Bruno Dalle

Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia

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 βE/β0-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The βE-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 βE-globin with partial instability. When this is compounded with a non-functional β0 allele, a profound decrease in β-globin synthesis results, and approximately half of βE/β0-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 βE/β0-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.

Stress-induced aberrant splicing of TSG101: association to high tumor grade and p53 status in breast cancers

The TSG101 gene, identified through insertional mutagenesis, is localized in a region that exhibits LOH in human cancers, suggesting that TSG101 might be a tumor suppressor gene. Numerous studies have then shown the presence of abnormal transcripts in various tumors which appear to result from aberrant splicing of the gene, rather than from intragenic deletions. Moreover, many studies demonstrated that these aberrantly spliced transcripts were not found in matched normal tissues. We have analysed TSG101 transcripts in 85 breast cancer samples and found that abnormal splicing of the gene is tightly correlated with tumor grade and p53 mutation. In addition, stress induced the appearance of these abnormal transcripts in primary lymphocytes. Hence, TSG101 splicing defects, while unrelated to the oncogenic process per se, could reflect the cellular environment of the tumor cells. The proposed role of stress and hypoxia to select p53 mutant cells could account for the tight association with p53 status.

Modulation of transduced erythropoietin expression by iron

OBJECTIVE Future prospects for gene therapy of chronic anemias involve expression of the erythropoietin transgene, which is regulated by oxygen tension. However, other factors such as cytokines or the iron load of erythropoietin-expressing cells can concomitantly modulate transgene expression, as shown for the expression of the endogenous erythropoietin gene in human cell lines and in animals. We tested the effects of iron overload or depletion on the expression of the mouse erythropoietin transgene (cDNA), driven by the hypoxia-regulated phosphoglycerate kinase 1 promoter. MATERIALS AND METHODS Retrovirally transduced mouse cells (C3H fibroblasts or C2C12 myoblasts) were cultured in normoxia (room air, O2: 21%) or hypoxia (O2: 1.5%) in the presence or absence of hemin (an iron donor) or deferiprone (an iron chelator), both of which easily enter the cell. RESULTS Hemin inhibited the hypoxia-induced expression of the transgene. In contrast, deferiprone enhanced the hypoxia-induced expression of the erythropoietin transgene and induced its expression in normoxia. CONCLUSION These results show that, in addition to oxygen partial pressure, the intracellular iron content is critical in the modulation of hypoxia-regulated erythropoietin transgene expression.

464. RCL-Pooling Assay: A Simplified Method for the Detection of Replication Competent Lentiviruses in Vector Batches Using Sequential Pooling

Non-replicative recombinant HIV-1 derived lentiviral vectors (LV) are increasingly used in gene therapy clinical trials for various genetic diseases, infectious diseases or cancer. Before they are used in man, preparations of LV must undergo extensive biochemical and biological quality control testing. In particular, the legislation stipulates that the absence of replication-competent lentiviruses (RCL) must be demonstrated with suitable methods, on representative fractions of batches. Current standard and widely used methods based on cell culture are challenging because high titers of vector batches achieved translate into high volumes of cell culture that have to be tested. Since vector batch titers and sizes are continuously-increasing due to the improvement of production and purification methods, it is necessary to modify the current cell culture method. Here, we propose a practical optimization of the p24-decrease-based culture assay developed by Escarpe et al. (2003) using a pairwise pooling strategy enabling the test of higher vector inoculum volumes. These modifications significantly decrease material handling, operator time, leading to a cost effective method, while maintaining optimal sensibility of the RCL testing. This optimized RCL-pooling assay ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.