Patent Application
20080279920
The invention relates to siRNA compositions that interfere with viral replication in respiratory viral infections, especially in respiratory syncytial virus and avian influenza A including the H5N1 strain. The invention further relates to uses of the siRNA compositions to inhibit expression of viral genes in respiratory virus-infected cells, and to uses in the treatment of respiratory virus infections in a subject.
Respiratory viral infections have been significant threats to human health and lives for centuries. Notorious episodes include infections caused by influenza strains, respiratory syncytial virus, and sever acute respiratory syndrome (SARS). These include the global influenza pandemic of 1918, which killed approximately 20-40 million people worldwide. There have been other influenza pandemics in more recent decades as well. A. SARS outbreak in 2002 claimed around 800 lives (2).
Respiratory Syncytial Virus
Respiratory syncytial virus (RSV) infection is the major cause of serious pediatric respiratory tract disease. About two-third of infants are infected with RSV during the first year of life and almost 100% have been infected by age 2. There is currently no specific and effective therapeutic available to treat RSV infection.
Respiratory syncytial virus (RSV) is an enveloped, non-segmented single-strand negative RNA virus (NNR) belonging to the family Paramyxoviradae, in the order mononegaviruses (14, 29). The paramyxoviruses share the following features. 1) They have a single stranded RNA genome that is tightly wrapped with the viral nucleocapsid protein (N)29, 30. 2) Sub-genomic mRNAs are transcribed from the negative genome by RdRP. 3) Virus replication takes place in the cytoplasm of host cells. The details of RSV life cycle from infection to release of progeny virions are well studied (15).
The RSV genome is a negative strand 15.5 kb long, containing the genes 3′-NS1, NS2, N, P, M, SL, G, F, M2, L-5′ (see
The incubation period for RSV infection is about 4 to 5 days; it first affects the nasopharynx, then in a few days it reaches the bronchi and bronchioles, with infection confined to the superficial layer of the respiratory epithelium.
Influenza A
Beginning in 1997, a new strain of avian influenza A, H5N1, has appeared. Although confined mostly to fowl, both wild populations and domesticated birds, the virus infects humans apparently only be direct contact with infected birds. In humans infection causes serious disease, leading to severe respiratory illness and death in human beings (3-12). Numerous cases and outbreaks have occurred in various nations of southeast Asia. In view of the ability of the avian virus to infect humans, there is increased risk of mutation to a contagious human variant, risking the emergence of a new influenza pandemic with efficient and sustained human-to-human transmission, and significant mortality.
Since avian flu H5N1 is a newly emerging infectious agent associated with pneumonia and its pathology and mechanism is not very clear, there is no specific and effective treatment for H5N1 avian flu in the human disease cases yet. Currently influenza infections are treated with antivirals, such as the two drugs (in the neuraminidase inhibitors class), oseltamivir (commercially known as Tamiflu) and zanamivir (commercially known as Relenza), or the older M2 inhibitors amantadine and rimantadine.
H5N1 is a subtype of influenza virus type A. As such it is an enveloped, fragmented, negative-single stranded RNA virus, belonging to the family Orthomyxoviridae. During the life cycle of the influenza A virus (including H5N1), the viral genome RNA (vRNA) serves as a template for complementary RNA (cRNA) production, which also serves as the template for messenger RNA (mRNA) production. Each of these three forms of RNA molecules arising during viral replication can all be targeted for siRNA-mediated degradation, using either sense or antisense siRNAs. The influenza A genome, consisting of 8 separate RNA segments containing at least 10 open reading frames (ORFs), serves as template for both viral genome replication and subgenomic or gene-directed mRNA synthesis.
