Abstract
Contract No.: 306201; EC contribution: 5,020,000; Total costs: 6,681,623.27; Starting date: 01/11/2012; Duration: 48 months
Background and Objectives
The β-thalassemia syndromes are a group of severe and rare anemias with monogenic inheritance, a complex systemic phenotype, and several treatment-related complications, caused by more than 300 mutations of the β-globin gene. Novel therapeutic protocols, most of which are based on still experimental treatments, show great promise but significant variability of success between patients. These strategies include chemical/molecular induction of the endogenous β-like γ-globin gene, restoration of clinically relevant β-globin levels by gene therapy, or genetic correction of the abnormal β-globin gene. In addition, a small number of modifiers with significant impact on disease penetrance, severity, and efficacy of treatments are known, but most remain elusive.
Improvements of existing treatment regimens and optimization and application of novel treatments will critically depend on the characterization of additional disease modifiers and the stratification of patients for customized clinical management. This requires extensive analyses based on “OMICS,” an English-language neologism that refers to different but connected fields in molecular biology and biochemistry, such as genomics, transcriptomics, exomics, proteomics, and metabolomics. The major objective of OMICS is a collective characterization of pools of biological molecules (gene sequences, transcripts, proteins, and protein domains) controlling biological structures, functions, and dynamics, including several involved in pathological conditions.
One of the most interesting observations of genomics in β-thalassemia is the association between genomic sequences and high fetal hemoglobin (HbF) levels, in consideration of the fact that high HbF levels are usually associated with milder forms of β-thalassemia. Related to this issue is the possibility to predict response to different therapeutic protocols on the basis of genomic analyses. For instance, three major loci (the Xmn1-HBG2 single-nucleotide polymorphism, the HBS1L-MYB intergenic region on chromosome 6q, and BCL11A) contribute to high HbF production. Pharmacogenomic analysis of the effects of hydroxyurea on HbF production in a collection of β-thalassemia and sickle cell disease patients allowed the identification of genomic signatures associated with high HbF. Therefore, it can be hypothesized that genomic studies might predict the response of patients to treatments based on hydroxyurea, which is at present the most used HbF inducer in pharmacological therapy of β-thalassemia.
Transcriptomic/proteomic studies allowed the identification of the zinc finger transcription factor B-cell lymphoma/leukemia 11A (BCL11A) as the major repressor of HbF expression. The field of research on γ-globin gene repressors (including BCL11A) is of top interest, since several approaches can lead to pharmacologically mediated inhibition of the expression of γ-globin gene repressors, leading to γ-globin gene activation. Among these strategies, we underline direct targeting of the transcription factors by aptamers or decoy molecules, as well as inhibition of the mRNA coding γ-globin gene repressors with shRNAs, antisense molecules, peptide nucleic acids, and microRNAs.
Approach and Methodology
THALAMOSS aims at developing a universal set of markers and techniques for stratification of β-thalassemia patients into treatment subgroups for (1) onset and frequency of blood transfusions, (2) choice of iron chelation, (3) induction of fetal hemoglobin, and (4) prospective efficacy of gene therapy. THALAMOSS is organized in the following work packages: WP1 (recruitment, patient characterization, and development of culture technologies for erythroid precursor cells); WP2 (OMICS analyses); WP3 (novel therapeutic approaches); WP4 (data management and analysis); WP5 (dissemination and exploitation); WP6 (regulatory and ethics issues); WP7 (program management).
Main Findings
The major findings and novel achievements are related to the work packages WP1–WP4. As far as WP1, the first list of recruited β-thalassemia patients with characterized genotype/phenotype was delivered. This is crucial for future activities employing blood sampling, culturing of erythroid cells, and isolation of genomic DNA, RNA, and protein. The list of patient to be recruited includes 1140 entries. The analysis of the most frequent genotypes and phenotypes indicates that homozygous patients are 138 β039/β039, 364 β+IVSI-110/β+IVSI-110, and 36 β+IVSI-6/β+IVSI-6. Double heterozygous patients are 55 β039/β+IVSI-110, 146 β+IVSI-6/β+IVSI-110, and 65 β0IVSI-1/β+IVSI-110. The list also includes 29 homozygous sickle-cell anemia (HbS/HbS) patients.
