miRNA PROFILING COMPOSITIONS AND METHODS OF USE

Abstract

Disclosed herein is a nanosensor of miRNA activity in a target cell, and methods of use, for detection and diagnostic applications. The nanosensor comprises a delivery particle comprising an iron oxide crystal coated with a polymer, and a sensor oligonucleotide covalently attached to the polymer. The sensor oligonucleotide comprises a seed region, comprising a nucleic acid sequence that is completely complementary to the target miRNA and comprises a cleavage site which can be engaged and cleaved by the target miRNA. The senor oligonucleotide also comprises two non-seed regions that each flank the seed region and are each comprised of a nucleic acid sequence that is complementary to the target miRNA to promote hybridization of the sensor oligonucleotide to the target miRNA, and members of a quencher-fluorophore pair. The quencher fluorophore pair members respectively flank the cleavage site and are separated by a distance that permits significant quenching of emitted fluorescent signal.

What is claimed: 
     
         1 . A method of identifying metastatic or pro-metastatic cells in a tumor tissue of a subject with breast cancer, comprising:
 a) identifying miRNA activity of one or more of let-7a, let-7d, let-7c, let-7i, miR-1, miR-100, miR-10a, miR-10b, miR-340, miR-155, miR-15b, miR-186, miR-222, miR-182, miR-210, miR-193b, miR-26a, miR-27a, miR-29a, miR-27b, miR-200c, miR-29c, miR-424, and miR-141 in tumor tissue of the subject, by delivering to the tumor tissue an effective amount of one or more nanosensors, comprising;
 i) a delivery particle comprising an iron oxide crystal coated with a polymer; and 
 ii) a sensor oligonucleotide covalently attached to the polymer, comprising:
 A) a seed region comprising a nucleic acid sequence that is completely complementary to the miRNA and comprises a cleavage site which can be engaged by the miRNA and cleaved by the miRNA in complex with RNA induced silencing complex; 
 B) two non-seed regions that each flank the seed region and are each comprised of nucleic acid sequences complementary to the miRNA to promote hybridization of the sensor oligonucleotide to the miRNA; and 
 C) members of a quencher-fluorophore pair; and 
 wherein the quencher fluorophore pair members respectively flank the cleavage site and are separated by a distance that permits quenching of emitted fluorescent signal; 
 
   b) detecting fluorescence emitted from the one or more nanosensors and assessing if the activity of the miRNA(s) is higher or lower than that of a control tissue; and   c) identifying metastatic or pro-metastatic cells as present in the tumor tissue if one or more of let-7a, let-7d, let-7c, let-7i, miR-1, miR-100, miR-10a, miR-10b, miR-340, miR-155, miR-15b, miR-186, miR-222 is assessed as overexpressed in the tissue, or if one or more of miR-182, miR-210, miR-193b, miR-26a, miR-27a, miR-29a, miR-27b, miR-200c, miR-29c, miR-424, and miR-141 is assessed as downregulated in the tissue.   
     
     
         2 . The method of  claim 1 , which is repeated periodically to monitor disease progression. 
     
     
         3 . The method of  claim 1 , further comprising treating the subject with an aggressive therapy for metastatic breast cancer when metastatic or pro-metastatic cells are identified. 
     
     
         4 . The method of  claim 3 , that is repeated periodically to monitor treatment effectiveness. 
     
     
         5 . The method of  claim 1 , wherein the miRNA is one or more of miR10b, miR-155, miR-200c, and miR-141. 
     
     
         6 . The method of  claim 1 , wherein the nanosensor further comprises a targeting ligand covalently attached to the polymer. 
     
     
         7 . The method of  claim 6 , wherein the targeting ligand is a peptide specific for an internalizing receptor located on the exterior plasma membrane of cells of the tumor tissue. 
     
     
         8 . The method of  claim 6 , wherein the targeting ligand is selected from the group consisting of arginine-glycine-aspartic acid, folic acid, peptide EPPT (SEQ ID NO: 11), polyarginine peptide, and chlorotoxin. 
     
     
         9 . The method of  claim 6 , wherein the polymer is selected from the group consisting of polyethylene glycol, dextran, polyvinylpyrrolidone, fatty acids, polypeptides, chitosan and gelatin, chitosan, polyethylenimine, and combinations thereof. 
     
     
         10 . The method of  claim 1 , wherein the iron oxide crystal is from about 20-30 nm in diameter and the polymer is dextran. 
     
     
         11 . The method of  claim 6 , wherein the sensor oligonucleotide and/or to the targeting ligand are covalently attached to the polymer by thiol crosslinking. 
     
     
         12 . The method of  claim 1 , wherein the entire sensor oligonucleotide nucleic acid sequence is completely complementary to the miRNA sequence. 
     
     
         13 . The method of  claim 1 , wherein the sensor oligonucleotide is RNA, or a combination of RNA and one or more other modified nucleotides that hybridize with RNA in a sequence dependent manner, wherein at least the entire seed region is RNA. 
     
     
         14 . The method of  claim 1 , wherein the sensor oligonucleotide is from about 18 to about 30 nucleotides in length or from about 20-25 nucleotides in length. 
     
     
         15 . The method of  claim 1 , wherein the members are separated by a distance of about 9 to about 30 nucleotides. 
     
