Patent Grant
8377901
The present disclosure relates to methods and compositions for treating viral infection by manipulating expression of a target sequence. The methods and compositions may, for example, be useful in treating virus infection and/or reducing virus replication and/or virus-mediated cytotoxicity.
Infections from influenza viruses pose potentially catastrophic global economic and health threats. Most current therapeutic strategies are directed at viral entry prevention, such as vaccination. These strategies face challenges because viruses display considerable antigenic malleability which enable then to circumvent innate and/or acquired immunity.
Influenza viruses pose a significant threat to the world population as causative agents in seasonal epidemics that claim many lives annually. Furthermore pandemic outbreaks of influenza can have a significant health and economic impact. The emergence of highly-pathogenic avian influenza virus H5N1 and the pandemic outbreak of swine-origin H1N1 influenza virus (SOIV) strongly signals the need for further investigation of this complex virus and its pathogenicity.
Consequently, developing strategies for preventing and treating viral infection is desirable. New and effective compositions are needed to prevent and treat influenza virus infection.
The present invention relates to methods and compositions for treating viral infection by manipulating expression of a target sequence. The methods and compositions may be useful in treating virus infection and/or reducing virus replication and/or virus-mediated cytotoxicity.
According to the present disclosure there is provided a method of reducing virus replication comprising contacting virus-infected cells with an effective amount of at least one inhibitor of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-INFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2.
The present disclosure also provides a method of reducing virus-mediated cytotoxicity comprising contacting virus-infected cells with an effective amount of at least one inhibitor of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2.
The present disclosure further provides a method for treating a viral infection in a subject comprising administering to the subject a composition comprising at least one inhibitor of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2, wherein the composition reduces expression or activity of the ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2 when administered to the subject.
In the methods as described above the inhibitor may be selected from siRNA, RNAi, shRNA, antisense RNA, antisense DNA, decoy molecule, decoy DNA, double stranded DNA, single-stranded DNA, complexed DNA, encapsulated DNA, viral DNA, plasmid DNA, naked RNA, encapsulated RNA, viral RNA, double stranded RNA, molecules capable of generating RNA interference, synthetic ligands, peptide ligands, antagonists, agonists, antibodies, small chemical molecules and combinations thereof.
Furthermore in the methods as described above the siRNA may have a sequence selected from the group of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID 29, SEQ ID NO: 36, SEQ ID 37, SEQ ID NO: 38, SEQ ID 39 and combinations thereof and the shRNA may have a sequence selected from the group of SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 31 to SEQ ID NO: 35, SEQ ID NO: 40 to SEQ ID NO: 45, SEQ ID NO: 47 to SEQ ID NO: 65, SEQ ID NO: 67 to SEQ ID NO: 98, and combinations thereof.
Furthermore the ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2 inhibitor may act on a polypeptide, DNA, or RNA.
In the methods as described above the cells may be contacted by the inhibitor in vivo and the subject may be a mammal. More specifically the mammal may be a human. Furthermore the virus in the methods described may be an influenza virus.
The present disclosure further provides a method of screening for an agent useful for reducing virus to production and/or reducing virus-mediated cytotoxicity in a cell comprising:
i) contacting a cell expressing ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2 with a test compound;
ii) comparing the amount of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2 in the cell in the presence and absence of the test compound; and
iii) selecting any test compound decreasing the amount of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2 as useful for reducing virus production and/or reducing virus-mediated cytotoxicity.
In addition a siRNA for treating virus infection is provided. The siRNA may comprise a sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID 29, SEQ ID NO: 36, SEQ ID 37, SEQ ID NO: 38, SEQ ID 39. A shRNA for treating virus infection is also provided. The shRNA may comprise a sequence represented by SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 31 to SEQ ID NO: 35, SEQ ID NO: 40 to SEQ ID NO: 45, SEQ ID NO: 47 to SEQ ID NO: 65, SEQ ID NO: 67 to SEQ ID NO: 98.
The present disclosure also provides a pharmaceutical formulation for treating a virus infection comprising at least one inhibitor of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2. At least one inhibitor may be a nucleic acid and the nucleic acid may be selected from siRNA, RNAi, shRNA, or combinations thereof or from nucleic acid molecules capable of encoding siRNA, RNAi, shRNA, or combinations thereof.
The siRNA in the pharmaceutical formulation may have a sequence selected from the group of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID 29, SEQ ID NO: 36, SEQ ID 37, SEQ ID NO: 38, SEQ ID 39 and combinations thereof and the shRNA may have a sequence selected from the group SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID NO: 11 to SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 31 to SEQ ID NO: 35, SEQ ID NO: 40 to SEQ ID NO: 45, SEQ ID NO: 47 to SEQ ID NO: 65, SEQ ID NO: 67 to SEQ ID NO: 98 and combinations thereof.
This summary does not necessarily describe all features of the invention. Other aspects, features and advantages of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of some specific embodiments of the invention.
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
In the description that follows, a number of terms are used, the following definitions are provided to facilitate understanding of various aspects of the disclosure. Use of examples in the specification, including examples of terms, is for illustrative purposes only and is not intended to limit the scope and meaning of the embodiments of the invention herein. Numeric ranges are inclusive of the numbers defining the range. In the specification, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to,” and the word “comprises” has a corresponding meaning.
The present disclosure provides, in part, target host factors. By target host factors is meant a host polypeptide, DNA or RNA that—when inhibited, decreased or otherwise interfered with—reduces virus replication and/or virus-mediated cytotoxicity in a host cell infected with the virus. Modulation of the expression level of host factor, or of host factor product activity, prevents and/or ameliorates disease progression. For example, virus replication and/or virus-mediated cytotoxicity and/or apoptosis may be affected. Thus, compounds that modulate the expression of a target host factor or the activity of a target host factor may be used in the diagnosis, treatment, and/or prevention of a viral infection. In particular, target host factors in the present disclosure include endogenous genes and gene products and their variants, as described herein.
Viral infections may include for example infections by respiratory viruses, including but not limited to, various types of influenza, such as influenza A, influenza B and numerous other strains of influenza, including seasonal, avian (e.g., H5N1 strains), and swine (e.g., H1N1 strains).
The present disclosure provides compositions that inhibit target host factors, for example nucleic acids, such as polynucleotides. More specifically, it provides siRNAs, such as shRNAs, that inhibit target host factors and are therefore useful in treating virus infection and/or reducing virus replication and/or virus-mediated cytotoxicity.
The novel target host factors are, for example, ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and SCG2 (see Table 1). The novel target host factor may be BAD, TNFSF12-TNFSF13, TNFSF13, MX2 or USP47. The target host factors may be used as targets for therapy. The target host factors can also can be used to identify compounds useful in the diagnosis, prevention, and/or therapy of virus infection, for example influenza virus infection.