RNA Interference
RNA interference (RNAi) is a sequence-specific RNA degradation process that provides a relatively easy and direct way to knockdown, or silence, theoretically any gene (17, 18, 19). In naturally occurring RNA interference, a double stranded RNA is cleaved by an RNase III/helicase protein, Dicer, into small interfering RNA (siRNA) molecules, a dsRNA of 19-23 nucleotides (nt) with 2-nt overhangs at the 3′ ends. These siRNAs are incorporated into a multicomponent-ribonuclease called RNA-induced-silencing-complex (RISC). One strand of siRNA remains associated with RISC, and guides the complex towards a cognate RNA that has sequence complementary to the guider ss-siRNA in RISC. This siRNA-directed endonuclease digests the RNA, thereby inactivating it. Recent studies have revealed that the use of chemically synthesized 21-25-nt siRNAs exhibit RNAi effects in mammalian cells 20, and the thermodynamic stability of siRNA hybridization (at terminals or in the middle) plays a central role in determining the molecule's function (21, 22). These and other characteristics of RISC, siRNA molecules and RNAi have been described (23-28).
Application of RNAi in mammalian cells in laboratory or potentially, in therapeutic applications, uses either chemically synthesized siRNAs or endogenously expressed molecules (2, 21). The endogenous siRNA is first expressed as a small hairpin RNAs (shRNAs) by an expression vector (plasmid or virus vector), and then processed by Dicer into siRNAs. It is thought that siRNAs hold great promise to be therapeutics for human diseases especially that caused by viral infections (19, 20, 27-30).
Importantly, it is presently not possible to predict with any degree of confidence which of many possible candidate siRNA sequences potentially targeting a viral genome sequence (e.g., oligonucleotides of about 16-30 base pairs) will in fact exhibit effective siRNA activity. Instead, individual specific candidate siRNA polynucleotide or oligonucleotide sequences must be generated and tested to determine whether the intended interference with expression of a targeted gene has occurred. Accordingly, no routine method exists in the art for designing a siRNA polynucleotide that is, with certainty, capable of specifically altering the expression of a given mRNA.
There remains a significant need for compositions and methods that inhibit expression of viral pathogen genes and their cognate protein products. In particular there is an urgent need for compositions and methods to inhibit expression of pathogenic respiratory viral genes in virus-infected-cells, and for treating a respiratory viral infection in a subject. There further is a need for compositions and methods addressing infection by RSV and avian influenza A, especially the H5N1 strain. There additionally is a need for compositions and methods for treatment that are highly effective, and do not rely on use or modification of known antiviral agents. The present invention addresses these and related needs.
The present invention provides compositions and methods related to use of RNA interference to inhibit viral infection and replication through disruption of viral RNA molecules of viral pathogens, such as those causing respiratory viral infections including influenza A H5N1 and RSV. These viruses are pathogens causing severe respiratory diseases in humans and other mammals. Inhibition of viral replication will combat the viral infection in cultured cells and in subjects infected with the virus, including relief from its symptoms.
In a first aspect, the invention provides an isolated polynucleotide whose length can be any number of nucleotides that is 200 or fewer, and 15 or greater. The polynucleotide includes a first nucleotide sequence that targets the genome of a respiratory syncytial virus or an influenza A virus. In the polynucleotide any T (thymidine) or any U (uridine) may optionally be substituted by the other. Additionally, in the polynucleotide the first nucleotide sequence consists of a) a sequence whose length is any number of nucleotides from 15 to 30, or b) a complement of a sequence given in a). Such a polynucleotide may be termed a linear polynucleotide herein.
In a related aspect of the invention, the polynucleotide described above further includes a second nucleotide sequence separated from the first nucleotide sequence by a loop sequence, such that the second nucleotide sequence
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, termed a hairpin polynucleotide, the first nucleotide sequence hybridizes with the second nucleotide sequence to form a hairpin whose complementary sequences are linked by the loop sequence.
In many embodiments of the linear polynucleotide and of the hairpin polynucleotide the first nucleotide sequence is either
In various embodiments of a linear polynucleotide or a hairpin polynucleotide the length of the first nucleotide sequence is any number of nucleotides from 21 to 25. In many embodiments a linear polynucleotide or a hairpin polynucleotide consists of a sequence chosen from SEQ ID NOS:1-263, and optionally includes a dinucleotide overhang bound to the 3′ of the chosen sequence. In yet additional embodiments of a linear polynucleotide or a hairpin polynucleotide the dinucleotide sequence at the 3′ end of the first nucleotide sequence is TT, TU, UT, or UU and includes either ribonucleotides or deoxyribonucleotides or both. In various further embodiments a linear or hairpin polynucleotide may be a DNA, or it may be an RNA, or it may be composed of both deoxyribonucleotides and ribonucleotides.
