Production of therapeutic satellite cells for treating muscular dystrophy

Abstract

The present disclosure provides compositions and methods for preparing autologous satellite cells that restore dysfunctional genes involved in muscular dystrophy in patients. Such transplanted satellite cells can promote myofibril and muscle repair and recovery in patients, thereby treating muscular dystrophy in patients. It is contemplated that muscular dystrophy characterized by mutant disease genes can be treated by intramuscular (IM) transplantation of satellite cells that do not have a defect in the disease gene, such as autologous satellite cells in which the disease gene has been corrected. Such transplanted satellite cells can promote myofibril and muscle repair and recovery in patients.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 388,187, filed July 11, 2022, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] background Limb-girdle muscular dystrophy type 2A (LGMD2A) is a condition characterized by non-lysosomal Ca 2+ It is an autosomal recessive LGMD syndrome caused by mutations in the calpain 3 (CAPN3) gene, which encodes a protease and plays a key role in sarcomere remodeling. CAPN3 mutations result in muscle cells unable to function properly and repair damage that occurs through normal physiological activity.

[0003] No drugs or biological products are specifically indicated for use in LGMD or LGMD2A, and no disease-modifying treatments are available. In humans, satellite cells are the driving force behind muscle regeneration and homeostasis. Upon muscle injury, quiescent satellite cells are activated and released from the basal lamina, undergoing asymmetric cell division to generate one identical satellite cell and one myoblast. The newly formed satellite cells return to the basal lamina and become quiescent, while the newly formed myoblasts increase in number and repair damaged muscle fibers by fusing with them or, in turn, fuse with other myoblasts to generate new muscle fibers.

[0004] In patients with LGMD2A, there is a defect in myoblasts, leading to a continuing decline in muscle structure and function. Furthermore, the proliferative capacity of satellite cells in LGMD2A patients is significantly reduced, impairing overall muscle regeneration. Summary of the Invention [Means for solving the problem]

[0005] overview It is contemplated that muscular dystrophies characterized by mutant disease genes may be treated by intramuscular (IM) transplantation of satellite cells lacking the disease gene defect, such as autologous satellite cells in which the disease gene has been corrected. Such transplanted satellite cells can promote myofibril and muscle repair and recovery in patients.

[0006] Thus, in one embodiment, the present disclosure provides an autologous cell product designed to restore normal expression of a disease gene in satellite cells and their progeny. These satellite cells not only replace and repair damaged muscle tissue, but also home to the satellite cell niche in a quiescent state to support long-lasting homeostasis and repair of future muscle damage. Treatment delivered to the muscle can promote locomotion, mobility, and improve a patient's functional status and ability to perform activities of daily living.

[0007] Also provided is a method for preparing autologous satellite cells with restored gene expression and activity in one embodiment. In one exemplary embodiment, the method involves: a) obtaining peripheral blood mononuclear cells (PBMCs) from a patient with muscular dystrophy; b) reprogramming the PBMCs into induced pluripotent stem cells (iPSCs); c) correcting the mutant gene in the iPSCs; c) differentiating the iPSCs into satellite cells; and d) enriching and expanding the satellite cells.

[0008] According to one embodiment of the present disclosure, a method for preparing satellite cells from stem cells containing a mutation in an endogenous gene is provided. In some embodiments, the method includes the steps of: introducing first and second vectors, each containing a first expression cassette encoding at least a portion of an unmutated gene, into stem cells such that each of the first expression cassettes is integrated into the locus of the endogenous gene in the genome to encode a wild-type version of the unmutated gene; differentiating the stem cells into satellite cells; and expanding the satellite cells into a population of satellite cells.

[0009] Also provided is a method for preparing satellite cells from stem cells containing a mutation in an endogenous gene, the method comprising: introducing first and second vectors, each containing a first expression cassette containing all of the coding sequence of a wild-type gene, into stem cells so that each of the first expression cassettes is integrated into a locus in the genome that is different from the endogenous gene; differentiating the stem cells into satellite cells; and expanding the satellite cells into a population of satellite cells.

[0010] In some embodiments, the locus is within a genomic safe harbor, such as an intron of the AAVS1 gene and one selected from the group consisting of human genome positions 195,338,589-195,818,588, 22,720,711-22,761,389, 145,090,941-145,219,513, 145,320,384-145,525,881, and 89,174,426-89,179,074 (GRCh38 coordinates). In some embodiments, the locus is within intron 1 of the AAVS1 gene.

[0011] In some embodiments, the expression cassette further comprises a promoter capable of initiating expression of the coding sequence in a muscle cell, hi some embodiments, the promoter is a muscle creatine kinase promoter (tMCK).

[0012] The mutation may be, but is not limited to, a homozygous mutation or a heterozygous mutation.

[0013] In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs) derived from peripheral blood mononuclear cells or fibroblasts isolated from patients with muscular dystrophy.

[0014] In some embodiments, the muscular dystrophy is selected from the group consisting of limb-girdle muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0015] In some embodiments, the gene is selected from the group consisting of CAPN3, DNAJB6, TNPO3, HNRPDL, CAPN3, COL6A1, COL6A2, COL6A3, DYSF, SGCA, SGCB, SGCG, SGCD, TCAP, TRIM32, FKRP, TTN, POMT1, ANO5, FKTN, POMT2, POMGNT1, DAG1, PLEC1, TRAPPC11, GMPPB, ISPD, POGLUT1, COL6A1, COL6A2, COL6A3, LAMA2, POMGNT2, POPDC1, POPDC3, JAG2, PYROXD1, DMD, LAMA-2, DMD, DYSF, DMPK, CNBP, DUX4, PABPN1, EMD, LMNA, SYNE1, SYNE2, FHL1, and TMEM43.

[0016] In some embodiments, the gene is CAPN3. For CAPN3, in some embodiments, at least a portion of the wild-type CAPN3 gene does not include exons 1 or 2, and the locus is downstream of exon 2 of the endogenous CAPN3 gene. In some embodiments, at least a portion of the wild-type CAPN3 gene includes exons 3-24, and the locus is 3' to the last nucleotide of exon 2 of the endogenous CAPN3 gene.

[0017] In some embodiments, the first vector further comprises a second expression cassette encoding a first selection marker, and the second vector further comprises a third expression cassette encoding a second selection marker, wherein the first expression cassette and the second expression cassette on the first vector are integrated into the genome together, and the first expression cassette and the third expression cassette on the second vector are integrated into the genome together.

[0018] In some embodiments, the second and third expression cassettes are excised from the genome after confirmation of integration. In some embodiments, the first vector and the second vector each further comprise a fourth expression cassette encoding a kill switch that is not integrated into the locus.

[0019] In some embodiments, the method further comprises culturing the stem cells in a container coated with an adhesion molecule prior to differentiation. In some embodiments, the adhesion molecule is laminin 511 or a fragment thereof. In some embodiments, the culturing is performed in a medium comprising fibroblast growth factor (FGF), a Rho kinase inhibitor, and N2 medium.

[0020] In some embodiments, differentiation is carried out in a medium containing a Wnt signaling activator. In some embodiments, differentiation is then further carried out in a medium containing a Notch signaling inhibitor. In some embodiments, differentiation is then further carried out in a medium containing a Notch signaling inhibitor and a TGF-β inhibitor.

