bims-muscge Biomed News
on Muscle stem cells and gene therapy
Issue of 2023–09–03
fourteen papers selected by
Chance Bowman, Dartmouth College
  1. An overview on androgen-mediated actions in skeletal muscle and adipose tissue.
  2. Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 System: Factors Affecting Precision Gene Editing Efficiency and Optimization Strategies.
  3. Disease modeling and gene correction of LGMDR21 iPSCs elucidates the role of POGLUT1 in skeletal muscle maintenance, regeneration, and the satellite cell niche.
  4. Ageing of skeletal muscle extracellular matrix and mitochondria: finding a potential link.
  5. Targeting Duchenne muscular dystrophy by skipping DMD exon 45 with base editors.
  6. Nanoparticles systemically biodistribute to regenerating skeletal muscle in DMD.
  7. Therapeutic approaches for Duchenne muscular dystrophy.
  8. Stimuli-Responsive Non-viral Nanoparticles for Gene Delivery.
  9. Transcriptional Profiling of Skeletal Muscle Stem Cells After In Vivo Engraftment into a Heterologous Niche Environment.
  10. Chromatographic Purification of Viral Vectors for Gene Therapy Applications.
  11. Programmable multi-kilobase RNA editing using CRISPR-mediated trans-splicing.
  12. A viable alternative for editor delivery.
  13. Long-term muscle-specific overexpression of DOK7 in mice using AAV9-tMCK-DOK7.
  14. Gpr18 agonist dampens inflammation, enhances myogenesis, and restores muscle function in models of Duchenne muscular dystrophy.
  1. Steroids. 2023 Aug 25. pii: S0039-128X(23)00134-4. [Epub ahead of print] 109306
    An overview on androgen-mediated actions in skeletal muscle and adipose tissue.
    Joe Rizk, Rajesh Sahu, Delphine Duteil.
      Androgens are a class of steroid hormones primarily associated with male sexual development and physiology, but exert pleiotropic effects in either sex. They have a crucial role in various physiological processes, including the regulation of skeletal muscle and adipose tissue homeostasis. The effects of androgens are mainly mediated through the androgen receptor (AR), a ligand-activated nuclear receptor expressed in both tissues. In skeletal muscle, androgens via AR exert a multitude of effects, ranging from increased muscle mass and strength, to the regulation of muscle fiber type composition, contraction and metabolic functions. In adipose tissue, androgens influence several processes including proliferation, fat distribution, and metabolism but they display depot-specific and organism-specific effects which differ in certain context. This review further explores the potential mechanisms underlying androgen-AR signaling in skeletal muscle and adipose tissue. Understanding the roles of androgens and their receptor in skeletal muscle and adipose tissue is essential for elucidating their contributions to physiological processes, disease conditions, and potential therapeutic interventions.
    Keywords:  Androgen receptor; adipose tissue; androgen signaling; pre-adipogenic progenitor; satellite cell; skeletal muscle
    DOI:  https://doi.org/10.1016/j.steroids.2023.109306
  2. Hum Gene Ther. 2023 Aug 29.
    Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 System: Factors Affecting Precision Gene Editing Efficiency and Optimization Strategies.
    Jiawen Li, Chuxi Tang, Guozheng Liang, Huiqun Tian, Guanxi Lai, Yixiang Wu, Shiwen Liu, Wenfeng Zhang, Song Liu, Hongwei Shao.
      The clustered regularly interspaced short palindromic repeat (CRISPR)- CRISPR-associated (Cas) system is a powerful genomic DNA editing tool. The increased applications of gene editing tools, including the CRISPR-Cas system, have contributed to recent advances in biological fields, such as genetic disease therapy, disease-associated gene screening and detection, and cancer therapy. However, the major limiting factor for the wide application of gene editing tools is gene editing efficiency. This review summarizes the recent advances in factors affecting the gene editing efficiency of the CRISPR-Cas9 system and the CRISPR-Cas9 system optimization strategies. The homology-directed repair efficiency-related signal pathways and the form and delivery method of the CRISPR-Cas9 system are the major factors that influence the repair efficiency of gene editing tools. Based on these influencing factors, several strategies have been developed to improve the repair efficiency of gene editing tools. This review provided novel insights for improving the repair efficiency of the CRISPR-Cas9 gene editing system, which may enable the development and improvement of gene editing tools.
