Beta-thalassemia is caused by a large spectrum of genetic mutations in the beta-globin gene. These mutations can be classified as beta 0 and beta +, depending upon whether the corresponding allele leads to no or low beta-globin chain synthesis respectively. Depending on the combination of these specific mutations, patients are classified into three principal groups with no, very low or moderately low beta-globin production (beta 0/0, 0/+ and +/+, respectively). Our research, along with that of others, showed that it is possible to rescue beta-thalassemia in mice by lentiviral-mediated transfer of the human beta-globin gene, its promoter, introns and large elements of the locus control region. Based on these studies, clinical trials have been proposed or are underway. However these original studies did not take in consideration the genotypic variability in beta-thalassemia patients. Our goal is to understand whether the outcome of gene transfer is influenced by the mutations in the beta-globin gene. Based on these studies, we will investigate the correlation between genotype and phenotype and generate more efficient gene transfer vectors for the cure of beta-thalassemia. The original studies utilized mice with deleted beta-globin genes. Therefore, these mice do not reproduce the large spectrum of mutations observed in beta-thalassemia patients. Our preliminary data demonstrate that the type of beta-globin gene mutation has a dramatic effect on the expression of the lentiviral mediated wild-type beta-globin. Specifically, we have found that transduction of erythroid progenitor cells (ErPC) from a subset of beta-thalassemic patients (mostly beta 0/0) leads to high beta- globin protein levels proportional to the amount of vector utilized. However, hemoglobin levels in transduced beta +/+ cells increased only slightly or not at all. In beta 0/+ we observed mixed results. We observed a good correlation between the presence of transcripts insensitive to non-sense mediated mRNA decay (NMD) and inhibition of the translation of the transgenic beta-globin mRNA. We propose to better understand the mechanism by which mutant beta-globin mRNAs regulate the expression or translation of normal beta-globin genes, and utilize this knowledge to generate more effective therapies to treat beta-thalassemia. Our preliminary data already suggests one mechanism which may bypass the deleterious effects of the mutated globin gene on a wild-type allele. As opposed to a parent lentiviral vector which does not increase globin expression in beta +/+ cells, modification of this vector with a genomic element from the ankyrin locus completely reversed the phenotype in beta +/+ ErPCs, achieving high level of hemoglobin synthesis. Based on this and other preliminary data, we hypothesize that mutant beta-globin mRNA compete with the normal beta-globin mRNA for translation. Therefore, we have formulated the following aims:
Aim 1 : To understand the effect of endogenous mutant beta-globin mRNAs on the expression of the transduced normal beta-globin gene.
Aim 2 : To develop novel expression vectors that restore the ability of the lentiviral mediated beta- globin mRNA to be translated.

Public Health Relevance

It is well established that beta-thalassemia is associated with the human beta-globin gene, and that certain mutations in this gene lead to the disease phenotype. There are over 200 known disease-associated mutations in the beta-globin gene. Through various molecular mechanisms (e.g. premature stop codons), these mutations affect the protein product yielding the disease phenotype. One potential approach for the treatment of beta-thalassemia is the introduction of non-mutant mRNAs through gene therapy (lentivirus). Promising results in animal models have shown that it is possible to rescue beta-globin knock-out mice using these approaches. However when this approach is translated to the clinic, only a certain subset of the patients respond to the treatment. We hypothesize that it is the presence of long-lived mutant mRNAs that compete with the viral mRNA for the translational machinery that reduces the effectiveness of the therapeutic approach. With this model, based on the genotype and analysis of the mRNA stability, we aim to predict the potential success of gene therapy given a specific mutation.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Erythrocyte and Leukocyte Biology Study Section (ELB)
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Goldsmith, Jonathan C
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Weill Medical College of Cornell University
Schools of Medicine
New York
United States
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Dong, Alisa C; Rivella, Stefano (2017) Gene Addition Strategies for ?-Thalassemia and Sickle Cell Anemia. Adv Exp Med Biol 1013:155-176
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