By “reduce,” “reduction”, or “reducing” is meant to destroy, prevent, control, decrease, slow, or otherwise interfere with the production, replication, and/or virus-mediated cytotoxicity of a virus by at least about 10% to about 100%, at least about 30% to about 100%, at least about 50% to about 100%, or any value therebetween for example about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% when compared to the production or replication of a virus in the absence of the inhibitor.
“Inhibitors” refers to molecules that inhibit and/or block an identified function. Any molecule or compound having potential to inhibit and/or block an identified function can be a “test molecule” or “test compound”, as described herein. For example, referring to anti-viral function or anti-apoptotic activity by acting on the ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 polypeptide, DNA or RNA such molecules or compounds may be identified using in vitro and in vivo assays of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139 USP47, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2, respectively. Inhibitors are molecules or compounds that partially or totally block ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139 USP47, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A 1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2 activity, decrease, prevent, or delay their activation, or desensitize the cellular response. This may be accomplished by binding to ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139 USP47, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2 factors directly or via other intermediate molecules. An antagonist or an antibody that blocks ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2 activity, including inhibition of pro-viral function or pro-apoptotic activity of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139 USP47, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2, is considered to be such an inhibitor.
Inhibitors may be for example siRNA, RNAi, shRNA, antisense RNA, antisense DNA, decoy molecules, decoy DNA, double stranded DNA, single-stranded DNA, complexed DNA, encapsulated DNA, viral DNA, plasmid DNA, naked RNA, encapsulated RNA, viral RNA, double stranded RNA, molecules capable of generating RNA interference, or combinations thereof. The group of inhibitors also includes genetically modified versions of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and SCG2, for example, versions with altered activity. The group thus is inclusive of the naturally occurring protein with altered activity, as well as synthetic ligands, peptide ligands, antagonists, agonists, antibodies, small chemical molecules and the like.
Screening for Inhibitors of Target Host Factors
A “test substance” or “test compound” is a compound or mixture of compounds, whose ability to modulate ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2 activity may be defined by various assays. A “test substance” is also referred to as a “candidate drug” or “candidate compound” in the present description.
Test substances may be screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from, e.g., Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., TIBTech, 1996; 14:60).
A modulatory effect may be determined by an in vitro method using a recombinant ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 reporter gene promoter activity system. Reporter genes encode detectable proteins, including, but not limited to, chloramphenicol transferase (CAT), beta-galactosidase (beta-gal), luciferase, green fluorescent protein (GFP) and derivatives thereof, yellow fluorescent protein and derivatives thereof, alkaline phosphatase, other enzymes that can be adapted to produce a detectable product, and other gene products that can be detected, e.g., immunologically (by immunoassay).
A screen involves detecting a change in the expression of the reporter gene by the host cell when contacted with a test substance. If there is no change in expression of the reporter gene, the test to substance may not be an effective modulator. If reporter gene expression is modified, in particular reduced or eliminated, the test substance has modulated, e.g., inhibited, ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2-mediated gene expression, and is thus a candidate for development as a ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 modulator, for use as inhibitor of virus replication and virus-mediated cytotoxicity. The reporter gene assay system described herein may be used in a high-throughput primary screen for antagonists, or it may be used as a secondary functional screen for candidate compounds identified by a different primary screen, e.g., a binding assay screen that identifies compounds that modulate ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 transcription activity.
Potential drugs may be identified by screening in high-throughput assays, including without limitation cell-based or cell-free assays. It will be appreciated by those skilled in the art that different types of assays can be used to detect different types of agents. Several methods of automated assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period of time. Such high-throughput screening methods are particularly useful when screening for candidates for further testing.
Examples of target host factor modulators include the polynucleotides such as siRNAs. “siRNA” refers to small interfering RNAs, which includes short hairpin RNA (“shRNA”) (Paddison et al., Genes & Dev. 16: 948-958, 2002), that are capable of causing interference (as described herein for RNAi) and can cause post-transcriptional silencing of specific genes in cells, for example, mammalian cells (including human cells) and in the body, for example, mammalian bodies (including humans). The phenomenon of RNA interference (RNAi) is described and discussed in Bass, Nature, 411:428-29, 2001; Elbashir et al., Nature, 411:494-98, 2001; and Fire et al., Nature, 391:806-11, 1998, wherein methods of making interfering RNA also are discussed. Exemplary siRNAs could have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integer thereabout or therebetween.
Expression of selected genes can be suppressed in human cells by transfecting with exogenous, short RNA duplexes (siRNA) where one strand corresponds to a target region of the mRNA of interest (Elbashir et al., Nature, 2001; 411:494-498). Upon entry into the cell, siRNA causes the degradation of single-stranded (ssRNAs) RNAs of with identical or near identical sequences, including endogenous mRNAs. siRNA is more potent than standard anti-sense technology since it acts through a catalytic mechanism. Effective strategies to deliver siRNAs to target cells, for example, include physical or chemical transfection. An alternative strategy uses the endogenous expression of siRNAs by various Pol III promoter expression cassettes that allow transcription of functional siRNAs or their precursors (Scherr et al., Curr. Med. Chem., 2003; 10(3):245-56). Recently, the RNA-polymerase III dependent promoter (H1-RNA promoter) was inserted in the lentiviral genome to drive the expression of a small hairpin RNA (shRNA) against enhanced green fluorescent protein (Abbas-Turki et al., Hum. Gene Ther., 2002; 13(18):2197-201). siRNA can also be delivered in a viral vector derived, e.g., from a lentivirus (Tiscornia et al., Proc. Natl. Acad. Sci. U.S.A., 2003; 100:1844-8).
Target Host Factors and siRNAs
BAD
According to an aspect of the present disclosure, it has been determined that BAD may be a suitable target when looking at inhibition of virus replication or virus-mediated cytotoxicity for example influenza virus.
The present disclosure provides polynucleotides that inhibit expression of a polypeptide encoded by a BAD coding region. As used herein a BAD coding region refers to the genomic nucleotide sequence disclosed under GeneID: 572. Examples of target mRNA encoding a BAD polypeptide are the sequences available at Genbank accession numbers NM—004322 (SEQ ID NO:10) or NM—032989 (SEQ ID NO: 103).
Polynucleotides of the present disclosure that will act to inhibit expression of a BAD polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of SEQ ID NO:10 or SEQ ID NO:103. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a BAD coding region include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4. SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.
TNFSF12-13 (TWE-PRIL)
The present disclosure provides polynucleotides that inhibit expression of a fusion polypeptide encoded by a TNFSF12-13 coding region. As used herein a TNFSF12-13 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 407977. An example of a target mRNA encoding a TNFSF12-13 fusion polypeptide is the sequence available at Genbank accession number NM—172089 (SEQ ID NO: 99).
