INFLUENZA POLYNUCLEOTIDES, EXPRESSION CONSTRUCTS, COMPOSITIONS, AND METHODS OF USE
The invention provides isolated RNA molecules containing a stretch of nucleotides from a conserved Influenza sequence, and provides complementary RNA molecules thereto. The RNA molecules of the invention are also substantially non-homologous to human sequences. The RNA molecules of the invention include double-stranded RNAs comprising a first region that is a conserved Influenza sequence, and a second region that is at least substantially complementary to the first region. Such double-stranded RNAs include single short hairpin RNAs (shRNAs) as well as multi-target hairpin RNAs containing a plurality, or several, stem-loop structures. The present invention further provides expression constructs that provide for expression of one, or a plurality, of RNA molecules of the invention. The RNA molecules, expression constructs, and compositions of the present invention find use in reducing levels of Influenza A RNA, in reducing Influenza A virus titer, and in treating or preventing Influenza virus infection. The invention is effective against at least human, swine and avian originating strains of Influenza A, and makes gene-silencing therapeutic strategies for combating Influenza A infection feasible.
1 . An isolated double-stranded polynucleotide consisting essentially of a conserved Influenza A sequence and its complementary sequence, said conserved Influenza A sequence being non-homologous to human sequences.
2 . An isolated RNA molecule consisting of 19 or more contiguous nucleotides of a sequence selected from the group consisting of:
(SEQ ID NO: 1)
GGGCAAGGAGACGURGUGUUGGUAAUGAAACG,
(SEQ ID NO: 2)
GACAACAUGACCAAGAAAAUGGUCACACAAAGAACAAUAGG,
(SEQ ID NO: 3)
CGYAGGCUUGCCGACCAAAGUCUCCC,
(SEQ ID NO: 4)
UUUAGAGCCUAUGUGGAUGGAUU,
(SEQ ID NO: 5)
AUGGCGUCYCAAGGCACCAAACGRUCUUAUGARCA,
(SEQ ID NO: 6)
AGGCCCCCUCAAAGCCGARAUCGCDCAGAVACUUGAA,
(SEQ ID NO: 7)
UUUGURUUCACGCUCACCGUGCCCAGUGAGCGR,
(SEQ ID NO: 8)
AGAUGAUCUUCUUGAAAAUUUGCAGVCCUAYCAGAAACGRAUGGG,
and
(SEQ ID NO: 9)
GAGGAUGUCAAAAAUGCAAUUGGGGUCCUCAUCVDAGGRHUUGAA
UGGA,
wherein:
R is A or G,
Y is U or C,
D is A, G, or U,
V is A, G, or C, and
H is C, A, or U.
3 . The RNA molecule of claim 2 , wherein the RNA molecule consists of 19 or more contiguous nucleotides of a sequence selected from the group consisting of:
(SEQ ID NO: 10)
GGGCAAGGAGACGUGGUGUUGGUAAUGAAACG
(SEQ ID NO: 11)
GGGCAAGGAGACGUGAUGUUGGUAAUGAAACG
(SEQ ID NO: 12)
CGCAGGCUUGCCGACCAAAGUCUCCC
(SEQ ID NO: 13)
CGUAGGCUUGCCGACCAAAGUCUCCC
(SEQ ID NO: 14)
AUGGCGUCUCAAGGCACCAAACG,
(SEQ ID NO: 15)
AUGGCGUCCCAAGGCACCAAACG,
(SEQ ID NO: 16)
AGGCCCCCUCAAAGCCGAGAUCGC
(SEQ ID NO: 17)
AGGCCCCCUCAAAGCCGAAAUCGC
(SEQ ID NO: 18)
UUUGUAUUCACGCUCACCGUGCCCAGUGAGCGA,
(SEQ ID NO: 19)
UUUGUGUUCACGCUCACCGUGCCCAGUGAGCG,
(SEQ ID NO: 20)
UUCACGCUCACCGUGCCCAGUGAGCG
(SEQ ID NO: 21)
AGAUGAUCUUCUUGAAAAUUUGCAGGCCUA
(SEQ ID NO: 22)
AGAUGAUCUUCUUGAAAAUUUGCAGACCUA
(SEQ ID NO: 23), SEQ ID NOs: 52-186
GAGGAUGUCAAAAAUGCAAUUGGGGUCCUCAUCG.
