Cas9-induced single cut enables highly efficient and template-free repair of a muscular dystrophy causing founder mutation.

With thousands of patients worldwide, CAPN3 c.550delA is the most frequent mutation causing severe, progressive, and untreatable limb girdle muscular dystrophy. We aimed to genetically correct this founder mutation in primary human muscle stem cells. We designed editing strategies providing CRISPR-Cas9 as plasmid and mRNA first in patient-derived induced pluripotent stem cells and applied this strategy then in primary human muscle stem cells from patients. Mutation-specific targeting yielded highly efficient and precise correction of CAPN3 c.550delA to wild type for both cell types. Most likely a single cut generated by SpCas9 resulted in a 5' staggered overhang of one base pair, which triggered an overhang-dependent base replication of an A:T at the mutation site. This recovered the open reading frame and the CAPN3 DNA sequence was repaired template-free to wild type, which led to CAPN3 mRNA and protein expression. Off-target analysis using amplicon sequencing of 43 in silico predicted sites demonstrates the safety of this approach. Our study extends previous usage of single cut DNA modification since our gene product has been repaired into the wild-type CAPN3 sequence with the perspective of a real cure.

Disintegration of the NuRD Complex in Primary Human Muscle Stem Cells in Critical Illness Myopathy.

Critical illness myopathy (CIM) is an acquired, devastating, multifactorial muscle-wasting disease with incomplete recovery. The impact on hospital costs and permanent loss of quality of life is enormous. Incomplete recovery might imply that the function of muscle stem cells (MuSC) is impaired. We tested whether epigenetic alterations could be in part responsible. We characterized human muscle stem cells (MuSC) isolated from early CIM and analyzed epigenetic alterations (CIM n = 15, controls n = 21) by RNA-Seq, immunofluorescence, analysis of DNA repair, and ATAC-Seq. CIM-MuSC were transplanted into immunodeficient NOG mice to assess their regenerative potential. CIM-MuSC exhibited significant growth deficits, reduced ability to differentiate into myotubes, and impaired DNA repair. The chromatin structure was damaged, as characterized by alterations in mRNA of histone 1, depletion or dislocation of core proteins of nucleosome remodeling and deacetylase complex, and loosening of multiple nucleosome-spanning sites. Functionally, CIM-MuSC had a defect in building new muscle fibers. Further, MuSC obtained from the electrically stimulated muscle of CIM patients was very similar to control MuSC, indicating the impact of muscle contraction in the onset of CIM. CIM not only affects working skeletal muscle but has a lasting and severe epigenetic impact on MuSC.

LMNA Co-Regulated Gene Expression as a Suitable Readout after Precise Gene Correction.

LMNA-related muscular dystrophy is an autosomal-dominant progressive disorder caused by mutations in LMNA. LMNA missense mutations are becoming correctable with CRISPR/Cas9-derived tools. Evaluating the functional recovery of LMNA after gene editing bears challenges as there is no reported direct loss of function of lamin A/C proteins in patient-derived cells. The proteins encoded by LMNA are lamins A/C, important ubiquitous nuclear envelope proteins but absent in pluripotent stem cells. We induced lamin A/C expression in induced pluripotent stem cells (iPSCs) of two patients with LMNA-related muscular dystrophy, NM_170707.4 (LMNA): c.1366A > G, p.(Asn456Asp) and c.1494G > T, p.(Trp498Cys), using a short three-day, serum-induced differentiation protocol and analyzed expression profiles of co-regulated genes, examples being COL1A2 and S100A6. We then performed precise gene editing of LMNA c.1366A > G using the near-PAMless (PAM: protospacer-adjacent motif) cytosine base editor. We show that the mutation can be repaired to 100% efficiency in individual iPSC clones. The fast differentiation protocol provided a functional readout and demonstrated increased lamin A/C expression as well as normalized expression of co-regulated genes. Collectively, our findings demonstrate the power of CRISPR/Cas9-mediated gene correction and effective outcome measures in a disease with, so far, little perspective on therapies.

Generation of hiPSC-Derived Skeletal Muscle Cells: Exploiting the Potential of Skeletal Muscle-Derived hiPSCs.

