Therapeutic haemoglobin synthesis in β-thalassaemic mice expressing lentivirus-encoded human β-globin

Abstract

The stable introduction of a functional beta-globin gene in haematopoietic stem cells could be a powerful approach to treat beta-thalassaemia and sickle-cell disease. Genetic approaches aiming to increase normal beta-globin expression in the progeny of autologous haematopoietic stem cells might circumvent the limitations and risks of allogeneic cell transplants. However, low-level expression, position effects and transcriptional silencing hampered the effectiveness of viral transduction of the human beta-globin gene when it was linked to minimal regulatory sequences. Here we show that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human beta-globin gene together with large segments of its locus control region. In long-term recipients of unselected transduced bone marrow cells, tetramers of two murine alpha-globin and two human betaA-globin molecules account for up to 13% of total haemoglobin in mature red cells of normal mice. In beta-thalassaemic heterozygous mice higher percentages are obtained (17% to 24%), which are sufficient to ameliorate anaemia and red cell morphology. Such levels should be of therapeutic benefit in patients with severe defects in haemoglobin production.

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

“…5 To overcome this obstacle, use of epigenetic regulatory elements such as insulators, matrix attachment regions and locus control regions have been attempted. 20,21,[30][31][32][33] In this study, we found that sea urchin ArsI protects an HIV-based vector from long-term silencing in cell type-and orientation-dependent manner. ArsI was effective in HL-60 and CCE cells, but not in other cell lines.…”

Physiological and tissue-specific vectors for treatment of inherited diseases

“…[98][99][100][101][102] However, the presence of large regulatory sequences in oncoretroviral vectors was limited due to its limited packaging capacity (8 kb) and to the presence of RNA sequences that favor RNA processing and therefore block the production of full-length vector-RNA molecules in producer cell lines. The first two encouraging examples of lineage-specific expression of the b-globin transgene and therapeutic efficacy were provided by May et al 103 for b-thalassemia and by Pawliuk et al 104 for sickle cell disease using HIV-1-based LVs. LVs allowed the incorporation of larger LCR and globin sequences (thanks to the Rev/R-responsive element) and a more efficient transduction of hematopoietic progenitors.…”

“…LVs allowed the incorporation of larger LCR and globin sequences (thanks to the Rev/R-responsive element) and a more efficient transduction of hematopoietic progenitors. Taking advantage of the LVs potential, May et al 103 Other modifications to improve safety and efficiency of the therapeutic vectors consisted in the deletion of 400 bp of the LTR U3 (this avoided vector expression from the 5¢ LTR after integration) to generate a self-inactivated lentiviral vector (SIN-LV) and the inclusion of the central polypurine tract_DNA of HIV-1 (cPPT), involved in facilitating gene transfer to quiescent cells 105 (Figure 2a). An additional improvement was provided by using SIN-LV containing the The latest vectors drive the expression of the globin gene using its own promoter and polyadenylation signals (blue), enhancers (red) and locus control regions (LCR).…”

Comparative analysis of FV vectors with human α- or β-globin gene regulatory elements for the correction of β-thalassemia

“…2,[25][26][27][28] The combination of core sequences from the HS2, HS3 and HS4 elements led to the construction of the first retroviral vectors with high titer, vector stability and efficient expression of the human b-globin transgene. 29,30 On another approach that was implemented during the same time, the HS40 regulatory element from the a-globin locus that drives tissue-specific globin gene expression, 31,32 was used to support expression of different globin genes. 8,33 In this study, we have used FV vectors to compare the efficiency of b-globin expression from a short (À127) b-globin gene promoter 33 regulated by either the HS40 a-globin enhancer or the HS2/HS3 regulatory sequences from the b-globin LCR.…”

“…Retroviral vectors were initially explored as therapeutic vehicles but were met with unsurpassed obstacles in titers, stability and expression. 3,29,39,40 Lentiviral globin vectors were then successfully explored in the preclinical mouse and more recently in the clinical arena; 18,30 however, globin expression was also accompanied by the unwanted overexpression of a gene (HMGA2) that may culminate to a malignant disorder. FV vectors are the less well explored genus of retroviruses and their therapeutic potential has not yet been explored in the clinic; however, we show that FV vectors can provide therapeutic levels of hemoglobin in the thal3 mouse model and, similarly to the lentiviral vectors, 4 with low VCNs (o2).…”