CN1719973A - Compositions and methods for cancer diagnosis and treatment - Google Patents

Abstract

The miR15 and miR16 small RNA genes are located at 13q14 within the 30kb region of loss of specific cancer cells, such as chronic lymphocytic leukemia or prostate cancer. Chronic lymphocytic leukemia or prostate cancer can be diagnosed by detecting a decrease in the copy number of the miR15 or miR16 genes, determining the status of miR15 or miR16 gene mutations, or by detecting a decrease in the RNA transcribed from these genes. When administered to patients with these diseases, the miR15 or miR16 gene products are capable of inhibiting neoplastic or tumor growth of cancers, such as chronic lymphocytic leukemia or prostate cancer cells.

Description

Translated fromChinese

用于癌症诊断和治疗的组合物和方法Compositions and methods for cancer diagnosis and treatment

政府许可government license

本发明部分受到国家癌症研究所的支持，许可号为P01CA76259，P01CA81534，和P30CA56036。美国政府对于本发明具有一定的权利。This invention was supported in part by the National Cancer Institute under grant numbers P01CA76259, P01CA81534, and P30CA56036. The US Government has certain rights in this invention.

发明领域field of invention

本发明涉及癌症的诊断，特别是通过检测miR15和miR16拷贝数目，突变情况，或基因表达来诊断慢性淋巴性白血病或前列腺癌。本发明还涉及癌症的治疗，包括miR15或miR16基因表达的减少或缺失，特别是通过施用miR15或miR16基因产物治疗慢性淋巴性白血病或前列腺癌。The present invention relates to the diagnosis of cancer, especially chronic lymphocytic leukemia or prostate cancer by detecting the number of miR15 and miR16 copies, mutations, or gene expression. The present invention also relates to the treatment of cancer, including the reduction or deletion of miR15 or miR16 gene expression, especially the treatment of chronic lymphocytic leukemia or prostate cancer by administering miR15 or miR16 gene products.

发明背景Background of the invention

在美国和全世界癌症都是死亡和发病的一个重要原因。特别是慢性淋巴性白血病(“CLL”)和前列腺癌在临床上是重要的成人肿瘤病。CLL是西方世界是最普遍的一种成人白血病形式。而且在美国的男人中，前列腺癌的年龄校正发病率目前超过了所有其它癌症，仅次于肺癌，是该国家所有男性癌症死亡的第二大原因。Cancer is a significant cause of death and morbidity in the United States and throughout the world. In particular, chronic lymphocytic leukemia ("CLL") and prostate cancer are clinically important adult neoplastic diseases. CLL is the most prevalent form of adult leukemia in the Western world. And among men in the United States, the age-adjusted incidence rate of prostate cancer now exceeds that of all other cancers and is second only to lung cancer as the second leading cause of cancer death among all men in the country.

在多于一半的报道CLL病例中都会发生13q14的半合子丢失和/或纯合子丢失，这构成了CLL中最频繁的染色体异常。CLL患者的组织样品的染色体组型相对具有较少的染色体异常，这表明所观察到的13q14缺失的特异性和频率具有病理学重要性。除此之外，60％的前列腺癌中也具有13q14缺失，表明CLL和前列腺癌的发病机理涉及位于13q14的一种或多种肿瘤抑制基因。Hemizygous and/or homozygous loss of 13q14 occurs in more than half of reported CLL cases and constitutes the most frequent chromosomal abnormality in CLL. The karyotypes of tissue samples from CLL patients have relatively few chromosomal abnormalities, suggesting that the specificity and frequency of the observed 13q14 deletions is of pathological importance. In addition, 13q14 deletions are also present in 60% of prostate cancers, suggesting that one or more tumor suppressor genes located at 13q14 are involved in the pathogenesis of CLL and prostate cancer.

CLL和前列腺癌中同时存在克隆性纯合和杂合性缺失，以及非常高的13q4丢失频率，表明该区域的缺失与特定癌症类型的病因有关。对几种基因进行位置克隆以鉴别缺失部位的基因。至今从突发性CLL和遗传性CLL的13q14的缺失区域共鉴别了八个基因，并检测了其DNA和/或RNA水平的改变：Leu1(BCMS或EST70/Leu1)，Leu2(ALT1或1B4/Leu2)，Leu5(CAR)，CLLD6，KPNA3，CLLD7，LOC51131(推测的锌指蛋白NY-REN-34抗原)和CLLD8。但是，详细的基因分析，包括大范围的杂合性丢失(LOH)，突变和表达研究，却没能证明任何这些基因与致癌作用的关系。The simultaneous presence of clonal homozygous and heterozygous deletions in CLL and prostate cancer, as well as a very high frequency of 13q4 loss, suggests that deletions in this region are associated with the etiology of specific cancer types. Positional cloning was performed on several genes to identify genes at deletion sites. So far, eight genes have been identified from the deletion region of 13q14 in sudden CLL and hereditary CLL, and their DNA and/or RNA level changes were detected: Leu1 (BCMS or EST70/Leu1), Leu2 (ALT1 or 1B4/ Leu2), Leu5 (CAR), CLLD6, KPNA3, CLLD7, LOC51131 (putative zinc finger protein NY-REN-34 antigen) and CLLD8. However, detailed gene analysis, including extensive loss of heterozygosity (LOH), mutation and expression studies, failed to demonstrate the relationship of any of these genes to carcinogenesis.

小分子RNA(miRNA)存在于超过一百种不同的生物体中，包括果蝇，线虫和人。认为在这些生物体中在多种发育调节过程中都涉及到miRNA，miRNA典型地是通过60到70个核苷酸的反馈RNA前体结构的处理得到的，该反馈RNA前体结构是由miRNA基因转录而来的。RNA前体或处理的miRNA产物很容易检测，缺少这些分子表明相应miRNA基因的缺失或功能丢失。Small RNAs (miRNAs) are present in more than a hundred different organisms, including Drosophila, C. elegans and humans. miRNAs are thought to be involved in a variety of developmental regulatory processes in these organisms, and miRNAs are typically obtained through the manipulation of feedback RNA precursor structures of 60 to 70 nucleotides that are formed by the miRNA transcribed from the gene. Pre-RNA or processed miRNA products are readily detectable, and the absence of these molecules indicates deletion or loss-of-function of the corresponding miRNA gene.

目前CLL的治疗典型地包括化学疗法，单独施用或与自体同源骨髓移植一起进行。所使用的化学治疗剂通常对患者具有毒性，在大部分患者中都只是达到部分缓解的效果。前列腺癌和其它癌症治疗的疗法也包括化学疗法，通常是在外科手术切除肿瘤以后。但是，对于CLL，化学治疗剂(与外科手术一起进行或不与其一起进行)的治疗受到限制。Current treatments for CLL typically include chemotherapy, administered alone or in conjunction with autologous bone marrow transplantation. The chemotherapeutic agents used are often toxic to the patient and only partial remission is achieved in the majority of patients. Treatment for prostate cancer and other cancers also includes chemotherapy, usually after surgery to remove the tumor. However, treatment with chemotherapeutic agents (with or without surgery) is limited for CLL.

前列腺癌也可以用外部射线照射或近距离放射疗法(例如使用具有放射活性的“种子”)治疗，也是单独使用或与外科手术一起使用。这种治疗的危险是将患者的正常组织暴露在放射线之中，以及可能不是完全有效。Prostate cancer can also be treated with external beam radiation or brachytherapy (for example using radioactive "seeds"), either alone or with surgery. This treatment risks exposing the patient's normal tissues to radiation and may not be fully effective.

CLL或前列腺癌需要有快速，经济和准确的诊断性检测。对于癌症还需要有经济和有效的并且对患者没有显著的负面影响的治疗，特别是CLL或前列腺癌。There is a need for rapid, affordable and accurate diagnostic tests for CLL or prostate cancer. There is also a need for treatments for cancer, especially CLL or prostate cancer, that are economical and effective and that do not have significant negative effects on the patient.

发明简述Brief description of the invention

现已发现在人中，miR15或miR16基因位于13q14，而在很大一部分CLL或前列腺癌患者中13q14区域都缺失。还发现miR15或miR16基因的RNA产物抑制CLL和前列腺癌细胞的赘生物或肿瘤生长。这些RNA产物可以用于癌症治疗，对miR15或miR16基因进行负调控。It has been found that in humans, the miR15 or miR16 gene is located at 13q14, and the 13q14 region is deleted in a large proportion of CLL or prostate cancer patients. The RNA products of the miR15 or miR16 genes were also found to inhibit neoplastic or tumor growth in CLL and prostate cancer cells. These RNA products can be used in cancer therapy to negatively regulate miR15 or miR16 genes.

miR15和miR16小分子RNA基因位于13q14，在CLL和前列腺癌丢失的30kb区域中，在大部分CLL和前列腺癌中这两个基因都缺失或受到负调控。因此，本发明提供了一种CLL或前列腺癌的诊断性检测，包括检测这些基因的基因产物，检测miR15或miR16基因的拷贝数，或测定其突变情况。The miR15 and miR16 small RNA genes are located at 13q14, a 30-kb region lost in CLL and prostate cancer, where both genes are either deleted or downregulated in most CLL and prostate cancers. Accordingly, the present invention provides a diagnostic test for CLL or prostate cancer comprising detecting the gene products of these genes, detecting the copy number of the miR15 or miR16 gene, or determining the mutation status thereof.

在一个实施方案中，所述诊断检测包括从怀疑患有CLL或前列腺癌的患者中分离RNA，用miR15或miR16前体或处理过的miRNA的探针用Northern blot杂交检测miR15或miR16基因产物，其中miR15或miR16前体或处理过的miRNA与正常样品对照相比有所减少则诊断为CLL或前列腺癌。In one embodiment, the diagnostic assay comprises isolating RNA from a patient suspected of having CLL or prostate cancer, detecting the miR15 or miR16 gene product by Northern blot hybridization with a probe for miR15 or miR16 precursor or processed miRNA, Where miR15 or miR16 precursor or processed miRNA is reduced compared with the normal sample control, it is diagnosed as CLL or prostate cancer.

在另一个实施方案中，所述诊断检测包括从怀疑患有miR15或miR16介导的癌症例如CLL或前列腺癌的患者中分离DNA，用miR15或miR16基因序列的探针用Southern blot杂交检测miR15或miR16基因拷贝数，其中基因拷贝数减少到1或0则诊断为CLL或前列腺癌。In another embodiment, the diagnostic test comprises isolating DNA from a patient suspected of having miR15 or miR16-mediated cancer, such as CLL or prostate cancer, and detecting miR15 or miR16 gene copy number, in which the gene copy number decreased to 1 or 0 was diagnosed as CLL or prostate cancer.

在另一个实施方案中，所述诊断检测包括通过评价D13S273和D13S272标记的杂合性丢失检测miR15或miR16基因拷贝数的减少，其中有这些标记的杂合性丢失诊断为CLL或前列腺癌。In another embodiment, the diagnostic assay comprises detection of a reduction in miR15 or miR16 gene copy number by evaluating loss of heterozygosity for D13S273 and D13S272 markers, wherein loss of heterozygosity for these markers is diagnostic of CLL or prostate cancer.

在另一个实施方案中，所述诊断检测包括从怀疑患有CLL或前列腺癌的患者中分离DNA，用PCR扩增miR15或miR16基因片段检测miR15或miR16基因的缺失或突变并将该扩增片段与正常样品对照的扩增片段相比较，其中在miR15或miR16基因的一个或多个拷贝中检测到突变则诊断为CLL或前列腺癌。可以用单链构象多态性方法比较扩增片段。在一方面，所述突变为miR15或miR16基因序列的部分缺失。In another embodiment, the diagnostic test comprises isolating DNA from a patient suspected of having CLL or prostate cancer, amplifying a fragment of the miR15 or miR16 gene by PCR to detect a deletion or mutation in the miR15 or miR16 gene and analyzing the amplified fragment A diagnosis of CLL or prostate cancer is made where a mutation is detected in one or more copies of the miR15 or miR16 gene compared to the amplified fragment of a normal sample control. Amplified fragments can be compared using the single-strand conformation polymorphism method. In one aspect, the mutation is a partial deletion of the miR15 or miR16 gene sequence.

在另一个实施方案中，所述诊断检测包括从怀疑患有CLL或前列腺癌的患者中分离RNA，检测到miR15或miR16基因产物的突变则诊断为CLL或前列腺癌。In another embodiment, the diagnostic assay comprises isolating RNA from a patient suspected of having CLL or prostate cancer, and detection of a mutation in the miR15 or miR16 gene product is diagnostic of CLL or prostate cancer.

在另一个实施方案中，所述诊断检测包括从怀疑患有CLL或前列腺癌的患者中分离RNA，用逆转录酶-聚合酶链式反应扩增miR15或miR16前体或处理过的miRNA从而检测miR15或miR16基因产物的水平，其中miR15或miR16前体或处理过的miRNA与内对照的扩增RNA相比有所减少则诊断为CLL或前列腺癌。In another embodiment, the diagnostic assay comprises isolating RNA from a patient suspected of having CLL or prostate cancer, amplifying miR15 or miR16 precursor or processed miRNA using reverse transcriptase-polymerase chain reaction to detect Levels of miR15 or miR16 gene products, in which miR15 or miR16 precursor or processed miRNA is reduced compared to internal control amplified RNA are diagnostic of CLL or prostate cancer.

本发明还提供了一种在需要治疗的患者中治疗miR15或miR16介导的癌症的方法，包括将有效量的miR15或miR16基因产物给予患者，由此癌细胞的增殖受到抑制。The present invention also provides a method of treating miR15 or miR16 mediated cancer in a patient in need thereof, comprising administering to the patient an effective amount of a miR15 or miR16 gene product, whereby proliferation of cancer cells is inhibited.

本发明还提供了一种在需要治疗的患者中治疗miR15或miR16介导的癌症的方法，其中分离患者的细胞并在离体条件下用有效量的含有编码miR15或miR16基因产物的序列的核酸转染。然后将这些细胞重新植入到患者体内，由此患者的癌细胞的增殖受到抑制。The present invention also provides a method of treating miR15 or miR16 mediated cancer in a patient in need thereof, wherein cells from the patient are isolated and treated ex vivo with an effective amount of a nucleic acid comprising a sequence encoding a miR15 or miR16 gene product transfection. These cells are then reimplanted into the patient, whereby the proliferation of the patient's cancer cells is inhibited.

本发明进一步提供了一种在患者中抑制miR15或miR16介导的癌症的增殖的方法，包括向细胞递送有效量的miR15或miR16基因产物。The present invention further provides a method of inhibiting the proliferation of miR15 or miR16 mediated cancer in a patient comprising delivering an effective amount of a miR15 or miR16 gene product to a cell.

本发明进一步还提供了一种治疗患有miR15或miR16介导的癌症的患者的药物组合物，包括分离的miR15或miR16基因产物，或编码miR15或miR16基因产物的核酸，以及药学可接收的载体。The present invention further provides a pharmaceutical composition for treating patients suffering from miR15 or miR16-mediated cancer, comprising isolated miR15 or miR16 gene product, or nucleic acid encoding miR15 or miR16 gene product, and a pharmaceutically acceptable carrier .

附图简述Brief description of the drawings

图1A和图1B分别是预测的miR15和miR16前体RNA的二级结构的示意图。用“mfold”程序，3.1版，Matthews et al.(1999)，J.Mol.Biol.288：911-940进行RNA二级结构预测，人工修饰以使螺旋区段内包含G/U摆动碱基对。处理的miR15和miR16miRNA以下划线表示。根据Lagos-Quintana et al.(2001)，Science294：853-858进行了修改。Figure 1A and Figure 1B are schematic diagrams of the predicted secondary structures of miR15 and miR16 precursor RNAs, respectively. RNA secondary structure prediction using the "mfold" program, version 3.1, Matthews et al. (1999), J. Mol. Biol. 288:911-940, artificially modified to include G/U wobble bases within the helical segment right. Processed miR15 and miR16 miRNAs are underlined. Modified from Lagos-Quintana et al. (2001), Science 294:853-858.

图2A是CLL中13q14肿瘤抑制子位点内的基因图谱，显示出miR15/16基因簇的定位。其中显示了遗传标记和基因在图谱上的位置。Figure 2A is a gene map within the 13q14 tumor suppressor locus in CLL showing the localization of the miR15/16 gene cluster. It shows the genetic markers and the location of the genes on the map.

图2B是已报道过的13q14缺失图谱，用水平条框标记。Figure 2B is a map of reported 13q14 deletions, marked with a horizontal bar.

图2C是D13S1150和D13S272标记之间的位点图谱。在此位点上每个基因的方向都在基因名称下方用箭头标出，彩色的垂直条表示每个基因相应外显子的位置。Figure 2C is a map of the site between the D13S1150 and D13S272 markers. The orientation of each gene at this locus is marked with an arrow below the gene name, and the colored vertical bars indicate the position of the corresponding exon for each gene.

图2D是Alu 18和D13S272标记之间的位点图谱。条和框表示LEU2/ALT1和LEU1外显子的位置。短垂直箭头表示miR15或miR16基因的位置。圆点表示用于筛选衍生自两种独立的白血病细胞(CLL-A和CLL-B)的融合细胞的体细胞杂交克隆的PCR引物的位置。填充框表示杂交体中的染色体13的部分。“←～31.4kb→”表示在来自患有具有t(2；13)(q12；q13)移位的CLL，两边眼癌，溃疡性结肠炎的患者的克隆CLL-A中大约31.4kb的缺失区域。长垂直箭头表示具有t(2；13)(q32；q14)移位的克隆CLL-B的断点位置，“←～29kb→”表示该克隆中大约29kb的缺失区域。Figure 2D is a map of the site between the Alu 18 and D13S272 markers. Bars and boxes indicate the positions of LEU2/ALT1 and LEU1 exons. Short vertical arrows indicate the location of miR15 or miR16 genes. Dots indicate the positions of PCR primers used to screen somatic cell hybrid clones derived from fusion cells of two independent leukemia cells (CLL-A and CLL-B). Filled boxes indicate the portion of chromosome 13 in the hybrid. "←~31.4kb→"indicates a deletion of approximately 31.4kb in clonal CLL-A from a patient with CLL with t(2;13)(q12;q13) shift, bilateral eye cancer, ulcerative colitis area. The long vertical arrow indicates the breakpoint position of clone CLL-B with t(2;13)(q32;q14) shift, and "←～29kb→"indicates about 29kb deletion region in this clone.

图3A是正常人肾，前列腺，肝脏，骨骼肌(“Sk肌”)，睾丸，CD5+B细胞(CD5+)，白血病细胞(“Per Bl Leuk”)和骨髓(“BM”)中miR15和miR16基因表达的Northern blot分析。Figure 3A is a graph of miR15 and miR16 in normal human kidney, prostate, liver, skeletal muscle ("Sk muscle"), testis, CD5+ B cells (CD5+), leukemia cells ("Per Bl Leuk") and bone marrow ("BM") Northern blot analysis of gene expression.

图3B是在18个CLL患者中微卫星标记D13S272和D13S273的杂合丢失(“LOH”)分析。用来自正常人CD5+细胞的DNA作为对照。样品的LOH状态用“+/+杂合”，“+/-LOH”，“-/-纯合缺失”，“NI”(无意义)，“？”(没有足够材料)和“ND”(未实施)。用溴化乙锭染色的Northern凝胶作为归一化对照。Figure 3B is a loss of heterozygosity ("LOH") analysis of microsatellite markers D13S272 and D13S273 in 18 CLL patients. DNA from normal human CD5+ cells was used as a control. The LOH status of the sample is represented by "+/+ heterozygous", "+/-LOH", "-/- homozygous deletion", "NI" (not meaningful), "?" (not enough material) and "ND" ( not implemented). Northern gels stained with ethidium bromide were used as normalization controls.

发明详述Detailed description of the invention

本文给出的所有核酸序列都是5’到3’方向的。此外，核酸序列中的所有脱氧核糖核苷酸都用大写字母表示(例如，脱氧胸腺嘧啶为“T”)，核酸序列中的核糖核苷酸用小写字母表示(例如尿嘧啶为“u”)。All nucleic acid sequences given herein are in the 5' to 3' orientation. In addition, all deoxyribonucleotides in nucleic acid sequences are capitalized (eg, deoxythymine is "T") and ribonucleotides in nucleic acid sequences are denoted by lowercase letters (eg, uracil is "u") .

CLL或前列腺癌可以通过检测miR15或miR16基因拷贝数的减少，或者通过检测miR15或miR16基因的一个或多个拷贝的突变来诊断。miR15或miR16基因拷贝数从双倍减少为单倍，或者减少到没有拷贝，则诊断为CLL或前列腺癌。同样地，miR15或miR16基因的一个或多个拷贝的突变表示基因功能的丢失，则诊断为CLL或前列腺癌。CLL or prostate cancer can be diagnosed by detecting a reduction in the copy number of the miR15 or miR16 gene, or by detecting a mutation in one or more copies of the miR15 or miR16 gene. A reduction in the copy number of the miR15 or miR16 gene from double to single, or to no copy, was diagnosed as CLL or prostate cancer. Likewise, mutations in one or more copies of the miR15 or miR16 genes, indicating loss of gene function, are diagnostic of CLL or prostate cancer.

这里所用的“CLL细胞”是来自患有或怀疑患有CLL的患者的淋巴细胞，其中淋巴细胞的“CLL分数”至少为4，这是根据Matutes et al.(1994)，Leukemia 8(10)：1640-1645所述的评分系统确定的，在此引入其全文作为参考。这里所用的“前列腺癌细胞”是来源于的前列腺的赘生物或肿瘤细胞，不管其是否位于前列腺中。本领域技术人员可以容易地鉴别CLL或前列腺癌细胞。As used herein, "CLL cells" are lymphocytes from patients with or suspected of having CLL, wherein the lymphocytes have a "CLL fraction" of at least 4 according to Matutes et al. (1994), Leukemia 8(10) : 1640-1645, the entire text of which is hereby incorporated by reference. As used herein, "prostate cancer cells" are neoplastic or tumor cells derived from the prostate, whether located in the prostate or not. Those skilled in the art can readily identify CLL or prostate cancer cells.

miR15/miR16基因簇的位置在13q14。这些基因的核酸序列包含在克隆317g11中，其核苷酸序列的GenBank记录号是AC069475。在此引入该记录的全部内容作为参考。可以通过测定怀疑患有CLL或前列腺癌的患者组织中的这些基因的结构或序列，并将其与该患者的未受影响的组织样品中，或正常对照组织的样品中的这些基因的结构或序列进行比较，从而检测miR15或miR16基因的缺失或突变。可以用任何适当的方法进行这种比较。The location of the miR15/miR16 gene cluster is at 13q14. The nucleic acid sequences of these genes are contained in clone 317g11, whose GenBank accession number of the nucleotide sequence is AC069475. The entire contents of this record are hereby incorporated by reference. This can be achieved by determining the structure or sequence of these genes in tissue from a patient suspected of having CLL or prostate cancer and comparing it to the structure or sequence of these genes in a sample of unaffected tissue from that patient, or in a sample of normal control tissue or The sequences were compared to detect deletions or mutations in the miR15 or miR16 genes. This comparison can be made using any suitable method.