[0021] In some embodiments, satellite cells are identified by an antibody specific for CD271. In some embodiments, the identified satellite cells are expanded in a medium comprising bFGF2, FGF8b and N2 medium.

[0022] In some embodiments, the method further comprises administering the prepared satellite cells to a patient carrying the mutation. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows an example process workflow for preparing genetically corrected satellite cells and their use to treat LGMD accordingly.

[0024] [Figure 2] Figure 2 shows the specific steps in the correction of the mutant CAPN3 gene in targeted iPSCs.

[0025] [Figure 3A]Figure 3A-B shows the organization of two genetic strategies for integrating the correct CAPN3. The first scheme (Figure 3A) shows vectors for integrating the correct CAPN3 coding sequence into both alleles of the CAPN3 locus. In the top vector, from left to right, the coding sequence contains thymidine kinase (TK), CAPN3, and the puromycin resistance gene (PuroR). Both TK and PuroR are preceded by constitutive promoters, and each coding sequence is followed by a poly(A) tail. Furthermore, the CAPN3 and PuroR cassettes together are flanked by CAPN3 homology arms, left arm (LA) and right arm (RA). In the bottom vector, the puromycin resistance gene is replaced with the neomycin resistance gene (NeoR). In both vectors, the selectable marker (PuroR or NeoR) is flanked by site-specific recombinase target sites (loxP), allowing for removal of these markers after successful integration and confirmation. The second scheme (Figure 3B) shows the vector for integrating the correct CAPN3 coding sequence into the AAVS1 locus driven by the tMCK promoter. The general design is the same as above. From left to right, the coding sequence contains thymidine kinase (TK) (in both vectors), and the selectable marker (PuroR or NeoR) is flanked by site-specific recombinase target sites (loxP), allowing for removal of these markers after successful integration and confirmation. Both TK and PuroR or NeoR are preceded by constitutive promoters, and each coding sequence is followed by a polyA tail. The tMCK promoter, CAPN3, and PuroR or NeoR cassettes are together flanked by AAVS1 homology arms, the left arm (LA) and the right arm (RA). [Figure 3B] Same as above

[0026] [Figure 4] Figure 4 shows how each of the CAPN3 modification strategies in Figure 2 leads to the production of functional CAPN3 protein.

[0027] [Figure 5A]5A-D show the design of a PCR strategy to confirm correct insertion of exogenous coding sequences (5A and 5C) and the results of testing such a strategy (5B and 5D). [Figure 5B] Same as above [Figure 5C] Same as above [Figure 5D] Same as above

[0028] [Figure 6] Figure 6 presents a chart showing the relative expression levels of functional CAPN3 mRNA in muscle fibers from a healthy donor (control) and two patients (P1 and P2) before and after implantation.

[0029] [Figure 7] Figure 7 shows a Western blot confirming the expression of functional CAPN3 protein in muscle fibers from a healthy donor (control) and two patients (P1 and P2) before and after insertion. DETAILED DESCRIPTION OF THE INVENTION

[0030] Detailed Description definition The following description details exemplary embodiments of the present technology, but it should be recognized that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of exemplary embodiments.

[0031] definition As used herein, the following words, phrases, and symbols are generally intended to have the meanings set forth below, except to the extent that the context in which they are used indicates otherwise.

[0032] As used herein, certain terms may have the following defined meanings. As used in the specification and claims, the singular forms "a," "an," and "the" include singular and plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a single cell as well as a plurality of cells, including mixtures thereof.

[0033] All numerical designations, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximations that are varied (+) or (-) by increments of 0.1. It is understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It is also understood, although not always explicitly stated, that the term "about" includes the exact value "X" as well as small increments of "X," such as "X+0.1" or "X-0.1." It is also understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0034] As used herein, "stem cell" defines a cell that has the ability to divide indefinitely in culture and generate specialized cells. Stem cells can be totipotent, pluripotent, multipotent, oligopotent, or unipotent. Non-limiting examples of stem cell types include somatic (adult) stem cells, embryonic stem cells, parthenogenetic stem cells, and / or induced pluripotent stem cells (iPS cells or iPSCs). As used herein, the term "pluripotent stem cells" refers to cells that (i) have the ability to proliferate indefinitely in vitro in an undifferentiated state; (ii) maintain normal karyotype throughout extended culture; and (iii) maintain the potential to differentiate into derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm) even after extended culture. Non-limiting examples of currently available pluripotent stem cells include embryonic stem cells and iPSCs. In some embodiments, the stem cell is iPSC, particularly human iPSCs.

[0035] By "muscle stem cells" is meant self-renewing mononuclear cells that produce mononuclear myoblasts as progeny, committed to forming multinucleated muscle fibers by cell-cell fusion. Included herein are muscle stem cells that produce skeletal, smooth, or cardiac muscle.

[0036] As used herein, the term "muscle cell" refers to any cell that contributes to muscle tissue. Myoblasts, satellite cells, myotubes, and myofibrillar tissue are all encompassed by the term "muscle cell" and can all be treated using the methods of the present invention. Myocyte effects can be induced in skeletal, cardiac, and smooth muscles. Muscle tissue in adult vertebrates regenerates from stored myoblasts called "satellite cells." Satellite cells are distributed throughout muscle tissue and are mitotically quiescent in the absence of injury or disease. After muscle injury or during recovery from disease, satellite cells re-enter the cell cycle, proliferate, and undergo differentiation into multinucleated myotubes, which either 1) enter existing muscle fibers or 2) form new muscle fibers. Myoblasts ultimately produce replacement muscle fibers or fuse with existing muscle fibers, thereby increasing fiber girth through the synthesis of contractile apparatus components. This process is illustrated, for example, by the nearly complete regeneration that occurs in mammals after induced muscle fiber degeneration; muscle precursor cells proliferate and fuse together to regenerate muscle fibers.

[0037] As used herein, "myogenic" cells are cells associated with the origin of muscle cells or fibers. Various molecular markers are known to be specific to the middle and late stages of myogenic differentiation. For example, in C2C12 cells, myosin and desmin mark the late stage of myogenesis and are mostly restricted to myotubes, whereas myogenin and MRF4 mark the middle stage of myogenesis and are found in all myotubes and in many committed myoblasts.

[0038] As used herein, "satellite cells" or "muscle satellite cells" refer to small, multipotent cells lacking the cytoplasm found in mature muscle. Satellite cells are precursors of skeletal muscle cells and can give rise to satellite cells or myoblasts, which give rise to skeletal muscle cells. They have the potential to provide additional myonuclei to their parent muscle fibers or to return to a quiescent state. Upon activation, satellite cells can re-enter the cell cycle, proliferate, and differentiate into myoblasts. Satellite cells may exhibit one or more characteristics that may be shared with endogenous satellite cells, including, but not limited to, the ability to repopulate the satellite cell niche, drive muscle regeneration, and may exhibit appropriate expression of genetic markers, appropriate expression of glycoproteins, and expandability in culture.

[0039] The terms "subject," "patient," "individual," etc. are not intended to be limiting and may generally be interchangeable; that is, an individual described as a "patient" does not necessarily have a given disease, but may simply be seeking medical advice.