     
         16 . The method of  claim 1 , wherein the fluorophore of the quencher-fluorophore pair has an emission maximal over 600 nm. 
     
     
         17 . The method of  claim 1 , wherein the delivery particle is functionalized with amines to thereby facilitate endosomal swelling and rupture upon cellular uptake. 
     
     
         18 . The method of  claim 1 , wherein quenching is with an efficiency of greater than 95%. 
     
     
         19 . The method of  claim 1 , wherein quenching is with an efficiency of about 99%. 
     
     
         20 . A method of treating breast cancer in a subject, comprising:
 a) analyzing tumor tissue obtained from a subject with breast cancer for the presence of metastatic or pro-metastatic breast cancer cells, by a method comprising identifying miRNA activity of one or more of let-7a, let-7d, let-7c, let-7i, miR-1, miR-100, miR-10a, miR-340, miR-15b, miR-186, miR-222, miR-182, miR-210, miR-193b, miR-26a, miR-27a, miR-29a, miR-27b, miR-29c, and miR-424 in a tissue of a subject;   b) identifying metastatic or pro-metastatic cells as present in the tumor tissue if one or more of let-7a, let-7d, let-7c, let-7i, miR-1, miR-100, miR-10a, miR-340, miR-15b, miR-186, miR-222 is assessed as overexpressed in the tissue, or if one or more of miR-182, miR-210, miR-193b, miR-26a, miR-27a, miR-29a, miR-27b, miR-29c, and miR-424 is assessed as downregulated in the tissue, as compared to that of a control tissue; and   c) treating the subject with an aggressive therapy for metastatic breast cancer when metastatic or pro-metastatic cells are identified.   
     
     
         21 . The method if  claim 20 , which is repeated periodically to monitor disease progression. 
     
     
         22 . The method of  claim 20 , wherein the analyzing step comprises delivering to the tumor tissue an effective amount of one or more nanosensors, comprising:
 a) a delivery particle comprising an iron oxide crystal coated with a polymer; and   b) a sensor oligonucleotide covalently attached to the polymer, comprising:
 i) a seed region comprising a nucleic acid sequence that is completely complementary to the miRNA and comprises a cleavage site which can be engaged by the miRNA and cleaved by the miRNA in complex with RNA induced silencing complex; 
 ii) two non-seed regions that each flank the seed region and are each comprised of nucleic acid sequences complementary to the miRNA to promote hybridization of the sensor oligonucleotide to the miRNA; and 
 iii) members of a quencher-fluorophore pair; and 
 wherein the quencher fluorophore pair members respectively flank the cleavage site and are separated by a distance that permits quenching of emitted fluorescent signal; 
   detecting fluorescence emitted from the one or more nanosensors, and assessing if the activity of the miRNA(s) is higher or lower than that of a control tissue.   
     
     
         23 . A method of identifying metastatic or pro-metastatic cells in a tumor tissue of a subject with cancer, comprising:
 a) identifying miRNA activity of one or more miRNA known to be overexpressed or downregulated in metastatic or pro-metastatic cells, in tumor tissue of the subject, by delivering to the tumor tissue an effective amount of one or more nanosensors, comprising;
 i) a delivery particle comprising an iron oxide crystal coated with a polymer; and 
 ii) a sensor oligonucleotide covalently attached to the polymer, comprising:
 A) a seed region comprising a nucleic acid sequence that is completely complementary to the miRNA and comprises a cleavage site which can be engaged by the miRNA and cleaved by the miRNA in complex with RNA induced silencing complex; 
 B) two non-seed regions that each flank the seed region and are each comprised of nucleic acid sequences complementary to the miRNA to promote hybridization of the sensor oligonucleotide to the miRNA; and 
 C) members of a quencher-fluorophore pair; and 
 wherein the quencher fluorophore pair members respectively flank the cleavage site and are separated by a distance that permits quenching of emitted fluorescent signal; 
 
   b) detecting fluorescence emitted from the one or more nanosensors and assessing if the activity of the miRNA(s) is higher or lower than that of a control tissue; and   c) identifying metastatic or pro-metastatic cells as present in the tumor tissue if one or more of the miRNA is assessed as overexpressed or downregulated in the tissue.   
     
     
         24 . The method of  claim 23 , wherein the one or more miRNA is selected from the group consisting of let-7a, let-7d, let-7c, let-7i, miR-1, miR-100, miR-10a, miR-10b, miR-340, miR-155, miR-15b, miR-186, miR-222, miR-182, miR-210, miR-193b, miR-26a, miR-27a, miR-29a, miR-27b, miR-200c, miR-29c, miR-424, and miR-141, and wherein detection of one or more of let-7a, let-7d, let-7c, let-7i, miR-1, miR-100, miR-10a, miR-10b, miR-340, miR-155, miR-15b, miR-186, miR-222 as overexpressed in the tissue, and/or detection of one or more of miR-182, miR-210, miR-193b, miR-26a, miR-27a, miR-29a, miR-27b, miR-200c, miR-29c, miR-424, and miR-141 as downregulated in the tissue, indicates the presence of metastatic or pro-metastatic cells in the tumor tissue. 
     
     
         25 . The method of  claim 23 , further comprising treating the subject with an aggressive therapy for metastatic cancer when metastatic or pro-metastatic cells are identified. 
     
     
         26 . The method of  claim 23 , that is repeated periodically to monitor treatment effectiveness.