JP2009526516A - Composition for treating respiratory viral infection and use thereof - Google Patents

Abstract

The present invention provides siRNA compositions that interfere with viral replication in respiratory viral infections, including RS virus and avian influenza A (including H5N1 strain). The present invention further provides the use of siRNA compositions for inhibiting the expression of viral genes in respiratory virus-infected cells and the use of siRNA compositions for use in the treatment of respiratory viral infections in a subject. . In general, the invention provides a polynucleotide comprising a first nucleotide sequence of 15-30 bases targeting the genome of RS virus or influenza A virus, its complement, a double-stranded polynucleotide or a hairpin polynucleotide. Furthermore, the present invention provides vectors, cells and pharmaceutical compositions comprising siRNA sequences.

Description

(Field of Invention)
The present invention relates to siRNA compositions that interfere with viral replication in respiratory viral infections, particularly RS virus and avian influenza A (including H5N1 strains). The present invention further relates to the use of siRNA compositions for inhibiting the expression of viral genes in respiratory virus-infected cells and the use of siRNA compositions for use in the treatment of respiratory viral infections in a subject. .

(Background of the Invention)
Respiratory viral infections have been a significant threat to human health and life for centuries. Infamous episodes include infections caused by influenza strains, RS virus, and severe acute respiratory syndrome (SARS). These include the 1918 global influenza pandemic that killed approximately 2-40 million people worldwide. In more recent ages, other influenza pandemics also existed. SARS occurred in 2002 and killed approximately 800 people (2).

(RS virus)
RS virus (RSV) infection is a major cause of serious pandemic airway disease. About two-thirds of infants are infected with RSV in the first year of life, and about 100% are infected by age two. Currently, there are no specific and effective therapeutic agents available for treating RSV infection.

  RS virus (RSV) is an enveloped, non-segmented single-stranded negative RNA virus (NNR), belonging to the genus Paramyxoviradae, Mononegaviruses (14, 29) . Paramyxoviruses share the following characteristics: 1) They have a single-stranded RNA genome that is tightly encapsulated by the viral nucleocapsid protein (N) (29, 30). 2) Subgenomic mRNA is transcribed from the normal genome by RdRP. 3) Viral replication occurs in the cytoplasm of the host cell. The details of the RSV life cycle from infection to the release of offspring virions are well studied (15).

  The RSV genome is a 15.5 kb long negative strand containing 3'-NS1, NS2, N, P, M, SH, G, F, M2, and L-5 'genes (see Figure 1). The products of these seven genes (ie, N, P, M, SH, G, F, and L) are in common with other paramyxoviruses 3. Several viral or host factors are involved in the regulation of RNA transcription and replication (20). In addition, there are viral cis-acting signals that play a regulatory role in mRNA transcription and RNA replication (3).

  The incubation period for RSV infection is about 4-5 days. RSV first affects the nasopharynx, then reaches the bronchi and bronchioles within a few days, and infection is confined to the surface layer of the respiratory epithelium.

(Influenza A)
At the beginning of 1997, H5N1, a new strain of avian influenza A, appeared. Although mostly confined to birds (both wild populations and poultry), the virus appeared to infect only humans in direct contact with infected birds. In humans, infection causes serious illness and leads to severe respiratory illness and death in humans (3-12). Many cases and outbreaks occurred in various countries in Southeast Asia. Given the ability of this avian virus to infect humans, there is an increased risk of mutation to a contact infectious human variant, which is due to efficient and continuous human-to-human transmission. It poses the emergence of a new influenza pandemic as well as many deaths.

  Since avian influenza H5N1 is a newly emerging infectious agent associated with pneumonia, its pathology and mechanism are less clear and there is a specific and effective treatment for H5N1 avian influenza in human disease cases do not do. Currently, influenza infections include two antiviral drugs, such as the neuraminidase inhibitor class of drugs: oseltamivir (known as Tamiflu in the market) and zanamivir (known as Relenza in the market), or older M2 inhibitors Is treated with amantadine and rimantadine.

  H5N1 is a subtype of influenza virus type A. Since this virus is an enveloped, fragmented, negative single-stranded RNA virus, it belongs to the Orthomyxoviridae family. During the life cycle of influenza A virus (including H5N1), viral genomic RNA (vRNA) serves as a template for complementary RNA (cRNA) production, which also serves for messenger RNA (mRNA) production. Work as a template. Each of these three forms of RNA molecules that occur during viral replication can all be targeted for siRNA-mediated degradation using either sense or antisense siRNA. Influenza A genome (consisting of 8 separate RNA segments and containing at least 10 open reading frames (ORFs)) as a template for both viral genome replication and subgenomic or gene-directed mRNA synthesis work. FIG. 2 shows a schematic diagram representing the structure of the influenza A virion. Polymerases PB2, PB1 (polymerase basic protein 1 and polymerase basic protein 2) and PA (polymerase acidic protein) were encoded by RNA1, RNA2 and RNA3, respectively. Four viral structural proteins, H (hemagglutinin), N (neuraminidase), M1 and M2 (matrix protein 1 and matrix protein 2) are encoded by RNA segment 4, RNA segment 6 and RNA segment 7, respectively. Whereas RNA5 encodes NP (nucleocapsid protein) and RNA8 encodes NS1 and NS2 (nonstructural protein 1 and nonstructural protein 2).

(RNA interference)
RNA interference (RNAi) is a sequence-specific RNA degradation process that theoretically provides a relatively easy and direct way to knock down or silence any gene (17, 18 19). In naturally occurring RNA interference, double-stranded RNA is cleaved into small interfering RNA (siRNA) molecules by Dicer, an RNase III / helicase protein. The siRNA molecule is a 19-23 nucleotide dsRNA with a 2 nucleotide (nt) overhang at the 3 'end. These siRNAs are incorporated into a multi-component ribonuclease called RNA-induced-silencing-complex (RISC). One strand of the siRNA remains bound to the RISC, leading this complex to a cognate RNA, which is directed against this guider ss-siRNA in the RISC. Sequence that is complementary to each other. The siRNA-directed endonuclease digests the RNA and thereby inactivates the RNA. Recent studies have shown that the use of chemically synthesized 21-25-nt siRNAs shows the action of RNAi in mammalian cells (20), and siRNA hybridization (terminal or central). Thermodynamic stability (in) has been shown to play a central role in determining the function of the molecule (21, 22). These and other features of RISC, siRNA molecules and RNAi have been described (23-28).

  Laboratory or potentially therapeutic uses of RNAi in mammalian cells use either chemically synthesized siRNAs or endogenously expressed molecules (2). Non-Patent Document 2 (21)). Endogenous siRNA is first expressed as a small hairpin RNA (shRNA) by an expression vector (plasmid or viral vector) and then processed into siRNA by Dicer. siRNA is considered to have great promise as a therapeutic agent for human diseases caused by viral infections in particular (Non-patent Document 3 (19), Non-patent Document 4 (20), 27-30).

Importantly, any of the many potential candidate siRNA sequences that currently target viral genome sequences (eg, about 16-30 base pair oligonucleotides) are actually effective siRNAs. It is impossible to predict with certainty whether activity will be exhibited. Instead, individual specific candidate siRNA polynucleotide sequences or specific candidate siRNA oligonucleotide sequences are generated and tested to determine whether the intended interference with the expression of the targeted gene occurs. There must be. Thus, there is no conventional method in the art to confidently design siRNA polynucleotides that allow specific modification of the expression of a given mRNA.
Abbott, A.M. Nature (2003) 424: 121-123 Reynolds, A.R. Nat. Biotechnology (2004) 22: 326-330 Schiwarz, D.C. S. Cell (2003) 115: 199-208. Khvorova, A .; Cell (2003) 115: 209-216.

  There continues to be a great need for compositions and methods that inhibit the expression of viral pathogen genes and their cognate protein products. In particular, there is an urgent need for compositions and methods for inhibiting the expression of pathogenic respiratory viral genes in virally infected cells, and compositions and methods for treating respiratory viral infections in subjects. To do. Furthermore, there is a further need for compositions and methods that address infection by RSV and avian influenza A (especially the H5N1 strain). There is a further need for compositions and methods for treatments that are highly effective and do not rely on the use of known anti-viral agents or modifications of known anti-viral agents. The present invention addresses these and the associated demands.

(Summary of the Invention)
The present invention relates to the use of RNA interference to inhibit viral infection and replication through disruption of viral RNA molecules of viral pathogens (eg, viral pathogens that cause respiratory viral infections, including influenza A H5N1 and RSV). Compositions and methods are provided. These viruses are pathogens that cause severe respiratory diseases in humans and other mammals. Inhibition of viral replication combats viral infection in cultured cells infected with the virus as well as subjects infected with the virus. This inhibition of viral replication includes a reduction in its symptoms.

  In a first aspect, the present invention provides an isolated polynucleotide that can be any number of nucleotides of 200 or less in length and 15 or more. The polynucleotide comprises a first nucleotide sequence that targets the genome of a sucking syncytial virus or influenza A virus. In this polynucleotide, any T (thymidine) or any U (uridine) can be substituted with others as necessary. Further, in this polynucleotide, the first nucleotide sequence consists of (a) a sequence that is any number of nucleotides 15 to 30 in length, or (b) the complement of the sequence given in (a) . Such a polynucleotide may be referred to herein as a linear polynucleotide.

In a related aspect of the invention, the polynucleotide further comprises a second nucleotide sequence that is separated from the first nucleotide sequence by a loop sequence, the second nucleotide sequence comprising:
(A) has substantially the same length as the first nucleotide sequence, and (b) is substantially complementary to the first nucleotide sequence. In this latter structure (referred to as a hairpin polynucleotide), the first nucleotide sequence hybridizes with the second nucleotide sequence, and the complementary sequence is linked by the loop sequence. Form.

In many embodiments of the linear polynucleotide and in many embodiments of the hairpin polynucleotide, the first nucleotide sequence is one of the following:
(A) a sequence selected from SEQ ID NOs: 1 to 263,
(B) a targeting sequence longer than the sequence given in item (a), the targeting sequence targeting the genome of respiratory viruses and selected from SEQ ID NO: 1-263 including,
(C) a fragment of a sequence selected from SEQ ID NOs: 1-263, wherein the fragment is at least 15 nucleotides in length and from a sequence of consecutive bases that is at least one base shorter than the selected sequence Become a fragment,
(D) a sequence that differs from the sequence selected from SEQ ID NO: 1-263 by 5 nucleotides, or (e) the complement of any sequence given in (a)-(d).

  In various embodiments of linear or hairpin polynucleotides, the length of the first nucleotide sequence is any number of 21-25 nucleotides. In many embodiments, the linear polynucleotide or hairpin polynucleotide consists of a sequence selected from SEQ ID NOs: 1-263, and optionally a dinucleotide overbinding that binds 3 ′ of the selected sequence. Includes hangs. In still further embodiments of the linear polynucleotide or hairpin polynucleotide, the dinucleotide sequence at the 3 ′ end of the first nucleotide sequence is TT, TU, UT, or UU, and ribonucleotides or deoxyribonucleotides Includes either or both. In various further embodiments, the linear polynucleotide or hairpin polynucleotide may be DNA, or it may be RNA, or it may be composed of both deoxyribonucleotides and ribonucleotides. Good.