根据本发明，要诊断miR15或miR16介导的癌症，要从患者中获取组织样品。然后制备样品并测定miR15或miR16基因产物的表达或miR15或miR16基因的缺失或突变。组织样品包括感兴趣的活组织，以及血液和体液样品。According to the present invention, to diagnose miR15 or miR16 mediated cancer, a tissue sample is obtained from a patient. Samples are then prepared and assayed for expression of the miR15 or miR16 gene product or deletion or mutation of the miR15 or miR16 gene. Tissue samples include living tissue of interest, as well as blood and body fluid samples.

这里所说的“miR15或miR16介导的癌症”是指在至少一部分与该癌症相关的肿瘤细胞或赘生物细胞中miR15或miR16基因减少或缺失的任何癌症。miR15或miR16介导的癌症的实例包括CLL和前列腺癌。The term "miR15 or miR16 mediated cancer" herein refers to any cancer in which miR15 or miR16 genes are reduced or deleted in at least a portion of tumor cells or neoplastic cells associated with the cancer. Examples of miR15 or miR16 mediated cancers include CLL and prostate cancer.

miR15或miR16缺失或突变的存在可以通过患者基因组DNA的Southern blot杂交来检测，使用miR15或miR16基因的探针，例如如下所述的。另外，可以从怀疑患有CLL或前列腺癌的患者中获取血液样品，分离白细胞进行DNA提取。优选血液或组织样品是从还没有开始放射疗法或化学疗法的患者中获得的。作为对照的相应的组织或血液样品可以从该患者的未受影响的组织中，或者从正常的人个体中获得。The presence of miR15 or miR16 deletions or mutations can be detected by Southern blot hybridization of the patient's genomic DNA using probes for the miR15 or miR16 genes, for example as described below. Alternatively, blood samples can be obtained from patients suspected of having CLL or prostate cancer, and white blood cells are separated for DNA extraction. Preferably the blood or tissue sample is obtained from a patient who has not yet started radiation therapy or chemotherapy. A corresponding tissue or blood sample as a control can be obtained from unaffected tissue of the patient, or from a normal human individual.

Southern blot杂交技术是本领域技术人员公知的。例如将从怀疑患有CLL或前列腺癌的患者的组织或血液中分离的基因组DNA用限制性内切酶消化。消化产生基因组DNA的限制性片段，可以用电泳，例如琼脂糖电泳分离。然后将限制性片段点样在杂交膜(例如硝酸纤维素或尼龙)上，并与miR15或miR16基因特异的标记探针进行杂交。与该患者的DNA样品进行了相同处理的对照DNA样品相比，杂交膜上限制性片段类型的改变表示这些基因发生缺失或突变。本领域普通技术人员可以容易地确定适合于检测miR15或miR16基因拷贝数或突变的探针标记和杂交条件。这里所用的术语“缺失”是指基因的部分缺失或全部基因缺失。Southern blot hybridization techniques are well known to those skilled in the art. For example, genomic DNA isolated from tissue or blood of a patient suspected of having CLL or prostate cancer is digested with restriction enzymes. Digestion produces restriction fragments of genomic DNA that can be separated by electrophoresis, eg, agarose electrophoresis. The restriction fragments are then spotted on a hybridization membrane (eg, nitrocellulose or nylon) and hybridized with a labeled probe specific for the miR15 or miR16 gene. Changes in the type of restriction fragments on the hybridization membrane compared to the same treated control DNA sample from the patient's DNA sample indicate deletions or mutations in these genes. Probe labels and hybridization conditions suitable for detecting miR15 or miR16 gene copy number or mutation can be readily determined by those of ordinary skill in the art. The term "deletion" as used herein refers to a partial deletion of a gene or a deletion of the entire gene.

用于Southern blot杂交的miR15和miR16核酸探针可以根据公开的miR15和miR16小分子RNA的序列用Lagos-Quintana etal.(2001)，Science 294：853-858所述的方法设计，在此引入其全文作为参考。miR15小分子RNA的核苷酸序列是uagcagcacauaaugguuugug(SEQ ID NO：3)。miR16小分子RNA的核苷酸序列是uagcagcacguaaauauuggcg(SEQ ID NO：4)。检测miR15和miR16 DNA的合适探针分别是：The miR15 and miR16 nucleic acid probes that are used for Southern blot hybridization can use Lagos-Quintana et al.(2001), the method design described in Science 294:853-858 according to the sequence of published miR15 and miR16 small molecule RNA, introduce its here The full text is provided by reference. The nucleotide sequence of miR15 small molecule RNA is uagcagcacauaauguuugug (SEQ ID NO: 3). The nucleotide sequence of miR16 small molecule RNA is uagcagcacguaaauauuggcg (SEQ ID NO: 4). Suitable probes for detecting miR15 and miR16 DNA are:

CACAAACCATTATGTGCTTGCTA(SEQ ID NO：5)CACAAAACCATTATGTGCTTGCTA (SEQ ID NO: 5)

GCCAATATTTACGTGCTGCTA(SEQ ID NO：6)GCCAATATTTACGTGCTGCTA (SEQ ID NO: 6)

SEQ ID NO：5和SEQ ID NO：6的互补序列也可以用作miR15和miR16 DNA的探针。The complementary sequences of SEQ ID NO: 5 and SEQ ID NO: 6 can also be used as probes for miR15 and miR16 DNA.

制备标记的DNA和RNA探针的方法，以及与靶核苷酸序列杂交的条件如Molecular Cloning：A Laboratory Manual，J.Sambrook et al.，eds.，2nd edition，Cold Spring Harbor LaboratoryPress，1989，Chapters 10 and 11所述，在此引入作为参考。Methods for preparing labeled DNA and RNA probes, and conditions for hybridization to target nucleotide sequences such as Molecular Cloning: A Laboratory Manual, J.Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapters 10 and 11, which are hereby incorporated by reference.

例如，核酸探针可以用例如放射性核素如³H，³²P，³³P，¹⁴C或³⁵S；重金属；或者能与标记的配体特异性结合的配体(例如生物素，抗生物素蛋白或抗体)，荧光分子，化学发光分子，酶等物质进行标记。For example, nucleic acid probes can be made of, for example, radionuclides such as³ H,³² P,³³ P,¹⁴ C or³⁵ S; heavy metals; or ligands that can specifically bind to labeled ligands (such as biotin, avidin proteins or antibodies), fluorescent molecules, chemiluminescent molecules, enzymes and other substances for labeling.

可以用Rigby et al.(1977)，J.Mol.Biol.113：237-251所述的缺口平移法或Fienberg et al.(1983)，Anal.Biochem.132：6-13所述的随机引物法获得高比活性的标记探针，在此引入其全文作为参考。后者则是从单链DNA或RNA模板合成高比活性³²P标记的探针的方法。例如，根据缺口平移法，通过用具有高放射活性的核苷酸替换已存在的核苷酸，可以制备得具有超过10⁸cpm/mg的比活性的³²P标记的核酸探针。然后将杂交膜曝光在胶片上，进行放射自显影检测。对杂交膜曝光的胶片进行光密度扫描，得到miR15或miR16基因拷贝数的精确测量结果。而且miR15或miR16基因拷贝数还可以用计算机成像系统，例如AmershamBiosciences，Piscataway，NJ的Molecular Dynamics 400-B 2DPhosphorimager进行定量分析。The gap translation method described in Rigby et al. (1977), J. Mol. Biol. 113: 237-251 or the random primer described in Fienberg et al. (1983), Anal. Biochem. 132: 6-13 can be used To obtain labeled probes with high specific activity, which is hereby incorporated by reference in its entirety. The latter is a method for synthesizing high specific activity³² P-labeled probes from single-stranded DNA or RNA templates. For example, according to the gap translation method,³² P-labeled nucleic acid probes having a specific activity in excess of 10⁸ cpm/mg can be prepared by replacing existing nucleotides with highly radioactive nucleotides. The hybridized membrane was then exposed to film for autoradiographic detection. Densitometric scanning of the film exposed to the hybridized membrane yields an accurate measurement of the copy number of the miR15 or miR16 gene. Moreover, the copy number of miR15 or miR16 gene can also be quantitatively analyzed with a computer imaging system, such as Molecular Dynamics 400-B 2DPhosphorimager from Amersham Biosciences, Piscataway, NJ.

如果无法用放射性核素对DNA或RNA探针进行标记，可以使用随机引物法将dTTP类似物5-(N-(N-生物素基--氨基己酸基)-3-氨基烯丙基)脱氧尿嘧啶三磷酸盐引入到探针分子中。生物素化的探针寡核苷酸可以通过与与荧光染色或能产生颜色反应的酶偶连的生物素结合蛋白如抗生物素蛋白，抗生物素蛋白链菌素，或抗生物素抗体反应来检测。If it is not possible to label the DNA or RNA probe with a radionuclide, the dTTP analogue 5-(N-(N-biotinyl-aminocaproyl)-3-aminoallyl) can be Deoxyuridine triphosphate was incorporated into the probe molecule. Biotinylated probe oligonucleotides can be reacted with a biotin-binding protein such as avidin, streptavidin, or an anti-biotin antibody coupled to a fluorescent stain or an enzyme that produces a color reaction to test.

miR15或miR16基因的缺失或突变还可以通过用聚合酶链式反应(PCR)扩增这些基因的片段，用测序或电泳分析扩增的片段以确定患者DNA样品的扩增片段的序列和/或长度是否和对照DNA样品有所不同来检测。本领域普通技术人员可以容易地确定DNA片段的PCR扩增的合适反应和循环条件。如下所述的实施例中使用的方法给出了典型的PCR反应和循环条件。Deletions or mutations in the miR15 or miR16 genes can also be determined by amplifying fragments of these genes by polymerase chain reaction (PCR) and analyzing the amplified fragments by sequencing or electrophoresis to determine the sequence and/or Whether the length is different from the control DNA sample is tested. Suitable reaction and cycling conditions for PCR amplification of DNA fragments can be readily determined by one of ordinary skill in the art. Typical PCR reactions and cycling conditions are given in the methods used in the Examples described below.

miR15或miR16介导的癌症的诊断可以通过检测不同染色体标记，例如图1A，1B，和2A-2D中所示的各标记之间13q14的缺失来进行。例如，在含有miR15或miR16的微卫星标记D13S272和D13S273之间的13q14区域的缺失表明存在miR15或miR16介导的癌症。另外，如果13q14的缺失是在微卫星标记D13S1150和D13S273之间或是在Alu18位点和微卫星标记D13S273之间，其中miR15或miR16缺失，则表明存在miR15或miR16介导的癌症。Diagnosis of miR15 or miR16 mediated cancers can be made by detecting deletion of 13q14 between different chromosomal markers, such as those shown in Figures 1A, 1B, and 2A-2D. For example, deletion of the 13q14 region between the microsatellite markers D13S272 and D13S273 containing miR15 or miR16 indicates the presence of miR15- or miR16-mediated cancer. In addition, if the deletion of 13q14 is between the microsatellite markers D13S1150 and D13S273 or between the Alu18 locus and the microsatellite marker D13S273, where miR15 or miR16 is missing, it indicates the presence of miR15- or miR16-mediated cancer.

另外一种确定组织样品中每个二倍体基因组的miR15或miR16基因数目的方法是基于miR15/miR16基因簇位于13q14并且与标记D13S272和D13S273相连的事实。在与D13S272和D13S273标记相连的位点杂合的个体的miR15或miR16基因拷贝的丢失可以从这些标记杂合的丢失推知。确定染色体标记杂合丢失的方法是本领域技术人员公知的。下述实施例3中给出了杂合丢失研究的实例。Another approach to determine the number of miR15 or miR16 genes per diploid genome in tissue samples is based on the fact that the miR15/miR16 gene cluster is located at 13q14 and is linked to markers D13S272 and D13S273. Loss of miR15 or miR16 gene copies in individuals heterozygous for loci linked to the D13S272 and D13S273 markers can be inferred from loss of heterozygosity for these markers. Methods for determining loss of heterozygosity for chromosomal markers are well known to those skilled in the art. An example of a loss of heterozygosity study is given in Example 3 below.

另一种确定怀疑患有CLL或前列腺癌的患者中miR15或miR16基因是否突变的方法是单链构象多肽性(SSCP)，例如Orita et al.(1989)，Genomics 5：874-879和Hayashi(1991)，PCRMethods and Applic.1：34-38中所述的，在此引入其全文作为参考。SSCP方法包括通过PCR扩增感兴趣的基因片段，使片段变性并在非变性条件下用两条变性单链进行电泳。单链呈现复杂的依赖于序列的链内二级结构，影响链的电泳迁移率。Another method for determining whether the miR15 or miR16 genes are mutated in patients suspected of having CLL or prostate cancer is single-strand conformational polypeptide (SSCP), e.g. Orita et al. (1989), Genomics 5:874-879 and Hayashi ( 1991), PCR Methods and Applic. 1:34-38, which is hereby incorporated by reference in its entirety. The SSCP method involves amplifying a gene fragment of interest by PCR, denaturing the fragment and electrophoresis with two denatured single strands under non-denaturing conditions. Single strands exhibit complex sequence-dependent intrachain secondary structures that affect the electrophoretic mobility of the strands.

miR15或miR16基因中一个或全部的缺失或突变还会引起miR15或miR16基因表达的减少。因此，CLL或前列腺癌还可以通过检测miR15或miR16基因产生的RNA的表达水平来诊断，miR15或miR16基因表达的减少则诊断为CLL或前列腺癌。Deletion or mutation of one or all of the miR15 or miR16 genes also results in decreased expression of the miR15 or miR16 genes. Therefore, CLL or prostate cancer can also be diagnosed by detecting the expression level of RNA produced by miR15 or miR16 gene, and the reduction of miR15 or miR16 gene expression can be diagnosed as CLL or prostate cancer.

miR15和miR16基因每个都转录产生～70kb的前体RNA，形成茎环结构。前体RNA不翻译成蛋白质，而是进一步处理成为“小分子RNA”或“miRNA”，认为这才是有功能的基因产物。The miR15 and miR16 genes are each transcribed to produce ~70 kb of precursor RNA, forming a stem-loop structure. Precursor RNA is not translated into protein, but is further processed into "small RNA" or "miRNA", which is considered to be a functional gene product.

这里所说的“miR15或miR16基因产物”是指由miR15和miR16基因而来的处理过的或是未处理的RNA转录物，下面将进一步说明。术语“RNA”，“RNA转录物”和“基因产物”在本文中述及miR15或miR16基因表达时可互换使用。As used herein, "miR15 or miR16 gene product" refers to processed or unprocessed RNA transcripts from miR15 and miR16 genes, as will be further described below. The terms "RNA", "RNA transcript" and "gene product" are used interchangeably herein when referring to miR15 or miR16 gene expression.

miR15和miR16前体RNA如Lagos-Quintana et al.(2001)，Science 294，853-858所述，在此引入其全文作为参考。miR15和miR16前体RNA的序列如SEQ ID NO：1和SEQ ID NO：2所示。预测的SEQ ID NO：1和SEQ ID NO：2的茎环结构分别如图1A和图1B所示。miR15 and miR16 precursor RNAs are described in Lagos-Quintana et al. (2001), Science 294, 853-858, which is hereby incorporated by reference in its entirety. The sequences of miR15 and miR16 precursor RNAs are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The predicted stem-loop structures of SEQ ID NO: 1 and SEQ ID NO: 2 are shown in Figure 1A and Figure 1B, respectively.

[SEQ ID NO：1]：[SEQ ID NO: 1]:

ccuuggaguaaaguagcagcacauaaugguuuguggauuuugaaaaggugcaggccauauugugcugccucaaaaauacaaggccuuggaguaaaguagcagcacauaauguuuguggauuuugaaaaggugcaggccaauauugugcugccucaaaaauacaagg

[SEQ ID NO：2]：[SEQ ID NO: 2]:

gucagcagugccuuagcagcacguaaauauuggcguuaagauucuaaaauuaucuccaguauuaacugug cugcugaagu aagguugacgucagcagugccuuagcagcacguaaauauuggcguuaagauucuaaaauuaucuccaguauuaacugug cugcugaagu aagguugac

不受任何理论的限制，认为miR15和miR16前体RNA是由miR15/miR16基因簇共表达的，由Dicer/Argonaute复合体处理成为功能性miRNA产物。参见，例如Lee et al.(2001)，Science 294：862。这些基因产生的两个功能性miRNA产物都是长度为22个核苷酸的单链RNA分子，5’端具有单磷酸盐，3’端具有羟基。处理过的miR15小分子RNA的核苷酸序列是uagcagcacauaaugguuugug(SEQ ID NO：3)。处理过的miR16小分子RNA的核苷酸序列是uagcagcacguaaauauuggcg(SEQ ID NO：4)。在本发明中，可以检测到由miR15或miR16基因产生的60-70nt的RNA前体分子。另外还可以检测到由Dicer和Argonaute蛋白对前体RNA进行处理产生的较短的miR15和miR16小分子RNA基因产物。Without being bound by any theory, it is believed that miR15 and miR16 precursor RNAs are co-expressed by the miR15/miR16 gene cluster and processed by the Dicer/Argonaute complex into functional miRNA products. See, eg, Lee et al. (2001), Science 294:862. Both functional miRNA products produced by these genes are single-stranded RNA molecules of 22 nucleotides in length with a monophosphate at the 5' end and a hydroxyl group at the 3' end. The nucleotide sequence of the processed miR15 small molecule RNA is uagcagcacauaauguuugug (SEQ ID NO: 3). The nucleotide sequence of the processed miR16 small molecule RNA is uagcagcacguaaauauuggcg (SEQ ID NO: 4). In the present invention, 60-70 nt RNA precursor molecules produced by miR15 or miR16 genes can be detected. In addition, shorter miR15 and miR16 small RNA gene products generated by the processing of precursor RNA by Dicer and Argonaute proteins were detected.

确定RNA表达水平的方法是本领域技术人员公知的。例如，如上所述从怀疑患有CLL或前列腺癌的患者中获得组织或血液样品。如上所述从患者的未受影响的组织中，或从正常人个体中获得相应的组织或血液样品作为对照。对照组织或血液样品与患者的样品一同处理。然后将患者中miR15或miR16基因的表达水平与患者中未受影响的组织的相比，或者与正常对照的组织或血液中的miR15或miR16表达水平相比。例如，CLL细胞或样品前列腺癌细胞中的相对miR15或miR16表达水平可以相对于一种或多种标准容易地确定。这些标准包括，例如，一种是表达水平为零，另一种是同一个患者的正常组织的基因表达水平，或正常对照组组织中的表达水平。所述标准还可以包括标准细胞系中的miR15或miR16表达水平。miR15或miR16表达与正常表达水平相比减少的量表示治疗后将具有的临床效果。Methods for determining RNA expression levels are well known to those skilled in the art. For example, a tissue or blood sample is obtained from a patient suspected of having CLL or prostate cancer, as described above. Corresponding tissue or blood samples were obtained as controls from unaffected tissues of patients, or from normal human subjects, as described above. A control tissue or blood sample is processed along with the patient's sample. The expression level of the miR15 or miR16 gene in the patient is then compared to that in unaffected tissue in the patient, or to the expression level of miR15 or miR16 in tissue or blood of a normal control. For example, relative miR15 or miR16 expression levels in CLL cells or sample prostate cancer cells can be readily determined relative to one or more standards. These criteria include, for example, one where the expression level is zero, and another that is the gene's expression level in normal tissue from the same patient, or the expression level in normal control tissue. The criteria can also include miR15 or miR16 expression levels in the standard cell lines. The amount by which miR15 or miR16 expression is reduced compared to normal expression levels is indicative of the clinical effect that treatment will have.

另外，还可以将怀疑患有CLL或前列腺癌的患者中的miR15或miR16基因表达水平与先前获得的正常对照人群的miR15或miR16基因表达的平均水平相比较。In addition, the expression level of miR15 or miR16 gene in patients suspected of having CLL or prostate cancer can also be compared with the average level of gene expression of miR15 or miR16 in a normal control population previously obtained.

确定细胞中特定基因的RNA转录物水平的适当方法是本领域技术人员公知的。根据其中一种方法。用核酸提取缓冲液进行匀浆从细胞中提取总细胞RNA，然后离心。使核酸沉淀，用DNA酶处理和沉淀除去DNA。然后根据标准方法用琼脂糖凝胶电泳分离RNA分子，用例如所谓的“Northern”blotting技术转移到硝酸纤维素膜上。然后通过加热将RNA固定在膜上。用与所述RNA互补的具有适当标记的DNA或RNA探针对特定的RNA进行检测和定量。参见，例如，Molecular Cloning：A LaboratoryManual，J.Sambrook et al.，eds.，2nd edition，Cold Spring HarborLaboratory Press，1989，Chapter 7，在此引入其全文作为参考。用于miR15或miR16 RNA的Northern blot杂交的适当探针包括SEQ ID NO：5和SEQ ID NO：6。Suitable methods for determining the level of RNA transcripts of a particular gene in a cell are well known to those skilled in the art. According to one of the methods. Total cellular RNA is extracted from cells by homogenization with nucleic acid extraction buffer, followed by centrifugation. Nucleic acids are precipitated, and the DNA is removed by DNase treatment and precipitation. The RNA molecules are then separated by agarose gel electrophoresis according to standard methods and transferred to nitrocellulose membranes, eg using the so-called "Northern" blotting technique. The RNA is then immobilized on the membrane by heating. Specific RNA is detected and quantified using appropriately labeled DNA or RNA probes complementary to the RNA. See, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7, which is hereby incorporated by reference in its entirety. Suitable probes for Northern blot hybridization of miR15 or miR16 RNA include SEQ ID NO:5 and SEQ ID NO:6.

将杂交膜曝光在胶片上，对与miR15或miR16 RNA杂交的探针进行放射自显影检测。对杂交膜曝光的胶片进行光密度扫描可以精确地测量RNA转录水平。另外，还可以通过杂交结果的计算机成像对RNA转录水平进行定量分析，例如用AmershamBiosciences，Piscataway，NJ的the Molecular Dynamics 400-B 2DPhosphorimager。The hybridized membrane was exposed on film, and the probe hybridized to miR15 or miR16 RNA was detected by autoradiography. Densitometric scanning of film exposed to hybridized membranes allows precise measurement of RNA transcript levels. Alternatively, RNA transcript levels can be quantified by computer imaging of the hybridization results, for example, with the Molecular Dynamics 400-B 2DPhosphorimager from Amersham Biosciences, Piscataway, NJ.

