Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature

Clinical Investigation Center in Biotherapy, Groupe Hospitalier Universitaire Ouest, Inserm/Assistance Publique-Hôpitaux de Paris, Paris 75015, France.
Nature (Impact Factor: 41.46). 09/2010; 467(7313):318-22. DOI: 10.1038/nature09328
Source: PubMed


The β-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of β-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound β(E)/β(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The β(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated β(E)-globin with partial instability. When this is compounded with a non-functional β(0) allele, a profound decrease in β-globin synthesis results, and approximately half of β(E)/β(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral β-globin gene transfer, an adult patient with severe β(E)/β(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded β-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

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    • "HSC-based gene therapy is gaining traction with a number of clinical trials targeting different conditions (X-SCID, ADA-SCID, ALD, CGD, Fanconi anemia, β-thalassemia) showing encouraging results [2], [3], [6], [27]-[30] and which have implications for curative lifelong treatment. Despite these clinical successes, problems of insertional mutagenesis and therapeutic gene silencing remain to be solved [16]. "
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    ABSTRACT: Hematopoietic Stem Cell (HSC) targeted gene transfer is an attractive treatment option for a number of hematopoietic disorders caused by single gene defects. However, extensive methylation of promoter sequences results in silencing of therapeutic gene expression. The choice of an appropriate promoter is therefore crucial for reproducible, stable and long-term transgene expression in clinical gene therapy. Recent studies suggest efficient and stable expression of transgenes from the ubiquitous chromatin opening element (UCOE) derived from the human HNRPA2B1-CBX3 locus can be achieved in murine HSC. Here, we compared the use of HNRPA2B1-CBX3 UCOE (A2UCOE)-mediated transgene regulation to two other frequently used promoters namely EF1α and PGK in human fetal liver-derived HSC (hflHSC). Efficient transduction of hflHSC with a lentiviral vector containing an HNRPA2B1-CBX3 UCOE-eGFP (A2UCOE-eGFP) cassette was achieved at higher levels than that obtained with umbilical cord blood derived HSC (3.1x; p<0.001). While hflHSC were readily transduced with all three test vectors (A2UCOE-eGFP, PGK-eGFP and EF1α-eGFP), only the A2-UCOE construct demonstrated sustained transgene expression in vitro over 24 days (p<0.001). In contrast, within 10 days in culture a rapid decline in transgene expression in both PGK-eGFP and EF1α-eGFP transduced hflHSC was seen. Subsequently, injection of transduced cells into immunodeficient mice (NOD/SCID/Il2rg-/-) demonstrated sustained eGFP expression for the A2UCOE-eGFP group up to 10 months post transplantation whereas PGK-eGFP and EF1α-eGFP transduced hflHSC showed a 5.1 and 22.2 fold reduction respectively over the same time period. We conclude that the A2UCOE allows a more efficient and stable expression in hflHSC to be achieved than either the PGK or EF1α promoters and at lower vector copy number per cell.
    PLoS ONE 08/2014; 9(8):e104805. DOI:10.1371/journal.pone.0104805 · 3.23 Impact Factor
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    • "Hematopoietic stem cell (HSC) targeted gene therapy is potentially curative for various hereditary and acquired diseases, and recent clinical trials have demonstrated efficacy in disorders in which a selective advantage is conferred upon corrected cells [1], [2], [3], [4], [5], [6]. However, further improvement of transduction strategies for human HSCs remains necessary before widespread application. "
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    ABSTRACT: Efficient ex vivo transduction of hematopoietic stem cells (HSCs) is encumbered by differentiation which reduces engraftment. We hypothesized that inhibiting DNA methyltransferase with decitabine would block differentiation of transduced CD34+ cells under cytokine stimulation and thus improve transduction efficiency for engrafting HSCs. Human CD34+ cells in cytokine-containing media were treated with or without decitabine for 24 or 48 hours, and then these cells were transduced with a GFP-expressing lentiviral vector. Utilizing decitabine pre-treatment for 48 hours, we observed an equivalent percentage of successfully transduced cells (GFP-positivity) and a higher percentage of cells that retained CD34 positivity, compared to no decitabine exposure. Cell proliferation was inhibited after decitabine exposure. Similar results were observed among CD34+ cells from six different donors. Repopulating activity was evaluated by transplantation into NOD/SCID/IL2Rγnull mice and demonstrated an equivalent percentage of GFP-positivity in human cells from decitabine-treated samples and a trend for higher human cell engraftment (measured 20–24 weeks after transplantation), compared to no decitabine exposure. In conclusion, ex vivo decitabine exposure inhibits both differentiation and proliferation in transduced human CD34+ cells and modestly increases the engraftment ability in xenograft mice, while the transduction efficiency is equivalent in decitabine exposure, suggesting improvement of lentiviral transduction for HSCs.
    PLoS ONE 08/2014; 9(8):e104022. DOI:10.1371/journal.pone.0104022 · 3.23 Impact Factor
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    • "As seen in previous gene-therapy clinical studies using murine leukemia virus (MLV) vectors, insertional mutagenesis may cause aberrant clonal proliferation or skewed differentiation. While low genotoxic effects of lentivirus vectors have been demonstrated in recent studies by VIS analysis for up to 2 years in humans (Aiuti et al., 2013; Biffi et al., 2013; Cartier et al., 2009; Cavazzana-Calvo et al., 2010), careful monitoring of the longterm effects by lentivirus vectors is required because genemodified HSCs will be maintained for a lifetime. Genomic VIS patterns of repopulating cells in our test animals largely reflect those of freshly transduced CD34+ HSPCs (acute infection) (Figure S7A). "
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    ABSTRACT: In mice, clonal tracking of hematopoietic stem cells (HSCs) has revealed variations in repopulation characteristics. However, it is unclear whether similar properties apply in primates. Here, we examined this issue through tracking of thousands of hematopoietic stem and progenitor cells (HSPCs) in rhesus macaques for up to 12 years. Approximately half of the clones analyzed contributed to long-term repopulation (over 3-10 years), arising in sequential groups and likely representing self-renewing HSCs. The remainder contributed primarily for the first year. The long-lived clones could be further subdivided into functional groups contributing primarily to myeloid, lymphoid, or both myeloid and lymphoid lineages. Over time, the 4%-10% of clones with robust dual lineage contribution predominated in repopulation. HSPCs expressing a CCR5 shRNA transgene behaved similarly to controls. Our study therefore documents HSPC behavior in a clinically relevant model over a long time frame and provides a substantial system-level data set that is a reference point for future work.
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