Gene Correction of LGMD2A Patient-Specific iPSCs for the Development of Targeted Autologous Cell Therapy.

Limb girdle muscular dystrophy type 2A (LGMD2A), caused by mutations in the Calpain 3 (CAPN3) gene, is an incurable autosomal recessive disorder that results in muscle wasting and loss of ambulation. To test the feasibility of an autologous induced pluripotent stem cell (iPSC)-based therapy for LGMD2A, here we applied CRISPR-Cas9-mediated genome editing to iPSCs from three LGMD2A patients to enable correction of mutations in the CAPN3 gene. Using a gene knockin approach, we genome edited iPSCs carrying three different CAPN3 mutations, and we demonstrated the rescue of CAPN3 protein in myotube derivatives in vitro. Transplantation of gene-corrected LGMD2A myogenic progenitors in a novel mouse model combining immunodeficiency and a lack of CAPN3 resulted in muscle engraftment and rescue of the CAPN3 mRNA. Thus, we provide here proof of concept for the integration of genome editing and iPSC technologies to develop a novel autologous cell therapy for LGMD2A.

Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells.

Some gene polymorphisms can lead to monogenic diseases, whereas other polymorphisms may confer beneficial traits. A well-characterized example is congenital erythrocytosis-the non-pathogenic hyper-production of red blood cells-that is caused by a truncated erythropoietin receptor. Here we show that Cas9-mediated genome editing in CD34+ human haematopoietic stem and progenitor cells (HSPCs) can recreate the truncated form of the erythropoietin receptor, leading to substantial increases in erythropoietic output. We also show that combining the expression of the cDNA of a truncated erythropoietin receptor with a previously reported genome-editing strategy to fully replace the HBA1 gene with an HBB transgene in HSPCs (to restore normal haemoglobin production in cells with a ß-thalassaemia phenotype) gives the edited HSPCs and the healthy red blood cell phenotype a proliferative advantage. Combining knowledge of human genetics with precise genome editing to insert natural human variants into therapeutic cells may facilitate safer and more effective genome-editing therapies for patients with genetic diseases.

In Vitro Maturation of Human Pluripotent Stem Cell-Derived Myotubes.

Pluripotent stem cells have a multitude of potential applications in the areas of disease modeling, drug screening, and cell-based therapies for genetic diseases, including muscular dystrophies. The advent of induced pluripotent stem cell technology allows for the facile derivation of disease-specific pluripotent stem cells for any given patient. Targeted in vitro differentiation of pluripotent stem cells into the muscle lineage is a key step to enable all these applications. Transgene-based differentiation using conditional expression of the transcription factor PAX7 leads to the efficient derivation of an expandable and homogeneous population of myogenic progenitors suitable for both in vitro and in vivo applications. Here, we describe an optimized protocol for the derivation and expansion of myogenic progenitors from pluripotent stem cells using conditional expression of PAX7. Importantly, we further describe an optimized procedure for the terminal differentiation of myogenic progenitors into more mature myotubes, which are better suited for in vitro disease modeling and drug screening studies.

High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition.

Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.

Establishment of Skeletal Myogenic Progenitors from Non-Human Primate Induced Pluripotent Stem Cells.

Pluripotent stem (PS) cells enable the scalable production of tissue-specific derivatives with therapeutic potential for various clinical applications, including muscular dystrophies. Given the similarity to human counterparts, the non-human primate (NHP) is an ideal preclinical model to evaluate several questions, including delivery, biodistribution, and immune response. While the generation of human-induced PS (iPS)-cell-derived myogenic progenitors is well established, there have been no data for NHP counterparts, probably due to the lack of an efficient system to differentiate NHP iPS cells towards the skeletal muscle lineage. Here, we report the generation of three independent Macaca fascicularis iPS cell lines and their myogenic differentiation using PAX7 conditional expression. The whole-transcriptome analysis confirmed the successful sequential induction of mesoderm, paraxial mesoderm, and myogenic lineages. NHP myogenic progenitors efficiently gave rise to myotubes under appropriate in vitro differentiation conditions and engrafted in vivo into the TA muscles of NSG and FKRP-NSG mice. Lastly, we explored the preclinical potential of these NHP myogenic progenitors in a single wild-type NHP recipient, demonstrating engraftment and characterizing the interaction with the host immune response. These studies establish an NHP model system through which iPS-cell-derived myogenic progenitors can be studied.