除了Northern和其它RNA blotting杂交技术，还可以用原位杂交技术检测RNA转录水平。这种技术比Northern blotting技术需要的细胞要少，包括将全细胞沉积在显微镜的盖片上，用含有放射性标记或其它标记的cDNA或cRNA探针的溶液检测细胞的核酸含量。这种技术特别适用于分析来自怀疑患有前列腺癌的患者的生物活组织样品。更详细的原位杂交的操作方法如美国专利序列号5,427,916所述，在此引入其全文作为参考。适用于miR15或miR16 RNA的原位杂交的探针包括SEQ ID NO：5和SEQ IDNO：6。In addition to Northern and other RNA blotting hybridization techniques, in situ hybridization can also be used to detect RNA transcript levels. This technique requires fewer cells than the Northern blotting technique, including depositing whole cells on a microscope cover slip, and detecting the nucleic acid content of the cells with a solution containing radiolabeled or other labeled cDNA or cRNA probes. This technique is particularly suitable for the analysis of biopsies from patients suspected of having prostate cancer. A more detailed procedure for in situ hybridization is described in US Patent Serial No. 5,427,916, which is hereby incorporated by reference in its entirety. Probes suitable for in situ hybridization of miR15 or miR16 RNA include SEQ ID NO: 5 and SEQ ID NO: 6.

miR15或miR16转录物的相对数目还可以通过miR15或miR16转录物的反向转录，然后进行聚合酶链式反应扩增(RT-PCR)来测定。miR15或miR16转录物水平可以与内标相比较定量，例如，相同样品中“持家”基因的mRNA水平。可用作内标的合适的“持家”基因包括肌球蛋白或甘油醛-3-磷酸酯脱氢酶(G3PDH)。适用于定量RT-PCR的方法以及其它方法是本领域普通技术人员公知的。The relative number of miR15 or miR16 transcripts can also be determined by reverse transcription of miR15 or miR16 transcripts followed by polymerase chain reaction amplification (RT-PCR). miR15 or miR16 transcript levels can be quantified in comparison to internal standards, eg, mRNA levels of "housekeeping" genes in the same sample. Suitable "housekeeping" genes that can be used as internal standards include myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Methods suitable for quantitative RT-PCR, as well as others, are well known to those of ordinary skill in the art.

测量miR15和miR16表达的其它方法也是本领域技术人员公知的，包括各种测量RNA转录和降解速度的方法。Other methods of measuring miR15 and miR16 expression are also known to those skilled in the art, including various methods of measuring RNA transcription and degradation rates.

可以将分离的miR15或miR16基因产物，单独或是组合给予miR15或miR16介导的癌症细胞，从而治疗miR15或miR16介导的癌症。不希望受到任何理论的限制，认为miR15或miR16基因产物能够抑制这些癌细胞的赘生物或肿瘤生长。The isolated miR15 or miR16 gene products, alone or in combination, can be administered to miR15 or miR16 mediated cancer cells, thereby treating miR15 or miR16 mediated cancer. Without wishing to be bound by any theory, it is believed that the miR15 or miR16 gene products are capable of inhibiting neoplastic or tumor growth of these cancer cells.

特别是，可以将分离的miR15或miR16基因产物单独或是组合给予CLL或前列腺癌细胞，来治疗CLL或前列腺癌。In particular, isolated miR15 or miR16 gene products, alone or in combination, can be administered to CLL or prostate cancer cells to treat CLL or prostate cancer.

这里所说的“miR15或miR16介导的癌症细胞”是指从患有miR15或miR16介导的癌症的患者中分离得到的肿瘤或是赘生物细胞。miR15或miR16介导的癌症细胞可以通过检测细胞中miR15或miR16基因产物的减少或缺失，或者通过检测细胞中的癌症或赘生物表型来识别。本领域技术人员可以容易地识别具有癌症或赘生物表型的细胞。例如，培养物中的细胞对于接触诱导的生长抑制不敏感，继续培养的时候聚集在一起。癌症或赘生物细胞还表现出特有的形态学变化，细胞集落无组织生长以及不贴壁生长。癌症或赘生物细胞还具有在易感动物中形成侵入性肿瘤的能力，这可以用本领域的技术通过将细胞注射到无胸腺小鼠中来评价。The term "miR15 or miR16-mediated cancer cells" herein refers to tumor or neoplastic cells isolated from patients suffering from miR15 or miR16-mediated cancers. miR15 or miR16 mediated cancer cells can be identified by detecting a reduction or absence of miR15 or miR16 gene products in the cells, or by detecting a cancer or neoplastic phenotype in the cells. Cells with a cancerous or neoplastic phenotype can readily be identified by those skilled in the art. For example, cells in culture are insensitive to contact-induced growth inhibition and aggregate when continued in culture. Cancer or neoplastic cells also exhibit characteristic morphological changes, disorganized growth of cell colonies, and non-adherent growth. Cancer or neoplastic cells also have the ability to form invasive tumors in susceptible animals, which can be assessed by injecting the cells into athymic mice using techniques in the art.

这里所说的“分离的”基因产物是通过人为介入使得与其自然状态不同或从其自然状态中分离出来的基因产物。例如，天然存在于活的动物中的RNA不是“分离的”。合成的RNA，或者从其自然状态的共存物中部分或全部分离出来的RNA是“分离的”。分离的RNA可以基本上纯的形式存在，也可以存在于RNA递送到其中的细胞中。因此，递送到细胞中，或者在细胞，例如CLL或前列腺癌细胞中表达的miR15或miR16基因产物认为是“分离的”基因产物。An "isolated" gene product as used herein is a gene product that has been made different from or separated from its natural state by human intervention. For example, RNA that occurs naturally in a living animal is not "isolated." Synthetic RNA, or RNA that has been partially or completely separated from its natural state coexistence, is "isolated." Isolated RNA can be present in substantially pure form or in a cell into which the RNA is delivered. Thus, a miR15 or miR16 gene product that is delivered to, or expressed in, a cell, such as a CLL or prostate cancer cell, is considered an "isolated" gene product.

可以用多种标准方法得到miR15或miR16基因产物。例如，基因产物可以化学合成或者用本领域公知的方法重组产生。优选地，所述RNA产物用适当保护的核糖核苷亚磷酰胺和传统DNA/RNA合成仪化学合成。合成RNA分子或合成试剂的供应商包括Proligo(Hamburg，Germany)，Dharmacon Research(Lafayette，CO，USA)，Pierce Chemical(part of Perbio Science，Rockford，IL，USA)，Glen Research(Sterling，VA，USA)，ChemGenes(Ashland，MA，USA)和Cruachem(Glasgow，UK)。A miR15 or miR16 gene product can be obtained using a variety of standard methods. For example, gene products can be chemically synthesized or produced recombinantly using methods well known in the art. Preferably, the RNA product is chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Suppliers of synthetic RNA molecules or synthetic reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA ), ChemGenes (Ashland, MA, USA) and Cruachem (Glasgow, UK).

另外，miR15和miR16基因产物可以用任何适当的启动子用重组环形或线性DNA质粒表达。用于在质粒中表达RNA的适当启动子包括U6或H1 RNA pol III启动子序列，或巨细胞病毒启动子。本领域技术人员熟知如何选择其它合适的启动子。本发明的重组质粒还可以包括诱导型或调节型启动子，用以在CLL，前列腺癌，或其它细胞中表达miR15和miR16基因产物。Alternatively, the miR15 and miR16 gene products can be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for expressing RNA in plasmids include the U6 or H1 RNA pol III promoter sequence, or the cytomegalovirus promoter. Those skilled in the art are familiar with how to select other suitable promoters. The recombinant plasmids of the present invention may also include inducible or regulatable promoters for expression of miR15 and miR16 gene products in CLL, prostate cancer, or other cells.

重组质粒表达的miR15和miR16基因产物可以用标准方法从培养的细胞表达系统中分离出来。重组质粒表达的miR15和miR16基因产物还可以递送到CLL或前列腺癌细胞中或直接在其中表达。用重组质粒将miR15和miR16基因产物递送到CLL或前列腺癌细胞中将在下面详细讨论。The miR15 and miR16 gene products expressed from the recombinant plasmids can be isolated from cultured cell expression systems using standard methods. The miR15 and miR16 gene products expressed from the recombinant plasmids can also be delivered to or directly expressed in CLL or prostate cancer cells. The use of recombinant plasmids to deliver miR15 and miR16 gene products into CLL or prostate cancer cells is discussed in detail below.

miR15和miR16基因产物可以分别在单独的重组质粒中表达，或者也可以在同一个重组质粒中表达。优选地，miR15和miR16基因产物在单个质粒中作为RNA前体分子表达，然后用适当的处理系统将前体分子处理成为功能性miRNA分子。适当的处理系统包括体外果蝇细胞溶出液系统，如Tuschl等的美国公开申请2002/0086356中所述的，在此引入其全文作为参考。miR15 and miR16 gene products can be expressed in separate recombinant plasmids, or can also be expressed in the same recombinant plasmid. Preferably, the miR15 and miR16 gene products are expressed in a single plasmid as RNA precursor molecules, which are then processed into functional miRNA molecules using an appropriate processing system. Suitable processing systems include the in vitro Drosophila cell lysate system as described in US Published Application 2002/0086356 by Tuschl et al., which is hereby incorporated by reference in its entirety.

适于表达miR15和miR16基因产物的质粒的选择，向质粒中插入核酸序列并表达基因产物的方法，以及将重组质粒递送到感兴趣细胞中的方法都是本领域技术人员公知的。参见，例如Zenget al.(2002)，Molecular Cell 9：1327-1333；Tuschl(2002)，Nat.Biotechnol，20：446-448；Brummelkamp et al.(2002)，Science 296：550-553；Miyagishi et al.(2002)，Nat.Biotechnol.20：497-500；Paddison et al.(2002)，Genes Dev.16：948-958；Lee et al.(2002)，Nat.Biotechnol.20：500-505；和Paul et al.(2002)，Nat.Biotechnol.20：505-508，在此引入其全文作为参考。Selection of plasmids suitable for expressing miR15 and miR16 gene products, methods for inserting nucleic acid sequences into plasmids and expressing gene products, and methods for delivering recombinant plasmids to cells of interest are well known to those skilled in the art. See, e.g., Zenget al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553; Miyagishi et al. al. (2002), Nat. Biotechnol. 20: 497-500; Paddison et al. (2002), Genes Dev. 16: 948-958; Lee et al. (2002), Nat. Biotechnol. 20: 500-505 and Paul et al. (2002), Nat. Biotechnol. 20:505-508, which is hereby incorporated by reference in its entirety.

在优选的实施方案中，用于表达miR15或miR16基因产物的质粒包括处于CMV中早期启动子控制之下的编码miR15或miR16前体RNA的序列。这里所述的“处于启动子控制之下”表示编码miRNA产物的的核酸序列位于该启动子的3’端，使得该启动子可以起始miRNA产物编码序列的转录。In a preferred embodiment, the plasmid used to express the miR15 or miR16 gene product includes a sequence encoding a miR15 or miR16 precursor RNA under the control of the CMV mid-early promoter. "Under the control of a promoter" as described herein means that the nucleic acid sequence encoding the miRNA product is located at the 3' end of the promoter, so that the promoter can initiate the transcription of the miRNA product coding sequence.

miR15或miR16基因产物还可以用重组病毒载体表达。miR15和miR16基因产物可以用两个单独的重组病毒载体表达，或者可以用同一个病毒载体表达。重组病毒载体表达的RNA可以用标准方法从培养的细胞表达系统中分离出来。或者可以直接在CLL或前列腺癌细胞中表达。使用重组病毒载体将miR15或miR16基因产物递送到CLL或前列腺癌细胞中将在下面详细讨论。miR15 or miR16 gene products can also be expressed using recombinant viral vectors. The miR15 and miR16 gene products can be expressed from two separate recombinant viral vectors, or can be expressed from the same viral vector. RNA expressed from recombinant viral vectors can be isolated from cultured cell expression systems by standard methods. Or it can be directly expressed in CLL or prostate cancer cells. The use of recombinant viral vectors to deliver miR15 or miR16 gene products into CLL or prostate cancer cells is discussed in detail below.

本发明的重组病毒载体包括编码miR15和miR16基因产物的序列和用于表达RNA序列的任何合适的启动子。合适的启动子包括，例如U6或H1 RNA pol III启动子序列，或巨细胞病毒启动子。本领域技术人员熟知如何选择其它合适的启动子。本发明的重组质粒还可以包括诱导型或调节型启动子，用以在CLL或前列腺癌细胞中表达miR15和miR16基因产物。The recombinant viral vectors of the present invention include sequences encoding the miR15 and miR16 gene products and any suitable promoter for expressing RNA sequences. Suitable promoters include, for example, the U6 or H1 RNA pol III promoter sequences, or the cytomegalovirus promoter. Those skilled in the art are familiar with how to select other suitable promoters. The recombinant plasmids of the present invention may also include inducible or regulatable promoters for expression of miR15 and miR16 gene products in CLL or prostate cancer cells.

可以使用能够接受miR15和miR16基因产物的编码序列的任何病毒载体；例如，来自腺病毒(AV)；腺相关病毒(AAV)；逆转录病毒(例如慢病毒(LV)，棒状病毒，鼠白血病毒)；疱疹病毒等的载体。还可以用包被蛋白或其它病毒的其它表面蛋白修饰载体从而改变病毒载体的趋向性。例如，可以用疱疹性口炎病毒(VSV)，狂犬病毒，埃博拉病毒，莫科拉病毒等修饰本发明的AAV载体。适用于本发明的重组病毒载体的选择，向载体中插入核酸序列并表达基因产物的方法，以及将病毒载体递送到感兴趣细胞中的方法都是本领域技术人员公知的。参见，例如，Dornburg(1995)，Gene Therap.2：301-310；Eglitis(1988)，Biotechniques 6：608-614；Miller(1990)，Hum.Gene Therap.1：5-14；和Anderson(1998)，Nature 392：25-30，在此引入其全文作为参考。Any viral vector capable of accepting the coding sequences of the miR15 and miR16 gene products can be used; e.g., from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g. lentivirus (LV), rhabdovirus, murine leukemia virus ); vectors of herpes virus, etc. The vector can also be modified with coat proteins or other surface proteins of other viruses to alter the tropism of the viral vector. For example, the AAV vectors of the invention can be modified with vesicular stomatitis virus (VSV), rabies virus, Ebola virus, Mokola virus, and the like. Selection of recombinant viral vectors suitable for use in the present invention, methods for inserting nucleic acid sequences into vectors and expressing gene products, and methods for delivering viral vectors to cells of interest are well known to those skilled in the art. See, e.g., Dornburg (1995), Gene Therap. 2: 301-310; Eglitis (1988), Biotechniques 6: 608-614; Miller (1990), Hum. Gene Therap. 1: 5-14; and Anderson (1998 ), Nature 392:25-30, which is hereby incorporated by reference in its entirety.

优选的病毒载体是来自于AV和AAV的载体。适用于表达本发明的miRNA的AV载体，构建重组AV载体的方法，和将载体递送到靶细胞中的方法，在Xia et al.(2002)，Nat.Biotech.20：1006-1010中有描述，在此引入其全文作为参考。适于表达本发明的miRNA的AAV载体，构建重组AAV载体的方法，和将载体递送到靶细胞中的方法，在Samulski et al.(1987)，J.Virol.61：3096-3101；Fisher et al.(1996)，J.Viral.，70：520-532；Samulski etal.(1989)，J.Virol.63：3822-3826；美国专利序列号5,252,479；美国专利序列号5,139,941；国际专利申请WO 94/13788；和国际专利申请WO 93/24641，在此引入其全文作为参考。优选地，miR15和miR16基因产物用含有CMV中早期启动子的单个重组AAV载体表达。Preferred viral vectors are those from AV and AAV. AV vectors suitable for expressing miRNAs of the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia et al. (2002), Nat.Biotech.20: 1006-1010 , which is hereby incorporated by reference in its entirety. AAV vectors suitable for expressing miRNAs of the present invention, methods for constructing recombinant AAV vectors, and methods for vector delivery into target cells, in Samulski et al. (1987), J.Virol.61: 3096-3101; Fisher et al. al. (1996), J.Viral., 70:520-532; Samulski et al. (1989), J.Virol.63:3822-3826; U.S. Patent Serial No. 5,252,479; U.S. Patent Serial No. 5,139,941; International Patent Application WO 94/13788; and International Patent Application WO 93/24641, which are hereby incorporated by reference in their entirety. Preferably, the miR15 and miR16 gene products are expressed from a single recombinant AAV vector containing the early promoter in CMV.

在一个实施方案中，本发明的重组AAV病毒载体包括编码miR15或miR16前体RNA的核酸序列，可操作地与polyT终止序列相连，并处于人U6 RNA启动子的控制之下。这里所述的“可操作地与polyT终止序列相连”是指编码有义链或反义链的核酸序列在5’方向紧邻polyT终止信号。在miR15或miR16序列在载体中转录的过程中，polyT终止信号用于终止转录。In one embodiment, the recombinant AAV viral vector of the present invention includes a nucleic acid sequence encoding miR15 or miR16 precursor RNA, operably linked to a polyT termination sequence, and under the control of a human U6 RNA promoter. The term "operably linked to a polyT termination sequence" as used herein means that the nucleic acid sequence encoding the sense strand or the antisense strand is adjacent to the polyT termination signal in the 5' direction. During transcription of the miR15 or miR16 sequence in the vector, the polyT termination signal is used to terminate transcription.

在本发明中，miR15或miR16基因产物用于抑制miR15或miR16介导的癌细胞，特别是CLL或前列腺癌细胞的赘生物或肿瘤生长。不希望受到任何理论的限制，认为处理过的miR15和miR16 miRNA与这些细胞中起始和/或维持赘生物或肿瘤细胞生长所必需的一个或多个靶mRNA中的互补序列相结合。因此，本发明提供了一种在需要这种治疗的患者中治疗miR15或miR16介导的癌症，例如CLL或前列腺癌的方法。所述方法包括给予患者有效量的miR15或miR16基因产物，使得miR15或miR16介导的癌细胞的增殖受到抑制。In the present invention, miR15 or miR16 gene products are used to inhibit miR15 or miR16-mediated neoplastic or tumor growth of cancer cells, especially CLL or prostate cancer cells. Without wishing to be bound by any theory, it is believed that the processed miR15 and miR16 miRNAs bind to complementary sequences in one or more target mRNAs necessary for the initiation and/or maintenance of neoplastic or tumor cell growth in these cells. Accordingly, the present invention provides a method of treating miR15 or miR16 mediated cancer, such as CLL or prostate cancer, in a patient in need of such treatment. The method includes administering to a patient an effective amount of miR15 or miR16 gene product, so that the proliferation of cancer cells mediated by miR15 or miR16 is inhibited.

如上所述，miR15或miR16介导的癌症是指其中在与该疾病相关的肿瘤或赘生物细胞的至少一部分中miR15或miR16基因中的任何一个或两者都减少或缺失的癌症。在CLL或前列腺癌的肿瘤或赘生物细胞中miR15或miR16基因的表达减少或缺失；因此，CLL和前列腺癌被认为是miR15或miR16介导的癌症。在其它癌症中也发现miR15或miR16基因表达的减少或缺失；这些癌症也被视为是miR15或miR16介导的癌症。As described above, a miR15 or miR16 mediated cancer refers to a cancer in which either or both of the miR15 or miR16 genes are reduced or deleted in at least a portion of the tumor or neoplastic cells associated with the disease. Expression of miR15 or miR16 genes is reduced or absent in tumor or neoplastic cells of CLL or prostate cancer; thus, CLL and prostate cancer are considered to be miR15- or miR16-mediated cancers. Reduced or absent miR15 or miR16 gene expression is also found in other cancers; these cancers are also considered to be miR15 or miR16 mediated cancers.

例如，至少在下述组织分型的癌症的初级或转移性肿瘤或赘生物细胞中miR15或miR16基因表达减少或缺失：肉瘤(中表层的结缔组织和其他组织癌症)；黑色素瘤(源于黑色素细胞的癌症)；癌(上皮癌症)；腺癌(腺上皮癌症)；神经系统癌症(神经胶质瘤/成胶质细胞瘤和星状细胞瘤)；和血液瘤，例如白血病和淋巴瘤(例如，急性淋巴细胞白血病和慢性骨髓性白血病)。For example, miR15 or miR16 gene expression is reduced or absent in at least primary or metastatic tumor or neoplastic cells of cancers of the following histological types: sarcomas (cancers of the connective and other tissues in the middle and superficial layers); cancers); carcinomas (epithelial cancers); adenocarcinomas (glandular epithelial cancers); nervous system cancers (gliomas/glioblastomas and astrocytomas); and hematological tumors, such as leukemias and lymphomas (e.g. , acute lymphoblastic leukemia and chronic myelogenous leukemia).

在来源于至少下述器官或组织的癌症中miR15或miR16基因表达也减少或缺失，不管其组织分型如何：乳腺；男性和女性泌尿生殖组织(例如输尿管，膀胱，前列腺，睾丸，卵巢，子宫颈，子宫，阴道)；肺；胃肠系统组织(例如，胃，大肠和小肠，结肠，直肠)；外分泌腺例如胰腺和肾上腺；口腔和食道组织；脑和脊髓；肾(肾脏)；胰腺；肝胆系统(例如，肝，胆囊)；淋巴系统；平滑肌和横纹肌；骨骼和骨髓；皮肤和眼部组织。miR15 or miR16 gene expression was also reduced or absent in cancers derived from at least the following organs or tissues, regardless of histologic type: breast; male and female urogenital tissues (e.g., ureter, bladder, prostate, testis, ovary, cervix, uterus, vagina); lungs; tissues of the gastrointestinal system (e.g., stomach, large and small intestines, colon, rectum); exocrine glands such as pancreas and adrenal glands; oral and esophageal tissues; brain and spinal cord; kidneys (kidneys); pancreas; Hepatobiliary system (eg, liver, gallbladder); lymphatic system; smooth and striated muscles; bone and bone marrow; skin and ocular tissues.