Polynucleotides of the present disclosure that will act to inhibit expression of a TNFSF12-13 fusion polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of SEQ ID NO: 99. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a TNFSF12-13 coding region include SEQ ID NO: 11, SEQ ID NO: 12. SEQ ID to NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
TNFSF13 (APRIL)
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a TNFSF13 coding region. As used herein a TNFSF13 coding region refers to the genomic nucleotide sequence disclosed for example under GeneID: 8741. Several splice variants of the TNFSF13 coding region are expressed and encode polypeptides including a TNFSF13 alpha polypeptide, a TNFSF13 beta polypeptide, and a TNFSF13 gamma polypeptide.
An example of a target mRNA encoding a TNFSF13 alpha polypeptide is the sequence available at Genbank accession number NM—003808 (SEQ ID NO: 100). An example of a target mRNA encoding a TNFSF13 beta polypeptide is the sequence available at Genbank accession number NM—172087 (SEQ ID NO:101). An example of a target mRNA encoding a TNFSF13 gamma polypeptide is the sequence available at Genbank accession number NM—172088 (SEQ ID NO: 102).
A preferred target mRNA includes a sequence that is present in all three splice variants. Polynucleotides of the present disclosure that will act to inhibit expression of a TNFSF13 alpha polypeptide, a TNFSF13 beta polypeptide, and a TNFSF13 gamma polypeptide include polynucleotides with a sense strand that is substantially identical or identical to a region of SEQ ID NO: 100.
Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a TNFSF13 coding region include SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.
MX2
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a MX2 coding region. As used herein a MX2 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 4600. An example of a target mRNA encoding a MX2 polypeptide is the sequence available at Genbank accession number NM—002463 (SEQ ID NO: 24).
Polynucleotides of the present disclosure that will act to inhibit expression of a MX2 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of SEQ ID NO: 24. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a MX2 coding region include SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27. SEQ ID NO: 28 and SEQ ID NO: 29.
USP47
The present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a USP47 coding region. As used herein a USP47 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 55031. An example of a target mRNA encoding a USP47 polypeptide is the sequence available at Genbank accession number NM—017944.3 (SEQ ID NO: 30).
Polynucleotides of the present disclosure that will act to inhibit expression of a USP47 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of SEQ ID NO:30. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a USP47 coding region include SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33. SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40.
MX1
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a MX1 coding region. As used herein a MX1 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 4599. An example of a target mRNA encoding a MX1 polypeptide is the sequence available at Genbank accession numbers NM—002462 and NM—001144925.
Polynucleotides of the present disclosure that will act to inhibit expression of a MX1 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—002462 or NM—001144925. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a MX1 coding region include SEQ ID NO: 41, SEQ ID NO: 42.
MALT1
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a MALT1 coding region. As used herein a MALT1 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 10892. An example of a target mRNA encoding a MALT1 polypeptide is the sequence available at Genbank accession numbers NM—006785 or NM—173844.
Polynucleotides of the present disclosure that will act to inhibit expression of a MALT1 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—006785 or NM—173844. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a MALT1 coding region include SEQ ID NO: 43.
RPS25
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a RPS25 coding region. As used herein a RPS25 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 6230. An example of a target mRNA encoding a RPS25 polypeptide is the sequence available at Genbank accession number NM—001028.
Polynucleotides of the present disclosure that will act to inhibit expression of a RPS25 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—001028. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a RPS25 coding region include SEQ ID NO: 44.
SH3BP4
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a SH3BP4 coding region. As used herein a SH3BP4 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 23677. An example of a target mRNA encoding a SH3BP4 polypeptide is the sequence available at Genbank accession number NM—014521.
Polynucleotides of the present disclosure that will act to inhibit expression of a SH3BP4 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—014521. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a SH3BP4 coding region include SEQ ID NO: 45.
HCG 1986447
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a HCG 1986447 coding region. As used herein a HCG 1986447 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 729324. An example of a target mRNA encoding a HCG 1986447 polypeptide is the sequence available at SEQ ID NO: 46.
Polynucleotides of the present disclosure that will act to inhibit expression of a HCG 1986447 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of SEQ ID NO: 46. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a HCG 1986447 coding region include SEQ ID NO: 47.
WNK2
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a WNK2 coding region. As used herein a WNK2 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 65268. An example of a target mRNA encoding a WNK2 polypeptide is the sequence available at Genbank accession number NM—006648.
Polynucleotides of the present disclosure that will act to inhibit expression of a WNK2 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of to NM—006648. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a WNK2 coding region include SEQ ID NO: 48.
TMC6
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a TMC6 coding region. As used herein a TMC6 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 11322. An example of a target mRNA encoding a TMC6 polypeptide are the sequences available at Genbank accession numbers NM—001127198 and NM—007267.
Polynucleotides of the present disclosure that will act to inhibit expression of a TMC6 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—001127198 or NM—007267. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a TMC6 coding region include SEQ ID NO: 49.
CFL1
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a CFL1 coding region. As used herein a CFL1 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 1072. An example of a target mRNA encoding a CFL1 polypeptide is the sequence available at Genbank accession number NM—005507.
Polynucleotides of the present disclosure that will act to inhibit expression of a CFL1 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—005507. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a CFL1 coding region include SEQ ID NO: 50.
ABI2
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a ABI2 coding region. As used herein a ABI2 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 10152. An example of a target mRNA encoding a ABI2 polypeptide is the sequence available at Genbank accession number NM—005759.
Polynucleotides of the present disclosure that will act to inhibit expression of a ABI2 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—005759. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a ABI2 coding region include SEQ ID NO: 51.
ARRDC3
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a ARRDC3 coding region. As used herein a ARRDC3 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 57561. An example of a target mRNA encoding a ARRDC3 polypeptide is the sequence available at Genbank accession number NM—020801.
Polynucleotides of the present disclosure that will act to inhibit expression of a ARRDC3 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—020801. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a ARRDC3 coding region include SEQ ID NO: 52.
BRCA1
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a BRCA1 coding region. As used herein a BRCA1 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 672. An example of a target mRNA encoding a BRCA1 polypeptide are the sequence available at Genbank accession numbers NM—007294, NM—007297, NM—007298, NM—007299 and NM—007300.
Polynucleotides of the present disclosure that will act to inhibit expression of a BRCA1 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—007294, NM—007297, NM—007298, NM—007299 or NM—007300. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a BRCA1 coding region include SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55.
C17orf85
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a C17orf85 coding region. As used herein a C17orf85 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 55421. An example of a target mRNA encoding a C17orf85 polypeptide are the sequences available at Genbank accession numbers NM—001114118 and NM—018553.
Polynucleotides of the present disclosure that will act to inhibit expression of a C17orf85 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—001114118 or NM—018553. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a C17orf85 coding region include SEQ ID NO: 56.
