The influence of genotype on the outcome of gene transfer in beta-thalassemia

描述（由申请人提供）：β-地中海贫血是由β-珠蛋白基因中的大量基因突变引起的。这些突变可以分为β 0和β +，这取决于相应的等位基因是否分别导致没有或低β-珠蛋白链合成。根据这些特定突变的组合，患者被分为三个主要组，没有，非常低或中度低β-珠蛋白生产（β 0/0，0/+和+/+，分别）。我们的研究与其他人的研究沿着表明，通过慢病毒介导的人β-珠蛋白基因、其启动子、内含子和基因座控制区的大元件的转移，有可能挽救小鼠中的β-地中海贫血。基于这些研究，已经提出或正在进行临床试验。然而，这些原始研究没有考虑β-地中海贫血患者的基因型变异性。我们的目标是了解基因转移的结果是否受到β-珠蛋白基因突变的影响。基于这些研究，我们将研究基因型和表型之间的相关性，并产生更有效的基因转移载体用于治疗β-地中海贫血。最初的研究使用了缺失β-珠蛋白基因的小鼠。因此，这些小鼠不会重现在β-地中海贫血患者中观察到的大范围突变。我们的初步数据表明，β-珠蛋白基因突变的类型对慢病毒介导的野生型β-珠蛋白的表达具有显着的影响。具体地，我们已经发现，来自β-地中海贫血患者亚组（主要是β 0/0）的红系祖细胞（ErPC）的转导导致与所用载体的量成比例的高β-珠蛋白水平。然而，转导的β +/+细胞中的血红蛋白水平仅略微增加或根本没有增加。在beta 0/+中，我们观察到混合结果。我们观察到对无义介导的mRNA衰变（NMD）不敏感的转录物的存在与转基因β-珠蛋白mRNA的翻译的抑制之间存在良好的相关性。我们建议更好地了解突变β-珠蛋白mRNA调节正常β-珠蛋白基因表达或翻译的机制，并利用这些知识来产生更有效的治疗β-地中海贫血的疗法。我们的初步数据已经提示了一种机制，它可以绕过突变的珠蛋白基因对野生型等位基因的有害影响。与不增加β +/+细胞中珠蛋白表达的亲本慢病毒载体相反，用来自锚蛋白基因座的基因组元件修饰该载体完全逆转了β +/+ ErPC中的表型，实现了高水平的血红蛋白合成。基于此和其他初步数据，我们假设突变的β-珠蛋白mRNA与正常的β-珠蛋白mRNA竞争翻译。因此，我们制定了以下目标：目标1：了解内源性突变β-珠蛋白mRNA对转导的正常β-珠蛋白基因表达的影响。目的2：开发新的表达载体，恢复慢病毒介导的β-珠蛋白mRNA的翻译能力。 公共卫生相关性：已经确定β-地中海贫血与人类β-珠蛋白基因相关，并且该基因中的某些突变导致疾病表型。在β-珠蛋白基因中有超过200种已知的疾病相关突变。通过各种分子机制（例如过早终止密码子），这些突变影响产生疾病表型的蛋白质产物。治疗β-地中海贫血的一种潜在方法是通过基因疗法（慢病毒）引入非突变mRNA。在动物模型中有希望的结果表明，使用这些方法可以拯救β-珠蛋白敲除小鼠。然而，当这种方法被转化为临床时，只有特定的患者子集对治疗有反应。我们假设，正是长寿突变mRNA的存在与病毒mRNA竞争翻译机制，从而降低了治疗方法的有效性。利用该模型，基于基因型和mRNA稳定性分析，我们的目标是预测给定特定突变的基因治疗的潜在成功。

DESCRIPTION (provided by applicant): 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.