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

Brusson, Mégane; Chalumeau, Anne; Martinucci, Pierre; Romano, Oriana; Félix, Tristan; Poletti, Venerino; Scaramuzza, Samantha; Ramadier, Sophie; Masson, Cécile; Ferrari, Giuliana; Mavilio, Fulvio; Cavazzana, Marina; Amendola, Mario; Miccio, Annarita

doi:10.1016/j.omtn.2023.03.012

Cited by 4 publications

(6 citation statements)

References 63 publications

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“… 13 Brusson et al. 25 reported a bifunctional LVV for SCD that expressed the same β AS3 -globin gene described here combined with an artificial miR to HbS. They observed a higher level of correction of parameters of SCD by this bifunctional vector compared with one expressing only the β AS3 -globin gene.…”

Section: Introductionmentioning

confidence: 72%

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

Hart,

Liu,

Brown

et al. 2024

Molecular Therapy - Methods & Clinical Development

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Section: Introductionmentioning

confidence: 72%

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

Hart,

Liu,

Brown

et al. 2024

Molecular Therapy - Methods & Clinical Development

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“…Furthermore, the development of dual-function LVs combining HBB-like transgene expression with potent shRNAmiRs designed in independent studies to selectively target the mRNA of (a) BCL11A to combine de-repression of HBG with expression of HBB [54,55], (b) HBA2 to emulate an α-thalassemia carrier background [56] and (c) HBB S (β S ) to reduce β S -encoding mRNA (HBB Glu6Val) in sickle cell disease patients [23,57], has paved the way for more targeted, mutation-specific gene therapy approaches to β-thalassemia. Earlier findings by our group based on the RNAi of aberrant HBB IVSI−110(G>A) mRNA in HSPCs from HBB IVSI−110(G>A) β-thalassemia individuals firmly established the aberrant transcript as a rational target for a mutation-specific gene therapy approach [22].…”

Section: Discussionmentioning

confidence: 99%

“…Recent publications have highlighted the design criteria and potential efficacy of same-vector combined transgene and RNApol(II)-based shRNAmiR expression, such as for the silencing of α-globin or of the sickling mutation concurrent with the overexpression of a β-like globin transgene. In the process, RNApol(II)-driven miRNA scaffolds have been studied extensively and been shown to reduce nonspecific toxicities related to shRNA overexpression [23][24][25][26]. Mutation-specific LV enhancement for HBB IVSI−110(G>A) -affected individuals may therefore be achieved by the expression of miRNA-embedded short hairpin RNA (shRNAmiR) sequences targeting HBB mRNA with a view to reducing the potential interference of aberrant mRNA and to enhancing incorporation of endogenous HBB or of vector-derived HBB AS3 into hemoglobin tetramers.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Mono- and Bi-Functional GLOBE-Based Vectors for Therapy of β-Thalassemia by HBBAS3 Gene Addition and Mutation-Specific RNA Interference

Koniali,

Flouri,

Kostopoulou

et al. 2023

Cells

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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.

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“…An interesting development of LV vectors proposed for gene therapy of β-thalassemia is the linkage of the therapeutic β-globin gene to other useful gene sequences. An example of such bifunctional vectors has been described by Brusson et al, using as experimental model system hematopoietic stem and progenitor cells (HSPCs) isolated from sickle-cells disease (SCD) patients ( Brusson et al, 2023 ). In this case, the employed bifunctional LV vector was expressing an anti-sickling β-globin (βAS3-globin) and an artificial microRNA specifically downregulating βS-globin expression ( Brusson et al, 2023 ).…”

Section: Combined Protocols Based On Gene Therapy and Gene Editingmentioning

confidence: 99%

“…An example of such bifunctional vectors has been described by Brusson et al, using as experimental model system hematopoietic stem and progenitor cells (HSPCs) isolated from sickle-cells disease (SCD) patients ( Brusson et al, 2023 ). In this case, the employed bifunctional LV vector was expressing an anti-sickling β-globin (βAS3-globin) and an artificial microRNA specifically downregulating βS-globin expression ( Brusson et al, 2023 ). The aim of this approach was to obtain a miRNA-mediated reduction of the HbS levels, favoring at the same time the incorporation of βAS3 into Hb tetramers.…”

Section: Combined Protocols Based On Gene Therapy and Gene Editingmentioning

confidence: 99%

Combined approaches for increasing fetal hemoglobin (HbF) and de novo production of adult hemoglobin (HbA) in erythroid cells from β-thalassemia patients: treatment with HbF inducers and CRISPR-Cas9 based genome editing

Finotti

Gambari

2023

Front. Genome Ed.

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Genome editing (GE) is one of the most efficient and useful molecular approaches to correct the effects of gene mutations in hereditary monogenetic diseases, including β-thalassemia. CRISPR-Cas9 gene editing has been proposed for effective correction of the β-thalassemia mutation, obtaining high-level “de novo” production of adult hemoglobin (HbA). In addition to the correction of the primary gene mutations causing β-thalassemia, several reports demonstrate that gene editing can be employed to increase fetal hemoglobin (HbF), obtaining important clinical benefits in treated β-thalassemia patients. This important objective can be achieved through CRISPR-Cas9 disruption of genes encoding transcriptional repressors of γ-globin gene expression (such as BCL11A, SOX6, KLF-1) or their binding sites in the HBG promoter, mimicking non-deletional and deletional HPFH mutations. These two approaches (β-globin gene correction and genome editing of the genes encoding repressors of γ-globin gene transcription) can be, at least in theory, combined. However, since multiplex CRISPR-Cas9 gene editing is associated with documented evidence concerning possible genotoxicity, this review is focused on the possibility to combine pharmacologically-mediated HbF induction protocols with the “de novo” production of HbA using CRISPR-Cas9 gene editing.

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

Cited by 4 publications

References 63 publications

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

Evaluation of Mono- and Bi-Functional GLOBE-Based Vectors for Therapy of β-Thalassemia by HBBAS3 Gene Addition and Mutation-Specific RNA Interference

Combined approaches for increasing fetal hemoglobin (HbF) and de novo production of adult hemoglobin (HbA) in erythroid cells from β-thalassemia patients: treatment with HbF inducers and CRISPR-Cas9 based genome editing

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