C1orf71 (CNST)
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a C1orf71 coding region. As used herein a C1orf71 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 163882. An example of a target mRNA encoding a C1orf71 polypeptide are the sequences available at Genbank accession numbers NM—001139459 and NM—152609.
Polynucleotides of the present disclosure that will act to inhibit expression of a C1orf71 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—001139459 or NM—152609. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a C1orf71 coding region include SEQ ID NO: 57.
C6orf162
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a C6orf162 coding region. As used herein a C6orf162 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 57150. An example of a target mRNA encoding a C6orf162 polypeptide are the sequences available at Genbank accession numbers NM—001042493 and NM—020425.
Polynucleotides of the present disclosure that will act to inhibit expression of a C6orf162 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—001042493 or NM—020425. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a C6orf162 coding region include SEQ ID NO: 58.
CCNJL
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a CCNJL coding region. As used herein a CCNJL coding region refers to the genomic nucleotide sequence disclosed under GeneID: 79616. An example of a target mRNA encoding a CCNJL polypeptide is the sequence available at Genbank accession number NM—024565.
Polynucleotides of the present disclosure that will act to inhibit expression of a CCNJL polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—024565. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a CCNJL coding region include SEQ ID NO: 59.
GON4L
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a GON4L coding region. As used herein a GON4L coding region refers to the genomic nucleotide sequence disclosed under GeneID: 54856. An example of a target mRNA encoding a GON4L polypeptide are the sequences available at Genbank accession numbers NM—001037533 and NM—032292.
Polynucleotides of the present disclosure that will act to inhibit expression of a GON4L polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—001037533 or NM—032292. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a GON4L coding region include SEQ ID NO: 60.
HIST1H2AB
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a HIST1H2AB coding region. As used herein a HIST1H2AB coding region refers to the genomic nucleotide sequence disclosed under GeneID: 8335. An example of a target mRNA encoding a HIST1H2AB polypeptide is the sequence available at Genbank accession number NM—003513.
Polynucleotides of the present disclosure that will act to inhibit expression of a HIST1H2AB polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—003513. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a HIST1H2AB coding region include SEQ ID NO: 61.
HPS4
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a HPS4 coding region. As used herein a HPS4 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 89781. An example of a target mRNA encoding a HPS4 polypeptide are the sequences available at Genbank accession numbers NM—022081 and NM—152841.
Polynucleotides of the present disclosure that will act to inhibit expression of a HPS4 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—022081 or NM—152841. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a HPS4 coding region include SEQ ID NO: 62.
LHX8
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a LHX8 coding region. As used herein a LHX8 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 431707. An example of a target mRNA encoding a LHX8 polypeptide is the sequence available at Genbank accession number NM—001001933.
Polynucleotides of the present disclosure that will act to inhibit expression of a LHX8 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—001001933. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a LHX8 coding region include SEQ ID NO: 63.
RPL23
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a RPL23 coding region. As used herein a RPL23 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 9349. An example of a target mRNA encoding a RPL23 polypeptide is the sequence available at Genbank accession number NM—000978.
Polynucleotides of the present disclosure that will act to inhibit expression of a RPL23 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—000978. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a RPL23 coding region include SEQ ID NO: 64.
RPL32
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a RPL32 coding region. As used herein a RPL32 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 6161. An example of a target mRNA encoding a RPL32 polypeptide are the sequences available at Genbank accession numbers NM—000994 and NM—001007073.
Polynucleotides of the present disclosure that will act to inhibit expression of a RPL32 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—000994 or NM—001007073. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a RPL32 coding region include SEQ ID NO: 65.
LOC730139
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a LOC730139 coding region. As used herein a LOC730139 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 730139. An example of a target mRNA encoding a LOC730139 polypeptide is the sequence of SEQ ID NO: 66.
Polynucleotides of the present disclosure that will act to inhibit expression of a LOC730139 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of SEQ ID NO: 66. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a LOC730139 coding region include SEQ ID NO: 67 and SEQ ID NO: 68.
LRRC39
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a LRRC39 coding region. As used herein a LRRC39 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 127495. An example of a target mRNA encoding a LRRC39 polypeptide is the sequence available at Genbank accession number NM—144620.
Polynucleotides of the present disclosure that will act to inhibit expression of a LRRC39 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—144620. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a LRRC39 coding region include SEQ ID NO: 69 and SEQ ID NO: 70.
NRG1
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a NRG1 coding region. As used herein a NRG1 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 3084. An example of a target mRNA encoding a NRG1 polypeptide is the sequence available at Genbank accession number NM—001159995, NM—001159996, NM—001159999, NM—001160001, NM—001160002, NM—001160004, NM—001160005, NM—001160007, NM—001160008, NM—004495, NM—013956, NM—013957, NM—013958, NM—013959, NM—013960, NM—013962 and NM—013964.
Polynucleotides of the present disclosure that will act to inhibit expression of a NRG1 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—001159995, NM—001159996, NM—001159999, NM—001160001, NM—001160002, NM—001160004, NM—001160005, NM—001160007, NM—001160008, NM—004495, NM—013956, NM—013957, NM—013958, NM—013959, NM—013960, NM—013962 or NM—013964. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a NRG1 coding region include SEQ ID NO: 71.
OR52A1
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a OR52A1 coding region. As used herein a OR52A1 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 23538. An example of a target mRNA encoding a OR52A1 polypeptide is the sequence available at Genbank accession number NM—012375.
Polynucleotides of the present disclosure that will act to inhibit expression of a OR52A 1 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—012375. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a OR52A1 coding region include SEQ ID NO: 72 and SEQ ID NO: 73.
PLEKHH1
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a PLEKHH1 coding region. As used herein a PLEKHH1 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 57475. An example of a target mRNA encoding a PLEKHH1 polypeptide is the sequence available at Genbank accession number NM—020715.
Polynucleotides of the present disclosure that will act to inhibit expression of a PLEKHH1 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—020715. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a PLEKHH1 coding region include SEQ ID NO: 74.
PTPN13
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a PTPN13 coding region. As used herein a PTPN13 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 5783. An example of a target mRNA encoding a PTPN13 polypeptide is the sequence available at Genbank accession number NM—006264, NM—080683, NM—080684 and NM—080685.
Polynucleotides of the present disclosure that will act to inhibit expression of a PTPN13 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—006264, NM—080683, NM—080684 or NM—080685. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a PTPN13 coding region include SEQ ID NO: 75.
PTPRJ
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a PTPRJ coding region. As used herein a PTPRJ coding region refers to the genomic nucleotide sequence disclosed under GeneID: 5795. An example of a target mRNA encoding a PTPRJ polypeptide is the sequence available at Genbank accession number NM—001098503 and NM—002843.
