ANTISENSE DRUG RESISTANT GENE THERAPY FOR CML

描述：(申请人摘要)慢性粒细胞白血病是一种恶性疾病。 以bcr/abl基因为特征的造血干细胞 重新编排。对于大多数慢性粒细胞白血病患者来说，唯一的治疗方法是 异基因供者的造血干细胞移植。虽然移植 自体、未净化或净化的骨髓或血祖细胞可以 一些患者恢复Ph-造血，大多数患者在一年内复发 移植后一年，部分原因是移植物受到Ph+污染 细胞，部分原因是疾病在宿主体内持续存在 准备养生法。因此，消除残留的新方法 疾病是必需的。一种可能的移植后治疗方法是 化疗的管理，如甲氨蝶呤(MTX)。然而，MTX 不仅会影响Ph+HSC，还会影响NL HSC。NL-HSC的转导作用 突变的二氢叶酸还原酶(DHFR)基因，如tyr22-DHFR，可能 允许移植后使用MTX消除残留白血病，但 选择性地保留转基因移植的HSC。自CML以来 祖细胞对MTX的敏感性至少与NL祖细胞一样敏感，转导 携带MTXr基因的自体移植物可能允许选择性地消除Ph+ 使用MTX移植后，克隆在宿主体内持续存在。申请人有 慢性粒细胞白血病慢性期骨髓CD34+人类白细胞抗原-DR-细胞筛选方法的建立 它们大量富含Ph原始祖细胞 足以进行移植。由于少量Ph+祖细胞可能 坚持FACS选择移植物，转导MTXr移植物 基因也会使Ph+HSC对MTX产生抗性。骨肉瘤的恶性表型 CML的祖细胞可以归因于BCR/ABL编码的存在 P210BCR/ABL酪氨酸激酶。自从bcr/abl基因被消除后 断点特异性反义寡核苷酸导致 Ph~+祖细胞的表型特征、转导 抗BCR/ABL反义序列(AS)可能导致表型正常 PH+CML祖细胞。根据申请人的初步研究，她 提议制造逆转录病毒载体，使其对MTX产生抗药性 输注的HSC人群允许移植后应用MTX 消除残留在宿主中的疾病。因为移植物可能 含有Ph+细胞，她会将MTXr基因与抗bcr/abl AS偶联 消除污染的Ph+HSC恶性表型的序列 移植物。在SA1中，她将构建包含这两种病毒的逆转录病毒载体 TYR22-DHFR基因和AS序列，以识别允许 MTXr组分和AS序列的表达相同。表达方式 DHFR和AS序列的水平以及容量和特异性 利用逆转录病毒载体消除bcr/abl mRNA和癌蛋白 与其相关的恶性表型将使用 32DBCR/ABL细胞株。AS序列的特异性和活性将 然后在原代CML祖细胞(SA2)体外和体内进行研究 慢性粒细胞白血病小鼠模型(SA3)。在SA2和SA3中的实验也将检验 向原代CML和NL 34+细胞的转导效率，转移和 在体外和体内持续表达耐药。一旦成为 已确定符合中规定标准的载体(S) CML自体移植物转导的应用及未来临床试验 而接受MTX治疗的患者将在移植后开始确认 该方法的临床有效性。

DESCRIPTION: (Applicant's Abstract) CML is malignant disease of the hematopoietic stem cell (HSC) characterized by the BCR/ABL gene rearrangement. The only curative treatment for most patients with CML is transplantation of HSC from an allogeneic donor. Although transplantation of autologous, unpurged or purged bone marrow or blood progenitors can restore Ph-hematopoiesis in some patients, most patients relapse within a year after transplant due in part to contamination of the graft with Ph+ cells and in part due to disease persisting in the host after the preparative regimen. Therefore, new approaches to eliminate residual disease are required. One possible post transplant therapy is the administration of chemotherapy, such as methotrexate (MTX). However, MTX will not only affect Ph+ HSC but also NL HSC. Transduction of NL HSC with mutant dihydrofolate reductase (DHFR) genes, such as the tyr22-DHFR, may allow use of MTX post-transplant to eliminate residual leukemia but selectively spare the genetically modified transplanted HSC. Since CML progenitors are at least as sensitive to MTX as NL progenitors, transduction of the autograft with a MTXr gene may allow selective elimination of the Ph+ clone persisting in the host after transplant with MTX. The applicant has developed methods to select CD34+HLA-DR-cells from chronic phase CML BM which are highly enriched in Ph- primitive progenitors in quantities sufficient for transplantation. Since small numbers of Ph+ progenitors may persist in the FACS selected graft, transduction of the graft with MTXr genes will also render Ph+ HSC MTX resistant. The malignant phenotype of CML progenitors can be attributed to the presence of the BCR/ABL encoded p210BCR/ABL tyrosine kinase. Since elimination of the BCR/ABL mRNA with breakpoint specific anti-sense oligonucleotides results in normalization of the phenotypic characteristics of Ph+ progenitors, transduction of anti-BCR/ABL antisense sequences (AS) may result in phenotypically normal Ph+ CML progenitors. Based on the applicant's preliminary studies, she proposes to generate retroviral vectors that will confer MTX resistance to the infused HSC population to allow administration of MTX post transplant to eliminate residual disease persisting in the host. Since the graft may contain Ph+ cells, she will couple the MTXr gene with anti-BCR/ABL AS sequences to eliminate the malignant phenotype in Ph+ HSC that contaminate the graft. In SA1, she will construct retroviral vectors containing both the tyr22-DHFR gene and AS sequences to identify the construct that allows equal expression of the MTXr component and the AS sequence. The expression level of the DHFR and AS sequences as well as the capacity and specificity with which a retroviral vector eliminates the BCR/ABL mRNA and oncoprotein and its associated malignant phenotype will be quantified using the 32DBCR/ABL cell line. The specificity and activity of the AS sequence will then be studied in vitro in primary CML progenitors (SA2) and in vivo in a murine model of CML (SA3). Experiments in SA2 and SA3 will also examine the transduction efficiency into primary CML and NL 34+ cells, transfer and persistent expression of drug-resistance in vitro and in vivo. Once a vector(s) has been identified which fulfills criteria set forth in the application, future clinical trials in which CML autografts are transduced and patients receive MTX post transplant will be initiated to confirm the clinical efficiency of this approach.