9
P06702
6280
ENSG00000163220
679

SAP30
O75446
8819
ENSG00000164105
680

SARS
P49591
6301
ENSG00000031698
681

SBSN
Q6UWP8
374897
ENSG00000189001
682

SCG3
Q8WXD2
29106
ENSG00000104112
683

SCGB2A2
Q13296
4250
ENSG00000110484
684

SCUBE1
Q8IWY4
80274
ENSG00000159307
685

SCUBE3
Q8IX30
222663
ENSG00000146197
686

SDC1
P18827
6382
ENSG00000115884
687

SDC4
P31431
6385
ENSG00000124145
688

SDCBP
O00560
6386
ENSG00000137575
689

SDF4
Q9BRK5
51150
ENSG00000078808
690

SEC13
P55735
6396
ENSG00000157020
691

SELENOP
P49908
6414
ENSG00000250722
692

SEMA3A
Q14563
10371
ENSG00000075213
693

SEMA3F
Q13275
6405
ENSG00000001617
694

SEMA4B
Q9NPR2
10509
ENSG00000185033
695

SEMA4D
Q92854
10507
ENSG00000187764
696

SEMA5A
Q13591
9037
ENSG00000112902
697

SEMA6A
Q9H2E6
57556
ENSG00000092421
698

SEMA6D
Q8NFY4
80031
ENSG00000137872
699

SEMG1
P04279
6406
ENSG00000124233
700

SEPT9
Q9UHD8
10801
ENSG00000184640
701

SERBP1
Q8NC51
26135
ENSG00000142864
702

SERPINA1
P01009
5265
ENSG00000197249
703

SERPINA3
P01011
12
ENSG00000196136
704

SERPINA5
P05154
5104
ENSG00000188488
705

SERPINA7
P05543
6906
ENSG00000123561
706

SERPINB12
Q96P63
89777
ENSG00000166634
707

SERPINB3
P29508
6317
ENSG00000057149
708

SERPINB9
P50453
5272
ENSG00000170542
709

SERPINC1
P01008
462
ENSG00000117601
710

SERPIND1
P05546
3053
ENSG00000099937
711

SERPINE1
P05121
5054
ENSG00000106366
712

SERPINE2
P07093
5270
ENSG00000135919
713

SERPINF1
P36955
5176
ENSG00000132386
714

SERPINF2
P08697
5345
ENSG00000167711
715

SERPING1
P05155
710
ENSG00000149131
716

SERPINH1
P50454
871
ENSG00000149257
717

SERPINI1
Q99574
5274
ENSG00000163536
718

SF3B1
O75533
23451
ENSG00000115524
719

SF3B2
Q13435
10992
ENSG00000087365
720

SF3B3
Q15393
23450
ENSG00000189091
721

SFPQ
P23246
6421
ENSG00000116560
722

SFRP1
Q8N474
6422
ENSG00000104332
723

SFRP2
Q96HF1
6423
ENSG00000145423
724

SH2B1
Q9NRF2
25970
ENSG00000178188
725

SHBG
P04278
6462
ENSG00000129214
726

SHMT1
P34896
6470
ENSG00000176974
727

SKIV2L2
L8E9T8

728

SLC1A5
Q15758
6510
ENSG00000105281
729

SLC2A14
Q8TDB8
144195
ENSG00000173262
730

SLC39A10
Q9ULF5
57181
ENSG00000196950
731

SLIT2
O94813
9353

732

SLIT3
O75094
6586
ENSG00000184347
733

SLPI
P03973
6590
ENSG00000124107
734

SLTM
Q9NWH9
79811
ENSG00000137776
735

SLURP1
P55000
57152
ENSG00000126233
736

SMARCC1
Q92922
6599
ENSG00000173473
737

SMARCD1
Q96GM5
6602
ENSG00000066117
738

SMARCD2
Q92925
6603
ENSG00000108604
739

SMC1A
Q14683
8243
ENSG00000072501
740

SMC3
Q9UQE7
9126
ENSG00000108055
741

SMOC1
Q9H4F8
64093
ENSG00000198732
742

SMOC2
Q9H3U7
64094
ENSG00000112562
743

SMPDL3B
Q92485
27293
ENSG00000130768
744

SMR3B
P02814
10879