    DOI:  https://doi.org/10.1089/hum.2023.115
  3. Mol Ther Nucleic Acids. 2023 Sep 12. 33 683-697
    Disease modeling and gene correction of LGMDR21 iPSCs elucidates the role of POGLUT1 in skeletal muscle maintenance, regeneration, and the satellite cell niche.
    Jose L Ortiz-Vitali, Jianbo Wu, Nasa Xu, Annie W Shieh, Nima Niknejad, Megumi Takeuchi, Carmen Paradas, Chunru Lin, Hamed Jafar-Nejad, Robert S Haltiwanger, Sidney H Wang, Radbod Darabi.
      Autosomal recessive limb-girdle muscular dystrophy 21 (LGMDR21) is caused by pathogenic variants in protein O-glucosyltransferase 1 (POGLUT1), which is responsible for O-glucosylation of specific epidermal growth factor (EGF) repeats found in ∼50 mammalian proteins, including Notch receptors. Previous data from patient biopsies indicated that impaired Notch signaling, reduction of muscle stem cells, and accelerated differentiation are probably involved in disease etiopathology. Using patient induced pluripotent stem cells (iPSCs), their corrected isotypes, and control iPSCs, gene expression profiling indicated dysregulation of POGLUT1, NOTCH, muscle development, extracellular matrix (ECM), cell adhesion, and migration as involved pathways. They also exhibited reduced in vitro POGLUT1 enzymatic activity and NOTCH signaling as well as defective myogenesis, proliferation, migration and differentiation. Furthermore, in vivo studies demonstrated significant reductions in engraftment, muscle stem cell formation, PAX7 expression, and maintenance, along with an increased percentage of mislocalized PAX7+ cells in the interstitial space. Gene correction in patient iPSCs using CRISPR-Cas9 nickase led to the rescue of the main in vitro and in vivo phenotypes. These results demonstrate the efficacy of iPSCs and gene correction in disease modeling and rescue of the phenotypes and provide evidence of the involvement of muscle stem cell niche localization, PAX7 expression, and cell migration as possible mechanisms in LGMDR21.
    Keywords:  CRISPR-Cas9; LGMDR21; MT: RNA/DNA Editing; POGLUT1; gene correction; iPSCs; muscle stem cells; skeletal muscle
    DOI:  https://doi.org/10.1016/j.omtn.2023.07.037
  4. Ann Med. 2023 ;55(2): 2240707
    Ageing of skeletal muscle extracellular matrix and mitochondria: finding a potential link.
    Lubing Cai, Luze Shi, Zhen Peng, Yaying Sun, Jiwu Chen.
      Aim: To discuss the progress of extracellular matrix (ECM) characteristics, mitochondrial homeostasis, and their potential crosstalk in the pathogenesis of sarcopenia, a geriatric syndrome characterized by a generalized and progressive reduction in muscle mass, strength, and physical performance.Methods: This review focuses on the anatomy and physiology of skeletal muscle, alterations of ECM and mitochondria during ageing, and the role of the interplay between ECM and mitochondria in the pathogenesis of sarcopenia.Results: Emerging evidence points to a clear interplay between mitochondria and ECM in various tissues and organs. Under the ageing process, the ECM undergoes changes in composition and physical properties that may mediate mitochondrial changes via the systematic metabolism, ROS, SPARC pathway, and AMPK/PGC-1α signalling, which in turn exacerbate muscle degeneration. However, the precise effects of such crosstalk on the pathobiology of ageing, particularly in skeletal muscle, have not yet been fully understood.Conclusion: The changes in skeletal muscle ECM and mitochondria are partially responsible for the worsened muscle function during the ageing process. A deeper understanding of their alterations and interactions in sarcopenic patients can help prevent sarcopenia and improve its prognoses.