  In a further aspect, the present invention provides a double-stranded polynucleotide. The double-stranded polynucleotide is complementary to and hybridizes to the first polynucleotide strand of claim 1 and at least the first nucleotide sequence of the first strand. A second polynucleotide strand that forms the strand composition. These polynucleotide structures can also be referred to as linear polynucleotides.

  In yet a further aspect, the present invention is a combination comprising two or more target polynucleotides according to claim 1, claim 2 or both, wherein each polynucleotide of the combination comprises a target virus Provide combinations that target different sequences in the genome.

  Due to the high degree of similarity and identity to respiratory viral pathogen targets and not wishing to be bound by theory, upon introduction into a virus-infected cell, this polynucleotide is Leading to the digestion of pathogen genomic RNA, complementary RNA and messenger RNA. In particular, in important embodiments of these aspects of the invention, the first nucleotide sequence in these polynucleotides or its complement forms an RNA-induced silencing complex (RISC), wherein the RISC is It is believed that a polynucleotide siRNA sequence is derived into a pathogen genomic RNA sequence, thereby facilitating cleavage of the pathogen genomic RNA.

  In a further aspect, the present invention provides a vector having a sequence provided by a linear polynucleotide or hairpin polynucleotide of the present invention. In various embodiments, any of these vectors can be a plasmid, a recombinant virus, a transposon, or a microchromosome. A still further aspect provides a cell transfected with one or more linear polynucleotides of the present invention, or one or more hairpin polynucleotides of the present invention.

  In yet a further aspect, the present invention provides a pharmaceutical composition comprising one or more linear or hairpin polynucleotides or mixtures thereof, and a pharmaceutically acceptable carrier, wherein Thus, each polynucleotide targets a different sequence in the genome of the target virus.

  In yet a further aspect, the present invention relates to a pharmaceutical comprising one or more vectors having a linear polynucleotide, a vector having a hairpin polynucleotide, or a mixture thereof, and a pharmaceutically acceptable carrier. A composition is provided wherein each vector has a polynucleotide that targets a different sequence in the genome of the target virus.

  In various embodiments of the pharmaceutical composition, the carrier includes a synthetic cationic polymer, a liposome, dextrose, a surfactant, or any combination of two or more thereof.

In yet a further aspect, the present invention provides a method of synthesizing a linear or hairpin polynucleotide having a sequence that targets the genome of RS virus or influenza A virus. This method includes the following steps:
(A) providing a nucleotide reagent comprising a reactive end and corresponding to a nucleotide at the first end of the sequence;
(B) additional nucleotide reagents, including reactive ends and corresponding to positions following the sequence, are added to react with the reactive ends of step (a) above, and the length of the growing polynucleotide sequence Adding 1 nucleotide, and removing unwanted products and excess reagents, and (c) adding the nucleotide reagent corresponding to the nucleotide at the second end of the sequence, whereby the complete polynucleotide is Repeating step (b) until provided.

  In yet a further aspect, the invention provides a method for transfecting a cell with an RNA inhibitor, wherein the method comprises transfecting the cell with one or more linear polynucleotides, or 1 Contacting with a composition containing a species or more hairpin polynucleotides. In many embodiments, cells transfected in this manner include respiratory viruses that are targeted by one or more polynucleotides.

  In yet a further aspect, the present invention provides a method of inhibiting replication of this virus in a cell infected with a respiratory virus, wherein the method comprises treating the cell with one or more linear polynucleotides, Or including contacting with a composition containing one or more hairpin polynucleotides, wherein the one or more polynucleotides target the virus.

  In yet a further aspect, the present invention provides a linear polynucleotide that targets this respiratory virus in the manufacture of a pharmaceutical composition effective for treating an infection caused by a respiratory virus in a subject, or these There is provided the use of two or more mixtures, or the use of a hairpin polynucleotide or a mixture of two or more of these that target this respiratory virus. In various embodiments of this use, the subject is a human.

  In yet a further aspect, the present invention provides a method of treating an infection resulting from a respiratory virus in a subject. This method involves the use of an effective dose of a linear polynucleotide targeting respiratory viruses or a mixture of two or more thereof, or a hairpin polynucleotide targeting respiratory viruses or two or more thereof. The method includes administering an effective dose of a number of mixtures to the subject. In various embodiments of this method, the subject is a human. In further embodiments, both linear polynucleotides and hairpin polynucleotides are administered to the subject.

(Detailed description of the invention)
All patents, patent application publications, and patent publications mentioned in this specification are hereby incorporated by reference in their entirety as if set forth verbatim in this specification. All technical publications mentioned herein are also incorporated by reference.

  In this specification, the articles “a”, “an”, and “the” represent equivalent meanings as a singular or plural. The specific meaning of these articles is clear from the context in which they are used.

  As used herein, the term “target” sequence and similar terms and phrases refer to a nucleotide sequence present in the genome of a pathogen to which a polynucleotide of the invention is directed. The polynucleotide comprises a pathogen sequence comprising (a) a sequence that is homologous or identical to a particular subsequence (referred to as a target sequence) contained within the genome of this pathogen, or (b) complement Is targeted either by containing a sequence that is homologous or identical to the target sequence. Any polynucleotide that targets a pathogen sequence has the ability to hybridize with the target sequence according to the RNA interference phenomenon, thereby initiating RNA interference.

  As used herein, the terms “complement”, “complementarity”, and similar terms and phrases relate to two sequences in which the base is base-to-base and forms a complementary base pair. As previously understood by those skilled in the art, such as biochemistry, molecular biology, genetics and similar fields related to the field of the present invention.

  As used herein, if a first sequence or subsequence has the same base at all positions in this sequence or subsequence as the second sequence or subsequence, the first sequence or subsequence A sequence is described by a term or phrase that is “identical”, has “100% identity”, or conveys the concept of 100% identity to a second or subsequence. In determining identity, any particular base position containing T (thymidine) or any derivative thereof or U (uridine) or any derivative thereof is considered equivalent to each other and therefore identical.

  As described herein, the sequence of the target polynucleotide, or its complement, may be completely identical to the target sequence, or it is mismatched at a particular position in the target sequence ( a mismatched base. Incorporation of mismatches is fully described herein. Without wishing to be bound by theory, mismatch incorporation provides the intended degree of hybridization stability under physiological conditions that optimizes the RNA interference phenomenon for the particular target sequence of interest. To do. The degree of identity determines the percentage of positions where the bases in the two sequences are identical to each other. “Percent sequence identity” is a comparison of two optimally aligned sequences over a region of comparison and the same nucleobase in both sequences (eg, A, T or U, C, G, Or I) to determine the number of positions present to obtain the number of matched positions, divide the number of matched positions by the total number of positions in the region of comparison (ie, the window size) and 100 Multiplied by to get the percentage of sequence identity. Sequences that are less than 100% identical to each other are “similar” or “homologous” to each other; the degree of homology or percent identity is between two sequences or subsequences as determined in the following sections Is a synonym, meaning the percentage of identity. For example, at least 60% identity to each other, or preferably at least 65% identity, or preferably at least 70% identity, or preferably at least 75% identity, or preferably at least 80% identity Or two sequences that exhibit at least 85% identity, or more preferably at least 90% identity, or even more preferably at least 95% identity, are “similar” to each other, or “Homologous”. Alternatively, with respect to the oligonucleotide sequence of the siRNA molecule, 5 or fewer bases, or 4 or fewer bases, or 3 or fewer bases, or 2 or fewer bases, or two sequences that differ by one base are , “Similar” or “homologous” to each other.

  “Identity” is an association between two or more polypeptide sequences or two or more polynucleotide sequences, as is known in the art, and is determined by comparing the sequences. Is done. In the art, “identity” also refers to the association of a sequence between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between the strings of such sequences. Also means degree. “Identity” and “similarity” can be readily calculated by known methods. These methods include, but are not limited to, the methods described below: Computational Molecular Biology, Lesk. A. M.M. , Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D .; W. Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I. Griffin, A.M. M.M. , And Griffin, H .; G. Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G .; , Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M .; And Devereux, J. et al. , Hen, M Stockton Press. New York, 1991; and Carillo, H .; , And Lipman, D .; , SIAM J. et al. Applied Math. (1988) 48: 1073. Preferred methods for determining identity are designed to give the greatest match between the sequences tested. The methods for determining identity and similarity are summarized in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to: GCG program package (Devercux, J., et al. (1984) Nucleic Acids Research 12 (1): 387), BLASTP, BLASTN, and FASTA (Atschul, SF et al. (1990) J. Molec. Biol. 215: 403-410). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al. (1990) J. MoI. Mol.Biol.215: 403-410 The well-known Smith Waterman algorithm can also be used to determine identity.

  Parameters for sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Mol Biol. 48: 443-453 (1970).

  Comparison Matrix: Hentikooff and Hentikoff, (1992) Proc. Natl. Acad. Sci. USA. 89: BLOSSSUM62 from 10915-10919.

  As used herein, the term “isolated”, and similar terms, when used to describe a nucleic acid, polynucleotide, or oligonucleotide, are in their native or original state. It is taken out from. Thus, if they exist in nature, they have been removed from their original environment. If they were prepared synthetically, they have been removed from the original product mixture obtained from the synthesis. For example, as the term is used herein, a naturally occurring polynucleotide that is naturally present in a living organism in its natural state is not “isolated” The same polynucleotide that has been separated from the material with which it is present in nature is “isolated”. In general, removal of at least one significant co-existing substance constitutes “isolation” of a nucleic acid, polynucleotide, oligonucleotide. In many cases, some, many, or most of the co-existing material can be removed to isolate the nucleic acid, polynucleotide, oligonucleotide, protein, polypeptide, or oligopeptide. As a non-limiting example, with respect to a polynucleotide, the term “isolated” can mean that the polynucleotide is separated from naturally occurring chromosomes and cells. Further, by way of example, “isolation” of a protein or polypeptide can mean separation from another component in a cell lysate or cell homogenate.

  A nucleic acid, polynucleotide, or oligonucleotide that is the product of an in vitro or chemical synthesis process is essentially isolated as a result of the synthesis process. In important embodiments, such synthetic products are treated to remove the reagents and precursors used, and by-products from the process.

  Similarly, polynucleotides and polypeptides are present in compositions such as, for example, formulations, compositions for introduction of polynucleotides into cells, compositions or solutions for chemical or enzymatic reactions. obtain. These compositions are not naturally occurring compositions, but maintain within them isolated polynucleotides or polypeptides that are within the meaning of the terms used herein.

  As used herein and in the claims, “nucleic acid” or “polynucleotide”, and similar terms based thereon, refer to polymers consisting of naturally occurring nucleotides and polymers consisting of synthetic or modified nucleotides. Say. Thus, as used herein, a polynucleotide that is RNA or a polynucleotide that is DNA can include naturally occurring moieties (eg, naturally occurring bases and ribose or deoxyribose rings). Or they can be composed of synthetic or modified moieties as described below. The linkage between nucleotides is generally a 3'-5 'phosphate linkage, whether it is a natural phosphodiester linkage, a phosphothioester linkage, or still other synthetic linkages. Good. Examples of modified backbones include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates (3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates). ), Phosphinates, phosphoramidates (including 3′-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates And boranophosphate. Additional linkages include phosphotriesters, siloxanes, carbonates, carboxymethyl esters, acetamidates, carbamates, thioethers, cross-linked phosphoramidates, cross-linked methylene phosphonates, cross-linked phosphorothioates and sulfone intranucleotide linkages. Can be mentioned. Other polymeric linkages include these 2'-5 'linked analogs. See US Pat. Nos. 6,503,754 and 6,506,735 and references cited therein, which are incorporated herein by reference.