ENSG00000171201
745

SNRPB
P14678
6628
ENSG00000125835
746

SNRPD1
P62314
6632
ENSG00000167088
747

SNRPD3
P62318
6634
ENSG00000100028
748

SOD3
P08294
6649
ENSG00000109610
749

SPARC
P09486
6678
ENSG00000113140
750

SPINT1
O43278
6692
ENSG00000166145
751

SPINT2
O43291
10653
ENSG00000167642
752

SPOCK1
Q08629
6695
ENSG00000152377
753

SPON1
Q9HCB6
10418
ENSG00000262655
754

SPP1
P10451
6696
ENSG00000118785
755

SRP14
P37108
6727
ENSG00000140319
756

SRPX
P78539
8406
ENSG00000101955
757

SRPX2
O60687
27286
ENSG00000102359
758

SRSF1
Q07955
6426
ENSG00000136450
759

SSB
P05455
6741
ENSG00000138385
760

SSC5D
A1L4H1
284297
ENSG00000179954
761

ST6GAL1
P15907
6480
ENSG00000073849
762

ST6GAL2
Q96JF0
84620
ENSG00000144057
763

STAG1
Q8WVM7
10274
ENSG00000118007
764

STC1
P52823
6781
ENSG00000159167
765

STC2
O76061
8614
ENSG00000113739
766

SUB1
P53999
10923
ENSG00000113387
767

SULF2
Q8IWU5
55959
ENSG00000196562
768

SUMF2
Q8NBJ7
25870
ENSG00000129103
769

SUPT16H
Q9Y5B9
11198
ENSG00000092201
770

SUPT6H
Q7KZ85
6830
ENSG00000109111
771

SVEP1
Q4LDE5
79987
ENSG00000165124
772

SYNCRIP
O60506
10492
ENSG00000135316
773

TAGLN2
P37802
8407
ENSG00000158710
774

TBL1XR1
Q9BZK7
79718
ENSG00000177565
775

TCN2
P20062
6948
ENSG00000185339
776

TCOF1
Q13428
6949
ENSG00000070814
777

TF
P02787
7018
ENSG00000091513
778

TFAM
Q00059
7019
ENSG00000108064
779

TFPI
P10646
7035
ENSG00000003436
780

TFRC
P02786
7037
ENSG00000072274
781

TGFB2
P61812
7042
ENSG00000092969
782

TGFBI
Q15582
7045
ENSG00000120708
783

THBS1
P07996
7057
ENSG00000137801
784

THBS2
P35442
7058
ENSG00000186340
785

THBS3
P49746
7059
ENSG00000169231
786

THBS4
P35443
7060
ENSG00000113296
787

THOC3
Q96J01
84321
ENSG00000051596
788

THSD7A
Q9UPZ6
221981
ENSG00000005108
789

TIMP1
P01033
7076
ENSG00000102265
790

TIMP2
P16035
7077
ENSG00000035862
791

TIMP3
P35625
7078
ENSG00000100234
792

TINAGL1
Q9GZM7
64129
ENSG00000142910
793

TJP1
Q07157
7082
ENSG00000104067
794

TNC
P24821
3371
ENSG00000041982
795

TNN
Q9UQP3
63923

796

TNXB
P22105
7148
ENSG00000168477
797

TOP1
P11387
7150
ENSG00000198900
798

TPM4
P67936
7171
ENSG00000167460
799

TPP2
P29144
7174
ENSG00000134900
800

TRIM28
Q13263
10155
ENSG00000130726
801

TRIP10
Q15642
9322
ENSG00000125733
802

TRMT1
Q9NXH9
55621
ENSG00000104907
803

TSKU
Q8WUA8
25987
ENSG00000182704
804

TTR
P02766
7276
ENSG00000118271
805

TUBB4A
P04350
10382
ENSG00000104833
806

TUFM
P49411
7284
ENSG00000178952
807

TWSG1
Q9GZX9
57045
ENSG00000128791
808

TXN
P10599
7295
ENSG00000136810
809

TXNDC16
Q9P2K2
57544
ENSG00000087301
810

TXNDC5
Q8NBS9
81567
ENSG00000239264
811

U2AF2
P26368
11338
ENSG00000063244
812

UBE20
Q9C0C9
