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Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences

机译:通过基因座控制区域增强子序列的高分辨率映射创造新的β-珠蛋白表达慢病毒载体

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摘要

Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis -regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic βsupAS3/sup-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types.
机译:造血干细胞基因治疗是治疗造血系统疾病的有希望的方法。当包括在慢病毒载体中,不妨碍包装或转导效率的CIS -Regulatory元素的组合已被证明是挑战性的。在这项研究中,我们部署了LV-MPRA(基于慢病毒载体的,大规模并行报告器测定),一种方法,其同时分析成千上万的合成DNA片段,以识别近碱基对的序列 - 内在和谱系特异性的增强子功能解析度。我们展示了LV-MPRA的力量阐明了人β-珠蛋白基因菌对照区域的先前未知的内在增强剂序列的界限。我们的方法促进了新型治疗性β As3 -globin慢病毒载体的快速组装,含有能够校正镰状细胞疾病的小鼠模型的强谱系特异性重组控制元素。 LV-MPRA可用于映射任何基因组基因座,用于增强剂活性,并促进治疗载体的快速发展,用于治疗造血系统或其他特异组织和细胞类型的疾病。

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