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，在出生时或出生后不久发生，并且是由于红细胞及其前体的生产失败，具有正常或接近正常的骨髓和血小板谱系。它在大约10%的病例中遗传，大多数是常染色体显性遗传。最近的遗传学研究已经导致在染色体19q13.2上的核糖体蛋白基因RP 219突变的令人惊讶的鉴定，在约25%的家族性和散发性病例（DBA 1）中，并且有证据表明涉及至少2个其他基因。患者可以完全缓解皮质类固醇或可能成为耐药治疗，然后需要定期输血，或骨髓移植，如果组织相容的同胞供体可用。本提案的长期目标是为不符合匹配同胞干细胞移植条件的严重DBA 1患者的基因治疗方案开发临床前数据。因此，具体的目的是（1）通过基于PCR的序列分析和通过使用针对RPS 19的抗体表征突变蛋白来鉴定RPS 19突变患者;（2）进一步表征这些患者中的体外红系缺陷，然后使用红系祖细胞和前体中的异常;和（3）“敲入”胚胎干（ES）细胞，这是在6个无关家族中独立发生的突变。将突变的ES细胞注射到胚泡中，并重新植入假孕雌性动物中，以产生嵌合动物，用于发育杂合子并繁殖至纯合性。将杂交传递杂合子以观察两个等位基因在体内突变的后果。本文的主要目的是创建可用于评估逆转录病毒和慢病毒RPS 19基因校正的DBA 1小鼠。这些目标的实现将导致进一步的体内评价和临床方案（不是本项目的一部分，而是研究计划的一部分）。除了对严重受影响的DBA 1患者的实际益处外，我们希望深入了解RPS 19突变如何导致早期红系细胞发育受阻。