Double Anti-neutrophil Cytoplasmic Antibody and Anti-glomerular Basement Membrane Antibody-positive Crescentic Glomerulonephritis, Following SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects kidneys mainly in the form of acute kidney injury but rarely can cause glomerular disease. On a very rare occasion, SARS-CoV-2 infection can be associated with anti-neutrophil cytoplasmic antigen (ANCA)-associated vasculitis and anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). We report a case of a 59-year-old man who presented with progressive renal failure 8 weeks after contracting the viral infection, which progressed slowly to severe renal dysfunction. Renal biopsy showed crescentic glomerulonephritis (CrGN) accompanied by interrupted linear IgG deposits along the glomerular basement membrane (GBM) on immunofluorescence (IF) staining with associated mild acute tubular injury. The serology for anti-myeloperoxidase (MPO), as well as anti-GBM antibodies, was positive. He was treated with steroid and pulse intravenous cyclophosphamide, following which there was a significant improvement in the renal function and serological resolution of both the antibodies 6 months post-treatment. To the best of our knowledge, this is the first reported case of "double-antibody" positive CrGN following SARS-CoV-2 infection.

Genomic Safe Harbor Expression of PAX7 for the Generation of Engraftable Myogenic Progenitors.

Inducible expression of PAX7 in differentiating pluripotent stem cells (PSCs) allows massively scalable generation of human myogenic progenitors, which upon transplantation into dystrophic muscles give rise to donor-derived myofibers and satellite cells. Therefore, PSC-derived PAX7+ myogenic progenitors represent an attractive therapeutic approach to promote muscle regeneration. Work to date has used lentiviral vectors (LVs) that randomly integrate inducible PAX7 transgenes. Here, we investigated whether equivalent induction of the myogenic program could be achieved by targeting the PAX7 transgene into genomic safe harbor (GSH) sites. Across multiple PSC lines, we find that this approach consistently generates expandable myogenic progenitors in vitro, although scalability of expansion is moderately reduced compared with the LV approach. Importantly, transplantation of GSH-targeted myogenic progenitors produces robust engraftment, comparable with LV counterparts. These findings provide proof of concept for the use of GSH targeting as a potential alternative approach to generate therapeutic PSC-derived myogenic progenitors for clinical applications.

Defective autophagy and increased apoptosis contribute toward the pathogenesis of FKRP-associated muscular dystrophies.

Fukutin-related protein (FKRP) is a glycosyltransferase involved in glycosylation of alpha-dystroglycan (α-DG). Mutations in FKRP are associated with muscular dystrophies (MD) ranging from limb-girdle LGMDR9 to Walker-Warburg Syndrome (WWS), a severe type of congenital MD. Although hypoglycosylation of α-DG is the main hallmark of this group of diseases, a full understanding of the underlying pathophysiology is still missing. Here, we investigated molecular mechanisms impaired by FKRP mutations in pluripotent stem (PS) cell-derived myotubes. FKRP-deficient myotubes show transcriptome alterations in genes involved in extracellular matrix receptor interactions, calcium signaling, PI3K-Akt pathway, and lysosomal function. Accordingly, using a panel of patient-specific LGMDR9 and WWS induced PS cell-derived myotubes, we found a significant reduction in the autophagy-lysosome pathway for both disease phenotypes. In addition, we show that WWS myotubes display decreased ERK1/2 activity and increased apoptosis, which were restored in gene edited myotubes. Our results suggest the autophagy-lysosome pathway and apoptosis may contribute to the FKRP-associated MD pathogenesis.

A universal gene correction approach for FKRP-associated dystroglycanopathies to enable autologous cell therapy.

Mutations in the fukutin-related protein (FKRP) gene result in a broad spectrum of muscular dystrophy (MD) phenotypes, including the severe Walker-Warburg syndrome (WWS). Here, we develop a gene-editing approach that replaces the entire mutant open reading frame with the wild-type sequence to universally correct all FKRP mutations. We apply this approach to correct FKRP mutations in induced pluripotent stem (iPS) cells derived from patients displaying broad clinical severity. Our findings show rescue of functional α-dystroglycan (α-DG) glycosylation in gene-edited WWS iPS cell-derived myotubes. Transplantation of gene-corrected myogenic progenitors in the FKRPP448L-NSG mouse model gives rise to myofiber and satellite cell engraftment and, importantly, restoration of α-DG functional glycosylation in vivo. These findings suggest the potential feasibility of using CRISPR-Cas9 technology in combination with patient-specific iPS cells for the future development of autologous cell transplantation for FKRP-associated MDs.