In an additional aspect the invention provides a double stranded polynucleotide that includes a first polynucleotide strand described in claim 1 and a second polynucleotide strand that is complementary to at least the first nucleotide sequence of the first strand and is hybridized thereto to form a double stranded composition. These polynucleotide structures may also be termed linear polynucleotides.
In still a further aspect the invention provides a combination that includes two or more targeting polynucleotides described in claim 1, claim 2, or both, such that each polynucleotide of the combination targets a different sequence in the genome of the target virus.
Because of the high degree of similarity or identity to the respiratory viral pathogen target, and not wishing to be bound by theory, it is believed that upon introduction within a virally infected cell the polynucleotide induces RNA interference, leading to digestion of the pathogen genomic RNA, complementary RNA, and messenger RNA. In particular, in important embodiments of these aspects of the invention it is believed that the first nucleotide sequence or its complement in these polynucleotides forms an RNA Induced Silencing Complex (RISC) that introduces the polynucleotide siRNA sequence to the pathogen genomic RNA sequence, thereby promoting cleavage of the pathogen genomic RNA.
In additional aspects the invention provides a vector that harbors a sequence given by a linear polynucleotide or a hairpin polynucleotide of the invention. In various embodiments any of these vectors may be a plasmid, a recombinant virus, a transposon, or a minichromosome. Still additional aspects provide cells transfected by one or more linear polynucleotides of the invention, or by one or more hairpin polynucleotides of the invention.
In still further aspects the invention provides a pharmaceutical composition that contains one or more linear polynucleotides or hairpin polynucleotides, or a mixture thereof, wherein each polynucleotide targets a different sequence in the genome of the target virus, and a pharmaceutically acceptable carrier.
In yet an additional aspect the invention provides a pharmaceutical composition containing one or more vectors harboring a linear polynucleotide, or a vector harboring a hairpin polynucleotide, or a mixture thereof, wherein each vector harbors a polynucleotide targeting a different sequence in the genome of the target virus, and a pharmaceutically acceptable carrier.
In various embodiments of the pharmaceutical compositions, the carrier includes a synthetic cationic polymer, a liposome, dextrose, a surfactant, or a combination of any two or more of them.
In still a further aspect the invention provides a method of synthesizing a linear polynucleotide or a hairpin polynucleotide having a sequence that targets the genome of a respiratory syncytial virus or an influenza A virus. The method includes the steps of
In still an additional aspect the invention provides methods for transfecting a cell with an RNA inhibitor wherein the method includes contacting the cell with a composition containing one or more linear polynucleotides, or one or more hairpin polynucleotides. In many embodiments the cell so transfected includes a respiratory virus that is targeted by the one or more polynucleotides.
In yet an additional aspect the invention provides a method of inhibiting replication of a respiratory virus in a cell infected with the virus that includes contacting the cell with a composition containing one or more linear polynucleotides, or one or more hairpin polynucleotides, wherein the one or more polynucleotides target the virus.
In still a further aspect the invention provides a use of a linear polynucleotide that targets a respiratory virus, or of a mixture of two or more of them, or the use of a hairpin polynucleotide targeting a respiratory virus, or of a mixture of two or more of them, in the manufacture of a pharmaceutical composition effective to treat an infection due to the respiratory virus in a subject. In various embodiments of the use, the subject is a human.
In yet an additional aspect the invention provides a method of treating an infection due to a respiratory virus in a subject. The method includes administering an effective dose of a linear polynucleotide targeting the respiratory virus, or of a mixture of two or more of them, or an effective dose of a hairpin polynucleotide targeting the respiratory virus, or a mixture of two or more of them, to the subject. In various embodiments of this method, the subject is a human. In additional embodiments both a linear polynucleotide and a hairpin polynucleotide is administered to the subject.
All patents, patent application publications, and patent applications identified herein are incorporated by reference in their entireties, as if appearing herein verbatim. All technical publications identified herein are also incorporated by reference.
In the present description, the articles “a”, “an”, and “the” relate equivalently to a meaning as singular or as plural. The particular sense for these articles is apparent from the context in which they are used.