    Keywords:  Ageing; extracellular matrix; mitochondria; reactive oxygen species; sarcopenia
    DOI:  https://doi.org/10.1080/07853890.2023.2240707
  5. Mol Ther Nucleic Acids. 2023 Sep 12. 33 572-586
    Targeting Duchenne muscular dystrophy by skipping DMD exon 45 with base editors.
    Michael Gapinske, Jackson Winter, Devyani Swami, Lauren Gapinske, Wendy S Woods, Shraddha Shirguppe, Angelo Miskalis, Anna Busza, Dana Joulani, Collin J Kao, Kurt Kostan, Anne Bigot, Rashid Bashir, Pablo Perez-Pinera.
      Duchenne muscular dystrophy is an X-linked monogenic disease caused by mutations in the dystrophin gene (DMD) characterized by progressive muscle weakness, leading to loss of ambulation and decreased life expectancy. Since the current standard of care for Duchenne muscular dystrophy is to merely treat symptoms, there is a dire need for treatment modalities that can correct the underlying genetic mutations. While several gene replacement therapies are being explored in clinical trials, one emerging approach that can directly correct mutations in genomic DNA is base editing. We have recently developed CRISPR-SKIP, a base editing strategy to induce permanent exon skipping by introducing C > T or A > G mutations at splice acceptors in genomic DNA, which can be used therapeutically to recover dystrophin expression when a genomic deletion leads to an out-of-frame DMD transcript. We now demonstrate that CRISPR-SKIP can be adapted to correct some forms of Duchenne muscular dystrophy by disrupting the splice acceptor in human DMD exon 45 with high efficiency, which enables open reading frame recovery and restoration of dystrophin expression. We also demonstrate that AAV-delivered split-intein base editors edit the splice acceptor of DMD exon 45 in cultured human cells and in vivo, highlighting the therapeutic potential of this strategy.
    Keywords:  AAV; CRISPR-Cas9; Duchenne muscular dystrophy; MT: RNA/DNA editing; adenine base editing; cytidine base editing; exon skipping; gene editing
    DOI:  https://doi.org/10.1016/j.omtn.2023.07.029
  6. J Nanobiotechnology. 2023 Aug 29. 21(1): 303
    Nanoparticles systemically biodistribute to regenerating skeletal muscle in DMD.
    Michael R Hicks, Xiangsheng Liu, Courtney S Young, Kholoud Saleh, Ying Ji, Jinhong Jiang, Michael R Emami, Ekaterina Mokhonova, Melissa J Spencer, Huan Meng, April D Pyle.
      Skeletal muscle disease severity can often progress asymmetrically across muscle groups and heterogeneously within tissues. An example is Duchenne Muscular Dystrophy (DMD) in which lack of dystrophin results in devastating skeletal muscle wasting in some muscles whereas others are spared or undergo hypertrophy. An efficient, non-invasive approach to identify sites of asymmetry and degenerative lesions could enable better patient monitoring and therapeutic targeting of disease. In this study, we utilized a versatile intravenously injectable mesoporous silica nanoparticle (MSNP) based nanocarrier system to explore mechanisms of biodistribution in skeletal muscle of mdx mouse models of DMD including wildtype, dystrophic, and severely dystrophic mice. Moreover, MSNPs could be imaged in live mice and whole muscle tissues enabling investigation of how biodistribution is altered by different types of muscle pathology such as inflammation or fibrosis. We found MSNPs were tenfold more likely to aggregate within select mdx muscles relative to wild type, such as gastrocnemius and quadriceps. This was accompanied by decreased biodistribution in off-target organs. We found the greatest factor affecting preferential delivery was the regenerative state of the dystrophic skeletal muscle with the highest MSNP abundance coinciding with the regions showing the highest level of embryonic myosin staining and intramuscular macrophage uptake. To demonstrate, muscle regeneration regulated MSNP distribution, we experimentally induced regeneration using barium chloride which resulted in a threefold increase of intravenously injected MSNPs to sites of regeneration 7 days after injury. These discoveries provide the first evidence that nanoparticles have selective biodistribution to skeletal muscle in DMD to areas of active regeneration and that nanoparticles could enable diagnostic and selective drug delivery in DMD skeletal muscle.
