Effects of Mini-Dystrophin on Dystrophin-Deficient, Human Skeletal Muscle-Derived Cells

BACKGROUND: We are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells. AIMS: Our aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells. METHODS: Skeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3-R13 spectrin-like repeats (ΔR3R13) or hinge2 to spectrin-like repeats R23 (ΔH2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression. RESULTS: Cell proliferation was not affected in cells transduced with ΔR3R13, but was significantly increased in cells transduced with ΔH2R23. The fusion index of myotubes derived from both ΔR3R13- and ΔH2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ΔH2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs. CONCLUSIONS: Ectopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter

Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne Muscular Dystrophy

Autologous stem cells that have been genetically modified to express dystrophin are a possible means of treating Duchenne Muscular Dystrophy (DMD). To maximize the therapeutic effect, dystrophin construct needs to contain as many functional motifs as possible, within the packaging capacity of the viral vector. Existing dystrophin constructs used for transduction of muscle stem cells do not contain the nNOS binding site, an important functional motif within the dystrophin gene. In this proof-of-concept study, using stem cells derived from skeletal muscle of a DMD patient (mdcs) transplanted into an immunodeficient mouse model of DMD, we report that two novel dystrophin constructs, C1 (ΔR3-R13) and C2 (ΔH2-R23), can be lentivirally transduced into mdcs and produce dystrophin. These dystrophin proteins were functional in vivo, as members of the dystrophin glycoprotein complex were restored in muscle fibres containing donor-derived dystrophin. In muscle fibres derived from cells that had been transduced with construct C1, the largest dystrophin construct packaged into a lentiviral system, nNOS was restored. The combination of autologous stem cells and a lentivirus expressing a novel dystrophin construct which optimally restores proteins of the dystrophin glycoprotein complex may have therapeutic application for all DMD patients, regardless of their dystrophin mutation

Lentiviral vectors can be used for full-length dystrophin gene therapy

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD

Quasi-BIC Resonant Enhancement of Second-Harmonic Generation in WS2 Monolayers.

Atomically thin monolayers of transition metal dichalcogenides (TMDs) have emerged as a promising class of novel materials for optoelectronics and nonlinear optics. However, the intrinsic nonlinearity of TMD monolayers is weak, limiting their functionalities for nonlinear optical processes such as frequency conversion. Here we boost the effective nonlinear susceptibility of a TMD monolayer by integrating it with a resonant dielectric metasurface that supports pronounced optical resonances with high quality factors: bound states in the continuum (BICs). We demonstrate that a WS2 monolayer combined with a silicon metasurface hosting BICs exhibits enhanced second-harmonic intensity by more than 3 orders of magnitude relative to a WS2 monolayer on top of a flat silicon film of the same thickness. Our work suggests a pathway to employ high-index dielectric metasurfaces as hybrid structures for enhancement of TMD nonlinearities with applications in nonlinear microscopy, optoelectronics, and signal processing

Absence of a Spin Liquid Phase in the Hubbard Model on the Honeycomb Lattice

A spin liquid is a novel quantum state of matter with no conventional order parameter where a finite charge gap exists even though the band theory would predict metallic behavior. Finding a stable spin liquid in two or higher spatial dimensions is one of the most challenging and debated issues in condensed matter physics. Very recently, it has been reported that a model of graphene, i.e., the Hubbard model on the honeycomb lattice, can show a spin liquid ground state in a wide region of the phase diagram, between a semi-metal (SM) and an antiferromagnetic insulator (AFMI). Here, by performing numerically exact quantum Monte Carlo simulations, we extend the previous study to much larger clusters (containing up to 2592 sites), and find, if any, a very weak evidence of this spin liquid region. Instead, our calculations strongly indicate a direct and continuous quantum phase transition between SM and AFMI.Comment: 15 pages with 7 figures and 9 tables including supplementary information, accepted for publication in Scientific Report

Chinese Expert Consensus on Critical Care Ultrasound Applications at COVID-19 Pandemic

The spread of new coronavirus (SARS-Cov-2) follows a different pattern than previous respiratory viruses, posing a serious public health risk worldwide. World Health Organization (WHO) named the disease as COVID-19 and declared it a pandemic. COVID-19 is characterized by highly contagious nature, rapid transmission, swift clinical course, profound worldwide impact, and high mortality among critically ill patients. Chest X-ray, computerized tomography (CT), and ultrasound are commonly used imaging modalities. Among them, ultrasound, due to its portability and non-invasiveness, can be easily moved to the bedside for examination at any time. In addition, with use of 4G or 5G networks, remote ultrasound consultation can also be performed, which allows ultrasound to be used in isolated medial areas. Besides, the contact surface of ultrasound probe with patients is small and easy to be disinfected. Therefore, ultrasound has gotten lots of positive feedbacks from the frontline healthcare workers, and it has played an indispensable role in the course of COVID-19 diagnosis and follow up

Mouse Regenerating Myofibers Detected as False-Positive Donor Myofibers with Anti-Human Spectrin