NAD+ enhances ribitol and ribose rescue of α-dystroglycan functional glycosylation in human FKRP-mutant myotubes.

Mutations in the fukutin-related protein (FKRP) cause Walker-Warburg syndrome (WWS), a severe form of congenital muscular dystrophy. Here, we established a WWS human induced pluripotent stem cell-derived myogenic model that recapitulates hallmarks of WWS pathology. We used this model to investigate the therapeutic effect of metabolites of the pentose phosphate pathway in human WWS. We show that functional recovery of WWS myotubes is promoted not only by ribitol but also by its precursor ribose. Moreover, we found that the combination of each of these metabolites with NAD+ results in a synergistic effect, as demonstrated by rescue of α-dystroglycan glycosylation and laminin binding capacity. Mechanistically, we found that FKRP residual enzymatic capacity, characteristic of many recessive FKRP mutations, is required for rescue as supported by functional and structural mutational analyses. These findings provide the rationale for testing ribose/ribitol in combination with NAD+ to treat WWS and other diseases associated with FKRP mutations.

Healthy muscles are complex machines that require a myriad of finely tuned molecules to work properly. For instance, a protein called alpha-DG sits at the surface of healthy muscle cells, where it strengthens the tissue by latching onto other proteins in the environment. To perform its role correctly, it first needs to be coated with sugar molecules, a complex process which requires over 20 proteins, including the enzyme FKRP. Faulty forms of FKRP reduce the number of sugars added to alpha-DG, causing the muscle tissue to weaken and waste away, potentially leading to severe forms of diseases known as muscular dystrophies. Drugs that can restore alpha-DG sugar molecules could help to treat these conditions. Previous studies on mice and fish have highlighted two potential candidates, known as ribitol and NAD+, which can help to compensate for reduced FKRP activity and allow sugars to be added to alpha-DG again. Yet no model is available to test these molecules on actual human muscle cells. Here, Ortiz-Cordero et al. developed such a model in the laboratory by growing muscle cells from naïve, undifferentiated cells generated from skin given by a muscular dystrophy patient with a faulty form of FKRP. The resulting muscle fibers are in essence identical to the ones present in the individual. As such, they can help to understand the effect various drugs have on muscular dystrophies. The cells were then put in contact with either NAD+, ribitol, or a precursor of ribitol known as ribose. Ortiz-Cordero et al. found that ribitol and ribose restored the ability of FKRP to add sugars to alpha-DG, reducing muscle damage. Combining NAD+ with ribitol or ribose had an even a bigger impact, further increasing the number of sugars on alpha-DG. The human muscle cell model developed by Ortiz-Cordero et al. could help to identify new compounds that can treat muscular conditions. In particular, the findings point towards NAD+, ribose and ribitol as candidates for treating FKRP-related muscular dystrophies. Further safety studies are now needed to evaluate whether these compounds could be used in patients.

Efficient engraftment of pluripotent stem cell-derived myogenic progenitors in a novel immunodeficient mouse model of limb girdle muscular dystrophy 2I.

BACKGROUND: Defects in α-dystroglycan (DG) glycosylation characterize a group of muscular dystrophies known as dystroglycanopathies. One of the key effectors in the α-DG glycosylation pathway is the glycosyltransferase fukutin-related protein (FKRP). Mutations in FKRP lead to a large spectrum of muscular dystrophies, including limb girdle muscular dystrophy 2I (LGMD2I). It remains unknown whether stem cell transplantation can promote muscle regeneration and ameliorate the muscle wasting phenotype associated with FKRP mutations. RESULTS: Here we transplanted murine and human pluripotent stem cell-derived myogenic progenitors into a novel immunodeficient FKRP-mutant mouse model by intra-muscular injection. Upon both mouse and human cell transplantation, we observe the presence of donor-derived myofibers even in absence of pre-injury, and the rescue of α-DG functional glycosylation, as shown by IIH6 immunoreactivity. The presence of donor-derived cells expressing Pax7 under the basal lamina is indicative of satellite cell engraftment, and therefore, long-term repopulation potential. Functional assays performed in the mouse-to-mouse cohort revealed enhanced specific force in transplanted muscles compared to PBS-injected controls. CONCLUSIONS: Altogether, our data demonstrate for the first time the suitability of a cell-based therapeutic approach to improve the muscle phenotype of dystrophic FKRP-mutant mice.