    DOI:  https://doi.org/10.1186/s12951-023-01994-0
  7. Nat Rev Drug Discov. 2023 Aug 31.
    Therapeutic approaches for Duchenne muscular dystrophy.
    Thomas C Roberts, Matthew J A Wood, Kay E Davies.
      Duchenne muscular dystrophy (DMD) is a monogenic muscle-wasting disorder and a priority candidate for molecular and cellular therapeutics. Although rare, it is the most common inherited myopathy affecting children and so has been the focus of intense research activity. It is caused by mutations that disrupt production of the dystrophin protein, and a plethora of drug development approaches are under way that aim to restore dystrophin function, including exon skipping, stop codon readthrough, gene replacement, cell therapy and gene editing. These efforts have led to the clinical approval of four exon skipping antisense oligonucleotides, one stop codon readthrough drug and one gene therapy product, with other approvals likely soon. Here, we discuss the latest therapeutic strategies that are under development and being deployed to treat DMD. Lessons from these drug development programmes are likely to have a major impact on the DMD field, but also on molecular and cellular medicine more generally. Thus, DMD is a pioneer disease at the forefront of future drug discovery efforts, with these experimental treatments paving the way for therapies using similar mechanisms of action being developed for other genetic diseases.
    DOI:  https://doi.org/10.1038/s41573-023-00775-6
  8. Handb Exp Pharmacol. 2023 Aug 30.
    Stimuli-Responsive Non-viral Nanoparticles for Gene Delivery.
    Liên S Reichel, Anja Traeger.
      Considering nucleic acids as the language of life and the genome as the instruction manual of cells, their targeted modulation promises great opportunities in treating and healing diseases. In addition to viral gene transfer, the overwhelming power of non-viral mRNA-based vaccines is driving the development of novel gene transporters. Thereby, various nucleic acids such as DNA (pDNA) or RNA (mRNA, siRNA, miRNA, gRNA, or ASOs) need to be delivered, requiring a transporter due to their high molar mass and negative charge in contrast to classical agents. This chapter presents the specific biological hurdles for using nucleic acids and shows how new materials can overcome these.
    Keywords:  Gene delivery; Gene therapy; Lipid; Nanomedicine; Non-viral; Polymer; Vaccination
    DOI:  https://doi.org/10.1007/164_2023_694
  9. Curr Protoc. 2023 Aug;3(8): e877
    Transcriptional Profiling of Skeletal Muscle Stem Cells After In Vivo Engraftment into a Heterologous Niche Environment.
    Felicia Lazure, Darren M Blackburn, Vahab D Soleimani.
      Adult stem cells play a critical role in the maintenance and repair of the organs in which they reside. However, their function is highly dependent on the crosstalk with their niche environment that changes during development and in disease states. The niche provides signals to stem cells to activate, proliferate, self-renew, or remain in quiescence. In skeletal muscle, the niche is perturbed in disease contexts such as aging, muscular dystrophies, and cachexia. Therefore, it is important to develop methods that permit the decoupling of niche-mediated from cell-intrinsic changes that occur in muscle stem cells (MuSCs) in development and disease contexts. With the purpose of determining the effect of the niche environment on the MuSC transcriptome, function, or health, we have coupled an allogeneic stem cell transplantation system, meaning the transplantation of MuSCs from a donor mouse into a recipient host mouse, with Switching Mechanism at 5' End of RNA Template (SMART-Seq) to quantify the effects of the niche on the MuSC transcriptome in vivo. Briefly, MuSCs are isolated from a GFP reporter donor mouse (Pax7-nGFP) and transplanted into the irradiated muscles of immunocompromised allogeneic hosts. The MuSCs are re-isolated by fluorescence-activated cell sorting (FACS) after three weeks of inhabiting the heterologous niche, defined as a niche that is different from their originating niche, and sequencing-ready libraries are created. This method allows for the direct comparison of the transcriptome of stem cells before and after transplantation into a host of a different age, disease status, or genetic background. This method can be used to accurately quantify the direct effect of the niche environment on the stem cell gene expression profile and to decouple cell-intrinsic versus niche-mediated alterations in the stem cell transcriptome. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Allogeneic muscle stem cell transplantation.