[0040] As used herein, "treating" or "treatment" of a condition, disease, or disorder, or symptoms associated with a condition, disease, or disorder, refers to an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, partial or complete relief or amelioration of one or more symptoms or conditions, attenuation of the severity of a condition, disorder, or disease, stabilization of the condition, disorder, or disease state, prevention of the development of a condition, disorder, or disease, prevention of the spread of a condition, disorder, or disease, delay or slowing of the progression of a condition, disorder, or disease, delay or slowing of the onset of a condition, disorder, or disease, remission or alleviation of the condition, disorder, or disease state, and remission. "Treating" can also mean inhibiting the progression of a condition, disorder, or disease, or temporarily slowing the progression of a condition, disorder, or disease, although in some instances, it also encompasses permanently halting the progression of a condition, disorder, or disease.

[0041] As used herein, the terms "treat" and "prevent" are not intended to be absolute terms. In embodiments, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of a disease or condition. In embodiments, a method for treating a disease is considered treatment when there is a 10% reduction in one or more symptoms of the disease in a subject compared to a control. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to the natural or control level. It is understood that treatment does not necessarily refer to a cure or complete elimination of a disease, condition, or symptom of a disease or condition. In embodiments, reference to decrease, reduction, or inhibition includes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater change compared to control levels, and such terms may, but do not necessarily, include complete elimination. In embodiments, the severity of the disease is reduced by at least 10%, for example, compared to the individual before administration or a control individual not undergoing treatment. In some embodiments, the severity of the disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, is no longer detectable using standard diagnostic techniques.

[0042] As used herein, the terms "effective amount," "effective dose," and the like refer to an amount of an agent that is sufficient to achieve a desired effect. In embodiments, the term "effective" when referring to an amount of cells or a therapeutic compound, as used in the present disclosure, may refer to an amount of cells or a compound that is sufficient to produce an improvement or desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic reaction), commensurate with a reasonable benefit / risk ratio. In embodiments, the term "effective" when referring to the production of a desired cell population may refer to an amount of one or more compounds that is sufficient to result in or promote the production of members of the desired cell population, particularly compared to culture conditions lacking one or more compounds.

[0043] Preparation of genetically modified satellite cells The present inventors have shown that muscular dystrophies characterized by mutant disease genes can be treated by intramuscular (IM) transplantation of satellite cells that are disease-gene-corrected, such as autologous satellite cells, but lacking the defect in the disease gene. These transplanted satellite cells can promote myofibril and muscle repair and recovery in patients.

[0044] Thus, in one embodiment, the present disclosure provides an autologous cell product designed to restore normal expression of disease genes in satellite cells and their progeny. These satellite cells not only replace and repair damaged muscle tissue, but also, upon homing to the quiescent satellite cell niche, support long-lasting homeostasis and repair of future muscle damage. Treatment delivered to muscles can promote locomotion, mobility, and improve a patient's functional condition and ability to perform activities of daily living.

[0045] Also provided in one embodiment is a method for preparing autologous satellite cells with restored expression and activity of a mutant disease gene. In one exemplary embodiment, with reference to Figure 1, the method of preparation comprises: a) obtaining peripheral blood mononuclear cells (PBMCs) from a patient with muscular dystrophy characterized by a mutant gene; b) reprogramming PBMCs into induced pluripotent stem cells (iPSCs); c) correcting the mutant gene in the iPSCs; c) differentiating the iPSCs into satellite cells; and d) enriching and expanding the satellite cells.

[0046] It is important to note that not all steps in this method are required, and the order of these steps can be adjusted. For example, mutant genes can be corrected before PBMC reprogramming. In another example, iPSCs can be expanded before differentiation.

[0047] Each of these steps is described in more detail below.

[0048] PBMC acquisition and reprogramming Methods for obtaining PBMCs are known in the art. For example, PBMCs can be generated from a patient's venous blood in accordance with 21 CFR Part 1271 Subpart C using density gradient centrifugation (steps 1 and 2 in FIG. 1).

[0049] PBMCs can be reprogrammed into induced pluripotent stem cells (iPSCs) by introducing PBMC factors that drive iPSC formation. Non-limiting examples of such factors include the Yamanaka factors Oct3 / 4, Sox-2, Klf-4, and L-Myc (step 3 in Figure 1). In some embodiments, these factors are transduced into PBMCs with a viral vector, such as a Sendai virus (SeV) vector.

[0050] In some embodiments, upon transduction, cells are monitored daily and iPSC colonies are observed after 14 days. It is also important to confirm negative test results by residual virus assay over two consecutive passages to ensure patient safety.

[0051] Mutant gene correction Mutant gene correction can be performed by known gene editing techniques. The target gene depends on the muscular dystrophy being treated. Table 1 lists some of the known mutant genes associated with various types of muscular dystrophy. [Table 1-1] [Table 1-2]

[0052] In the following description, CAPN3 is used as an example mutant gene in muscular dystrophy (Figure 2), but it is understood that the present technology can be applied to other mutant genes and corresponding types of muscular dystrophy as well.

[0053] In one embodiment, iPSC is subjected to CRISPR gene editing by introducing Cas9 protein, CAPN3 gene locus specific guide RNA and the template that contains the optimized CAPN3 coding sequence that is inserted downstream of endogenous CAPN3 promoter.The nucleic acid sequence that codes normal CAPN3 protein is provided in GenBank Accession No: NM_000070.3.

[0054] In some embodiments, the endogenous CAPN3 gene contains one or more mutations, so that the gene is unable to express a functional CAPN3 protein. If the mutation is not within exon 1 or the first few exons, the first few exons do not need to be replaced with an exogenous coding sequence.

[0055] Furthermore, the present inventors have discovered that intron 1 of the CAPN3 gene is important for expression of a functional CAPN3 protein. Thus, in one embodiment, the exogenous CAPN3 coding sequence need not include exon 1 or even exon 2, and the insertion locus is after exon 2 of the endogenous CAPN3 gene. In some embodiments, the inserted CAPN3 coding sequence begins in exon 3 (e.g., includes exons 3-24), and insertion occurs at a locus immediately after (3' to) the last nucleotide of exon 2 of the endogenous gene.

[0056] In some embodiments, the insertion occurs at a locus further downstream from exon 2, such as, but not limited to, in intron 2, exon 3, intron 3, or exon 4, so long as the retained exon does not contain a mutation. The corresponding exogenous coding sequence comprises a downstream exon that is complementary to the exon upstream of the insertion locus.

[0057] In an alternative embodiment, the CAPN3 coding sequence can be inserted into the target genome at a locus different from the endogenous CAPN3 gene, such as a "genomic safe harbor" (GSH). A genomic safe harbor (GSH) is a site within the genome that can accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic element: (i) functions as expected, and (ii) does not cause alterations to the host genome that pose a risk to the host cell or organism. A known GSH is the human AAVS1 gene (PPP1R12C; Ensembl ID ENSG00000125503).

[0058] Other GSHs and methods for identifying new GSHs are also known. For example, in Aznauryan et al., Cell Rep Methods. 2022 Jan 14;2(1):100154, the authors reported the discovery of several new GSHs, including GSH1 (GRCh38 coordinates 195,338,589–195,818,588), GSH2 (GRCh38 coordinates 22,720,711–22,761,389), GSH7 (GRCh38 coordinates 145,090,941–145,219,513), GSH8 (GRCh38 coordinates 145,320,384–145,525,881), and GSH31 (GRCh38 coordinates 89,174,426–89,179,074).