Stem cell transplantation is being tested as a potential therapy for a number of diseases. Stem cells isolated directly from tissue specimens or generated via reprogramming of differentiated cells require rigorous testing for both safety and efficacy in preclinical models. The availability of mice with immune-deficient background that carry additional mutations in specific genes facilitates testing the efficacy of cell transplantation in disease models. The muscular dystrophies are a heterogeneous group of disorders, of which Duchenne muscular dystrophy is the most severe and common type. Cell-based therapy for muscular dystrophy has been under investigation for several decades, with a wide selection of cell types being studied, including tissue-specific stem cells and reprogrammed stem cells. Several immune-deficient mouse models of muscular dystrophy have been generated, in which human cells obtained from various sources are injected to assess their preclinical potential. After transplantation, the presence of engrafted human cells is detected via immunofluorescence staining, using antibodies that recognize human, but not mouse, proteins. Here we show that one antibody specific to human spectrin, which is commonly used to evaluate the efficacy of transplanted human cells in mouse muscle, detects myofibers in muscles of NOD/Rag1nullmdx5cv, NOD/LtSz-scid IL2Rγnull mice, or mdx nude mice, irrespective of whether they were injected with human cells. These “reactive” clusters are regenerating myofibers, which are normally present in dystrophic tissue and the spectrin antibody is likely recognizing utrophin, which contains spectrin-like repeats. Therefore, caution should be used in interpreting data based on detection of single human-specific proteins, and evaluation of human stem cell engraftment should be performed using multiple human-specific labeling strategies

Increasing the sensitivity of terahertz split ring resonator metamaterials for dielectric sensing by localized substrate etching

We demonstrate a significant enhancement in the sensitivity of split ring resonator terahertz metamaterial dielectric sensors by the introduction of etched trenches into their inductive-capacitive gap area, both through finite element simulations and in experiments performed using terahertz time-domain spectroscopy. The enhanced sensitivity is demonstrated by observation of an increased frequency shift in response to overlaid dielectric material of thicknesses up to 18 μm deposited on to the sensor surface. We show that sensitivity to the dielectric is enhanced by a factor of up to ~2.7 times by the incorporation of locally etched trenches with a depth of ~3.4 μm, for example, and discuss the effect of the etching on the electrical properties of the sensors. Our experimental findings are in good agreement with simulations of the sensors obtained using finite element methods

Myostatin inhibition in combination with antisense oligonucleotide therapy improves outcomes in spinal muscular atrophy

BACKGROUND Spinal muscular atrophy (SMA) is caused by genetic defects in the survival motor neuron 1 (SMN1) gene that lead to SMN deficiency. Different SMN‐restoring therapies substantially prolong survival and function in transgenic mice of SMA. However, these therapies do not entirely prevent muscle atrophy and restore function completely. To further improve the outcome, we explored the potential of a combinatorial therapy by modulating SMN production and muscle‐enhancing approach as a novel therapeutic strategy for SMA. METHODS The experiments were performed in a mouse model of severe SMA. A previously reported 25‐mer morpholino antisense oligomer PMO25 was used to restore SMN expression. The adeno‐associated virus‐mediated expression of myostatin propeptide was used to block the myostatin pathway. Newborn SMA mice were treated with a single subcutaneous injection of 40 μg/g (therapeutic dose) or 10 μg/g (low‐dose) PMO25 on its own or together with systemic delivery of a single dose of adeno‐associated virus‐mediated expression of myostatin propeptide. The multiple effects of myostatin inhibition on survival, skeletal muscle phenotype, motor function, neuromuscular junction maturation, and proprioceptive afferences were evaluated. RESULTS We show that myostatin inhibition acts synergistically with SMN‐restoring antisense therapy in SMA mice treated with the higher therapeutic dose PMO25 (40 μg/g), by increasing not only body weight (21% increase in male mice at Day 40), muscle mass (38% increase), and fibre size (35% increase in tibialis anterior muscle in 3 month female SMA mice), but also motor function and physical performance as measured in hanging wire test (two‐fold increase in time score) and treadmill exercise test (two‐fold increase in running distance). In SMA mice treated with low‐dose PMO25 (10 μg/g), the early application of myostatin inhibition prolongs survival (40% increase), improves neuromuscular junction maturation (50% increase) and innervation (30% increase), and increases both the size of sensory neurons in dorsal root ganglia (60% increase) and the preservation of proprioceptive synapses in the spinal cord (30% increase). CONCLUSIONS These data suggest that myostatin inhibition, in addition to the well‐known effect on muscle mass, can also positively influence the sensory neural circuits that may enhance motor neurons function. While the availability of the antisense drug Spinraza for SMA and other SMN‐enhancing therapies has provided unprecedented improvement in SMA patients, there are still unmet needs in these patients. Our study provides further rationale for considering myostatin inhibitors as a therapeutic intervention in SMA patients, in combination with SMN‐restoring drugs