  Nucleic acids and polynucleotides are 20 or more nucleotides in length, or 30 or more nucleotides in length, or 50 or more nucleotides in length, or 100 or more in length, or It is 1000 or more, or 10,000 or more, or 100,000 or more in length. The siRNA can be a polynucleotide as defined herein. As used herein, “oligonucleotide” and similar terms based thereon are derived from short polymers of naturally occurring nucleotides, as well as synthetic or modified nucleotides, as described in the immediately preceding paragraph. Is a polymer. Oligonucleotides are 10 or more nucleotides in length, or 15, or 16, or 17, or 18, or 19, or 20 or more nucleotides in length, or 21, or 22, or 23 Or 24 or more nucleotides in length, or 25, or 26, or 27, or 28 or 29, or 30 or more nucleotides in length, 35 or more, 40 or more 45 or more and up to about 50 nucleotides in length. Oligonucleotides that are siRNAs can have any number of nucleotides between 15 and 30. In many embodiments, the siRNA can have any number of nucleotides between 21 and 25.

  It is understood from the definitions given above that due to overlapping size ranges, the terms “polynucleotide” and “oligonucleotide” can be used similarly herein to refer to the siRNA of the invention. Is done.

  As used herein and in the claims, “nucleotide sequence”, “oligonucleotide sequence” or “polynucleotide sequence”, and similar terms include the sequence of bases an oligonucleotide or polynucleotide has, Both are interchangeably related to the structure of an oligonucleotide or polynucleotide having this sequence. The nucleotide sequence or polynucleotide sequence further relates to any natural polynucleotide or oligonucleotide, or any synthetic polynucleotide or oligonucleotide, in which the sequence of bases is defined in the art. , As conventionally used in, defined by the description or reference of the specific sequence of letters indicating the base.

  Bases in oligonucleotides and polynucleotides include “unmodified” bases, including purine bases (adenine (A) and guanine (G)) and pyrimidine bases (thymidine (T), cytosine (C) and uracil (U)). It can be a “natural” base. In addition, these can be bases with modifications or substitutions. As used herein, non-limiting examples of modified bases include other synthetic bases and natural bases. These bases include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and 6-methyl derivatives of guanine and other alkyl derivatives, adenine 2-propyl derivatives of guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymidine and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine and other alkynyl derivatives of the pyrimidine base 6-azouracil, 6-azocytosine and 6-azothymidine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adducts And guanine, 5-halo (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-fluoro-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido [5,4-b] [1,4] benzoxazin-2 (3H) -one), phenothiazine cytidine (1-pyrimido [5 , 4-b] [1,4] benzothiazin-2 (3H) -one)), G-clamp (eg substituted phenoxazine cytidine (eg 9- (2-aminoethoxy) -H-pyrimido [ 5,4-b] [1,4] benzoxazin-2 (3H) -one), carbazole cytidine (2H-pyrimido [4,5-b] indol-2-one), pyridoindole cytidine (H-pyrido [ 3 ′, 2 ′: 4,5] pyrrolo [2,3-d] pyrimidin-2-one)). Modified bases also include modified bases (eg, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone) in which purine or pyrimidine bases are replaced with other heterocycles. Additional bases include those disclosed in U.S. Pat. No. 3,687,808, The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. et al. L, John Wiley & Sons, 1990, the base disclosed by Englisch et al., Angelwandte Chemie, International Edition (1991) 30, 613, and Sanghvi, Y. et al. S. , Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. et al. T.A. And Lebleu, B .; , Ed., CRC Press, 1993. Certain of these bases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines and O-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine) Is mentioned. 5-Methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2 ° C (Sangvi, YS, Crooke, ST, and Lebleu, B. et al. , Hen, Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), where preferred base substitutions, and even more preferred when combined with 2'-O-methoxyethyl sugar modification It is. See US Pat. Nos. 6,503,754 and 6,506,735 and references cited therein, which are hereby incorporated by reference. The use of any modified base is equivalent to the use of a naturally occurring base with the same base-pairing properties, as will be appreciated by those skilled in the art.

  As used herein and in the claims, the term “complementary” and similar terms based thereon include the nucleic acid of the first nucleobase in one strand of a nucleic acid, polynucleotide or oligonucleotide, The ability to specifically interact only with a specific second nucleobase in one strand of a polynucleotide or oligonucleotide. As a non-limiting example, when a naturally occurring base is considered, A and T or U interact with each other and G and C interact with each other. As used in the present invention and claims, “complementary” means “fully complementary”, ie one strand when two polynucleotide strands are juxtaposed to each other. There is at least a portion of the strand in which each base in the sequence of consecutive bases in is complementary to the interacting base in the sequence of consecutive bases of the same length in the opposite strand.

  As used herein, “hybridize”, “hybridization” and like terms are used to refer to nucleic acids, polynucleotides, or oligonucleotides by interacting with each other a strand and a complementary sequence. This refers to the process of forming a chain. This interaction occurs when complementary bases in each strand interact specifically to form a pair. The ability of the strands to hybridize to each other depends on various conditions as indicated below. Nucleic acid strands hybridize to each other if a sufficient number of corresponding positions in each strand are occupied by nucleotides that can interact with each other. It is understood by those skilled in the art of the present invention (as non-limiting examples, that the sequences of the strands forming the duplex do not require 100% complementarity with each other in order to be able to hybridize specifically. Molecular biologists and cell biologists).

  As used herein, “fragment” and similar terms based thereon relate to a portion of a nucleic acid, polynucleotide or oligonucleotide that is shorter than the entire reference sequence. The sequence of the bases in the fragment is not altered from the sequence of the corresponding part of the molecule in which this fragment occurs; there is no insertion or deletion in the fragment compared to the sequence of the corresponding part of the molecule in which this fragment occurs . As contemplated herein, a nucleic acid or polynucleotide fragment (eg, an oligonucleotide) is 15 or more bases in length, or 16 or more, 17 or more, 18 Or more, or 19 or more, or 20 or more, or 21 or more, or 22 or more, or 23 or more, or 24 or more, or 25 or More, or 26 or more, or 27 or more, or 28 or more, or 29 or more, 30 or more, 50 or more, 75 or more, 100 too Ku is the length of more bases in length up to 1 base shorter in length than the full-length sequence. Oligonucleotides can be chemically synthesized and used as siRNA, PCR primers, or probes.

  Detection and labeling. Targeting polynucleotides, such as polynucleotides containing siRNA sequences, as well as viral polynucleotide targets can be detected by a number of means. Detection can include one or more of any process that provides the ability to observe the presence or amount of the target polynucleotide. In one embodiment, the sample nucleic acid containing the targeting polynucleotide or viral target can be detected prior to expansion. In an alternative embodiment, the targeting polynucleotide in the sample can be expanded to provide an expanded targeting polynucleotide or an expanded viral target, and is this expanded polynucleotide detected? Alternatively, it can be quantified. Physical, chemical or biological methods can be used to detect and quantify the target polynucleotide. Physical methods include, as non-limiting examples, surface plasmon resonance (SPR) detection (eg, binding of the probe to the surface) and use of the SPR to detect binding of the target polynucleotide to the immobilized probe. Or having the probe in a chromatographic medium and detecting binding of the target polynucleotide in the chromatographic medium. The physical method further includes a gel electrophoresis format or a capillary electrophoresis format in which the target polynucleotide is separated from other polynucleotides and the separated polynucleotide is detected. Chemical methods include polymerase chain reaction (PCR) methods and hybridization methods, where the target polynucleotide generally hybridizes to the probe. Biological methods include causing a target polynucleotide or target polynucleotide to exert a biological effect in a cell and detect this effect. The present invention discloses examples of biological effects that can be used in biological assays. These include virion counting by counting particles, plaque assay, evaluation of cytopathic effects in infected cells, and the like. In many embodiments, the polynucleotide is labeled as described below to aid detection and quantification. For example, in embodiments that do not include expansion, the sample nucleic acid can be labeled by chemical or enzymatic addition of a labeling moiety such as a labeled nucleotide or a labeled oligonucleotide linker.

  Extended polynucleotides can be detected directly and / or quantified. For example, the expanded polynucleotide can be subjected to electrophoresis in a gel (separated by size) and stained with a dye to reveal its presence and amount. Alternatively, the expanded target polynucleotide can be detected upon exposure of the probe nucleic acid under hybridization conditions (see below), and binding by hybridization is detected and / or quantified. Detection is accomplished by any means that allows the determination that the target polynucleotide is bound to the probe. This can be accomplished by detecting changes in the physical properties of the probe caused by hybridizing the fragments. A non-limiting example of such a physical detection method is SPR.

  An alternative way of achieving detection is to use this extended labeled form of the polynucleotide and to detect the bound label. The label can be a radioisotope label such as, for example, 125I, 35S, 32P, 14C, or 3H, which can be detected by its radioactivity. Alternatively, the label can be selected such that it can be detected using spectroscopic methods (eg, by fluorescence, phosphorescence or chemiluminescence). Thus, fluorescent or phosphorescent labels or labels that induce chemiluminescent reactions can be used. The label can still be a ligand in a specific ligand-receptor pair; the presence of this ligand is then detected by secondary binding of a specific receptor that is normally itself labeled for detection. .

(Interfering RNA)
In accordance with the present invention, gene expression of respiratory viral targets is reduced by RNA interference. The expression product of the viral gene is targeted by a specific double stranded siRNA nucleotide sequence that is complementary to at least one segment of the viral target, which segment can be any number between 15 and 30. Contains nucleotides, or often contains any number of nucleotides between 21 and 25. This target may be present in the 5 ′ untranslated (UT) region, in the coding sequence, or in the 3 ′ UT region. For example, PCT applications WO00 / 44895, WO99 / 32619, WO01 / 75164, WO01 / 92513, WO01 / 29058, WO01 / 89304, WO02 / 16620, and WO02 / 29858 (each of which is herein incorporated by reference in its entirety) See incorporated).

  In accordance with the method of the present invention, respiratory viral gene expression and thereby respiratory viral replication is suppressed using siRNA. Targeting polynucleotides according to the present invention include siRNA oligonucleotides. Such siRNAs can also be prepared by chemical synthesis of nucleotide sequences that are the same or similar to viral sequences. See, for example, Tuschl, Zamore, Lehmann, Bartel and Sharp (1999), Genes & Dev. 13: 3191-3197 (incorporated herein by reference in its entirety). Alternatively, the targeted siRNA can be obtained using a target polynucleotide sequence, for example, by digesting a respiratory viral ribopolynucleotide sequence in a cell-free system (such as, but not limited to, Drosophila extract) or It can be obtained by transcription of a replacement double-stranded viral cRNA.