[0059] When an exogenous CAPN3 coding sequence is inserted into a locus different from that of the endogenous gene, the native CAPN3 promoter is unavailable. Thus, in some embodiments, the exogenous CAPN3 coding sequence is operably linked to a promoter capable of expressing the CAPN3 gene. In some embodiments, expression occurs within muscle cells. In some embodiments, the promoter is the muscle creatine kinase promoter (tMCK). tMCK is composed of a core sequence from the endogenous muscle creatine kinase (Ensembl ID ENSMUSG00000030399) promoter. It was generated by ligating a triple tandem repeat of the MCK enhancer to the basal promoter, thus generating a strong muscle-specific promoter.

[0060] In some embodiments, the CAPN3 coding sequence is followed by a selection marker (after a polyA tail). In one embodiment, the selection marker is a puromycin or neomycin resistance gene driven by a constitutive promoter (e.g., a PGK (3-phosphoglycerate kinase) promoter). In some embodiments, it is preferable to correct both alleles of the mutant CAPN3 gene; thus, two copies of the exogenous CAPN3 coding sequence are integrated into the genome of the target iPSCs. This can be achieved by transfecting two vectors, each containing a CAPN3 coding sequence but with different selection markers (see, e.g., Figures 3A-B). For example, one vector contains a puromycin resistance gene and the other contains a neomycin resistance gene. In addition to resistance genes for puromycin and neomycin, those providing resistance to other drugs, such as gentamicin, blasticidin, and zeocin, can also be used.

[0061] Upon transfection of the two vectors, iPSCs that have integrated both copies of the CAPN3 coding sequence can be identified under both selection conditions, e.g., in culture medium with both puromycin and neomycin added (e.g., see step 2 in Figure 2).

[0062] In each vector, the integrated sequence can be flanked by CAPN3 gene-specific homology arms to allow target-specific editing. For each selectable marker, the expression cassette can be flanked by site-specific recombinase target sites to allow removal of the cassette (e.g., loxP sites to allow Cre excision) after drug selection is completed (e.g., see step 3 in Figure 2).

[0063] In some embodiments, it is desirable to remove the incorrectly integrated sequence. In one example, a constitutively expressed kill switch (e.g., a thymidine kinase gene cassette) is included in the vector, but is located outside the CAPN3 gene homology arm. If the CAPN3 cassette is correctly integrated into the target genome, the kill switch will necessarily be removed during homology-directed integration. If the kill switch is not removed, it means that the integration is incorrect. In this situation, cells can be killed by treating with a drug (e.g., ganciclovir) that is the corresponding kill switch.

[0064] In one example, after puromycin and G418 (neomycin analog) selection, cells are treated with ganciclovir. The resulting iPSCs are then expanded and subjected to a round of treatment with the TAT-Cre enzyme, which removes the puromycin / neomycin selection cassette (containing the Cre site), leaving only the CAPN3 coding sequence insert in place in the genome.

[0065] In some embodiments, the iPSCs remaining after Cre-lox excision are expanded at low density for clonal isolation. Isolated clones are tested for the presence of the inserted CAPN3 coding sequence in both copies of the genome, the absence of the puromycin / neomycin selection cassette, and the absence of randomly integrated vector backbone to create a master cell bank (MCB). Cells can be expanded and cryopreserved for later use (e.g., see step 4 in Figure 2).

[0066] For repair of the CAPN3 locus, for example, the original mutant CAPN3 (exons 3-24) coding sequence is preceded by the newly integrated exogenous CAPN3 (exons 3-24) coding sequence (Figure 3A). The endogenous CAPN3 promoter drives expression of the exogenous CAPN3 coding sequence, while the endogenous / mutant CAPN3 is inactivated (e.g., by a polyA tail / stop codon from the inserted cassette).

[0067] The process of inserting or incorporating nucleic acids into cells can be performed through known techniques, including, but not limited to, transformation, transfection, or transduction. Transformation introduces recombinant plasmid DNA into competent cells that incorporate extracellular DNA from the environment. This process is amenable to plasmid DNA propagation, protein production, and other uses. Transfection is the process of incorporating foreign nucleic acids into eukaryotic cells. Transduction refers to the introduction of recombinant viral vector particles into target cells.

[0068] The term "vector" refers to a nucleic acid molecule capable of mediating the transport or expression of heterologous nucleic acids. Plasmids are a species of the genus encompassed by the term "vector." A vector typically refers to a nucleic acid sequence containing an origin of replication and other entities necessary for replication and / or maintenance within a host cell. A vector capable of directing the expression of a gene and / or nucleic acid sequence to which it is operably linked is referred to herein as an "expression vector." In general, useful expression vectors are often in the form of "plasmids," which, in vector form, are not bound to a chromosome and typically refer to circular double-stranded DNA molecules containing entities for stable or transient expression of encoded DNA. Other expression vectors that can be used in the methods disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophage, or viral vectors, although such vectors can be integrated into the host genome or replicate autonomously within the cell. The vector can be a DNA or RNA vector. Other forms of expression vectors known to those skilled in the art which serve equivalent functions may also be used, such as self-replicating extrachromosomal vectors or vectors capable of integrating into a host genome. Exemplary vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked.

[0069] In vitro differentiation of CAPN3-corrected iPSCs The CAPN3 gene-corrected iPSCs can be further cultured, differentiated, enriched and expanded to produce satellite cells.

[0070] In some embodiments, iPSCs are seeded as single cells for cell culture by adherent culture without the use of feeder cells. For culture, culture vessels such as dishes, flasks, microplates, or cell culture sheets such as OptiCell (Nalge Nunc International) are used.

[0071] In some embodiments, the culture vessel is surface-treated to improve cell adhesion (hydrophilicity) or coated with a substrate for cell adhesion, such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, extracellular matrix (e.g., BD Matrigel (Becton Dickinson), Geltrex (Gibco)), or vitronectin. The culture vessel may be coated with type I collagen, Matrigel, fibronectin, vitronectin, or poly-D-lysine. The culture medium includes mouse embryonic fibroblast-conditioned medium. In some embodiments, the vessel is coated with a suitable adhesion molecule. Non-limiting examples of adhesion molecules include laminin-511, -521, -332, and -111, and fragments thereof. Laminin-511 is a major component of the basement membrane used as a scaffold for pluripotent stem cells (ES / iPS cells) because it binds to integrins on the cell surface. Laminin-511 is a large protein (approximately 800 kDa) composed of three chains (α-, β-, and γ-) that form supramolecular aggregates. In addition to the whole protein, fragments of the protein can be conveniently used. One such fragment is the E8 fragment (Miyazaki et al., Nature Commun. 3;1236 (2012)). Recombinant laminin-511 E8 is commercially available as iMatrix 511.