As used herein the term “target” sequence and similar terms and phrases relate to a nucleotide sequence that occurs in the genome of a pathogen against which a polynucleotide of the invention is directed. A polynucleotide targets a pathogen sequence either a) by including a sequence that is homologous or identical to a particular subsequence (termed a target sequence) contained within the genome of the pathogen, or b) by including a sequence whose complement is homologous or identical to the target sequence. It is believed that any polynucleotide so targeting 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 whose bases form complementary base pairs, base by base, as conventionally understood by workers of skill in fields such as biochemistry, molecular biology, genomics, and similar fields related to the field of the invention.
As used herein, a first sequence or subsequence is “identical”, or has “100% identity”, or is described by a term or phrase conveying the notion of 100% identity, to a second sequence or subsequence when the first sequence or subsequence has the same base as the second sequence or subsequence at every position of the sequence or subsequence. In determining identity, any particular base position containing a T (thymidine) or any derivative thereof, or a U (uridine) or any derivative thereof, are equivalent to each other, and so considered identical.
As described herein, a sequence of a targeting polynucleotide, or its complement, may be completely identical to the target sequence, or it may include mismatched bases at particular positions in the sequence. Incorporation of mismatches is described fully herein. Without wishing to be bound by theory, it is believed that incorporation of mismatches provides an intended degree of stability of hybridization under physiological conditions to optimize the RNA interference phenomenon for the particular target sequence in question. The extent of identity determines the percent of the positions in the two sequences whose bases are identical to each other. The “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T or U, C, G, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield 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 the percent similarity are synonymous terms relating to the percent of identity between two sequences or subsequences as determined in the following paragraphs. For example, two sequences displaying at least 60% identity, 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 more preferably at least 85% identity, or more preferably at least 90% identity, or still more preferably at least 95% identity, to each other are “similar” or “homologous” to each other. Alternatively, with reference to the oligonucleotide sequence of an siRNA molecule, two sequences that differ by 5 or fewer bases, or by 4 or fewer bases, or by 3 or fewer bases, or by two or fewer bases, or by one base, are termed “similar” or “homologous” to each other.
“Identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by, comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk. A. 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., and Griffin, H. G., eds. 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., eds., M Stockton Press. New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math. (1988) 48: 1073. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devercux, J., et al. (1984) Nucleic Acids Research 12(1): 387), BLASTP, BLASTN, and FASTA (Atschul, S. F. 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. Mol. Biol. 215: 403-410. The well known Smith Waterman algorithm may also be used to determine identity.
Parameters for sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970).
Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, (1992) Proc. Natl. Acad. Sci. USA. 89:10915-10919.
As used herein, the term “isolated”, and similar words, when used to describe a nucleic acid, a polynucleotide, or an oligonucleotide relate to being removed from its natural or original state. Thus, if it occurs in nature, it has been removed from its original environment. If it has been prepared synthetically, it has been removed from an original product mixture resulting from the synthesis. For example, a naturally occurring polynucleotide naturally present in a living organism in its natural state is not “isolated,” but the same polynucleotide separated from materials with which it coexists in its natural state is “isolated”, as the term is employed herein. Generally, removal of at least one significant coexisting material constitutes “isolating” a nucleic acid, a polynucleotide, an oligonucleotide. In many cases several, many, or most coexisting materials may be removed to isolate the nucleic acid, a polynucleotide, an oligonucleotides, a protein, a polypeptide, or an oligopeptide. By way of nonlimiting example, with respect to polynucleotides, the term “isolated” may mean that it is separated from the chromosome and cell in which it naturally occurs. Further by way of example, “isolating” a protein or polypeptide may mean separating it from another component in a cell lysate or cell homogenate.
A nucleic acid, a polynucleotide, or an oligonucleotide that is the product of an in vitro synthetic process or a chemical synthetic process is essentially isolated as the result of the synthetic process. In important embodiments such synthetic products are treated to remove reagents and precursors used, and side products produced, by the process.
Similarly, the polynucleotides and polypeptides may occur in a composition, such as a formulation, a composition for introduction of polynucleotides into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein.