  Efficient silencing is generally observed with siRNA duplexes composed of 16-30 nt sense strands and 16-30 nt antisense strands of the same length. In many embodiments, each strand of the siRNA pairing duplex has a 2-nt overhang addition at the 3 'end. The sequence of the 2-nt 3 'overhang further contributes slightly to the specificity of siRNA target recognition. In one embodiment, the nucleotide in the 3 'overhang is a ribonucleotide. In an alternative embodiment, the nucleotide in the 3 'overhang is a deoxyribonucleotide. The use of 3 'deoxynucleotides provides enhanced intracellular stability.

  The recombinant expression vectors of the invention are processed to provide RNA comprising an siRNA sequence that targets a respiratory virus when introduced into a cell. Such vectors are DNA molecules cloned into an expression vector containing operably linked control sequences that are flanked by viral targeting sequences in a manner that allows expression. From this vector, an RNA molecule that is antisense to viral RNA is transcribed by a first promoter (eg, the 3 ′ promoter sequence of the cloned DNA) and is an sense molecule for the viral RNA target. Is transcribed by a second promoter (eg, the 5 ′ promoter sequence of the cloned DNA). The sense and antisense strands then hybridize in vivo to produce siRNA constructs that target the virus for silencing of respiratory viral genes. Alternatively, two constructs can be used to create the sense and antisense strands of the siRNA construct. In addition, the cloned DNA can encode a transcript with secondary structure, where one transcript has both a sense sequence and a complementary antisense sequence from the target gene. In the example of this embodiment, the hairpin RNAi product is similar to all or part of the target gene. In another example, the hairpin RNAi product is an siRNA. The regulatory sequences that are flanking the viral sequence may be the same or different, so that their expression may be regulated independently or regulated in a temporal or spatial manner. May be.

  In certain embodiments, siRNAs are produced intracellularly, for example, by cloning a viral gene template into a vector comprising RNA pol III transcription units from smaller nuclear RNA (snRNA) U6 or human RNase P RNA H1. Transcribed. One example of a vector system is the GeneSuppressor ™ RNA interference kit (commercially available from Imgenex). The U6 promoter and the H1 promoter are members of the type III class of the Pol III promoter. The +1 nucleotide of the U6-like promoter is always guanosine, while the +1 for the H1 promoter is adenosine. Termination signals for these promoters are defined by five consecutive thymidines. This transcript is typically cleaved after the second uridine. Cleavage at this position produces a 3 'UU overhang in the expressed siRNA, which is similar to the 3' overhang of the synthetic siRNA. Since any sequence less than 400 nucleotides in length can be transcribed by these promoters, they are ideally suited for expression of, for example, about 21 nucleotides siRNA in an RNA stem-loop transcript of about 50 nucleotides. . Factors affecting the characteristics of RNAi and the efficacy of siRNA have been studied (eg, Elbashir, Lendeckel and Tuschl (2001) Genes & Dev. 15: 188-200).

  An initial BLAST homology search for the selected siRNA sequences is performed on the available nucleotide sequence library to confirm that only viral genes are targeted and host genes are not targeted. Elbashir et al., 2001 EMBO J. et al. 20 (23): 6877-88.

(Synthesis of polynucleotide)
An oligonucleotide corresponding to the target nucleotide sequence and a polynucleotide comprising the target sequence can be prepared by standard synthetic techniques, eg, using an automated DNA synthesizer. Methods for synthesizing oligonucleotides include well-known chemical processes, including the sequential addition of nucleotide phosphoramidites to surface derivatized particles (T, Brown and Dorcas J. S. Brown, Oligonucleotides and Analogues A Practical Approach, F. Eckstein, Ed., Oxford University Press, Oxford, pp. 1-24 (1991), which is incorporated herein by reference), but is not limited thereto.

  An example of a synthetic procedure uses Expedite RNA phosphoramidites and thymidine phosphoramidites (Proligo, Germany). Synthetic oligonucleotides are deprotected and gel purified (Elbashir et al. (2001) Genes & Dev. 15, 188-200) followed by purification on Sep-Pak C18 cartridges (Waters, Milford, Mass., USA) ( Tuschl et al. (1993) Biochemistry, 32: 11658-11668). Complementary ssRNA was incubated in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH (pH 7.4), 2 mM magnesium acetate) for 1 minute at 90 ° C. and then incubated for 1 hour at 37 ° C. Hybridize with ds-siRNA.

  Other methods of oligonucleotide synthesis include, but are not limited to: the phosphotriester method and the phosphodiester method (Narang et al. (1979) Meth. Enzymol. 68:90) and the H-phosphonate method ( In particular, Garegg, PJ et al., (1985) “Formation of intermediate bonds via phosphate intermediates”, Chem. Scripta 25, 280-282; and Froehler, B. C, et al. (1986). -Phospho intermediates ", Nucleic Acid Res , 14, 5399-5407), and on-support synthesis (Beaucage et al. (1981) Tetrahedron Letters 22: 1859-1862), as well as phosphoramidate technology (Caruthers, MH, et al. , “Methods in Enzymology”, Vol. 154, pp. 287-314 (1988)), US Pat. Nos. 5,153,319, 5,132,418, 4,500,707, 4,458,066, 4,973,679, 4,668,777, and 4,415,732 and “Synthesis and Applications of DNA and RNA”, S.A. A. Narang, Ed., Academic Press, New York, 1987, and the techniques described in the references contained therein, as well as non-phosphoramidite techniques. Solid phase synthesis helps isolate the oligonucleotide from impurities and excess reagents. Once cleaved from the solid support, the oligonucleotide can be further isolated by known techniques.

(Inhibitory polynucleotide of the present invention)
The present invention broadly provides oligonucleotides that are intended to cause RNA interference events when entering cells infected with respiratory viral pathogens. The present invention is not limited to the nature of respiratory virus targets, but emphasizes oligonucleotides that target several strains of RSV and influenza A. RNA interference occurs in cells with appropriate double stranded RNA, one of which is identical or highly similar to the sequence in the viral target gene. In general, oligonucleotides that target respiratory viruses can be DNA or RNA, or can include a mixture of ribonucleotides and deoxyribonucleotides. An example of the latter is an RNA sequence that terminates at the 3 ′ end with a deoxydinucleotide sequence (eg, d (TT), d (UU), d (TU), d (UT) and other possible dinucleotides). It is. In further embodiments, the 3 ′ overhang may be composed of ribonucleotides and the like having the bases specified above. Further, the oligonucleotide agent can be single stranded or double stranded. Some embodiments of the therapeutic oligonucleotides of the invention are expected to be oligoribonucleotides or oligoribonucleotides having a 3′d (TT) terminus. Single-stranded targeting polynucleotides are readily converted to double-stranded molecules upon entry into mammalian cells by endogenous enzyme activity present in the cells.

  Most generally, the present invention provides oligonucleotides or polynucleotides, which can range anywhere from 15 to 200 nucleotides in length. The polynucleotide comprises a first nucleotide sequence that targets the genome of RS virus or influenza A virus. This first nucleotide sequence consists of either (a) a sequence that is 15-30 nucleotides in length, or (b) its complement. Such polynucleotides are referred to herein as linear polynucleotides.

  FIG. 3 provides a schematic diagram of a particular embodiment of the polynucleotide of the present invention. The present invention discloses target sequences in various strains of RSV or influenza A, or in certain cases, siRNA sequences that are slightly incompatible with the target sequence, all of which are SEQ ID NO: 1 263. The sequence provided in SEQ ID NOs: 1-263 ranges in length from 19 nucleotides to 25 nucleotides. This targeting sequence is represented by the lightly shaded blocks in FIG. FIG. 3, panel A, a) shows that the disclosed sequence shown as “SEQ” can be included within a larger polynucleotide as needed, and the total length of this polynucleotide can range up to 200 nucleotides. Figure 1 illustrates an embodiment.

  The present invention relates to longer targeting in a targeting polynucleotide such that a sequence selected from SEQ ID NOs: 1-263 targets a sequence in the viral genome that is longer than the first nucleotide sequence represented by SEQ. It is further provided that it can be part of the sequence. This is illustrated in FIG. 3, panel A b), where the complete targeting sequence is indicated by a horizontal line above the polynucleotide and a dark shadow around the SEQ block. In all embodiments of the polynucleotide, this longer sequence can optionally be included in a longer (200 or less base length) polynucleotide (FIG. 3, Panel A, b). ).

  The invention is illustrated in any fragment of SEQ ID NO: 1-263, which is at least 15 nucleotides in length (and at most one base shorter than the reference SEQ ID NO; FIG. 3, panel A, c). As well as a sequence (illustrated in FIG. 3, panel A, d) that can differ from the target sequence given in SEQ ID NOs: 1-263). (Represented by a dark vertical bar).

  Still further, the present invention provides a sequence that is the complement of any of the above sequences (shown in FIG. 3, panel A e) and named “COMPL”). Any of these sequences are included in the oligonucleotides or polynucleotides of the invention. Any linear polynucleotide of the present invention may be constituted only by the sequences described in a) to e) above, or may contain additional bases up to an upper limit of 200 nucleotides, if necessary. Since RNA interference requires double stranded RNA, the targeting polynucleotide itself comprises at least a second strand that is complementary to and hybridizes to the sequence given in SEQ ID NOs: 1-263. It can be double stranded, or can produce complementary strands depending on intracellular processing.

  Accordingly, the polynucleotide described above may be single-stranded or double-stranded. In still further embodiments, the polynucleotide comprises only deoxyribonucleotides, only ribonucleotides, or both deoxyribonucleotides and ribonucleotides. In important embodiments of the polynucleotides described herein, the target sequence is 15 nucleotides (nt), or 16 nt, or 17 nt, or 18 nt, or 19 nt, or 20 nt, or 21 nt, or 22 nt, or 23 nt. Or 24 nt, or 25 nt, or 26 nt, or 27 nt, or 28 nt, or 29, or 30 nt. In yet another advantageous embodiment, the targeting sequence can differ by up to 5 bases from the target sequence in the viral pathogen genome.

  In some embodiments of the invention, the polynucleotide is a siRNA consisting of a targeting sequence and optionally comprising a 3 'dinucleotide overhang as described herein.

  Alternatively, in recognition of the need for double stranded RNA in RNA interference, the oligonucleotide or polynucleotide can be prepared to form an intramolecular hairpin loop double stranded molecule. Such a molecule is complementary to the first sequence described in any of the embodiments of the above paragraph, the short loop sequence behind it, and the second sequence behind it. Formed from a typical array. Such a structure forms the desired intramolecular hairpin. Furthermore, and also has a length of up to 200 nucleotides such that the three desired structures listed can be constructed in any oligonucleotide or polynucleotide having any overall length up to 200 nucleotides Polynucleotides are disclosed. Hairpin loop polynucleotides are illustrated in FIG.

(RSV strain as a target)
For RSV, any part of the viral genome can be targeted, but theoretically, a targeted gene encoding a virus-specific enzymatic function should provide a potential siRNA candidate. These genes include L gene, F gene, G gene and P gene. The L gene located at the 5 ′ end of the viral genome is expressed only in small amounts, so effective RNAi silencing may require only small amounts of RNAi. Structural genes can also be targeted.

