Erythroid lineage-specific lentiviral RNAi vectors suitable for molecular functional studies and therapeutic applications

Numerous genes exert multifaceted roles in hematopoiesis. Therefore, we generated novel lineage-specific RNA interference (RNAi) lentiviral vectors, H23B-Ery-Lin-shRNA and H234B-Ery-Lin-shRNA, to probe the functions of these genes in erythroid cells without affecting other hematopoietic lineages. The lineage specificity of these vectors was confirmed by 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 any gene, and they also provide superior knockdown of the target gene expression with a single shRNA integration per cell. High-level lineage-specific downregulation of BCL11A and ZBTB7A, two 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 >80% reduction in the target protein levels and up to 40% elevation in the gamma-chain levels in the differentiated erythroid cells. Xenotransplantation of the human CD34(+) cells transduced with H23B-Ery-Lin-shBCL11A LV in immunocompromised mice showed similar to 60% reduction in BCL11A protein expression with similar to 40% elevation of gamma-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 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.

Automated summary

This study presents novel lineage-specific lentiviral RNAi vectors that efficiently reduce target gene expression in erythroid cells, making them suitable for gene therapy of hemoglobinopathies and high-throughput RNAi screening for studying normal and pathological erythropoiesis.