Polynucleotides of the present disclosure that will act to inhibit expression of a PTPRJ polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—001098503 or NM—002843. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a PTPN13 coding region include SEQ ID NO: 76 and SEQ ID NO: 77.
RLN1
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a RLN1 coding region. As used herein a RLN1 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 6013. An example of a target mRNA encoding a RLN1 polypeptide is the sequence available at Genbank accession number NM—006911.
Polynucleotides of the present disclosure that will act to inhibit expression of a RLN1 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—006911. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a RLN1 coding region include SEQ ID NO: 78.
RNF19A
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a RNF19A coding region. As used herein a RNF19A coding region refers to the genomic nucleotide sequence disclosed under GeneID: 25897. An example of a target mRNA encoding a RNF19A polypeptide are the sequence available at Genbank accession numbers NM—015435 and NM—183419.
Polynucleotides of the present disclosure that will act to inhibit expression of a RNF19A polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—015435 or NM—183419. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a RLN1 coding region include SEQ ID NO: 79.
SLC7A 14
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a SLC7A14 coding region. As used herein a SLC7A14 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 57709. An example of a target mRNA encoding a SLC7A14 polypeptide is the sequence available at Genbank accession number NM—020949.
Polynucleotides of the present disclosure that will act to inhibit expression of a SLC7A14 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—020949. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a SLC7A14 coding region include SEQ ID NO: 80.
ST8SL43
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a ST8SIA3 coding region. As used herein a ST8SIA3 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 51046. An example of a target mRNA encoding a ST8SIA3 polypeptide is the sequence available at Genbank accession number NM—015879.
Polynucleotides of the present disclosure that will act to inhibit expression of a ST8SIA3 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—015879. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a SLC7A14 coding region include SEQ ID NO: 81 and SEQ ID NO: 82.
TMTC4
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a TMTC4 coding region. As used herein a TMTC4 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 84899. An example of a target mRNA encoding a TMTC4 polypeptide are the sequences available at Genbank accession numbers NM—001079669 and NM—032813.
Polynucleotides of the present disclosure that will act to inhibit expression of a TMTC4 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—001079669 or NM—032813. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a TMTC4 coding region include SEQ ID NO: 84 and SEQ ID NO: 85.
TTN
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a TTN coding region. As used herein a TTN coding region refers to the genomic nucleotide sequence disclosed under GeneID: 7273. An example of a target mRNA encoding a TTN polypeptide is the sequence available at Genbank accession numbers NM—003319, NM—133378, NM—133379, NM—133432 and NM—133437.
Polynucleotides of the present disclosure that will act to inhibit expression of a TTN polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—003319, NM—133378, NM—133379, NM—133432 or NM—133437. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a TTN coding region include SEQ ID NO: 86 and SEQ ID NO: 87.
UBXN7
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a UBXN7 coding region. As used herein a UBXN7 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 26043. An example of a target mRNA encoding a UBXN7 polypeptide is the sequence available at Genbank accession number NM—015562.
Polynucleotides of the present disclosure that will act to inhibit expression of a UBXN7 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—015562. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a UBXN7 coding region include SEQ ID NO: 88.
YPEL2
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a YPEL2 coding region. As used herein a YPEL2 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 388403. An example of a target mRNA encoding a YPEL2 polypeptide is the sequence available at Genbank accession number NM—001005404.
Polynucleotides of the present disclosure that will act to inhibit expression of a YPEL2 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—001005404. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a YPEL2 coding region include SEQ ID NO: 89 and SEQ ID NO: 90.
ZNF251
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a ZNF251 coding region. As used herein a ZNF251 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 90987. An example of a target mRNA encoding a ZNF251 polypeptide is the sequence available at Genbank accession number NM—138367.
Polynucleotides of the present disclosure that will act to inhibit expression of a ZNF251 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM—138367. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a ZNF251 coding region include SEQ ID NO: 91 and SEQ ID NO: 92.
MERTK
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a MERTK coding region. As used herein a MERTK coding region refers to the genomic nucleotide sequence disclosed under GenelD: 10461. An example of a target mRNA encoding a MERTK polypeptide is the sequence available at Genbank accession number NM_006343.
Polynucleotides of the present disclosure that will act to inhibit expression of a MERTK polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM_006343. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a MERTK coding region include SEQ ID NO: 93, SEQ ID NO: 94 and SEQ ID NO: 95.
SCG2
In another aspect, the present disclosure includes polynucleotides that inhibit expression of a polypeptide encoded by a SCG2 coding region. As used herein a SCG2 coding region refers to the genomic nucleotide sequence disclosed under GeneID: 7857. An example of a target mRNA encoding a SCG2 polypeptide is the sequence available at Genbank accession number NM_003469.
Polynucleotides of the present disclosure that will act to inhibit expression of a SCG2 polypeptide, include polynucleotides with a sense strand that is substantially identical or identical to a region of NM_003469. Examples of such polynucleotides that will act to inhibit expression of a polypeptide encoded by a SCG2 coding region include SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98.
A person skilled in the art will appreciate that other polynucleotides can be designed to inhibit the target host factor polypeptides.
Methods for designing such molecules are known in the art. For instance, polynucleotides that inhibit the expression of one of the polypeptides described herein may be identified by the use of cell lines including, but not limited to, HT29 and KM20. A candidate polynucleotide is the polynucleotide that is being tested to determine if it decreases expression of one of the polypeptides described herein. The candidate polynucleotide can be identical to nucleotides located in the region encoding the polypeptide, or located in the 5′ or 3′ untranslated regions of the mRNA. Other methods are known in the art and used for designing and selecting candidate polynucleotides. Candidate polynucleotides are typically screened using publicly available algorithms (e.g., BLAST) to compare the candidate polynucleotide sequences with coding sequences. Those that are likely to form a duplex with an mRNA expressed by a non-target coding region are typically eliminated from further consideration. The remaining candidate polynucleotides may then be tested to determine if they inhibit expression of one of the polypeptides described herein.
In general, candidate polynucleotides are individually tested by introducing a candidate polynucleotide into a cell that expresses the appropriate polypeptide. The candidate polynucleotides may be prepared in vitro and then introduced into a cell. Methods for in vitro synthesis include, for instance, chemical synthesis with a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotides and reagents for such synthesis are well known. Methods for in vitro synthesis also include, for instance, in vitro transcription using a circular or linear vector in a cell free system.
When evaluating whether a candidate polynucleotide functions to inhibit expression of one of the polypeptides described herein, the amount of target mRNA in a cell containing a candidate polynucleotide can be measured and compared to the same type of cell that does not contain the candidate polynucleotide. Methods for measuring mRNA levels in a cell are known in the art. Such methods include quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). Primers and specific conditions for amplification of an mRNA vary depending upon the mRNA, and can be readily determined by the skilled person. Other methods include, for instance, Northern blotting, and array analysis.
