GENETIC STRATEGIES FOR CORRECTING SICKLE CELL DISEASE

这项提议的主要目标是开发和测试新的、遗传的 纠正镰状细胞病的方法。这些协议最初将 在转基因小鼠模型上进行测试。我们最近培育了成年小鼠 它能合成99%的人HbS和1%的人HbF；没有老鼠的血红蛋白 在这些成年动物身上产生的。小鼠贫血，10%的小鼠 外周血中的红细胞呈镰状。初步研究表明 重要的活体病理学；然而，这些动物是活的和 有生育能力。我们计划对这些动物进行纵向研究，以 评估疾病的进展情况。当哈佛商学院的动物们完全 将从这些小鼠中提纯出具有特征性的造血干细胞 并感染重组AAV(腺相关病毒)和逆转录病毒 包含抗病态(β/AS)珠蛋白基因的股票。这些基因是 旨在有效地抑制HBS聚合，因此 抑制红细胞病态。当有效率所需的条件 干细胞的转导是指从HBS小鼠中分离纯化的干细胞 会被感染并移植到小鼠模型中。这些老鼠会 然后进行评估，以确定体内病理是否减少或 被淘汰了。一种治疗镰状细胞病的替代基因疗法将 也被开发出来。一种修饰的转录因子(红系Krupple样 因子；EKLF)与增量珠蛋白基因结合并激活 都是设计好的。将该因子转导到含有 人β和β/S珠蛋白基因或转入人造血干细胞 细胞将刺激三角洲珠蛋白基因的表达，该基因具有 强大的抗病性能。这个系统的优点是 相对较低水平的转录因子表达可以模拟 相对较高水平的β-珠蛋白基因表达。一个主要的障碍 成功的基因治疗一直是对基因表达的抑制 在病毒转导细胞中。在许多情况下，立即高表达 在基因被转化但合成随后被抑制后 有时完全沉默。我们最近证明了钠 丁酸盐和曲古抑素A戏剧性地重新激活沉默的病毒 转导的基因。我们建议测试这些药物以重新激活 病毒转导后的β/AS-珠蛋白和Delta-EKLF基因 造血干细胞移植。当描述的协议 上述方法在转基因小鼠模型中被证明是安全有效的 分离的人造血干细胞，我们计划评估这三种方法 在人体临床试验中。

The major goal of this proposal is to develop and test novel, genetic methods for correcting sickle cell disease. The protocols will initially be tested in a transgenic mouse model. We recently produced adult mice that synthesize 99% human HbS and 1% human HbF; no mouse hemoglobin is produced in these adult animals. The mice are anemic and 10% of erythrocytes in peripheral blood are sickled. Preliminary studies suggest significant in vivo pathology; however, the animals are viable and fertile. We plan to examine these animals in longitudinal studies to evaluate the progression of the disease. When the HbS animals are fully characterized, hematopoietic stem cells will be purified from these mice and infected with recombinant AAV (Adeno-Associated Viral) and retroviral stocks that contain anti-sickling (beta/AS) globin genes. These genes are designed to effectively inhibit HbS polymerization and, therefore, to inhibit erythrocyte sickling. When conditions required for efficient transduction of stem cells are defined, purified stem cells from HbS mice will be infected and transplanted into the mouse model. These mice will then be evaluated to determine whether in vivo pathology is reduced or eliminated. An alternative genetic therapy for sickle cell disease will also be developed. A modified transcription factor (Erythroid Krupple Like Factor; EKLF) that binds to and activates the delta-globin gene will be designed. Transduction of this factor into transgenic mice that contain human delta-and beta/s- globin genes or into human hematopoietic stem cells will stimulate expression of the delta-globin gene which has powerful anti-sickling properties. The advantage of this system is that relatively low levels of transcription factor expression can simulate relatively high levels of delta-globin gene expression. A major impediment to successful genetic therapy has been the suppression of gene expression in virally transduced cells. In many cases, expression is high immediately after genes are transformed but synthesis is subsequently suppressed and sometimes completely silenced. We recently demonstrated that sodium butyrate and trichostatin A dramatically reactivate silenced, virally transduced genes. We propose to test these drugs for reactivation of beta/AS-globin and delta-EKLF genes after viral transduction and transplantation of hematopoietic stem cells. When the protocols described above are proven safe and effective in the transgenic mouse model and in isolated human hematopoietic stem ells, we plan to evaluate thee methods in human clinical trials.