在癌症或肿瘤的预后阶段miR15或miR16基因表达也减少或缺失，例如可用“Overall Stage Groupings”(也称为“RomanNumeral”)或“Tumor，Nodes，and Metastases”(TNM)分期系统测量。对于给定癌症的适合的预后分期系统和分期描述是本领域公知的，例如在National Cancer Institute的“CancerNet”国际互联网站上所述的。miR15 or miR16 gene expression is also reduced or absent at prognostic stages of cancer or tumors, as measured, for example, by the "Overall Stage Groupings" (also known as "RomanNumeral") or "Tumor, Nodes, and Metastases" (TNM) staging systems. Suitable prognostic staging systems and staging descriptions for a given cancer are well known in the art, for example at the National Cancer Institute's "CancerNet" Internet site.

需要治疗miR15或miR16介导的癌症的患者可以通过下述方法鉴别：从该患者获得肿瘤或赘生物细胞(或者怀疑是肿瘤或赘生物的细胞)样品，测定其中至少一部分细胞与得自该患者正常组织的细胞相比miR15或miR16的表达是否减少或缺失。检测细胞中miR15或miR16基因表达水平的方法是本领域技术人员公知的(如上所述)。另外，得自患者的细胞中的miR15或miR16表达还可以与这些基因在正常人群的细胞中的平均表达水平相比较。医生可以容易地用标准诊断技术鉴别需要治疗CLL的患者。参见，例如″Chronic lymphocytic leukemia：recommendationsfor diagnosis，staging，and response criteria.International Workshopon Chronic Lymphocytic Leukemia，″(1989)Annals of InternalMedicine110(3)：236-238，在此引入其全文作为参考。例如，患有CLL的患者具有循环的CLL细胞，表现出淋巴细胞增多(即血液中的淋巴细胞数目等于或大于每立方毫米10,000个细胞)，以及CLL细胞在骨髓和淋巴组织中逐渐积累。A patient in need of treatment for a miR15- or miR16-mediated cancer can be identified by obtaining a sample of tumor or neoplastic cells (or cells suspected of being a tumor or neoplasm) from the patient, and assaying at least a portion of the cells for Whether the expression of miR15 or miR16 is reduced or absent in cells of normal tissues. Methods for detecting the expression levels of miR15 or miR16 genes in cells are well known to those skilled in the art (as described above). In addition, miR15 or miR16 expression in cells from patients can also be compared to the average expression levels of these genes in cells from a normal population. Physicians can readily identify patients in need of treatment for CLL using standard diagnostic techniques. See, e.g., "Chronic lymphocytic leukemia: recommendations for diagnosis, staging, and response criteria. International Workshop on Chronic Lymphocytic Leukemia," (1989) Annals of Internal Medicine 110(3):236-238, which is hereby incorporated by reference in its entirety. For example, patients with CLL have circulating CLL cells, exhibit lymphocytosis (ie, the number of lymphocytes in the blood is equal to or greater than 10,000 cells per cubic millimeter), and a progressive accumulation of CLL cells in the bone marrow and lymphoid tissues.

患者血液或其它组织中CLL细胞的鉴定可以通过直接目测观察血液样品，和/或测定淋巴细胞的“CLL分数”来验证。CLL分数表示是否存在CLL细胞的五个淋巴细胞表面标记：CD5+，CD23+，FMC7-，和表面免疫球蛋白(SmIg)的微弱表达(+/-)和CD22。该评分系统对这五个标记中的每一个都给出1或0的值，依据是其对CLL是典型的还是非典型的。CLL细胞的CLL分数为4或5，而其它白血病的淋巴细胞的CLL分数为＜1到3。参见Matutes et al.(1994)，Leukemia 810：1640-1645 and Moreauet al.(1997)，American Journal of Clinical Pathology，108：378-82，在此引入其全文作为参考。CLL细胞与正常外周血B细胞相比膜表面的免疫球蛋白水平较低。淋巴细胞膜表面的免疫球蛋白水平可以容易地用标准方法检测；参见，例如Rozman et al.(1995)，New England Journal of Medicine 333：1052-1057，在此引入其全文作为参考。Identification of CLL cells in a patient's blood or other tissue can be verified by direct visual inspection of a blood sample, and/or determination of a "CLL fraction" of lymphocytes. The CLL score indicates the presence or absence of five lymphocyte surface markers of CLL cells: CD5+, CD23+, FMC7-, and weak expression (+/-) of surface immunoglobulin (SmIg) and CD22. This scoring system gives each of these five markers a value of 1 or 0, depending on whether it is typical or atypical for CLL. CLL cells have a CLL score of 4 or 5, whereas lymphocytes from other leukemias have a CLL score of <1 to 3. See Matutes et al. (1994), Leukemia 810:1640-1645 and Moreau et al. (1997), American Journal of Clinical Pathology, 108:378-82, which are hereby incorporated by reference in their entirety. CLL cells have lower levels of immunoglobulins on the membrane surface than normal peripheral blood B cells. Immunoglobulin levels on the membrane surface of lymphocytes can be readily detected by standard methods; see, eg, Rozman et al. (1995), New England Journal of Medicine 333:1052-1057, which is hereby incorporated by reference in its entirety.

医生也可以容易地根据标准诊断技术鉴别出需要治疗前列腺癌的患者，所用的标准例如患者年龄，用肛门触诊检查出前列腺增大，前列腺特异的抗原(“PSA”)水平，组织的格里森分数，以及存在在免疫组织化学水平上可检测到的遗传标记，例如前列腺组织细胞上的p53，p21，和细胞周期蛋白。血清PSA水平为20ng/ml或更高并且前列腺组织分化不佳(例如格里森分数为8或更高)表示患有前列腺癌。患者中前列腺肿瘤的存在也可以通过非侵入性的成像技术验证，例如CT扫描，对前列腺的经直肠的超声检查(“TRUSP”)，以及磁共振成像(“MRI”)，这些技术都是本领域公知的。Physicians can also readily identify patients in need of treatment for prostate cancer based on standard diagnostic techniques, such as patient age, detection of enlarged prostate by anal palpation, prostate-specific antigen ("PSA") levels, Gerry Sen score, and the presence of genetic markers detectable at the immunohistochemical level, such as p53, p21, and cyclins on prostate tissue cells. A serum PSA level of 20 ng/ml or higher and poorly differentiated prostate tissue (eg, a Gleason score of 8 or higher) is indicative of prostate cancer. The presence of a prostate tumor in a patient can also be verified by non-invasive imaging techniques such as CT scans, transrectal ultrasonography of the prostate ("TRUSP"), and magnetic resonance imaging ("MRI"), which are all well known in the field.

这里所述的miR15或miR16基因产物的“有效量”是指足以抑制患有miR15或miR16介导的癌症的患者中miR15或miR16介导的癌细胞的量。例如，miR15或miR16基因产物的有效量可以是足以抑制患有CLL的患者中CLL细胞增殖的量，或患有前列腺癌的患者中前列腺癌细胞增殖的量。应当理解，“前列腺肿瘤细胞”不一定存在于前列腺中，而是包括来源于前列腺的转移肿瘤的细胞。An "effective amount" of a miR15 or miR16 gene product as used herein refers to an amount sufficient to inhibit miR15 or miR16 mediated cancer cells in a patient suffering from miR15 or miR16 mediated cancer. For example, an effective amount of a miR15 or miR16 gene product can be an amount sufficient to inhibit the proliferation of CLL cells in a patient with CLL, or the proliferation of prostate cancer cells in a patient with prostate cancer. It should be understood that "prostate tumor cells" are not necessarily present in the prostate, but include cells derived from metastatic tumors of the prostate.

这里所说的“抑制miR15或miR16介导的癌细胞的增殖”是指杀死细胞，或永久性或暂时性地使细胞停止生长。如果在施用了miR15或miR16产物以后患者中的这些细胞保持不变或减少，那么则可以推断出miR15或miR16介导的癌细胞的增殖受到了抑制。如果这些细胞的绝对数量增加，但是肿瘤生长的速度降低了，那么也认为miR15或miR16介导的癌细胞的增殖受到了抑制。The term "inhibiting the proliferation of cancer cells mediated by miR15 or miR16" refers to killing cells, or permanently or temporarily stopping the growth of cells. If these cells remain unchanged or decrease in a patient following administration of miR15 or miR16 products, it can be concluded that miR15 or miR16 mediated proliferation of cancer cells is inhibited. If the absolute number of these cells is increased, but the rate of tumor growth is reduced, then miR15- or miR16-mediated proliferation of cancer cells is also considered to be inhibited.

患者体内miR15或miR16介导的癌细胞的数量可以通过直接测量确定，或者从原始或转移肿瘤块的尺寸估计确定。The number of miR15- or miR16-mediated cancer cells in a patient can be determined by direct measurement or estimated from the size of the original or metastatic tumor mass.

例如，患者CLL细胞的数目可以容易地确定，例如通过全血或白细胞计数。CLL细胞的数目还可以通过免疫组织化学方法，流式细胞术，或其它用于检测CLL细胞的特征性表面标记容易地测定。For example, the number of CLL cells in a patient can be readily determined, eg, by complete blood or white blood cell count. The number of CLL cells can also be readily determined by immunohistochemical methods, flow cytometry, or other characteristic surface markers for detection of CLL cells.

前列腺肿瘤块可以通过直接目测观察，或者通过诊断性成像方法例如X射线，磁共振成像，超声波，和闪烁照相法来测定。用于确定肿瘤块尺寸的诊断性成像方法可以与对照试剂一起使用或者不与其一起使用，如本领域公知的。肿瘤块的尺寸还可以用物理方法测定，例如组织块的触诊或者用测量仪器例如测径器对组织块进行测量。对于前列腺肿瘤，优选的确定肿瘤块尺寸的物理方法是肛门触诊。Prostate tumor mass can be determined by direct visual observation, or by diagnostic imaging methods such as X-ray, magnetic resonance imaging, ultrasound, and scintigraphy. Diagnostic imaging methods for determining tumor mass size may be used with or without control agents, as is known in the art. The size of the tumor mass can also be determined by physical means, such as palpation of the mass of tissue or measurement of the mass of tissue with a measuring instrument such as a caliper. For prostate tumors, the preferred physical method for determining the size of a tumor mass is anal palpation.

本领域技术人员可以容易地确定给特定患者施用的miR15或miR16基因产物的有效量，其是根据下列参数，如患者的个头和体重；疾病的程度；患者的年龄，健康状况和性别；给药方式；以及给药是局部的还是全身性的。Those skilled in the art can readily determine the effective amount of miR15 or miR16 gene product to be administered to a particular patient based on parameters such as the size and weight of the patient; the extent of the disease; the age, health and sex of the patient; mode; and whether the administration is local or systemic.

例如，本发明的化合物的有效量可以是基于要治疗的肿瘤块的近似重量。肿瘤块的近似重量可以通过计算肿瘤块的近似体积测定，其中一立方厘米的体积重约一克。基于肿瘤块重量的miR15或miR16基因产物的有效量可以是至少10mg/g肿瘤块，优选是10-500mg/g肿瘤块。更优选地，有效量是至少60mg/g肿瘤块。特别优选的是，有效量是至少100mg/g肿瘤块。优选将基于肿瘤块重量的有效量直接注射到肿瘤中。For example, an effective amount of a compound of the invention may be based on the approximate weight of the tumor mass to be treated. The approximate weight of a tumor mass can be determined by calculating the approximate volume of the tumor mass, where a cubic centimeter of volume weighs approximately one gram. An effective amount of miR15 or miR16 gene product based on the weight of the tumor mass may be at least 10 mg/g tumor mass, preferably 10-500 mg/g tumor mass. More preferably, the effective amount is at least 60 mg/g tumor mass. It is particularly preferred that the effective amount is at least 100 mg/g tumor mass. An effective amount based on the weight of the tumor mass is preferably injected directly into the tumor.

miR15或miR16基因产物的有效量可以是基于要治疗的患者的近似或估计体重。优选地，该有效量是肠胃外或肠给药，如下所述。例如，给予患者的miR15或miR16基因产物的有效量是5-3000mg/kg体重，优选是700-1000mg/kg体重，更优选是大于1000mg/kg体重。An effective amount of a miR15 or miR16 gene product can be based on the approximate or estimated body weight of the patient to be treated. Preferably, the effective amount is administered parenterally or enterally, as described below. For example, the effective amount of miR15 or miR16 gene product administered to a patient is 5-3000 mg/kg body weight, preferably 700-1000 mg/kg body weight, more preferably greater than 1000 mg/kg body weight.

本领域技术人员可以容易地确定对给定患者给予miR15或miR16基因产物的合适的给药方案。例如miR15或miR16基因产物可以一次性给予患者(例如单次注射或沉积)。另外，该基因产物还可以每天给予患者一到两次，给药时间持续大约三到大约二十八天，更优选是大约七到大约十天。在优选的给药方案中，miR15或miR16基因产物每天给药一次，持续七天。如果给药方案是多次给药，应当理解给予患者的miR15或miR16基因产物的有效量是在整个给药方案中给予的基因产物的总量。An appropriate dosing regimen for administering the miR15 or miR16 gene product to a given patient can readily be determined by one of skill in the art. For example, the miR15 or miR16 gene product can be administered to a patient at one time (eg, a single injection or deposit). Alternatively, the gene product may be administered to the patient once or twice daily for a period of about three to about twenty-eight days, more preferably about seven to about ten days. In a preferred dosing regimen, the miR15 or miR16 gene product is administered once daily for seven days. If the dosing regimen is multiple doses, it is understood that the effective amount of the miR15 or miR16 gene product administered to the patient is the total amount of the gene product administered throughout the dosing regimen.

miR15或miR16基因产物可以通过任何适于将该基因产物递送到患者细胞，例如造血干细胞(HSC)，CLL细胞或前列腺癌细胞中的方式给予患者。例如miR15或miR16基因产物可以用适合于用miR15或miR16基因产物，或者用含有编码miR15或miR16基因产物的序列的核酸转染患者细胞的方式给药。细胞可以直接用miR15或miR16基因产物(当其是核酸的时候)转染，或者可以用含有编码miR15或miR16基因产物的序列的核酸转染。优选地，细胞用含有编码miR15或miR16基因产物的序列的质粒或病毒载体转染，如上所述。The miR15 or miR16 gene product can be administered to the patient by any means suitable for delivering the gene product to the patient's cells, eg, hematopoietic stem cells (HSC), CLL cells or prostate cancer cells. For example, the miR15 or miR16 gene product can be administered in a manner suitable for transfection of patient cells with the miR15 or miR16 gene product, or with a nucleic acid containing a sequence encoding the miR15 or miR16 gene product. Cells can be transfected directly with the miR15 or miR16 gene product (when it is a nucleic acid), or can be transfected with a nucleic acid containing a sequence encoding the miR15 or miR16 gene product. Preferably, the cells are transfected with a plasmid or viral vector containing sequences encoding the miR15 or miR16 gene products, as described above.

真核细胞的转染方法是本领域公知的，包括将核酸直接注射到细胞核或细胞原核中；电穿孔；脂质体转化或者由亲脂性物质介导的转化；受体介导的核酸递送，生物弹或粒子加速；磷酸钙沉淀，病毒载体介导的转染。Transfection methods for eukaryotic cells are well known in the art and include direct injection of nucleic acid into the nucleus or pronucleus; electroporation; liposome transformation or transformation mediated by lipophilic substances; receptor-mediated nucleic acid delivery, Biolistic or particle acceleration; calcium phosphate precipitation, viral vector-mediated transfection.

例如，细胞可以用脂质体转化化合物，例如DOTAP(N-[1-(2，3-二油酰氧)丙基]-N，N，N-三甲基-甲硫酸铵，Boehringer-Mannheim)或其等同物例如LIPOFECTIN转染。所使用的核酸的量对于本发明的实施并不重要；用0.1-100mg核酸/10⁵个细胞都能得到满意的结果。例如，使用的量可以是每10⁵个细胞3mg DOTAP，其中含有0.5mg质粒载体。For example, cells can be transformed with liposomes with compounds such as DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methosulfate, Boehringer-Mannheim ) or its equivalent such as LIPOFECTIN transfection. The amount of nucleic acid used is not critical to the practice of the invention; satisfactory results are obtained with 0.1-100 mg nucleic acid/¹⁰⁵ cells. For example, the amount used may be 3 mg DOTAP per¹⁰⁵ cells containing 0.5 mg of the plasmid vector.

在一个实施方案中，从患者中分离出miR15或miR16介导的癌细胞，例如CLL或前列腺癌，用编码miR15或miR16基因产物的核酸转染，并重新植入患者中。在优选的实施方案中，转染的和重新植入的细胞是CLL细胞。在更优选的实施方案中，转染的和重新植入的细胞是诊断为CLL的患者的HSC。In one embodiment, miR15 or miR16 mediated cancer cells, such as CLL or prostate cancer, are isolated from a patient, transfected with a nucleic acid encoding a miR15 or miR16 gene product, and reimplanted into the patient. In preferred embodiments, the transfected and reimplanted cells are CLL cells. In a more preferred embodiment, the transfected and re-implanted cells are HSCs from a patient diagnosed with CLL.

从患者中提取CLL细胞的技术是本领域公知的，例如下述实施例7中所述的。从患者中提取、鉴别、分离和培养HSC的技术也是本领域公知的，例如美国专利序列号5,635,387和5,643,741，以及Campana et al.(1995)Blood 85：1416-1434中所述的，在此引入其全文作为参考。优选地，在转染HSC之前从收集的骨髓中提取出肿瘤或赘生物细胞。合适的提取技术包括，例如固定化外周血细胞的白细胞去除，基于免疫亲和的选择或杀死肿瘤细胞，或者使用细胞毒性或者光敏试剂选择性地杀死肿瘤细胞，都是本领域公知的。参见，例如，Bone Marrow Processing andPurging，Part 5(A.Gee，ed.)，CRC Press，Boca Raton，Fla.，1991；Lydaki et al.(1996)J.Photochem.and Photobiol.32：27-32；和Gazitt et al.(1995)，Blood，86：381-389，在此引入其全文作为参考。Techniques for extracting CLL cells from patients are well known in the art, such as described in Example 7 below. Techniques for extracting, identifying, isolating and culturing HSCs from patients are also well known in the art, such as described in U.S. Patent Serial Nos. 5,635,387 and 5,643,741, and Campana et al. (1995) Blood 85:1416-1434, incorporated herein The full text of which is incorporated by reference. Preferably, tumor or neoplastic cells are extracted from the harvested bone marrow prior to transfection of HSCs. Suitable extraction techniques include, for example, leukapheresis of immobilized peripheral blood cells, immunoaffinity-based selection or killing of tumor cells, or selective killing of tumor cells using cytotoxic or photosensitizing agents, all known in the art. See, e.g., Bone Marrow Processing and Purging, Part 5 (A. Gee, ed.), CRC Press, Boca Raton, Fla., 1991; Lydaki et al. (1996) J. Photochem. and Photobiol. 32:27-32 and Gazitt et al. (1995), Blood, 86: 381-389, which are hereby incorporated by reference in their entirety.

分离的CLL细胞或HSC可以用任何适当的技术转染，如上所述。转染之后检查一部分CLL细胞或HSC以验证所述基因产物是否具有适当的表达水平。一旦验证了miR15或miR16基因产物具有适当的表达，就将其余的转染细胞重新引入到患者中。转染的CLL细胞或HSC可以通过肠胃外方法重新引入到患者中，包括静脉输注或向骨髓中直接注射。转染细胞优选以盐溶液或其它药学可接收的载体重新引入患者。重新引入的转染细胞的合适的数量是每kg患者体重大约10⁵到大约10⁸个细胞。在转染之前可在培养基中扩张细胞从而增加可用于重新引入的转染细胞的数量。Isolated CLL cells or HSCs can be transfected using any suitable technique, as described above. A subset of CLL cells or HSCs is examined after transfection to verify that the gene product has appropriate expression levels. Once proper expression of the miR15 or miR16 gene product was verified, the remaining transfected cells were reintroduced into the patient. Transfected CLL cells or HSCs can be reintroduced into the patient by parenteral methods, including intravenous infusion or direct injection into the bone marrow. Transfected cells are preferably reintroduced to the patient in saline or other pharmaceutically acceptable carrier. A suitable number of reintroduced transfected cells is about¹⁰⁵ to about¹⁰⁸ cells per kg of patient body weight. Cells can be expanded in culture prior to transfection to increase the number of transfected cells available for reintroduction.

优选地，CLL细胞或HSC用含有编码miR15或miR16基因产物的序列的核酸，例如能稳定整合到CLL细胞或HSC基因组中以长期表达该化合物的质粒表达载体转染。稳定的整合和表达可以用本领域公知的技术验证，例如用miR15或miR16 cDNA(或其片段)作为探针对基因组DNA进行Southern blot。miR15或miR16基因产物的表达还可以用标准Northern blot技术来检测。用编码miR15或miR16基因产物的序列稳定转染的CLL细胞或HSC在重新植入患者中以后会继续表达该化合物。从患者中分离HSC，用表达miR15或miR16基因产物的质粒转染，并将转染的HSC重新植入到患者中的示例性方法如下述实施例8所示。Preferably, CLL cells or HSCs are transfected with a nucleic acid containing a sequence encoding the miR15 or miR16 gene product, eg, a plasmid expression vector capable of stably integrating into the CLL cell or HSC genome for long-term expression of the compound. Stable integration and expression can be verified by techniques known in the art, for example, using miR15 or miR16 cDNA (or a fragment thereof) as a probe to carry out Southern blot on genomic DNA. Expression of miR15 or miR16 gene products can also be detected using standard Northern blot techniques. CLL cells or HSCs stably transfected with sequences encoding miR15 or miR16 gene products will continue to express the compound after re-implantation into the patient. An exemplary method for isolating HSCs from a patient, transfecting with a plasmid expressing the miR15 or miR16 gene product, and reimplanting the transfected HSCs into the patient is shown in Example 8 below.

miR15或miR16基因产物还可以通过任何适当的肠或肠胃外给药方式给予患者。适用于本发明的适当的肠给药方式包括口服，直肠，或鼻内递送。适当的肠胃外给药方式包括血管内给药(例如静脉高剂量注射，静脉输注，动脉内高剂量注射，动脉内输注和经导管输注到脉管系统中)；组织外和组织内注射(例如肿瘤外和肿瘤内注射，视网膜内注射，或视网膜下注射)；皮下注射或沉积，包括皮下输注(例如渗透泵)；直接应用到感兴趣的组织上，例如通过导管或其它放置装置(例如视网膜颗粒或栓剂或含有多孔，无孔，或胶状物质的植入物)；以及吸入。优选地，miR15或miR16基因产物通过注射或输注给药。对于miR15或miR16介导的含有肿瘤块的癌症，优选miR15或miR16基因产物通过直接注射到肿瘤中给药。The miR15 or miR16 gene product can also be administered to a patient by any suitable enteral or parenteral administration. Suitable modes of enteral administration for use in the present invention include oral, rectal, or intranasal delivery. Appropriate modes of parenteral administration include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intraarterial bolus injection, intraarterial infusion, and catheter infusion into the vasculature); extratissue and intratissue Injection (eg, extratumoral and intratumoral injection, intraretinal injection, or subretinal injection); subcutaneous injection or deposition, including subcutaneous infusion (eg, osmotic pump); direct application to tissue of interest, eg, via catheter or other placement devices (such as retinal pellets or suppositories or implants containing porous, non-porous, or gel-like substances); and inhalations. Preferably, the miR15 or miR16 gene product is administered by injection or infusion. For miR15 or miR16 mediated cancers containing tumor masses, it is preferred that the miR15 or miR16 gene product is administered by direct injection into the tumor.