4 . The RNA molecule of claim 2 , wherein said RNA molecule includes no more than one nucleotide designated as R, Y, D, V, or H.
5 - 7 . (canceled)
8 . The RNA molecule of claim 2 , wherein said RNA molecule is hybridized to a substantially complementary RNA molecule.
9 - 26 . (canceled)
27 . An isolated double-stranded RNA comprising:
(1) a first region having 19 or more contiguous nucleotides of a sequence selected from the group consisting of:
(SEQ ID NO: 1)
GGGCAAGGAGACGURGUGUUGGUAAUGAAACG,
(SEQ ID NO: 2)
GACAACAUGACCAAGAAAAUGGUCACACAAAGAACAAUAGG,
(SEQ ID NO: 3)
CGYAGGCUUGCCGACCAAAGUCUCCC,
(SEQ ID NO: 4)
UUUAGAGCCUAUGUGGAUGGAUU,
(SEQ ID NO: 5)
AUGGCGUCYCAAGGCACCAAACGRUCUUAUGARCA,
(SEQ ID NO: 6)
AGGCCCCCUCAAAGCCGARAUCGCDCAGAVACUUGAA,
(SEQ ID NO: 7)
UUUGURUUCACGCUCACCGUGCCCAGUGAGCGR,
(SEQ ID NO: 8)
AGAUGAUCUUCUUGAAAAUUUGCAGVCCUAYCAGAAACGRAUGGG,
and
(SEQ ID NO: 9)
GAGGAUGUCAAAAAUGCAAUUGGGGUCCUCAUCVDAGGRHUUGAA
UGGA,
wherein:
R is A or G,
Y is U or C,
D is A, G, or U,
V is A, G, or C, and
H is C, A, or U; and
(2) a second region being substantially complementary to the first region.
28 - 32 . (canceled)
33 . The double-stranded RNA of claim 27 , further comprising a single-stranded hairpin region connecting said first and second regions, and/or single-stranded 5′ and/or 3′ end(s).
34 . The double-stranded RNA of claim 27 , wherein the first region consists of 19 or more contiguous nucleotides of a sequence selected from the group consisting of:
(SEQ ID NO: 10)
GGGCAAGGAGACGUGGUGUUGGUAAUGAAACG
(SEQ ID NO: 11)
GGGCAAGGAGACGUGAUGUUGGUAAUGAAACG
(SEQ ID NO: 12)
CGCAGGCUUGCCGACCAAAGUCUCCC
(SEQ ID NO: 13)
CGUAGGCUUGCCGACCAAAGUCUCCC
(SEQ ID NO: 14)
AUGGCGUCUCAAGGCACCAAACG,
(SEQ ID NO: 15)
AUGGCGUCCCAAGGCACCAAACG,
(SEQ ID NO: 16)
AGGCCCCCUCAAAGCCGAGAUCGC
(SEQ ID NO: 17)
AGGCCCCCUCAAAGCCGAAAUCGC
(SEQ ID NO: 18)
UUUGUAUUCACGCUCACCGUGCCCAGUGAGCGA,
(SEQ ID NO: 19)
UUUGUGUUCACGCUCACCGUGCCCAGUGAGCG,
(SEQ ID NO: 20)
UUCACGCUCACCGUGCCCAGUGAGCG
(SEQ ID NO: 21)
AGAUGAUCUUCUUGAAAAUUUGCAGGCCUA
(SEQ ID NO: 22)
AGAUGAUCUUCUUGAAAAUUUGCAGACCUA
(SEQ ID NO:23), SEQ ID NOs: 52-186
GAGGAUGUCAAAAAUGCAAUUGGGGUCCUCAUCG.
35 . The double-stranded RNA of claim 27 , wherein the first region includes no more than one nucleotide designated as R, Y, D, V, or H.
