Treatment options for patients with CADASIL and large-scale cerebral infarction: mechanical thrombectomy and antiplatelet therapy-A case report.

Background: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an autosomal dominant inherited arterial disease, with lacunar infarction resulting from intracranial small vessel lesions being the most prevalent clinical manifestation of CADASIL. However, large-scale cerebral infarction caused by intracranial non-small vessels occlusion is relatively uncommon, and reports of vascular intervention and long-term antiplatelet drug treatment for patients with CADASIL and large-scale cerebral infarction are rarer. Methods: We reported a 52 year-old male who experienced a significant cerebral infarction due to an occlusion in the second segment of the left middle cerebral artery, 4 months subsequent to being diagnosed with CADASIL. Following the benefit and risk assessment, the patient underwent intracranial vascular thrombectomy and balloon dilation angioplasty. Subsequently, he was administered dual antiplatelet therapy for 3 months, followed by mono antiplatelet therapy. Results: After undergoing intracranial vascular intervention and receiving antiplatelet therapy, significant improvement in the symptoms were observed. The National Institutes of Health Stroke Scale score decreased from 6 to 2 points, and no bleeding lesions were detected on the head computed tomography during regular follow-up visits after discharge. Conclusion: Our case highlights the possibility that patients with CADASIL may also encounter extensive cerebral infarction resulting from stenosis or occlusion of intracranial non-small vessels. Considering the specific circumstances of the patient, intravascular intervention and antiplatelet therapy can be regarded as viable treatment options for individuals with CADASIL.

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.

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.

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.