Methods and compositions for cell therapies

Abstract

Described herein are methods and compositions for producing clinical grade myogenic progenitor cells from stem cells.

Description

[0001] F&RRefNo.: 09531-0562WO1

[0002] METHODS AND COMPOSITIONS FOR CELL THERAPIES

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority under 35 U.S.C. 119(e) to U.S. Application No. 63 / 749,405 filed on January’ 24, 2025, which is incorporated by reference herein in its entirety.

[0005] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under W81XWH-17-1 -0659 awarded by the Department of the Army and under AR055299 awarded by National Institutes of Health. The government has certain rights in the invention.

[0007] TECHNICAL FIELD

[0008] This disclosure generally relates to cell therapies.

[0009] BACKGROUND

[0010] Muscular dystrophies are a genetically and clinically heterogeneous group of more than 30 disorders in which progressive muscle weakness due to skeletal muscle degeneration leads to atrophy and loss of function. The most prevalent form is Duchenne Muscular Dystrophy (DMD), an X-linked lethal disorder affecting 1 in 5000 male live births. DMD is caused by mutations in DYSTROPHIN, the largest gene in the human genome. In the early phase of the disease, skeletal muscle is characterized by a continual process of degeneration and regeneration, ultimately leading to exhaustion of regenerative capacity', fibrosis, and disruption of the muscle tissue architecture. Clinically, DMD patients initially experience muscle weakness and atrophy that is progressive, leading to eventual wheelchair dependence by the teenage years and early death a decade or two later usually due to cardiorespiratory' failure. Although several therapeutic approaches for DMD have recently been approved by the Food and Drug Administration (FDA), including exon skipping and adeno-associated virus (AAV)-mediated gene therapy, outcomes remain sub-optimal and several obstacles remain. For example, exon skipping is restricted to certain mutations; immune responses against the AAV capsids and / or transgenes are common; and microdystrophins do not fully recapitulate full-length dystrophin function. Furthermore, low rates of delivery to the stem cell compartment areF&RRefNo.: 09531-0562WO1

[0011] predicted to limit the duration of effect, as muscle fibers turn over and are replaced with fibers carrying the original mutation. While these strategies have helped ameliorate disease progression in some patients, they are not curative. Therefore, novel therapeutic approaches for DMD as well as other forms of muscular dystrophies are still urgently needed.

[0012] SUMMARY

[0013] Methods and compositions are described for producing clinical grade myogenic progenitor cells from stem cells.

[0014] In one aspect, methods of producing clinical grade myogenic progenitor cells are provided. Such methods typically include the steps of: (a) providing stem cells that comprise an exogenous nucleic acid encoding PAX7 under control of an inducible promoter; (b) culturing the stem cells under conditions that cause the stem cells to undergo myogenic specification, wherein the conditions comprise exposing the stem cells to an inhibitor of GSK3, and an inhibitor of BMP and an inhibitor of TGF-beta; (c) contacting the stem cells undergoing myogenic specification with a compound that induces expression of PAX7; (d) contacting the stem cells undergoing myogenic specification with FGF to cause the stem cells undergoing myogenic specification to differentiate into myogenic progenitor cells; (e) dispersing cell aggregates into single cells; (1) isolating the myogenic progenitor cells using a CD54 antibody; (g) expanding the isolated myogenic progenitor cells in a large-scale culture system under appropriate conditions, thereby isolating clinical grade myogenic progenitor cells.

[0015] In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the stem cells are passage 16 to passage 22 stem cells.

[0016] In some embodiments, the stem cells are exposed to the inhibitor of GSK.3 from about day 2 to about day 4. In some embodiments, the inhibitor of GSK3 is CHIR99021. In some embodiments, the stem cells are exposed to the inhibitor of BMP and an inhibitor of TGF-beta from about day 4 to about day 6. In some embodiments, the inhibitor of BMP is LDN193189. In some embodiments, the inhibitor of TGF-beta is SB431542.

[0017] In some embodiments, the stem cells undergoing myogenic specification are contacted with the compound that induces expression of PAX7 starting on about day 5. In some embodiments, the compound that induces expression of PAX7 is doxycycline.F&RRefNo.: 09531-0562WO1

[0018] In some embodiments, the stem cells undergoing myogenic specification are contacted with FGF starting on about day 8.

[0019] In some embodiments, the dispersing occurs on about day 12.

[0020] In some embodiments, the isolating occurs on about day 16.

[0021] In some embodiments, the large-scale culture system is a 2-dimensional culture system. In some embodiments, the large-scale culture system is a 3-dimensional culture system. In some embodiments, the large-scale culture system is a cell stack.

[0022] In some embodiments, the appropriate conditions for expanding the isolated myogenic progenitor cells comprises IMDM media (e.g., bFGF, hyclone, defined FBS, knockout serum replacement, insulin-transferrin-selenium, glutamax).

[0023] In some embodiments, the method further includes performing a quality control test to determine the number of cells. In some embodiments, the method further includes performing a quality control test to determine the quality of cells. In some embodiments, the method further includes exposing the stem cells to an inhibitor of ROCK (e g., Y27632) from about day 0 to about day 2.

[0024] In some embodiments, the clinical grade myogenic progenitor cells mature into skeletal muscle.

[0025] In another aspect, clinical grade myogenic progenitor cells are provided. Such cells are made by the methods described herein.

[0026] In still another aspect, methods of treating a muscle disease are provided. Such methods typically include administering the clinical grade myogenic progenitor cells described herein to an individual suffering from a muscle disease.

[0027] In some embodiments, the muscle disease is muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy.

[0028] In some embodiments, the cells are allogeneic to the individual. In some embodiments, the cells are autologous to the individual.

[0029] In some embodiments, the cells produce healthy myofibers in the individual. In some embodiments, the cells self-renew in the individual.

[0030] In one aspect, methods of producing clinical grade myogenic progenitor cells are provided. Such methods typically include the steps of: (a) providing pluripotent stem cells that comprise an exogenous nucleic acid encoding PAX7 under control of an inducible promoter; (b) culturing the pluripotent stem cells under conditions that cause the pluripotent stem cells to undergo myogenic specification, wherein the conditionsF&RRefNo.: 09531-0562WO1

[0031] comprise exposing the pluripotent stem cells to an inhibitor of GSK3, and an inhibitor of BMP and an inhibitor of TGF-beta; (c) contacting the pluripotent stem cells undergoing myogenic specification with a compound that induces expression of PAX7; (d) contacting the pluripotent stem cells undergoing myogenic specification with bFGF to cause the pluripotent stem cells undergoing myogenic specification to differentiate into myogenic progenitor cells; (e) dispersing cell aggregates into single cells; (1) isolating the myogenic progenitor cells on about day 16 using a CD54 antibody; (g) expanding the isolated myogenic progenitor cells in a large-scale culture system under appropriate conditions, thereby isolating clinical grade myogenic progenitor cells.

[0032] In some embodiments, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are passage 16 to passage 22 pluripotent stem cells.

[0033] In some embodiments, clinical grade includes at least 50 million cells per vial (e.g., thawing and expansion of 1 vial of WCB containing 35 million cells from pl to p4 produced 7.5 billion myogenic progenitors (MyoPAXon), calculated as 50 million cells per vial x 150 vials) or 300-500 million cells per 100-200 million myogenic progenitors with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells expressing CD54.

[0034] In some embodiments, the pluripotent stem cells are exposed to the inhibitor of GSK3 from about day 2 to about day 4. A representative inhibitor of GSK3 is CHIR99021.

[0035] In some embodiments, the pluripotent stem cells are exposed to the inhibitor of BMP and an inhibitor of TGF-beta from about day 4 to about day 6. A representative inhibitor of BMP is LDN193189. A representative inhibitor of TGF-beta is SB431542.

[0036] In some embodiments, the pluripotent stem cells undergoing myogenic specification are contacted with the compound that induces expression of PAX7 starting on about day 5. A representative compound that induces expression of PAX7 is doxycycline.

[0037] In some embodiments, the pluripotent stem cells undergoing myogenic specification are contacted with bFGF starting on about day 8. In some embodiments, the dispersing occurs on about day 12. In some embodiments, the isolating occurs on about day 16.F&RRefNo.: 09531-0562WO1

[0038] In some embodiments, the large-scale culture system is a 2-dimension culture system. In some embodiments, the large-scale culture system is a 3-dimensional culture system. In some embodiments, the large-scale culture system is a cell stack.

[0039] In some embodiments, the appropriate conditions for expanding the isolated myogenic progenitor cells includes IMDM media (e.g., bFGF, hyclone, defined FBS, knockout serum replacement, insulin-transferrin-selenium, glutamax).

[0040] In some embodiments, the methods described herein further include performing a quality control test to determine the number of cells. In some embodiments, the methods described herein further include performing a quality control test to determine the quality' of cells. In some embodiments, the methods described herein further include exposing the pluripotent stem cells to an inhibitor of ROCK (e.g.. Y27632) from about day 0 to about day 2.

[0041] In some embodiments, the clinical grade myogenic progenitor cells mature into skeletal muscle.

[0042] In another aspect, clinical grade myogenic progenitor cells are provided that are made by any of the methods described herein.

[0043] In still another aspect, methods of treating a muscle disease are provided. Such methods typically include administering the clinical grade myogenic progenitor cells described herein to an individual suffering from a muscle disease.

[0044] In some embodiments, the muscle disease is muscular dystrophy (e.g., Duchenne muscular dystrophy).

[0045] In some embodiments, the cells are allogeneic to the individual. In some embodiments, the cells are autologous to the individual.

[0046] In some embodiments, the cells produce healthy myofibers in the individual. In some embodiments, the cells self-renew in the individual.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.F&RRefNo.: 09531-0562WO1

[0048] DESCRIPTION OF DRAWINGS FIG. 1 shows MyoPAXon cGMP manufacturing and characterization. (1A) Overview of MyoPAXon cGMP manufacturing and testing. (IB) Detailed outline of the cGMP manufacturing process. (1C) Scalability of MyoPAXon, as indicated by growth curves of three independent production batches from passage 1 to 4 (P1-P4). VNC:

[0049] Viable Nucleated Cells. (1D-1F) Identity of MyoPAXon DP. Representative flow cytometry plots show the expression of PAX7 (ID) and the surface markers CD54 and a901 (IE), and the absence of a double-positive population for the pluripotent markers SSEA-4 and TRA-1-81 (IF), in the MyoPAXon DP. (1G) Quantification of flow cytometry' data from 3 MyoPAXon productions (including panels 1D-1F). Error bars indicate the mean ± SEM (n = 3). (1H) In vitro myogenic differentiation potential of MyoPAXon DP. Representative image shows staining for MYHC (red). DAPI (blue). Scale bar: 500 pm. (II) MyoPAXon stability' over a period of 27 months. Graph shows viability' of cryopreserved MyoPAXon at 3, 6, 9, 12, 18, 21, 24 and 27 months. X axis indicates hours post-thaw. (1J) Confirmation of MyoPAXon DP in vivo regenerative potential. Representative images of transplanted muscles. Immunostaining shows LMNA (red), DYS (gray), and DAPI (blue). Scales bar: 500 pm (lower magnification) and 10 pm (close-up). (IK) Fiber type composition in non-injected (Ctrl), CTX-injured PBS-injected (Ctrl CTX), and MyoPAXon DP-injected mouse TA muscles. Graph reports the percentages of each fiber type (mean ± SEM) in Ctrl (n = 4), Ctrl CTX (n = 4) and cGMP2 (n = 8) muscles. (IL) In vivo regenerative potential of three independent MyoPAXon productions. 1 x 106cells were transplanted in CTX-injured TA muscles of NSG mice. Data are shown as mean ± SEM (n = 14 for PV2, n = 24 for cGMPl, and n = 32 for DP).