Other methods for evaluating whether a candidate polynucleotide functions to inhibit expression of one of the polypeptides described herein include monitoring the polypeptide. For instance, assays can be used to measure a decrease in the amount of polypeptide encoded by the mRNA, or to measure a decrease in the activity of the polypeptide encoded by the mRNA. Methods for measuring a decrease in the amount of a polypeptide include assaying for the polypeptide present in cells containing a candidate polynucleotide and comparing to the same type of cell that does not contain the candidate polynucleotide. For instance, antibody to one of the polypeptides described herein can be used in Western immunoblot, immunoprecipitation, or immunohistochemistry.
Methods for measuring a decrease in the activity of one of the polypeptides, e.g., ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2, vary depending upon the polypeptide. In general, methods for measuring a decrease in the activity of a polypeptide include assaying the appropriate activity present in a cell containing a candidate polynucleotide and comparing to the same type of cell that does not contain the candidate polynucleotide. Methods for measuring the activity of a ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 polypeptide are known in the art.
A candidate polynucleotide that is able to decrease the expression of a polypeptide encoded by a ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF25 or SCG2 coding region, a polypeptide encoded by a ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 coding region, or a target mRNA by at least 80%, or at least 90%, or up to 100%, is considered to be a polynucleotide of the present disclosure.
An inhibiting polynucleotide of the present disclosure can be present in a vector. A vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989). A vector can provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polynucleotide, i.e., an expression vector. The term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, or artificial chromosome vectors. Examples of viral vectors include, for instance, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, and herpes virus vectors. A vector may result in integration into a cell's genomic DNA. Typically, a vector is capable of replication in a bacterial host, for instance E. coli. Preferably the vector is a plasmid. A polynucleotide of the present disclosure can be present in a vector as two separate complementary polynucleotides, each of which can be expressed to yield a sense and an antisense strand of a dsRNA, or as a single polynucleotide containing a sense strand, a loop region, and an antisense strand, which can be expressed to yield an RNA polynucleotide having a sense and an antisense strand of the dsRNA.
Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like. Suitable host cells for cloning or expressing the vectors herein are prokaryotic or eukaryotic cells. Suitable eukaryotic cells include mammalian cells, such as murine cells and human cells. Suitable prokaryotic cells include eubacteria, such as gram-negative organisms, for example, E. coli.
An expression vector optionally includes regulatory sequences operably linked to the polynucleotide of the present disclosure. Typically, the promoter results in the production of an RNA polynucleotide. Examples of such promoters include, but are not limited to, those that cause binding of an RNA polymerase III complex to initiate transcription of an operably linked polynucleotide of the present disclosure. Examples of such promoters include U6 and H1 promoters. Vectors may also include inducible or regulatable promoters for expression of a polynucleotide of the present disclosure in a particular tissue or intracellular environment. The polynucleotide of the present disclosure also typically includes a transcription terminator. Suitable transcription terminators are known in the art and include, for instance, a stretch of 5 consecutive thymidine nucleotides.
Polynucleotides of the present disclosure can be produced in vitro or in vivo. For instance, methods for in vitro synthesis include, but are not limited to, chemical synthesis with a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotides and reagents for such synthesis are well known. Methods for in vitro synthesis also include, for instance, in vitro transcription using a circular or linear expression vector in a cell free system. Expression vectors can also be used to produce a polynucleotide of the present disclosure in a cell, and the polynucleotide then isolated from the cell.
The present disclosure is further directed to methods of treating viral infection and virus-mediated cytotoxicity by inhibiting the target host factors of the present disclosure.
RNAi, antisense, ribozyme and other nucleic acid therapeutics can be used to inhibit expression of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 or a combination thereof in patients suffering from virus infection. For example, a ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 and/or SCG2 antisense strand (either RNA or DNA) may be directly introduced into the cells in a form that is capable of binding to the mRNA transcripts. Alternatively, a vector containing a sequence which once within the target cells, is transcribed into the appropriate antisense mRNA, may be administered. Antisense nucleic acids which hybridize to target mRNA decrease or inhibit production of the polypeptide product encoded by a gene by associating with the normally single-stranded mRNA transcript, thereby interfering with translation and thus, expression of the protein. For example, DNA containing a promoter, e.g., a tissue-specific is operably linked to a DNA sequence (an antisense template), which is transcribed into an antisense RNA. By “operably linked” is meant that a coding sequence and a regulatory sequence(s) (i.e., a promoter) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
Oligonucleotides complementary to various portions of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 can be determined in vitro for their ability to decrease production of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 in human cells according to standard methods. A reduction in ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 gene product in cells contacted with the candidate antisense composition compared to cells cultured in the absence of the candidate composition is detected using ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2-specific antibodies or other detection strategies. Sequences which decrease production of ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 in vitro cell-based or cell-free assays are then be tested in vivo in rats or mice to confirm decreased ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 production in animals with virus infection.
Antisense therapy may be carried out by administering to a patient an antisense nucleic acid by standard vectors and/or gene delivery systems. Suitable gene delivery systems may include liposomes, polymers, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses and adeno-associated viruses, among others. A therapeutic nucleic acid composition is formulated in a pharmaceutically acceptable carrier. The therapeutic composition may also include a gene delivery system as described above.
The present disclosure is also directed to compositions including one or more inhibitors of the target host factor polypeptide of the present disclosure. Such compositions typically include a pharmaceutically acceptable carrier. As used herein “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Additional active compounds can also be incorporated into the compositions.
Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal: e.g., physiological saline. A therapeutically effective amount of a compound is an amount which is capable of producing a medically desirable result such as reduced production of a ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2 gene product in a treated animal.
Parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal delivery routes, may be used to deliver nucleic acids or ABI2, ARRDC3, BAD, BRCA1, C17orf85, C1orf71, C6orf162, CCNJL, CFL1, GON4L, HCG 1986447, HIST1H2AB, HPS4, LHX8, RPS25, RPL23, RPL32, LOC730139, LRRC39, MALT1, MX1, MERTK, MX2, NRG1, OR52A1, PLEKHH1, PTPN13, PTPRJ, RLN1, RNF19A, SH3BP4, SLC7A14, ST8SIA3, STX3, TMC6, TMTC4, TNFSF12-TNFSF13, TNFSF13, TTN, UBXN7, USP47, WNK2, YPEL2, ZNF251 or SCG2-inhibitory peptides on non-peptide compounds. Liposome formulations of therapeutic compounds may also facilitate activity.
Dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular nucleic acid to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
A list of sequence identification numbers of the present disclosure is given in Table 7.
