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In this final of a 5-part Focus Seminar series on precision medicine, we focus on catecholaminergic polymorphic ventricular tachycardia (CPVT). This focus on CPVT allows us to take a “deep dive” and explore the full extent of the precision medicine opportunities for a single cardiovascular condition at a level that was not possible in the preceding articles. As a new paradigm presented in this article, it has become clear that CPVT can occur as either a typical or atypical form. Although there is a degree of overlap between the typical and atypical forms, it is notable that they arise due to different underlying genetic changes, likely exhibiting differing mechanisms of action, and presenting with different phenotypic features. The recognition of these differing forms of CPVT and their different etiologies and mechanisms is an important step toward implementing rapidly emerging precision medicine approaches that will tailor novel therapies to specific gene defects.  相似文献   
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目的:研究不同血清型腺相关病毒 (adeno-associated virus,AAV) 载体介导的外源基因在视网膜中的表达效率, 同时比较AAV载体和两种眼科常用启动子组合后转染小鼠视网膜的表达效率高低,为视网膜色素变性基因治疗选择合适的AAV载体与启动子提供依据。方法:AAV病毒根据衣壳蛋白不同可分为不同血清型,本课题选取视网膜疾病基因治疗中常用的AAV2/2、AAV2/5、AAV2/8和AAV2/9四种血清型AAV载体,并以绿色荧光蛋白 (green fluorescent protein, GFP) 作为报告基因,用GFP的表达强度判断AAV载体介导的外源基因在视网膜中的表达效率。AAV载体纯化后滴度为1.00×10 13 mg/L,注射1 μL至C57BL/6J小鼠视网膜下腔, 于2周取眼球做成冰冻切片,在共聚焦显微镜下观察 GFP在小鼠视网膜各层的表达情况。选取在感光细胞内特异性表达最强的AAV2/8于第4周取眼球冰冻切片继续观察是否能持续稳定表达。随后选取眼科基因治疗最常用的广谱启动子CMV和由CMV增强子与鸡β-肌动蛋白启动子组成的CAG启动子,并构建AAV2/8-GFP-CMV和AAV2/8-GFP-CAG两种不同启动子的病毒载体注射至视网膜下腔,于2周取眼球做成冰冻切片,在共聚焦显微镜下观察不同启动子的AAV2/8在小鼠视网膜各层的表达情况。结果: 注射AAV-GFP后未见典型的术后细菌感染及明显免疫反应。AAV2/2、AAV2/5、AAV2/8和AAV2/9四种血清型AAV载体视网膜下腔注射2周后,AAV2/8和AAV2/9在小鼠视网膜的GFP绿色荧光明显,说明这两种AAV载体转染小鼠视网膜后的表达效率高,而在这两种血清型中,AAV2/8的GFP绿色荧光主要集中在感光细胞内,AAV2/9 在视网膜全层均有表达,说明AAV2/8对视网膜感光细胞特异性更强。对AAV2/8的进一步实验表明在视网膜下腔注射4周后小鼠视网膜的GFP绿色荧光明显,说明AAV2/8载体介导的外源基因能在体内稳定表达。使用CMV启动子时GFP在感光细胞与视网膜色素上皮细胞均有表达,而使用CAG启动子时GFP主要在感光细胞表达。结论:视网膜下腔注射AAV病毒载体可在视网膜细胞内稳定表达报告基因;AAV2/2、AAV2/5、AAV2/8和AAV2/9四种血清型AAV载体中,AAV2/8和AAV2/9在视网膜表达能力最强,AAV2/8对视网膜感光细胞特异性最好;CMV和CAG两种启动子,CAG启动子对感光细胞特异性更高。  相似文献   
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《Molecular therapy》2020,28(9):2073-2082
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《Molecular therapy》2020,28(6):1432-1441
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Vision is the sense that we use to navigate the world around us. Thus it is not surprising that blindness is one of people's most feared maladies. Heritable diseases of the retina, such as age-related macular degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world, collectively affecting as many as one-third of all people over the age of 75, to some degree. For decades, scientists have dreamed of preventing vision loss or of restoring the vision of patients affected with retinal degeneration through drug therapy, gene augmentation or a cell-based transplantation approach. In this review we will discuss the use of the induced pluripotent stem cell technology to model and develop various treatment modalities for the treatment of inherited retinal degenerative disease. We will focus on the use of iPSCs for interrogation of disease pathophysiology, analysis of drug and gene therapeutics and as a source of autologous cells for cell transplantation and replacement.  相似文献   
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Adeno-associated virus(AAV) is an essential instrument in the neuroscientist's toolkit, which allows delivery of DNA to provide labeling with fluorescent proteins or genetic instructions to regulate gene expression. In the field of neural regeneration, the transduction of neurons enables the observation and regulation of axon growth and regeneration, and in the future will likely be a mechanism for delivering molecular therapies to promote sprouting and regeneration after central nervous system injury. Traditional formulations of AAV preparations permit efficient viral transduction under physiologic conditions, but an improved understanding of the mechanistic limitations of AAV transduction may facilitate production of more resilient AAV strains for investigative and therapeutic purposes. We studied AAV transduction in the context of prior exposure of AAV serotype 8(AAV8) to environmental p H within the range encountered during endosomal endocytosis(p H 7.4 to p H 4.4), during which low p H-triggered structural and autoproteolytic changes to the viral capsid are believed to be necessary for endosome escape and virus uncoating. Due to the fundamental nature of these processes, we hypothesized that premature exposure of AAV8 particles to acidic p H would decrease viral transduction of HT1080 cells in vitro, as measured by fluorescent reporter gene expression using high-content imaging analysis. We found that increasingly acidic incubation conditions were associated with concomitant reductions in transduction efficiency, and that quantitative levels of reporter gene expression in transduced cells were similarly decreased. The biggest decrease in transduction occurred between p H 7.4 and p H 6.4, suggesting the possible co-occurrence of a p H-associated event and viral inactivation within that range. Taken together, these findings indicate that exposure of AAV8 to acidic p H for as little as 1 hour is deleterious to transduction ability. Future studies are necessary to understand the p H-associated causative mechanisms involved. This study was approved by the University of Miami Institutional Animal Care and Use Committee, USA(Protocol #18-108-LF) on July 12, 2018.  相似文献   
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《Molecular therapy》2020,28(7):1717-1730
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