[0050] FIG. 2 shows MyoPAXon efficacy in NSG-mdx4Cvand NSG-FKRPP448Lmice. (2A) Overview' of transplantation studies in NSG-mdx4Cvmice. Two independent MyoPAXon cGMP batches were assessed. (2B) MyoPAXon in vivo regenerative potential inNSG-mdx4Cvmice. Representative image of the engrafted area following the injection of 1 x 106cells into irradiated TA muscles. LMNA (red), DYS (gray), and DAPI (blue). Scale bar: 500 pm. (2C) Engraftment quantification for both cGMP batches of MyoPAXon (cGMPl in red dots and DP in blue dots). Graph reports mean ± SEM (n = 16). (2D) Functional effect changes following cell transplantation in TA muscles of NSG-mdx4Cvmice. Specific force (sFO: F0 normalized to CSA) of muscles transplanted withF&RRefNo.: 09531-0562WO1

[0051] cGMPl or DP MyoPAXon or injected with PBS (control). Graph reports mean ± SEM (n= 16). **p < 0.01 by the Student's t test. (2E) Muscle satellite cell contribution in MyoPAXon DP-injected NSG-mdx4CvTA muscles. Donor-derived satellite cells (arrowhead) identified as cells located below the basal lamina (LAM, gray) co-expressing LMNA (red) and Pax7 (green). DAPI in blue stains nuclei. Scale bar is 20 pm. (2F) Overview of transplantation studies inNSG-FKRPP448Lmice. (2G) MyoPAXon in vivo regenerative potential in NSG-FKRPP448Lmice. Representative image of the engrafted area following the injection of 1 x 106cells into irradiated TA muscles. LMNA (red), DYS (gray), and DAPI (blue). Scale bar: 500 pm. (2H) Engraftment quantification in MyoPAXon -injected NSG-FKRPP448Lmice. Graph reports mean ± SEM (n = 13). (21) MyoPAXon rescues a-DG glycosylation in NSG-FKRPP448Lmice. Representative immunostaining of cell-injected TA muscles. LMNA (red), IIH6 (green), and DAPI (blue). Scale bar: 500 pm. (2J) Functional changes following cell transplantation into TA muscles of NSG-FKRPP448Lmice. Specific force of muscles transplanted with MyoPAXon or injected with PBS (control). Graph reports mean ± SEM (n= 9). *p < 0.05 by the Student’s t test.

[0052] (2K) Muscle satellite cell contribution in MyoPAXon DP -injected NSG-FKRPP448LTA muscles. LAM (gray); LMNA (red) and Pax7 (green). DAPI (blue). Scale bar: 20 pm.

[0053] FIG. 3 shows GLP safety study to assess toxicity, tumorigenicity, and biodistribution. (3 A) Overview of GLP safety studies. Mice were divided into 4 cohorts, receiving intramuscular administration of either vehicle or the MFD of 3.5 x 106MyoPAXon DP, and assessed at 11 weeks (toxicity; group 1) or at 25-26 weeks (tumorigenicity ; group 2) post-administration. (3B-3C) Histopathology of selected tissues (Table 4) from the toxicity (3B) and tumorigenicity (3C) studies. Representative images of hematoxylin and eosin (H&E) staining in the targeted muscle quad, lung, liver, and kidney of NSG mice following treatment with vehicle or MyoPAXon. Scale bar: 200 pm. (3D-3E) MyoPAXon biodistribution. PCR-based quantification of human Alu DNA in tissues from animals treated with cells (3D) or vehicle (3E). MyoPAXon: n = 44, except for the ovaries and testis, n = 22. Vehicle: n = 8 (4 male and 4 female). ILN, Inguinal lymph node; MLN, Mesenteric lymph node.

[0054] FIG. 4 demonstrates that long-term studies show7persistent contribution of MyoPAXon to muscle regeneration. (4A-4B) Persistent engraftment of MyoPAXon PV2 at 13-month post-injection. Representative images of muscle sections stained to visualizeF&RRefNo.: 09531-0562WO1

[0055] LMNA (red). DYS (gray), and DAPI (blue). Scale bars: 500 gm (4A) and 100 gm (4B). (4C) Engraftment quantification (from 4A-4B). Data are shown as mean ± SEM (n = 7). (4D-4E) Persistent engraftment of MyoPAXon DP at 6-month post-injection.

[0056] Representative images of muscle sections stained to visualize LMNA (red), DYS (gray), and DAPI (blue). Scale bars: 500 pm (4D) and 100 pm (4E). (4F) Engraftment quantification (from 4D-4E). Data are shown as mean ± SEM (n = 8). (4G) Representative images of muscle sections stained to visualize Ki67 (green), LMNA (red), and DAPI (blue). The white star indicates a LMNA+Ki67+ cell. Scale bar: 50 pm. (4H) Quantification of donor-derived proliferative cells. Graph reports the percentage of LMNA+Ki67+ nuclei among the LMNA+ cell population (from 4G). Data are shown as a percentage of Ki67+ nuclei (PV2: n = 637 nuclei for 1-month and n = 446 nuclei for 13-month; DP: n = 1566 nuclei for 1-month and n = 1393 nuclei for 6-month).

[0057] FIG. 5 shows transplantation of large doses of MyoPAXon into muscles of nonhuman primate recipients. (5 A) Overview of experimental design. (5B) Representative images of the transplanted EDB muscle in the NHP recipient 19JP7 show RFP (red) and TRA-1-85 (gray) co-staining. Saline-injected APB muscle served as negative control (Ctrl). DAPI (blue). Scale bar: 100 pm. (5C) Abundant and widespread engraftment in the transplanted EDB muscle of NHP recipient 20AP25. Representative images show immunostaining for RFP (red), TRA-1-85 (gray) and DAPI (blue). Scale bar: 500 pm. (5D) Representative images show myofiber engraftment, as indicated by staining for RFP (red) and DYS (gray), in transplanted EDB muscles from NHP recipients 19JP7, 20HP6 and 20AP25. DAPI stains nuclei (in blue). Scale bar is 100 pm. (5E) Engraftment quantification in EDB muscles injected with 37-40 x 106cells (3 distinct NHPs). Data show the total number of DYS+RFP+ donor-derived myofibers. (5F) Engraftment quantification in EDB muscles injected with 60 x 106cells (2 distinct NHPs). Data show the total number of DYS+RFP+ donor-derived myofibers. (5G) Transplanted cells contribute to the muscle satellite cell pool of NHP EDB. Donor-derived satellite cell (arrowhead) identified based on the localization below- the basal lamina (LAM, gray) and the co-expression of RFP (red) and PAX7 (green). DAPI (blue). Scale bar: 10 pm. (5H) Biodistribution of H2B-RFP-labelled MyoPAXon in NHP recipients. PCR-based quantification of RFP transcripts in negative control (RFP-), positive control (RFP+), heart, kidney, lung, liver, APB and diaphragm muscles from the 2 distinct engrafted NHP recipients (20HP6 and 20AP25).F&RRefNo.: 09531-0562WO1

[0058] FIG. 6 shows the phase 1 clinical trial overview. Potential participants will be screened 1 month prior to dosing. Those who are determined eligible to participate and consent to participate will begin immunosuppression 1 week before dosing. Each participant will receive a single intramuscular dose of MyoPAXon in one EDB muscle depending on the dose escalation plan. After 3 months, muscle biopsies will be assessed for the presence of dystrophin.

[0059] FIG. 7 shows fiber type characterization of MyoPAXon DP-injected TA muscles (related to FIG. 1). (7 A) Embryonic MYHC (Emb)-stained muscle. LMNA (red), MYHC-Emb (green), LAM (grey), DAPI (blue). Scale bar: 100 pm. (7B) MYHC type I (I)-stained muscle. LMNA (red). MYHC -I (green), LAM (grey), DAPI (blue). Scale bar: 100 pm. (7C) MYHC type II A (II Abstained muscle. LMNA (red), MYHC-IIA (green), LAM (grey), DAPI (blue). Scale bar: 100 pm. (7D) MYHC type IIX (IIX) fibers (negative for ty pe I+IIA+IIB). LMNA (red), MYHC-I+IIA+IIB (green), LAM (grey), DAPI (blue). Scale bar: 100 pm. Data not shown for the MYHC type IIB.

[0060] FIG. 8 shows in vivo regenerative potential of MyoPAXon inNSG-mdx4Cv and NSG-FKRPP448L mice (related to FIG. 2). (8A) MyoPAXon DP engrafted area in NSG-mdx4Cv mice. Representative image of muscle sections (marked in white) with DYS+LMNA+ myofibers (marked in red). DAPI (blue). Scale bar: 500 pm. (8B) Quantification of the percentage of engrafted area in cell-injected NSG-mdx4Cv mouse TA muscles. Data are shown as mean ± SEM (n=16). (8C) Effect of cell transplantation on absolute (F0) force in NSG-mdx4Cv mouse TA muscles injected with cells or PBS. Data are shown as mean ± SEM (n= 16). **p < 0.01 by the Student’s ttest. (8D) MyoPAXon DP engrafted area in NSG-FKRPP448L mice. Representative image of muscle sections (marked in white) with DYS+LMNA+ myofibers (marked in red). DAPI (blue). Scale bar: 500 pm. (8E) Quantification of the percentage of engrafted area in cell-injected NSG-FKRPP448L mouse TA muscles. Data are shown as mean ± SEM (n=12). (8F) Lack of IIH6 immunoreactivity7in PBS-injected TA muscles of NSG-FKRPP448L mice. Representative immunostaining of PBS-injected TA muscles. LMNA (red), IIH6 (green), and DAPI (blue). Scale bar: 200 pm.(8G) Functional changes following cell transplantation into TA muscles of NSG-FKRPP448L mice. Graph reports absolute (F0) force of muscles injected with cells or PBS. Data are shown as mean ± SEM (n= 9). **p < 0.01 by the Student’s t test.F&RRefNo.: 09531-0562WO1

[0061] FIG. 9 shows detection of RFP -labeled MyoPAXon in vitro and in vivo (related to FIG. 5). (9 A) FACS plots show RFP expression levels in iPAX7 iPSCs after their transduction with H2B-RFP (left panel) and in MyoPAXon at passage 4 (right panel). (9B) Representative images show engraftment of RFP-labeled MyoPAXon in TA muscles of NSG mice. Immunostaining shows LMNA (red), DYS (gray), and DAPI (blue). Scale bar: 200 pm.

[0062] FIG. 10 shows the safety profile of MyoPAXon transplantation in NHPs (related to FIG. 5). (10A) IgG binding assay performed at different times using post-transplant serum samples for the 4 NHP recipients. (10B) IgM binding assay performed at different time using post-transplant serum samples for the 4 NHP recipients. (10C-10E) Proliferation assay for CD4+ (10C), CD8+ (10D). and CD20+ (10E) cells performed with NHP PBMC samples collected at serial time points after cell injection. (10F) Body weight remained stable in all 4 NHP MyoPAXon recipients. (10G) Biodistribution of RFP-labeled My oPAXon in NHP recipients. PCR-based quantification of human LINE1 transcripts in organs and tissues from the 2 distinct engrafted NHP recipients (20HP6 and 20AP25). (10H) Biodistribution of RFP-labeled MyoPAXon in NHP recipients. PCR-based quantification of RFP (left) and human LINE1 (right) transcripts in organs and tissues from the non-engrafted NHP recipient (20AP21).

[0063] DETAILED DESCRIPTION

[0064] Muscular dystrophies (MDs) are still incurable genetic diseases characterized by progressive muscle wasting and loss of muscle function. Transplantation of regenerative myogenic progenitors to replace defective fibers and populate the stem cell pool with healthy cells represents an attractive therapy for MDs. Described herein is a manufacturing process to generate clinical grade, including large-scale numbers, cryopreserved PAX7-induced myogenic progenitors from induced pluripotent stem cells.

[0065] As used herein, a clinical grade preparation of cells refers to cells that have been cultured in a strictly controlled environment with a validated method by properly trained individuals with detailed records of all compositions, methods, and conditions. In addition, a clinical grade preparation of cells refers to cells and culture conditions that undergo regular testing and checks to ensure the product meets all uality control specifications, including extensive testing for pathogens. Further, as described herein, a clinical grade preparation of cells refers to at least 50 million cells per vial (e.g., thawing and expansion of 1 vial of WCB containing 35 million cells from pl to p4 produces 7.5F&RRefNo.: 09531-0562WO1

[0066] billion myogenic progenitors, calculated as 50 million cells per vial x 150 vials) or SOO-SOO million cells per 100-200 million myogenic progenitors with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells expressing CD54.

[0067] This clinical-grade cell product, MyoPAXon, was subjected to extensive quality' control, including biodistribution, toxicity, and tumorigenicity in mice under GLP conditions with no adverse effects, and demonstrated long-term engraftment (> 1 year) and efficacy in dystrophic mice. Transplantation of large cell numbers of MyoPAXon in immunosuppressed non-human primate recipients showed human contribution to muscle fibers and the satellite cell pool, with no safety concerns. MyoPAXon is a fully characterized off-the-shelf cGMP product, recently authorized by the FDA for testing in a first-in-human clinical trial in patients with Duchenne MD.