在本文的方法中，miR15或miR16基因产物可以作为裸露的RNA，或者和递送试剂一起，或者作为含有能表达该基因产物的序列的核酸(例如重组质粒或病毒载体)给药。适用于miR15或miR16基因产物给药的合适的递送试剂包括Mirus TransitTKO亲脂性试剂；lipofectin；lipofectamine；cellfectin；或聚阳离子(例如聚赖氨酸)，或脂质体。In the methods herein, the miR15 or miR16 gene product can be administered as naked RNA, either with a delivery agent, or as a nucleic acid (eg, a recombinant plasmid or viral vector) containing sequences capable of expressing the gene product. Suitable delivery agents for administration of miR15 or miR16 gene products include Mirus TransitTKO lipophilic reagents; lipofectin; lipofectamine; cellfectin; or polycations (eg, polylysine), or liposomes.

含有能表达miR15或miR16基因产物的序列的重组质粒如上所述。含有能表达miR15或miR16基因产物的序列的重组病毒载体也如上所述，将这些载体递送到患者的CLL或前列腺癌细胞中的方法是本领域技术人员公知的。Recombinant plasmids containing sequences capable of expressing miR15 or miR16 gene products are described above. Recombinant viral vectors containing sequences capable of expressing miR15 or miR16 gene products are also described above, and methods for delivering these vectors to CLL or prostate cancer cells in patients are well known to those skilled in the art.

在优选的实施方案中，脂质体可用于将miR15或miR16基因产物，或含有编码该基因产物的序列的核酸递送到患者中。脂质体还可以增加该基因产物或核酸在血液中的半衰期。在本发明的该实施方案的实践中，所述的miR15或miR16基因产物，或含有编码该基因产物的序列的核酸可以在给患者施用之前装入到脂质体中。In a preferred embodiment, liposomes are used to deliver the miR15 or miR16 gene product, or a nucleic acid containing a sequence encoding the gene product, to a patient. Liposomes can also increase the half-life of the gene product or nucleic acid in the blood. In the practice of this embodiment of the invention, the miR15 or miR16 gene product, or a nucleic acid containing a sequence encoding the gene product, may be incorporated into liposomes prior to administration to a patient.

适用于本发明的脂质体可以用标准的可形成载体的脂类制备，通常包括中性的或具有负电荷的磷脂和甾醇例如胆固醇。脂类的选择通常可以依据各种因素例如所希望的脂质体大小和脂质体在血管中的半衰期。已知多种制备脂质体的方法，例如Szokaet al.(1980)，Ann.Rev.Biophys.Bioeng.9：467；和美国专利序列号4,235,871，4,501,728，4,837,028，和5,019,369中所述的，在此引入其全文作为参考。Liposomes suitable for use in the invention can be prepared using standard carrier-forming lipids, and generally include neutral or negatively charged phospholipids and a sterol such as cholesterol. The choice of lipid can generally be based on various factors such as the desired liposome size and the half-life of the liposomes in blood vessels. Various methods of preparing liposomes are known, such as those described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9: 467; It is incorporated by reference in its entirety.

包含有miR15或miR16基因产物，或含有编码该基因产物的序列的核酸的脂质体可以包含能够将所述脂质体靶定到miR15或miR16介导的癌细胞，例如CLL或前列腺癌细胞上的配体分子。在这些癌细胞中普遍能与受体结合的配体，例如能与肿瘤细胞抗原或CLL细胞表面标记的单克隆抗体是优选的。Liposomes comprising the miR15 or miR16 gene product, or a nucleic acid comprising a sequence encoding the gene product, can comprise a miR15 or miR16 mediated targeting of the liposome to cancer cells, such as CLL or prostate cancer cells ligand molecules. Ligands that bind to receptors commonly found in these cancer cells, such as monoclonal antibodies that bind to tumor cell antigens or CLL cell surface markers, are preferred.

还可以对包含有miR15或miR16基因产物，或含有编码该基因产物的序列的核酸的脂质体进行修饰以避免被单核巨噬细胞系统(“MMS”)和网状内皮组织系统(“RES”)清除。这种修饰的脂质体的表面上或其脂质体结构中具有调理作用抑制分子。在特定的优选的实施方案中，本发明的脂质体可以同时包含调理作用抑制分子和配体。Liposomes containing miR15 or miR16 gene products, or nucleic acids containing sequences encoding such gene products, can also be modified to avoid being detected by the mononuclear macrophage system ("MMS") and the reticuloendothelial system ("RES"). ') to clear. Such modified liposomes have opsonization-inhibiting molecules on the surface or within the liposome structure. In certain preferred embodiments, the liposomes of the invention may comprise both an opsonization-inhibiting molecule and a ligand.

用于制备本发明的脂质体的调理作用抑制分子典型地是能与脂质体膜结合的大分子的亲水性聚合物。这里所述的调理作用抑制分子与脂质体膜“结合”是指其与膜发生化学或物理连接，例如脂溶性锚定物插入到膜中，或者直接与膜脂的活性基团结合。这些调理作用抑制亲水性聚合物形成具有保护作用的表面层，能显著减少脂质体被MMS和RES吸收，例如美国专利序列号4,920,016中所述的，在此引入其全文作为参考。Opsonization-inhibiting molecules useful in preparing liposomes of the invention are typically macromolecular hydrophilic polymers capable of associating with the liposome membrane. The term "association" of an opsonization-inhibiting molecule to a liposome membrane means that it is chemically or physically linked to the membrane, for example, a lipid-soluble anchor is inserted into the membrane, or it is directly bound to an active group of a membrane lipid. These opsonizations inhibit the formation of a protective surface layer by the hydrophilic polymer and can significantly reduce liposome uptake by MMS and RES, such as described in US Patent Serial No. 4,920,016, which is incorporated herein by reference in its entirety.

适用于修饰脂质体的调理作用抑制分子优选是水溶性聚合物，其平均分子量是大约500到大约40,000Da，更优选是大约2,000到大约20,000Da。这种聚合物包括聚乙二醇(PEG)或聚丙二醇(PPG)衍生物；例如甲氧PET或PPG，以及PET或PPG硬脂酸盐；合成聚合物例如聚丙烯酰胺或聚N-聚乙烯吡咯烷酮；直链，枝链和分枝聚酰胺基胺；聚丙烯酸；多元醇，例如聚乙烯醇和聚木糖醇，其中羧基和氨基以化学方法连接，以及神经节苷脂，例如神经节苷脂GM1。也可以使用PEG，甲氧PEG，或甲氧PPG，或其衍生物的共聚物。此外，调理作用抑制聚合物可以是PEG和聚氨基酸，多聚糖，聚酰胺，聚乙烯胺，或聚核苷酸中的任一个的嵌段共聚物。所述调理作用抑制聚合物还可以是天然的含有氨基酸或羧酸，例如半乳糖醛酸，葡萄糖醛酸，甘露糖醛酸，透明质酸，果胶酸，神经氨酸，褐藻酸，角叉胶的多糖；胺化多糖或寡糖(直链或枝链)；或羧化多糖或寡糖，例如可与碳酸衍生物反应最终连接羧基基团。优选地，所述调理作用抑制分子是PEG，PPG，或其衍生物。用PEG或PEG衍生物修饰的脂质体有时被称为“PEG化的脂质体”。Opsonization-inhibiting molecules suitable for modifying liposomes are preferably water-soluble polymers having an average molecular weight of about 500 to about 40,000 Da, more preferably about 2,000 to about 20,000 Da. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; such as methoxyPET or PPG, and PET or PPG stearate; synthetic polymers such as polyacrylamide or poly-N-polyethylene Pyrrolidones; linear, branched and branched polyamidoamines; polyacrylic acids; polyols, such as polyvinyl alcohol and xylitol, in which the carboxyl and amino groups are chemically linked, and gangliosides, such as gangliosides GM1. Copolymers of PEG, methoxyPEG, or methoxyPPG, or derivatives thereof, may also be used. Additionally, the opsonization-inhibiting polymer can be a block copolymer of PEG and any of polyamino acids, polysaccharides, polyamides, polyvinylamines, or polynucleotides. The opsonization inhibiting polymer may also be a natural amino acid or carboxylic acid containing such as galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectinic acid, neuraminic acid, alginic acid, carrageenan Gum polysaccharides; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, such as can be reacted with carbonic acid derivatives to finally attach carboxyl groups. Preferably, the opsonization-inhibiting molecule is PEG, PPG, or a derivative thereof. Liposomes modified with PEG or PEG derivatives are sometimes referred to as "PEGylated liposomes."

调理作用抑制分子可以通过多种已知方法与脂质体膜结合。例如，PEG的N-羟基琥珀酰亚胺酯可与磷脂酰乙醇胺的脂溶性锚定物相结合，然后结合到膜上。类似地，右旋糖苷聚合物可以与十八胺脂溶性锚定物通过用Na(CN)BH和溶剂混合物如30：12的四氢呋喃：水在60℃进行的还原性胺化作用来衍生化。Opsonization-inhibiting molecules can be associated with liposome membranes by a variety of known methods. For example, the N-hydroxysuccinimide ester of PEG can be conjugated to a lipid-soluble anchor of phosphatidylethanolamine and then bound to the membrane. Similarly, dextran polymers can be derivatized with stearylamine liposoluble anchors by reductive amination with Na(CN)BH and a solvent mixture such as 30:12 THF:water at 60°C.

用调理作用抑制分子修饰的脂质体在循环系统中保留的时间比未修饰的脂质体长。鉴于此原因，这些脂质体有时称为“隐形”脂质体。隐形脂质体已知可在由多孔的或“有渗漏的”微脉管系统反馈的组织中积累。因此，具有微脉管系统毛病的组织，例如肿瘤块，可有效积累这些脂质体；参见Gabizon，et al.(1988)，Proc.Natl.Acad.Sci.，USA，18：6949-53。此外，RES吸收的减少由于阻止了该脂质体在肝和脾中的积累从而降低了隐形脂质体的毒性。因此，用调理作用抑制分子修饰的脂质体适用于将miR15或miR16基因产物，或含有编码该基因产物的序列的核酸递送到肿瘤细胞中。Liposomes modified with opsonization-inhibiting molecules remain in the circulation longer than unmodified liposomes. For this reason, these liposomes are sometimes called "stealth" liposomes. Stealth liposomes are known to accumulate in tissues fed back by porous or "leaky" microvasculature. Thus, tissues with microvasculature disorders, such as tumor masses, can efficiently accumulate these liposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., USA, 18:6949-53. In addition, the reduction in RES uptake reduces the toxicity of stealth liposomes by preventing the accumulation of the liposomes in the liver and spleen. Thus, liposomes modified with opsonization-inhibiting molecules are suitable for the delivery of miR15 or miR16 gene products, or nucleic acids containing sequences encoding such gene products, into tumor cells.

miR15或miR16基因产物优选在给患者施用之前根据本领域公知的技术制成药物组合物。本发明的药物组合物至少是无菌的和不含热源的。这里所说的“药物制剂”包括用于人的和兽医使用的制剂。制备本发明的药物组合物的方法是本领域技术人员公知的，例如Remington′s Pharmaceutical Science，17th ed.，MackPublishing Company，Easton，Pa.(1985)中所述的，在此引入其全文作为参考。The miR15 or miR16 gene product is preferably formulated into a pharmaceutical composition according to techniques well known in the art prior to administration to a patient. The pharmaceutical compositions of the present invention are at least sterile and pyrogen-free. The term "pharmaceutical preparation" herein includes preparations for human and veterinary use. Methods for preparing the pharmaceutical compositions of the present invention are well known to those skilled in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), which is hereby incorporated by reference in its entirety .

本发明的药物制剂含有miR15或miR16基因产物，或含有编码该基因产物的序列的核酸(例如按重量计0.1到90％)，或其生理学可接收的盐，并与药学可接收的载体相混和。本发明的药物制剂还可以包括包含在脂质体中的miR15或miR16基因产物，或含有编码该基因产物的序列的核酸，以及药学可接收的载体。The pharmaceutical preparation of the present invention contains miR15 or miR16 gene product, or nucleic acid (for example, 0.1 to 90% by weight) containing the sequence encoding the gene product, or a physiologically acceptable salt thereof, and mixed with a pharmaceutically acceptable carrier . The pharmaceutical formulation of the present invention may also include the miR15 or miR16 gene product contained in liposomes, or a nucleic acid containing a sequence encoding the gene product, and a pharmaceutically acceptable carrier.

优选药学可接收的载体是水，缓冲液，生理盐水，0.4％盐水，0.3％甘氨酸，透明质酸等。Preferred pharmaceutically acceptable carriers are water, buffer, physiological saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

在优选的实施方案中，本发明的药物组合物包括能抵抗核酸酶降解的miR15或miR16基因产物。本领域技术人员可以容易地合成具有核酸酶抗性的miR15和miR16基因产物，例如在miR15和miR16基因产物的2’位置加入一种或多种核糖核苷酸。合适的2’修饰的核糖核苷酸包括在2’位置具有氟，氨基，烷基，烷氧基，和O-烯丙基修饰的核糖核苷酸。In preferred embodiments, the pharmaceutical compositions of the invention include miR15 or miR16 gene products that are resistant to nuclease degradation. Those skilled in the art can easily synthesize miR15 and miR16 gene products with nuclease resistance, such as adding one or more ribonucleotides at the 2' position of miR15 and miR16 gene products. Suitable 2' modified ribonucleotides include ribonucleotides with fluorine, amino, alkyl, alkoxy, and O-allyl modifications at the 2' position.

例如，本发明的药物组合物含有加入了一个或多个具有2′AR-核苷酸的通式的2’修饰的核糖核苷酸的miR15或miR16基因产物，其中：For example, the pharmaceutical compositions of the invention contain the miR15 or miR16 gene product to which one or more 2'-modified ribonucleotides having the general formula 2'AR-nucleotides have been added, wherein:

A是氧或卤素(优选氟，氯或溴)；以及A is oxygen or halogen (preferably fluorine, chlorine or bromine); and

R是氢或直链或枝链C_1-6烷基；R is hydrogen or straight or branched C_1-6 alkyl;

其中当A是卤素的时候，没有X和R。优选的修饰的2-核糖核苷酸是2’-O甲基核糖核苷酸。优选地，本发明的药物组合物包括其中每个核糖核苷酸都是2’-修饰的核糖核苷酸的miR15或miR16基因产物。Wherein when A is halogen, X and R are absent. A preferred modified 2-ribonucleotide is a 2'-Omethyl ribonucleotide. Preferably, the pharmaceutical composition of the invention comprises a miR15 or miR16 gene product wherein each ribonucleotide is a 2&apos;-modified ribonucleotide.

本发明的药物组合物还可以包括传统的药物赋形剂和/或添加剂。合适的药物赋形剂包括稳定剂，抗氧化剂，调节摩尔渗透压浓度的试剂，缓冲液，和pH调节试剂。合适的添加剂包括生理学的生物相容的缓冲液(例如盐酸缓血酸胺)，加入螯合剂(例如DTPA或DTPA-双酰胺)或者钙螯合复合物(例如DTPA钙，CaNaDTPA-双酰胺)，或者，可选择地，加入钙盐或钠盐(例如，氯化钙，抗坏血酸钙，葡萄糖酸钙或乳酸钙)。本发明的药物组合物可以以液体形式使用，也可以以冻干形式使用。The pharmaceutical composition of the present invention may also include conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolarity adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (such as tromethamine hydrochloride), adding chelating agents (such as DTPA or DTPA-bisamide) or calcium chelating complexes (such as calcium DTPA, CaNaDTPA-bisamide), Or, alternatively, calcium or sodium salts (eg, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate) are added. The pharmaceutical composition of the present invention can be used in liquid form or in freeze-dried form.

对于本发明的固体药物组合物，可以使用传统的无毒的固体的药学可接收的载体；例如药物级的甘露醇，乳糖，淀粉，硬脂酸镁，糖精钠，纤维素，葡萄糖，蔗糖，碳酸镁等。For the solid pharmaceutical composition of the present invention, conventional nontoxic solid pharmaceutically acceptable carriers can be used; for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, glucose, sucrose, Magnesium carbonate, etc.

例如，用于口服给药的固体药物组合物可以含有上述的任何载体以及10-95％，优选25％-75％的miR15或miR16基因产物。用于喷雾(吸入)给药的药物组合物可以含有按重量计0.01-20％，优选按重量计1％-10％的包含于上述脂质体中的miR15或miR16基因产物，和推进剂。还可以包含其它载体；例如用于鼻内递送的卵磷脂。For example, a solid pharmaceutical composition for oral administration may contain any of the above-mentioned carriers and 10-95%, preferably 25%-75%, of the miR15 or miR16 gene product. A pharmaceutical composition for spray (inhalation) administration may contain 0.01-20% by weight, preferably 1%-10% by weight, of the miR15 or miR16 gene product contained in the above liposome, and a propellant. Other carriers may also be included; for example lecithin for intranasal delivery.

下述非限制性实施例将对本发明作出说明。The following non-limiting examples illustrate the invention.

实施例Example

在实施例中使用下述方法：The following method is used in the examples:

患者样品和细胞系-患者样品是在得到了被CLL ResearchConsortium institutions诊断为CLL的患者的知情同意后获取的。简短来说，外周血是从CLL患者中获得的，单核细胞是通过Ficoll-Hypaque梯度离心(Amersham Pharmacia Biotech，Piscataway，NJ)分离的，然后进行处理以便按标准方法提取RNA和DNA，如Sambrook J et al.(1989)，Molecular cloning：ALaboratory Manual(Cold Spring Harbor Laboratory Press，ColdSpring Harbor，NY)中所述的，在此引入其全文作为参考。将相应患者的口腔粘膜DNA放置在小片(1-2mm²)纸上，作为LOH研究中的正常对照。Patient samples and cell lines - Patient samples were obtained after informed consent was obtained from patients diagnosed with CLL by the CLL Research Consortium institutions. Briefly, peripheral blood was obtained from CLL patients and mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation (Amersham Pharmacia Biotech, Piscataway, NJ) and processed for RNA and DNA extraction by standard methods such as Sambrook J et al. (1989), Molecular cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), which is hereby incorporated by reference in its entirety. The oral mucosal DNA of the corresponding patients was placed on a small piece (1-2 mm² ) of paper as a normal control in the LOH study.

三十个人的细胞系是得自美国典型培养物保藏中心(ATCC；Manassas，VA)并根据ATCC的说明培养。这些细胞系是AS283，BL2，Bla，BJAB，CA46，Namalva，P3HRI，PAPB 682，PABm，Raji(Burkitt′s淋巴瘤)，Dell，SKDHL，ST486(T-细胞淋巴瘤)，JM(免疫母细胞B细胞淋巴瘤)，MC116(未分化的淋巴瘤)，Molt3，Supt 11(T-ALL)，U266(多发性骨髓瘤)，A549，H1299(肺癌)，TE2，TE10(食道癌)，HeLa(子宫颈癌)，RC48(肾癌)和2220，2221，11609，11611，LNCAP，TSUR(前列腺癌)。Thirty human cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured according to ATCC instructions. These cell lines are AS283, BL2, Bla, BJAB, CA46, Namalva, P3HRI, PAPB 682, PABm, Raji (Burkitt's lymphoma), Dell, SKDHL, ST486 (T-cell lymphoma), JM (immunoblastic B cell lymphoma), MC116 (undifferentiated lymphoma), Molt3, Supt 11 (T-ALL), U266 (multiple myeloma), A549, H1299 (lung cancer), TE2, TE10 (esophageal cancer), HeLa ( Cervical cancer), RC48 (kidney cancer) and 2220, 2221, 11609, 11611, LNCAP, TSUR (prostate cancer).

CD5+B-细胞的分离一从儿科组(3-9岁)通过常规的扁桃腺切除术获得扁桃腺。用唾液酸苷酶处理过的绵羊红血球使单核细胞散开，得到纯化的B细胞。将B细胞用不连续Percoll梯度(Pharmacia Biotech，Uppsala，Sweden)进一步分级，如Dono M etal.(2000)，J.Immunol.164：5596-5604中所述，在此引入其全文作为参考。从50％的Percoll级分中收集B细胞，并与抗CD5 mAb一起培育，然后与和微磁珠结合的羊抗鼠Ig一起培育。收集细胞并置于磁性柱MS上，用MiniMACS系统(Miltenyi Biotec)通过正选择获得CD5+B细胞。Isolation of CD5+ B-cells - Tonsils were obtained from a pediatric group (3-9 years old) by conventional tonsillectomy. Mononuclear cells were dispersed with sialidase-treated sheep erythrocytes to obtain purified B cells. B cells were further fractionated using a discontinuous Percoll gradient (Pharmacia Biotech, Uppsala, Sweden) as described in Dono M et al. (2000), J. Immunol. 164:5596-5604, which is hereby incorporated by reference in its entirety. B cells were harvested from a 50% Percoll fraction and incubated with anti-CD5 mAb followed by goat anti-mouse Ig bound to micromagnetic beads. Cells were collected and placed on magnetic column MS, and CD5+ B cells were obtained by positive selection with MiniMACS system (Miltenyi Biotec).

