Microrna-based compositions and methods used in disease treatment

Abstract

The present disclosure provides a miRNA hairpin that, when processed by a virus-infected cell, increases the therapeutic effectiveness of the virus. The present disclosure also provides a virus encoding such a miRNA hairpin. The present disclosure also provides pre-miRNAs that, when processed by a virus-infected cell, increase extracellular vesicle secretion of the encoded miRNA hairpin by the virus-infected cell.

1 . A miRNA-hairpin for encoding by a virus that, when processed by a virus-infected cell, increases the therapeutic effectiveness of the virus. 
     
     
         2 . The miRNA-hairpin according to  claim 1 , wherein the miRNA-hairpin includes:
 a sense sequence that is at least 60% identical to SEQ ID NO: 1 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 1, and an antisense sequence that is at least 60% identical to SEQ ID NO: 2 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 2;   a sense sequence that is at least 60% identical to SEQ ID NO: 3 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 3, and an antisense sequence that is at least 60% identical to SEQ ID NO: 4 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 4;   a sense sequence that is at least 60% identical to SEQ ID NO: 5 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 5, and an antisense sequence that is at least 60% identical to SEQ ID NO: 6 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 6;   a sense sequence that is at least 60% identical to SEQ ID NO: 1 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 7, and an antisense sequence that is at least 60% identical to SEQ ID NO: 8 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 8;   a sense sequence that is at least 60% identical to SEQ ID NO: 9 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 9, and an antisense sequence that is at least 60% identical to SEQ ID NO: 10 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 10;   a sense sequence that is at least 60% identical to SEQ ID NO: 11 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 11, and an antisense sequence that is at least 60% identical to SEQ ID NO: 12 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 12;   a sense sequence that is at least 60% identical to SEQ ID NO: 13 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 13, and an antisense sequence that is at least 60% identical to SEQ ID NO: 14 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 14; or   a sense sequence that is at least 60% identical to SEQ ID NO: 15 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 15, and an antisense sequence that is at least 60% identical to SEQ ID NO: 16 and at least 70% identical over the middle seven nucleotides of SEQ ID NO: 16.   
     
     
         3 . The miRNA-hairpin according to  claim 1 , wherein the hairpin includes a loop sequence according to SEQ ID NO: 17. 
     
     
         4 . A virus whose genome includes a nucleic acid sequence that encodes at least one miRNA-hairpin according to  claim 1 . 
     
     
         5 . A plasmid that comprises a nucleotide sequence that, when inserted into a virus, encodes at least one miRNA hairpin according to  claim 1 . 
     
     
         6 . A cassette encoded by a virus that, when processed by a cell that is infected by the virus, increases extracellular vesicle secretion of at least one miRNA hairpin encoded by the cassette. 
     
     
         7 . The cassette according to  claim 6 , wherein the cassette includes at least one sequence according to: [Z]-[A]-[X]-[Y]-[X′]-[B]-[Z′], wherein [Z] and [Z′] represent optional flanking sequences; [X] and [X] represent sense and antisense sequences of the miRNA hairpin stem; [Y] represents a loop sequence of the miRNA hairpin; and [A] and [B] represent, respectively:
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 20 and 21; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 23 and 24; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 27 and 28; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 30 and 31; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 33 and 34; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 36 and 37; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 39 and 40; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 42 and 43; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 45 and 46; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 48 and 49; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 51 and 52; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 54 and 55; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 57 and 58; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 60 and 61; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 63 and 64; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 66 and 67; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 69 and 70; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 72 and 73; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 75 and 76; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 78 and 79; or 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 81 and 82, and wherein, when the cassette includes more than one sequence according to: [Z]-[A]-[X]-[Y]-[X′]-[B]-[Z], each sequence is independently selected. 
 
     
     
         8 . The cassette according to  claim 6 , wherein the cassette includes at least one sequence according to: [Z]-[A]-[X]-[Y]-[X′]-[B]-[Z ‘], wherein [Z] and [Z’] represent optional flanking sequences; [X] and [X] represent sense and antisense sequences of the miRNA hairpin; [Y] represents a loop sequence of the miRNA hairpin; and [A] and [B] represent, respectively:
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 84 and 85; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 87 and 88; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 27 and 28; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 90 and 91; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 93 and 94; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 96 and 97; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 99 and 100; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 102 and 103; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 105 and 106; or 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 108 and 109, and wherein, when the cassette includes more than one sequence according to: [Z]-[A]-[X]-[Y]-[X′]-[B]-[Z], each sequence is independently selected. 
 
     
     
         9 . The cassette according to  claim 6 , wherein the cassette includes at least one sequence according to: [Z]-[A]-[X]-[Y]-[X′]-[B]-[Z ‘], wherein [Z] and [Z’] represent optional flanking sequences; [X] and [X] represent sense and antisense sequences of the miRNA hairpin; [Y] represents a loop sequence of the miRNA hairpin; and [A] and [B] represent, respectively:
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 93 and 94; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 111 and 112; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 22 and 23; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 114 and 115; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 117 and 118; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 120 and 121; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 123 and 124; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 126 and 127; or 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 129 and 130, and wherein, when the cassette includes more than one sequence according to: [Z]-[A]-[X]-[Y]-[X′]-[B]-[Z], each sequence is independently selected. 
 
     
     
         10 . The cassette according to  claim 6 , wherein the cassette includes at least one sequence according to: [Z]-[A]-[X]-[Y]-[X′]-[B]-[Z ‘], wherein [Z] and [Z’] represent optional flanking sequences; [X] and [X] represent sense and antisense sequences of the miRNA hairpin; [Y] represents a loop sequence of the miRNA hairpin; and [A] and [B] represent, respectively:
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 132 and 133; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 135 and 136; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 138 and 139; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 141 and 142; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 144 and 145; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 147 and 148; 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 150 and 151; or 
 sequences that include up to 5 different nucleotides from SEQ ID NOs: 153 and 154, and wherein, when the cassette includes more than one sequence according to: [Z]-[A]-[X]-[Y]-[X′]-[B]-[Z], each sequence is independently selected. 
 
     
     
         11 . The cassette according to  claim 7 ,
 wherein [A] and [B] represent sequences that are identical to the sequences identified by SEQ ID NO.   
     
     
         12 . The cassette according to  claim 7 ,
 wherein [Z] and [Z′] are present and have sequences that are at least 80% identical to SEQ ID NOs: 18 and 19, respectively.   
     
     
         13 . The cassette according to  claim 7 ,
 wherein [Y] has a sequence that is at least 80% identical to SEQ ID NO: 17.   
     
     
         14 . A virus whose genome comprises a nucleotide sequence that encodes one or more cassettes according to  claim 7 . 
     
     
         15 . The virus according to  claim 14 , wherein the one or more cassettes encode 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 independently selected sequences according to [Z]-[A]-[X]-[Y]-[X′]4B1-[Z]. 
     
     
         16 . The virus according to  claim 14 , wherein the one or more cassettes encode about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100, independently selected sequences according to [Z]-[A]-[X]-[Y]-[X′]4B1-[Z]. 
     
     
         17 . A plasmid that comprises a nucleotide sequence that, when inserted into a virus, encodes one or more cassettes according to  claim 6 . 
     
     
         18 . (canceled) 
     
     
         19 . (canceled)