[0068] There are several ongoing P SC-based cell therapy clinical trials involving both allogeneic and autologous derivatives for other diseases, including macular degeneration, Parkinson’s disease, as well as immune therapies to hematological malignancies and solid tumors. Results to date demonstrate safety, supporting the further exploration of PSC-derivatives for other clinical applications. In the case of muscular dystrophy, the optimal cell therapy would provide: i) healthy myofibers that replace the diseased muscle tissue, and ii) donor-derived muscle stem cells that can self-renew and ensure long-term regenerative potential.

[0069] Following extensive proof-of-concept studies demonstrating the regenerative capacity of mouse PSC-derived myogenic progenitors in mouse models of DMD, including dystrophin-deficient (mdx) and dystrophin and utrophin double knockout (dko) mice, we have developed a corresponding yet unique strategy for manufacturing myogenic progenitor cells from human PSCs. The strategy is based on the controlled in vitro myogenic differentiation of human PSCs, including conditional expression of PAX7, in the appropriate developmental windows, which results in highly efficient production and expansion of myogenic progenitor cells at a scale that has previously not been reported. We identified surface markers enabling the purification of human myogenic progenitors from PAX7-induced differentiating PSC cultures and developed a cGMP manufacturing protocol that is specific for the generation of a clinical-grade human myogenic progenitor cell product, which we named MyoPAXon. We have characterized this cell product through xenotransplantation into mouse and non-humanF&RRefNo.: 09531-0562WO1

[0070] primate (NHP) recipients and subjected it to an extensive battery' of IND-enabling safety studies. Our FDA-approved plan for the first-in-human clinical trial of MyoPAXon is described herein.

[0071] After over a decade since the inception of our method to generate large numbers of human PAX7-induced myogenic progenitors from PSCs endowed with in vivo regenerative potential, here we describe the development of a reproducible cGMP protocol for the manufacturing of MyoPAXon. Clinical-grade MyoPAXon passed extensive QC and a battery' of IND-enabling safety studies in mice and non-human primates (NHP) recipients that culminated in the recent FDA authorization for testing in a first-in-human clinical trial in DMD patients.

[0072] While there is now a long list of registered interventional clinical trials for iPSC-based therapies, none of the recently reported trials involve a skeletal muscle cell product. MyoPAXon will be the first iPSC-derived skeletal muscle product to be tested in a clinical trial. Previous cell therapy attempts for DMD involved distinct cell populations. Early clinical trials that occurred between 1990 and 1997 focused on the transplantation of adult myoblasts from a first-degree relative donor, mostly in the presence of a calcineurin inhibitor for immunosuppression. Whereas these studies demonstrated that intramuscular transplants were well-tolerated, engraftment results were variable, and clinical effects limited or non-existent. This could be attributed to several reasons, including the substantial death and limited migratory ability of injected myoblasts, as well as the lack of sternness of these cells, limiting their regenerative potential. Another possibility is inadequate immunosuppression, as myoblast clinical trials led by Skuk and colleagues, a decade later, showed higher levels of dystrophin rescue using tacrolimus for immunosuppression. More recently, other cell types have been investigated as a therapy for DMD. Cossu and colleagues completed a first-in-human trial that consisted of multiple intra-arterial infusions of HLA-matched donor mesoangioblasts (a subset of pericytes) in five pediatric DMD patients, and although it was proven to be feasible and relatively safe, there was no demonstration of efficacy as DNA chimerism analysis showed minimal donor cell engraftment (< 0.69%). Clinical trials involving allogeneic cardiosphere-derived cells (CPCs) have also been reported for DMD. Outcomes show benefit on some metrics, but this intervention does not seek muscle replacement, but to improve muscle function by the release of exosomes.F&RRefNo.: 09531-0562WO1

[0073] Although it has recently been suggested that it would not be feasible to generate cGMP myogenic cells in sufficient quantity for clinical applications using 2D cultures, the cGMP 2D-myogenic differentiation protocol described here undoubtedly shows this is not the case, as one single vial of the WCB (pl) expands exponentially, giving rise to 7.5 billion MyoPAXon cells at p4 (FIG. 1 C; 150 vials wi th 50 million cells / vial). Considering the MCB contains 108 vials and one vial of the MCB gave rise to 68 vials of WCB, this means the total amount of DP that could be produced reaches approximately 55 trillion myogenic progenitor cells.

[0074] Importantly, transplantation of off-the-shelf MyoPAXon in multiple animal models showed both myofiber and satellite cell engraftment. The latter ensures long-term regenerative potential as indicated by the lasting engraftment (> 1 year) observed after one-time transplants. Although PSC-derivatives are notorious for their more immature nature, myofiber type composition of human grafts revealed a trend to adult phenotype, as we previously described using transcriptomics. In accordance, the engraftment reported here is devoid of fibers expressing embryonic MYHC and is enriched for type II fibers In fact, donor-derived human graft mimicked the mouse recipient muscle as no differences were observed between transplanted and control muscles. Thus, transplanted cells assimilate into the tissue and contribute to adult fiber formation. Whether they form new fibers on their own or fuse to strengthen existing fibers, they seem to be functionally mature.

[0075] Having demonstrated the safety profile of MyoPAXon in GLP mouse and non-GLP NHP studies, we have recently received FDA approval to test this DP in patients with DMD. This first-in-human clinical trial will focus on safety, and the EDB muscle of non-ambulatory patients was chosen for intramuscular transplantation of MyoPAXon. Several factors influenced this choice, but mainly the fact that the EDB muscle in DMD is characterized by a slower disease progression, as monitored by MRI. This muscle has been successfully used to test the effect of exon 51 skipping in DMD patients, where it was demonstrated that localized delivery of a morpholino splice-switching oligonucleotide (AVI-4658) could restore DYS protein levels to 42% of normal. Notably, the first study of myoblast transfer in DMD was performed in the EDB muscle of a 9-year-old boy immunosuppressed with cyclosporine and transplanted with his adoptive father’s cells. This early study showed only scant results of DYS expression at 3-months post-transplant, but a follow-up study 6 years later showed indication of persistent DYSF&RRefNo.: 09531-0562WO1

[0076] expression in the re-biopsied EDB muscle. While encouraging, unfortunately some key information is missing from these early reports, such as the particular DMD mutation and whether the patient received immunosuppression beyond the initial three months.

[0077] In the FDA-approved MyoPAXon clinical trial, the whole EBD muscle will be excised at the end of the study for assessment of safety' and DYS expression. We tested a similar approach in NHP preclinical studies, in which we transplanted EDB muscles with large numbers of MyoPAXon cells (37 to 60 x 106). The results from this study showed high levels of engraftment and a favorable safety profile. It is important to note that the first two escalating doses to be tested in the clinical trial are below the maximal dose tested in NHPs (25 x 106and 50 x 106), and that the NHP EDB is about one fifth the size of a patient's EDB. Therefore, the relative dose will still be lower than the maximum tested in the NHP. Because MyoPAXon is a universal off-the-shelf allogeneic stem cell product, if the results from this upcoming first-in-human clinical trial demonstrate safety, tolerability, and engraftment, we envision this DP can advance relatively quickly to the next phases in DMD and other types of muscular dystrophies.

[0078] MyoPAXon and Methods of Making and Using

[0079] This disclosure describes methods of producing clinical grade myogenic progenitor cells. As used herein, clinical grade refers to at least about 5 billion (e.g., at least about 6 billion, at least about 7 billion) cells produced by the culturing and expansion described herein. For example, culturing a vial of WCB containing about 35 million cells from the first passage (pl) to the fourth passage (p4) using the method described herein followed by expansion produced about 7.5 billion myogenic progenitors (MyoPAXon) (e.g., calculated as 50 million cells per vial x 150 vials or >200-fold increase in the number of progenitor cells from stem cells). For example, 300-500 million myogenic progenitors (MyoPAXon) can be produced using the methods described herein from 100-200 million myogenic progenitors, where at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the cells expressing CD54.

[0080] The methods described herein begin by providing stem cells that contain an exogenous nucleic acid encoding PAX7 under control of an inducible promoter. While the methods are exemplified herein using induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs) or stem cells that have started differentiating down the mesoderm lineage (i.e., mesenchymal stem cells (MSCs)) can be used. In some instances,F&RRefNo.: 09531-0562WO1

[0081] the methods described herein are used with pluripotent stem cells that have been passaged between 16 and 22 times.

[0082] The pluripotent stem cells are cultured under conditions that result in them undergoing myogenic specification. Conditions under which pluripotent stem cells undergo myogenic specification include, for example, exposing the cells to inhibitors of GSK3, BMP. and TGF-beta. Inhibitors of GSK3, BMP, and TGF-beta are known in the art and include, without limitation. CHIR99021. LDN193189, and SB431542, respectively. Inhibiting GSK3 in the pluripotent stem cells can occur between about day 2 and about day 4 of the culture, while inhibiting BMP and TGF-beta can occur between about day 4 and about day 6 of the culture. Optionally, the pluripotent stem cells also can be exposed to an inhibitor of ROCK (e.g., Y27632) between about day 0 and about day 2.

[0083] While the pluripotent stem cells are undergoing myogenic specification, the stem cells are contacted with a compound that induces expression of PAX7. A compound that induces expression of PAX7 is doxycycline, and the pluripotent stem cells undergoing myogenic specification can be contacted with doxycycline or a similar compound that induces expression of PAX7 starting on about day 5 of the culture.

[0084] After inducing PAX7 in the cells undergoing myogenic specification, the cells are contacted with FGF to initiate differentiation of the cells into myogenic progenitor cells. The FGF can be basic FGF (bFGF), and the cells undergoing myogenic specification can be contacted with the FGF starting on about day 8 of the culture.

[0085] Any cell aggregates that are present are dispersed into single cells, usually on about day 12 of the culture, and the myogenic progenitor cells are isolated using a CD54 antibody, usually on about day 16 of the culture.

[0086] Next, the isolated myogenic progenitor cells are expanded in a large-scale culture system under appropnate conditions. Large-scale culture systems are known in the art and include, for example, a 2-dimensional culture system, a 3 -dimensional culture system, and / or a cell stack. Appropriate conditions for expanding the isolated myogenic progenitor cells also are known in the art and include, without limitation, IMDM media, which can contain bFGF. hy clone, defined FBS. knockout serum replacement, insulin-transferrin-selenium, and glutamax. Expansion ultimately results in clinical grade myogenic progenitor cells that are capable of maturing into skeletal muscle.

[0087] In some instances, it may be desirable to further perform a quality control test to determine, for example, the number of cells and / or the quality of cells.F&RRefNo.: 09531-0562WO1

[0088] Clinical grade myogenic progenitor cells made by the methods described herein can be used to treat a muscle disease. Any number of muscle diseases can be treated with the myogenic progenitor cells described herein including, without limitation, muscular dystrophy (e.g., Duchenne muscular dystrophy). The pluripotent stem cells used in the methods described herein can be allogeneic or autologous to the individual suffering from a muscle disease. After the clinical grade myogenic progenitor cells described herein have been administered to an individual suffering from a muscle disease, the cells can selfrenew and produce healthy myofibers in the individual.

[0089] Embodiments

[0090] Embodiment 1 is a method of producing clinical grade myogenic progenitor cells, the method comprising the steps of: (a) providing stem cells that comprise an exogenous nucleic acid encoding PAX7 under control of an inducible promoter; (b) culturing the stem cells under conditions that cause the stem cells to undergo myogenic specification, wherein the conditions comprise exposing the stem cells to an inhibitor of GSK3, and an inhibitor of BMP and an inhibitor of TGF-beta; (c) contacting the stem cells undergoing myogenic specification with a compound that induces expression of PAX7; (d) contacting the stem cells undergoing myogenic specification with FGF to cause the stem cells undergoing myogenic specification to differentiate into myogenic progenitor cells; (e) dispersing cell aggregates into single cells; (f) isolating the myogenic progenitor cells using a CD54 antibody; (g) expanding the isolated myogenic progenitor cells in a large-scale culture system under appropriate conditions, thereby isolating clinical grade myogenic progenitor cells.

[0091] Embodiment 2 is the method of Embodiment 1, wherein the stem cells are pluripotent stem cells.

[0092] Embodiment 3 is the method of Embodiment 2, wherein the pluripotent stem cells are induced pluripotent stem cells.

[0093] Embodiment 4 is the method of any of the preceding Embodiments, wherein the stem cells are passage 16 to passage 22 stem cells.