体细胞杂交-用传统方法进行体细胞杂交，在次黄嘌呤-氨喋呤-胸苷(HAT)介质上选择，如Negrini M et al.(1994)，CancerRes.54：1818-1824中所述的，在此引入其全文作为参考。从单个细胞克隆和亚克隆中得到的DNA用DNeasy组织试剂盒(Qiagen)分离并用PCR检测是否存在染色体13和染色体2标记(引物序列见下述表1)。从一种带有t(2；13)(q32；q14)移位的CLL细胞(CLL-B)中分离得到十五个克隆，从另一种带有t(2；13)(q12；q13)移位的CLL细胞(CLL-A)中分离得到一个克隆。十二个来自CLL-B的克隆带有染色体13和2的完全互补序列，另外三个带有del(13q)和染色体2的完全互补序列。来自CLL-A的一个克隆含有在13q14位置具有染色体13的小缺失并且不含部分染色体2。Somatic cell hybridization - somatic cell hybridization was performed by conventional methods, with selection on hypoxanthine-aminopterin-thymidine (HAT) medium, as described in Negrini M et al. (1994), Cancer Res. 54:1818-1824 , which is hereby incorporated by reference in its entirety. DNA from individual cell clones and subclones was isolated using the DNeasy tissue kit (Qiagen) and tested for the presence of chromosome 13 andchromosome 2 markers by PCR (see Table 1 below for primer sequences). Fifteen clones were isolated from one type of CLL cell with t(2;13)(q32;q14) shift (CLL-B), and from another type with t(2;13)(q12;q13 ) was isolated from a translocated CLL cell (CLL-A). Twelve clones from CLL-B carried the perfect complement ofchromosomes 13 and 2, and three others had del(13q) and the perfect complement ofchromosome 2. One clone from CLL-A contained a small deletion of chromosome 13 at position 13q14 and contained no part ofchromosome 2.

Northern blotting-用Tri Reagent方法(Molecular ResearchCenter，Inc)分离总RNA。将RNA样品(每个30g)在15％丙烯酰胺变形(脲)Criterion预制凝胶(Bio-Rad Laboratories，Hercules，CA)上分离并转移到Hybond-N+膜(AmershamPharmacia Biotech)上。在42℃在7％SDS，0.2M Na₂PO₄ pH7.0的条件下与-³²P ATP杂交过夜。在42℃将膜用2x SSPE，0.1％SDS洗涤两次，用0.5x SSPE，0.1％SDS洗涤两次。用于检测miR15和miR16 RNA的探针分别是：Northern blotting - total RNA was isolated using the Tri Reagent method (Molecular Research Center, Inc). RNA samples (30 g each) were separated on 15% acrylamide deformed (urea) Criterion precast gels (Bio-Rad Laboratories, Hercules, CA) and transferred to Hybond-N+ membranes (AmershamPharmacia Biotech). Hybridization with^-32P ATP was performed overnight at 42°C in 7% SDS, 0.2M Na₂ PO₄ pH 7.0. Membranes were washed twice with 2x SSPE, 0.1% SDS and twice with 0.5x SSPE, 0.1% SDS at 42°C. The probes used to detect miR15 and miR16 RNA were:

CACAAACCATTATGTGCTTGCTA(SEQ ID NO：5)CACAAAACCATTATGTGCTTGCTA (SEQ ID NO: 5)

GCCAATATTTACGTGCTGCTA(SEQ ID NO：6)GCCAATATTTACGTGCTGCTA (SEQ ID NO: 6)

在0.1％SDS水溶液/0.1x SSC中煮沸10分钟剥离杂交膜并重新杂交几次。用溴化乙锭染色的5S rRNA作为对照。Boil in 0.1% SDS in water/0.1x SSC for 10 minutes to peel off the hybridized membrane and rehybridize several times. 5S rRNA stained with ethidium bromide served as a control.

反向转录酶聚合酶链式反应(RT-PCR)-通过RT-PCR分析正常CD5+细胞和23个B-CLL样品中的基因表达水平。用Advantage2 PCR试剂盒(Clontech)每次扩增反应用一微升cDNA，每种基因特异性引物10pmol进行35次循环，94℃20秒，65℃30秒，68℃1分钟(引物列表见下述表1)。为了保证RT-PCR中使用的RNA具有足够的纯度，用G3PDH cDNA(Clontech，PaloAlto，CA)特异的引物进行PCR检测。用上述Sambrook J et al.(1989)中的标准方法用琼脂糖凝胶电泳分离RT-PCR产物。Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) - Gene expression levels in normal CD5+ cells and 23 B-CLL samples were analyzed by RT-PCR. Use Advantage2 PCR Kit (Clontech) to use one microliter of cDNA per amplification reaction, and carry out 35 cycles of 10 pmol of each gene-specific primer, 94°C for 20 seconds, 65°C for 30 seconds, and 68°C for 1 minute (primer list see below Table 1). In order to ensure that the RNA used in RT-PCR has sufficient purity, PCR detection was performed with G3PDH cDNA (Clontech, Palo Alto, CA) specific primers. RT-PCR products were separated by agarose gel electrophoresis using the standard method described in Sambrook J et al. (1989) above.

Western blotting-对9个B-CLL患者的细胞溶解物的SDS/PAGE凝胶产物用GST-SLUG Middle抗体(Dr.Thomas Look-Harvard，MA赠与)和SNX2(N17)抗体(Santa Cruz Biotechnology，CA)检测。用ECL Western Blotting检测试剂盒(AmershamPharmacia，UK)进行检测，根据制造商的说明操作。Western blotting-GST-SLUG Middle antibody (gifted by Dr. Thomas Look-Harvard, MA) and SNX2 (N17) antibody (Santa Cruz Biotechnology, CA ) detection. Detection was performed with ECL Western Blotting Detection Kit (AmershamPharmacia, UK) according to the manufacturer's instructions.

数据库分析-用由National Institutes of Health and theNational Library of Medicine维护的National Center forBiotechnology Information网站提供的BLAST排列工具搜索“nr”和“dbEST”数据库，搜索短的几乎完全匹配的序列。还可参见Altschul et al.(1990)，J.Mol.Biol.215：403-10和Altschul et al.(1997)，Nucleic Acids Res.25：33 89-3402，在此引入其全文作为参考。也可以用Biology workBench网站提供的FASTA排列工具搜索短序列的同源序列。Database Analysis - The 'nr' and 'dbEST' databases were searched for short near-exact matches using the BLAST alignment tool provided by the National Center for Biotechnology Information website maintained by the National Institutes of Health and the National Library of Medicine. See also Altschul et al. (1990), J. Mol. Biol. 215: 403-10 and Altschul et al. (1997), Nucleic Acids Res. 25: 33 89-3402, which are hereby incorporated by reference in their entirety. You can also use the FASTA alignment tool provided by the Biology workBench website to search for homologous sequences of short sequences.

表1-用于检测体细胞杂交株的引物Table 1 - Primers used to detect somatic hybrid strains

名称                   引物序列                    SEQName Primer Sequence SEQ

                                                   IDID

                                                   NO：NO:

D2S396L    ATA CAC CTC TAA ATA TCT GTT CCA G       7D2S396L ATA CAC CTC TAA ATA TCT GTT CCA G 7

D2S396R    AAG TAG GAC CAT TCT AAT AGC C           8D2S396R AAG TAG GAC CAT TCT AAT AGC C 8

D2S112L    GAG TGG CGG TGA GAA GGT AT              9D2S112L GAG TGG CGG TGA GAA GGT AT 9

D2S112R    AGC CAT TGCTAT CTT TGA GG               10D2S112R AGC CAT TGCTAT CTT TGA GG 10

D2S2243L   TGG GAT ATG CTT CAG GGA C               11D2S2243L TGG GAT ATG CTT CAG GGA C 11

D2S2243R   AGC TGA CCT TGG AAT CTG GTT             12D2S2243R AGC TGA CCT TGG AAT CTG GTT 12

D13S260L   AGA TAT TGT CTC CGT TCC ATG A           13D13S260L AGA TAT TGT CTC CGT TCC ATG A 13

D13S260R   CCC AGA TAT AAGl GAC CTG GCT A          14D13S260R CCC AGA TAT AAGl GAC CTG GCT A 14

D13S263L   CCT GGC CTG TTA GTT TTT ATT GTT A       15D13S263L CCT GGC CTG TTA GTT TTT ATT GTT A 15

D13S263R   CCC AGT CTT GGG TAT GTT TTT A           16D13S263R CCC AGT CTT GGG TATGTT TTT A 16

D13S165L   GTT TCG CCAAGC CTG TT                   17D13S165L GTT TCG CCAAGC CTG TT 17

D13S165R   GTT GAC AAT AAA ATA CGC CAC A           18D13S165R GTT GAC AAT AAA ATACGC CAC A 18

D13S273L   CTG NGG CAA AAA CAA CTC TT              19D13S273L CTG NGG CAA AAA CAA CTC TT 19

D13S273R   ATC TGT ATG TCC TCC TTT CAA TG          20D13S273R ATC TGT ATG TCC TCCTTT CAA TG 20

D13S1168L  AAC CTC ATT TAA ATG TAA AGC ATC A       21D13S1168L AAC CTC ATT TAA ATG TAA AGC ATC A 21

D13S1168R  GTAATG TCA TTG CTT TTG ATT TGC          22D13S1168R GTAATG TCA TTG CTT TTG ATT TGC 22

D13S1150L  CTC TTG AGG GAA AAA AAA AAT CA          23D13S1150L CTC TTG AGG GAA AAA AAA AAT CA 23

D13S1150R  CCA GGC AAC CAA CCA GTC                 24D13S1150R CCA GGC AAC CAA CCA GTC 24

D13S272L   ATA CAG ACT TCC CAG TGG CT              25D13S272L ATA CAG ACT TCC CAG TGG CT 25

D13S272R     AGC TAT TAA AGT TCC CTG GAT AAA T   26D13S272R AGC TAT TAA AGT TCC CTG GAT AAA T 26

GCT16C05L    AAG GAA TCA GAG AAA TGG GG          27GCT16C05L AAG GAA TCA GAG AAA TGG GG 27

GCT16CO5R    GCT GAG TCA GAG GGA TTT GA          28GCT16CO5R GCT GAG TCA GAG GGA TTT GA 28

D13S25FOR    AGA GGT AAA CAA ACC AAA CCC         29D13S25FOR AGA GGT AAA CAAACC AAA CCC 29

D13S25REV    GCT GAC AAT CAA GAG AAG ATG         30D13S25REV GCT GAC AAT CAA GAG AAG ATG 30

D13S284L     AAA ATC AGG TGG AAA CAG AAT         31D13S284L AAA ATC AGG TGG AAA CAG AAT 31

D13S284R     AAA GGC TAA CAT CGA AGG GA          32D13S284R AAA GGC TAA CAT CGA AGG GA 32

01ALU18      CAG AAC CAG AGA AAC AGC             3301ALU18 CAG AAC CAG AGA AAC AGC 33

02ALU18      ATG GCA CAA CAG CTT AAC             3402ALU18 ATG GCA CAA CAG CTT AAC 34

AFMA301WB5   GAA TGC AGG TGT ACC TAT CAA C       35AFMA301WB5 GAA TGC AGG TGT ACC TAT CAA C 35

AFMA301WB5   ACT GAG TGA CTG CTA CCC AG          36AFMA301WB5 ACT GAG TGA CTG CTA CCC AG 36

D13S272L1    AGC TAG CCC TAT CAG GGT             37D13S272L1 AGC TAG CCC TAT CAG GGT 37

D13S272R1    GTA AGT GGA GGT TAC CTG             38D13S272R1 GTA AGT GGA GGT TAC CTG 38

5279F        GAA TCA TTC GTG CTA AGT GGA T       395279F GAA TCA TTC GTG CTA AGT GGA T 39

5451R        TGC CAA CTG CTT GAA GAA TCT C       405451R TGC CAA CTG CTT GAA GAA TCT C 40

7130F        ACA CCT AAC TCC TGG GTT GTT C       417130F ACA CCT AAC TCC TGG GTT GTT C 41

7371R        ACT AAA TGC CAG CGT TTG CAT G       427371R ACT AAA TGC CAG CGT TTG CAT G 42

9530F        GGT CTT ACT CTG GTT AAA TCT         439530F GGT CTT ACT CTG GTT AAA TCT 43

9757R        CAT TGG TAG CTA AGG AAA CAC         449757R CAT TGG TAG CTA AGG AAA CAC 44

11521F       CCA TTC AAG CCT GGA CAA TCT T       4511521F CCA TTC AAG CCT GGA CAA TCT T 45

11802R       GAA ACT TGA GAC AAT AAG GAG C       4611802R GAA ACT TGA GAC AAT AAG GAG C 46

12440F       CAT GTA ACC AAG ATA AAT CCG T       4712440F CAT GTA ACC AAG ATA AAT CCG T 47

12558R       CTG GAA AAT GTA TGT GAT GAG G       4812558R CTG GAA AAT GTA TGT GAT GAG G 48

17261F       CTG TTG CTA TCT GTA ATA ACA C       4917261F CTG TTG CTA TCT GTA ATA ACA C 49

17494R       CTT GGA ATT TTC CAC TGA ATC         5017494R CTT GGA ATT TTC CAC TGA ATC 50

18701R    TCA TCA GAA GAA ATC AAG GCA G      5118701R TCA TCA GAA GAA ATC AAG GCA G 51

18560F    CAG TGT TAG GAA TAC GCA TTC A      5218560F CAG TGT TAG GAA TAC GCA TTC A 52

GSP2F4    CCT TGC CAG TAC GCC CAC AAG CTG    53GSP2F4 CCT TGC CAG TAC GCC CAC AAG CTG 53

GSP1R1    CCC CAC CTA TGG TTG TAG TGA GCA    54GSP1R1 CCC CAC CTA TGG TTG TAG TGA GCA 54

          TCCTCC

实施例1-CLL患者的体细胞杂交株中的一段30kb的缺失区域Example 1-A 30kb deletion region in a somatic cell hybrid strain of a CLL patient

至今还没有确定CLL患者13q14处丢失的最小区域。已经描述了CLL中13q14处缺失的多种以及相对较大的(在130到550kb)区域(参见图2B)。用LOH和Southern blot分析鉴别Alu18位点纯合子丢失的着丝粒边界，该位点位于D13S1150和D13S272之间小于65kb着丝粒到LEU2基因的外显子5处。但是，没有发现能使靶肿瘤抑制子定位的小的或重叠的纯合子缺失。The minimal region of loss at 13q14 in CLL patients has not been identified to date. Multiple and relatively large (between 130 and 550 kb) regions of deletion at 13q14 have been described in CLL (see Figure 2B). LOH and Southern blot analysis were used to identify the centromeric boundary of the homozygous loss of the Alu18 locus, which is located between D13S1150 and D13S272 less than 65 kb centromere toexon 5 of the LEU2 gene. However, no small or overlapping homozygous deletions that would enable localization of target tumor suppressors were found.

为了更好的定义CLL中的丢失区域，制备鼠LM-TK-的体细胞杂交株和带有13q14移位和/或缺失的CLL细胞。对得到的杂交克隆的PCR检测可以分开肿瘤中存在的染色体13的两个拷贝。用这种方法可以识别一种细胞中的31.4kb的缺失，以及另一种细胞中染色体断点的精确定位(图2D)。这些结果表明13q14肿瘤抑制基因位于LEU2基因的外显子2和5之间的29kb的区域中。用于检测体细胞杂交株的引物如表1所示。To better define the lost regions in CLL, somatic hybrids of murine LM-TK- and CLL cells with 13q14 translocation and/or deletion were generated. PCR detection of the resulting hybridizing clones can separate the two copies of chromosome 13 present in the tumor. A 31.4 kb deletion in one cell and the precise location of the chromosomal breakpoint in another could be identified with this approach (Fig. 2D). These results indicate that the 13q14 tumor suppressor gene is located in a 29 kb region betweenexons 2 and 5 of the LEU2 gene. The primers used to detect somatic cell hybrid strains are listed in Table 1.

如图2所示，体细胞杂交株中缺失的区域与所有已报道的丢失区域相同，包括几年前由Liu et al.(1997)Oncogene 15：2463-2473报道的10kb的区域。LEU2的外显子1和2也在此区域内，并且也在本文所定义的区域内。但是LEU2并不是B-CLL的可能的候选肿瘤抑制子基因(参见Bullrich et al.(2001)，CancerRes.61：6640-6648；Migliazza et al.(2001)，Blood 97：2098-2104；Wolf et al.(2001)，Hum.Mol.Genet.10：1275-1285；和Mertens etal.(2002)Blood 99：4116-4121)As shown in Figure 2, the region deleted in the somatic hybrid is identical to all reported loss regions, including a 10 kb region reported several years ago by Liu et al. (1997) Oncogene 15:2463-2473. Exons 1 and 2 of LEU2 are also within this region, and also within the region defined herein. But LEU2 is not a possible candidate tumor suppressor gene of B-CLL (see Bullrich et al. (2001), CancerRes.61: 6640-6648; Migliazza et al. (2001), Blood 97: 2098-2104; al. (2001), Hum. Mol. Genet. 10: 1275-1285; and Mertens et al. (2002) Blood 99: 4116-4121)

实施例2-miR15和miR16基因位于染色体13的最小缺失区域内，并且在CD5+细胞中高表达Example 2 - The miR15 and miR16 genes are located within the minimally deleted region of chromosome 13 and are highly expressed in CD5+ cells

在已公开可用的序列信息和数据库中筛选13q14处的最小丢失区域中的新的调控基因。最近被克隆的两个miRNA基因簇，miR15和miR16正是位于该缺失区域内(图2A)。为了评价miR15和miR16在正常组织中的表达水平，对一组正常组织，包括分离自正常个体的扁桃腺的CD5+B细胞进行miR15和miR16 RNA的Northern blot分析(图3A)。用CD5+B细胞做对照，这是因为B-CLL具有逐渐积累CD5+B淋巴细胞的特性。发现普遍存在miR15和miR16基因的表达，并且在正常CD5+淋巴细胞中表达量最高。此外，在正常组织中miR16始终比miR15表达水平高。这些数据表明miR15和miR16基因在正常CD5+B细胞的动态平衡中具有重要作用。Publicly available sequence information and databases were screened for novel regulatory genes in the minimally lost region at 13q14. Two recently cloned miRNA gene clusters, miR15 and miR16, are located within this deletion region (Fig. 2A). In order to evaluate the expression levels of miR15 and miR16 in normal tissues, Northern blot analysis of miR15 and miR16 RNA was performed on a group of normal tissues, including CD5+ B cells isolated from tonsils of normal individuals (Fig. 3A). CD5+ B cells were used as a control because B-CLL has the characteristic of gradually accumulating CD5+ B lymphocytes. Expression of miR15 and miR16 genes was found to be ubiquitous and highest in normal CD5+ lymphocytes. Furthermore, miR16 was consistently expressed at higher levels than miR15 in normal tissues. These data suggest that the miR15 and miR16 genes play an important role in the homeostasis of normal CD5+ B cells.

实施例3-在13q14缺失的CLL样品中miR15和miR16基因常常缺失或受到负调控Example 3 - miR15 and miR16 genes are frequently absent or negatively regulated in 13q14-deleted CLL samples

为了研究miR15和miR16基因是否与CLL的发病有关，用Northern blotting分析了60个CLL样品和30个人癌细胞系的miR15和miR16基因表达(图3A)。CLL患者中的68％(41/60)，以及分析的6个前列腺癌细胞系中的5个与其正常组织对应物相比显示出表达显著减少。这些发明证明miR15和miR16基因在所检测的大部分B-CLL和前列腺癌细胞中受到负调控。To investigate whether miR15 and miR16 genes are related to the pathogenesis of CLL, the expression of miR15 and miR16 genes in 60 CLL samples and 30 human cancer cell lines were analyzed by Northern blotting (Fig. 3A). 68% (41/60) of the CLL patients, and 5 of the 6 prostate cancer cell lines analyzed showed significantly reduced expression compared to their normal tissue counterparts. These findings demonstrate that the miR15 and miR16 genes are negatively regulated in most of the B-CLL and prostate cancer cells examined.

此外，60个CLL样品中的23个(38％)显示出明显可识别的代表miR15前体RNA的大约70nt的条带。而除了骨髓之外的任何所分析的正常组织中都没有发现70nt的miR15条带(图3A)，这表明CLL细胞对miR15前体RNA的处理效率较低。Furthermore, 23 of 60 CLL samples (38%) showed a clearly identifiable band of approximately 70 nt representing miR15 precursor RNA. However, no 70nt miR15 band was found in any of the normal tissues analyzed except bone marrow (Fig. 3A), suggesting that CLL cells process miR15 precursor RNA less efficiently.

为了确定所观察到的表达的负调控是否与CLL的等位基因缺失有关，用微卫星标记D13S272和D13S273对46个CLL患者进行了LOH研究，从这些患者可以获得正常DNA(图3B)。我们发现有义样品的68％都在至少一个标记中表现出LOH(35个中的24个)。在除了4个样品以外的所有样品(75％)中miR15/16基因产物的表达都有所减少。有12个样品由于起始物质的问题不能得到可重复的结果。此外，在没有明显LOH的11个样品中的6个(55％)中表达水平也有所减少。在这些实验中，可能由于缺失片段太小而不能被所用的标记检测到。To determine whether the observed negative regulation of expression was related to the allelic loss in CLL, a LOH study was performed using the microsatellite markers D13S272 and D13S273 in 46 CLL patients from whom normal DNA could be obtained (Fig. 3B). We found that 68% of the sense samples exhibited LOH in at least one marker (24 of 35). Expression of the miR15/16 gene product was reduced in all but 4 samples (75%). There were 12 samples where reproducible results could not be obtained due to starting material issues. In addition, expression levels were also reduced in 6 of 11 samples (55%) without overt LOH. In these experiments, it may be that the deletion fragment was too small to be detected by the markers used.

Northern blot分析表明在在13q14处具有已知的较大纯合子缺失的细胞和少于5％的正常细胞中都表达miR15和miR16基因产物，表明在基因组中存在其它高度类似的小分子RNA基因。实际上，已有报道有一个与miR15/miR16基因簇非常类似的基因簇位于染色体3q25-26.1处(参见Lagos-Quintana et al.(2002)，Curr.Biol.12：735-739)。为了表明miR15/16基因表达的变化确实与染色体13q的缺失有关，设计了对染色体13上的miR16前体RNA特异的和对染色体3上的基因产生的miRNA前体RNA特异的探针，用于进行Northern blot。Northern blot analysis showed that both miR15 and miR16 gene products were expressed in cells with a known large homozygous deletion at 13q14 and in less than 5% of normal cells, suggesting the presence of other highly similar small RNA genes in the genome. In fact, a very similar gene cluster to the miR15/miR16 gene cluster has been reported at chromosome 3q25-26.1 (see Lagos-Quintana et al. (2002), Curr. Biol. 12:735-739). In order to show that the changes in miR15/16 gene expression are indeed associated with the deletion of chromosome 13q, probes specific to the miR16 precursor RNA on chromosome 13 and to the miRNA precursor RNA produced by genes on chromosome 3 were designed for Perform Northern blot.

