GENETIC STRATEGIES FOR CORRECTING SICKLE CELL DISEASE

这项提案的主要目标是开发和测试新的， 用于矫正镰状细胞病的方法。这些协议最初将 在转基因小鼠模型中进行测试。我们最近培育了成年老鼠 合成99%的人类HbS和1%的人类HbF;没有小鼠血红蛋白 在这些成年动物中产生。小鼠贫血，10%的 外周血中的红细胞呈镰状。初步研究表明 显著的体内病理学;然而，动物是存活的， 多产我们计划对这些动物进行纵向研究， 评估疾病的进展。当HbS动物完全 造血干细胞将从这些小鼠中纯化 并感染重组AAV（腺相关病毒）和逆转录病毒 含有抗镰状化（β/AS）珠蛋白基因的股票。这些基因 设计成有效抑制HbS聚合，因此， 抑制红细胞镰状化。当需要高效的 干细胞的转导是来自HbS小鼠的定义的纯化干细胞 将被感染并移植到小鼠模型中。这些老鼠会 然后进行评价以确定体内病理学是否减少或 淘汰镰状细胞病的另一种遗传疗法将 也要发展。一种修饰的转录因子（红细胞Krupple样 因子（EKLF）结合并激活δ-珠蛋白基因， 设计了将该因子转导到转基因小鼠中， 人δ-和β/s-珠蛋白基因或人造血干细胞 细胞将刺激δ-珠蛋白基因的表达， 强大的抗镰状化特性。该系统的优点在于， 相对低水平的转录因子表达可以刺激 相对高水平的δ-珠蛋白基因表达。一个主要障碍 成功的基因治疗是抑制基因表达 在病毒转导的细胞中。在许多情况下， 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 丁酸和抑制素A显著地重新激活沉默的病毒， 转导基因。我们建议测试这些药物是否能重新激活 β/AS-珠蛋白和δ-EKLF基因， 造血干细胞移植当协议描述 以上在转基因小鼠模型中被证明是安全有效的， 分离人造血干细胞，我们计划评估这些方法 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.