36 . (canceled)
37 . (canceled)
38 . A multi-target double-stranded RNA comprising:
(1) two or more segments each consisting of 19 or more contiguous nucleotides of a sequence selected from the group consisting of:
(SEQ ID NO: 1)
GGGCAAGGAGACGURGUGUUGGUAAUGAAACG,
(SEQ ID NO: 2)
GACAACAUGACCAAGAAAAUGGUCACACAAAGAACAAUAGG,
(SEQ ID NO: 3)
CGYAGGCUUGCCGACCAAAGUCUCCC,
(SEQ ID NO: 4)
UUUAGAGCCUAUGUGGAUGGAUU,
(SEQ ID NO: 5)
AUGGCGUCYCAAGGCACCAAACGRUCUUAUGARCA,
(SEQ ID NO: 6)
AGGCCCCCUCAAAGCCGARAUCGCDCAGAVACUUGAA,
(SEQ ID NO: 7)
UUUGURUUCACGCUCACCGUGCCCAGUGAGCGR,
(SEQ ID NO: 8)
AGAUGAUCUUCUUGAAAAUUUGCAGVCCUAYCAGAAACGRAUGGG,
and
(SEQ ID NO: 9)
GAGGAUGUCAAAAAUGCAAUUGGGGUCCUCAUCVDAGGRHUUGAA
UGGA,
wherein:
R is A or G,
Y is U or C,
D is A, G, or U,
V is A, G, or C, and
H is C, A, or U; and
(2) a substantially complementary region for each of said two or more segments,
wherein each of said two or more segments is connected to its complementary region through a single-stranded hairpin region.
39 - 43 . (canceled)
44 . An expression construct containing a DNA segment that encodes the RNA molecule of claim 2 , said DNA segment being operably linked to one or more promoter(s).
45 . The expression construct of claim 44 , wherein said expression construct is a plasmid.
46 . The expression construct of claim 45 , wherein the plasmid encodes at least 2 said double-stranded RNA molecules.
47 - 54 . (canceled)
55 . A composition comprising:
(A) two or more RNA molecules each consisting of 19 or more contiguous nucleotides of a sequence selected from the group consisting of:
(SEQ ID NO: 1)
GGGCAAGGAGACGURGUGUUGGUAAUGAAACG,
(SEQ ID NO: 2)
GACAACAUGACCAAGAAAAUGGUCACACAAAGAACAAUAGG,
(SEQ ID NO: 3)
CGYAGGCUUGCCGACCAAAGUCUCCC,
(SEQ ID NO: 4)
UUUAGAGCCUAUGUGGAUGGAUU,
(SEQ ID NO: 5)
AUGGCGUCYCAAGGCACCAAACGRUCUUAUGARCA,
(SEQ ID NO: 6)
AGGCCCCCUCAAAGCCGARAUCGCDCAGAVACUUGAA,
(SEQ ID NO: 7)
UUUGURUUCACGCUCACCGUGCCCAGUGAGCGR,
(SEQ ID NO: 8)
AGAUGAUCUUCUUGAAAAUUUGCAGVCCUAYCAGAAACGRAUGGG,
and
(SEQ ID NO: 9)
GAGGAUGUCAAAAAUGCAAUUGGGGUCCUCAUCVDAGGRHUUGAA
UGGA,
wherein:
R is A or G,
Y is U or C,
D is A, G, or U,
V is A, G, or C, and
H is C, A, or U; and
(B) an RNA molecule complementary to each of said two or more RNA molecules, and
(C) a pharmaceutically acceptable carrier.
56 . A composition comprising an expression vector encoding at least 2 RNA molecules of claim 2 , and a pharmaceutically acceptable carrier.
57 . (canceled)
58 . A method of preventing Influenza A replication in a cell, comprising:
introducing the double-stranded RNA of claim 27 , into a cell susceptible to Influenza A virus.
59 . The method of claim 58 , wherein said Influenza A is a human, swine or avian strain.
60 . A method of reducing an Influenza A virus RNA, comprising: introducing the double-stranded RNA of claim 27 , into a cell susceptible to Influenza A virus.
61 . (canceled)
62 . A method of treating a subject having an Influenza A viral infection, comprising introducing into said subject the double-stranded RNA of claim 27 .
63 . The method of claim 62 , wherein or the double-stranded RNA, is introduced into the subject by administering an expression vector providing for expression in said subject of said double-stranded RNA molecule.
64 . (canceled)
65 . (canceled)