Gene Therapy of the Hemoglobinopathies

Abstract

Sickle cell disease and the ß-thalassemias are caused by mutations of the ß-globin gene and represent the most frequent single gene disorders worldwide. Even in European countries with a previous low frequency of these conditions the prevalence has substantially increased following large scale migration from Africa and the Middle East to Europe. The hemoglobin diseases severely limit both, life expectancy and quality of life and require either life-long supportive therapy if cure cannot be achieved by allogeneic stem cell transplantation. Strategies for ex vivo gene therapy aiming at either re-establishing normal ß-globin chain synthesis or at re-activating fetal γ-globin chain and HbF expression are currently in clinical development. The European Medicine Agency (EMA) conditionally licensed gene addition therapy based on lentiviral transduction of hematopoietic stem cells in 2019 for a selected group of patients with transfusion dependent non-ß° thalassemia major without a suitable stem cell donor. Gene therapy thus offers a relevant chance to this group of patients for whom cure has previously not been on the horizon. In this review, we discuss the potential and the challenges of gene addition and gene editing strategies for the hemoglobin diseases.

Figure 1

Ex vivo gene therapy for hemoglobinopathies. Stem cells are mobilized by G-CSF and/or plerixafor (1) and harvested by leukapheresis (2). Alternatively, bone marrow without preceding mobilization can be harvested. Stem cells are enriched from the apheresis product by positive selection for CD34 (3). The vector is manufactured under GMP-conditions (4) and incubated with the purified stem cells (5). After the product has been subjected to rigorous quality controls and is released (6), the patient is treated with myeloablative chemotherapy (7). The manipulated stem cell product is applied either intravenously or intraosseously (8).

Figure 2

Selected milestones of ex vivo gene therapy for hematologic disorders.

Figure 3

Vectors used for gene addition. A: The vector used for the first clinical application of gene transfer into human hematopoietic stem cells. This vector was based on the Moloney murine leukemia virus. LTR- long terminal repeat, ψ⁺ extended packaging signal; ADA, human ADA cDNA; SV, SV40 early region promoter; NEO, neomycin resistance gene. The viral LTR sequencing mediate high-level expression of the transgene and genomic integration in the vicinity of transcriptionally active genes, resulting in the risk of oncogene activation. B: The self-inactivating lentiviral vector used for manufacturing of Zynteglo, the first approved gene therapy for ß-thalassemia.^, Hybrid LTR - hybrid long terminal repeat: the CMV enhancer and promoter replace the HIV U3 enhancer and promoter (allowing for Tat-independent translation), HIV R/U5 regions are preserved; ψ⁺ - extended packaging signal (carrying 2 stop codons to prevent readthrough from the CMV promoter); cPPT - central polypurine track (facilitates nuclear import of the viral preintegration complex); RRE – Rev responsive element (facilitates nuclear export of viral RNA in the packaging cell lines); I, II, III – human ß globin exons (the reverse orientation enables maintenance of the intron structure of the transgene; the Thr87Gln mutation confers anti-sickling properties without affecting hemoglobin function and enables tracking of therapeutic gene expression; a deletion in intron 2 is indicated by a triangle); HS2, HS3, HS4 – hypersensitive sites (HS) of the human ß-globin locus control region (stimulating ß-globin expression via looping to the ß-globin promoter); ppt – polypurine tract; ΔU3 – HIV LTR U3 region carrying a 400 bp deletion that results in self-inactivation after transduction (because the 3’ UTR serves as template for both LTRs); R – HIV LTR R region (including viral polyA signal); pA - rabbit ß-globin polyA signal (as additional safety measure).

Figure 4

Alternatives to gene addition for correction of hemoglobinopathies. A: In cells with active HDR (homology directed repair) double strand breaks introduced by nucleases can be repaired with the help of a template, resulting in the correction of pathogenic mutations. Because a specific template is needed for each mutation and because HDR is not active in hematopoietic stem cells, this approach is not in clinical use for the hemoglobinopathies. An alternative technique of direct correction of pathogenic mutations is “base editing” by deaminases that result in C to T or G to A changes in a sequence context defined by a guide RNA. While this technique does not require any endogenous DNA repair activity, it suffers from limited specificity and thus from off target effects. B: Induction of γ-globin expression by introduction of mutations that activate the expression of γ-globin as can be naturally observed in HPFH (hereditary persistence of fetal hemoglobin), presumably by abrogating BCL11A binding.^, C: Induction of γ-globin expression by disruption of an erythroid-specific BCL11A enhancer.^,– Approaches B and C can be used for all ß-hemoglobinopathies and rely on non-homologous end joining or microhomology-mediated end joining that are active in hematopoietic stem cells. D: Induction of γ-globin expression by depletion of BCL11A via shRNA expressed from a lentiviral vector.^, E: Induction of γ-globin expression by forced chromatin looping mediated by a Zink-Finger/Ldb1-fusion protein expressed from a lentiviral vector. Approaches D and E can be used in principle for all ß-hemoglobinopathies and do not rely on endogenous DNA repair activity.