[0072] In a specific embodiment, iPSCs are cultured in a medium containing a growth factor, such as fibroblast growth factor 2 (FGF-2), and / or a ROCK inhibitor, such as Y-27632. The term "ROCK inhibitor" refers to a substance that inhibits Rho kinase (ROCK: Rho-associated, coiled-coil-containing protein kinase), and may be a substance that inhibits either ROCKI or ROCKII. The ROCK inhibitor is not particularly limited, as long as it has the above-mentioned function. Examples of ROCK inhibitors that can be used include: N-(4-pyridinyl)-4β-[(R)-1-aminoethyl]cyclohexane-1α-carboxamide (Y-27632), fasudil (HA1077), (2S)-2-methyl-1-[(4-methyl-5-isoquinolinyl]sulfonyl]hexahydro-1-H-1,4-diazepine (H-1152), 4β-[(1R)-1-aminoethyl]-N-(4-pyridyl)benzenecarboxamide (Wf-536), N-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4PER(R)-1-aminoethyl]cyclohexane-carboxamide (Y-30141), N-(3-{[2-(4-amino-1,2, 5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]oxy}benzamide (GSK269962A) and N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A); antibodies (including functional fragments), antisense nucleic acids, and siRNA against ROCK; ROCK antagonists and dominant negative forms; and other ROCK inhibitors known in the art.

[0073] In some embodiments, the culture medium comprises a base stem cell medium and an N2 supplemented medium. The N2 medium may contain components such as bovine or recombinant human insulin, transferrin, putrescine, selenite, and progesterone. In some embodiments, the base stem cell medium and the N2 medium are used in a ratio of 20:80 to 45:55 (v / v), or 30:70 to 45:55, 35:65 to 45:55, or approximately 40:60 (v / v).

[0074] In the coated vessel with a suitable stem cell culture medium, the iPSCs can be cultured to reach a suitable target confluency (e.g., at least 40%, 50%, 60%, 70%, 80%, or 90%, which can be determined by microscopy). The iPSCs are then subjected to differentiation.

[0075] In one embodiment, the differentiation medium comprises a Wnt signaling activator (e.g., CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidin-yl]amino]ethyl]amino]nicotinonitrile)), a ROCK inhibitor (e.g., Y-27632 (N-(4-pyridinyl)-4β-[(R)-1-aminoethyl]cyclohexane-1α-carboxamide)), a Notch signaling inhibitor (e.g., Y-27632), a TGF-β signaling inhibitor (e.g., SB431542 (4-[4-(2H-1,3-benzodioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl]benzamide) and / or N2 medium, in various combinations (see, e.g., Table 2).

[0076] CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidin-yl]amino]ethyl]amino]nicotinonitrile) is a Wnt signaling activator and GSK3β inhibitor. GSK3β (glycogen synthase kinase 3) is a serine / threonine protein kinase involved in various signaling pathways related to glycogen production, apoptosis, and stem cell maintenance. GSK3 contains two isoforms, α and β. Examples of GSK3β inhibitors include CHIR98014 (2-[[2-[(5-nitro-6-aminopyridin-2-yl)amino]ethyl]amino]-4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidine), CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidin-yl]amino]ethyl]amino]nicotinonitrile), Kenpaullone, AR-A0144-18, and TDZD-8 (4-benzyl-2-methyl-1,2,4 -thiadiazolidine-3,5-dione), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), BIO (6-bromoindirubin-3-oxime), TWS-119 (3-[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yloxy]phenol), and SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione). In addition, antisense oligonucleotides, siRNAs, etc. against GSK3β mRNA can also be used as GSK3β inhibitors, and are commercially available or can be synthesized according to methods known in the art.

[0077] In some embodiments, the concentration of the Wnt signaling activator / GSK3β inhibitor (e.g., CHIR99021) may be at least 0.2 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, or 3 μM. In some embodiments, the concentration may not be higher than 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 40 μM. In some embodiments, the concentration may be, but is not limited to, 0.2-10 μM, 0.5-8 μM, 1-7 μM, 2-5 μM, or 2-4 μM.

[0078] In some examples, iPSCs are cultured with a γ-secretase inhibitor and / or a Notch signaling inhibitor. An example of a γ-secretase inhibitor is N-[N-(3,5-difluorophenacetyl)- L [-alanyl]-S-phenylglycine t-butyl ester (DAPT). DAPT is a potent and specific inhibitor of γ-secretase, a multimeric membrane protein complex that catalyzes the proteolytic cleavage of amyloid precursor protein (APP), leading to the accumulation of amyloid β (Aβ) peptides associated with early-onset familial Alzheimer's disease (AD), blocking Notch signaling. It binds directly to the C-terminal fragment of the catalytic center of γ-secretase presenilin (PS), specifically within the C-terminal region of transmembrane domain 7 or more, resulting in the synthesis of a photoactivatable DAPT derivative. DAPT indirectly inhibits Notch, a substrate of γ-secretase.

[0079] Notch signaling inhibitors are drugs that inhibit the Notch signaling pathway, such as chemical compounds or antibodies. For example, inhibitors against γ-secretase can inhibit the Notch signaling pathway. Such γ-secretase inhibitors are, for example, natural peptides, non-peptides, or semi-peptides, and are preferably small molecules. Examples include DAPT (N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). In addition, compounds from chemical classes AS (arylsulfonamides), DBZ (dibenzazepines (DBZ)), BZ (benzodiazepines), LY-411,575, and many others have also been tested for their γ-secretase inhibitory activity. γ-secretase inhibitors are divided into sulfonamides / sulfones and benzodiazepines / benzolactams.

[0080] In certain embodiments, iPSCs are cultured in medium containing at least a Notch signaling inhibitor (e.g., DAPT) for at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days. In certain embodiments, the cells are cultured in medium containing at least a Notch signaling inhibitor (e.g., DAPT) for, but not limited to, 1 to 20 days, 2 to 15 days, 3 to 12 days, 4 to 11 days, 5 to 10 days, 6 to 9 days, 7 to 9 days, 7 to 8 days, or 8 to 10 days.

[0081] In some embodiments, the medium contains TGF-β inhibitor.TGF-β inhibitors include, for example, SB431542 (4-[4-(2H-1,3-benzodioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl]benzamide), SB202190 (Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124G (GlaxoSmithKline), Lefty-1 (e.g., NM_020997), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A-83-01 (WO2009146408) and their derivatives.

[0082] In some embodiments, the concentration of the TGF-β inhibitor in the medium is 1 μM to 50 μM, for example, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, and 50 μM. In some embodiments, the concentration is 2 μM to 10 μM, for example, 5 μM.

[0083] In one example procedure, iPSC differentiation is carried out as shown in Table 2. On each specified day, the medium is completely replaced with the addition of the indicated supplements (e.g., Wnt signaling activators). [Table 2-1] [Table 2-2]

[0084] Selection and expansion Differentiated cells can be sorted by magnetically assisted cell sorting (MACS). In some embodiments, sorted cells are identified with an antibody that binds to CD271, which can be coupled to magnetic beads.

[0085] CD271 belongs to the low-affinity neurotrophin receptor and tumor necrosis factor receptor superfamily. Human CD271 (LNGFR) was initially described as being expressed in cells of the central and peripheral nervous system and was suggested to be involved in cell development, survival, and differentiation. Herein, CD271 has been discovered to be a suitable marker for identifying differentiated satellite cells suitable for treatment.

[0086] Sorted cells can be expanded for 2-3 weeks in a suitable medium, such as N2 medium supplemented with 10% FBS, bFGF2, and FGF8b.