Cell therapies for muscle wasting disorders are on the verge of becoming a realistic clinical perspective. Muscle precursor cells derived from human induced pluripotent stem cells (hiPSCs) represent the key to unrestricted cell numbers indispensable for the treatment of generalized muscle wasting such as cachexia or intensive care unit (ICU)-acquired weakness. We asked how the cell of origin influences efficacy and molecular properties of hiPSC-derived muscle progenitor cells. We generated hiPSCs from primary muscle stem cells and from peripheral blood mononuclear cells (PBMCs) of the same donors (n = 4) and compared their molecular profiles, myogenic differentiation potential, and ability to generate new muscle fibers in vivo. We show that reprogramming into hiPSCs from primary muscle stem cells was faster and 35 times more efficient than from blood cells. Global transcriptome comparison revealed significant differences, but differentiation into induced myogenic cells using a directed transgene-free approach could be achieved with muscle- and PBMC-derived hiPSCs, and both cell types generated new muscle fibers in vivo. Differences in myogenic differentiation efficiency were identified with hiPSCs generated from individual donors. The generation of muscle-stem-cell-derived hiPSCs is a fast and economic method to obtain unrestricted cell numbers for cell-based therapies in muscle wasting disorders, and in this aspect are superior to blood-derived hiPSCs.

mRNA-mediated delivery of gene editing tools to human primary muscle stem cells.

Muscular dystrophies are approximately 50 devastating, untreatable monogenic diseases leading to progressive muscle degeneration and atrophy. Gene correction of transplantable cells using CRISPR/Cas9-based tools is a realistic scenario for autologous cell replacement therapies to restore organ function in many genetic disorders. However, muscle stem cells have so far lagged behind due to the absence of methods to isolate and propagate them and their susceptibility to extensive ex vivo manipulations. Here, we show that mRNA-based delivery of SpCas9 and an adenine base editor results in up to >90% efficient genome editing in human muscle stem cells from many donors regardless of age and gender and without any enrichment step. Using NCAM1 as an endogenous reporter locus expressed by all muscle stem cells and whose knockout does not affect cell fitness, we show that cells edited with mRNA fully retain their myogenic marker signature, proliferation capacity, and functional attributes. Moreover, mRNA-based delivery of a base editor led to the highly efficient repair of a muscular dystrophy-causing SGCA mutation in a single selection-free step. In summary, our work establishes mRNA-mediated delivery of CRISPR/Cas9-based tools as a promising and universal approach for taking gene edited muscle stem cells into clinical application to treat muscle disease.

Base editing repairs an SGCA mutation in human primary muscle stem cells.

Skeletal muscle can regenerate from muscle stem cells and their myogenic precursor cell progeny, myoblasts. However, precise gene editing in human muscle stem cells for autologous cell replacement therapies of untreatable genetic muscle diseases has not yet been reported. Loss-of-function mutations in SGCA, encoding α-sarcoglycan, cause limb-girdle muscular dystrophy 2D/R3, an early-onset, severe, and rapidly progressive form of muscular dystrophy affecting both male and female patients. Patients suffer from muscle degeneration and atrophy affecting the limbs, respiratory muscles, and heart. We isolated human muscle stem cells from 2 donors, with the common SGCA c.157G>A mutation affecting the last coding nucleotide of exon 2. We found that c.157G>A is an exonic splicing mutation that induces skipping of 2 coregulated exons. Using adenine base editing, we corrected the mutation in the cells from both donors with > 90% efficiency, thereby rescuing the splicing defect and α-sarcoglycan expression. Base-edited patient cells regenerated muscle and contributed to the Pax7+ satellite cell compartment in vivo in mouse xenografts. Here, we provide the first evidence to our knowledge that autologous gene-repaired human muscle stem cells can be harnessed for cell replacement therapies of muscular dystrophies.

Generation of two human induced pluripotent stem cell lines derived from myoblasts (MDCi014-A) and from peripheral blood mononuclear cells (MDCi014-B) from the same donor.

We describe the generation and characterization of two human induced pluripotent stem cell (hiPSCs) lines reprogrammed from myoblasts and from peripheral blood mononuclear cells (PBMCs) from the same donor. The donor was free of neuromuscular disorders, male and 18 years of age. For reprogramming we used Sendai-virus delivery of the four Yamanaka factors. The pluripotent identity of the hiPSC lines was confirmed by the expression of pluripotency markers and their capacity to differentiate into all three germ layers. These hiPSCs constitute a tool to study tissue of origin specific differences in the identity of hiPSCs.

Generation of three age and gender matched pairs of human induced pluripotent stem cells derived from myoblasts (MDCi011-A, MDCi012-A, MDCi013-A) and from peripheral blood mononuclear cells (MDCi011-B, MDCi012-B, MDCi013-B) from the same donor.