[0094] Embodiment 5 is the method of any of the preceding Embodiments, wherein the stem cells are exposed to the inhibitor of GSK3 from about day 2 to about day 4.

[0095] Embodiment 6 is the method of Embodiment 5, wherein the inhibitor of GSK3 is CHIR99021.F&RRefNo.: 09531-0562WO1

[0096] Embodiment 7 is the method of any of the preceding Embodiments, wherein the stem cells are exposed to the inhibitor of BMP and an inhibitor of TGF-beta from about day 4 to about day 6.

[0097] Embodiment 8 is the method of Embodiment 7, wherein the inhibitor of BMP is LDN193189.

[0098] Embodiment 9 is the method of Embodiment 7, wherein the inhibitor of TGF-beta is SB431542.

[0099] Embodiment 10 is the method of any of the preceding Embodiments, wherein the stem cells undergoing myogenic specification are contacted with the compound that induces expression of PAX7 starting on about day 5.

[0100] Embodiment 11 is the method of Embodiment 10, wherein the compound that induces expression of PAX7 is doxycycline.

[0101] Embodiment 12 is the method of any of the preceding Embodiments, wherein the stem cells undergoing myogenic specification are contacted with FGF starting on about day 8.

[0102] Embodiment 13 is the method of any of the preceding Embodiments, wherein the dispersing occurs on about day 12.

[0103] Embodiment 14 is the method of any of the preceding Embodiments, wherein the isolating occurs on about day 16.

[0104] Embodiment 15 is the method of any of the preceding Embodiments, wherein the large-scale culture system is a 2-dimensional culture system.

[0105] Embodiment 16 is the method of any of the preceding Embodiments, wherein the large-scale culture system is a 3-dimensional culture system.

[0106] Embodiment 17 is the method of any of the preceding Embodiments, wherein the large-scale culture system is a cell stack.

[0107] Embodiment 18 is the method of any one of the preceding Embodiments, wherein the appropriate conditions for expanding the isolated myogenic progenitor cells comprises IMDM media (e.g., bFGF, hyclone, defined FBS, knockout serum replacement, insulin-transferrin-selenium. glutamax).

[0108] Embodiment 19 is the method of any one of the preceding Embodiments, further comprising performing a quality control test to determine the number of cells.

[0109] Embodiment 20 is the method of any one of the preceding Embodiments, further comprising performing a quality control test to determine the quality of cells.F&RRefNo.: 09531-0562WO1

[0110] Embodiment 21 is the method of any one of the preceding Embodiments, further comprising exposing the stem cells to an inhibitor of ROCK (e.g., Y27632) from about day 0 to about day 2.

[0111] Embodiment 22 is the method of any one of the preceding Embodiments, wherein the clinical grade myogenic progenitor cells mature into skeletal muscle.

[0112] Embodiment 23 is clinical grade myogenic progenitor cells made by the method of any of the preceding Embodiments.

[0113] Embodiment 24 is a method of treating a muscle disease, comprising administering the clinical grade myogenic progenitor cells of Embodiment 23 to an individual suffering from a muscle disease.

[0114] Embodiment 25 is the method of Embodiment 24, wherein the muscle disease is muscular dystrophy.

[0115] Embodiment 26 is the method of Embodiment 24 or 25, wherein the muscular dystrophy is Duchenne muscular dystrophy.

[0116] Embodiment 27 is the method of any one of Embodiments 24-26, wherein the cells are allogeneic to the individual.

[0117] Embodiment 28 is the method of any one of Embodiments 24-27, wherein the cells are autologous to the individual.

[0118] Embodiment 29 is the method of any one of Embodiments 24-28, wherein the cells produce healthy myofibers in the individual.

[0119] Embodiment 30 is the method of any one of Embodiments 24-29, wherein the cells self-renew in the individual.

[0120] In accordance with the present invention, there may be employed molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims.F&RRefNo.: 09531-0562WO1

[0121] EXAMPLES

[0122] 1 — Cell Line

[0123] LiPSC-GRl.l iPSCs were obtained from samples. Passage 14 cGMP grade iPSCs (ND50039) were used to generate the MCB. Passage 20 research grade iPSC vial (ND50038) from the same cell line was used for a practice run.

[0124] cGMP iPAX7 human iPSCs (Master cell bank (MCB)), PV2 myogenic progenitors, cGMPl myogenic progenitors, DP myogenic progenitors were produced at the University of Minnesota Molecular and Cellular Therapeutics (MCT) Facility.

[0125] For NHP studies, iPAX7 H2B-RFP iPSCs and H2B-RFP labelled MyoPAXon were generated in the Perlingeiro Lab, University of Minnesota, USA.

[0126] 2 — GLP Mouse Studies

[0127] The GLP toxicity' and tumorigenicity studies were performed at the University of Minnesota Center for Translational Medicine (CTM). A total of eighty seven (forty three females and forty four males) 6-8 weeks old NOD.Cg-PrkdcscidI12rgtmlwji / SzJ (NSG, RRID:IMSR_J AX: 005557, The Jackson Laboratory) mice were used for these two studies. They were conducted according to the study protocol and University of Minnesota - Experimental Surgical Services and the University of Minnesota Center for Translational Medicine, Standard Operating Procedures (SOPs). These non-clinical studies were conducted in accordance with United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) for Non-Clinical Laboratory Studies, Title 21 Code of Federal Regulations (CFR) Part 58 and were audited by the Quality Assurance Unit (QAU) of Experimental Surgical Services.

[0128] Example 3 — Non-GLP Mouse Studies

[0129] Non-GLP studies were performed in the laboratory of Professor Rita Perlingeiro, University of Minnesota. All animal procedures were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC) and followed NIH guidelines. 6-12 week old NSG mouse (males only, The Jackson Laboratory), 6-8 week old NSG-mdx4cv(males only) and 6-8 week old NSG-FKRPP448L(males only) mice were used for these studies.F&RRefNo.: 09531-0562WO1

[0130] Example 4 — Non-GLP Non-Human Primate Studies

[0131] All procedures conducted with cynomolgus macaques (Macaca fascicularis) were approved by the University of Minnesota Institutional Animal Care and Use Committee, in compliance with the Animal Welfare Act, adhered to principles stated in the National Institutes of Health Guide for Care and Use of Laboratory Animals and were performed and reported in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. All animals were purpose-bred and purchased from institutionally- approved commercial vendors.

[0132] A total of four male Mauritian origin cynomolgus macaques Macaca fascicularis) were enrolled in the study. All enrolled animals were healthy and confirmed TB negative and viral negative (macaque herpes B virus, simian retrovirus D, simian immunodeficiency virus, and Simian T-Cell Leukemia Virus- 1). Animals were aged 5.3- 8.1 years and weighed between 5.6-8.9 kg at the time of transplant. Animals were fed a standardized diet of either 2055C Certified Teklad Global 25% Protein Primate Diet or 7195 Teklad High Fiber Primate Diet (Envigo, Madison, WI, USA). Nutritional enrichment was provided daily, including fresh fruits and vegetables, grains, beans, and nuts, as well as a children’s multivitamin. To realize the need for frequent blood draws while avoiding confounding effects from restraint, sedation, and pain, all animals were implanted with a central vascular access ports as previously described. All animals were trained to cooperate with examination, blood collection, and general husbandry activities as part of the behavioral management program.

[0133] Animal behavior and clinical status were evaluated at least twice daily. Scheduled physical examinations per protocol and semi-annual comprehensive veterinary examinations were performed on all animals. Animals were housed in socially compatible same-sex pairs. An environmental enrichment program including social play, toys, music, and regularly scheduled access to a large exercise and swimming area was provided to encourage sensory engagement, enhance foraging behavior and novelty seeking, promote mental stimulation, increase exploration, play, and activity levels, and strengthen social behaviors, increasing the proportion of time animals spent on species-typical behaviors.

[0134] Example 5 — MyoPAXon Cells Manufacturing

[0135] Master Cell Bank (MCB): Passage 16 GMP grade LiPSC-GRl.l iPSCs (Sampled) were thawed and cultured with the mTeSR-1 medium (mTeSR-1 basal medium andF&RRefNo.: 09531-0562WO1

[0136] mTeSR-1 5x Supp, Stem Cell Tech) on CTS™ Vitronectin (0.9 pg / cm2, Thermo Fisher Scientific) coated culture vessels and incubated in a 37°C incubator with 5% CO2.

[0137] Medium was changed to fresh mTeSR-1 even’ day, and cells were passaged with CTS™ Versene (Thermo Fisher Scientific). For transduction at passage 18 w ith pCCL-rtTA and pCCL-PAX7 lentivirus (Indiana University7VPF), iPSCs were single cell harvested using TrypLE™ Select Enzyme (Thermo Fisher Scientific) and seeded at a density of 2.5 - 3.0 x 104cells / cm2with mTeSR-1 medium and 10 pM of EZSolution™ Y-27632 (BioVision) in a well from a 48 well plate. Two hours (+ / - 15 min) before transduction, mTeSR-1 medium was replaced by fresh medium. pCCL-rtTA and pCCL-PAX7 viral supernatant were thawed at RT and equilibrated in the hood for 5 minutes. Before adding the viruses, the medium was aspirated, viruses were mixed (1:1 ratio) and 100 pl of this mix was diluted with 900 pl mTeSR-1 medium and added to the well. The plate was then spun down for 1.5 hours at 1100 RCF and 30°C. After centrifugation, the virus mTeSR-1 mix was replaced by fresh mTeSR-1 medium and placed in 37°C incubator with 5% CO2. Cells were maintained in mTeSR-1 medium changed daily and passed when they were 70-80% confluent. PAX7 analysis was performed by FACS to ensure transduction efficiency. iPAX7 iPSCs were then expanded to generate the Master Cell Bank (MCB) at passage 22. MCB cells were collected with CTS™ Versene and cryopreserved in 1 ml CryoStor® CS10 (Biolife Solutions) at 1-2.7 x 106cells / vial, in 108 vials total.

[0138] Working Cell Bank (WCB): Before starting embryoid body (EB) formation, passage 22 iPAX7 iPSC from the MCB were thawed and passaged 3 times using CTS™ Versene. For the 4thpassage, they were passaged w ith Try pLE™ Select Enzy me (Thermo Fisher Scientific) and plated in a 150 mm dish with mTeSR-1 medium and 10 pM EZSolution™ Y-27632 and rotated at 60 RPM in a 37°C incubator with 5% CO? to induce EB formation. Two days after, mTeSR-1 medium w as replaced by my ogenic medium composed of IMDM without Glutamax (Thermo Fisher Scientific) supplemented with 11% KnockOut™ Serum Replacement (Thermo Fisher Scientific), 4% HyClone™ Defined Fetal Bovine Serum (FBS, Cytiva), 1% Coming® Insulin-Transferrin-Selenium (100X, Life Sciences) and 1% GlutaMAX™ Supplement (Thermo Fisher Scientific). At this point, 10 pM of the GSK3 inhibitor: EZSolution™ CHIR99021 (BioVision) was added to myogenic medium. 2 days later, medium w as replaced with fresh myogenic medium with 200 nM of LDN 193189 dihydrochloride (Biotechne) and 10 pM of SB-431542 (Biotechne). After one day, 1 pg / mL of doxycycline hyclate (dox, MylanF&RRefNo.: 09531-0562WO1

[0139] Institutional) was added to the medium to induce PAX7 expression. The next day, medium was changed to myogenic medium with 1 pg / mL of dox. Two days later, EBs are plated on culture treated dish without agitation with myogenic medium supplemented with 1 pg / mL dox and 10 ng / ml PeproGMP® Recombinant Human FGF-basic (bFGF, Peprotech). 72 hours later, the medium is changed again with myogenic medium plus dox and bFGF. The following day, which correspond to the 12thday of EBs formation, EBs are passaged with TrypLE™ Select Enzyme and plated at 3.07 x 104 / cm2with the same medium. Medium was changed twice in 4 days and, on the 16thday of EB formation, EB were dissociated with TrypLE™ Select Enzyme. CD54+ cells were purified with a CliniMACS Plus (Miltenyi Biotec) and plated at 2.88 x 104 / cm2with myogenic medium plus dox and bFGF. Medium was changed 2 days later, 4 days after purification, cells were harvested with TrypLE™ Select Enzyme and frozen with CryoStor® CS10. 68 vials of 35 x 106cells w ere generated at passage 1 after CliniMACS purification, which correspond to the WCB.

