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将基于基因治疗的SOD1沉默引入人体试验：一种高效、无靶点和生物标记物支持的fALS策略
Bringing gene therapy based SOD1 silencing towards human trials: A highly efficacious, off-target free and biomarker supported strategy for fALS |
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| 课程网址: |
http://videolectures.net/encals2017_scarrott_gene_therapy/
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| 主讲教师: |
Joseph M. Scarrott
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| 开课单位: |
雪菲尔大学
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| 开课时间: |
2017-07-21
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| 课程语种: |
英语
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| 中文简介: |
目标：1。在SOD1-G93A小鼠模型中使用临床就绪载体评估scAAV9 shRNA介导的SOD1沉默的治疗效果。2.研究CSF SOD1蛋白水平的测量作为有效剂量和SOD1敲除效果的生物标志物。3.研究和评估靶向hSOD1的治疗性shRNA对基因表达的miRNA样序列特异性脱靶效应。方法：在出生后第1天（P1，发病前）和第40天（发病前）对动物进行治疗。scAAV9-hSOD1si或加扰对照scAAV9-hSOD1si病毒载体通过大池注射递送。使用行为测试对小鼠进行测试，包括每周的rotarod跑步、神经评分和猫道步态分析。每周收集体重。通过ELISA分析CSF，确定治疗后SOD1蛋白水平。通过全基因组微阵列分析从表达野生型或G93A hSOD1转基因的HEK293T细胞系中转导的等基因TET诱导FLP中提取的总RNA，以阐明潜在的脱靶效应。结果：在p1和P40大池注射scAAV9-hSOD1si可改善小鼠模型的运动性能，并使中位生存期分别延长42%和13%。与乱序对照组相比，P1治疗也延迟了疾病的发病，并显著降低了ELISA检测到的CSF中的SOD1蛋白。使用人类细胞进行的体外试验表明，该构建物不会产生靶向效应。结论：这些发现显然是研究的重要转化方面，1）体外人类细胞缺乏可观察到的靶外效应，表明治疗性shRNA结构对hSOD1具有特异性，不太可能在患者中引发有害的基因调控；2）大多数临床方法缺乏与治疗益处相关的可靠生物标记物。由于从患者收集和分析脑脊液是一个相对简单的过程，因此测量接受SOD1基因敲除治疗的患者脑脊液中的SOD1蛋白有可能成为非常有用和有用的治疗效果生物标志物。因此，我们的方法加强了已经有希望的基因治疗策略在SOD1连锁家族性ALS中的临床应用，在以前的研究中缺乏额外的翻译方面。
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| 课程简介: |
Aims: 1. To evaluate the therapeutic efficacy of scAAV9-shRNA mediated SOD1 silencing in the SOD1-G93A mouse model using a clinic ready vector. 2. To investigate the measurement of CSF SOD1 protein levels as a biomarker of effective dosing and efficacy of SOD1 knockdown. 3. To investigate and evaluate miRNA-like sequence specific off-target effects on gene expression resulting from the viral delivery of a therapeutic shRNA targeting hSOD1. Methods: Animals were treated at postnatal day 1 (P1, pre-onset) and P40 (onset). scAAV9-hSOD1si or scrambled control scAAV9-hSOD1ssi viral vectors were delivered via cisterna magna injection. Mice were tested using behavioural tests including weekly rotarod runs, neurological scoring and CatWalk gait analysis. Weekly body weight was also collected. Analysis of CSF by ELISA was used to determine SOD1 protein levels after treatment. Total RNA extracted from transduced isogenic Tetinducible FLP-In HEK293T cell lines expressing either wildtype or G93A hSOD1 transgenes was analysed by whole genome microarray to elucidate potential off target effects. Results: Cisterna magna injection of scAAV9-hSOD1si at P1and P40 improves motor performance and extends median survival in the mouse model by 42% and 13% respectively. Treatment at P1 also delays disease onset and significantly reduces SOD1 protein in the CSF as detected by ELISA, compared to scrambled controls. An in vitro assay using human cells demonstrated that the construct generated no off-target effects. Conclusions: These findings are evidently important translational aspects of the study, in as much as: 1) a lack of observable off-target effects in human cells in vitro suggest the therapeutic shRNA construct is specific to hSOD1 and unlikely to elicit detrimental gene regulation in patients and 2) most clinical approaches suffer from lack of reliable biomarkers that correlate with treatment benefit. As the collection and analysis of CSF from patients is a relatively simple procedure, the measurement of SOD1 protein in CSF from patients undergoing SOD1 knockdown therapy has the potential to be a very useful and informative biomarker of therapeutic efficacy. Our approach therefore reinforces an already promising gene therapy strategy for clinical application in SOD1- linked familial ALS with additional translational aspects absent from previous studies.
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| 关 键 词: |
miRNA; 全基因组; 靶向
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| 课程来源: |
视频讲座网
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| 数据采集: |
2021-12-25：zkj
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| 最后编审: |
2021-12-25：zkj
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| 阅读次数: |
37
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