Exonuclease editor promotes precision of gene editing in mammalian cells.

BACKGROUND: Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging. RESULTS: Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control. CONCLUSIONS: Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.

Enhancing prime editor flexibility with coiled-coil heterodimers.

BACKGROUND: Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency. RESULTS: Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol. CONCLUSIONS: Our innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.

Interleukin 4 improved adipose-derived stem cells engraftment via interacting with fibro/adipogenic progenitors in dystrophic mice.

Adipose-derived stem cells (ADSC) therapy shows promise as an effective treatment for dystrophinopathy. Fibro-/adipogenic progenitors (FAPs) play an essential role in the myogenesis of muscle satellite cells and contribute to muscle fibrosis and adipocyte infiltration. The interleukin 4 (IL-4) pathway acts as a switch that regulates the functions of FAPs. The interaction between FAPs and engrafted cells remains unclear. In this study, we used a co-culture system to investigate possible crosstalk between the FAPs of dystrophic mice and ADSC overexpressing IL4 (IL4-ADSC) and control ADSC. Systemic transplantation of IL4-ADSC and control ADSC in dystrophic mice was conducted for 16 weeks, after which motor function and molecular improvements were evaluated. Overexpression of IL4 in ADSC significantly promoted myogenesis in vitro, increasing the expression of Pax7, Myogenin, and MyHC. Co-culture indicated that although myoblasts derived from control ADSC promoted adipogenic and fibrogenic differentiation of FAPs, FAPs did not significantly affect myogenesis of ADSC-derived myoblasts. However, overexpression of IL4 in ADSC inhibited their myotube-dependent promotion of FAPs differentiation on the one hand and promoted FAPs to enhance myogenesis on the other. Dystrophic mice administered with IL4-ADSC-derived myoblasts displayed significantly better motor ability, more engrafted cells showing dystrophin expression, and less muscle fibrosis, intramuscular adipocytes, and macrophage infiltration than mice administered control-ADSC-derived myoblasts. In conclusion, IL4 activation enhanced the therapeutic potential of ADSC transplantation in dystrophic mice, possibly by improving the myogenesis of IL4-ADSC and altering the crosstalk between engrafted stem cells and resident FAPs.

iPSCs ameliorate hypoxia-induced autophagy and atrophy in C2C12 myotubes via the AMPK/ULK1 pathway.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disorder for which there is no effective treatment. Previous studies have shown that stem cell transplantation into mdx mice can promote muscle regeneration and improve muscle function, however, the specific molecular mechanisms remain unclear. DMD suffers varying degrees of hypoxic damage during disease progression. This study aimed to investigate whether induced pluripotent stem cells (iPSCs) have protective effects against hypoxia-induced skeletal muscle injury. RESULTS: In this study, we co-cultured iPSCs with C2C12 myoblasts using a Transwell nested system and placed them in a DG250 anaerobic workstation for oxygen deprivation for 24 h. We found that iPSCs reduced the levels of lactate dehydrogenase and reactive oxygen species and downregulated the mRNA and protein levels of BAX/BCL2 and LC3II/LC3I in hypoxia-induced C2C12 myoblasts. Meanwhile, iPSCs decreased the mRNA and protein levels of atrogin-1 and MuRF-1 and increased myotube width. Furthermore, iPSCs downregulated the phosphorylation of AMPKα and ULK1 in C2C12 myotubes exposed to hypoxic damage. CONCLUSIONS: Our study showed that iPSCs enhanced the resistance of C2C12 myoblasts to hypoxia and inhibited apoptosis and autophagy in the presence of oxidative stress. Further, iPSCs improved hypoxia-induced autophagy and atrophy of C2C12 myotubes through the AMPK/ULK1 pathway. This study may provide a new theoretical basis for the treatment of muscular dystrophy in stem cells.

iPSCs ameliorate hypoxia-induced autophagy and atrophy in C2C12 myotubes via the AMPK/ULK1 pathway