染色体13的miR16前体RNA只检测到很低的水平，在相同样品中用染色体3探针却没有发生特异性的杂交。此外，用两个跨越位于该基因簇着丝粒的2Mb区域的微卫星标记进行LOH研究。17个有义样品中的4个在至少一个标记中显示出了LOH，并且没有发现其与miR15/16的表达水平之间的关系。这些数据明显证明CLL中miR15和miR16基因表达的负调控与13q14处的等位基因丢失相关，表明miR15和miR16基因产物在CLL的发病中具有重要作用。Chromosome 13 miR16 precursor RNA was detected at very low levels, but no specific hybridization occurred with the chromosome 3 probe in the same samples. In addition, LOH studies were performed with two microsatellite markers spanning a 2 Mb region located at the centromere of this gene cluster. Four of the 17 sense samples showed LOH in at least one marker, and no relationship was found with miR15/16 expression levels. These data clearly demonstrate that the negative regulation of miR15 and miR16 gene expression in CLL is associated with allelic loss at 13q14, suggesting that miR15 and miR16 gene products have important roles in the pathogenesis of CLL.

实施例4-在小鼠中miR15和miR16也与CLL发病有关Example 4 - miR15 and miR16 are also associated with CLL pathogenesis in mice

为了进一步研究miR15和miR16基因是否与CLL发病有关，继续用患有CLL的E-TCL1转基因小鼠进行研究(Bichi et al.，(2002)，Proc.Natl.Acad.Sci.USA 99：10：6955-6960)。检测E-TCL1转基因小鼠中细胞和基因的变化。如上所述进行Northern blot分析(参见实施例-“Northern blotting”，和实施例3)。In order to further study whether miR15 and miR16 genes are related to the pathogenesis of CLL, continue to use E-TCL1 transgenic mice with CLL for research (Bichi et al., (2002), Proc.Natl.Acad.Sci.USA 99: 10: 6955-6960). Detection of cellular and genetic changes in E-TCL1 transgenic mice. Northern blot analysis was performed as described above (see Example - "Northern blotting", and Example 3).

在大约80％的转基因小鼠中，与正常鼠脾淋巴细胞相比，CLL细胞中miR15和miR16的鼠同系物都有所降低。这些结果与实施例3中所述的人CLL与正常人细胞相比较的结果一致。In approximately 80% of transgenic mice, the murine homologues of both miR15 and miR16 were reduced in CLL cells compared with normal murine splenic lymphocytes. These results are consistent with the results described in Example 3 comparing human CLL to normal human cells.

还将小鼠染色体15和人染色体12进行了比较。转基因小鼠的白血病的比较基因杂交(CGH)显示出大约35％扩增出了小鼠染色体15的一个区域，相应于人染色体12的区域。这些小鼠白血病的细胞分析也表明小鼠染色体15具有三倍体性或四倍体性。已知大约有25％的人CLL的染色体12具有三倍体性。Mouse chromosome 15 was also compared to human chromosome 12. Comparative genetic hybridization (CGH) of leukemias in transgenic mice showed approximately 35% amplification of a region of mouse chromosome 15 corresponding to that of human chromosome 12. Cellular analysis of these mouse leukemias also showed triploidy or tetraploidy for chromosome 15 in the mice. Chromosome 12 is known to be triploid in approximately 25% of human CLL.

比较基因杂交还显示了小鼠染色体14(51.6-78.5Mb)具有丢失的区域，相应于人的13q14区域。Comparative genetic hybridization also revealed that mouse chromosome 14 (51.6-78.5 Mb) has a lost region corresponding to the human 13q14 region.

这些研究结果表明CLL小鼠模型重现了人CLL发病中发生的事件。综合来说，实施例1-4的数据表明在哺乳动物中miR15和miR16在CLL发病中具有重要作用。These findings suggest that the CLL mouse model recapitulates events that occur in human CLL pathogenesis. Taken together, the data of Examples 1-4 suggest that miR15 and miR16 play an important role in the pathogenesis of CLL in mammals.

实施例5-突变分析没有显示出在CLL和胃肠癌中miR15和miR16基因具有点突变Example 5 - Mutation analysis did not reveal point mutations in miR15 and miR16 genes in CLL and gastrointestinal cancer

为了进一步评价miR15和miR16基因在CLL中的作用，用对PCR扩增产物直接测序的方法检测了120个B-CLL以及80个结肠直肠癌和胃癌中是否具有突变。扩增了含有全部基因簇的720 bp的染色体区域。在这三种情况下，发现miR16前体RNA具有同样的变化；在位点2具有T到C的置换。这种变化不会改变miRNA的发夹结构。还发现了几种非遗传性的多肽性。由于miR16基因较小(70bp)，miR16基因仅具有极少的突变并不令人惊讶。In order to further evaluate the role of miR15 and miR16 genes in CLL, 120 B-CLL and 80 colorectal and gastric cancers were detected for mutations by direct sequencing of PCR amplification products. A 720 bp chromosomal region containing all gene clusters was amplified. In all three cases, the miR16 precursor RNA was found to have the same change; with a T to C substitution atposition 2. This change does not alter the hairpin structure of the miRNA. Several non-inherited polypeptides have also been found. Since the miR16 gene is small (70bp), it is not surprising that the miR16 gene has only few mutations.

为了识别在显示出LOH的CLL中其余的等位基因失活的可能机制，用“in silico”克隆鉴别位于miR16基因下游大约215bp的可能的启动子区域。已报道有几种癌相关基因具有启动子高度甲基化反应的负调控，包括p16^INK4a，p73，hMLH1，或VHL(参见Esteller(2002)，Oncogene 21：5427-5440)。因此使用甲基化特异的PCR分析位于可能的miR16启动子的5’的一个富含CpG区域的甲基化状态。在十个CLL样品中，与miR15或miR16基因表达的水平(八个表达减少，两个具有高表达)无关，在任何所分析的CpG位点都没有检测到甲基化模式的差别。但是，也不能排除甲基化特异的PCR没有检测到CpG位点的不同区域或较小区域的甲基化。To identify possible mechanisms of remaining allelic inactivation in CLL exhibiting LOH, "in silico" cloning was used to identify a possible promoter region located approximately 215 bp downstream of the miR16 gene. Several cancer-associated genes have been reported to have negative regulation of promoter hypermethylation responses, including pl6^INK4a , p73, hMLH1, or VHL (see Esteller (2002), Oncogene 21:5427-5440). Methylation-specific PCR was therefore used to analyze the methylation status of a CpG-rich region located 5' to the probable miR16 promoter. In ten CLL samples, no difference in methylation pattern was detected at any of the CpG sites analyzed, regardless of the level of miR15 or miR16 gene expression (eight had reduced expression, two had high expression). However, it cannot be excluded that methylation-specific PCR did not detect methylation in different or smaller regions of the CpG sites.

实施例6-miR15和miR16基因产物在人细胞中的表达Example 6 - Expression of miR15 and miR16 gene products in human cells

将编码全长70个核苷酸的miR15和miR16 RNA前体的cDNA序列分别克隆到无关的mRNA中，该mRNA处于巨细胞立即早期(CMV-IE)启动子的控制之下，根据Zeng et al.(2002)，Mol.Cell 9：1327-1333中所述的方法操作，在此引入其全文作为参考。The cDNA sequences encoding the full-length 70 nucleotide miR15 and miR16 RNA precursors were cloned separately into unrelated mRNAs under the control of the giant cell immediate early (CMV-IE) promoter according to Zeng et al (2002), Mol. Cell 9: 1327-1333, which is hereby incorporated by reference in its entirety.

简短来说，将Xho I连接子置于编码miR15和miR16 RNA前体的双链cDNA序列的末端，并将这些构建体分别克隆到pBC12/CMV质粒的Xho I位点。pBC12/CMV质粒如Cullen，(1986)，Cell 46：973-982所述，在此引入其全文作为参考。含有miR15前体RNA序列的所述质粒称为pCMV-miR15，含有miR16前体RNA序列的所述质粒称为pCMV-miR16。Briefly, Xho I linkers were placed at the ends of double-stranded cDNA sequences encoding miR15 and miR16 RNA precursors, and these constructs were cloned into the Xho I sites of pBC12/CMV plasmids, respectively. The pBC12/CMV plasmid is described in Cullen, (1986), Cell 46: 973-982, which is hereby incorporated by reference in its entirety. The plasmid containing the miR15 precursor RNA sequence was named pCMV-miR15, and the plasmid containing the miR16 precursor RNA sequence was named pCMV-miR16.

用FuGene 6试剂(Roche)用标准方法将pCMV-miR15和pCMV-miR16分别转染培养的人293T细胞。如上所述提取总RNA，用miR15和miR16特异的探针用Northern blot分析检测是否存在处理过的miR15或miR16 RNA。Cultured human 293T cells were transfected with pCMV-miR15 and pCMV-miR16 using FuGene 6 reagent (Roche) using standard methods. Total RNA was extracted as described above, and the presence of processed miR15 or miR16 RNA was detected by Northern blot analysis using probes specific for miR15 and miR16.

再使pCMV-miR15和pCMV-miR16分别转染培养的人前列腺癌细胞系2220，2221，11609，11611，LNCAP，TSUR。如上所述提取总RNA，用miR15和miR16特异的探针用Northern blot分析检测前列腺癌细胞中是否存在处理过的miR15或miR16RNA。同时评估转染的前列腺癌细胞在形态上的变化，克服接触抑制的能力，以及能指示转化表型的其它标记。Then pCMV-miR15 and pCMV-miR16 were transfected into cultured human prostate cancer cell lines 2220, 2221, 11609, 11611, LNCAP, TSUR, respectively. Total RNA was extracted as described above, and the presence of processed miR15 or miR16 RNA in prostate cancer cells was detected by Northern blot analysis using miR15 and miR16-specific probes. Transfected prostate cancer cells were also assessed for changes in morphology, ability to overcome contact inhibition, and other markers indicative of a transformed phenotype.

实施例7-用miR15和miR16基因产物转染CLL细胞Example 7 - Transfection of CLL cells with miR15 and miR16 gene products

从诊断患有CLL的患者中分离CLL细胞，如下所述用编码miR15和miR16小分子RNA的质粒转染。CLL cells were isolated from patients diagnosed with CLL and transfected with plasmids encoding miR15 and miR16 small RNAs as described below.

如上所述分离CD5+B细胞，用目测观察或者通过用Matuteset al.(1994)，Leukemia 8(10)：1640-1645的评分系统测定CLL分数来鉴别CLL细胞，在此引入其全文作为参考。CLL分数至少为4的CD5+B细胞视为是CLL细胞。证实了在分离的CLL细胞中具有13q14区域的缺失，从而除去了miR15/miR16基因簇。CD5+ B cells were isolated as described above, and CLL cells were identified visually or by measuring the CLL fraction using the scoring system of Matute et al. (1994), Leukemia 8(10): 1640-1645, which is hereby incorporated by reference in its entirety. CD5+ B cells with a CLL score of at least 4 were considered CLL cells. Deletion of the 13q14 region was demonstrated in isolated CLL cells, thereby removing the miR15/miR16 gene cluster.

通过标准方法用pCMV-miR15和pCMV-miR16转染分离的CLL细胞。如上所述提取总RNA，用miR15和miR16特异的探针用Northern blot分析检测是否存在处理过的miR15或miR16RNA。用miR15和miR16基因序列特异的探针进行Southern blot杂交也证实了miR15和miR16基因的稳定整合。Isolated CLL cells were transfected with pCMV-miR15 and pCMV-miR16 by standard methods. Total RNA was extracted as described above, and the presence of processed miR15 or miR16 RNA was detected by Northern blot analysis using probes specific for miR15 and miR16. Southern blot hybridization with probes specific to miR15 and miR16 gene sequences also confirmed the stable integration of miR15 and miR16 genes.

实施例8-用miR15和miR16基因产物转染造血干细胞Example 8 - Transfection of hematopoietic stem cells with miR15 and miR16 gene products

如下所述从诊断为CLL的患者的骨髓中获得造血干细胞(HSC)。Hematopoietic stem cells (HSCs) were obtained from the bone marrow of patients diagnosed with CLL as described below.

在手术室用标准方法在常规麻醉状态下在从患者的髂骨中收集骨髓。用肝素化注射器进行多次抽吸，得到总量为大约750到1000ml的骨髓。将抽出的骨髓立刻转移到运送介质(TC-199，Gibco，Grand Island，New York)中，每100ml该介质含有10,000单位的不含防腐剂的肝素。将抽出的骨髓通过三次逐渐精细的分筛过滤得到不含细胞聚集物，细胞碎片和骨骼颗粒的细胞悬液。然后将过滤得到的骨髓进一步用自动细胞分离器(例如Cobe2991 Cell Processor)得到“血沉棕黄层”(即不含红细胞和血小板的白细胞)。Bone marrow was collected from the patient's iliac crest using standard methods under general anesthesia in the operating room. Multiple aspirations were performed with a heparinized syringe to obtain a total bone marrow volume of approximately 750 to 1000 ml. The aspirated bone marrow was immediately transferred to transport medium (TC-199, Gibco, Grand Island, New York) containing 10,000 units of preservative-free heparin per 100 ml. The aspirated bone marrow was filtered through three progressively finer sieves to obtain a cell suspension free of cell aggregates, cell debris and bone particles. Then the filtered bone marrow is further used in an automatic cell separator (such as Cobe2991 Cell Processor) to obtain "buffy coat" (ie, white blood cells without red blood cells and platelets).

如下所述用免疫磁珠(Dynal A.S.，Oslo，Norway)进行CD34+细胞的正选择使血沉棕黄层制备物部分富集造血干细胞(HSC)。将血沉棕黄层制备物重悬于加入添加物的介质中，并与小鼠抗HPCA-I抗体以1∶20的稀释比率共培育，在4℃轻轻颠倒试管45分钟。用加入添加物的介质洗涤细胞三次，然后与包覆有羊抗鼠IgG₁(75 1免疫珠/10⁷ CD34+细胞)的Fc片段的微珠共培育。在4℃培育45分钟以后，用磁性微粒集中器对粘附在微珠上的细胞进行正选择，根据制造商的说明书操作。Positive selection of CD34+ cells using immunomagnetic beads (Dynal AS, Oslo, Norway) as described below partially enriched the buffy coat preparations for hematopoietic stem cells (HSCs). The buffy coat preparation was resuspended in media with supplements and co-incubated with mouse anti-HPCA-I antibody at a dilution ratio of 1:20 by gently inverting the tubes for 45 minutes at 4°C. Cells were washed three times with supplemented media and then co-incubated with microbeads coated with the Fc fragment of goat anti-mouse IgG₁ (75 1 immunobeads/10⁷ CD34+ cells). After incubation at 4°C for 45 minutes, cells adhered to the beads were positively selected using a magnetic particle concentrator according to the manufacturer's instructions.

将富集HSC的制备物中的2×10⁴个细胞在5ml聚丙烯管(Fisher Scientific，Pittsburgh，PA)中的含有2％人AB血清和10mM Hepes缓冲液的总体积为0.4ml的Iscove′s修改的Dulbecco′s培养基(IMDM)中培育，并用标准方法用pCMV-miR15和pCMV-miR16转染。用一部分转染的HSC通过Northern blot分析证实其中表达miR15或miR16 RNA，用一部分HSC通过Southern blot分析证实其中稳定整合了miR15或miR16基因序列。将其余的转染细胞以大约4×10⁸/kg体重到大约8×10⁸/kg体重的比率用标准骨髓移植方法重新植入到患者体内。2 x¹⁰⁴ cells from the HSC-enriched preparation were placed in a total volume of 0.4 ml of Iscove' containing 2% human AB serum and 10 mM Hepes buffer in a 5 ml polypropylene tube (Fisher Scientific, Pittsburgh, PA). s modified Dulbecco's medium (IMDM) and transfected with pCMV-miR15 and pCMV-miR16 using standard methods. A part of transfected HSCs was used to confirm the expression of miR15 or miR16 RNA through Northern blot analysis, and a part of HSCs was used to confirm the stable integration of miR15 or miR16 gene sequence through Southern blot analysis. The remaining transfected cells are re-implanted into the patient at a rate of about 4 x¹⁰⁸ /kg body weight to about 8 x¹⁰⁸ /kg body weight using standard bone marrow transplantation procedures.

重复该实验，除了用在转染和重新移植之前用电离辐射除去骨髓中的赘生物细胞，如下所述。调整血沉棕黄层制备物中的细胞至细胞浓度为在含有大约20％的自体血浆的TC-199中是2×10⁷/ml。向细胞悬液中加入重组人造血生长因子rH IL-3或rHGM-CSF以刺激造血赘生物的生长，从而增加其对电离辐射的敏感性。然后将这些细胞暴露于5-10Gy的电离辐射中，在4℃用含有大约20％的自体血浆的TC-199洗涤一次，然后如上所述用pCMV-miR15和pCMV-miR16转染。The experiment was repeated except that neoplastic cells in the bone marrow were removed by ionizing radiation prior to transfection and reimplantation, as described below. The cell-to-cell concentration in the buffy coat preparation was adjusted to be 2 x¹⁰⁷ /ml in TC-199 containing approximately 20% autologous plasma. Recombinant human hematopoietic growth factor rH IL-3 or rHGM-CSF is added to the cell suspension to stimulate the growth of hematopoietic neoplasms, thereby increasing their sensitivity to ionizing radiation. These cells were then exposed to 5-10 Gy of ionizing radiation, washed once at 4°C with TC-199 containing approximately 20% autologous plasma, and then transfected with pCMV-miR15 and pCMV-miR16 as described above.

实施例9-含有miR15或miR16的脂质体的制备Example 9 - Preparation of liposomes containing miR15 or miR16

脂质体的制备1-由乳酰脑苷酯，磷脂酰甘油，卵磷脂和胆固醇以摩尔比率1∶1∶4∶5组成的脂质体通过美国专利序列号4,235,871中所述的反相蒸发方法制备，在此引入其全文作为参考。在100g/ml的处理的miR15或miR16 RNA或500g/ml的pCMV-miR15或pCMV-miR16水溶液中制备脂质体，因此制备得到的脂质体含处理的miR15或miR16 RNA，或pCMV-miR15或pCMV-miR16质粒。Preparation of Liposomes 1 - Liposomes composed of lactoyl cerebroside, phosphatidylglycerol, lecithin and cholesterol in a molar ratio of 1:1:4:5 were evaporated by reverse phase as described in US Patent Serial No. 4,235,871 method of preparation, which is hereby incorporated by reference in its entirety. Prepare liposomes in 100g/ml processed miR15 or miR16 RNA or 500g/ml pCMV-miR15 or pCMV-miR16 aqueous solution, so the liposomes prepared contain processed miR15 or miR16 RNA, or pCMV-miR15 or pCMV-miR16 plasmid.

然后将脂质体通过0.4聚碳酸酯膜，重悬于盐水中，用135mM氯化钠，10mM磷酸钠pH7.4通过柱层析与未包覆的物质分离开来。所述脂质体无需修饰即可进一步使用，或者如下所述进行修饰。Liposomes were then passed through a 0.4 polycarbonate membrane, resuspended in saline, and separated from uncoated material by column chromatography with 135 mM sodium chloride, 10 mM sodium phosphate pH 7.4. The liposomes were used further without modification, or modified as described below.

将一些如上所述制备的脂质体加入到适当的反应容器中，并边搅拌边加入含有20mM偏高碘酸钠，135mM氯化钠和10mM磷酸钠(pH7.4)的溶液。将得到的混合物在黑暗中在温度为大约20℃的条件下放置90分钟。将反应混合物用250ml盐水缓冲液(135mM氯化钠，10mM磷酸钠，pH7.4)透析2小时除去过量的高碘酸盐。得到的产品是表面碳水化合物的羟基被氧化成乙醛基的脂质体。目标基团或调理作用抑制分子可以通过这些乙醛基团与脂质体表面结合。A number of liposomes prepared as described above were added to a suitable reaction vessel and a solution containing 20 mM sodium metaperiodate, 135 mM sodium chloride and 10 mM sodium phosphate (pH 7.4) was added with stirring. The resulting mixture was left at a temperature of about 20° C. for 90 minutes in the dark. The reaction mixture was dialyzed against 250 ml saline buffer (135 mM sodium chloride, 10 mM sodium phosphate, pH 7.4) for 2 hours to remove excess periodate. The resulting product is a liposome in which the hydroxyl groups of the surface carbohydrates have been oxidized to acetaldehyde groups. Target groups or opsonization-inhibiting molecules can be bound to the liposome surface via these acetaldehyde groups.

脂质体的制备2-如下所述制备由马来酰亚胺苯甲酰-磷脂酰乙醇胺(MBPE)，卵磷脂和胆固醇组成的第二种脂质体。MBPE是能使含有巯基的化合物，包括蛋白质与脂质体偶合的活性磷脂。Liposome Preparation 2 - A second liposome consisting of maleimide benzoyl-phosphatidylethanolamine (MBPE), lecithin and cholesterol was prepared as follows. MBPE is an active phospholipid that can couple thiol-containing compounds, including proteins, to liposomes.

将十四酰磷脂酰乙醇胺(DMPE)(100mmol)溶解于含有2当量的三乙胺和50mg m-马来酰亚胺苯甲酰N-羟基琥珀酰亚胺酯的5ml无水甲醇中，如Kitagawa et al.(1976)，J.Biochem.79：233-236中所述，在此引入其全文作为参考。在氮气气氛下在室温继续反应过夜，然后用氯仿/甲醇/水(65/25/4)在硅胶H上进行薄层层析，这可以定量显示出DMPE向更快的迁移产物的转变。减压除去甲醇，将产物重新溶于氯仿中。用1％氯化钠提取氯仿相两次，用氯仿/甲醇(4/1)作为溶剂用硅酸层析纯化马来酰亚胺苯甲酰-磷脂酰乙醇胺(MBPE)。纯化以后，薄层层析指示出含有茚三酮阴性斑点的纯磷酸盐。Dissolve myristylphosphatidylethanolamine (DMPE) (100 mmol) in 5 ml of anhydrous methanol containing 2 equivalents of triethylamine and 50 mg of m-maleimidebenzoyl N-hydroxysuccinimide ester, as Described in Kitagawa et al. (1976), J. Biochem. 79: 233-236, which is hereby incorporated by reference in its entirety. The reaction was continued overnight at room temperature under a nitrogen atmosphere, followed by thin layer chromatography on silica gel H with chloroform/methanol/water (65/25/4), which quantitatively revealed the shift of DMPE to the more rapidly migrating product. Methanol was removed under reduced pressure and the product was redissolved in chloroform. The chloroform phase was extracted twice with 1% sodium chloride, and the maleimidobenzoyl-phosphatidylethanolamine (MBPE) was purified by chromatography on silicic acid using chloroform/methanol (4/1) as solvent. After purification, thin layer chromatography indicated pure phosphate containing a ninhydrin negative spot.