    Keywords:  SMART-Seq; muscle stem cell; niche environment; transcriptomics; transplantation
    DOI:  https://doi.org/10.1002/cpz1.877
  10. Methods Mol Biol. 2023 ;2699 51-60
    Chromatographic Purification of Viral Vectors for Gene Therapy Applications.
    Aoife Mairéad Kearney.
      Chromatography has been a mainstay in the downstream processing and purification of biopharmaceutical medicines. Until now, this has largely involved the purification of protein products such as recombinant enzymes and monoclonal antibodies using large-scale column chromatography methods. The development of advanced therapeutic medicinal products (ATMP) is heralding in a new era of therapeutics for a range of indications. These new therapeutics use diverse substances ranging from live stem cell preparations to fragments of nucleic acid enclosed in a viral delivery system. With these new technologies come new challenges in their purification. In this chapter, the challenges faced in producing and purifying viral vectors capable of delivering life-altering gene therapy to the patient will be discussed. Current methods of chromatography capable of adaptation to meet these new challenges and advancements that may be needed to increase the purification capabilities for these new products will also be discussed.
    Keywords:  Adeno-associated virus; Adenovirus; Gene therapy; Lentivirus; Viral vector
    DOI:  https://doi.org/10.1007/978-1-0716-3362-5_4
  11. bioRxiv. 2023 Aug 18. pii: 2023.08.18.553620. [Epub ahead of print]
    Programmable multi-kilobase RNA editing using CRISPR-mediated trans-splicing.
    Jacob Borrajo, Kamyab Javanmardi, James Griffin, Susan J St Martin, David Yao, Kaisle Hill, Paul C Blainey, Basem Al-Shayeb.
      Current gene editing approaches in eukaryotic cells are limited to single base edits or small DNA insertions and deletions, and remain encumbered by unintended permanent effects and significant challenges in the delivery of large DNA cargo. Here we describe Splice Editing, a generalizable platform to correct gene transcripts in situ by programmable insertion or replacement of large RNA segments. By combining CRISPR-mediated RNA targeting with endogenous cellular RNA-splicing machinery, Splice Editing enables efficient, precise, and programmable large-scale editing of gene targets without DNA cleavage or mutagenesis. RNA sequencing and measurement of spliced protein products confirm that Splice Editing achieves efficient and specific targeted RNA and protein correction. We show that Splice Editors based on novel miniature RNA-targeting CRISPR-Cas systems discovered and characterized in this work can be packaged for effective delivery to human cells and affect different types of edits across multiple targets and cell lines. By editing thousands of bases simultaneously in a single reversible step, Splice Editing could expand the treatable disease population for monogenic diseases with large allelic diversity without the permanent unintended effects of DNA editing.
    One-sentence summary: CRISPR-guided trans-splicing enables efficient and specific recombination of large RNA molecules in mammalian cells, with broad applications in therapeutic development for genetic diseases and as a research tool for the study of basic RNA biology.
    DOI:  https://doi.org/10.1101/2023.08.18.553620
  12. Blood. 2023 Aug 31. 142(9): 755-756
    A viable alternative for editor delivery.
    Feyisayo Eweje, Daniel E Bauer.
      
    DOI:  https://doi.org/10.1182/blood.2023021309
  13. Mol Ther Nucleic Acids. 2023 Sep 12. 33 617-628
    Long-term muscle-specific overexpression of DOK7 in mice using AAV9-tMCK-DOK7.
