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: 8240460
• 负责人: STEFANO RIVELLA
• 金额: $ 41.83万
• 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/8240460
• 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 Decay; RNA Splicing; RNA chemical synthesis; 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(申请人提供)：β-地中海贫血是由β-珠蛋白基因的大量遗传突变引起的。这些突变可分为β0和β+，这取决于相应的等位基因分别导致不合成或低合成β-珠蛋白链。根据这些特定突变的组合，患者被分为三个主要的组，即没有、非常低或中等低的β-珠蛋白产生(分别为0/0、0/+和+/+)。我们和其他人的研究表明，通过慢病毒介导的人β-珠蛋白基因、其启动子、内含子和基因位点控制区的大元件的转移，有可能拯救小鼠的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到β-地中海贫血患者的基因变异性。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。在这些研究的基础上，我们将研究基因型和表型之间的相关性，并建立更有效的基因转移载体来治疗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，这些小鼠不会复制在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-珠蛋白基因突变类型对慢病毒介导的野生型β-珠蛋白的表达有显著影响。具体地说，我们发现转导一部分β-地中海贫血患者的红系祖细胞(ERPC)(主要是β0/0)会导致高β-珠蛋白水平，与所使用的载体数量成比例。然而，转导的β+/+细胞中的血红蛋白水平仅略有增加或根本没有增加。在测试版0/+中，我们观察到了混合结果。我们观察到转录本的存在与抑制转基因β-珠蛋白mRNA的翻译之间有很好的相关性。我们建议更好地了解突变的β-珠蛋白mRNAs调节正常β-珠蛋白基因表达或翻译的机制，并利用这一知识产生更有效的治疗β-地中海贫血的方法。我们的初步数据已经表明，有一种机制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+/+细胞珠蛋白表达的亲本慢病毒载体不同，用来自ankyrin基因座的基因组元件修饰该载体后，完全逆转了β+/+ErPC的表型，实现了高水平的血红蛋白合成。基于这一点和其他初步数据，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：目的1：了解内源性突变的β-珠蛋白mRNAs对转导的正常β-珠蛋白基因表达的影响。目的2：构建新型表达载体，恢复慢病毒介导的β-珠蛋白基因的翻译能力。 公共卫生相关性：众所周知，β-地中海贫血与人类β-珠蛋白基因有关，该基因的某些突变会导致疾病表型。β-珠蛋白基因有200多个已知的与疾病相关的突变。通过不同的分子机制(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-地中海贫血的一个潜在方法是通过基因治疗(慢病毒)引入非突变的mRNAs。动物模型中有希望的结果表明，使用这些方法拯救β-珠蛋白基因敲除小鼠是可能的。然而，当这种方法被转化到临床上时，只有一定一部分患者对治疗有反应。我们假设，正是长寿突变mRNAs的存在，与病毒mRNAs竞争翻译机制，降低了治疗方法的有效性。在这个模型的基础上，基于基因分型和对基因稳定性的分析，我们旨在预测特定突变情况下基因治疗的潜在成功。