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disorder for which there is no effective treatment. Previous studies have shown that stem cell transplantation into mdx mice can promote muscle regeneration and improve muscle function, however, the specific molecular mechanisms remain unclear. DMD suffers varying degrees of hypoxic damage during disease progression. This study aimed to investigate whether induced pluripotent stem cells (iPSCs) have protective effects against hypoxia-induced skeletal muscle injury. RESULTS: In this study, we co-cultured iPSCs with C2C12 myoblasts using a Transwell nested system and placed them in a DG250 anaerobic workstation for oxygen deprivation for 24 h. We found that iPSCs reduced the levels of lactate dehydrogenase and reactive oxygen species and downregulated the mRNA and protein levels of BAX/BCL2 and LC3II/ LC3I in hypoxia-induced C2C12 myoblasts. Meanwhile, iPSCs decreased the mRNA and protein levels of atrogin-1 and MuRF-1 and increased myotube width. Furthermore, iPSCs downregulated the phosphorylation of AMPKA and ULK1 in C2C12 myotubes exposed to hypoxic damage. CONCLUSIONS: Our study showed that iPSCs enhanced the resistance of C2C12 myoblasts to hypoxia and inhibited apoptosis and autophagy in the presence of oxidative stress. Further, iPSCs improved hypoxia-induced autophagy and atrophy of C2C12 myotubes through the AMPK/ULK1 pathway. This study may provide a new theoretical basis for the treatment of muscular dystrophy in stem cells.

In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers.

BACKGROUND: Mutations in the DMD gene encoding dystrophin-a critical structural element in muscle cells-cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. METHODS: In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46-54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. RESULTS: Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member ß-dystroglycan. CONCLUSIONS: We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

CCR2 improves homing and engraftment of adipose-derived stem cells in dystrophic mice.

BACKGROUND: Dystrophinopathy, a common neuromuscular disorder caused by the absence of dystrophin, currently lacks effective treatments. Systemic transplantation of adipose-derived stem cells (ADSCs) is a promising treatment approach, but its low efficacy remains a challenge. Chemokine system-mediated stem cell homing plays a critical role in systemic transplantation. Here, we investigated whether overexpression of a specific chemokine receptor could improve muscle homing and therapeutic effects of ADSC systemic transplantation in dystrophic mice. METHODS: We analysed multiple microarray datasets from the Gene Expression Omnibus to identify a candidate chemokine receptor and then evaluated the protein expression of target ligands in different tissues and organs of dystrophic mice. The candidate chemokine receptor was overexpressed using the lentiviral system in mouse ADSCs, which were used for systemic transplantation into the dystrophic mice, followed by evaluation of motor function, stem cell muscle homing, dystrophin expression, and muscle pathology. RESULTS: Chemokine-profile analysis identified C-C chemokine receptor (CCR)2 as the potential target for improving ADSC homing. We found that the levels of its ligands C-C chemokine ligand (CCL)2 and CCL7 were higher in muscles than in other tissues and organs of dystrophic mice. Additionally, CCR2 overexpression improved ADSC migration ability and maintained their multilineage-differentiation potentials. Compared with control ADSCs, transplantation of those overexpressing CCR2 displayed better muscle homing and further improved motor function, dystrophin expression, and muscle pathology in dystrophic mice. CONCLUSIONS: These results demonstrated that CCR2 improved ADSC muscle homing and therapeutic effects following systemic transplantation in dystrophic mice.

Critical Role of E1623 Residue in S3-S4 Loop of Nav1.1 Channel and Correlation Between Nature of Substitution and Functional Alteration.

