Human artificial chromosomes for Duchenne muscular dystrophy and beyond: challenges and hopes.

Safe and efficacious vectors able to carry large or several transgenes are of key importance for gene therapy. Human artificial chromosomes can fulfil this essential requirement; moreover, they do not integrate into the host genome. However, drawbacks such as the low efficiency of chromosome transfer and their relatively complex engineering still limit their widespread use. In this article, I summarise the key steps that brought human artificial chromosomes into preclinical research for Duchenne muscular dystrophy, an X-linked, monogenic disorder. I will also review possible future pre-clinical and clinical perspectives for this technology

Generation of two genomic-integration-free DMD iPSC lines with mutations affecting all dystrophin isoforms and potentially amenable to exon-skipping

Duchenne muscular dystrophy (DMD) is the most common paediatric muscular dystrophy and is caused by mutations in the DYSTROPHIN gene. We generated two induced pluripotent stem cell (iPSC) lines from DMD patients with nonsense mutations in exons 68 (UCLi011-A) or 70 (UCLi012-A) by transfecting reprogramming mRNAs. Both mutations affect expression of all dystrophin isoforms. iPSCs expressed pluripotency-associated markers, differentiated into cells of the three germ layers in vitro and had normal karyotypes. The selected mutations are potentially amenable to read-through therapies, exon-skipping and gene-editing. These new iPSCs are also relevant to study DYSTROPHIN role in tissues other than skeletal muscle

Advanced models of human skeletal muscle differentiation, development and disease: Three-dimensional cultures, organoids and beyond

Advanced in vitro models of human skeletal muscle tissue are increasingly needed to model complex developmental dynamics and disease mechanisms not recapitulated in animal models or in conventional monolayer cell cultures. There has been impressive progress towards creating such models by using tissue engineering approaches to recapitulate a range of physical and biochemical components of native human skeletal muscle tissue. In this review, we discuss recent studies focussed on developing complex in vitro models of human skeletal muscle beyond monolayer cell cultures, involving skeletal myogenic differentiation from human primary myoblasts or pluripotent stem cells, often in the presence of structural scaffolding support. We conclude with our outlook on the future of advanced skeletal muscle three-dimensional cultures (e.g. organoids and biofabrication) to produce physiologically and clinically relevant platforms for disease modelling and therapy development in musculoskeletal and neuromuscular disorders

Cellular dynamics of myogenic cell migration: molecular mechanisms and implications for skeletal muscle cell therapies

Directional cell migration is a critical process underlying morphogenesis and post‐natal tissue regeneration. During embryonic myogenesis, migration of skeletal myogenic progenitors is essential to generate the anlagen of limbs, diaphragm and tongue, whereas in post‐natal skeletal muscles, migration of muscle satellite (stem) cells towards regions of injury is necessary for repair and regeneration of muscle fibres. Additionally, safe and efficient migration of transplanted cells is critical in cell therapies, both allogeneic and autologous. Although administration of various myogenic cell types has been done intramuscularly or intravascularly, functional restoration has not been achieved yet in patients with degenerative diseases affecting multiple large muscle. One of the key reasons for this negative outcome is the limited migration of donor cells, which hinders the overall cell engraftment potential. Here, we review mechanisms of myogenic stem/progenitor cell migration during skeletal muscle development and post‐natal regeneration. Furthermore, strategies utilised to improve migratory capacity of myogenic cells are examined in order to identify potential treatments that may be applied to future transplantation protocols

Challenging the "chromatin hypothesis" of cardiac laminopathies with LMNA mutant iPS cells

Lamins A and C are intermediate filaments that provide structural support to the nuclear envelope and regulate gene expression. In this issue, Bertero et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201902117) report that although lamin A/C haploinsufficient cardiomyocytes show disease-associated phenotypes, those changes cannot be explained by alterations in chromatin compartmentalization

Generation of an MTM1-mutant iPSC line (CRICKi008-A) from an individual with X-linked myotubular myopathy (XLMTM)

Centronuclear myopathies (CNMs) are a group of inherited rare muscle disorders characterised by the abnormal position of the nucleus in the center of the muscle fiber. One of CNM is the X-Linked Myotubular Myopathy, caused by mutations in the myotubularin (MTM1) gene (XLMTM), characterised by profound muscle hypotonia and weakness, severe bulbar and respiratory involvement. Here, we generated an induced pluripotent stem cell (iPSC) line from a patient with a severe form of XLMTM. Dermal fibroblasts were reprogrammed to pluripotency using a non-integrating mRNA-based protocol. This new MTM1-mutant iPSC line could facilitate disease-modelling and therapy development studies for XLMTM