[0087] Enriched and expanded CD271+ satellite cells have excellent repopulation and engraftment capacity, and the repopulation and engraftment capacity of CD271+ satellite cells requires a minimum cell concentration.

[0088] Thus, according to one embodiment of the present disclosure, a population of cells (e.g., mammalian cells, or more particularly human cells) is provided, wherein at least 30% of the cells are CD271+ satellite cells derived from in vitro or ex vivo iPSCs. In some embodiments, the cell population comprises at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD271+ satellite cells.

[0089] In some embodiments, the cell population comprises at least 100, 1000, 10,000, 100,000, 1x10 6 , 1x10 7 , 1x10 8 , or 1x10 9In some embodiments, a substantial portion of the cells of the population are cultured with CD271+ satellite cells during differentiation. For example, at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% of the cells of the population are cultured with CD271+ satellite cells during differentiation.

[0090] In addition to CD271, differentiated cells also express NCAM + , HNK1 - , CD271+, MyoD+, CD54+, integrin α9β1+, and / or SDC2+. Further, suitable markers for identifying these cells include, but are not limited to, CHRNA1+, NTSR1+, FZD1+, FZD5-, GPR37-, and GPR27-. In some embodiments, differentiated CD271+ satellite cells are characterized by at least two, three, four, or five of CHRNA1+, NTSR1+, FZD1+, FZD5-, GPR37-, and GPR27-.

[0091] Whether a cell surface protein is positive (+) or negative (-) can be assessed by an agent that recognizes the marker, such as an antibody. However, it is readily understood by those skilled in the art that such positivity and negativity may not be absolute. In some embodiments, a positive marker is one that has higher expression on the cell than on the reference cell. In some embodiments, a negative marker is one that has lower expression on the cell than on the reference cell. For example, the reference cell is a cell that has been similarly differentiated from a pluripotent stem cell but is unable to regenerate muscle tissue in vivo.

[0092] In some embodiments, the produced cells may be cryopreserved for later use.

[0093] treatment Also provided are compositions, uses, therapies, medicaments and methods for treating limb-girdle muscular dystrophy type 2A (LGMD2A).

[0094] Satellite cells may be administered to a patient in a pharmaceutically or physiologically acceptable preparation or composition containing a physiologically acceptable carrier, excipient, or diluent, and administered to the tissue of a recipient organism of interest, including humans and non-human animals.

[0095] Satellite cell compositions can be prepared by resuspending the cells in a suitable liquid or solution, such as sterile saline or other physiologically acceptable injectable aqueous liquids. The amounts of components to be used in such compositions can be routinely determined by one skilled in the art.

[0096] In an example, for injectable administration, a composition (e.g., a composition containing satellite cells) may be in a sterile solution or suspension or resuspended in a pharmaceutically and physiologically acceptable aqueous or oily vehicle, and may contain preservatives, stabilizers, and materials to render the solution or suspension isotonic with the recipient's body fluids (i.e., blood). Non-limiting examples of excipients suitable for use include water, phosphate-buffered saline, pH 7.4, 0.15 M aqueous sodium chloride solution, dextrose, glycerol, dilute ethanol, etc., and mixtures thereof. Exemplary stabilizers include polyethylene glycol, proteins, sugars, amino acids, inorganic acids, and organic acids, which may be used either by themselves or in mixtures. The amount or quantity, as well as the route of administration used, are determined on an individual basis and correspond to amounts used for similar types of applications or indications known to those skilled in the art.

[0097] Consistent with the present invention, satellite cells can be administered to body tissues, including muscles. The number of satellite cells in suspension and the mode of administration can vary depending on the site and condition to be treated.

[0098] In an embodiment, a therapeutically effective amount of a composition (e.g., a composition comprising satellite cells) can be administered to a human. In one embodiment, the composition (e.g., a composition comprising satellite cells) is administered three times daily, twice daily, once daily, 14 days (four times daily, three times daily, or twice daily, or once daily) in a three-week cycle with seven days off, up to five or seven days (four times daily, three times daily, or twice daily, or once daily) in a three-week cycle with 14 to 16 days off, or once every two days, or once a week, or once every two weeks, or once every three weeks.

[0099] In one embodiment, the composition (e.g., a composition comprising satellite cells) is administered once a week, or once every two weeks, or once every three weeks, or once every four weeks for at least one week, and in some embodiments, for 1 to 4 weeks, 2 to 6 weeks, 2 to 8 weeks, 2 to 10 weeks, or 2 to 12 weeks, 2 to 16 weeks, or longer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 36, 48 weeks, or more). [Example]

[0100] Example 1 Insertion of an exogenous CAPN3 coding sequence In this example, the insertion of an exogenous CAPN3 coding sequence into a targeted human genome was designed and tested, allowing for the expression of active CAPN3 protein.

[0101] A targeting vector encoding the human CAPN3 coding sequence (CDS) was constructed as a double-stranded DNA plasmid and designed for integration into a specific genomic locus using a DNA-cleaving nuclease. The encoded CAPN3 CDS nucleotide sequence was defined by the NCBI reference sequence NM_000070.3, with transcription terminated by a transgenic polyadenylation signal (pA) following the stop codon of the inserted CAPN3 CDS.

[0102] Each targeting vector contains a loxP-flanked excisable drug selectable marker (PuroR and NeoR) that is expressed by an internal promoter (EFS; elongation factor 1a short) (Figure 3A) or the endogenous hAAVS1 locus. The selectable marker is spliced into the translational open reading frame of the hAAVS1 CDS via a splicing (SA) and self-cleaving peptide sequence (T2A) (Figure 3B). After integration of the targeting vector, the selectable marker can be excised from the genome by treating the cells with Cre recombinase.

[0103] The region of the vector intended for integration into the genome is flanked by homology arms (LA; left arm, RA; right arm) 500–100 nucleotides in length that are homologous to the adjacent region of the genome where insertion occurs. A short sequence within the genome located between the homology arms defines the site at which a nuclease will cleave the DNA in the presence of an RNA oligonucleotide complementary to the genomic nuclease target site. The double-strand break induces cells to integrate the region of the target vector flanked by the homology arms into the genome. Each target vector encodes a herpes simplex virus type 1 thymidine kinase (HSV-TK) suicide gene, which is expressed by an upstream promoter (EF1a; full-length elongation factor 1a). This allows for the elimination of cells that have integrated vector backbone elements not contained within the homology arms using the prodrug ganciclovir.

[0104] Here, we tested two integration strategies. First, as shown in Figure 3A, exons 3–24 of the CAPN3 CDS encoded by the targeting vector were inserted in frame after the last nucleotide of exon 2 from the endogenous CAPN3 gene (Ensembl ID; ENSG00000092529). Therefore, the inserted CAPN3 exons 3–24 were transcribed as part of the final CAPN3 mRNA, including endogenous exons 1 and 2, under the control of the endogenous CAPN3 promoter. The entire transcript was terminated by a transgenic pA signal encoded within the targeting vector.