63893
ENSG00000175931
813

UBR4
Q5T4S7
23352
ENSG00000127481
814

UCHL1
P09936
7345
ENSG00000154277
815

UCHL3
P15374
7347
ENSG00000118939
816

UFL1
O94874
23376
ENSG00000014123
817

UGP2
Q16851
7360
ENSG00000169764
818

USP11
P51784
8237
ENSG00000102226
819

USP14
P54578
9097
ENSG00000101557
820

USP43
Q70EL4
124739
ENSG00000154914
821

UTP4
Q969X6
84916
ENSG00000141076
822

VARS
P26640
7407
ENSG00000096171
823

VASN
Q6EMK4
114990
ENSG00000168140
824

VCAN
P13611
1462
ENSG00000038427
825

VCP
P55072
7415
ENSG00000165280
826

VEGFA
P15692
7422
ENSG00000112715
827

VIT
Q6UXI7
5212
ENSG00000205221
828

VNN1
O95497
8876
ENSG00000112299
829

VPS35
Q96QK1
55737
ENSG00000069329
830

VTN
P04004
7448
ENSG00000109072
831

VWF
P04275
7450
ENSG00000110799
832

WDR3
Q9UNX4
10885
ENSG00000065183
833

WDR36
Q8NI36
134430
ENSG00000134987
834

WDR4
P57081
10785
ENSG00000160193
835

WDR43
Q15061
23160
ENSG00000163811
836

WFIKKN1
Q96NZ8
117166
ENSG00000127578
837

WFIKKN2
Q8TEU8
124857
ENSG00000173714
838

XRCC5
P13010
7520
ENSG00000079246
839

XYLT1
Q86Y38
64131
ENSG00000103489
840

XYLT2
Q9H1B5
64132
ENSG00000015532
841

YBX1
P67809
4904
ENSG00000065978
842

YBX3
P16989
8531
ENSG00000060138
843

ZG16B
Q96DA0
124220
ENSG00000162078
844

ZNF207
O43670
7756
ENSG00000010244
845

ZNF326
Q5BKZ1
284695
ENSG00000162664
846

ZNF706
Q9Y5V0
51123
ENSG00000120963
847

APLP1
P51693
333
ENSG00000105290
848

APP
P05067
351
ENSG00000142192
849

NPNT
Q6UXI9
255743
ENSG00000168743
850

RPL22
Q6UXI9
6146
ENSG00000116251
851

FGF19
O95750
9965
ENSG00000162344
852

BTC
P35070
685
ENSG00000174808
853

IL13RA2
Q14627
3598
ENSG00000123496
854

CD170
O15389

ENSG00000105501
855

IL15
P40933
3600
ENSG00000164136
856

WAP
Q8TEU8

ENSG00000173714
857

MFRP
Q9BY79
83552
ENSG00000235718
858

IL10Ra
Q13651
3587
ENSG00000110324
859

ChemR23
Q99788
1240
ENSG00000174600
860

HBEGF
Q99075
1839
ENSG00000113070
861

IL16
Q14005
3603
ENSG00000172349
862

IL7Ra
P16871
3575
ENSG00000168685
863

TNFSF10C
O14798
8794
ENSG00000173535
864

BMP6
P22004
654
ENSG00000153162
865

IL36g
P14778
56300
ENSG00000136688
866

IL1RA
P18510
3557
ENSG00000136689
867

KREMEN2
Q8NCW0
79412

868

TNFSF10D
Q9UBN6
8793
ENSG00000173530
869

CXCCR1
P49238
1524
ENSG00000168329
870

CCL23
P55773
6368
ENSG00000276114
871

Catenin
P35222
1499
ENSG00000168036
872

TNFSF10
P50591
8743
ENSG00000121858
873

CCL14
Q16627
6358
ENSG00000276409
874

IL2
P60568
3558
ENSG00000109471
875

FGF1
P05230
2246
ENSG00000113578
876

	Number	Date	Country
	62953425	Dec 2019	US
	62967393	Jan 2020	US

Heparin-Associated Polypeptides and Uses Thereof

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