Abstract:
The present invention is directed to assay methods in which the levels of certain specific microRNAs are determined in the cerebrospinal fluid of a subject. These methods may be used in the diagnosis or monitoring of neurological diseases, especially brain tumors.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application claims priority to, and the benefit of, U.S. provisional application 60/924,600, filed on May 22, 2007, the contents of which is hereby incorporated by reference in its entirety. 
     
    
     STATEMENT OF GOVERNMENT FUNDING 
       [0002]    The United States Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others under reasonable terms as provided for by the terms of NIH grant R21CA116141, awarded by the Department of Health and Human Services. 
     
    
     FIELD OF THE INVENTION 
       [0003]    The present invention is directed to methods for diagnostically evaluating cerebrospinal fluid (CSF) based upon the relative amount of specific microRNAs that are present. These methods can be used in the detection and monitoring of neurological diseases. 
       BACKGROUND OF THE INVENTION 
       [0004]    MicroRNAs (miRNA) are small, single-stranded nucleic acids that bind to partly complementary sequences in mRNAs and thereby prevent them from being translated into protein. In this manner, miRNAs are believed to play a major role in regulating gene expression (Bartel,  Cell  116:281-297 (2004); He, et al.,  Nat. Rev. Genet.  5:522-531(2004)). It has been found that miRNA levels are often altered in tumor cells and there have been suggestions that such alterations may contribute directly to the formation of some cancers (Meltzer,  Nature  435:745-746 (2005)). As a result, there has been a great deal of interest in the diagnostic and therapeutic use of these molecules (see e.g., US 2007/0089196; 2005/0120415; 2005/0227934; 2005/02222067; 2006/0105360; 2006/0200878; 2006/0185025; and 2006/0019286). 
         [0005]    Although, the diagnostic potential miRNA has been suggested, the use of cerebrospinal fluid (CSF) as a source for test samples has been largely overlooked. This is unfortunate since assays of CSF might be of considerable value in the diagnosis and monitoring of neurological diseases and, especially, brain tumors. 
       SUMMARY OF THE INVENTION 
       [0006]    General Summary 
         [0007]    The present invention is based upon the discovery that some microRNAs are expressed at different levels in the normal and pathological brain and that these differences are reflected in the cerebrospinal fluid (CSF) of patients. These observations provide a basis for the concept that assays of CSF microRNA levels may be used in diagnosing brain diseases and in the post-therapy monitoring of patients. In particular, a comparison can be made between the levels of microRNA in the CSF of a test subject and in that of one or more control subjects. Comparisons can either be made directly or a comparison of the ratio of two microRNAs can be made. In the latter case, one would: a) measure the expression of two microRNAs; b) calculate the ratio of their expression levels; and c) determine the presence or progression of a disease if the ratio exceeds a certain threshold. For example, in the case of brain tumors, a ratio of miR-21 to mir-125b of greater than about 4 is indicative of a high grade glioma. Measurements of microRNA levels may be carried out using singleplex (involving one set of primers) or multiplex (involving more than one set of primers) qRT-PCR as described by, for example, Chen, et al. ( Nucleic Acids Res.  33(20):e179 (2005), incorporated herein by reference in its entirety). 
         [0008]    Specific Aspects of the Invention 
         [0009]    In its first aspect, the invention is directed to a method of diagnosing or monitoring a neurological disease in a subject. The term “diagnosing” refers to the detection of disease in an individual that either has not previously had the disease or that has had the disease but who was treated and is believed to be cured. The term “monitoring” refers to tests performed on patients known to have a disease for the purpose of measuring its progress or for measuring the response of a patient to therapy. The method involves obtaining a test sample of cerebrospinal fluid (CSF) from the subject and assaying this sample to determine the concentration or amount of one or more microRNAs. The results obtained are compared with those obtained using control samples of CSF. The control samples may be from subjects known to be free of the disease or they may be from the general population. In cases where the method is being used to monitor a patient who has a disease or to test for the recurrence of a disease, the “control” sample may be test results obtained from the same patient at an earlier time, i.e., the patient may be examined for changes in microRNA levels before and after surgery or treatment. 