The present invention will be further illustrated in the following examples. However it is to be understood that these examples are for illustrative purposes only, and should not be used to limit the scope of the present invention in any manner.
Drosophila-based screens' have been used to identify genes involved in influenza replication, and two very recent studies employed genome-wide siRNA arrays to identify mammalian host proteins involved in various stages of the influenza virus lifecycle.6,7 Severe influenza pathology has been seen with pandemic 1918 virus8 and SOIV-infected patient lung pathology showed alveolar damage and hemorrhage suggestive of atypical immune responses.9 Studies in pigtailed macaques reported that the activation of apoptotic pathways may contribute to tissue damage during infection10.
Human genes (target host factors) required for viral replication and cytotoxicity were identified. Interference with these proteins provided a basis for inhibiting viral production while protecting infected cells from virus-induced death.
Multiple genome-wide lentiviral-based shRNAmir screens consistently identified 35 annotated candidate genes associated with cell survival despite infection (see Table 1). Initial analysis of 4 candidate genes (BAD, INFSF12-13/INFSF13, MX2, and USP47), using lentiviral-based and synthetic RNAi silencing confirmed protective roles with dramatic inhibition of virus replication after challenge with influenza viruses of multiple H and N types, including the contemporary pandemic swine-origin H1N1 virus.
High Throughput Genome-Wide Screens
The availability of human genome-wide lentiviral-based shRNAi (short hairpin RNA interference) libraries provides the ability to identify host genes important in influenza pathology. RNAi regulates gene expression through sequence-specific mRNA targeting, and genome-wide RNAi screens produce genome-wide loss-of-function phenotypes.11 This allows for a system-level understanding of the host cellular processes and comprehensive identification of molecular components underlying influenza pathogenicity. The lentiviral-based Decode™ RNAi library (Open Biosystems), consisting of 7 pools, each with ˜10,000 shRNAmir constructs, was used to establish stably transduced genome-wide knockdown of every host gene in human lung adenocarcinoma A549 cells (
Target Host Factor Validation
As an initial confirmation that BAD, TNFSF12-TNFSF13/TNFSF13, MX2, and USP47 knockdown eliminate influenza virus-mediated cytotoxicity, sets of A549 cells were individually transduced with shRNA-lentiviruses specifically targeting each transcript. The shRNA constructs contained puromycin markers for positive selection of transduced cells. Stably transduced cells were passaged in puromycin at least twice to remove non-transduced cells, followed by infection with various influenza A viruses. Non-transduced cells, as well as cells transduced with an irrelevant non-silencing shRNA, were killed after being infected with NY55 at an MOI of 1. In contrast, there was no observable cytopathic effect (CPE) in influenza virus infected cells that had been transduced with BAD-, TNFSF12-TNFSF13/TNFSF13-, MX2-, or USP47-specific shRNAs (
As further validation, to ensure that lack of cytotoxicity and reduced virus replication were not caused by off-target effects or other artifacts, protein knockdowns were repeated with siRNA duplexes (Dharmacon®). Sets of A549 cells were treated with each of four distinct siRNAs that target each of the four proteins (plus an irrelevant non-silencing control) twice, 24 h apart, and after a further 24 h, were infected with influenza virus. Influenza virus replication was dramatically reduced by each of the four BAD-, TNFSF12-TNFSF13/TNFSF13-, MX2-, and USP47-specific siRNAs (
Since NY55 is an H3N2 virus (like the 1968 pandemic “Hong Kong” virus), it was then determined whether these observations could be extended more broadly to other influenza virus subtypes. Therefore, the effects of knocking down these genes were examined on cytopathology and replication of the A/Puerto Rico/8/1934 (H1N1) (PR8) and contemporary pandemic SOIV H1N1 isolates. Similar to effects observed with NY55, knockdown of BAD, TNFSF12-TNFSF13/TNFSF13, MX2, and USP47 also protected the host cell from virus-induced cytopathology during PR8 (H1N1) and SOIV (H1N1) infections (
TNFSF13 and the fusion protein TNFSF12-13—also known as APRIL and TWE-PRIL, respectively—belong to the tumour necrosis factor (TNF) ligand superfamily.12,13 Influenza virus infection up-regulates TNFSF13 expression in lung tissues and affects recruitment of macrophages to the site of infection in TNFSF13 deficient mice.14 Human MX2 protein has no anti-influenza virus activity described to date; however, it localizes to the nuclear membrane where it may play a role in nucleocytoplasmic trafficking.15,16,17,18 USP47 is a deubiquitinase, whose specific role in cellular processes remains relatively unknown.19 Although there are previous reports indicating the importance of the ubiquitin-proteasome pathway in other viruses, the involvement of deubiquitylation in influenza remains relatively unknown. More studies on the cellular functions and their involvement in influenza infection are warranted to delineate the specific molecular mechanisms to which TNFSF12-13, TNFSF13 and USP47 proteins contribute.
In order to delineate host biomolecular interactions that may function in conferring protection against viral infection, network analysis20,21 was performed. The genes identified in this study participated in 175 binary interactions and three regulatory protein complexes (
Knockdown of host MX2, BAD, TNFSF12-13, TNFSF13, or USP47 genes confers broad, non-lethal and long-lasting virus type-independent protection against influenza virus-mediated cytotoxicity and promotes host cell survival.
Target Host Factors
Cytopathology induced during influenza infection is a contributing factor to tissue damage and has been suggested to be a catalyst for aberrant host immune response during disease progression, but the underlying molecular mechanisms remain obscure.
BAD is a pro-apoptotic protein that belongs to the BH3-only subfamily. Its interaction with Bcl-2 and Bcl-XL inhibits the function of these two anti-apoptotic molecules to promote apoptosis.1,2 In West Nile virus (WNV)3,4, the human immunodeficiency virus (HIV)5,6, and Hepatitis B and C viruses7, viral-induction of apoptosis are significant contributing factors to disease pathology. It also has been recently reported that influenza viruses initiate and require cellular apoptosis for efficient virus replication, and that the anti-apoptotic Bcl-2 protein negatively effects influenza virus replication.8,9,10 Similarly, a study with the 1918 pandemic virus in macaques indicate up-regulation of cell death and inflammatory related genes11. Without whishing to be bound by theory the results suggest a synergistic role of BAD in the virus' lifecycle by sequestering Bcl-2 and promoting apoptosis.
