Morgan Richard A, Ma Feiyang, Unti Mildred J, Brown Devin, Ayoub Paul George, Tam Curtis, Lathrop Lindsay, Aleshe Bamidele, Kurita Ryo, Nakamura Yukio, Senadheera Shantha, Wong Ryan L, Hollis Roger P, Pellegrini Matteo, Kohn Donald B
Charles R. Drew University of Medicine and Science, Los Angeles, CA 90059, USA.
Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Mol Ther Methods Clin Dev. 2020 Apr 18;17:999-1013. doi: 10.1016/j.omtm.2020.04.006. eCollection 2020 Jun 12.
Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of -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 β-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.
造血干细胞基因治疗是治疗造血系统疾病的一种很有前景的方法。事实证明，识别包含在慢病毒载体中时不会阻碍包装或转导效率的调控元件组合具有挑战性。在本研究中，我们采用了LV-MPRA（基于慢病毒载体的大规模平行报告基因检测），该方法可同时并行分析数千个合成DNA片段，以近碱基对分辨率识别序列内在和谱系特异性增强子功能。我们展示了LV-MPRA在阐明人类β-珠蛋白基因座控制区先前未知的内在增强子序列边界方面的能力。我们的方法促进了新型治疗性β-珠蛋白慢病毒载体的快速组装，这些载体含有能够纠正镰状细胞病小鼠模型的强大谱系特异性重组控制元件。LV-MPRA可用于绘制任何基因组位点的增强子活性图谱，并有助于快速开发用于治疗造血系统或其他特定组织和细胞类型疾病的治疗载体。
