CORRECTION OF RPS 19 DEFECTS IN DIAMOND BLACKFAN ANEMIA

Diamond Blackfan贫血（DBA）是一种先天性贫血，发生在出生时或出生后不久，是由于红细胞及其前体的生产失败，骨髓和血小板谱系正常或接近正常。约10%的病例遗传，多数为常染色体显性。最近的遗传学研究令人惊讶地发现，在大约25%的家族性和散发性病例（DBA1）中，染色体19q13.2上的核糖体蛋白基因RP219发生了突变，并且有证据表明至少有2个其他基因参与其中。患者可以完全缓解皮质类固醇，或者可能对治疗产生耐药性，然后需要定期输血，或者如果有组织相容的兄弟姐妹供体，则需要骨髓移植。该提案的长期目标是为不符合匹配兄弟姐妹干细胞移植条件的严重DBA1患者的基因治疗方案开发临床前数据。因此，具体目标是：(1)通过基于pcr的序列分析和使用RPS19抗体对突变蛋白进行表征来鉴定RPS19突变患者；(2)进一步表征这些患者的体外红细胞缺陷，然后利用红细胞祖细胞和前体细胞的异常；(3)在胚胎干细胞中“敲入”一种在6个不相关的家族中独立发生的突变。突变的胚胎干细胞将被注射到囊胚中，并重新植入到假怀孕的雌性体内，以产生嵌合动物，以发育杂合子并繁殖到纯合子。将传递杂合子进行杂交，在体内观察两个等位基因突变的结果。这里的主要目标是创建一个DBA1小鼠，可用于评估逆转录病毒和慢病毒RPS19基因校正。这些目标的实现将导致进一步的体内评估和临床方案（不是本项目的一部分，而是研究计划的一部分）。除了对严重影响DBA1患者的实际益处外，我们希望深入了解RPS19突变如何导致早期红系细胞发育受阻。

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.