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Series GSE252157 Query DataSets for GSE252157
Status Public on May 01, 2024
Title Large-scale discovery of potent, compact and lineage specific enhancers for gene therapy vectors [CUT&RUN]
Organism Homo sapiens
Experiment type Genome binding/occupancy profiling by high throughput sequencing
Summary 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.
Contributor(s) Psatha N, Sova P, Georgolopoulos G, Paschoudi K, Iwata M, Bloom J, Ulyanova T, Wang H, Kirtsou A, Vasiloudis N, Wilken MS, Stamatoyannopoulos JA, Yannaki E, Papayanopoulou T, Stamatoyannopoulos G, Vierstra J
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Submission date Dec 27, 2023
Last update date May 01, 2024
Contact name Jeff Vierstra
Organization name Altius Institute for Biomedical Sciences
Street address 2211 Elliot Ave
City Seattle
State/province WA
ZIP/Postal code 98121
Country USA
Platforms (1)
GPL18573 Illumina NextSeq 500 (Homo sapiens)
Samples (2)
GSM7995329 HUDEP2 WT H3K27ac CUT&RUN
This SubSeries is part of SuperSeries:
GSE252163 Large-scale discovery of potent, compact and lineage specific enhancers for gene therapy vectors
BioProject PRJNA1057938

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Supplementary file Size Download File type/resource
GSE252157_RAW.tar 403.7 Mb (http)(custom) TAR (of BW)
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Raw data are available in SRA

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