Evaluation of Mono- and Bi-Functional GLOBE-Based Vectors for Therapy of β-Thalassemia by HBBAS3 Gene Addition and Mutation-Specific RNA Interference
Author:
Koniali Lola1ORCID, Flouri Christina2, Kostopoulou Markela I.1, Papaioannou Nikoletta Y.1ORCID, Papasavva Panayiota L.1ORCID, Naiisseh Basma1, Stephanou Coralea1ORCID, Demetriadou Anthi1ORCID, Sitarou Maria3, Christou Soteroula4, Antoniou Michael N.2ORCID, Kleanthous Marina1ORCID, Patsali Petros1ORCID, Lederer Carsten W.1ORCID
Affiliation:
1. Department of Molecular Genetics Thalassaemia, The Cyprus Institute of Neurology & Genetics, 6 Iroon Avenue, 2371 Nicosia, Cyprus 2. Gene Expression and Therapy Group, Department of Medical and Molecular Genetics, King’s College London, Guy’s Hospital, London SE1 9RT, UK 3. Thalassemia Clinic Larnaca, Larnaca General Hospital, 6301 Larnaca, Cyprus 4. Thalassemia Clinic Nicosia, Archbishop Makarios III Hospital, 1474 Nicosia, Cyprus
Abstract
Therapy via the gene addition of the anti-sickling βAS3-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 βAS3-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 HBBIVSI−110(G>A) 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 HBBIVSI−110(G>A)-specific miRNA30-embedded shRNAs (shRNAmiR). This included bifunctional LVs, allowing for concurrent βAS3-globin expression. LVs were initially compared for their ability to achieve high β-like globin expression in HBBIVSI−110(G>A)-transgenic cells, before the evaluation of shortlisted candidate LVs in HBBIVSI−110(G>A)-homozygous HSPCs. The latter revealed that β-globin promoter-driven designs for monotherapy with HBBIVSI−110(G>A)-specific shRNAmiRs only marginally increased β-globin levels compared to untransduced cells, whereas bifunctional LVs combining miR30-shRNA with βAS3-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 HBB transcription terminator, emphasize the importance of the HBB terminator for the high-level expression of HBB-like transgenes, qualify the therapeutic utility of late-erythroid HBBIVSI−110(G>A)-specific miR30-shRNA expression and highlight the exceptional potential of GLV2-βAS3 for the treatment of severe β-hemoglobinopathies.
Funder
the European Regional Development Fund and the Republic of Cyprus through the Research and Innovation Foundation the Cyprus Institute of Neurology & Genetics project “New Infrastructure for the Diagnosis and Therapy of Patients” was funded by Norway Grants
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