  The sequences of representative strains of the following RSV subgroups A and B are common between subgroup A and subgroup B (Table 1), or subgroup A (Table 2) or subgroup B ( Used to select target sequences that are specific to Table 3).

    Subgroup A: strain A2 (GenBank M74568) and strain Long (P-mRNA, GenBank M22644, and F-mRNA, GenBank M22643 only).

    Subgroup B: strain B1 (GenBank NC — 001781) and strain 9320 (GenBank AY353550).

  Viral gene sequences were aligned in order to search for common or unique regions. For each targeted gene or region, at least two targets were selected unless not applicable (NA). Finding a common target sequence for both subgroups was more difficult than finding a unique target for either of these. This is because very few sequences are available that are homologous or identical. In this case (Table 1), it was sometimes necessary to introduce a mismatch at the 5 'end of one end of the RNA duplex.

（表２．サブグループＡ（株Ａ２ ＆ Ｌｏｎｇ株のＦ／Ｐ）に特異的な遺伝子標的）

(Table 2. Specific gene targets for subgroup A (F / P of strain A2 & Long strain))

（表３．サブグループＢ（株Ｂ１および株９３２０）に特異的な遺伝子標的）

(Table 3. Specific gene targets for subgroup B (strain B1 and strain 9320))

ウイルスゲノムの５’末端に対して指向されるｓｉＲＮＡ因子を提供するために、株Ｂ１もしくは株９３２０のいずれか個々に独特の尾部（ｔｒａｉｌ）標的が、以下に示される（表４）。（＋）鎖（アンチ−ゲノムＲＮＡ）の５’末端が標的化され、すなわち、ポジティブＲＮＡ鎖（アンチゲノムＲＮＡ）をテンプレートとして用いてウイルスゲノムが複製を開始する際、新生リーダー配列が産生される。

In order to provide siRNA elements directed against the 5 ′ end of the viral genome, individual tail targets, either strain B1 or strain 9320, are shown below (Table 4). A nascent leader sequence is produced when the 5 'end of the (+) strand (anti-genomic RNA) is targeted, ie when the viral genome begins to replicate using the positive RNA strand (antigenomic RNA) as a template .

  (Table 4. Tail targets for two strains of RSV subgroup B)

上の表１〜表４に列挙した標的に基づき、ｓｉＲＮＡは、以下のように作製され得る：ａ）二本鎖ｓｉＲＮＡの各鎖に、３’−ｄＴｄＴオーバーハングが提供される、ｂ）「ミスマッチ」が必要な場合、Ｇ：Ｃは、Ｇ：ＴもしくはＧ：Ａに変えられ、Ａ：Ｔは、Ａ：ＣもしくはＡ：Ｇに変えられ、そしてＣ：ＧはＣ：ＴもしくはＣ：Ａに変えられる。

Based on the targets listed in Tables 1 to 4 above, siRNAs can be made as follows: a) Each strand of a double stranded siRNA is provided with a 3′-dTdT overhang, b) “ When “mismatch” is required, G: C is changed to G: T or G: A, A: T is changed to A: C or A: G, and C: G is C: T or C: It can be changed to A.

(Influenza A strain as a target)
The 25 and 19 nucleotide long siRNA target sequences within the (+) strand mRNA sequence have been identified in all eight segments. This sequence is shown in Tables 5-12. siRNA sequences are derived from each of the eight segments. The entry for each sequence also indicates the starting nucleotide position in the mRNA from the 5 'end.

  (Table 5. siRNA sequences targeting the hemagglutinin gene of influenza A virus (A / chicken / Thailand / CH-2 / 2004 (H5N1); GenBank AY649382).)

（表６．インフルエンザＡウイルスのマトリックスタンパク質２およびマトリックスタンパク質１（Ｍ）遺伝子（Ａ／Ｔｈａｉｌａｎｄ／１（ＫＡＮ−１）／２００４（Ｈ５Ｎ１）；ＧｅｎＢａｎｋ ＡＹ６２６１４４）を標的化するｓｉＲＮＡ配列。）

(Table 6. siRNA sequences targeting the matrix protein 2 and matrix protein 1 (M) genes of influenza A virus (A / Thailand / 1 (KAN-1) / 2004 (H5N1); GenBank AY626144)).

（表７．インフルエンザＡウイルスのノイラミニダーゼ遺伝子（Ａ／ｃｈｉｃｋｅｎ／Ｔｈａｉｌａｎｄ／ＣＨ−２／２００４（Ｈ５Ｎ１）；ＧｅｎＢａｎｋ ＡＹ６４９３８３）を標的化するｓｉＲＮＡ配列。）

(Table 7. siRNA sequences targeting the neuraminidase gene of influenza A virus (A / chicken / Thailand / CH-2 / 2004 (H5N1); GenBank AY649383)).

（表８．インフルエンザＡウイルスのヌクレオカプシドタンパク質（ＮＰ）遺伝子（Ａ／Ｔｈａｉｌａｎｄ／１（ＫＡＮ−１）／２００４（Ｈ５Ｎ１）；ＧｅｎＢａｎｋ ＡＹ６２６１４５）を標的化するｓｉＲＮＡ配列。）

(Table 8. siRNA sequences targeting the nucleocapsid protein (NP) gene of influenza A virus (A / Thailand / 1 (KAN-1) / 2004 (H5N1); GenBank AY626145)).

（表９．インフルエンザＡウイルスの非構造タンパク質１および非構造タンパク質２（ＮＳ）の遺伝子（Ａ／Ｔｈａｉｌａｎｄ／１（ＫＡＮ−１）／２００４（Ｈ５Ｎ１）；ＧｅｎＢａｎｋ ＡＹ６２６１４６）を標的化するｓｉＲＮＡ配列。）

(Table 9. siRNA sequences targeting the nonstructural protein 1 and nonstructural protein 2 (NS) genes of influenza A virus (A / Thailand / 1 (KAN-1) / 2004 (H5N1); GenBank AY626146).)

（表１０．インフルエンザＡウイルスのポリメラーゼ酸性タンパク質（ＰＡ）遺伝子（Ａ／Ｔｈａｉｌａｎｄ／１（ＫＡＮ−１）／２００４（Ｈ５Ｎ１）；ＧｅｎＢａｎｋ ＡＹ６２６１４７）を標的化するｓｉＲＮＡ配列。）

(Table 10. siRNA sequences targeting the polymerase acidic protein (PA) gene of influenza A virus (A / Thailand / 1 (KAN-1) / 2004 (H5N1); GenBank AY626147)).

（表１１．インフルエンザＡウイルスのポリメラーゼ塩基性タンパク質１（ＰＢ１）遺伝子（Ａ／Ｔｈａｉｌａｎｄ／１（ＫＡＮ−１）／２００４（Ｈ５Ｎ１）；ＧｅｎＢａｎｋ ＡＹ６２６１４８）を標的化するｓｉＲＮＡ配列。）

(Table 11. siRNA sequences targeting the polymerase A protein 1 (PB1) gene of influenza A virus (A / Thailand / 1 (KAN-1) / 2004 (H5N1); GenBank AY626148)).

（表１２．インフルエンザＡウイルスのポリメラーゼ塩基性タンパク質２（ＰＢ２）遺伝子（Ａ／Ｔｈａｉｌａｎｄ／１（ＫＡＮ−１）／２００４（Ｈ５Ｎ１）；ＧｅｎＢａｎｋ ＡＹ６２６１４９）を標的化するｓｉＲＮＡ配列。）

(Table 12. siRNA sequences targeting the polymerase basic protein 2 (PB2) gene of influenza A virus (A / Thailand / 1 (KAN-1) / 2004 (H5N1); GenBank AY626149)).

インフルエンザＡを標的化する潜在的ｓｉＲＮＡ候補配列の選択のための別のアプローチは、２つの主要なウイルス表面糖タンパク質であるヘマグルチニン（ＨＡ）およびノイラミニダーゼ（ＮＡ）のサブタイプ特異性に着目することである。ヒト感染を引き起こすことが報告されているサブタイプは、Ｈ５Ｎ１、Ｈ７Ｎ７、およびＨ９Ｎ２である。以下は、サブタイプ特異的なＨＡ標的およびＮＡ標的の例である。

Another approach for the selection of potential siRNA candidate sequences that target influenza A is by focusing on the subtype specificity of two major viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). is there. Subtypes that have been reported to cause human infection are H5N1, H7N7, and H9N2. The following are examples of subtype-specific HA and NA targets.

  (Table 13. siRNA targeting sequence of H5N1 hemagglutinin (HA) (based on GenBank DQ023145, GI: 66777524):

表１３における配列は、中国において２０００年と２００３年との間にＨＰＡＩウイルスによって感染したニワトリから単離された６種のＨ５Ｎ１株によって共有される。これらのＨ５Ｎ１株は、表１４に列挙される。

The sequences in Table 13 are shared by six H5N1 strains isolated from chickens infected with HPAI virus between 2000 and 2003 in China. These H5N1 strains are listed in Table 14.

  (Table 14)

（表１５．Ｈ５Ｎ１ノイラミニダーゼ（ＮＡ）のｓｉＲＮＡ標的化配列（ＧｅｎＢａｎｋ ＤＱ０２３１４７，ＧＩ：６６７７５６２８に基づく）：

(Table 15. SiRNA targeting sequence of H5N1 neuraminidase (NA) (based on GenBank DQ023147, GI: 66777528):

上の配列は、中国において２００１年と２００３年との間に感染したニワトリもしくはブタから単離された６種のＨ５Ｎ１株によって共有される。これらのＨ５Ｎ１株は、以下の表１６に列挙される。

The above sequence is shared by six strains of H5N1 isolated from chickens or pigs infected between 2001 and 2003 in China. These H5N1 strains are listed in Table 16 below.

  (Table 16)

（表１７．Ｈ７Ｎ７ヘマグルチニン（ＨＡ）のｓｉＲＮＡ標的化配列（ＧｅｎＢａｎｋ ＡＹ９９９９８６，ＧＩ：６６３９４８３７に基づく）：

(Table 17. siRNA targeting sequence of H7N7 hemagglutinin (HA) (based on GenBank AY999986, GI: 66394837):

上の全ての配列は、スウェーデンにおいて２００２年にマガモから単離された６種のＨ７Ｎ７株によって共有される。これらのＨ７Ｎ７株は、以下の表１８に列挙される。

All the above sequences are shared by six H7N7 strains isolated from mallard ducks in 2002 in Sweden. These H7N7 strains are listed in Table 18 below.

  Comparing the six HA sequences, it has been found that the HA1-related domain has a higher frequency of point mutations, thus 10 targets are selected from the HA2 domain.

  (Table 18)

いくつかのインフルエンザＡ Ｈ５Ｎ１ ｍＲＮＡ配列に対するさらなるｓｉＲＮＡ二本鎖が、同定された。これらを表１９に示す。これらの位置は図４に図示される。

Additional siRNA duplexes for several influenza A H5N1 mRNA sequences were identified. These are shown in Table 19. These positions are illustrated in FIG.

