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?T87Q-Globin Gene Therapy Reduces Sickle Hemoglobin Production, Allowing for Ex Vivo Anti-sickling Activity in Human Erythroid Cells.

ABSTRACT: Lentiviral addition of ?T87Q-globin, a modified ?-globin with an anti-sickling mutation, is currently being used in gene therapy trials for sickle cell disease (SCD) and ?-thalassemia patients. ?T87Q-globin interferes with sickle hemoglobin (HbS) polymerization. Here, we generated the SCD mutation in an immortalized human erythroid cell line (HUDEP-2) to investigate the anti-sickling activity of ?T87Q-globin. Sickle HUDEP-2 (sHUDEP-2) cells produced robust HbS after differentiation and sickled under deoxygenated conditions, comparable with SCD CD34+ progeny. Lentiviral transduction provided 9.5-26.8 pg/cell ?T87Q-globin (R2 = 0.83) in a vector copy number (VCN)-dependent manner, resulting in a significant reduction of sickling ratios (R2 = 0.92). Interestingly, ?T87Q-globin transduction markedly reduced endogenous ?S-globin (R2 = 0.84) to an undetectable level (0.4-16.8 pg/cell) in sHUDEP-2 cells, as well as endogenous ?-globin in human CD34+ cell-derived erythroid cells. RNA sequencing (RNA-seq) analysis with ?T87Q-transduced sHUDEP-2 and human CD34+-derived cells revealed activation of inflammation- and proliferation-related programs, suggesting minimal changes in background gene expression except for ?T87Q-globin expression and endogenous ?/?S-globin suppression. In summary, using sHUDEP-2 and CD34+-derived cells, we demonstrated that lentiviral addition of ?T87Q-globin strongly reduced endogenous ?-/?S-globin expression, resulting in an anti-sickling effect. Our findings should be helpful to understand the anti-sickling effects of therapeutic genes in SCD gene therapy.

SUBMITTER: Demirci S

PROVIDER: S-EPMC7210457 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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βT87Q-Globin Gene Therapy Reduces Sickle Hemoglobin Production, Allowing for <i>Ex Vivo</i> Anti-sickling Activity in Human Erythroid Cells.

Demirci Selami S Gudmundsdottir Bjorg B Li Quan Q Haro-Mora Juan J JJ Nassehi Tina T Drysdale Claire C Yapundich Morgan M Gamer Jackson J Seifuddin Fayaz F Tisdale John F JF Uchida Naoya N

Molecular therapy. Methods & clinical development 20200418

Lentiviral addition of βT87Q-globin, a modified β-globin with an anti-sickling mutation, is currently being used in gene therapy trials for sickle cell disease (SCD) and β-thalassemia patients. βT87Q-globin interferes with sickle hemoglobin (HbS) polymerization. Here, we generated the SCD mutation in an immortalized human erythroid cell line (HUDEP-2) to investigate the anti-sickling activity of βT87Q-globin. Sickle HUDEP-2 (sHUDEP-2) cells produced robust HbS after differentiation and sickled u ...[more]

PMID: 32405513

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Project description:Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild-type beta- or gamma-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel nonviral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild-type beta-globin expression cassette into the human genome for sustained expression of beta-globin. We initially constructed a beta-globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken beta-actin promoter (CAGGS) and full-length beta-globin cDNA, as well as truncated forms lacking either the 3' or 3' and 5' untranslated regions (UTRs), to optimize expression of beta-globin. Beta-globin with its 5' UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn-beta-globin plasmids using a minimal beta-globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, ankyrin-1 promoter), IHbetap (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, beta-globin promoter), or HS3betap (HS3 core element from human betaLCR, beta-globin promoter) to establish erythroid-specific expression of beta-globin. Stable genomic insertion of the minimal gene and expression of the beta-globin transgene for >5 months at a level comparable to that of the endogenous gamma-globin gene were achieved using a SB-Tn beta-globin cis construct. Interestingly, erythroid-specific expression of beta-globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient genomic insertion conferring stable, persistent erythroid-specific expression of beta-globin.

Dataset Information

?T87Q-Globin Gene Therapy Reduces Sickle Hemoglobin Production, Allowing for Ex Vivo Anti-sickling Activity in Human Erythroid Cells.

Publications

βT87Q-Globin Gene Therapy Reduces Sickle Hemoglobin Production, Allowing for <i>Ex Vivo</i> Anti-sickling Activity in Human Erythroid Cells.

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