Objective: An overwhelming majority of the genetic variants associated with genetic disorders are missense. The association between the nature of substitution and the functional alteration, which is critical in determining the pathogenicity of variants, remains largely unknown. With a novel missense variant (E1623A) identified from two epileptic cases, which occurs in the extracellular S3-S4 loop of Nav1.1, we studied functional changes of all latent mutations at residue E1623, aiming to understand the relationship between substitution nature and functional alteration. Methods: Six latent mutants with amino acid substitutions at E1623 were generated, followed by measurements of their electrophysiological alterations. Different computational analyses were used to parameterize the residue alterations. Results: Structural modeling indicated that the E1623 was located in the peripheral region far from the central pore, and contributed to the tight turn of the S3-S4 loop. The E1623 residue exhibited low functional tolerance to the substitutions with the most remarkable loss-of-function found in E1623A, including reduced current density, less steady-state availability of activation and inactivation, and slower recovery from fast inactivation. Correlation analysis between electrophysiological parameters and the parameterized physicochemical properties of different residues suggested that hydrophilicity of side-chain at E1623 might be a crucial contributor for voltage-dependent kinetics. However, none of the established algorithms on the physicochemical variations of residues could well predict changes in the channel conductance property indicated by peak current density. Significance: The results established the important role of the extracellular S3-S4 loop in Nav1.1 channel gating and proposed a possible effect of local conformational loop flexibility on channel conductance and kinetics. Site-specific knowledge of protein will be a fundamental task for future bioinformatics.

Genetic Modifiers of Duchenne Muscular Dystrophy in Chinese Patients.

Background: Duchenne muscular dystrophy (DMD) is a fatal, X-linked recessive muscle disorder characterized by heterogeneous progression and severity. We aimed to study the effects of single nucleotide polymorphisms (SNPs) in SPP1 and LTBP4 on DMD progression in Chinese patients. Methods: We genotyped LTBP4 haplotypes and the SPP1 promoter SNPs rs28357094, rs11730582, and rs17524488 in 326 patients registered in the neuromuscular database of The First Affiliated Hospital of Sun Yat-sen University. Kaplan-Meier curves and log-rank tests were used to estimate and compare median age at loss of ambulation, while Cox proportional hazard regression models were used as to analyze the effects of glucocorticoids treatments, DMD genotype, and SPP1/LTBP4 SNPs on loss of ambulation. Results: The CC/CT genotype at rs11730582 was associated with a 1.33-year delay in ambulation loss (p = 0.006), with hazard ratio 0.63 (p = 0.008), in patients with truncated DMD genotype and undergoing steroid treatment. On the other hand, rs17524488 in SPP1 and the IAAM/IAAM haplotype in LTBP4 were not associated with time to ambulation loss. Conclusions: SPP1 rs11730582 is a genetic modifier of the long-term effects of steroid treatment in Chinese DMD patients. Thus, any future clinical study in DMD should adjust for glucocorticoids use, DMD genotype, and SPP1 polymorphisms.

Engraftment of human induced pluripotent stem cell-derived myogenic progenitors restores dystrophin in mice with duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a devastating genetic muscular disorder with no effective treatment that is caused by the loss of dystrophin. Human induced pluripotent stem cells (hiPSCs) offer a promising unlimited resource for cell-based therapies of muscular dystrophy. However, their clinical applications are hindered by inefficient myogenic differentiation, and moreover, the engraftment of non-transgene hiPSC-derived myogenic progenitors has not been examined in the mdx mouse model of DMD. METHODS: We investigated the muscle regenerative potential of myogenic progenitors derived from hiPSCs in mdx mice. The hiPSCs were transfected with enhanced green fluorescent protein (EGFP) vector and defined as EGFP hiPSCs. Myogenic differentiation was performed on EGFP hiPSCs with supplementary of basic fibroblast growth factor, forskolin, 6-bromoindirubin-3'-oxime as well as horse serum. EGFP hiPSCs-derived myogenic progenitors were engrafted into mdx mice via both intramuscular and intravenous injection. The restoration of dystrophin expression, the ratio of central nuclear myofibers, and the transplanted cells-derived satellite cells were accessed after intramuscular and systemic transplantation. RESULTS: We report that abundant myogenic progenitors can be generated from hiPSCs after treatment with these three small molecules, with consequent terminal differentiation giving rise to mature myotubes in vitro. Upon intramuscular or systemic transplantation into mdx mice, these myogenic progenitors engrafted and contributed to human-derived myofiber regeneration in host muscles, restored dystrophin expression, ameliorated pathological lesions, and seeded the satellite cell compartment in dystrophic muscles. CONCLUSIONS: This study demonstrates the muscle regeneration potential of myogenic progenitors derived from hiPSCs using non-transgenic induction methods. Engraftment of hiPSC-derived myogenic progenitors could be a potential future therapeutic strategy to treat DMD in a clinical setting.