[0140] MyoPAXon productions: To proceed with a GMP run, passage 1 MyoPAXon myogenic progenitors from the WCB were thawed into myogenic medium with dox and bFGF at 2.75xlO4 / cm2Cells were passaged twice for expansion (2.9 x 104 / cm2and 3.14 x 104 / cm2, respectively) and were frozen at passage 4 in CryoStor® CS10. Two cGMP full expansion batches were performed at MCT and are referred to as cGMPl and DP (cGMP2). 150 vials of 50 x 106MyoPAXon DP were frozen and stored at the University of Minnesota cGMP MCT facility.

[0141] Example 6 — Generation of H2B-RFP Labelled MyoPAXon forNHP Studies

[0142] For labeling with H2B-RFP, MCB iPSCs were thawed in the Perlingeiro lab and transduced with the lentiviral vector pLKO-H2B-RFP (Addgene). Labeled iPSCs were then differentiated into myogenic progenitors following the protocol described for the GMP production at a smaller scale, except for the use of antibiotics with 1% Penicillin -Streptomycin (Pen-Strep, 5,000 U / mL, Thermo Fisher Scientific) and non cGMP reagents listed in the key resource table. Passage 4 H2B-RFP labelled MyoPAXon were frozen in CryoStor® CS10 at a concentration of 100 x 106cells / ml.F&RRefNo.: 09531-0562WO1

[0143] 7 — In Vitro Terminal Differentiation

[0144] Assessment of in vitro terminal differentiation for the different cell lines generated was performed in the Perlingeiro Lab. As described previously (Selvaraj, Mondragon et al.), MyoPAXon DP were allowed to grow 3 days in myogenic medium plus dox and bFGF in a well of a 24 well. Once confluent, the myogenic medium was changed to differentiation medium consisting of KnockOut™ DMEM (Thermo Fisher Scientific), 20% KnockOut™ Serum Replacement, 1% MEM Non-Essential Amino Acids Solution (100X, Thermo Fisher Scientific), 1% GlutaMAX™ Supplement, 1% Pen-Strep complemented with 10 pM of SB-431542 hydrate, 10 pM of DAPT (Selleck Chemicals), 10 pM of Dexamethasone (Cayman Chemical) and 10 pM of Forskolin (Cayman Chemical) and kept in culture for 5 days to derive myotubes.

[0145] 8 — CliniMACS Purification

[0146] To proceed with cells purification based on CD54 with a cliniMACS apparatus, EBs were dissociated with TrypLE™ Select Enzyme. Cells were then resuspended in culture medium. Cells were filtered with a standard blood filter (170-260 pm) if clumps were observed. Cells are then spun down and resuspended in CliniMACS Buffer to a concentration of 5.0 x 106cells per ml. 2.5 pl per million cells of Biotin-conjugated CD54 antibody (Miltenyi Biotec) and 300 pl per hundred million cells of 10% GAMMAGARD (Baxalta) were added and incubated 20 (+ / - 2) min on a standard blood tube rocker at 2-8°C. Cells were then washed with CliniMACS Buffer and resuspended in CliniMACS Buffer supplemented with 0.6 pl per million cells of CliniMACS® Anti-Biotin Reagent CR / GMP (Miltenyi Biotec) and incubated 15 (+ / - 2) min on a standard blood tube rocker at 2-8°C. After incubation cells were washed again with CliniMACS Buffer resuspended in CliniMACS Buffer and enriched for CD54+ cells using the CliniMACS Plus instrument (Enrichment program 1.1). CD54+ cells w ere collected into aTeruflex Transfer Bag (Terumo Corporation) and used to generate the WCB after plating.

[0147] 9 — Flow Cytometry

[0148] PAX7 analysis'. Myogenic progenitors were collected with TrypLE™ Select Enzyme and spun down at 400 RCF for 3 minutes. Supernatant was removed, the pellet resuspended in 4% PFA (Sigma) and incubated for 10 minutes at RT. The pellet was then washed twice with PBS. Cells were then spun down at 400 RCF for 5 minutes,F&RRefNo.: 09531-0562WO1

[0149] supernatant was discarded, and pellet resuspended in FACS Buffer composed of IX PBS, 4% FBS and 2 mM EDTA (Thermo Fisher Scientific) complemented with 0.5% Saponin (Thermo Fisher Scientific) and 0.2% TWEEN® 20 (Sigma) for 30 min at RT for permeabilization. Cell were then spun down and resuspended in FACS Buffer with 0.5% Saponin and the PAX7 antibody (mouse 1:50, DSHB) and incubated 45 min at RT. The cells were then washed twice with PBS and resuspended in FACS Buffer with 0.5% Saponin and the secondary antibody, Alexa Fluor 555 anti-mouse IgG (1:500, Thermo Fisher Scientific). Cells were incubated with the secondary antibody for 45 min at RT in the dark. After incubation, cells w ere spun down, and the supernatant was replaced by FACS Buffer with 0.5% Saponin. Cells were washed twice with PBS and resuspended in FACS Buffer for analysis with a FACSAria (BD). BD and FlowJo software were used to analyze the results.

[0150] CD54 and a9fl marker profile after purification: CD54+ purified cells are resuspended in staining buffer composed of PBS with 0.5% BSA (Thermo Fisher Scientific) and EDTA 2 mM. 1 pl per million cells of biotin-conjugated CD54 antibody (Thermo Fisher Scientific) and 1 pl per million cells of PE-conjugated Integrin a9pi antibody (BioLegend) were added to the cells and incubated 20 min on ice. Cells are then washed with staining buffer, spun down and resuspended with staining buffer for the secondary' antibody staining with PE-Cy7-conjugated streptavidin (0.5 pl per million cell; Thermo Fisher Scientific). Cells are incubated with the antibody for 5 min on ice, washed again with staining buffer, and resuspended with staining buffer supplemented with 50 pg / ml propidium iodide (Sigma) and then analyzed with the relevant isotype controls with a MACSQuant Analyzer. The Beckman Coulter Kaluza software was used to analyze the results.

[0151] Pluripotent markers analysis: Cells were collected with TrypLE™ Select Enzyme and spun down at 400 RCF for 5 minutes. They were then resuspended with FITC conjugated SSEA-4 antibody (mouse 1:20, BioLegend), Alexa Fluor 647 conjugated TRA-1-81 antibody (mouse 1:20, BioLegend) and 7AAD (1:10, BioLegend) in PBS and incubated 20 min on ice. Cells were then washed with PBS and analyzed with a MACSQuant Analyzer. The Beckman Coulter Kaluza software was used to analyze the results.F&RRefNo.: 09531-0562WO1

[0152]

[0153] GLP mouse studies: MyoPAXon DP generated at MCT was shipped to CTM facility and stored in vapor phase of liquid nitrogen. Vials were thawed by rolling them between the palms of gloved hands just until ice was no longer visible in the vial, and then they were placed on wet ice until transplant. Cryopreserved myogenic progenitors were used directly from the vial without further manipulation of the cell therapy product. MyoPAXon DP was stored and handled under conditions adequate to maintain sterility. Animals were injected with cells based on the cell concentration of the MyoPAXon at cry opreservation (concentration 100 x 106cells / ml) and the volume transplanted in the quadriceps muscle was documented.

[0154] Non-GLP mouse studies: MyoPAXon PV2, cGMPl and DP generated at the MCT were shipped to the Perlingeiro Lab and stored in vapor phase of liquid nitrogen. Passage 4 cryopreserved myogenic progenitors were thawed in a 37°C water bath just until a small chip of ice was visible, then they w ere placed on ice until transplantation. Cells were directly used from the vials without any further manipulation.

[0155] Non-GLP NHP studies: H2B-RFP labelled MyoPAXon were stored in the vapor phase of liquid nitrogen until the day of administration. Prior to administration, MyoPAXon vials were kept on dry ice, and before injection, were thawed using a 37°C water bath, then syringes were loaded using aseptic technique in a biosafety cabinet. Syringes were kept on ice, on average 55 m. prior to injection.

[0156]

[0157] GLP mouse studies: For both CTX injury and cell injection surgeries, mice were anesthetized with a mix of Ketamine (80-120 mg / kg) and Xylazine (5-10 mg / kg) by intraperitoneal (IP) injection. On the day before implant of either MyoPAXon DP or CryoStor® CS10 (VWR), the right middle rectus femoris quadriceps (Quad) muscle was pre-injured with 35 pL of 10 pM CTX (Sigma) injected by intramuscular injection (IM). The next day, mice randomized to the MyoPAXon DP group received a dose of 3.5 x 106cells suspended in CryoStor® CS-10 in a 35 pL volume at a concentration of 100 x 106cells / mL. Mice randomized to the control group received 35pL of CryoStor® CS-10. All injections were performed by a single surgeon supported by assistants and were delivered by IM with a 25g Hamilton syringe. Mice were terminated after 25-26 weeks for the tumorigenicity study and 11 weeks for the toxicity study.F&RRefNo.: 09531-0562WO1

[0158] Non-GLP mouse studies: Prior to transplantation, mice were anesthetized with a mix of Ketamine (80-120 mg / kg) and Xylazine (5-10 mg / kg) by IP injection. Cell transplantation was performed in TA or Quad muscles. 6-12 weeks NSG, NSG-mdx4Cvor 6-8 weeks old NSG-FKRPP448Lmouse targeted muscles were pre-injured 24 hours before cell injection with CTX (15 pl for the TA, 35 pl for the Quad of 10 pM stock; Latoxan) or irradiated with 12 Grays for NSG-mdx4Cvand NSG-FKRPP448Lmice. Different cell preparation, dose and volume were injected as described in Table 7. All the injections in the TA and Quad muscles were performed by IM with a 25 g Hamilton syringe. Human donor-derived engraftments were analyzed at different times points going from 4 to 57 weeks (Table 7).

[0159] Non-GLP NHP studies: Anti-CD20, provided by the Nonhuman Primate Reagent Resource (NHPRR), was given by intravenous injection (IV) at 50 mg / kg on days -7 and 0 in relation to cell transplantation. Anti-CD154 (NHPRR) was given IV at 20 mg / kg on days -7, 0, 7, and 14 in relation to cell transplantation. Tacrolimus (Astellas Pharma, Inc) was given IM at 0.01-0.05 mg / kg twice daily on days -2 to 28 in relation to cell transplantation. Solid food was withheld overnight prior to surgical induction. Water was available at all times. Anesthesia was induced with Ketamine / Midazolam (8-15 mg / kg and 0.01 mg / kg; IM / IV, respectively) and 0.5-5% isoflurane inhaled for maintenance anesthesia. Preoperative antibiotics and analgesics included: cefazolin (50 mg / kg, IM), buprenorphine (0.01-0.03 mg / kg, IM) and ketoprofen (1-2 mg / kg, IM). Following sedation, NHPs were intubated, the surgical site was shaved and prepped with chlorhexidine scrub and draped following sterile technique. During surgery, thermal support was provided by a Bair Hugger® (forced hot air). Local anesthesia (2% lidocaine diluted 1:2 with NS) was administered subcutaneously to the injection sites: EDB and APB muscles. Primary muscle sites were exposed from the medial to the lateral side using blunt dissection. Using a > 27g needle a total of 150 pl of 10 pM CTX (Sigma) was injected among five different sites (30 pl per site) to each exposed muscle. CTX had been allowed to dwell for an average time of 25 minutes, followed by injection of either saline or MyoPAXon (Table 6).

[0160]

[0161] and Tissue Collection

[0162] GLP mouse studies: Mice from the toxicity study were euthanized 11 weeks after transplantation while the mice from the tumorigenicity study were euthanized after 25-26F&RRefNo.: 09531-0562WO1

[0163] weeks. Mice were euthanized using CO2 asphyxiation and were subjected to an extensive necropsy performed by a board-certified veterinary pathologist. Findings were recorded and abnormalities were described and photographed to supplement documentation. 37 organs and tissues (Table 4) were collected and sent to Scientific Solutions (Minneapolis, USA), a GLP-certified contract research organization (CRO), for histopathology7. 14 organs and tissues (Table 4) were sent to Charles River Laboratories (a GLP-certified CRO; Montreal ULC, Laval Site, Canada) for biodistribution analysis.

[0164] Non-GLP mouse studies: Endpoints for the different non-GLP studies are detailed in Table 7. Mice were euthanized using CO2. Muscles were collected and embedded in OCT for immunofluorescence analysis.

