Evaluation of Mono- and Bi-Functional GLOBE-Based Vectors for Therapy of β-Thalassemia by Gene Addition and Mutation-Specific RNA Interference.

Therapy via the gene addition of the anti-sickling β-globin transgene is potentially curative for all β-hemoglobinopathies and therefore of particular clinical and commercial interest. This study investigates GLOBE-based lentiviral vectors (LVs) for β-globin addition and evaluates strategies for an increased β-like globin expression without vector dose escalation. First, we report the development of a GLOBE-derived LV, GLV2-βAS3, which, compared to its parental vector, adds anti-sickling action and a transcription-enhancing 848-bp transcription terminator element, retains high vector titers and allows for superior β-like globin expression in primary patient-derived hematopoietic stem and progenitor cells (HSPCs). Second, prompted by our previous correction of thalassemia based on RNApol(III)-driven shRNAs in mono- and combination therapy, we analyzed a series of novel LVs for the RNApol(II)-driven constitutive or late-erythroid expression of -specific miRNA30-embedded shRNAs (shRNAmiR). This included bifunctional LVs, allowing for concurrent β-globin expression. LVs were initially compared for their ability to achieve high β-like globin expression in -transgenic cells, before the evaluation of shortlisted candidate LVs in -homozygous HSPCs. The latter revealed that β-globin promoter-driven designs for monotherapy with -specific shRNAmiRs only marginally increased β-globin levels compared to untransduced cells, whereas bifunctional LVs combining miR30-shRNA with β-globin expression showed disease correction similar to that achieved by the parental GLV2-βAS3 vector. Our results establish the feasibility of high titers for LVs containing the full transcription terminator, emphasize the importance of the terminator for the high-level expression of -like transgenes, qualify the therapeutic utility of late-erythroid -specific miR30-shRNA expression and highlight the exceptional potential of GLV2-βAS3 for the treatment of severe β-hemoglobinopathies.