Homozygous NOTCH3 p.R587C mutation in Chinese patients with CADASIL: a case report.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small vessel disease caused by mutations in NOTCH3 gene with remarkable phenotypic heterogeneity. Cases of CADASIL associated with homozygous NOTCH3 mutations are rare and subsequently understudied. In this study, we investigate the genetic and phenotypic features within patients of CADASIL with homozygous NOTCH3 mutations. CASE PRESENTATION: We recruited two affected individuals with CADASIL from a mainland Chinese family. The proband (Patient 1), a 60-year-old male, presented with slow progressive gait instability, severe cognitive impairment, and emotional disorder for more than 2 years with a history of ischemic stroke and hypertension. His younger brother (Patient 2) presented with apparent gait difficulties, dysarthria as well as cognitive decline at 59 years old. Brain magnetic resonance imaging (MRI) showed diffused white matter lesions involving bilateral periventricular white matter, semioval center region, and anterior temporal lobes. Molecular genetic testing identified a homozygous variant, c.1759C > T (p.R587C), in NOTCH3 gene in both patients. Pathological analysis revealed granular osmiophilic material (GOM) deposits in small arterial walls of skin from the proband. The diagnosis of CADASIL was confirmed. CONCLUSIONS: Our cases of CADASIL with homozygous mutation c.1759C > T (p.R587C) in NOTCH3 share similar manifestation to the patients with heterozygous same mutation reported previously. Other than genetic factors, vascular risk factors or environmental factors might contribute to the phenotypic variation of CADASIL.

Engraftment of human induced pluripotent stem cell-derived myogenic progenitors restores dystrophin in mice with duchenne muscular dystrophy

BACKGROUND: Duchenne muscular dystrophy (DMD) is a devastating genetic muscular disorder with no effective treatment that is caused by the loss of dystrophin. Human induced pluripotent stem cells (hiPSCs) offer a promising unlimited resource for cell-based therapies of muscular dystrophy. However, their clinical applications are hindered by inefficient myogenic differentiation, and moreover, the engraftment of non-transgene hiPSC-derived myogenic progenitors has not been examined in the mdx mouse model of DMD. METHODS: We investigated the muscle regenerative potential of myogenic progenitors derived from hiPSCs in mdx mice. The hiPSCs were transfected with enhanced green fluorescent protein (EGFP) vector and defined as EGFP hiPSCs. Myogenic differentiation was performed on EGFP hiPSCs with supplementary of basic fibroblast growth factor, forskolin, 6-bromoindirubin-3'-oxime as well as horse serum. EGFP hiPSCs-derived myogenic progenitors were engrafted into mdx mice via both intramuscular and intravenous injection. The restoration of dystrophin expression, the ratio of central nuclear myofibers, and the transplanted cells-derived satellite cells were accessed after intramuscular and systemic transplantation. RESULTS: We report that abundant myogenic progenitors can be generated from hiPSCs after treatment with these three small molecules, with consequent terminal differentiation giving rise to mature myotubes in vitro. Upon intramuscular or systemic transplantation into mdx mice, these myogenic progenitors engrafted and contributed to human-derived myofiber regeneration in host muscles, restored dystrophin expression, ameliorated pathological lesions, and seeded the satellite cell compartment in dystrophic muscles. CONCLUSIONS: This study demonstrates the muscle regeneration potential of myogenic progenitors derived from hiPSCs using non-transgenic induction methods. Engraftment of hiPSC-derived myogenic progenitors could be a potential future therapeutic strategy to treat DMD in a clinical setting.

HLA Polymorphism Affects Risk of de novo Mutation of dystrophin Gene and Clinical Severity of Duchenne Muscular Dystrophy in a Southern Chinese Population.