  Table 19. RNAi sequences targeting H5N1 avian influenza

（ｓｉＲＮＡの組み合わせ）
本発明のいくつかの実施形態は、２種もしくはそれより多くのオリゴヌクレオチドまたはポリヌクレオチドを含有する薬学的組成物を提供し、これらのオリゴヌクレオチドまたはポリヌクレオチドの各々は、呼吸性ウイルスのゲノムにおける遺伝子を標的化する配列を含む。関連の実施形態は、この組み合わせを用いて細胞を処置する方法、および呼吸性ウイルス感染を処置する方法を提供し、そしてこのような組み合わせ組成物の、呼吸性ウイルス感染を処置することを意図される薬学的組成物の製造における使用を提供する。この組み合わせの個々のポリヌクレオチド成分は、ウイルス病原体のゲノムにおける同じ遺伝子の異なる部分、または異なる遺伝子、あるいは１つの遺伝子および１つより多くの遺伝子のいくつかの部分を、標的化し得る。オリゴヌクレオチドもしくはポリヌクレオチドの組み合わせを用いる利点は、所定の遺伝子の発現を阻害する利益が、この組み合わせにおいて大きくなることである。多数の標的性配列の使用により、遺伝子のノックダウンもしくはウイルスゲノムのサイレンシングにおいて、より大きな効力が、達成される。ウイルス複製の阻害における増大した効力は、ウイルスゲノムにおける１種より多くの遺伝子を標的化することによって達成される。

(Combination of siRNA)
Some embodiments of the invention provide a pharmaceutical composition containing two or more oligonucleotides or polynucleotides, each of these oligonucleotides or polynucleotides in the genome of a respiratory virus Contains sequences that target the gene. Related embodiments provide methods of treating cells with this combination and methods of treating respiratory viral infections, and such combination compositions are intended to treat respiratory viral infections. For use in the manufacture of a pharmaceutical composition. The individual polynucleotide components of this combination may target different parts of the same gene in the viral pathogen genome, or different genes, or several parts of one gene and more than one gene. An advantage of using a combination of oligonucleotides or polynucleotides is that the benefit of inhibiting the expression of a given gene is greater in this combination. Through the use of multiple targeting sequences, greater efficacy is achieved in gene knockdown or viral genome silencing. Increased efficacy in inhibiting viral replication is achieved by targeting more than one gene in the viral genome.

(Pharmaceutical composition)
The targeting polynucleotides of the invention are described herein as “active compounds” or “therapeutic agents”. These therapeutic agents can be incorporated into pharmaceutical compositions suitable for administration to a subject.

  As used herein, “pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are compatible with pharmaceutical administration. It is intended to include tonicity and absorption delaying agents and the like. Suitable carriers include Remington's Pharmaceutical Sciences, Gennaro AR (ed.), 20th edition (2000) Williams & Wilkins Principal Organic, and Wilson and Gisold's Textbook of Ord. In a textbook (incorporated herein by reference). Preferred examples of ingredients that can be used in such carriers or diluents include, but are not limited to: water, saline, phosphate, carboxylate, amino acid solution, Ringer's solution, dextrose (Synonymous for glucose) solution, and 5% human serum albumin. As a non-limiting example, dextrose can be used as a 5% or 10% aqueous solution. Liposomes and non-aqueous vehicles such as non-volatile oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Additional active compounds can also be incorporated into the compositions.

  The pharmaceutical compositions of the invention are formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral administration (eg, intravenous administration, intradermal administration, subcutaneous administration), oral administration, nasal administration, inhalation administration, transdermal administration (topical administration), transmucosal administration, and rectal administration. Can be mentioned. Solutions or suspensions used for parenteral, intravenous, intradermal or subcutaneous application may contain the following components: water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, Sterile diluents such as propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; Buffers such as citrate or phosphate, and agents for adjusting tonicity (eg, sodium chloride or dextrose).

  For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressurized container or dispenser which contains a suitable nebulizer (eg, a gas such as carbon dioxide) or a nebulizer.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid removal from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Suitable examples of sustained release preparations include semi-permeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of shaped products (eg films or microcapsules). Examples of sustained release matrices include polyesters, hydrogels (eg poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (US Pat. No. 3,773,919), L-glutamic acid and gamma ethyl- Copolymers with L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (eg LUPRON DEPOT ^™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)), and poly- D-(-)-3-hydroxybutyric acid is mentioned. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules over 100 days, and certain hydrogels release pharmaceutically active agents over a shorter period of time. Useful polymers are biodegradable or biocompatible. Liposomal suspensions (including liposomes that target infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811. Sustained release preparations (eg, microspheres) having beneficial forms can be prepared from materials as described above.

  The siRNA polynucleotides of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by any of a number of routes (eg, as described in US Pat. No. 5,703,055). Accordingly, delivery also includes, for example, intravenous injection, local administration (see US Pat. No. 5,328,470) or stereotactic injection (eg, Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the therapeutic vector in an acceptable diluent or can include a sustained release matrix in which the gene delivery vehicle is embedded. Alternatively, where a complete gene delivery vector can be produced intact from recombinant cells (eg, a retroviral vector), the pharmaceutical preparation includes one or more cells that produce the gene delivery system. obtain.

  The pharmaceutical composition may be included in the kit together with instructions for administration, for example, in a container, pack or dispenser.

  The use of a therapeutic agent in the manufacture of a pharmaceutical composition or medicament for treating respiratory viral infections in a subject is also within the scope of the present invention.

(Delivery)
In some embodiments, the siRNA polynucleotides of the invention can be obtained by liposome-mediated transfection, for example, using commercially available reagents or techniques (eg, Oligofectamine ^™ , LipofectAmine ^™ reagent, LipofectAmine 2000 ^™ (Invitrogen), and electroporation and Similar technology) is used to deliver to cells in culture. In addition, siRNA polynucleotides are delivered to animal models (eg, rodents or non-human primates) via inhalation or instillation into the respiratory tract. Additional routes for use in animal models include intravenous (IV), subcutaneous (SC), and related routes of administration. The pharmaceutical composition containing the siRNA further protects the stability of the siRNA, extends the duration of the siRNA, promotes siRNA function, or targets the siRNA to specific tissues / cells. Contains ingredients. These components include various biodegradable polymers, cationic polymers (eg, polyethyleneimine), cationic copolypeptides such as histidine-lysine (HK) polypeptides (eg, PCT Publication No. WO 01/47496 to Mixson et al., Positively charged polypeptides, PolyTran polymers (natural polysaccharides, scleros, such as WO 02/096941 to Biomerieux and WO 99/42091 to Massachusetts Institute of Technology), pegylated cationic polypeptides, and polymers incorporating ligands. Also known as glucan), nanoparticles composed of polymers conjugated to targeting ligands (TargetTran variants), surfactants (In asurf; Forest Laboratories, Inc;. ONY Inc.), as well as cationic polymers (e.g., polyethyleneimine). Infasurf® (calfactant) is a natural pulmonary surfactant isolated from calf lung for use in intratracheal instillation; it is a phospholipid, neutral Including lipids, and hydrophobic surfactant-related protein B and hydrophobic surfactant-related protein C. The polymer can be either a one-dimensional polymer or a multi-dimensional polymer and can also be a microparticle or nanoparticle with a diameter of less than 20 microns, between 20 and 100 microns, or more than 100 microns. The polymer can have a ligand molecule specific for a specific tissue or cellular receptor or molecule and can therefore be used to target the delivery of siRNA. siRNA polynucleotides are also delivered by cationic liposome-based carriers (eg, DOTAP, DOTAP / cholesterol (Qbiogene, Inc.)) and other types of aqueous lipids. Further, aqueous glucose solution and Infasurf low percentage (5-10%) are effective carriers for airway delivery of siRNA ^30.

  Using fluorescently labeled siRNA suspended in oral airway delivery solution of 5% glucose and Infasurf and examined under a fluorescence microscope, siRNA was delivered to mice via the nostril or via the oral-airway route and lung tissue After washing, this siRNA has been shown to be widely distributed in the lung (see shared WO 2005/01940, which is hereby incorporated by reference in its entirety). Delivery of siRNA to mouse nasal passages and lungs (the upper and deep respiratory tracts) successfully silences the indicator gene (GFP or luciferase) delivered in the plasmid simultaneously with the siRNA. (This plasmid had a fusion of this indicator gene with the siRNA target) (see WO 2005/01940 in common). Furthermore, experiments reported by the inventors (researched with others) showed that siRNA species inhibit SARS coronavirus replication, thereby alleviating lung pathology in SARS infected rhesus monkeys. .

(SiRNA recombination vector)
Another aspect of the present invention relates to a vector, preferably an expression vector, having the siRNA polynucleotide of the present invention. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. A particular vector may direct the expression of an operably linked gene. Such vectors are referred to herein as “expression vectors”. In general, expression vectors used in recombinant DNA technology are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which provide equivalent functions.

  The recombinant expression vector of the present invention comprises the nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, wherein the recombinant expression vector is transformed into a host cell used for expression. It is meant to include one or more regulatory sequences selected on the basis of, and these regulatory sequences are operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” means that the nucleotide sequence of interest is the nucleotide sequence of this nucleotide sequence (eg, in an in vitro transcription / translation system or when the vector is introduced into a host cell). It is meant to be linked to regulatory sequences in a manner that allows expression (in a host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel (1990) GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. Described in. Regulatory sequences include regulatory sequences that direct constitutive expression of nucleotide sequences in many types of host cells, as well as regulatory sequences that direct constitutive expression of nucleotide sequences only in specific host cells (eg, tissue-specific regulatory sequences). ). In yet another embodiment, the nucleic acids of the invention are expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. Further vectors include microchromosomes such as bacterial artificial chromosomes, yeast artificial chromosomes or mammalian artificial chromosomes. Other suitable expression systems are expression systems for both prokaryotic and eukaryotic cells. For example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.A. Y. , 1989, chapters 16 and 17.

  In another embodiment, the recombinant mammalian expression vector can selectively direct the expression of the nucleic acid in a particular cell type (eg, airway cells). Tissue specific regulatory elements are known in the art. The present invention further provides an expression vector comprising the DNA molecule of the present invention cloned into a recombinant expression vector. The DNA molecule is operably linked to regulatory sequences in a manner that allows expression of the RNA molecule, including siRNA that targets viral RNA (by transcription of the DNA molecule). Regulatory sequences (eg, viral promoters and / or enhancers) operably linked to the nucleic acid that direct continuous expression of the RNA molecule in various cell types can be selected, or construction of antisense RNA Regulatory sequences can be selected that direct constitutive expression, tissue specific expression or cell type specific expression.

  Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are recognized in various arts for introducing exogenous nucleic acid (eg, DNA) into a host cell. It is intended to refer to a technique, which includes coprecipitation of calcium phosphate or calcium chloride, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (2001), Ausubel et al. (2002), and other laboratory manuals.

(Method of assay for virus titer or amount)
In the practice of the present invention, assays for viruses can be performed by several procedures. Among these, non-limiting examples include immunoblot (Western blot), immunoprecipitation (IP), and complex reverse transcription polymerase chain reaction (RT-PCR) assays. Such a procedure measures the decrease in synthesis of the targeted mRNA or its protein product that may be present in the lysate or supernatant in the transfected tissue culture. Similarly, these assays may be used as a diagnostic procedure to target mRNA or its mRNA that may be present in homogenized tissue samples, nasopharyngeal lavage fluids, secretions obtained from infected animal or human subjects. It can be used to measure the decrease in protein product synthesis.