         [0010]    It will be understood that it is not absolutely essential that an actual control sample be run at the same time that assays are being performed on a test sample. Once “normal,” i.e., control, levels of the microRNAs (or of microRNA ratios) have been established, these levels can provide a basis for comparison without the need to rerun a new control sample with each assay. The comparison between the test and control samples provides a basis for a conclusion as to whether a subject has a neurological disease (in cases where the method is being used diagnostically) or whether the disease is progressing or regressing in response to therapy (in cases where the method is being used for monitoring). In general, the greater the difference between the test sample and the control, the stronger the indication for the presence or progression of disease. At a minimum, a difference of 25% should be seen to conclude that a disease is present or progressing with higher differences (50%, 75%, 100% or more) being more conclusive. 
         [0011]    The specific microRNAs that are tested for in the method discussed above include one or more of the following: miR-21; miR-17-5p; miR-18; miR-19; miR-20; miR-92; mir-10a; mir-10b; mir-96; mir-182/182*; mir-183; mir-15a; mir-15b; mir-16; mir-125b; mir-124; mir-1; mir-7; mir-103; mir-134; mir-137; mir-345; mir-200a; mir-330; mir-485-5p; mir-151; mir-22; mir-181; mir-219; mir-30; mir-128; mir-29a; mir-29b; mir-29c; mir-139; mir-338; mir-324-3p; mir-135; mir-296; mir-467; mir-521; and mir-155. The designations provided are standard in the art and are associated with specific sequences that can be found at the microRNA registry (http://microrna.sanger.ac.uk/sequences/). In all cases, they refer to human sequences as shown in Table 1. In some cases, there are additional family members of these microRNAs that are recognized in the art and which should be considered equivalents of the specific sequences listed herein. Although all sequences are shown as RNA sequences, it will be understood that, when referring to hybridizations or other assays, corresponding DNA sequences can be used as well. For example, RNA sequences may be reverse transcribed and amplified using the polymerase chain reaction (PCR) in order to facilitate detection. In these cases, it will actually be DNA and not RNA that is directly quantitated. It will also be understood that the complement of the reverse transcribed DNA sequences can be analyzed instead of the sequence itself. In this context, the term “complement” refers to an oligonucleotide that has an exactly complementary sequence, i.e. for each adenine there is a thymine, etc. Although assays may be performed for the microRNAs individually, it is generally preferable to assay several microRNAs or to compare the ratio of two of the microRNAs. 
         [0012]    The microRNAs above will be particularly useful in the diagnosis and monitoring of cancers of the brain (e.g., gliomas, meningiomas, medulloblastomas, pituitary tumors, nerve sheath tumors, ependymomas, or CNS lymphomas). The most preferred of these microRNAs are miR-21 (increased levels being indicative of the presence or progression of a glioma) and mir-125b (decreased levels being indicative of the presence or progression of a glioma). The ratio of these two microRNAs may also be used to detect and monitor gliomas with a ratio of miR-21 to mir-125b of 3 or 4 being dispositive. Alternative microRNAs that may be used are mir-10b (increased in glioma) and mir-124 (decreased in glioma). 
         [0013]    Specific neurological diseases that may be tested for using the methods described above include Alzheimer&#39;s disease; Huntington&#39;s disease; Parkinson&#39;s disease; amyotrophic lateral sclerosis; multiple sclerosis; stroke; and brain tumors. Of these, brain tumors are especially preferred with gliomas being the most preferred brain tumor. For Alzheimer&#39;s disease, the most preferred diagnostic microRNAs are mir-132 (decreased in AD), mir-212 (decreased in AD), mir-30a (increased in AD) and mir-26b (increased in AD). 
         [0014]    In order to facilitate the testing of multiple microRNAs with the limited amounts of total RNA available from CSF, one of the following methods can be used: 1) multiplex and/or singleplex real-time RT-PCR (reagents available from, e.g., Applied Biosystems and System Biosciences (SBI)); 2) single-molecule detection (Neely, et al.,  Nat. Methods.  