Immune-mediated pathology has been thought to be an important factor contributing to Duchenne muscular dystrophy (DMD). Allele frequencies of certain HLA types are known to differ between patients with dystrophinopathies and healthy controls with low-resolution HLA gene typing data in limit reports. Using Polymerase chain reactionsequence based typing (PCR-SBT) to genotype 64 children with DMD in HLA-A, -B,-C, -DRB1, and -DQB1 locus and 503 healthy controls in HLA-A, -B, -DRB1 locus, this study aimed to investigate associations of specific HLA alleles with, and their possible roles in the development and clinical phenotypic severity of DMD. The χ2 test was used to evaluate the distribution of allele frequencies in HLA-A, -B, -DRB1 locus between the patients and healthy controls. A significantly higher frequency of HLA-B * 07:05 was found in children with DMD compared to that in controls (OR = 16.2, 95%CI = 2.9-89.3, P < 0.046). More importantly, significantly higher frequencies of HLA-A * 29:01 (OR = 77.308, 95%CI = 6.794-879.731, P < 0.0160) and HLA-B *07:05 (OR = 60.240, 95%CI = 9.637-376.535, P < 2.41*10-3) was found in patients with de novo mutations (n = 14) compared to controls while no difference of HLA alleles frequency ware indicated between patients with inherited mutation and control. The result indicates that HLA alleles is associated with pathogenesis of DMD especially DMD with de novo mutation. We use Vignos scale to estimate the lower limb motor function of patients. The impact of HLA alleles on score of Vignos scale of DMD children was estimated by multiple linear regression. Our study indicates that HLA-A *02:01 may have a dampening effect on the clinical phenotypic severity of DMD, evidenced by the presence of HLA-A *02:01 being associated with lower Vignos score. Our study demonstrates that certain HLA alleles are indeed associated with the pathogenesis and clinical phenotypic severity of DMD.

Ratio of Creatine Kinase to Alanine Aminotransferase as a Biomarker of Acute Liver Injury in Dystrophinopathy.

OBJECTIVE: To investigate the ratios of creatine kinase (CK) to aminotransferases as biomarkers of acute liver injury in dystrophinopathy. METHODS: C57 and mdx (dystrophic) mice were treated with a hepatotoxic reagent D-galactosamine (D-GalN). The degrees of liver and muscle injury were assessed using histological examinations. To examine whether serum CK-adjusted aminotransferase levels could indicate liver status in dystrophic mice, the CK/alanine aminotransferase (ALT) and CK/aspartate aminotransferase (AST) ratios were analyzed. Furthermore, we enrolled 658 male patients with dystrophinopathy and 378 male patients without muscle and liver injury as control, whose serum ALT, AST, and CK levels were examined. RESULTS: Animal experiments indicated that D-GalN treatment could induce acute liver injury but not muscle injury. Additionally, D-GalN decreased the CK/ALT and CK/AST ratios in both C57 mice and mdx mice (P < 0.001). However, there was an overlap of the CK/AST ratio between dystrophic mice with and without acute liver injury. In patients with dystrophinopathy, CK-adjusted ALT diminished the variability associated with age, genotype, clinical phenotype, and motor function (P > 0.05). CONCLUSIONS: CK/ALT is a potential biomarker for the differential evaluation of acute liver injury in dystrophic mice, which highlights the value to further evaluate the practice of CK/ALT in dystrophinopathy patients.

[Analysis of SMN1 gene mutations in 78 patients with spinal muscular atrophy].

OBJECTIVE: To explore the significance of SMN1 gene mutations among patients with spinal muscular atrophy (SMA) and the value of multiplex ligation dependent probe amplification (MLPA) for its diagnosis. METHODS: Potential mutations of the SMN1 gene were detected among 78 SMA patients with a MLPA assay. RESULTS: Homozygous deletion of SMN1 exons 7 and 8 was detected in 70 (89.7%) of all patients. Homozygous deletion of exons 7 and heterozygous deletion of exon 8 was detected in 3 patients (3.8%). Homozygous deletion of SMN1 exons 7 alone was detected in 3 patients (3.8%). Heterozygous deletion of SMN1 exons 7 and 8 was detected in 2 patients (2.6%). For 77 of the patients, both parents were found to carry heterozygous deletion of the SMN1 gene, which was consistent with the recessive inheritance of SMA. One patient with SMA type I was found to be rather rare. The patient was found to carry homozygous deletion of SMN1 exons 7 and 8, for which her mother was heterozygous, while no mutation was found in her father. CONCLUSION: Homozygous deletion of the SMN1 gene have been detected in more than 95% of SMA patients. No homozygous deletion of exon 8 has been found. Homozygous deletion of exon 7 is more significant in the pathogenesis of SMA.