[0105] In the second strategy, the entire CAPN3 CDS (exons 1–24) was inserted into intron 1 of the AAVS1 gene (PPP1R12C; Ensembl ID ENSG00000125503), as shown in Figure 3B. The CAPN3 CDS is expressed under the control of a synthetic muscle-specific promoter (tMCK), which consists of a core sequence derived from the endogenous muscle creatine kinase (Ensembl ID ENSMUSG00000030399) promoter. The promoter, CAPN3 CDS, and pA site are flanked by a minimal chicken hypersensitive site 4 (cHS4) insulator, which alleviates transcriptional silencing of the CAPN3 CDS.

[0106] Genotyping was used to confirm integration of the exogenous CAPN3 coding sequence at the designated sites. The genotyping design for the first and second integration strategies (Figures 3A and 3B, respectively) is illustrated in Figures 5A and 5C, respectively. PCR primers were selected so that the amplified sequence from the integrated site was 1522 bp in length (middle panel) and that from the wild-type site was 3622 bp in length, as shown in Figure 5A.

[0107] More specifically, in Figure 5A, boxes (not drawn to scale) depicting the left and right homology arms of the targeting vector indicate the positions relative to the genotyping primers. Cells were electroporated with the CAPN3 targeting vector, Cas9 nuclease, and an RNA oligonucleotide complementary to the region at the end of endogenous exon 2 of the CAPN3 gene. Cells that did not incorporate the targeting vector were eliminated by adding antibiotics (puromycin and G418; resistance is encoded by the PuroR and NeoR transgenes on the targeting vector) to the cell culture after electroporation. Antibiotic-resistant cells were treated with Cre recombinase (TAT-Cre) to excise the loxP-flanked selection marker. Cells were then sparsely seeded to allow for the isolation and expansion of single-cell-derived colonies for genotyping.

[0108] A series of polymerase chain reactions were performed on genomic DNA purified from each clonal line to determine the correct insertion. First, a primer (CAPN3 WT F+R, 30-second extension) was used to detect the endogenous CAPN3 allele without integration to distinguish between clones with hemizygous (one copy) and homozygous (two copies) integration of the target vector. Next, primers were used to amplify the 5' and 3' regions of the genomic insertion junction (CAPN3 5'F+R and CAPN3 3'F+R, respectively). In the 5' reaction, the forward primer was located outside the genomic region encoded by the left homology arm, while the reverse primer was located inside the target vector. In the 3' reaction, the forward primer was located inside the target vector, while the reverse primer was located outside the genomic region encoded by the right homology arm. Finally, the CAPN3 WT F+R primer was used with a 3-minute PCR extension time to amplify the entire inserted CAPN3 CDS exons 3–24.

[0109] All PCR reactions were analyzed by gel electrophoresis (Figure 5B) to confirm that for all reactions, clones contained the expected amplification of each band, the size of which was predicted based on the location of primer binding within the genome. Clones 5 and 12 were homozygous clones (2 copies); clones 9 and 17 were hemizygous (1 copy).

[0110] As shown in Figure 5C, cells were electroporated with the hAAVS1 targeting vector, Cas9 nuclease, and an RNA oligonucleotide complementary to a region of intron 1 within the hAAVS1 locus. Cells that did not incorporate the targeting vector were eliminated by adding antibiotics (puromycin and G418; resistance is encoded by the PuroR and NeoR transgenes on the targeting vector) to the cell culture after electroporation. Antibiotic-resistant cells were treated with Cre recombinase (TAT-Cre) to excise the loxP-flanked selection marker. Cells were then sparsely seeded to allow for the isolation and expansion of clonal single-cell-derived colonies for genotyping.

[0111] A series of polymerase chain reactions were performed on genomic DNA purified from each clonal line to determine the correct insertion. First, a primer for detecting the unintegrated AAVS1 wild-type allele (AAVS1 WT F+R) was used to distinguish between clones with hemizygous (one copy) and homozygous (two copies) integration of the target vector. Next, primers were used to amplify the 5' and 3' regions of the genomic insertion junction (AAVS1 5'F+R and AAVS1 3'F+R, respectively). In the 5' reaction, the forward primer was located outside the genomic region encoded by the left homology arm, and the reverse primer was located inside the target vector. In the 3' reaction, the forward primer was located inside the target vector, and the reverse primer was located outside the genomic region encoded by the right homology arm. Finally, primers were used to amplify the entire CAPN3 CDS (tMCK CAPN3 F+R), where the forward primer was located inside the tMCK promoter and the reverse primer was located inside the pA sequence.

[0112] All PCR reactions were analyzed by gel electrophoresis (Figure 5D) to confirm that for all reactions, clones contained the expected amplification of each band, the size of which was predicted based on the location of primer binding within the genome. The boxes around clones 42 and 44 indicate that these clones contained the correct hemizygous (one copy) insertion of the targeting vector encoding CAPN3.

[0113] Example 2 Expression of active CAPN3 protein in engineered cells In this example, we determined whether human cells engineered to incorporate an exogenous CAPN3 coding sequence can express functional CAPN3 mRNA and protein.

[0114] material and method myotube formation iPSC-derived satellite cells were cultured at 3x10 4Cells were seeded at 1000 x 1000 cells per well and cultured in growth medium (N2 medium + 10% FBS + FGF2 + FGF8) for 2 days, then the medium was switched to N2 medium supplemented with 10 μM TGFβ inhibitor (SB431542), 10 μM forskolin, 10 μM DAPT, and 10 μM dexamethasone, and the cells were cultured for an additional 5 days to form mature myotubes.

[0115] QPCR Cells were lysed in buffer RLT (RNasey Mini Kit, Qiagen) and homogenized using a QIAshredder. RNA was extracted from the lysate using an RNasey Mini Kit (Qiagen) with on-column DNAse treatment according to the manufacturer's instructions. RNA concentration was quantified using a Nanodrop. For quantitative RT-PCR analysis, reverse transcription was performed using iScript RT Supermix (bio-Rad). qPCR was performed using TaqMan probes (Thermo Scientific) and TaqMan Fast Advanced Master Mix. QPCR was performed using a QuantStudio™ 5 Real-Time PCR System. The TaqMan probes used were CAPN3 (Hs01115989_m1), GAPDH (Hs99999905_m1), and ACTB (Hs99999903_m1).

[0116] Western blot analysis Cells were lysed in lysis buffer consisting of 20 mM Tris HCl, 0.1 mM EDTA, 1 mM DTT, 20 mg / mL soybean trypsin inhibitor, 28 mM E64, and 2 mM phenylmethylsulfonyl fluoride (PMSF) plus 1x Laemmli sample buffer. Lysates were collected using a cell scraper, boiled at 95°C for 5 minutes, and centrifuged at 10,000 rpm for 10 minutes to remove cell debris. Total protein concentration was measured using a Bradford assay. A total of 30 mg of protein was electrophoresed in a 10% SDS-PAGE gel. After electrophoresis, proteins were transferred to nitrocellulose using a Transblot Turbo Transfer system. After transfer, blots were blocked with 5% BSA in PBS-T (phosphate-buffered saline and 0.1% Tween® 20) for 1 hour at room temperature (RT). After blocking, the blots were incubated with primary antibodies (CAPN3: NCL-CALP-12A2; desmin: 5332S, GAPDH: 2118S) diluted in 3% BSA in PBS-T and incubated overnight at 4° C. The next day, the membranes were incubated with secondary antibodies and imaged using a Li-cor Odyssey imager.