  In the RT-PCR assay method, viral genomic RNA synthesis is detected using a primer that is complementary to a sequence spanning the joint between the NS1 and NS2 ORFs. This RT-PCR result with NS1 / NS2 primers reflects the synthesis of total genomic RNA.

  This method is based on quantitative real-time PCR (RTQ-PCR), TCID50, viral plaque assay, immunity, as siRNA-induced interference of virus replication in tissue culture is known to those skilled in the art. Further included are assays as measured by fluorescence assays and immunohistochemistry. In addition, the method further comprises an assay for the presence of virus that monitors inhibition of virus replication in tissue culture using the TCID50 method described above. Here, the virus titer is measured by any type of cytopathogenic endpoint (non-limiting examples include cell fusion, cytopathic effect (CPE), cell adsorption, etc.).

  The methods further include assays wherein the interference of the siRNA-mediated viral replication in the test animal is measured by RTQ-PCR, pathology, immunohistochemistry and virus reisolation.

  Primers are designed for RT-PCR detection used to measure the reduction of mRNA synthesis by RNAi. This RT reaction is initiated by hexamer or poly-dT primers; and PCR is performed using upstream or downstream primers specific for each gene targeted by the siRNA.

  For detection of genomic RNA synthesis, a pair of primers (top and bottom) are designed corresponding to sequences in the NS1 ORF and NS2 ORF. The purpose of using a “joint-crossing” primer rather than a primer complementary to either end of the viral genome is the large, difficult-to-handle large RNA transcript (approximately 15 K-nt long) This is to avoid this). This RT-PCR product reflects the synthesis of the entire genomic RNA by this design.

  The identified primers and RT-PCR products are listed in Table 20, Table 21 and Table 22 below.

  (Table 20. Primers for RSV strain A2 (subgroup A))

（表２１．ＲＳＶ株Ｂ１（サブグループＢ）についてのプライマー）

(Table 21. Primers for RSV strain B1 (subgroup B))

（表２２．ＰＣＲ産物の大きさ）

(Table 22. Size of PCR product)

Example 1. Effect of anti-H5N1 influenza A siRNA molecules in cell culture.
This example reports the evaluation of the inhibitory effect of siRNA on H5N1-infected cells in culture.

(Design and synthesis of siRNA)
Developed two siRNAs targeting the NP genes (NP-1 and NP-2; Table 19, SEQ ID NO) and M2 genes (M2-1 and M2-2; Table 19, SEQ ID NO), respectively, of H5N1 did. These siRNA target sequences are conserved in several strains of H5N1 virus (eg, gi | 47834945_10-1506, gi | 8452827_1-1497, gi | 13952758_46-1542, gi | 61927237_46-1542, gi | 59940391_44- > 1535, gi | 9802277_46-1542). Fluorescein-labeled ds-siRNA in the 5 'sense strand was chemically synthesized by Proligo BioTech Ltd (Paris, France). Its anti-H5N1 effect was detected in MDCK cells infected with H5N1 virus, transfected with siRNA (Madin-Darby canine kidney; ATCC accession number: CCL-34, Manassas, VA). Inhibition of H5N1 was determined by cytopathic effect (CPE), back titration of virus released into culture medium according to standard TCID ₅₀ (50% tissue culture infectious dose) protocol, and real-time RT-, as described below. Determined by quantification of intracellular viral RNA using PCR.

(Cell culture, siRNA transfection and H5N1 virus infection)
MDCK cells were cultured and maintained in MEM medium containing 10% fetal bovine serum (FBS, Invitrogen). Approximately 5000 cells were placed in each well of a 96 well plate for virus infection and replication assays. Cells were transfected with 100 nM, 50 nM, 25 nM and 12.5 nM siRNA mixed with 0.5 μl of Lipofectamine 2000 (Invitrogen, CA). An irrelevant siRNA targeting luciferase (GL2i), or an siRNA targeting SARS coronavirus (C-1), and transfectant Lipofectamine 2000 alone (C-2) as negative controls in this experiment included. Six hours after transfection, the culture medium was removed and the cells were washed twice with PBS prior to H5N1 virus infection. 100 μl of 100 TCID ₅₀ H5N1 virus (strain: A / Hong Kong / 486/97) ⁷ diluted in MEM containing 1% FBS was added to the transfected cells. Culture supernatants and cells were collected for detection of released virus titer and intracellular viral RNA copy number at 12, 16, and 24 hours after infection, respectively. 24 hours after infection, CPE was observed and recorded under a phase contrast microscope. This experiment was performed in triplicate and repeated at least three times.

(Quantitative RT-PCR)
Q-RT-PCR is performed as follows. Total intracellular RNA was isolated using the RNeasy Mini kit (Qiagen, Germany) according to the manufacturer's instructions. Reverse transcription experiments were performed by oligo-dT priming using a ThermoScript RT-PCR system (Invitrogen, CA). Real-time PCR was then performed using the primers shown in Table 23.

  (Table 23. H5N1 primers for Q-RT-PCR)

５μｌのＲＴ産物（テンプレート）、各１μｌの順（ｆｏｒｗａｒｄ）プライマーおよび逆（ｒｅｖｅｒｓｅ）プライマー（最終濃度５００ｎＭ）、２μｌの２５ｍＭ ＭｇＣｌ_２、ならびに９μｌのＨ_２Ｏを、２μｌのＳＹＢＲ Ｇｒｅｅｎ Ｉ Ｍａｓｔｅｒ Ｍｉｘ（Ｒｏｃｈｅ，ＵＳＡ）と混合し、そしてリアルタイム定量を、ＡＢＩ７９００ Ｓｅｑｕｅｎｃｅ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍを用いて実施した。ＰＣＲ条件は、以下であった：５０℃を５分間、９５℃を１０分間、次いで９５℃を１０秒間、６１℃を５秒間および７２℃を５秒間を４０サイクル。β−アクチンのコピー数もまた、内部コントロールとして測定した。１０００コピーのβ−アクチンあたりの細胞内ウイルスＲＮＡのコピー数を、計算し、そして未処理コントロールと比較した相対的ウイルスＲＮＡコピー数として表した（処理したコピー数／未処理のコピー数×１００％）。

5 μl RT product (template), 1 μl each forward and reverse primer (final concentration 500 nM), 2 μl 25 mM MgCl ₂ , and 9 μl H ₂ O, 2 μl SYBR Green I Master Mix ( Roche, USA) and real-time quantification was performed using the ABI 7900 Sequence Detection System. The PCR conditions were as follows: 50 ° C for 5 minutes, 95 ° C for 10 minutes, then 95 ° C for 10 seconds, 61 ° C for 5 seconds and 72 ° C for 5 seconds for 40 cycles. β-actin copy number was also measured as an internal control. The number of copies of intracellular viral RNA per 1000 copies of β-actin was calculated and expressed as relative viral RNA copy number compared to untreated control (treated copy number / untreated copy number × 100% ).

(Measurement of virus titer)
Virus titers in cultures with or without siRNA treatment were tested as follows. Briefly, conditioned medium from infected cells was diluted in a 10-fold sequential process in MEM containing 1% FBS. Each dilution was used to infect cells according to the standard TCID ₅₀ protocol. Briefly, cells were placed in 96 well dishes 16 hours prior to infection. 72 hours after infection, CPE was observed under a phase contrast microscope and CPE positive (CPE +) cells were determined. The TCID ₅₀ was then evaluated as follows:

ここで、ｈ＝希釈工程の補間されたｌｏｇ_１０値、これを５０％を超える値の工程のｌｏｇ_１０に加えた。感染性ウイルス力価を、計算しそして未処理コントロールと比較した相対的ウイルス収率として表した（処理した力価／未処理の力価×１００％）。

Where h = interpolated log ₁₀ value of the dilution step, which was added to log ₁₀ of the step with a value greater than 50%. Infectious virus titers were calculated and expressed as relative virus yield compared to untreated controls (treated titer / untreated titer × 100%).

(result)
siRNA NP-1 (Table 19) treatment resulted in H5N1 virus production in culture medium (Figure 5, Panel A) and replication of viral RNA copies in cells (Figure 5, Panel B) at concentrations of 12.5-100 nM. Reduced by more than 99%. On the other hand, treatment with siRNA targeting NP-2 (Table 19) inhibited only about 60% of virus growth in cell culture at a concentration of 100 nM. In contrast, irrelevant siRNA-treated control (C-1) and carrier-treated control (C-2) did not significantly inhibit virus growth compared to untreated controls.

  Treatment with siRNA (Table 19) directed against M2-1 (Table 19) also resulted in viral growth (Figure 5, Panel C) and viral RNA replication (Figure 5, Panel D) at various concentrations. On the other hand, it showed an inhibitory effect of about 80% to 94%. Here, M2-2 showed an inhibitory effect of about 60% at a concentration of 100 nM, but lower concentrations were not tested.

In a second experiment, cells in culture were treated with 50 nM of various siRNAs. Virus produced in the culture supernatant medium was collected at three time points. Samples were titrated by determining the tissue culture infectious dose (TCID ₅₀ ) required to infect 50% of the cells in the culture (FIG. 6, panel A; this is shown on a log coordinate scale) Note that The number of viral RNA copies / 1000 copies of β-actin was determined by real-time RT-PCR (Figure 6, Panel B). Irrelevant siRNA (C-1) and transfectant (C-2) were applied to the experiment as controls. NP-1 and NP-2 siRNA are shown to be very effective in inhibiting viral particle growth and viral RNA expansion. The use of M1-1 and M2-2 siRNAs is partially effective in preventing viral replication.

  The results in this example demonstrate that various siRNAs directed against different genes in the H5N1 genome are very effective in inhibiting viral replication in infected cell growth in culture.

(Example 2. Combination of siRNAs directed against H5N1 influenza A.)
A combination of siRNAs directed against various genes in the H5N1 genome was identified to provide effective anti-H5N1 activity. This example shows such a combination of the two.

（実施例３．インビトロの細胞中のｓｉＲＮＡによる標的遺伝子およびウイルス複製の阻害）
この実施例において、種々の呼吸性ウイルス遺伝子を標的化する、同族（ｃｏｇｎａｔｅ）の標的遺伝子をサイレンシングする能力もしくはウイルス複製を阻害する能力において最適に有効なｓｉＲＮＡおよび最適に有効なｓｉＲＮＡの組み合わせが、細胞培養物中の実験によって同定される。インビトロで有効なｓｉＲＮＡは、インビボでさらに試験されそして使用される候補である。

Example 3. Inhibition of target genes and viral replication by siRNA in cells in vitro
In this example, a combination of siRNA optimally effective in the ability to target various respiratory viral genes, silence cognate target genes or inhibit viral replication and optimally effective siRNA , Identified by experiments in cell culture. In vitro effective siRNAs are candidates for further testing and use in vivo.