    Yu-Ting Huang, Hannah R Crick, Helena Chaytow, Dinja van der Hoorn, Abrar Alhindi, Ross A Jones, Ralph D Hector, Stuart R Cobb, Thomas H Gillingwater.
      Neuromuscular junction (NMJ) dysfunction underlies several diseases, including congenital myasthenic syndromes (CMSs) and motor neuron disease (MND). Molecular pathways governing NMJ stability are therefore of interest from both biological and therapeutic perspectives. Muscle-specific kinase (MuSK) is necessary for the formation and maintenance of post-synaptic elements of the NMJ, and downstream of tyrosine kinases 7 (DOK7) is crucial for activation of the MuSK pathway. Overexpression of DOK7 using AAV9 has been shown to ameliorate neuromuscular pathology in pre-clinical disease models of CMS and MND. However, long-term consequences of DOK7 expression have been sparsely investigated and targeted overexpression of DOK7 in skeletal muscle yet to be established. Here, we developed and characterized a novel AAV9-DOK7 facilitating forced expression of DOK7 under a skeletal muscle-specific promoter. AAV9-tMCK-DOK7 was systemically delivered to newborn mice that were monitored over 6 months. DOK7 overexpression was restricted to skeletal muscles. Body weight, blood biochemistry, and histopathological assessments were unaffected by AAV9-tMCK-DOK7 treatment. In contrast, forced expression of DOK7 resulted in enlargement of both the pre- and post-synaptic components of the NMJ, without causing denervation. We conclude that muscle-specific DOK7 overexpression can be achieved in a safe manner, with the capacity to target NMJs in vivo.
    Keywords:  AAV9; DOK7; MT: Delivery strategies; NMJ; gene therapy; mouse; neuromuscular junction
    DOI:  https://doi.org/10.1016/j.omtn.2023.07.036
  14. Front Cell Dev Biol. 2023 ;11 1187253
    Gpr18 agonist dampens inflammation, enhances myogenesis, and restores muscle function in models of Duchenne muscular dystrophy.
    Junio Dort, Zakaria Orfi, Melissa Fiscaletti, Philippe M Campeau, Nicolas A Dumont.
      Introduction: Muscle wasting in Duchenne Muscular Dystrophy is caused by myofiber fragility and poor regeneration that lead to chronic inflammation and muscle replacement by fibrofatty tissue. Our recent findings demonstrated that Resolvin-D2, a bioactive lipid derived from omega-3 fatty acids, has the capacity to dampen inflammation and stimulate muscle regeneration to alleviate disease progression. This therapeutic avenue has many advantages compared to glucocorticoids, the current gold-standard treatment for Duchenne Muscular Dystrophy. However, the use of bioactive lipids as therapeutic drugs also faces many technical challenges such as their instability and poor oral bioavailability. Methods: Here, we explored the potential of PSB-KD107, a synthetic agonist of the resolvin-D2 receptor Gpr18, as a therapeutic alternative for Duchenne Muscular Dystrophy. Results and discussion: We showed that PSB-KD107 can stimulate the myogenic capacity of patient iPSC-derived myoblasts in vitro. RNAseq analysis revealed an enrichment in biological processes related to fatty acid metabolism, lipid biosynthesis, small molecule biosynthesis, and steroid-related processes in PSB-KD107-treated mdx myoblasts, as well as signaling pathways such as Peroxisome proliferator-activated receptors, AMP-activated protein kinase, mammalian target of rapamycin, and sphingolipid signaling pathways. In vivo, the treatment of dystrophic mdx mice with PSB-KD107 resulted in reduced inflammation, enhanced myogenesis, and improved muscle function. The positive impact of PSB-KD107 on muscle function is similar to the one of Resolvin-D2. Overall, our findings provide a proof-of concept that synthetic analogs of bioactive lipid receptors hold therapeutic potential for the treatment of Duchenne Muscular Dystrophy.
    Keywords:  bioactive lipid; inflammation; muscle; muscle stem cell (MuSC); muscular dystrophies
    DOI:  https://doi.org/10.3389/fcell.2023.1187253
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