The HbS genotype was associated in 24 cases with β039, in 49 cases with β+IVSI-110, in 9 cases with β+IVSI-6, and in 12 cases with β0IVSI-1. Within WP1 we validated three protocols for isolation and culturing of erythroid precursor cells, named protocol A, protocol B, and protocol C. These cell culture strategies will be used by the THALAMOSS network for in vitro production of erythroid cells. The steps common to all three methods are drawing of peripheral blood (20–40 ml), Ficoll separation of mononuclear cells, and washing. In protocol A, the mononuclear cells are cultured according to the two-stage procedure first published by the Fibach's group. 1 In THALAMOSS protocol B, the isolation of CD34+ cells is performed using the BIOCEP advanced platform for cell separation and its patented Cell Enrichment Process.
Finally, THALAMOSS protocol C was validated for the best efficient culturing, expansion, storage (frozen), subculturing, transduction with therapeutic vectors, and differentiation with HbF inducers. Using this protocol, dry-ice shipments have been performed for centralized wide-genomics, transcriptomics, and proteomics analysis. Protocol C is based on CD34 cell separation using immunomagnetic beads, plating, and expanding at low density in StemSpan medium supplemented with StemSpan CC-100 cytokine cocktail, erythropoietin, dexamethasone, and penicillin/streptomycin. This protocol allowed freezing of cells from β-thalassemia patients to generate BioBanks. The first release of the THALAMOSS β-thal BioBank has been completed and validated. At present, the BioBank is located at Department of Life Sciences and Biotechnology, University of Ferrara, and consists of a total of more than 700 vials, comprising cryo-preserved cells from more than 70 patients. The most common genotypes are β039/β039, β039/β+IVSI-110, and β+IVSI-110/β+IVSI-110.
As far as WP2, genomic DNA samples from 52 β-thalassemia patients have been used for identification of β-globin gene mutations, polymorphisms of the β-globin genes, the XmnI polymorphism of the promoter of the fetal G-γ-globin gene, and polymorphisms of the BCL11A and HBS1L-MYB loci, which are disease modifiers through their role in fetal γ-globin expression. Standardized protocols for functional assays were developed, as they are critical to avoid center-to-center differences, which might create problems in the analysis of results obtained not only in studies based on transcriptomic and proteomic analyses, but also in studies focusing on the screening and characterization of novel inducers of fetal hemoglobin and of gene therapy interventions. The standardization of functional analyses has been carried out and includes the isolation of biomaterials for central analyses as well as the common local use of RT-qPCR for mRNA quantification, HPLC for hemoglobin determination, and the lentiviral transduction procedure itself as a prerequisite for the analysis of gene therapy efficacy.
As far as WP3, results were already achieved of key interest for the THALAMOSS network. The major results were obtained in the following fields of investigation: (1) results relevant for studies on HbF induction; (2) results relevant for the development of gene therapy; (3) results relevant for studies on read-through correction of stop-codon mutations; (4) novel options/trials.
As far as WP4, an analysis of data management in the THALAMOSS project was provided. We focused on the initial stage that encompasses data acquisition by the partner medical facilities, data de-identification, export from the facilities of origin to a central database, and distributed data presentation for later research on β-thalassemia. The following issues have been addressed: (1) external and internal requirements, (2) patient privacy; (3) collected data structure; (4) data-collecting application. First, we have proceeded to create a fully functional and ready-to-use gathering application. Based on the THALAMOSS workflow, the next step was to centralize the obtained data into one major database and provide a suitable interface for data presentation and distribution.
Expected Outcome
The final outcome and major impact of THALAMOSS are the provision of novel biomarkers for distinct treatment subgroups in β-thalassemia (500–1000 samples from participating medical centers), identified by combined genomics, proteomics, transcriptomics, and tissue culture assays; the development of new or improved products for the cell isolation; characterization and treatment of β-thalassemia patients; and the establishment of routine techniques for detection of these markers and stratification of patients into treatment groups. Translation of these activities into the product portfolio and R&D methodology of participating SMEs will be a major boost for them as well as for the field. THALAMOSS tools and technologies will (1) facilitate identification of novel diagnostic tests, drugs, and treatments specific to patient subgroups and (2) guide conventional and novel therapeutic approaches for β-thalassemia, including personalized medical treatments.