Pluripotent Stem Cell-Based Therapeutics for Muscular Dystrophies.

Pluripotent stem cells (PSCs) represent an attractive cell source for treating muscular dystrophies (MDs) since they easily allow for the generation of large numbers of highly regenerative myogenic progenitors. Using reprogramming technology, patient-specific PSCs have been derived for several types of MDs, and genome editing has allowed correction of mutations, opening the opportunity for their therapeutic application in an autologous transplantation setting. However, there has been limited progress on preclinical studies that validate the therapeutic potential of these gene corrected PSC-derived myogenic progenitors. In this review, we highlight the major research advances, challenges, and future prospects towards the development of PSC-based therapeutics for MDs.

Transplantation studies reveal internuclear transfer of toxic RNA in engrafted muscles of myotonic dystrophy 1 mice.

BACKGROUND: Stem cell transplantation represents a potential therapeutic option for muscular dystrophies (MD). However, to date, most reports have utilized mouse models for recessive types of MD. Here we performed studies to determine whether myotonic dystrophy 1 (DM1), an autosomal dominant type of MD, could benefit from cell transplantation. METHODS: We injected human pluripotent stem (PS) cell-derived myogenic progenitors into the muscles of a novel mouse model combining immunodeficiency and skeletal muscle pathology of DM1 and investigated transplanted mice for engraftment as well as for the presence of RNA foci and alternative splicing pattern. FINDINGS: Engraftment was clearly observed in recipient mice, but unexpectedly, we detected RNA foci in donor-derived engrafted myonuclei. These foci proved to be pathogenic as we observed MBNL1 sequestration and abnormal alternative splicing in donor-derived transcripts. INTERPRETATION: It has been assumed that toxic CUG repeat-containing RNA forms foci in situ in the nucleus in which it is expressed, but these data suggest that CUG repeat-containing RNA may also exit the nucleus and traffic to other nuclei in the syncytial myofiber, where it can exert pathological effects. FUND: This project was supported by funds from the LaBonte/Shawn family and NIH grants R01 AR055299 and AR071439 (R.C.R.P.). R.M-G. was funded by CONACyT-Mexico (#394378).

Screening identifies small molecules that enhance the maturation of human pluripotent stem cell-derived myotubes.

Targeted differentiation of pluripotent stem (PS) cells into myotubes enables in vitro disease modeling of skeletal muscle diseases. Although various protocols achieve myogenic differentiation in vitro, resulting myotubes typically display an embryonic identity. This is a major hurdle for accurately recapitulating disease phenotypes in vitro, as disease commonly manifests at later stages of development. To address this problem, we identified four factors from a small molecule screen whose combinatorial treatment resulted in myotubes with enhanced maturation, as shown by the expression profile of myosin heavy chain isoforms, as well as the upregulation of genes related with muscle contractile function. These molecular changes were confirmed by global chromatin accessibility and transcriptome studies. Importantly, we also observed this maturation in three-dimensional muscle constructs, which displayed improved in vitro contractile force generation in response to electrical stimulus. Thus, we established a model for in vitro muscle maturation from PS cells.

PAX7 Targets, CD54, Integrin α9ß1, and SDC2, Allow Isolation of Human ESC/iPSC-Derived Myogenic Progenitors.

Pluripotent stem (PS)-cell-derived cell types hold promise for treating degenerative diseases. However, PS cell differentiation is intrinsically heterogeneous; therefore, clinical translation requires the development of practical methods for isolating progenitors from unwanted and potentially teratogenic cells. Muscle-regenerating progenitors can be derived through transient PAX7 expression. To better understand the biology, and to discover potential markers for these cells, here we investigate PAX7 genomic targets and transcriptional changes in human cells undergoing PAX7-mediated myogenic commitment. We identify CD54, integrin α9ß1, and Syndecan2 (SDC2) as surface markers on PAX7-induced myogenic progenitors. We show that these markers allow for the isolation of myogenic progenitors using both fluorescent- and CGMP-compatible magnetic-based sorting technologies and that CD54+α9ß1+SDC2+ cells contribute to long-term muscle regeneration in vivo. These findings represent a critical step toward enabling the translation of PS-cell-based therapies for muscle diseases.