3(1):41-46 (2006); 3) bead-based flow cytometric methods (Lu, et al.,  Nature  435:7043 (2005); systems or reagents available from Luminex, Austin, Tex.); 4) array-based methods (e.g., Nelson, et al.,  Nat. Methods  1(2):155-61 (2004); Wu, et al.,  RNA  13(1): 151-159 (2007), all references being hereby incorporated by reference in their entirety). Microarrays can be prepared in which oligonucleotides having complementary sequences (or oligonucleotides with sequences matching the microRNAs themselves) are immobilized on the surface of a solid support. Materials that can be used as supports include membranes, and plates dishes or slides made of glass or plastic. At least 5 (and preferably, 10, 30 or more) of the microRNAs described above should be recognized by the immobilized oligonucleotides, with each different oligonucleotide occupying a distinct and known position on the support. Microarrays of this type may be made using methodology well known in the art or appropriate microRNA arrays can purchased commercially (e.g., from Ambion (Applied Biosystems), Foster City, Calif., Agilent or Exiqon). MicroRNA can then be isolated from the CSF (e.g., using Ambion&#39;s mirVana™ miRNA Isolation Kit) of a test subject, amplified using the polymerase chain reaction, and analyzed by hybridizations performed under stringent conditions. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0015]    The present invention is based upon the identification of microRNAs that are present in CSF and that can be used to identify patients with neurological diseases. These are shown in Table 1 and are all known in the art. References providing methods that can be used for amplifying and quantitating miRNA sequences include: Chen, et al., ( Nucl. Ac. Res.  33(20):e179 (2005); Lao, et al.,  Biochem. Biophys. Res. Commun.  343(1):85-9 (2006); Lao, et al.,  Biotechnol. J.  2(1):33-5 (2007); Tang, et al.,  Nat. Protoc.  1(3):1154-1159 (2006); Neely, et al.,  Nat. Methods.  3(1):41-6 (2006) all incorporated by reference herein in their entirety). 
         [0016]    Although an increased level of any of these microRNAs in the CSF of a subject is suggestive of the presence of disease, especially a brain tumor, a much better assessment can be made by examining many, preferably all, of the microRNAs. Many United States patents have issued describing techniques that can be used for detecting and quantitating microRNA and which may be used to analyze cerebrospinal fluid. These techniques include the following: detection by quantitative real time reverse transcriptase PCR (qRT-PCR) as described in patents owned by Applied Biosystems (U.S. Pat. Nos. 5,928,907; and 6,015,674), single-molecule detection as described in patents owned by US Genomics (U.S. Pat. Nos. 6,355,420; 6,916,661; and 6,632,526), bead-based assays as described in patents owned by Luminex (e.g., U.S. Pat. No. 6,524,793) and in assays using arrays of nucleic acids as described in patents owned by Ambion, Agilent, and Exiqon (U.S. Pat. Nos. 6,057,134; 6,891,032; 7,122,303; 6,458,583; 6,465,183; 6,461,816; 6,458,583; 7,026,124; 7,052,841; 7,060,809; 6,436,640; and 7,060,809). Other references providing guidance helpful in conducting assays include: patents generally describing techniques for producing microarray plates, slides and related instruments (U.S. Pat. No. 6,902,702; U.S. Pat. No. 6,594,432; U.S. Pat. Nos. 5,622,826; 5,556,752; 6,600,031; 6,576,424; 5,566,495; 6,551,784; and 6,887,655) and for carrying out assays (U.S. Pat. No. 6,902,900; U.S. Pat. No. 6,759,197). All of these patent references are hereby incorporated by reference herein in their entirety. 
         [0017]    When microarray supports are used in assays they may be membranes or glass or plastic plates, slides or dishes having a series of distinct, immobilized oligonucleotides recognizing some or all of the microRNA sequences shown Table 1. The immobilized oligonucleotides must hybridize under stringent conditions to one of the microRNA sequences. The term “stringent conditions” indicates conditions that essentially only permit hybridization to occur with the exact complementary sequence of the immobilized oligonucleotide. In general, these hybridizations are performed in buffers of about neutral pH containing 0.1-0.5 NaCl and at a temperature of between 37-50° C. It is also possible to carry out incubations under conditions of low stringency and then to use high stringency wash conditions to cause the dissociation of hybridized sequences that are not exact matches. 
         [0018]    One way to carry out microarray assays would involve amplifying microRNA in the presence of a detectable label, e.g., a nucleotide bound to a dye or other marker and present in a PCR primer. Thus, a population of labeled cDNAs may be obtained that can be used directly in hybridizations with oligonucleotides immobilized on a microarray plate or slide. After hybridizations are completed, plates may be analyzed using an automated reader to determine the amount of label associated with each immobilized sequence, which, in turn, reflects the abundance of the hybridized sequence in the original microRNA population. Many variations of this basic procedure have been described in the art and are compatible with the present invention. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 MicroRNA Sequences 
               
