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

基因型对β-地中海贫血基因转移结果的影响

• 批准号: 7866331
• 负责人: STEFANO RIVELLA
• 金额: $ 42.25万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-05 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7866331
• 关键词: Acute Erythroblastic Leukemia; Address; Affect; Alleles; Alternative Splicing; Animal Model; Ankyrins; Beta Cell; Bypass; Cells; Clinic; Clinical Trials; Code; Data; Disease; Effectiveness; Elements; Erythroid Cells; Erythroid Progenitor Cells; Fetal Hemoglobin; Gene Mutation; Gene Transfer; Genes; Genetic; Genetic Transcription; Genetic Translation; Genomics; Genotype; Globin; Goals; Hemoglobin concentration result; Hereditary Disease; Human; Individual; Introns; Knockout Mice; Knowledge; Lead; Lentivirus Vector; Life; Locus Control Region; Mediating; Messenger RNA; Modality; Modeling; Modification; Molecular; Mus; Mutate; Mutation; Mutation Spectra; Nonsense Codon; Outcome; Parents; Patients; Phenotype; Polyribosomes; Production; Protein Biosynthesis; Proteins; RNA; RNA Decay; RNA Splicing; Research; Role; Small Nuclear RNA; Subfamily lentivirinae; Testing; Transcript; Transgenic Organisms; Translating; Translations; Viral; alpha Globin; base; beta Globin; beta Thalassemia; cellular transduction; disease phenotype; effective therapy; expression vector; gene therapy; gene transfer vector; improved; lentiviral-mediated; mRNA Decay; mRNA Stability; mutant; novel; promoter; public health relevance; small hairpin RNA; success; therapeutic effectiveness; vector

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.

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。这些突变可以分类为β0和β +，具体取决于相应的等位基因是否分别导致beta-Globin链合成。根据这些特定突变的组合，将患者分为三个主要群体，这些主要群体，非常低或中度低的β-珠蛋白产生（分别为beta 0/0、0/+和+/+）。我们的研究以及其他的研究表明，可以通过慢病毒介导的人β-珠蛋白基因的转移，其启动子，内含子和大量元素来挽救小鼠中的β-甲性贫血性。基于这些研究，已经提出或正在进行临床试验。然而，这些原始研究并未考虑到β-核心症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，我们将研究基因型与表型之间的相关性，并为治愈β-氢质量治愈而产生更有效的基因转移量。原始研究利用了带有β-珠蛋白基因的小鼠。因此，这些小鼠不会再现在β-地中海贫血患者中观察到的大量突变。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-珠蛋白的表达具有巨大影响。具体而言，我们发现从β-丘脑症患者子集（主要是β0/0）的子集的红细胞祖细胞（ERPC）的转导导致高β-环球蛋白蛋白水平与所使用的载体的数量成比例。但是，转导的β +/ +细胞中的血红蛋白水平仅略有或根本不增加。在Beta 0/+中，我们观察到混合的结果。我们观察到对非义介导的mRNA衰变（NMD）不敏感的转录本的存在与转基因β-珠蛋白mRNA的翻译不敏感之间存在良好的相关性。我们建议更好地理解突变β-珠蛋白mRNA调节正常β-珠蛋白基因的表达或翻译的机制，并利用这些知识来产生更有效的疗法来治疗β-甲硫0-甲硫0-甲基硫酸盐。我们的初步数据已经提出了一种机制，该机制可以绕过突变的球蛋白基因对野生型等位基因的有害作用。与不会增加β +/ +细胞中球蛋白表达的母体慢病毒载体相反，用arnkyrin locus的基因组元素修饰该载体完全逆转了β +/ + ERPC中的表型，从而达到了高水平的血红蛋白合成水平。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竞争进行翻译。因此，我们提出了以下目的：目标1：了解内源性突变体β-珠蛋白mRNA对转导的正常β-珠蛋白基因表达的影响。目标2：开发新型表达矢量，以恢复慢病毒介导的β球蛋白mRNA的能力。 公共卫生的相关性：众所周知，β-地中海贫血与人β-珠蛋白基因有关，并且该基因中的某些突变导致疾病表型。 β-珠蛋白基因中有200多个已知的疾病相关突变。通过各种分子机制（例如，过早的终止密码子），这些突变会影响产生疾病表型的蛋白质产物。治疗β-甲性甲性贫困的一种潜在方法是通过基因治疗（慢病毒）引入非突变mRNA。动物模型中有希望的结果表明，可以使用这些方法来挽救β-珠蛋白敲除小鼠。但是，当将这种方法转化为诊所时，只有一定部分患者对治疗做出反应。我们假设正是长寿突变mRNA的存在与病毒mRNA竞争转化机制，从而降低了治疗方法的有效性。通过该模型，基于MRNA稳定性的基因型和分析，我们旨在预测特定突变的基因治疗的潜在成功。