[0165] Non-GLP NHP studies: At four weeks post-transplantation, NHPs were deeply sedated with Ketamine / Midazolam (8-15 mg / kg and 0.01 mg / kg; IM / IV) and were heparinized (Heparin 400 p / kg). Animals were then humanely euthanized by injection of Euthasol solution (1 mL (390 mg pentobarbital + 50 mg phenytoin) per 4.5 kg (~86 mg / kg pentobarbital)) IV (or equivalent)). A full post-mortem examination was performed. Tissues were collected for histological review, assessment of engraftment and biodistribution of transplanted cells.

[0166] Example 13 — Biodistribution

[0167] GLP mouse studies (Charles River): Right quadriceps (injury and injections), left quadriceps (uninjured control), adrenal glands, blood, brain, heart, kidneys liver, lungs, inguinal lymph nodes, mesenteric lymph nodes, spleen, ovaries and testes collected from the toxicity and tumorigenicity GLP studies were processed by Charles River Laboratories for biodistribution analysis based on the detection of human Alu sequences by qPCR. Tissue DNA extraction was performed following Charles River’s ASP.190 for tissues and ASP.270 for blood. DNA from human PBMC (Zenbio) was used as a positive control and to establish the standard curve (ASP.267). Mouse DNA and water were used as negative controls. The Taqman™ Fast Advanced Master Mix (Thermo Fisher Scientific) was used to run the qPCR, the list of the primers used are presented in Table 8 and the detailed procedure for the quantification of human cells in mouse tissues and blood by qPCR using ALU Sequences is presented in Charles River’s ASP.267.

[0168] Non-GLP NHP studies: Flash frozen tissue specimens (lung, liver, kidney, heart, diaphragm, and APB muscles) were collected from NHPs 20AP21, 20AP25, and 20HP6.F&RRefNo.: 09531-0562WO1

[0169] DNA was extracted using Qiagen’s DNeasy Blood & Tissue kit (Qiagen) following manufacturer's protocol. DNA from human cells (PV2) and NHP cells (CyMN.2 myogenic progenitors, as previously reported) were used as positive and negative control, respectively. Each qPCR reaction was composed of 100 ng of DNA, a TaqMan Gene Expression Master Mix (Thermo Fisher Scientific), and amplicon-specific primer / probe sets (Table 8. RFP1 (donor specific), LINE1 (human specific) and primate-specific AluYB8 (internal control). Invitrogen). Each sample was run in duplicate on a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems, Waltham, MA, USA). Cycling conditions used for these reactions were: (1) 50°C for 2 min; (2) 95°C for 10 min; (3) 95°C for 15 s, followed by 61°C for 1 min repeated for 39 more cycles; and (4) 61°C for 3 min. RFP or LINE1 Ct values were normalized by AluYB8.

[0170] 14 — Immunofluorescence

[0171] In vitro terminal differentiation: Five days after induction of the terminal differentiation in vitro iPSCs derived mvotubes were fixed for 30 min at room temperature (RT) with 4% PF A, permeabilized with 0.3% Triton XI 00 (Sigma) in PBS for 15 minutes at RT, blocked for 30 min with 3% BSA in PBS, and incubated with MF 20 (MYHC, mouse 1:50, DSHB) antibody overnight at 4°C. The following day, samples were rinsed with PBS and then incubated with Alexa Fluor 555 goat anti-mouse IgG (1:500. Thermo Fisher Scientific) secondary antibody and 4,6-Diamidino-2-phenylindole (DAPI, Santa Cruz) for 1 h at RT and rinsed and kept protected from light at 4°C in PBS. Samples were analyzed by inverted microscopy (Zeiss inverted microscope). Image processing was performed with the Fiji software.

[0172] Non-GLP mouse studies: Muscles were embedded in Tissue-Tek O.C.T. compound (Sakura), and snap frozen on isopentane pre-cooled with liquid nitrogen. 14 pm Cryosections were collected on glass slides and immunostained followed the protocol detailed in the previous section. Primary' antibodies used on mouse in vivo samples included human DYSTROPHIN (DYS, mouse 1:100, DSHB) in combination with human LAMIN A / C (LMNA, rabbit 1:500, Abeam) to detect the human donor-derived myofibers. PAX7 (mouse 1:10, DSHB) in combination with laminin-a2 (LAM, rat 1:200, Santa Cruz), and human LMNA (rabbit 1:500, Abeam) have been used for the detection of donor-derived satellite cell. To study the engrafted cell proliferative status, a staining with human LMNA (rabbit 1:500, Abeam) and Ki67 (mouse 1 : 100. BD Biosciences) wasF&RRefNo.: 09531-0562WO1

[0173] performed to detect the presence of Ki67 positive donor-derived myofibers. For the fiber type analysis, samples were stained with an anti-MYHC-Embryonic (Emb, mouse 1:10, DSHB) or an anti-MYHC-I (mouse 1 : 100, DSHB) or an anti-MYHC-IIA (mouse 1 :30, DSHB) or an anti-MYHC-IIX (mouse 1:20, DSHB) or an anti-MYHC-IIB (mouse 1:20, DSHB) antibody all in combination with LAM (rat 1 :200, Santa Cruz), and human LMNA (rabbit 1:500, Abeam). To show the functional rescue of the a-DG glycosylation in NSG-FKRPP448Lmouse, a staining with the anti-dystroglycan IIH6C4 (IIH6. mouse 1:200, Sigma) and the human LMNA (rabbit 1:500, Abeam) antibodies was performed. Regarding the secondary antibodies, Alexa Fluor 488 anti-rat IgG (1:500, Thermo Fisher Scientific), Alexa Fluor 555 anti-rabbit IgG (1:500, Thermo Fisher Scientific), and Alexa Fluor 647 goat anti-mouse IgG (1:500, Thermo Fisher Scientific) and 4,6-Diamidino-2-phenylindole (DAPI, Santa Cruz) were used for the fiber type quantification staining 1 hour at RT. Samples were incubated 1 hour at RT with Alexa Fluor 488 anti-rat IgG, Alexa Fluor 555 anti-rabbit IgG, Alexa Fluor 647 goat anti-mouse IgG and DAPI for the satellite cell and fiber type staining. For the Ki67 staining, Alexa Fluor 555 anti-rabbit IgG, Alexa Fluor 647 goat anti-mouse IgG and DAPI were used. Alexa Fluor 555 antirabbit IgG, Alexa Fluor 647 goat anti-mouse IgM (1:500, Thermo Fisher Scientific) and DAPI were used for the IIH6 staining. After washing three times with PBS, sections were dried and mounted with Prolong Gold with DAPI (Invitrogen). Samples were analyzed by confocal (NikonNiE C2 upright confocal microscope) and upright (Zeiss upright) microscopy. Image processing and quantification were performed with the Fiji software.

[0174] Merged images of DAPI, DYS, and LMNA were used to quantify human donor-derived fibers. The two best engraftment from two different slides were quantified and averaged to get the number of fibers of one sample. For the fiber type composition merge images of the fiber type analyzed with LAM, LMNA and DAPI were used for quantification. More than 1000 fibers were analyzed per control samples and all the donor-derived myofibers in the cell injected samples. Merged images of LMNA and Ki67 were used to determine the percentage of human engrafted nuclei positive for the proliferation marker K167. The number of nuclei analyzed range from 446 to 1566.

[0175] Non-GLP NHP studies: O C T. embedded NHP muscles were processed as described in the previous section. Primary antibodies used on NHP muscles cryosection included human DYS (mouse 1:100, DSHB) in combination with RFP (rabbit 1:500, Abeam) to detect and quantify the human donor-derived myofibers, and human TRA-1-F&RRefNo.: 09531-0562WO1

[0176] 85 (mouse 1:50, DSHB) combined with RFP (rabbit 1:500, Abeam) to stain with two injected cell specific makers and Pax7 (mouse 1:10, DSHB) in combination with LAM (rat 1:200, SantaCruz), and RFP (rabbit 1:500, Abeam) for the satellite cell staining. Secondary antibodies used were Alexa Fluor 555 anti-rabbit IgG (1:500, Thermo Fisher Scientific), and Alexa Fluor 647 goat anti-mouse IgG (1:500, Thermo Fisher Scientific) and DAPI (Santa Cruz) for the fiber quantification and human specificity staining 1 hour at RT and Alexa Fluor 488 anti-rat IgG, Alexa Fluor 555 anti-rabbit IgG (1:500, Thermo Fisher Scientific), Alexa Fluor 647 goat anti-mouse IgG (1 :500, Thermo Fisher Scientific) and DAPI (Santa Cruz) 1 hour at RT for the satellite cells staining. Slides were mounted, analyzed, and quantified as previously described for mouse samples.

[0177] GLP study histopathology: At the time of necropsy at CTM. tissues collected (Table 4) for histopathology from the toxicity and tumorigenicity GLP studies were placed in neutral-buffered formalin and sent to Scientific Solutions (CRO). Upon receipt, tissues were processed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. All tissues were evaluated by a board-certified veterinary’ pathologist. The histopathologist was blind to all endpoints, treatment groups and procedures except the gender of each animal.

[0178] Example 15 — In Situ Force Measurements in Dystrophic Mice

[0179] A 3-in-l animal system (Aurora Scientific) was used to perform the measurements of TA muscle force in NSG-mdx4cvand NSG-FKRPP448L. During the procedure, anesthesia was maintained with isoflurane. The TA muscle was carefully exposed, and the knee stabilized with a knee pin and clamp. After connection of the distal tendon to the force transducer with a silk loop, the tendon was cut, and two electrodes were positioned under the TA muscle. A series of rectangular unipolar pulses of 0.2 ms at different muscle base tensions to stimulate the TA muscles were used to determine the optimal stimulation length. The maximal tetanic force (Fo) was recorded at 150 Hz using the same rectangular unipolar pulses of 0.2 ms for 300 ms. After the measurement of muscle optimal length and weight for each muscle, the cross-sectional area and the specific force (sFo) were calculated. At the end of the procedure, TA muscles were embedded in O.C.T for immunofluorescence analysis.F&RRefNo.: 09531-0562WO1

[0180] Example 16 — Flow Crossmatch (IgG / IgM Binding)

[0181] Recipient serum was incubated with donor cell lines as outlined to assess the presence of preformed donor-specific antibodies, as indicated by binding of the antibodies to donor cells. Briefly, serum from the recipient is mixed w ith donor cells, stained with anti-IgG (Bio-Rad) and anti-IgM (BD Biosciences). Binding of donor specific antibody to the donor cells is measured as mean fluorescence intensity using multiparametric flow cytometry. A 3-fold increase in IgG / IgM is considered a significant development of donor specific antibodies that can contribute to transplant rejection in the allotransplantation scenario, though the cut-off for xenotransplantation is not yet defined.

[0182]

[0183] Recipient PBMCs were stimulated with irradiated donor cell lines to assess the presence of pre-existing cellular immune responses, as indicated by proliferation of T and B cells using a one-w ay carboxyfluorescein succinimidyl ester (CFSE) mixed lymphocyte reaction assay (ThermoFisher). Briefly, CFSE-labelled PBMCs from the recipient is stimulated with irradiated violet proliferation dye labeled donor cells for six days, stained with fluorochrome conjugated antibodies specific for CD4 (BD Biosciences), CD8a (Tonbo Bioscience) and CD20 (BD Biosciences), acquired on a flow cytometer. Donor specific proliferation of cells is measured by quantitating the frequencies of CFSElowCD4. CD8 and CD20. In the allotransplantation scenario, a 5% frequency of proliferating CD4, CD8, and CD20 when stimulated with donor cells indicates the development of donor specific cellular immune responses that can contribute to the rejection of transplanted cells. A cutoff for xenotransplantation has not been thoroughly defined.

[0184] Example 18 — Quantification and Statistical Analysis

[0185] Engraftment, fiber type and Ki 67 quantifications are detailed in the Immunofluorescence section. Statistical analysis was performed with GraphPad Prism (v.9.1.2). Data are presented as means ± SEM. Statistical analysis was conducted using unpaired Student's t test, p < 0.05 was considered to be significant in all experiments.