TNFSF13 and the fusion protein TNFSF12-13 belong to the tumour necrosis factor (TNF) ligand superfamily.12,13 Intergenic splicing between exon 6 of TNFSF12, another member of the TNF superfamily, and exon 2 of TNFSF13 leads to the production of a fusion protein called TNFSF12-13, which displays the same receptor specificity as TNFSF13.12 TNFSF13 and TNFSF12-13 are reported to induce cellular proliferation and be involved in innate and adaptive immune responses.14 TNFSF13 is a proliferation inducing ligand located in the cytosol and reportedly involved in class switch recombination in B cell responses.15 Influenza virus infection has been shown to upregulate TNFSF13 expression in lung tissues and affect recruitment of macrophages to the site of infection in TNFSF13 deficient mice.16
Human MX2 protein has no anti-influenza virus activity described to date; however, it localizes to the nuclear membrane where it may play a role in nucleocytoplasmic trafficking.17,18,19 Without whishing to be bound by theory the results suggest that MX2 has a role in trafficking specific viral proteins or protein complexes between the cytoplasm and the nucleus.20,21
USP47 is a deubiquitinase, whose specific role in cellular processes remains relatively unknown.22 Recently, USP47 has been reported to play a function in scattering responses in epithelial cells.23 Although there are previous reports indicating the importance of the ubiquitin-proteasome pathway in other viruses, the involvement of deubiquitylation in influenza remains relatively unknown.
31 of the 35 gene candidates identified in the Illumina® screen (4 genes had no known functions) were used for bioinformatics interrogation in order to identify host biomolecular interactions that these candidates participated in employing a open source interaction database analysis platform24,25. Computational network analysis demonstrated that several members of the two regulatory pathways Nuclear factor (NF)-κB and the mitogen activated protein kinase (MAPK) pathways were direct interactors of the identified gene candidates. For example, (i) the gene product of MALT-1 interacts with IκBκG (NF-κB essential modulator, NEMO), TRAF-6 (regulator of TLRNF-κB pathway), MAP3K and MAKP9, and TNFAIP3 (a negative regulator of NF-κB), (ii) RPL23 and PTPNI3 directly interacts with NF-κBIA (a regulator of NF-κB), (iii) RPL23 participated in direct interactions with members of the MAPK pathway and TRAF-2 (TRAF-2 is required for the activation of both MAPK/JNK and NF-κB pathways), (iv) Members of MAPK family and AKT, known to phosphorylate NF-κB subunits26 were demonstrated to be direct to interacting protein partner with BAD, (v) 14-3-3 family of proteins, known to be involved in protection against viral infections27, including in AKT signalling pathway28, were found to be direct interacting partners for four of the 31 candidates, BAD, SH3BP4, CFL1 and RPL23 (Table 9). (vi) TNFSF-12-13 (TWE-PRIL) a critical node in the analyses was previously demonstrated to be involved in stimulating lymphocyte proliferation. Further, the 31 identified gene candidates were analyzed for the overrepresentation of transcription factor binding sites. Over represented transcription factors (TFs) were defined if the binding sites for the factors were predicted in the promoter region of at least three of the candidate genes. This analysis revealed 36 transcription factor binding sites to be enriched within the promoter regions of the submitted genes, among which several TFs were known to be active in host responses downstream of the NF-κB and MAPK pathways. Over represented TFs such as MEF2A, AIRE, SRF, CREB1 and IRF1, all are known to be involved in host responses against pathogenic infections, including viral infections29,30,31,32,33,34. This analysis was consistent with the interaction network analysis which also demonstrated the involvement of both NF-κB and MAPK pathways in the activity of the identified candidates. The human genome-wide screen identified genes that play significant roles in protecting host cells from virus-induced cytopathic effect, and also are important in influenza propagation.
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Methods Summary
Human whole-genome screen: A549 cells were transduced at MOI 0.3 with each of 7 Decode RNA GIPZ Lentiviral Positive Screening Library pools according to manufacturer's protocol (Open Biosystems). After 72 h, cells were washed twice with 1× phosphate-buffered saline (PBS) and infected with NY55 at an MOI of 7 PFU per cell. At 72 hpi, cells were washed twice with 1×PBS and harvested. Genomic DNA was isolated by phenol/chloroform extraction followed by ethanol precipitation.
PCR: PCR was carried out on isolated genomic DNA using Expand High Fidelity polymerase mix (Roche) and product was purified from polyacrylamide gels. Pooled cDNA was sequenced by high-throughput Illumina® sequencing technology at Canada's Michael Smith Genome Sciences Centre (Vancouver, British Columbia).
Lentivirus packaging and transduction: Individual human shRNAmir lentiviral clones (Thermo Scientific Open Biosystems; 3-4 for each gene target of interest) were prepared and isolated according to manufacturer's protocol. Individual shRNAs were packaged into lentivirus particles by co-transfection of each shRNAmir with pMD2.G and psPAX2 (Addgene plasmid 12260) in individual sets of HEK-293T cells according to Open Biosystems' Trans-lentiviral Packaging protocol. A549 cells were transduced with lentivirus at an MOI of 0.5. At 72 h post transduction, 3 μg/ml puromycin (Sigma) was added to the media. Cells were passaged twice in puromycin-supplemented completed media to select transductants before they were infected with virus (described below).
siRNA Transfection: Sets of A549 cells were treated with 25 nM of each of 40N-Targetplus siRNA (Dharmacon) targeting each of the USP47, TNFSF12-13, TNFSF13, and BAD genes. siRNAs were introduced into cells with Lipofectamine RNAiMAX (Invitrogen). Each cell set was re-treated with the same siRNA 24 h later. After a further 24 h, cells were infected with virus at an MOI 0.1 and harvested for analysis 48 hpi.
Influenza virus infections: Sets of transduced or transfected A549 cells were infected with influenza virus strains A/NY/55/2004(H3N2) at an MOI of 1 PFU/cell, or with A/PR/8/34(H1N1) at an MOI of 0.01, or with SOIV at an MOI of 0.1 and harvested at 48 hpi for virus titration by plaque assay.
Influenza plaque assay: Influenza plaque assay was carried out on MDCK cells as previously described22.
Bioinformatics Analysis: Sequences were analysed by an in-house computer algorithm. Genes were functionally categorized using PANTHER ontology system23,24. Network analysis were done with InnateDB20 database and visualization employing Cerebral21.
All citations are herein incorporated by reference, as if each individual publication was specifically and individually indicated to be incorporated by reference herein and as though it were fully set forth herein. Citation of references herein is not to be construed nor considered as an admission that such references are prior art to the present invention.
One or more currently preferred embodiments of the invention have been described by way of example. The invention includes all embodiments, modifications and variations substantially as hereinbefore described and with reference to the examples and figures. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims. Examples of such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way.
References
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| 5856445 | Korsmeyer | Jan 1999 | A |
| 6573079 | Palese et al. | Jun 2003 | B1 |
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| Hong et al., Induction of apoptotic death in cells via Bad gene expression by infectious pancreatic necrosis virus infection, 2002, Cell Death and Differentiation, vol. 9, pp. 113-124. |
| Number | Date | Country | |
|---|---|---|---|
| 20120009202 A1 | Jan 2012 | US |