             
          
           
               
                 MicroRNA 
                 Sequence 
                 SEQ ID NO: 
               
               
                   
               
               
                 miR-21 
                 uagcuuaucagacugauguuga 
                  1 
               
               
                   
               
               
                 miR-17-5p 
                 caaagugcuuacagugcagguagu 
                  2 
               
               
                   
               
               
                 miR-18b 
                 uaaggugcaucuagugcaguua 
                  3 
               
               
                   
               
               
                 miR-19 
                 ugugcaaaucuaugcaaaacuga 
                  4 
               
               
                   
               
               
                 miR-20 
                 uaaagugcuuauagugcagguag 
                  5 
               
               
                   
               
               
                 miR-92 
                 uauugcacuugucccggccug 
                  6 
               
               
                   
               
               
                 mir-10a 
                 uacccuguagauccgaauuugu 
                  7 
               
               
                   
               
               
                 mir-10b 
                 uacccuguagaaccgaauuugu 
                  8 
               
               
                   
               
               
                 mir-96 
                 uuuggcacuagcacauuuuugc 
                  9 
               
               
                   
               
               
                 mir-182 
                 uuuggcaaugguagaacucaca 
                 10 
               
               
                   
               
               
                 mir-182* 
                 ugguucuagacuugccaacua 
                 11 
               
               
                   
               
               
                 mir-183 
                 uauggcacugguagaauucacug 
                 12 
               
               
                   
               
               
                 mir-15a 
                 uagcagcacauaaugguuugug 
                 13 
               
               
                   
               
               
                 mir-15b 
                 uagcagcacaucaugguuuaca 
                 14 
               
               
                   
               
               
                 mir-16 
                 uagcagcacguaaauauuggcg 
                 15 
               
               
                   
               
               
                 mir-125b 
                 ucccugagacccuaacuuguga 
                 16 
               
               
                   
               
               
                 mir-124 
                 uuaaggcacgcggugaaugcca 
                 17 
               
               
                   
               
               
                 mir-1 
                 uggaauguaaagaaguaugua 
                 18 
               
               
                   
               
               
                 mir-7 
                 uggaagacuagugauuuuguug 
                 19 
               
               
                   
               
               
                 mir-103 
                 agcagcauuguacagggcuauga 
                 20 
               
               
                   
               
               
                 mir-134 
                 ugugacugguugaccagaggg 
                 21 
               
               
                   
               
               
                 mir-137 
                 uauugcuuaagaauacgcguag 
                 22 
               
               
                   
               
               
                 mir-345 
                 ugcugacuccuaguccagggc 
                 23 
               
               
                   
               
               
                 mir-200a 
                 caucuuaccggacagugcugga 
                 24 
               
               
                   
               
               
                 mir-330 
                 gcaaagcacacggccugcagaga 
                 25 
               
               
                   
               
               
                 mir-485-5p 
                 agaggcuggccgugaugaauuc 
                 26 
               
               
                   
               
               
                 mir-151 
                 acuagacugaagcuccuugagg 
                 27 
               
               
                   
               
               
                 mir-22 
                 aagcugccaguugaagaacugu 
                 28 
               
               
                   
               
               
                 mir-181 
                 aacauucaacgcugucggugagu 
                 29 
               
               
                   
               
               
                 mir-219 
                 ugauuguccaaacgcaauucu 
                 30 
               
               
                   
               
               
                 mir-30a 
                 uguaaacauccucgacuggaag 
                 31 
               
               
                   
               
               
                 mir-128 
                 ucacagugaaccggucucuuuu 
                 32 
               
               
                   
               
               
                 mir-29a 
                 uagcaccaucugaaaucgguu 
                 33 
               
               
                   
               
               
                 mir-29b 
                 uagcaccauuugaaaucaguguu 
                 34 
               
               
                   
               
               
                 mir-29c 
                 uagcaccauuugaaaucggu 
                 35 
               
               
                   
               
               
                 mir-139 
                 ucuacagugcacgugucu 
                 36 
               
               
                   
               
               
                 mir-338 
                 uccagcaucagugauuuuguuga 
                 37 
               
               
                   
               
               
                 mir-324-3p 
                 ccacugccccaggugcugcugg 
                 38 
               
               
                   
               
               
                 mir-135 
                 uauggcuuuuuauuccuauguga 
                 39 
               
               
                   
               
               
                 mir-296 
                 agggcccccccucaauccugu 
                 40 
               
               
                   
               
               
                 mir-467 
                 auauacauacacacaccuacac 
                 41 
               
               
                   
               
               
                 mir-521 
                 aacgcacuucccuuuagagugu 
                 42 
               
               
                   
               
               
                 mir-155 
                 uuaaugcuaaucgugauagggg 
                 43 
               
               
                   
               
               
                 mir-26b 
                 uucaaguaauucaggauagguu 
                 44 
               
               
                   
               
               
                 mir-132 
                 uaacagucuacagccauggucg 
                 45 
               
               
                   
               
               
                 mir-212 
                 uaacagucuccagucacggcc 
                 46 
               
               
                   
               
             
          
         
       
     
         [0019]    All references cited herein are fully incorporated by reference in their entirety. Having now fully described the invention, it will be understood by those of skill in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.