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

基本信息

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
项目类别：
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

项目摘要

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-珠蛋白基因中的大范围基因突变引起的。这些突变可分为 β 0 和 β +，具体取决于相应的等位基因是否分别导致不合成或导致低 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，不产生β-珠蛋白、极低或中低β-珠蛋白产生（分别为β 0/0、0/+和+/+）。我们和其他人的研究表明，通过慢病毒介导的人类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，可以挽救小鼠的β-地中海贫血。基于这些研究，临床试验已被提议或正在进行中。然而，这些最初的研究没有考虑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来治疗β-地中海贫血。最初的研究使用了删除了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-珠蛋白基因突变的类型对慢病毒介导的野生型β-珠蛋白的表达具有显着影响。具体来说，我们发现来自β-地中海贫血患者亚群（主要是β 0/0）的红系祖细胞（ErPC）的转导导致高β-珠蛋白水平，其与所使用的载体的量成比例。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 beta 0/+ 中，我们观察到了不同的结果。我们观察到对无义介导的 mRNA 衰减 (NMD) 不敏感的转录物的存在与转基因 β-珠蛋白 mRNA 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型 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方法来治疗 β-地中海贫血。我们的初步数据已经表明了一种机制，可以绕过突变珠蛋白基因对野生型等位基因的有害影响。与不增加 β +/+ 细胞中球蛋白表达的亲本慢病毒载体相反，用锚蛋白基因座的基因组元件对该载体进行修饰，完全逆转了 β +/+ ErPC 中的表型，实现了高水平的血红蛋白合成。基于这一数据和其他初步数据，我们假设突变型 β-珠蛋白 mRNA 与正常 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。目标 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：众所周知，β 地中海贫血与人类 β 珠蛋白基因相关，并且该基因的某些突变会导致疾病表型。 β-珠蛋白基因中有超过 200 种已知的与疾病相关的突变。通过各种分子机制（例如提前终止密码子），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。动物模型中的有希望的结果表明，使用这些方法可以拯救 β-珠蛋白敲除小鼠。然而，当这种方法转化为临床时，只有一部分患者对治疗有反应。我们假设，长寿命突变体 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。通过该模型，基于基因型和 mRNA 稳定性分析，我们的目标是预测特定突变的基因治疗的潜在成功率。