Serum Creatinine Distinguishes Duchenne Muscular Dystrophy from Becker Muscular Dystrophy in Patients Aged ≤3 Years: A Retrospective Study.

Here, we investigated correlations between serum creatinine (SCRN) levels and clinical phenotypes of dystrophinopathy in young patients. Sixty-eight patients with dystrophinopathy at the Neuromuscular Clinic, The First Affiliated Hospital, Sun Yat-sen University, were selected for this study. The diagnosis of dystrophinopathy was based on clinical manifestation, biochemical changes, and molecular analysis. Some patients underwent muscle biopsies; SCRN levels were tested when patients were ≤3 years old, and reading frame changes were analyzed. Each patient was followed up, and motor function and clinical phenotype were assessed when the same patients were ≥4 years old. Our findings indicated that in young patients, lower SCRN levels were associated with increased disease severity (p < 0.01) and that SCRN levels were the highest in patients exhibiting mild Becker muscular dystrophy (BMD) (p < 0.001) and the lowest in patients with Duchenne muscular dystrophy (DMD) (p < 0.01) and were significantly higher in patients carrying in-frame mutations than in patients carrying out-of-frame mutations (p < 0.001). SCRN level cutoff values for identifying mild BMD [18 µmol/L; area under the curve (AUC): 0.947; p < 0.001] and DMD (17 µmol/L; AUC: 0.837; p < 0.001) were established. These results suggest that SCRN might be a valuable biomarker for distinguishing DMD from BMD in patients aged ≤3 years and could assist in the selection of appropriate treatment strategies.

Abnormal Neural Progenitor Cells Differentiated from Induced Pluripotent Stem Cells Partially Mimicked Development of TSC2 Neurological Abnormalities.

Tuberous sclerosis complex (TSC) is a disease featuring devastating and therapeutically challenging neurological abnormalities. However, there is a lack of specific neural progenitor cell models for TSC. Here, the pathology of TSC was studied using primitive neural stem cells (pNSCs) from a patient presenting a c.1444-2A>C mutation in TSC2. We found that TSC2 pNSCs had higher proliferative activity and increased PAX6 expression compared with those of control pNSCs. Neurons differentiated from TSC2 pNSCs showed enlargement of the soma, perturbed neurite outgrowth, and abnormal connections among cells. TSC2 astrocytes had increased saturation density and higher proliferative activity. Moreover, the activity of the mTOR pathway was enhanced in pNSCs and induced in neurons and astrocytes. Thus, our results suggested that TSC2 heterozygosity caused neurological malformations in pNSCs, indicating that its heterozygosity might be sufficient for the development of neurological abnormalities in patients.

Long-term engraftment of myogenic progenitors from adipose-derived stem cells and muscle regeneration in dystrophic mice.

Stem cell therapy is a promising approach for treating Duchenne muscular dystrophy (DMD); however, its application is hindered by poor cell engraftment. There have been no reports to date describing the efficient generation of myogenic progenitors from adipose-derived stem cells (ADSCs) that can contribute to muscle regeneration. In this study, we examined the in vivo myogenic potential of progenitors differentiated from ADSCs using forskolin, basic fibroblast growth factor, the glycogen synthase kinase 3ß inhibitor 6-bromoindirubin-3'-oxime as well as the supernatant of ADSC cultures. The results indicate that a proliferative population of myogenic progenitors can be derived from ADSCs that have characteristics similar to muscle satellite cells and are capable of terminal differentiation into multinucleated myotubes. When transplanted into DMD model mdx mice either by intramuscular injection or systemic delivery, progenitors were successfully engrafted in skeletal muscle for up to 12 weeks, and generated new muscle fibers, restored dystrophin expression and contributed to the satellite cell compartment. These findings highlight the potential application of myogenic progenitors derived from ADSCs to the treatment of muscular dystrophy.