ANTISENSE DRUG RESISTANT GENE THERAPY FOR CML

描述：（申请人摘要）CML是慢性粒细胞白血病（CML）的恶性疾病。 BCR/ABL基因造血干细胞 重排 大多数CML患者的唯一治愈性治疗是 移植来自同种异体供体的HSC。 虽然移植 自体的、未净化的或净化的骨髓或血液祖细胞可以 部分患者恢复Ph-造血功能，大部分患者在一周内复发。 移植后1年，部分原因是移植物被Ph+污染 部分原因是由于疾病在宿主体内持续存在， 准备方案。 因此，消除残留的新方法 疾病是必须的。 一种可能的移植后治疗是 施用化疗，如甲氨蝶呤（MTX）。 然而，MTX 不仅影响Ph + HSC，还影响NL HSC。 NL HSC的转导与 突变的二氢叶酸还原酶（DHFR）基因，例如tyr22-DHFR，可以 允许在移植后使用MTX来消除残留的白血病， 选择性地保留转基因移植的HSC。 自从慢性粒细胞白血病 祖细胞至少与NL祖细胞一样对MTX敏感，转导 MTXr基因的自体移植物可以选择性消除Ph+， MTX移植后在宿主体内持续存在的克隆。 申请人已经 开发了从慢性期CML BM中选择CD34 + HLA-DR-细胞的方法 其大量富含Ph-原始祖细胞 足以移植。 由于少量的Ph+祖细胞可能 在FACS选择的移植物中持续存在，用MTXr转导移植物 基因也将使Ph + HSC MTX具有抗性。 的恶性表型 CML祖细胞可以归因于BCR/ABL编码的BCR/ABL基因的存在。 p210 BCR/ABL酪氨酸激酶。 由于BCR/ABL mRNA的消除， 断点特异性反义寡核苷酸导致 Ph+祖细胞的表型特征， 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 提出产生逆转录病毒载体，将赋予MTX抗性， 输注的HSC群体，以允许在移植后施用MTX， 消灭宿主体内残留的疾病。 由于移植物可能 含有Ph+细胞，她将MTXr基因与抗BCR/ABL AS偶联 消除Ph + HSC中污染的恶性表型的序列 移植物 在SA1中，她将构建含有两种基因的逆转录病毒载体， tyr22-DHFR基因和AS序列，以鉴定允许 MTXr组分和AS序列的相等表达。 表达 DHFR和AS序列的水平以及能力和特异性 逆转录病毒载体消除BCR/ABL mRNA和癌蛋白 及其相关的恶性表型将使用 32DBCR/ABL细胞系。 AS序列的特异性和活性将 然后在原代CML祖细胞（SA2）中进行体外研究， CML小鼠模型（SA3）。 SA2和SA3中的实验还将检查 转导效率进入原代CML和NL 34+细胞，转移和 体外和体内耐药性的持续表达。 一次 已经识别出满足在 应用，未来的临床试验，其中CML自体移植物被转导 移植后接受MTX的患者将开始确认 这种方法的临床效果。

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.