[0117] result qPCR was used to detect the relative CAPN3 mRNA in the engineered cells (see Example 1). The results are shown in Figure 6.

[0118] CAPN3 mRNA levels in muscle fibers from healthy donors were used as a control (=1.0). Among all samples, the one with the exogenous complete CAPN3 coding sequence inserted (Patient 1 (P1): AAVS1-tMCK P1-bulk) had the highest expression. All three clones (5, 9, and 12) with modified CAPN3 at the CAPN3 locus exhibited similarly high expression levels (Patient 2 (P2)). In contrast, no functional mRNA was detected in muscle fibers with unmodified CAPN3 (P1 without modification).

[0119] Western blot was used to detect CAPN3 protein levels in these muscle fibers. As shown in Figure 7, left panel (P1), the control (C, from a healthy donor) and AAVS1 insertion samples showed positive CAPN3 expression, whereas the uncorrected P1 showed no expression. Similarly, in Figure 7, right panel, the control (C) and three corrected samples (within the CAPN3 locus) showed positive CAPN3 expression, whereas the uncorrected P2 showed no expression.

[0120] Therefore, this example demonstrated that targeted insertion of an exogenous CAPN3 coding sequence using the present technology can restore functional expression of the CAPN3 protein.

[0121] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0122] The invention illustratively described herein may be conveniently practiced in the absence of any element or elements, or limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc., shall be read expansively and without limitation. Furthermore, the terms and expressions used herein are used as terms of description and not limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or portions thereof, but recognizes that various modifications are possible within the scope of the claimed invention.

[0123] Thus, while the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that alterations, modifications, and variations of the invention disclosed herein and embodied therein may be resorted to by those skilled in the art, and that such alterations, modifications, and variations are considered to be within the scope of the present invention. The materials, methods, and examples provided herein are representative of preferred embodiments and are illustrative and are not intended as limitations on the scope of the invention.

[0124] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a provisos or negative limitation removing any subject matter from the genus, regardless of whether the omitted material is specifically set forth herein.

[0125] Furthermore, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also described in terms of any individual member or subgroup of members of the Markush group.

[0126] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0127] While the present disclosure has been described in conjunction with the above-described embodiments, it will be understood that the foregoing descriptions and examples are intended to illustrate, but not limit, the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

Claims

1. 1. A method for preparing satellite cells from stem cells containing a mutation in an endogenous gene, comprising: introducing first and second vectors, each comprising a first expression cassette encoding at least a portion of a wild-type gene, into said stem cells such that each of said first expression cassettes integrates into the locus of said endogenous gene in the genome to enable encoding of a wild-type version of said gene lacking said mutation; differentiating the stem cells into satellite cells; and Expanding the satellite cells into a population of satellite cells. A method comprising:

2. 1. A method for preparing satellite cells from stem cells containing a mutation in an endogenous gene, comprising: introducing first and second vectors, each comprising a first expression cassette that includes the entire coding sequence of a wild-type gene, into the stem cells such that each of the first expression cassettes is integrated into a locus in the genome that is different from that of the endogenous gene; differentiating the stem cells into satellite cells; and Expanding the satellite cells into a population of satellite cells. A method comprising:

3. 3. The method of claim 2, wherein the locus is selected from the group consisting of an intron of the AAVS1 gene and human genome coordinates 195,338,589-195,818,588, 22,720,711-22,761,389, 145,090,941-145,219,513, 145,320,384-145,525,881, and 89,174,426-89,179,074 (GRCh38 coordinates).

4. 4. The method of claim 3, wherein the locus is within intron 1 of the AAVS1 gene.

5. The method of any one of claims 2 to 4, wherein the expression cassette further comprises a promoter capable of initiating expression of the coding sequence in a muscle cell.

6. 6. The method of claim 5, wherein the promoter is a muscle creatine kinase promoter (tMCK).

7. 10. The method of any preceding claim, wherein the mutation is a homozygous mutation.

8. 10. The method of any preceding claim, wherein the stem cells are induced pluripotent stem cells (iPSCs) derived from peripheral blood mononuclear cells or fibroblasts isolated from a patient with muscular dystrophy.

9. 9. The method of claim 8, wherein the muscular dystrophy is selected from the group consisting of limb-girdle muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.

10. The genes are CAPN3, DNAJB6, TNPO3, HNRPDL, CAPN3, COL6A1, COL6A2, COL6A3, DYSF, SGCA, SGCB, SGCG, SGCD, TCAP, TRIM32, FKRP, TTN, POMT1, ANO5, FKTN, POMT2, POMGNT1, DAG1, PLEC1, TRAPPC11, GMPPB, ISPD, POGLU 10. The method of any preceding claim, wherein the polypeptide is selected from the group consisting of T1, COL6A1, COL6A2, COL6A3, LAMA2, POMGNT2, POPDC1, POPDC3, JAG2, PYROXD1, DMD, LAMA-2, DMD, DYSF, DMPK, CNBP, DUX4, PABPN1, EMD, LMNA, SYNE1, SYNE2, FHL1, and TMEM43.

11. The method of claim 10, wherein the gene is CAPN3.

12. 12. The method of claim 11, wherein said at least a portion of said wild-type CAPN3 gene does not include exon 1 or 2, and said locus is downstream of exon 2 of said endogenous CAPN3 gene.

13. 13. The method of claim 12, wherein the at least a portion of the wild-type CAPN3 gene comprises exons 3 to 24, and the locus is 3' to the last nucleotide of exon 2 of the endogenous CAPN3 gene.

14. 10. The method of any preceding claim, wherein the first vector further comprises a second expression cassette encoding a first selectable marker, and the second vector further comprises a third expression cassette encoding a second selectable marker, wherein the first expression cassette and the second expression cassette on the first vector are integrated together into the genome, and the first expression cassette and the third expression cassette on the second vector are integrated together into the genome.

15. 15. The method of claim 14, wherein the second and third expression cassettes are excised from the genome after the integration is confirmed.

16. 10. The method of any preceding claim, wherein the first and second vectors each further comprise a fourth expression cassette encoding a kill switch that does not integrate into the locus.

17. 10. The method of any preceding claim, further comprising culturing the stem cells in a container coated with adhesion molecules prior to differentiation.

18. The method of claim 17, wherein the adhesion molecule is laminin 511 or a fragment thereof.

19. 19. The method of claim 17 or 18, wherein the culturing step is carried out in a medium comprising fibroblast growth factor (FGF), a Rho kinase inhibitor, and N2 medium.

20. 10. The method of any preceding claim, wherein the differentiation is carried out in a medium comprising a Wnt signalling activator.

21. 21. The method of claim 20, wherein the differentiation is then further carried out in a medium comprising a Notch signaling inhibitor.

22. 22. The method of claim 21, wherein the differentiation is then further carried out in a medium comprising a Notch signaling inhibitor and a TGF-β inhibitor.

23. 10. The method of any preceding claim, wherein the satellite cells are identified by an antibody specific for CD271.

24. 24. The method of claim 23, wherein the identified satellite cells are expanded in a medium comprising bFGF2, FGF8b and N2 medium.

25. 10. The method of any preceding claim, further comprising administering the prepared satellite cells to a patient carrying the mutation.

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