We describe the generation and characterization of three pairs of human induced pluripotent stem cell (hiPSC) lines reprogrammed from myoblasts and from peripheral blood mononuclear cells (PBMCs) of the same donor. All donors were free of neuromuscular disorders, female and between 47 and 50 years of age. For reprogramming we used Sendai-virus delivery of the four Yamanaka factors. The pluripotent identity of the hiPSC lines was confirmed by the expression of pluripotency markers and their capacity to differentiate into all three germ layers. These hiPSCs constitute a tool to study tissue of origin specific differences in the identity of hiPSCs.

Human muscle-derived CLEC14A-positive cells regenerate muscle independent of PAX7.

Skeletal muscle stem cells, called satellite cells and defined by the transcription factor PAX7, are responsible for postnatal muscle growth, homeostasis and regeneration. Attempts to utilize the regenerative potential of muscle stem cells for therapeutic purposes so far failed. We previously established the existence of human PAX7-positive cell colonies with high regenerative potential. We now identified PAX7-negative human muscle-derived cell colonies also positive for the myogenic markers desmin and MYF5. These include cells from a patient with a homozygous PAX7 c.86-1G > A mutation (PAX7null). Single cell and bulk transcriptome analysis show high intra- and inter-donor heterogeneity and reveal the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7-negative cell populations, including PAX7null, form myofibers after transplantation into mice, and regenerate muscle after reinjury. Transplanted PAX7neg cells repopulate the satellite cell niche where they re-express PAX7, or, strikingly, CLEC14A. In conclusion, transplanted human cells do not depend on PAX7 for muscle regeneration.

Exon Skipping in a Dysf-Missense Mutant Mouse Model.

Limb girdle muscular dystrophy 2B (LGMD2B) is without treatment and caused by mutations in the dysferlin gene (DYSF). One-third is missense mutations leading to dysferlin aggregation and amyloid formation, in addition to defects in sarcolemmal repair and progressive muscle wasting. Dysferlin-null mouse models do not allow study of the consequences of missense mutations. We generated a new mouse model (MMex38) carrying a missense mutation in exon 38 in analogy to a clinically relevant human DYSF variant (DYSF p.Leu1341Pro). The targeted mutation induces all characteristics of missense mutant dysferlinopathy, including a progressive dystrophic pattern, amyloid formation, and defects in membrane repair. We chose U7 small nuclear RNA (snRNA)-based splice switching to demonstrate a possible exon-skipping strategy in this new animal model. We show that Dysf exons 37 and 38 can successfully be skipped in vivo. Overall, the MMex38 mouse model provides an ideal tool for preclinical development of treatment strategies for dysferlinopathy.

Localized irradiation of mouse legs using an image-guided robotic linear accelerator.

BACKGROUND: To investigate the potential of human satellite cells in muscle regeneration small animal models are useful to evaluate muscle regeneration. To suppress the inherent regeneration ability of the tibialis muscle of mice before transplantation of human muscle fibers, a localized irradiation of the mouse leg should be conducted. We analyzed the feasibility of an image-guided robotic irradiation procedure, a routine treatment method in radiation oncology, for the focal irradiation of mouse legs. METHODS: After conducting a planning computed tomography (CT) scan of one mouse in its customized mold a three-dimensional dose plan was calculated using a dedicated planning workstation. 18 Gy have been applied to the right anterior tibial muscle of 4 healthy and 12 mice with immune defect in general anesthesia using an image-guided robotic linear accelerator (LINAC). The mice were fixed in a customized acrylic mold with attached fiducial markers for image guided tracking. RESULTS: All 16 mice could be irradiated as prevised without signs of acute radiation toxicity or anesthesiological side effects. The animals survived until scarification after 8, 21 and 49 days as planned. The procedure was straight forward and the irradiation process took 5 minutes to apply the dose of 18 Gy. CONCLUSIONS: Localized irradiation of mice legs using a robotic LINAC could be conducted as planned. It is a feasible procedure without recognizable side effects. Image guidance offers precise dose delivery and preserves adjacent body parts and tissues.

Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle.

Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysf(prmd) Prkdc(scid)/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy.

Human satellite cells have regenerative capacity and are genetically manipulable.

Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7+ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7+ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon-mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7+ satellite cells have potential to restore gene function in muscular dystrophies.

Detección del cannabinoide sintético JWH-210 en la Comunidad Valenciana / Detection of the synthetic cannabinoid JWH-210 in the Valencia region

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