Pluripotent Stem Cells for Gene Therapy of Degenerative Muscle Diseases

Human pluripotent stem cells represent a unique source for cell-based therapies and regenerative medicine. The intrinsic features of these cells such as their easy accessibility and their capacity to be expanded indefinitely overcome some limitations of conventional adult stem cells. Furthermore, the possibility to derive patient-specific induced pluripotent stem (iPS) cells in combination with the current development of gene modification methods could be used for autologous cell therapies of some genetic diseases. In particular, muscular dystrophies are considered to be a good candidate due to the lack of efficacious therapeutic treatments for patients to date, and in view of the encouraging results arising from recent preclinical studies. Some hurdles, including possible genetic instability and their efficient differentiation into muscle progenitors through vector/transgene-free methods have still to be overcome or need further optimization. Additionally, engraftment and functional contribution to muscle regeneration in pre-clinical models need to be carefully assessed before clinical translation. This review offers a summary of the advanced methods recently developed to derive muscle progenitors from pluripotent stem cells, as well as gene therapy by gene addition and gene editing methods using ZFNs, TALENs or CRISPR/Cas9. We have also discussed the main issues that need to be addressed for successful clinical translation of genetically corrected patient-specific pluripotent stem cells in autologous transplantation trials for skeletal muscle disorders

Dystrophin deficiency affects human astrocyte properties andresponse to damage

In addition to progressive muscular degeneration due to dystrophin mutations, 1/3 of Duchenne muscular dystrophy (DMD) patients present cognitive deficits. However, there is currently an incomplete understanding about the function of the multiple dystrophin isoforms in human brains. Here, we tested the hypothesis that dystrophin deficiency affects glial function in DMD and could therefore contribute to neural impairment. We investigated human dystrophin isoform expression with development and differentiation and response to damage in human astrocytes from control and induced pluripotent stem cells from DMD patients. In control cells, short dystrophin isoforms were up-regulated with development and their expression levels changed differently upon neuronal and astrocytic differentiation, as well as in 2-dimensional versus 3-dimensional astrocyte cultures. All DMD-astrocytes tested displayed altered morphology, proliferative activity and AQP4 expression. Furthermore, they did not show any morphological change in response to inflammatory stimuli and their number was significantly lower as compared to stimulated healthy astrocytes. Finally, DMD-astrocytes appeared to be more sensitive than controls to oxidative damage as shown by their increased cell death. Behavioral and metabolic defects in DMD-astrocytes were consistent with gene pathway dysregulation shared by lines with different mutations as demonstrated by bulk RNA-seq analysis. Together, our DMD model provides evidence for altered astrocyte function in DMD suggesting that defective astrocyte responses may contribute to neural impairment and might provide additional potential therapeutic targets

Efficient derivation and inducible differentiation of expandable skeletal myogenic cells from human ES and patient-specific iPS cells.

Skeletal muscle is the most abundant human tissue; therefore, an unlimited availability of myogenic cells has applications in regenerative medicine and drug development. Here we detail a protocol to derive myogenic cells from human embryonic stem (ES) and induced pluripotent stem (iPS) cells, and we also provide evidence for its extension to human iPS cells cultured without feeder cells. The procedure, which does not require the generation of embryoid bodies or prospective cell isolation, entails four stages with different culture densities, media and surface coating. Pluripotent stem cells are disaggregated to single cells and then differentiated into expandable cells resembling human mesoangioblasts. Subsequently, transient Myod1 induction efficiently drives myogenic differentiation into multinucleated myotubes. Cells derived from patients with muscular dystrophy and differentiated using this protocol have been genetically corrected, and they were proven to have therapeutic potential in dystrophic mice. Thus, this platform has been demonstrated to be amenable to gene and cell therapy, and it could be extended to muscle tissue engineering and disease modeling

Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study

BACKGROUND: Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is a skin cancer therapy that still has limitations due to the low penetration of this drug into the skin. We have proposed in this work a delivery system for 5-ALA based on liposomes having lipid composition similar to the mammalian stratum corneum (SCLLs) in order to optimize its skin delivery in Photodynamic Therapy (PDT) of skin cancers. METHODS: SCLLs were obtained by reverse phase evaporation technique and size distribution of the vesicles was determinated by photon correlation spectroscopy. In vitro permeation profile was characterized using hairless mouse skin mounted in modified Franz diffusion cell. RESULTS: Size exclusion chromatography on gel filtration confirmed vesicle formation. SCLLs obtained by presented a degree of encapsulation of 5-ALA around 5.7%. A distribution of vesicle size centering at around 500 nm and 400 nm respectively for SCLLs and SCLLs containing 5-ALA was found. In vitro 5-ALA permeation study showed that SCLLs preparations presented higher skin retention significantly (p < 0.05) on the epidermis without SC + dermis, with a decreasing of skin permeation compared to aqueous solution. CONCLUSIONS: The in vitro delivery performance provided by SCLLs lead to consider this systems adequate for the 5-ALA-PDT of skin cancer, since SCLLs have delivered 5-ALA to the target skin layers (viable epidermis + dermis) to be treated by topical PDT of skin cancer