@article {3341,
	title = {Erythroid lineage-specific lentiviral RNAi vectors suitable for molecular functional studies and therapeutic applications.},
	journal = {Sci Rep},
	volume = {12},
	year = {2022},
	month = {2022 08 18},
	pages = {14033},
	abstract = {<p>Numerous genes exert multifaceted roles in hematopoiesis. Therefore, we generated novel\&nbsp;lineage-specific RNA interference\&nbsp;(RNAi) lentiviral\&nbsp;vectors, H23B-Ery-Lin-shRNA\&nbsp;and H234B-Ery-Lin-shRNA, to probe the functions of these genes in erythroid cells\&nbsp;without affecting other hematopoietic lineages. The lineage specificity of these vectors was confirmed by\&nbsp;transducing multiple hematopoietic cells to express a fluorescent protein. Unlike the previously reported erythroid lineage RNAi vector, our vectors were designed for cloning the short hairpin RNAs (shRNAs) for\&nbsp;any gene, and they also provide superior knockdown of the target gene expression with a\&nbsp;single shRNA integration per cell. High-level lineage-specific downregulation of BCL11A and ZBTB7A,\&nbsp;two\&nbsp;well-characterized transcriptional repressors of HBG in adult erythroid cells, was achieved with substantial induction of fetal hemoglobin with a single-copy lentiviral vector integration. Transduction of primary healthy donor CD34 cells with these vectors resulted in\&nbsp;\&gt;80\% reduction in the target\&nbsp;protein levels and up to 40\% elevation in the γ-chain levels in the differentiated erythroid cells. Xenotransplantation of the human CD34 cells transduced with H23B-Ery-Lin-shBCL11A\&nbsp;LV in immunocompromised mice showed ~ 60\% reduction in BCL11A protein expression with ~\&nbsp;40\% elevation of γ-chain levels in the erythroid cells derived from the transduced CD34 cells. Overall, the novel erythroid lineage-specific lentiviral RNAi vectors described in this study provide a\&nbsp;high-level knockdown of target gene expression in the erythroid cells, making them suitable for their use in gene therapy for hemoglobinopathies. Additionally, the design of these vectors also makes them ideal for high-throughput RNAi screening for studying normal and pathological erythropoiesis.</p>
},
	keywords = {Animals, Cell Line, Tumor, Cell Lineage, DNA-Binding Proteins, Genetic Vectors, Humans, Lentivirus, Mice, RNA Interference, RNA, Small Interfering, Transcription Factors, Transduction, Genetic},
	issn = {2045-2322},
	doi = {10.1038/s41598-022-13783-0},
	author = {Bagchi, Abhirup and Devaraju, Nivedhitha and Chambayil, Karthik and Rajendiran, Vignesh and Venkatesan, Vigneshwaran and Sayed, Nilofer and Pai, Aswin Anand and Nath, Aneesha and David, Ernest and Nakamura, Yukio and Balasubramanian, Poonkuzhali and Srivastava, Alok and Thangavel, Saravanabhavan and Mohankumar, Kumarasamypet M and Velayudhan, Shaji R}
}