[0186] Example 19 — cGMP Manufacturing and Quality Control of MyoPAXon

[0187] We manufactured a master cell bank (MCB) with 108 cryovials of iPAX7 human induced PSCs (iPSCs) via a source cell bank (SCB) from the cGMP -grade cell iPSC lineF&RRefNo.: 09531-0562WO1

[0188] LiPSC-GRl.1, which was generated by Lonza under the auspices of the NIH. This MCB, generated upon transduction of SCB cells with cGMP third generation lentiviral vectors encoding PAX7 and rtTA (FIG. 1A), underwent extensive quality control (QC), including pluripotency characterization by flow cytometry, assessment of karyotype, absence of adventitious agents and replication-competent lentivirus (RCL), among others (Table 2). To manufacture MyoPAXon, the MCB was subjected to the myogenic differentiation protocol (FIG. IB; see STAR Methods), which includes an intermediate working cell bank (WCB) containing 68 vials of CD54+myogenic progenitors, purified using CliniMACS and cryopreserved at 35 million cells / vial (FIG. 1A). The WCB also underwent QC testing (Table 3). Once thawed, one vial of the WCB (pl) expands exponentially, giving rise to the final drug product (DP) MyoPAXon at p4 (cGMP2; hereafter referred as DP): 150 vials with 50 million cells / vial (total of 7.5 billion myogenic progenitors per run; 214-fold cell expansion). This large-scale production allowed for the same batch of DP to be used in safety IND-enabling studies and the first- in-human clinical trial. Also importantly, as shown in FIG. 1C, multiple large scale batches of MyoPAXon (engineering run PV2 and two independent cGMP runs: cGMPl and DP) show comparable expansion potential.

[0189] MyoPAXon DP, the clinical product, was extensively characterized for purity, identity, and safety, and passed all QC measurements (Table 1). In agreement with our prior experience. MyoPAXon DP is homogenously positive for PAX7, CD54, and a9pi integrin (FIG. ID, IE and 1G), and produces myosin heavy-chain (MYHC)-expressing myotubes when subjected to in vitro terminal differentiation conditions (FIG. 1H).

[0190] Importantly, MyoPAXon DP is devoid of potentially undifferentiated cells, as evidenced by the absence of an SSEA4+TRA-1-81+ double positive population (FIG. IF and 1G), and its viability after cryopreservation has been maintained in batches of cGMP myogenic progenitors up to 27 months to date, demonstrating the stability of the cryopreserved DP (FIG. II).

[0191] Table 1. MyoPAXon DP release specification and results <> < < <>

[0192]

[0193] > F&RRefNo.: 09531-0562WO1

[0194] <

[0195] > >

[0196]

[0197] All tests presented in the table were performed on the MyoPAXon DP (cGMP2) for FDA approval. Specification, Test Result and PASS / FAIL are reported for each Lot Release Testing. USP, United State Pharmacopeia; LAL, limulus amebocyte lysate; TD-PCR, touchdown polymerase chain reaction; RCL, Replication-Competent Lentivirus.

[0198] Before performing IND-enabling safety studies, we confirmed the in vivo regenerative potential of these myogenic progenitors following their local transplantation into cardiotoxin (CTX)-injured tibialis anterior (TA) and quadriceps muscles ofNSG mice (FIG. 1 J). Assessment of myofiber type composition of human grafts revealed that donor-derived human myofiber type composition mirrored the mouse recipient muscle (FIG. 1 K and 7), as no differences were observed when we compared the DP cohort with non-injured and CTX controls (Ctrl and Ctrl CTX, respectively). Importantly, the engraftment observed with the DP, measured by the numbers of donor-derived myofibers expressing human DYSTROPHIN (DYS) and human LAMIN A / C (LMNA), was comparable to previous productions of MyoPAXon (PV2 and cGMPl), corroborating the reproducibility and consistency of outcomes of our cGMP differentiation protocol (FIG. IL).

[0199] Example 20 — MyoPAXon Ameliorates the Dystrophic Phenotype Upon Transplantation inNSG-mdx4Cvand NSG-FKRPP448LMice

[0200] Next, we confirmed the in vivo regenerative potential of MyoPAXon in the context of DMD using NSG-mdx4Cvmice as recipients (FIG. 2A, 2B, 8A and 8B). As observed with NSG mice (FIG. IL), we detected comparable levels of engraftment between cGMPl and DP MyoPAXon batches (FIG. 2C), with an average of approximately 200 human donor-derived DYS+LMNA+ myofibers following the transplantation of I x 106my ogenic progenitors. In situ force measurements showedF&RRefNo.: 09531-0562WO1

[0201] functional improvement of dystrophic muscle weakness in transplanted muscles, as evidenced by both superior isometric tetanic force (FIG. 8C) and specific force (FIG. 2D) when compared to their respective contralateral PBS-injected TA muscles. Consistent with our previous studies, we detected the presence of human donor-derived muscle stem cells in transplanted mice, as indicated by the presence of LMNA+cells co-expressing PAX7, residing at the satellite stem cell position under the basal lamina (FIG. 2E).

[0202] Similar rescue of dystrophic phenotype was observed when we transplanted MyoPAXon in FKRP-mutant mice (FIG. 2F), a model for limb girdle muscular dystrophy type 21 (LGMD2I / R9) that is associated with defects in a-dystroglycan (a-DG) glycosylation and laminin binding. Engraftment of MyoPAXon was confirmed by the detection of human DYS+LMNA+ myofibers (FIGs. 2G, 2H, 8D and 8E) as well as by the immunoreactivity' to the IIH6 antibody (FIG. 21), which specifically recognizes the laminin binding domain of a-DG, which is absent in muscles of FKRPP448Lmice (FIG.

[0203] 8F). Consistently, functional measurements revealed improvements in muscle strength in muscles that had been transplanted with MyoPAXon when compared to their respective contralateral PBS-injected muscles (FIGs. 2J and 8G), and assessment of satellite cell engraftment confirmed the presence of LMNA+PAX7+ cells under the basal lamina (FIG. 2K).

[0204] Example 21 — GLP Safety Assessment in Mice Shows No Abnormalities Nor Biodistribution Outside the Transplanted Muscle

[0205] To determine the safety' profile of MyoPAXon, we performed toxicity', tumorigenicity, and biodistribution studies of the DP in NSG mice under full GLP conditions. These studies involved a total of 87 randomized male and female mice, in which target muscles, the rectus femoris of the quadriceps, were administered with vehicle (CryoSt or; n = 43) or cryopreserved MyoPAXon cells (n = 44) at the maximal feasible dose (MFD), 3.5 x 106myogenic progenitors, which was established as the maximum single dose that could feasibly be administered without leakage, taking in consideration cell density, and volume of the cells and target muscle. One day before vehicle or cell administration, target muscle (right leg) was pre-injured with CTX injection. Monitoring consisted of daily cage observation, w eekly body weight, and biweekly detailed clinical observation. As outlined in FIG. 3A, animals w ere sacrificed at 11 weeks post-administration for toxicity and biodistribution (group 1). and at 25-26F&RRefNo.: 09531-0562WO1

[0206] weeks post-administration for tumorigenicity and biodistribution (group 2). Assessment included histopathology, hematology, blood chemistry, and biodistribution.

[0207] No treatment-related effects on mortality, body weight, body temperature, or organ weight were observed. No deaths were observed in the toxicity cohort (group 1). There was 1 early death and 2 mice prematurely euthanized for welfare reasons in the tumorigenicity cohort (3 out of 44 mice; 6.8%), but these 3 mice had each been injected with vehicle. Therefore, none of these adverse events were ascribed to MyoPAXon. Detailed clinical observations were performed prior to necropsies at the end of the study, and all 41 surviving mice were in good health at the 25-26 week endpoint. Hematology7, serum chemistry, and bone marrow (BM) smear evaluations were performed in 86 of the 87 animals (as per protocol, no necropsy was performed in the mouse found dead). The results showed no apparent effects of the MyoPAXon treatment on hematological and biochemical parameters as well as bone marrow cellularity, morphology, maturation, or cell populations, in either cohort. There were no statistically significant differences between the vehicle and MyoPAXon administered groups (data not shown). Nonspecific inter-group rare variations included the development of BM neoplasia but these were not attributed to the graft by the study pathologist as they occurred in one mouse receiving vehicle and another mouse receiving MyoPAXon. The SCID mutation does predispose these immunodeficient mice to develop lymphomas, therefore BM neoplasia at low frequency is not unexpected in this mouse strain.

[0208] Interpretation of clinicopathologic and gross necropsy findings, performed by third party individuals blinded to treatment, indicate that MyoPAXon DP administration in mice resulted in zero tumors. Histopathology and biodistribution studies, conducted by independent GLP-certified contract research organizations (CROs), further confirmed the safety7of MyoPAXon. The histopathology report describes no significant findings in all the 37 tissues and organs (Table 4) analyzed for both toxicity (FIG. 3B) and tumorigenicity (FIG. 3C) cohorts. To assess biodistribution, qPCR for human-specific Alu sequences was used to detect the presence of human DNA in the collected organs and tissues (FIG. 3 A). These studies confirmed the presence of human DNA only in the quadriceps muscles that had been administered MyoPAXon (R Quad DP), whereas human DNA was undetected in the contra-lateral non-injected quadriceps counterparts as well as other organs and tissues from these mice (FIG. 3D). Human DNA was not detected in any tissue or organ of vehicle administered mice (FIG. 3E).F&RRefNo.: 09531-0562WO1

[0209] Example 22 — Long-Term Engraftment and Proliferative Status of MyoPAXon Grafts To determine whether the engraftment of MyoPAXon is sustained, as previously documented for PAX7-induced myogenic progenitors produced from other PSC lines, we performed long-term studies by transplanting multiple batches of MyoPAXon (PV2 and DP) into CTX-injured muscles of NSG mice. As shown in FIGs. 4A-B, the presence of human donor-derived myofibers was evident at 13 months following the injection of PV2 cells. Engraftment quantification showed an average of 125 myofibers, with 350 fibers detected in one recipient (FIG. 4C). Similarly, engraftment of the DP assessed at 6 months post-transplantation produced high numbers of human myofiber grafts (FIGs. 4D-E), with an average of 200 donor-derived DYS+LMNA+ myofibers (FIG. 4F). These results are equivalent to the engraftment levels observed at 1 -month post-transplantation (FIG. IL), thus confirming the durability of human muscle grafts, which is in agreement with the presence of human donor-derived satellite cells in engrafted muscles (FIG. 2E and 2K). Of note, these studies further provide evidence for safety as long-term grafts showed no signs of teratoma or any abnormal tissue formation.

[0210] Next, we investigated MyoPAXon grafts for the presence of human donor-derived proliferating cells. We used Ki67 as a marker of proliferation in combination with humanspecific LMNA and DAPI (FIG. 4G). Quantification of LMNA+Ki67+nuclei revealed very low rates of human proliferating cells (average below 1%) at short- and long-term, and this frequency did not change over time (FIG. 1H). These results are consistent with the interpretation that human donor-derived satellite cells behave similarly to primary muscle stem cells, which are not highly proliferative in the absence of injury.

[0211] Example 23 — Transplantation of MyoPAXon into Non-Human Primate (NHP) Muscles To address the feasibility7and safety of transplanting large numbers of MyoPAXon, we established a model of skeletal muscle injury7in cynomolgus macaques (Macaca fasciculciris). We focused on the extensor digitorum brevis (EDB), the same muscle that will be targeted in the Phase I Clinical Trial. Following screening for the absence of donor-specific antibodies (Table 5), we qualified four NHP recipients for the transplantation of MyoPAXon into CTX pre-injured EDB muscles (pilot 19JP7, followed by 20HP6, 20AP25, and 20AP21). Recipients received induction and maintenance immunosuppression, as outlined in FIG. 5 A. To facilitate the detection of transplantedF&RRefNo.: 09531-0562WO1

[0212] human myogenic progenitors, we labeled these cells with a lentiviral vector encoding the nuclear-localized H2B-RFP fusion protein (RFP), which gives robust expression in vivo (FIG. 9). In a pilot recipient, we evaluated two methods of cell delivery to the EDB. A dose of 53 x 106myogenic progenitor cells was delivered to the right EDB using a 22g catheter, and a dose of 40 x 106cells was delivered contralaterally by direct intramuscular injection using a grid pattern. While both strategies resulted in engraftment (Table 6), the direct injection produced more consistent and higher levels of engraftment. as indicated by a greater number of RFP+ cells in injected muscles (FIG. 5B and Table 6).