在100g/ml处理的miR15或miR16 RNA水溶液或500g/ml pCMV-miR15或pCMV-miR16溶液中，用上述美国专利序列号4,235,871中的反相蒸发方法制备由MBPE，卵磷脂和胆固醇以摩尔比为1∶9∶8组成的脂质体。用100mM氯化钠-2mM磷酸钠(pH6.0)通过柱层析使脂质体与未包覆的物质分离开来。In 100g/ml treated miR15 or miR16 RNA aqueous solution or 500g/ml pCMV-miR15 or pCMV-miR16 solution, use the reverse phase evaporation method in the above-mentioned U.S. Patent Serial No. 4,235,871 to prepare MBPE, lecithin and cholesterol in a molar ratio of Liposomes with a composition of 1:9:8. Liposomes were separated from uncoated material by column chromatography using 100 mM sodium chloride-2 mM sodium phosphate (pH 6.0).

实施例10-使抗CD5+或抗前列腺肿瘤抗体与含有miR15或miR16的脂质体结合Example 10 - Conjugation of anti-CD5+ or anti-prostate tumor antibodies to liposomes containing miR15 or miR16

将1.1ml(含有大约10mmol)的带有活性乙醛基团的脂质体制备物1，或上述脂质体制备物2装入适当的容器中。向该制备物中边搅拌边加入0.2ml的200mM氰硼氢钠溶液和1.0ml的3mg/ml的直接针对CD5+细胞表面标记或前列腺肿瘤细胞抗原的单克隆抗体溶液。在室温继续反应过夜。将得到的混合物在BiogelA5M琼脂糖柱(Biorad，Richmond，Ca.；1.5×37cm)上分离。1.1 ml (containing approximately 10 mmol) ofliposome preparation 1 with active acetaldehyde groups, orliposome preparation 2 above, was filled into a suitable container. To this preparation was added with stirring 0.2 ml of 200 mM sodium borocyanide solution and 1.0 ml of a 3 mg/ml monoclonal antibody solution directed against CD5+ cell surface markers or prostate tumor cell antigens. The reaction was continued overnight at room temperature. The resulting mixture was separated on a Biogel A5M agarose column (Biorad, Richmond, Ca.; 1.5 x 37 cm).

实施例11-在体内用miR15或miR16基因产物抑制人前列腺肿瘤Example 11 - Inhibition of human prostate tumors in vivo by miR15 or miR16 gene products

给裸鼠接种激素抗性人前列腺腺癌细胞系(PC-3)，将小鼠分为处理组和对照组。当小鼠中的肿瘤达到100到250立方毫米的时候，将包裹在脂质体中的处理过的miR15和miR16直接注射到处理组的肿瘤中。对照组的肿瘤中只注射包裹有载体溶液的脂质体。研究过程中一直测量肿瘤体积。还评价了miR15和miR16基因产物在Dunning R-3327鼠前列腺腺癌模型中抑制前列腺肿瘤生长的功效，如下所述。将Dunning R-3327前列腺腺癌细胞的高度转移性的和恶性克隆(RT-3.1)接种到Copenhagen鼠中，然后将其分为处理组和对照组。大约一周后两组都形成实体肿瘤块。然后向处理组的肿瘤中注射包裹在脂质体中的处理过的miR15和miR16，两周一次，一共注射5周。对照组的肿瘤只注射含有载体溶液的脂质体。研究过程中一直测量肿瘤体积。Nude mice were inoculated with hormone-resistant human prostate adenocarcinoma cell line (PC-3), and the mice were divided into treatment group and control group. When tumors in mice reached 100 to 250 mm3, treated miR15 and miR16 encapsulated in liposomes were injected directly into tumors in the treatment group. Only liposomes encapsulated with carrier solution were injected into the tumors of the control group. Tumor volumes were measured throughout the study. The efficacy of the miR15 and miR16 gene products to inhibit prostate tumor growth in the Dunning R-3327 murine prostate adenocarcinoma model was also evaluated, as described below. A highly metastatic and malignant clone (RT-3.1) of Dunning R-3327 prostate adenocarcinoma cells was inoculated into Copenhagen mice, which were then divided into treatment and control groups. Solid tumor masses formed in both groups after about a week. The treated miR15 and miR16 encapsulated in liposomes were then injected into the tumors of the treatment group once every two weeks for a total of 5 weeks. Tumors in the control group were injected only with liposomes containing vehicle solution. Tumor volumes were measured throughout the study.

所有本文所涉及的参考文献在此都引入作为参考。本领域技术人员应当容易理解，本发明非常适于实施并达到本文所提及的以及其所固有的发明目的和优点。本发明还可以以其它特定方式实施而不背离其精神和基本属性，相应地可作为下述权利要求而不是上述说明书的参考，表征本发明的保护范围。All references mentioned herein are hereby incorporated by reference. It will be readily appreciated by those skilled in the art that the present invention is well adapted to be practiced and attained the objects and advantages of the invention herein mentioned as well as inherent therein. The present invention can also be implemented in other specific ways without departing from its spirit and essential properties, and accordingly, the following claims can be used as a reference instead of the above specification to characterize the protection scope of the present invention.

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<110>托马斯杰斐逊大学<110> Thomas Jefferson University

     卡洛·M·克罗西Carlo M. Crosi

     乔治·A·卡林  George A. Carlin

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<210>23<210>23

<211>23<211>23

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>23<400>23

ctcttgaggg aaaaaaaaaa tca                                        23ctcttgaggg aaaaaaaaaa tca 23

<210>24<210>24

<211>18<211>18

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>24<400>24

ccaggcaacc aaccagtc                                              18ccaggcaacc aaccagtc 18

<210>25<210>25

<211>20<211>20

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>25<400>25

atacagactt cccagtggct                                            20atacagactt cccagtggct 20

<210>26<210>26

<211>25<211>25

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>26<400>26

agctattaaa gttccctgga taaat                                      25agctattaaa gttccctgga taaat 25

<210>27<210>27

<211>20<211>20

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>27<400>27

aaggaatcag agaaatgggg                                            20aaggaatcag agaaatgggg 20

<210>28<210>28

<211>20<211>20

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>28<400>28

gctgagtcag agggatttga                                            20gctgagtcag agggattga 20

<210>29<210>29

<211>21<211>21

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>29<400>29

agaggtaaac aaaccaaacc c                                          21agaggtaaac aaaccaaacc c 21

<210>30<210>30

<211>21<211>21

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>30<400>30

gctgacaatc aagagaagat g                                          21gctgacaatc aagagaagat g 21

<210>31<210>31

<211>21<211>21

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>31<400>31

aaaatcaggt ggaaacagaa t                                        21aaaatcaggt ggaaacagaa t 21

<210>32<210>32

<211>20<211>20

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>32<400>32

aaaggctaac atcgaaggga                                          20aaaggctaac atcgaaggga 20

<210>33<210>33

<211>18<211>18

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>33<400>33

cagaaccaga gaaacagc                                            18cagaaccaga gaaacagc 18

<210>34<210>34

<211>18<211>18

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>34<400>34

atggcacaac agcttaac                                            18atggcacaac agcttaac 18

<210>35<210>35

<211>22<211>22

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>35<400>35

gaatgcaggt gtacctatca ac                                        22gaatgcaggt gtacctatca ac 22

<210>36<210>36

<211>20<211>20

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> primer

<400>36<400>36

actgagtgac tgctacccag                                           20actgagtgac tgctacccag 20

<210>37<210>37

<211>18<211>18

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> primer

<400>37<400>37

agctagccct atcagggt                                             18agctagccct atcagggt 18

<210>38<210>38

<211>18<211>18

<212>DNA<212> DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>38<400>38

gtaagtggag gttacctg                                             18gtaagtggag gttacctg 18

<210>39<210>39

<211>22<211>22

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>39<400>39

gaatcattcg tgctaagtgg at                                        22gaatcattcg tgctaagtgg at 22

<210>40<210>40

<211>22<211>22

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>40<400>40

tgccaactgc ttgaagaatc tc                                        22tgccaactgc ttgaagaatc tc 22

<210>41<210>41

<211>22<211>22

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>41<400>41

acacctaact cctgggttgt tc                                        22acacctaact cctgggttgt tc 22

<210>42<210>42

<211>22<211>22

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>42<400>42

actaaatgcc agcgtttgca tg                                        22actaaatgcc agcgtttgca tg 22

<210>43<210>43

<211>21<211>21

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>43<400>43

ggtcttactc tggttaaatc t                                        21ggtcttactc tggttaaatc t 21

<210>44<210>44

<211>21<211>21

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>44<400>44

cattggtagc taaggaaaca c                                        21cattggtagc taaggaaaca c 21

<210>45<210>45

<211>22<211>22

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>45<400>45

ccattcaagc ctggacaatc tt                                       22ccattcaagc ctggacaatc tt 22

<210>46<210>46

<211>22<211>22

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>46<400>46

gaaacttgag acaataagga gc                                       22gaaacttgag acaataagga gc 22

<210>47<210>47

<211>22<211>22

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>47<400>47

catgtaacca agataaatcc gt                                        22catgtaacca agataaatcc gt 22

<210>48<210>48

<211>22<211>22

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>48<400>48

ctggaaaatg tatgtgatga gg                                        22ctggaaaatg tatgtgatga gg 22

<210>49<210>49

<211>22<211>22

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>49<400>49

ctgttgctat ctgtaataac ac                                        22ctgttgctat ctgtaataac ac 22

<210>50<210>50

<211>21<211>21

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>50<400>50

cttggaattt tccactgaat c                                         21cttggaattt tccactgaat c 21

<210>51<210>51

<211>22<211>22

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>51<400>51

tcatcagaag aaatcaaggc ag                                        22tcatcagaag aaatcaaggc ag 22

<210>52<210>52

<211>22<211>22

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>52<400>52

cagtgttagg aatacgcatt ca                                        22cagtgttagg aatacgcatt ca 22

<210>53<210>53

<211>24<211>24

<212>DNA<212> DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>53<400>53

ccttgccagt acgcccacaa gctg                                      24ccttgccagt acgcccacaa gctg 24

<210>54<210>54

<211>27<211>27

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物<223> Primer

<400>54<400>54

ccccacctat ggttgtagtg agcatcc                                   27ccccacctat ggttgtagtg agcatcc 27

Claims (74)

1. a method for the treatment of miR15 or miR16 cancers mediated in the patient of needs treatment comprises miR15 or the miR16 gene outcome of using the separation of effective dose to the patient, and the propagation of miR15 or miR16 cancers mediated cell is suppressed thus.

2. the process of claim 1 wherein that described miR15 or miR16 cancers mediated are chronic lymphocytic leukemia or prostate cancer.

3. the process of claim 1 wherein the mode administration of the miR15 of described separation or miR16 gene outcome by transfection patient cell.

4. the method for claim 3, wherein said cell is a candidate stem cell, chronic lymphocytic leukemia cell or prostate gland cancer cell.

5. the process of claim 1 wherein that the miR15 or the miR16 gene outcome of described separation use to the patient with the form in parenteral or the intestines.

6. administering mode is oral in the method for claim 5, wherein said intestines, rectum, or in the nose.

7. the method for claim 5, wherein said parenteral mode is selected from intravascular administration, organizes injection in periphery or the tissue, and hypodermic injection or deposition comprise h inf, directly application, and suck.

8. the method for claim 7, wherein said intravascular administration is selected from the quick high dose infusion of intravenous, intravenous infusion, the quick high dose infusion of intra-arterial, endoarterial infusion and instil to the conduit of vascular system.

9. the method for claim 7 is injected to be selected from outside the tumour and in the tumour outside the wherein said tissue and in organizing and is injected, and inject in the retina, or the retina rising pouring is penetrated.

10. the method for claim 7, wherein said hypodermic injection or deposition comprise uses the osmotic pump infusion.

11. the method for claim 7, wherein said direct application comprises by conduit, the retina ball, and suppository contains the implant of porous mass, contains non-porous material, perhaps contains the implant of the material of spawn.

12. the process of claim 1 wherein the miR15 of described separation or miR16 gene outcome with naked rna, or and delivery of agents together, perhaps to contain the nucleic acid administration of the sequence of expressing miR15 or miR16 gene outcome.

13. the method for claim 12, the wherein said nucleic acid that contains the sequence of expressing miR15 or miR16 gene outcome is recombinant plasmid or recombinant viral vector.

14. the method for claim 13, wherein said recombinant plasmid or recombinant viral vector comprise the U6 promotor, H1 promotor, or cytomegalovirus promoter.

15. the method for claim 13, wherein said recombinant viral vector is an adenovirus vector, gland relevant viral vector, retroviral vector, or herpesvirus vector.

16. the method for claim 15, wherein said retrovirus is selected from slow virus carrier, rhabdovirus carrier and murine leukemia poisonous carrier.

17. the process of claim 1 wherein miR15 or the miR16 gene outcome and the lipophilicity reagent of described separation, lipofectin, lipofectamine, cellfectin, polycation, or liposome administration together.

18. the method for claim 17, wherein said liposome contain opsonic action and suppress molecule.

19. the method for claim 18, wherein said liposome contain can be with the fixed part on chronic lymphocytic leukemia cell or prostate gland cancer cell of this liposome target.

20. the process of claim 1 wherein that described patient suffers from tumour, the miR15 of the separation of described effective dose or miR16 gene outcome are about at least 10mg/g tumor mass.

21. the method for claim 20, the miR15 of the separation of wherein said effective dose or miR16 gene outcome are about at least 60mg/g tumor mass.

22. the method for claim 20, the miR15 of the separation of wherein said effective dose or miR16 gene outcome are about at least 100mg/g tumor mass.

23. the method for claim 20, the miR15 of the separation of wherein said effective dose or miR16 gene outcome are about 10-500mg/g tumor mass.

24. the process of claim 1 wherein that the miR15 of separation of described effective dose or miR16 gene outcome are about 5 to the 3000mg/kg weight in patients.

25. the preferably about 700-1000mg/kg weight in patients of the method for claim 24, the miR15 of the separation of wherein said effective dose or miR16 gene outcome.

26. the process of claim 1 wherein that the miR15 of separation of described effective dose or miR16 gene outcome are greater than about 1000mg/kg weight in patients.

27. a method for the treatment of miR15 or miR16 cancers mediated in the patient of needs treatment comprises the steps:

1) isolated cell from the patient;

2) with the described cell of nucleic acid transfection of the sequence that contains the miR15 of the effective dose of encoding or miR16 gene outcome; And

3) cells transfected is implanted in patient's body again, miR15 among the patient or miR16 cancers mediated cell are suppressed thus.

28. the method for claim 27, wherein said miR15 or miR16 cancers mediated are chronic lymphocytic leukemia or prostate cancer.

29. the method for claim 28, wherein conclusive evidence is expressed described miR15 or miR16 gene outcome in transfectional cell before implanting cells transfected in patient's body again.

30. the method for claim 27, wherein the nucleic acid stability that conclusive evidence contains the sequence of the miR15 of the effective dose of encoding or miR16 gene outcome before implanting cells transfected in patient's body again is incorporated in the genome of transfectional cell.

31. the method for claim 27, the nucleic acid of the sequence of wherein said miR15 that contains the effective dose of encoding or miR16 gene outcome comprises recombinant plasmid or recombinant viral vector.

32. the method for claim 31, wherein said recombinant plasmid or recombinant viral vector comprise the U6 promotor, H1 promotor, or cytomegalovirus promoter.

33. the method for claim 32, wherein said recombinant viral vector is an adenovirus vector, gland relevant viral vector, retroviral vector, or herpesvirus vector.

34. the method for claim 33, wherein said retrovirus is selected from slow virus carrier, rhabdovirus carrier and murine leukemia poisonous carrier.

35. the method for claim 27, wherein said cells transfected are chronic lymphocytic leukemia cell or prostate gland cancer cell.

36. the method for claim 27, wherein said cells transfected is a candidate stem cell.

37. a method that suppresses miR15 or the cell proliferation of miR16 cancers mediated in the patient comprises to miR15 or miR16 cancers mediated cell and uses the miR15 or the miR16 gene outcome of the separation of effective dose.

38. the method for claim 37, wherein said miR15 or miR16 cancers mediated cell comprise chronic lymphocytic leukemia cell or prostate gland cancer cell.

39. the method for claim 37, the miR15 of wherein said separation or miR16 gene outcome are with the mode administration of transfection miR15 or miR16 cancers mediated cell.

40. the method for claim 37, the miR15 of wherein said separation or miR16 gene outcome be with naked rna, or and delivery of agents together, perhaps to contain the nucleic acid administration of the sequence of expressing miR15 or miR16 gene outcome.

41. the method for claim 40, the wherein said nucleic acid that contains the sequence of expressing miR15 or miR16 gene outcome is recombinant plasmid or recombinant viral vector.

42. the method for claim 41, wherein said recombinant plasmid or recombinant viral vector comprise the U6 promotor, H1 promotor, or cytomegalovirus promoter.

43. the method for claim 41, wherein said recombinant viral vector is an adenovirus vector, gland relevant viral vector, retroviral vector, or herpesvirus vector.

44. the method for claim 43, wherein said retrovirus is selected from slow virus carrier, rhabdovirus carrier and murine leukemia poisonous carrier.

45. the method for claim 37, the miR15 of wherein said separation or miR16 gene outcome and lipophilicity reagent, lipofectin, lipofectamine, cellfectin, polycation, or liposome administration together.

46. the method for claim 45, wherein said liposome contain opsonic action and suppress molecule.

47. the method for claim 45, wherein said liposome contain can be with the fixed part on chronic lymphocytic leukemia cell or prostate gland cancer cell of this liposome target.

48. a pharmaceutical composition contains miR15 or the miR16 gene outcome and the pharmaceutically acceptable carrier of separation.

49. the pharmaceutical composition of claim 48, the miR15 of wherein said separation or miR16 gene outcome are wrapped in the liposome.

50. the pharmaceutical composition of claim 49, wherein said liposome contain opsonic action and suppress molecule.

51. the pharmaceutical composition of claim 49, wherein said liposome contain can be with the fixed part on chronic lymphocytic leukemia cell or prostate gland cancer cell of this liposome target.

52. the pharmaceutical composition of claim 48, the degraded that the miR15 of wherein said separation or miR16 gene outcome can be resisted nuclease.

53. the pharmaceutical composition of claim 52, the miR15 of wherein said separation or miR16 gene outcome contain one or more ribonucleotides that has modification in 2 ' position.

54. the pharmaceutical composition of claim 53, containing wherein, each ribonucleotide all is the miR15 or the miR16 gene outcome of the 2 ' ribonucleotide of modifying.

55. the pharmaceutical composition of claim 53, the one or more ribonucleotides that wherein have modification in 2 ' position have fluorine in 2 ' position, amino, alkyl, alkoxyl, or the modification of O-allyl group.

56. the pharmaceutical composition of claim 53, the one or more ribonucleotides that wherein have modification in 2 ' position have the general formula of 2 ' AR-nucleotide, wherein:

A is oxygen or halogen (preferred fluorine, chlorine or bromine); And

R is hydrogen or straight chain or branch chain C_1-6Alkyl;

Wherein when A is halogen, then there are not X and R.

57. the pharmaceutical composition of claim 53, the one or more ribonucleotides that wherein have modification in 2 ' position are 2 '-O methyl ribonucleotides.

58. a pharmaceutical composition contains the miR15 of coding separation or the nucleic acid of miR16 gene outcome, and pharmaceutically acceptable carrier.

59. the pharmaceutical composition of claim 58, wherein said nucleic acid is wrapped in the liposome.

60. the pharmaceutical composition of claim 59, wherein said liposome contain opsonic action and suppress molecule.

61. the pharmaceutical composition of claim 59, wherein said liposome contain the part that this liposome target can be arrived surely CLL or prostate gland cancer cell.

62. method of diagnosing miR15 among the patient or miR16 cancers mediated, comprise the level of mensuration from miR15 in patient's the sample or miR16 gene outcome, wherein the level of miR15 or miR16 gene outcome is lower with respect to miR15 in the control sample or miR16 gene outcome in the sample, shows to have miR15 or miR16 cancers mediated.

63. the method for claim 62, wherein the detection method to miR15 or miR16 gene outcome level is the detection method that is selected from down group: northern blot analyzes, in situ hybridization and quantitative reverse transcriptase polymerase chain reaction (PCR).

64. being selected from, the method for claim 62, wherein said miR15 or miR16 gene outcome have sequence SEQ ID NO:1, SEQ ID NO:2, the gene outcome of SEQ ID NO:3 and SEQ ID NO:4.

65. the method for claim 62, wherein said miR15 or miR16 cancers mediated are chronic lymphocytic leukemia or prostate cancer.

66. method of diagnosing miR15 among the patient or miR16 cancers mediated, comprise that analysis is from miR15 in patient's the sample or miR16 gene, the miR15 or the miR16 gene that wherein detect in the sample that comes from the patient have one or more disappearances or sudden change with respect to miR15 in the control sample or miR16 gene, show to have miR15 or miR16 cancers mediated.

67. the method for claim 66, the detection method of wherein analyzing described one or more disappearance or sudden change are the detection methods that is selected from down group: Southern blot hybridization, sequence analysis and single stranded conformational polypeptide.

68. the method for claim 66, wherein said miR15 or miR16 cancers mediated are chronic lymphocytic leukemia or prostate cancer.

69. method of diagnosing miR15 among the patient or miR16 cancers mediated, comprise miR15 or the miR16 gene copy number measured in the sample that derives from the patient, wherein miR15 or miR16 gene copy number reduce to 1 or 0, then show to have miR15 or miR16 cancers mediated.

70. the method for claim 69, the minimizing of wherein said gene copy number is to lose mensuration by the D13S273 that analyzes the 13q14 place or the heterozygosis of D13S272 microsatellite marker, and wherein the heterozygosis of D13S273 or D13S272 microsatellite marker is lost and shown and have miR15 or miR16 cancers mediated.

71. the method for claim 70, wherein heterozygosis is lost and is to lose mensuration by the heterozygosis of analyzing D13S1150 or D13S273 microsatellite marker.

72. the method for claim 71, wherein heterozygosis is lost and is to lose mensuration by the heterozygosis of site of analysis Alu18 or D13S273 microsatellite marker.

73. the method for claim 69, wherein miR15 or miR16 gene copy number are by Southern blot hybridization assays.

74. the method for claim 68, wherein said miR15 or miR16 cancers mediated are chronic lymphocytic leukemia or prostate cancer.

Images