Large-scale discovery of potent, compact and lineage specific enhancers for gene therapy vectors [DNAseq]. Large-scale discovery of potent, compact and lineage specific enhancers for gene therapy vectors [DNAseq]

Regulation of gene expression during cell development and differentiation is chiefly orchestrated by distal noncoding regulatory elements that precisely modulate cell selective gene activity. Gene therapy vectors rely on the cellular and context specificity of regulatory DNA elements to express therapeutic transgenes in the correct location and time. Here, we develop a straight-forward, one-shot approach to screen putative regulatory sequences identified in large-scale epigenomics profiling experiments for precise and programmable control of transgenes encoded within gene therapy viral vectors. We designed a library of 15,000 short sequences (~200bp) derived from a set of developmentally active DHS elements during human ex vivo erythropoiesis and cloned them into a GFP reporter lentiviral vector. In an erythroid progenitor cell line, these elements display a gradient of transcriptional enhancer activity, with some demonstrating equivalent activity to the canonical β-globin μLCR despite a 9-fold smaller size. We show that these elements are both highly cell type restricted and developmental stage specific both in vitro and in vivo. Finally, we replace the μLCR element with one of the novel short enhancers in a β-thalassemia lentiviral therapeutic vector and efficiently correct the thalassemic phenotype in patient-derived HSPCs. More broadly, our approach provides further insights into enhancer biology with wider implications into the development of highly cell type specific and efficacious viral vectors for human gene therapy. Overall design: DNAseq of HUDEP-2 cells transduced with a library of 15,000 short DNA sequences cloned in a GFP-reporter lentiviral vector for assaying transcriptional enhancers. Additionally, We then performed DNase I seq as well as CUT&RUN experiments targeting GATA1, TAL1, H3K27ac and H3K9me3 to identify chromatin features associated with transcriptional enhancer acrivity Finally we performed genetic deletion experiments of two identified enhancer elements and performed DNaseI and RNA-seq to validate deletion of the hypersensitive sites and identify gene expression changes in cis as a result of the element deletion.

细胞发育与分化过程中的基因表达调控，主要由远端非编码调控元件主导，这类元件可精准调控细胞选择性基因活性。基因治疗载体依赖调控DNA元件的细胞与语境特异性，实现在正确的时空位置表达治疗性转基因。本研究开发了一种简便易行的单步筛选方法，用于从大规模表观基因组谱分析实验中鉴定出的推定调控序列中，筛选可精准、可编程调控基因治疗病毒载体所编码转基因的序列。我们设计了一个包含15000条短序列（约200bp）的文库，这些序列源自人类离体红细胞生成过程中一组发育活跃的DNase I超敏位点（DHS）元件，并将其克隆至绿色荧光蛋白（GFP）报告基因慢病毒载体中。在红系祖细胞系中，这些元件展现出梯度化的转录增强子活性，其中部分元件的活性可与经典β球蛋白μ位点控制区（μLCR）相当，尽管其尺寸仅为后者的1/9。研究证实，这些元件在体外与体内环境中均表现出高度的细胞类型限制性与发育阶段特异性。最后，我们在β地中海贫血慢病毒治疗载体中，用一种新型短增强子替换了原有的μLCR元件，并在患者来源的造血干细胞与祖细胞（HSPCs）中有效纠正了地中海贫血表型。从更广泛的视角来看，本研究的方法为增强子生物学研究提供了新的见解，同时对开发高度细胞类型特异性且高效的人类基因治疗病毒载体具有重要应用价值。实验整体设计：将克隆于绿色荧光蛋白（GFP）报告基因慢病毒载体中的15000条短DNA序列文库转导至HUDEP-2细胞后进行DNA测序，以检测转录增强子活性。此外，我们还开展了靶向GATA1、TAL1、H3K27ac及H3K9me3的DNase I测序与CUT&RUN实验，以鉴定与转录增强子活性相关的染色质特征。最后，我们对两个已鉴定的增强子元件进行基因敲除实验，并通过DNase I测序与RNA测序验证超敏位点的敲除情况，同时鉴定元件敲除后顺式作用的基因表达变化。