[0213] Importantly, we also demonstrated that a human-specific anti-TRA-1-85 antibody, reported not to cross-react with NHP, can be used to identify human cells that have successfully engrafted in the NHP muscle. We confirmed co-localization of RFP and TRA-1-85 in NHP EDB muscles transplanted with MyoPAXon, whereas saline-injected abductor pollicis brevis (APB) control muscles (Table 6) were negative for RFP and TRA-1-85 (FIG. 5B). Having validated our model, we transplanted the three additional NHP recipients (20HP6, 20AP25, and 20AP21) with cell escalation doses ranging from 37 to 60 x 106myogenic progenitors using the direct injection approach. Our findings confirmed the presence of human donor-derived myofibers in six of a total of seven cell-injected EDB muscles (Table 6) and, as shown in FIG. 5C, abundant and widespread engraftment of MyoPAXon was observed. There was no evidence of engraftment in recipient 20AP21 (Table 6), which may be attributed to issues during preconditioning, administration, and / or tissue collection, rather than immune rejection. This recipient met all enrollment criteria (Table S4) and did not develop a humoral or cellular immune response during the study (FIG. 10A-E). Importantly, serial immune monitoring revealed no donor-specific humoral or cellular immune responses in any recipient (FIG. 10A-E). This was indicated by the lack of IgG / IgM binding and the absence of donor antigenspecific proliferation of CD4, CD8 and CD20 populations within peripheral blood mononuclear cells (PBMC) stimulated with MyoPAXon.

[0214] Engraftment of MyoPAXon was quantified by enumeration of RFP and DYS positive muscle fibers present in target muscles (FIG. 5D). The lowest dose tested in a single muscle (37-40 x 106cells) resulted in engraftment of 400 to 700 donor-derived myofibers (FIG. 5E and Table 6), while the highest dose tested (60 x 106cells) resulted in engraftment ranging from 500-1800 myofibers (FIG. 5F and Table 6). Of note, we also detected the presence of human donor-derived satellite stem cells in transplanted NHPF&RRefNo.: 09531-0562WO1

[0215] recipients, as shown by the presence of RFP+PAX7+ cells residing under the basal lamina (FIG. 5G).

[0216] In addition to evaluating muscle regeneration, we also evaluated safety in NHP recipients. All recipients displayed a favorable safety profde, characterized by stable body weight (FIG. 1 OF), normal clinical pathology results (data not shown), and no treatment-associated adverse events. Macroscopic and histological assessment of the graft sites were unremarkable with no evidence of inflammation. Comprehensive histopathological analysis revealed no abnormalities associated with MyoPAXon therapy (data not shown). Accordingly, biodistribution analysis for RFP and the human-specific marker LINE1 transcripts performed in several organs and tissues, including the heart, kidney, lung, liver, and the APB and diaphragm muscles, from two of the three engrafted NHP recipients (i.e., 20HP6, 20AP25; tissues were not collected from the pilot recipient, 19JP7) show undetectable expression of both RFP (FIG. 5H) and LINE1 (FIG. 10G). RFP and human transcripts were also absent in recipient 20AP21 (FIG. 10H).

[0217] Collectively, these findings demonstrate in a stringent NHP model the ability of MyoPAXon to successfully engraft in muscle tissue, remain localized at the target tissue, and convey a favorable safety profile, underscoring its potential for therapeutic applications.

[0218] Example 24 — MyoPAXon Clinical Trial Design

[0219] The Phase 1 clinical trial of MyoPAXon will have a primary objective of assessing safety and determining the maximum tolerated dose in the context of an intramuscular injection. The secondary objectives are to assess engraftment, immunological responses (if any), and feasibility and tolerability of the treatment. Study participants will receive intramuscular injections into the EDB muscle according to a fast-track dose escalation schedule (FIG. 6). Successive participants will be dose-escalated to 100 x 106cells unless dose limiting toxicity (DLT) occurs, with a possibility of escalating one more step to 200 x 106cells if no toxicity is encountered at the lower doses. Five additional participants will be assigned to the highest dose. Assuming the highest dose is achieved, this schedule requires 9 participants, all of whom will receive tacrolimus to suppress immune rejection of the graft. After a 3-month monitoring period, an excisional muscle biopsy will be performed on the treated muscle to assess the presence ofF&RRefNo.: 09531-0562WO1

[0220] DYSTROPHIN, and tacrolimus will be discontinued. Less frequent monitoring will be continued after the 3 -month timepoint.

[0221] Example 25 — Tables

[0222] Table 2 shows the release specification and results for the Master Cell Bank (MCB) used for MyoPAXon manufacture (related to FIG. 1). All tests presented in the table were performed on the MCB for FDA approval. Specification, Test Result and PASS / FAIL are reported for each Lot Release Testing. LAL, limulus amebocyte lysate; TD-PCR, touchdown polymerase chain reaction; RCL, Replication Competent Lentivirus; MAP, Mouse Antibody Production, a All tests performed in compliance with 21 CFR § 210 and § 211, MHRA GMP (Rules and Guidance for pharmaceutical Manufacturers and Distributors), and applicable ICH Q7 standards for Phase I through Commercial Material.

[0223] Table 2

[0224] < < <> >

[0225] >

[0226]

[0227] F&RRefNo.: 09531-0562WO1

[0228]

[0229] Table 3 shows the release specification and results for the Working Cell Bank (WCB) used for MyoPAXon manufacture (related to FIG. 1). All tests presented in the table were performed on the WCB for FDA approval. Specification, Test Result and PASS / FAIL are reported for each Lot Release Testing. LAL, limulus amebocyte lysate; TD-PCR, touchdown polymerase chain reaction, a All tests performed in compliance with 21 § CFR 210 and § 211, MHRA GMP (Rules and Guidance for Pharmaceutical Manufacturers and Distributors), and applicable ICH Q7 standards for Phase I through Commercial Material.

[0230] Table 3

[0231] < < <> >

[0232]

[0233] Table 4 shows a summary of tissues and organs collected for histopathology and biodistribution (related to FIG. 3). This table details the organs and tissues collected for histopathology7and / or biodistribution analysis. 36 organs and tissues were processed for histopathology for each mouse from the toxicity and tumorigenicity cohorts (all MyoPAXon and vehicle injected mice). 14 organs and tissues were analyzed for biodistribution (all 44 mice injected with MyoPAXon from the toxicity andF&RRefNo.: 09531-0562WO1

[0234] tumorigenicity cohorts, 4 mice injected with the vehicle from the toxicity cohorts, and 4 mice injected with the vehicle from the tumorigenicity cohorts).

[0235] Table 4

[0236]

[0237] Table 5 shows a summary of NHP pre-enrollment crossmatch analysis (related to FIG. 5). Table reports the results of the donor-specific humoral immune response and theF&RRefNo.: 09531-0562WO1

[0238] donor-specific cellular immune response at the time of pre-enrolment for the 4 NHPs selected for the study. NHP, non-human primate; IgG. Immunoglobulin G; IgM, Immunoglobulin M; MFI, Mean fluorescence intensity; CFSE, Carboxyfluorescein succinimidyl ester; MLR, Mixed lymphocyte reaction.

[0239] Table 5

[0240]

[0241] Table 6 shows a summary of NHP transplantation and engraftment results (related to FIG. 5). Engraftment results are reported for each APB and EDB muscle from the 4 treated NHP recipients (0, or CTX + saline or CTX + Cells). The table details both the total number of donor-derived myofibers present in the muscle a month after injection and the number of fibers detected in each analyzed block. NHP, non-human primate;

[0242] APB, Abductor pollicis brevis muscle; EDB, Extensor digitorum brevis muscle, 0, no treatment; CTX, cardiotoxin.

[0243] Table 6

[0244]

[0245] F&RRefNo.: 09531-0562WO1

[0246]

[0247] Table 7 shows a summary of mouse transplantation outline for all experiments.

[0248] The figure panels reporting the data from these experiments are listed along with the mouse strain, target muscle, ty pe of pre-injury, cell batch, cell dose and endpoint. NSG, NOD.Cg-PrkdcscidI12rgtmlwjVSzJ; TA, Tibialis anterior muscle, Quad, quadriceps muscles; CTX. cardiotoxin; DP. MyoPAXon drug product.

[0249] Table 7

[0250]

[0251] F&RRefNo.: 09531-0562WO1

[0252]

[0253] Table 8 shows the primer sequences used for biodistribution studies. Forward, reverse and probe sequences used in the mouse GLP and NHP biodistribution studies. NHP. non-human primate.

[0254] Table 8

[0255]

[0256] It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

[0257] Disclosed are methods and compositions that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference to each various individual and collectiveF&RRefNo.: 09531-0562WO1

[0258] combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

Claims

F&RRefNo.: 09531-0562WO1WHAT IS CLAIMED IS:

1. A method of producing clinical grade myogenic progenitor cells, the method comprising the steps of:(a) providing stem cells that comprise an exogenous nucleic acid encoding PAX7 under control of an inducible promoter;(b) culturing the stem cells under conditions that cause the stem cells to undergo myogenic specification, wherein the conditions comprise exposing the stem cells to an inhibitor of GSK3, and an inhibitor of BMP and an inhibitor of TGF-beta;(c) contacting the stem cells undergoing myogenic specification with a compound that induces expression of PAX7;(d) contacting the stem cells undergoing myogenic specification with FGF to cause the stem cells undergoing myogenic specification to differentiate into myogenic progenitor cells;(e) dispersing cell aggregates into single cells;(f) isolating the myogenic progenitor cells using a CD54 antibody;(g) expanding the isolated myogenic progenitor cells in a large-scale culture system under appropriate conditions,thereby isolating clinical grade myogenic progenitor cells.

2. The method of claim 1, wherein the stem cells are pluripotent stem cells.

3. The method of claim 2, wherein the pluripotent stem cells are induced pluripotent stem cells.

4. The method of any of the preceding claims, wherein the stem cells are passage 16 to passage 22 stem cells.

5. The method of any of the preceding claims, wherein the stem cells are exposed to the inhibitor of GSK3 from about day 2 to about day 4.

6. The method of claim 5, wherein the inhibitor of GSK3 is CHIR99021.F&RRefNo.: 09531-0562WO17. The method of any of the preceding claims, wherein the stem cells are exposed to the inhibitor of BMP and an inhibitor of TGF-beta from about day 4 to about day 6.

8. The method of claim 7, wherein the inhibitor of BMP is LDN 193189.

9. The method of claim 7. wherein the inhibitor of TGF-beta is SB431542.

10. The method of any of the preceding claims, wherein the stem cells undergoing myogenic specification are contacted with the compound that induces expression of PAX7 starting on about day 5.

11. The method of claim 10, wherein the compound that induces expression of PAX7 is doxycycline.

12. The method of any of the preceding claims, wherein the stem cells undergoing myogenic specification are contacted with FGF starting on about day 8.

13. The method of any of the preceding claims, wherein the dispersing occurs on about day 12.

14. The method of any of the preceding claims, wherein the isolating occurs on about day 16.

15. The method of any of the preceding claims, wherein the large-scale culture system is a 2-dimensional culture system.

16. The method of any of the preceding claims, wherein the large-scale culture system is a 3-dimensional culture system.

17. The method of any of the preceding claims, wherein the large-scale culture system is a cell stack.F&RRefNo.: 09531-0562WO118. The method of any one of the preceding claims, wherein the appropriate conditions for expanding the isolated myogenic progenitor cells comprises IMDM media (e.g., bFGF, hyclone, defined FBS, knockout serum replacement, insulin-transferrin-selenium, glutamax).

19. The method of any one of the preceding claims, further comprising performing a quality control test to determine the number of cells.

20. The method of any one of the preceding claims, further comprising perfonning a quality control test to determine the quality of cells.

21. The method of any one of the preceding claims, further comprising exposing the stem cells to an inhibitor of ROCK (e.g., Y27632) from about day 0 to about day 2.

22. The method of any one of the preceding claims, wherein the clinical grade myogenic progenitor cells mature into skeletal muscle.

23. Clinical grade myogenic progenitor cells made by the method of any of the preceding claims.

24. A method of treating a muscle disease, comprising administering the clinical grade myogenic progenitor cells of claim 23 to an individual suffering from a muscle disease.

25. The method of claim 24, wherein the muscle disease is muscular dystrophy.

26. The method of claim 24 or 25, wherein the muscular dystrophy is Duchenne muscular dystrophy.

27. The method of any one of claims 24-26, wherein the cells are allogeneic to the individual.F&RRefNo.: 09531-0562WO128. The method of any one of claims 24-27, wherein the cells are autologous to the individual.

29. The method of any one of claims 24-28, wherein the cells produce healthy myofibers in the individual.

30. The method of any one of claims 24-29, wherein the cells self-renew in the individual.

Images