CORRECTION OF RPS 19 DEFECTS IN DIAMOND BLACKFAN ANEMIA

修正钻石黑扇贫血症中的 RPS 19 缺陷

• 批准号: 6368218
• 负责人: COLIN A SIEFF
• 金额: $ 28.42万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6368218
• 关键词: Lentivirus; Retroviridae; animal breeding; biotechnology; clinical research; congenital aplastic anemia; disease /disorder model; embryonic stem cell; family genetics; gene mutation; gene targeting; gene therapy; hematopoietic stem cells; human subject; immunologic assay /test; laboratory mouse; model design /development; transfection /expression vector

Diamond Blackfan anemia (DBA) is a congenital anemia that develops at birth or soon after, and is due to failure of production of erythrocytes and their precursors, with normal or near normal myeloid and platelet lineages. It is inherited in about 10% of cases, mostly as an autosomal dominant. Recent genetic studies have led to the surprising identification of mutations in a ribosomal protein gene, RP219, on chromosome 19q13.2, in about 25% of both familial and sporadic cases (DBA1), and there is evidence for involvement of at least 2 other genes. Patients can remit completely on corticosteroids or may become resistant to treatment, and then require regular blood transfusions, or bone marrow transplant if a histocompatible sibling donor is available. The long term objective of this proposal is to develop preclinical data for a gene therapy protocol for severe DBA1 patients who are not eligible for matched sibling stem cell transplantation. Therefore the specific aims are (1) to identify RPS19 mutant patients by PCRT-based sequence analysis and by characterization of mutant proteins using antibodies to RPS19; (2) to further characterize the in vitro erythroid defect in these patients and then use abnormality in the erythroid progenitor cells and precursors; and (3), to "knock-in" to embryonic stem (ES) cells a mutation that has occurred independently in 6 unrelated families. The mutant ES cells will be injected into blastocysts and reimplanted into pseudopregnant females to generate chimeric animals for developing heterozygotes and breeding to homozygosity. Transmitting heterozygotes will be cross-bred to observe the consequences of mutation of both alleles in vivo. The major objective here is to create a DBA1 mouse that can be used to evaluate retrovirus and lentivirus RPS19 gene correction. Accomplishment of these goals will lead to further in vivo evaluation and a clinical protocol (not part of this project but part of the research program). In addition to the practical benefit to severely affected DBA1 patients, we hope to gain insight into how mutations in RPS19 lead to a block in the development of early erythroid cells.

Diamond Blackfan贫血（DBA）是一种先天性贫血，在出生时或之后不久就会发育，这是由于红细胞及其前体的产生失败，其正常或接近正常的髓样和血小板谱系。它在大约10％的病例中继承，主要是常染色体显性。最近的遗传研究导致了核糖体蛋白基因RP219，在19q13.2上的突变令人惊讶的鉴定，大约25％的家族性和零星病例（DBA1），并且有证据表明至少有至少2个其他2个基因。患者可以完全递减皮质类固醇或可能对治疗有抵抗力，然后需要定期输血，或者如果有组织兼容的同胞供体，则需要骨髓移植。该提案的长期目标是为不符合匹配的兄弟姐妹干细胞移植的严重DBA1患者开发临床前数据。因此，具体目的是（1）通过基于PCRT的序列分析和使用抗RPS19抗体来鉴定RPS19突变患者； （2）进一步表征这些患者的体外红细胞缺陷，然后在红细胞祖细胞和前体中使用异常； （3），将“敲入”到胚胎茎（ES）细胞中，在6个无关家族中独立发生的突变。突变体ES细胞将被注入胚泡中，并将其重新植入伪久的雌性，以生成嵌合动物，以开发杂合子和繁殖至纯合性。传输杂合子将被交叉繁殖，以观察体内两个等位基因突变的后果。这里的主要目的是创建可用于评估逆转录病毒和慢病毒RPS19基因校正的DBA1小鼠。实现这些目标将导致进一步的体内评估和临床方案（不是该项目的一部分，而是研究计划的一部分）。除了对严重影响DBA1患者的实际好处外，我们还希望深入了解RPS19中的突变如何导致早期红细胞细胞的发展。