Coordinated β-globin expression and α2-globin reduction in a multiplex lentiviral gene therapy vector for β-thalassemia

Abstract

A primary challenge in lentiviral gene therapy of b-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVb-sha2, that allows coordinated expression of the therapeutic b A-T87Q -globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the a2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of a-globin chain synthesis ameliorates disease severity in b-thalassemia. We demonstrate that LVb-sha2 reduces a2-globin mRNA expression in erythroid cells while keeping a1-globin mRNA levels unchanged and b A-T87Q -globin gene expression identical to the parent vector. Compared with the first b A-T87Q -globin lentiviral vector that has received conditional marketing authorization, BB305, LVb-sha2 shows 1.7-fold greater potency to improve a/b ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of b-thalassemia and provide an improved benefit/risk ratio regardless of the b-thalassemia genotype.

Interplay between α‐thalassemia and β‐hemoglobinopathies: Translating genotype–phenotype relationships into therapies

“…For instance, pharmacological activation of UPS and autophagy pathways have further exemplified the role of clearing toxic α‐globin protein aggregates to alleviate the manifestation of disease 110 . Gene therapy/RNAi/gene editing strategies have been used to alter the expression of α‐globin while increasing β‐globin expression and reversing the α/β‐globin chain imbalance in β‐thalassemia 146,147,151 . Pharmacological silencing of α‐globin and induction of γ‐globin gene expression has demonstrated synergistic activity without affecting erythroid cell viability and differentiation in cord blood‐derived HSCs 141 .…”

“… 80 , 148 Alternatively, the transduction of HSC with lentiviral (LV) vectors harboring short hairpin RNA (shRNAs) has emerged as an effective delivery strategy for the clinical translation of RNAi‐based therapies. 149 , 150 , 151 , 152 In this strategy, the artificial miRNA scaffold is used to express a shRNA in the cell type of interest. 151 , 152 , 153 , 154 , 155 Such a design principle permits the shRNA to be processed in the same pathway as natural miRNAs and mediate silencing of target mRNA.…”

Novel lentiviral vectors for gene therapy of sickle cell disease combining gene addition and gene silencing strategies

“…Interestingly, our results are in line with a recent study describing a similar LV expressing simultaneously the T87Q β-globin (carrying only one anti-sickling amino acid) and an amiR targeting BCL11A-XL (Pires Lourenco et al, 2021). Of note, in the case of β-thalassemia, this strategy could be combined with the introduction of a second amiR targeting the α-globin, which was recently shown to improve the α/β ratio in erythrocytes derived from β-thalassemia HSPCs (Nualkaew et al, 2021). Another miR-based gene therapy strategy has recently been developed to further boost HbF expression by expressing two amiRs targeting both BCL11A or ZNF410 γ-globin repressors (Liu et al, 2022).…”

The Future of Gene Therapy for Transfusion-Dependent Beta-Thalassemia: The Power of the Lentiviral Vector for Genetically Modified Hematopoietic Stem Cells

“…LVβ-shα2 demonstrates reduction of α2-globin expression while maintaining expression of the therapeutic β A−T87Q -globin gene, which improves α/β-globin balance and decreases cellular damage from unbound α-globin chains in erythroid cells (Figure 2B). Compared with LentiGlobin BB305, LVβ-shα2 also requires a lower VCN of the viral vector for equivalent efficacy, which could improve safety (Nualkaew et al, 2021). This gene therapy approach is promising for curative treatment of the β0/β0 disease, but further studies are required to explore efficacy and reliability in a clinical setting.…”