A cultured permissive cell line (for example, as a non-limiting example, A549 (ATCC registration number: CCL-185 ^™ , type II alveolar epithelial lung carcinoma cell line)) is transformed into an RSV virus strain or influenza A strain (eg, H5N1). And then transfected individually or in combination with various siRNAs. In combination, two siRNAs that target one signal gene or siRNA that target two or more genes are used. Keep the total siRNA dose of a single siRNA or a combination siRNA constant. In different experimental protocols, siRNA transfection is performed hours before RSV infection or influenza A infection, is performed simultaneously with viral infection, or is performed several hours after infection. These different procedures provide information about whether the tested siRNAs exhibit a prophylactic and / or therapeutic effect at the cellular level.

The degree of target gene inhibition or inhibition of viral replication is assayed by various methods. Non-limiting examples of assays include the following procedure:
(1) An immunoblot (Western) is performed using cell lysates and RSV specific antibodies or influenza A specific antibodies against a given viral antigen.

  (2) Immunoprecipitation (IP) is performed using cell lysates and RSV specific antibodies or influenza A specific antibodies against the one gene product described above.

  (3) Perform rvtr-PCR to demonstrate inhibition of mRNA transcription or viral RNA replication. Special primers are designed to detect transcripts of targeted genes or to detect whether the tested siRNA oligos can also target genomic RNA.

  (4) Cell fusion-based TCID50 measurements can be used to monitor inhibition of viral replication as compared to cell cultures treated with specific siRNA of an irrelevant control siRNA.

  (5) Immunofluorescence or immunohistochemistry can also be used to titrate virus titer, thereby monitoring siRNA-mediated inhibition of virus replication.

(Example 4. Inhibition of target gene by siRNA in a small animal model)
In this example, various siRNAs (individually or in combination) are tested to determine their efficacy in treating RSV or avian influenza H5N1 in animal models. The RSV strain or influenza H5N1 strain is used to infect test animals through the respiratory tract by inhalation or instillation. The siRNA is delivered through the same route and is applied before, simultaneously with, or after infection, RSV infection or influenza H5N1 infection. Information on the efficacy of siRNA in inhibiting RSV or influenza H5N1 replication in test animals, reducing pathology induced by RSV or influenza H5N1 and reducing RSV or influenza H5N1-like symptoms by varying siRNA delivery time And it is possible to obtain information on whether siRNA specific for RSV or influenza H5N1 exhibits a preventive or therapeutic effect on experimental RSV infection or influenza H5N1 infection in animals. is there.

  Although RSV and influenza H5N1 can infect a wide range of animal species, mice and cotton rats are conventionally used in RSV or influenza H5N1 animal model studies. Rodent infections usually result in low to moderate viral replication, which peaks on day 4 and is cleared rapidly. These animals show no obvious airway disease but show pulmonary pathology. In high dose viral infections, they show weight loss, changes in lung function, and ruffled fur, which are indicators of disease.

  The same diagnostic assay as described in Example 3 above monitors siRNA-mediated gene silencing and inhibition of RSV infection or influenza H5N1 infection in samples taken from animals (eg, nasal, buccal and pharyngeal swabs) Use for. In addition, lung pathology, lung immunohistochemistry and symptom observation will be performed to determine the efficacy of siRNA in RSV infection or influenza H5N1 infection in vivo.

(Example 5. Inhibition of target gene by siRNA in non-human primate model)
This example describes the study of siRNA-mediated inhibition of RSV or influenza H5N1 viral replication in non-human primates. The efficacy and safety of dose administration is the main objective of this study.

Based on our protocol, using a rhesus monkey model for SARS, another respiratory disease, doses of siRNA up to 30 mg / kg body weight are tolerable and show no signs of toxicity in animals. ⁽²²⁾ Respiratory delivery of siRNA (by inhalation or instillation) that has been previously screened by non-human primate mammals is substantial for inhibition of virus replication and reduction of virus-induced pathology and disease-like symptoms. It has been shown that For this study of RSV or influenza H5N1, the same delivery route is used and similar dosages of siRNA are delivered.

  In addition to the experiments described for small animal studies (Example 4), the following assays are readily performed in primates and contribute to a more direct assessment of therapeutic efficacy.

  (1) Virus dispersibility: Virus dispersibility is measured using a nasopharyngeal wash sample of an infected monkey. Viral yield is titrated by either TCID50 (cell fusion based) and / or immunofluorescence assay.

  (2) RTQ-PCR of nasopharyngeal lavage samples: monitoring changes in viral genome copy number.

  (3) Symptom monitoring: It is easy to find bronchiole inflammation as observed in human infant patients.

  Although the invention has been described and illustrated with reference to various exemplary embodiments of the invention, equivalent embodiments and various other modifications, additions and deletions are contemplated in these embodiments and implementations thereof. Changes may be made to the form without departing from the spirit and scope of the invention.

  (References)

Schematic of the human RS virus (hRSV, RSV) (−) ssRNA genome. Based on GenBank registration number NC_001781. Schematic of influenza A virus structure. This figure shows that eight segments of the viral genome are integrated into virions. Schematic of various embodiments of the polynucleotide of the present invention. Panel A is an embodiment of a linear polynucleotide. The length is 200 nucleotides or less and 15 nucleotides or more. In (b), the described targeting sequence is contained within a larger targeting sequence. In (d), dark vertical bars illustrate substituted nucleotides. Panel B is an embodiment of a hairpin polynucleotide having an overall length of 200 nucleotides or less. It is the schematic which shows arrangement | positioning in the H5N1 genome of a siRNA sequence. Inhibition of H5N1 virus growth in MDCK cell cultures. Inhibition of H5N1 virus growth in MDCK cell cultures. Inhibitory effects of siRNA determined at different time points.

Claims (30)

An isolated polynucleotide that is 200 nucleotides or less in length, wherein the polynucleotide comprises a first nucleotide sequence, the first nucleotide sequence comprising an RS virus or an influenza A virus Wherein any T (thymidine) or any U (uridine) can be optionally substituted with others, and wherein the first nucleotide sequence is
(A) a polynucleotide comprising a sequence which is any number of nucleotides having a length of 15 to 30, or (b) a complement of the sequence described in (a) The polynucleotide of claim 1, further comprising a second nucleotide sequence separated from the first nucleotide sequence by a loop sequence, wherein the second nucleotide sequence is
(A) has substantially the same length as the first nucleotide sequence, and (b) is substantially complementary to the first nucleotide sequence;
Polynucleotide. The polynucleotide of claim 1 or claim 2, wherein the first nucleotide sequence is
(A) a sequence selected from SEQ ID NOs: 1 to 263,
(B) a targeting sequence that is longer than the sequence set forth in item (a), wherein the targeting sequence targets the respiratory virus genome and comprises a sequence selected from SEQ ID NOs: 1-263 , Targeting sequence,
(C) a fragment of a sequence selected from SEQ ID NOs: 1-263, wherein the fragment is at least 15 nucleotides long and is at least one base shorter than the selected sequence A fragment consisting of a sequence (d) up to 5 nucleotides, a sequence different from the sequence selected from SEQ ID NOs: 1 to 263, or (e) consisting of a complement of the sequence described in (a) to (d) nucleotide. The polynucleotide according to claim 1 or 2, wherein the first nucleotide sequence has a length of 21 to 25 arbitrary numbers. The polynucleotide according to claim 1 or 2, comprising a sequence selected from SEQ ID NOs: 1 to 263, and optionally, a dinucleotide overhang that binds to 3 'of the selected sequence. A polynucleotide comprising. The polynucleotide of claim 1 or claim 2, wherein the dinucleotide sequence at the 3 'end of the first nucleotide sequence is TT, TU, UT, or UU, and wherein A dinucleotide is a polynucleotide comprising ribonucleotides or deoxyribonucleotides or both. The polynucleotide according to claim 1 or 2, wherein the polynucleotide is DNA. The polynucleotide of claim 1 or claim 2, wherein the polynucleotide is RNA. The polynucleotide of claim 1 or claim 2, wherein the polynucleotide comprises both deoxyribonucleotides and ribonucleotides. The first polynucleotide strand of claim 1 and a second polynucleotide strand that is at least complementary to and hybridizes to the first nucleotide sequence of the first polynucleotide strand. A double-stranded polynucleotide comprising A combination comprising a plurality of targeting polynucleotides according to claim 1, 2 or both, wherein each polynucleotide targets a different sequence in the genome of the target virus. A vector comprising the polynucleotide of claim 1. A vector comprising the polynucleotide of claim 2. 14. A vector according to claim 12 or claim 13, wherein the vector is a plasmid, a recombinant virus, a transposon, or a microchromosome. A cell transfected with one or more polynucleotides according to claim 1. A cell transfected with one or more polynucleotides according to claim 2. A pharmaceutical composition comprising one or more polynucleotides according to claim 1 or claim 2, or a mixture thereof, and a pharmaceutically acceptable carrier, wherein each polynucleotide comprises A pharmaceutical composition that targets different sequences in the genome of a target virus. 14. A pharmaceutical composition comprising one or more vectors according to claim 12 or claim 13 or a mixture thereof and a pharmaceutically acceptable carrier, each vector comprising a genome of said target virus. Pharmaceutical compositions carrying polynucleotides that target different sequences in 19. The pharmaceutical composition according to claim 17 or claim 18, wherein the carrier comprises a synthetic polymer, liposome, dextrose, surfactant, or any combination of two or more thereof. Composition. A method of synthesizing a polynucleotide of claim 1 or claim 2 having a sequence that targets the genome of RS virus or influenza A virus, the method comprising:
(A) providing a nucleotide reagent comprising a reactive end and corresponding to the nucleotide at the first end of the sequence;
(B) adding an additional nucleotide reagent, including a reactive end and corresponding to the position following the sequence, to react with the reactive end of the step (a) and of the polynucleotide sequence being extended Increasing the length by one nucleotide, and removing undesired products and excess reagents, and (c) adding the nucleotide reagent corresponding to the nucleotide at the second end of the sequence so that the complete poly Repeating the step (b) until a nucleotide is provided. A method of transfecting a cell with an RNA inhibitor, the method comprising contacting the cell with a composition comprising one or more polynucleotides of claim 1. Method. 24. The method of claim 21, wherein the cell comprises a respiratory virus that is targeted by the one or more polynucleotides. A method of transfecting a cell with an RNA inhibitor, the method comprising contacting the cell with a composition comprising one or more polynucleotides of claim 2. Method. 24. The method of claim 23, wherein the cell comprises a respiratory virus that is targeted by the one or more polynucleotides. A method of inhibiting replication of said virus in cells infected with a respiratory virus, said method comprising contacting said cell with a composition comprising one or more polynucleotides according to claim 1. A method wherein the one or more polynucleotides target the virus. A method of inhibiting replication of the virus in a cell infected with a respiratory virus, comprising contacting the cell with a composition comprising one or more polynucleotides according to claim 2. Wherein the one or more polynucleotides target the virus. The use of a polynucleotide according to claim 1, or two or more thereof, which targets said respiratory virus in the manufacture of a pharmaceutical composition effective for treating an infection caused by a respiratory virus in a subject. Use of a mixture of more said polynucleotide. 28. Use according to claim 27, wherein the subject is a human. The use of a polynucleotide according to claim 2, or two or more thereof, which targets said respiratory virus in the manufacture of a pharmaceutical composition effective for treating an infection caused by a respiratory virus in a subject. Use of a mixture of more said polynucleotide. 30. Use according to claim 29, wherein the subject is a human.

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