As used herein and in the claims, a “nucleic acid” or “polynucleotide”, and similar terms based on these, refer to polymers composed of naturally occurring nucleotides as well as to polymers composed of synthetic or modified nucleotides. Thus, as used herein, a polynucleotide that is a RNA, or a polynucleotide that is a DNA may include naturally occurring moieties such as the naturally occurring bases and ribose or deoxyribose rings, or they may be composed of synthetic or modified moieties as described in the following. The linkages between nucleotides is commonly the 3′-5′ phosphate linkage, which may be a natural phosphodiester linkage, a phosphothioester linkage, and still other synthetic linkages. Examples of modified backbones include, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates. Additional linkages include phosphotriester, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate and sulfone internucleotide linkages. Other polymeric linkages include 2′-5′ linked analogs of these. See U.S. Pat. Nos. 6,503,754 and 6,506,735 and references cited therein, incorporated herein by reference.
Nucleic acids and polynucleotides may be 20 or more nucleotides in length, or 30 or more nucleotides in length, or 50 or more nucleotides in length, or 100 or more, or 1000 or more, or tens of thousands or more, or hundreds of thousands or more, in length. An siRNA may be a polynucleotide as defined herein. As used herein, “oligonucleotides” and similar terms based on this relate to short polymers composed of naturally occurring nucleotides as well as to polymers composed of synthetic or modified nucleotides, as described in the immediately preceding paragraph. Oligonucleotides may be 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, up to about 50, nucleotides in length. An oligonucleotide that is an siRNA may have any number of nucleotides between 15 and 30 nucleotides. In many embodiments an siRNA may have any number of nucleotides between 21 and 25 nucleotides.
It is understood from the definitions just provided that, because of the overlap in size ranges the term “polynucleotide” and “oligonucleotide” may be used synonymously herein to refer to an siRNA of the invention.
As used herein and in the claims “nucleotide sequence”, “oligonucleotide sequence” or “polynucleotide sequence”, and similar terms, relate interchangeably both to the sequence of bases that an oligonucleotide or polynucleotide has, as well as to the oligonucleotide or polynucleotide structure possessing the sequence. A nucleotide sequence or a polynucleotide sequence furthermore relates to any natural or synthetic polynucleotide or oligonucleotide in which the sequence of bases is defined by description or recitation of a particular sequence of letters designating bases as conventionally employed in the field.
The bases in oligonucleotides and polynucleotides may be “unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). In addition they may be bases with modifications or substitutions. As used herein, nonlimiting examples of modified bases include other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-fluoro-adenine, 2-amino-adenine, 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-clamps such as a substituted phenoxazine cytidine (e.g. 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 may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further bases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition (1991) 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. 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, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications. See U.S. Pat. Nos. 6,503,754 and 6,506,735 and references cited therein, incorporated herein by reference. Use of any modified base is equivalent to use of a naturally occurring base having the same base-pairing properties, as understood by a worker of skill in the art.
As used herein and in the claims, the term “complementary” and similar words based on this, relate to the ability of a first nucleic acid base in one strand of a nucleic acid, polynucleotide or oligonucleotide to interact specifically only with a particular second nucleic acid base in a second strand of a nucleic acid, polynucleotide or oligonucleotide. By way of nonlimiting example, if the naturally occurring bases are considered, A and T or U interact with each other, and G and C interact with each other. As employed in this invention and in the claims, “complementary” is intended to signify “fully complementary”, namely, that when two polynucleotide strands are aligned with each other, there will be at least a portion of the strands in which each base in a sequence of contiguous bases in one strand is complementary to an interacting base in a sequence of contiguous bases of the same length on the opposing strand.
As used herein, “hybridize”, “hybridization” and similar words relate to a process of forming a nucleic acid, polynucleotide, or oligonucleotide duplex by causing strands with complementary sequences to interact with each other. The interaction occurs by virtue of complementary bases on each of the strands specifically interacting to form a pair. The ability of strands to hybridize to each other depends on a variety of conditions, as set forth below. Nucleic acid strands hybridize with each other when a sufficient number of corresponding positions in each strand are occupied by nucleotides that can interact with each other. It is understood by workers of skill in the field of the present invention, including by way of nonlimiting example molecular biologists and cell biologists, that the sequences of strands forming a duplex need not be 100% complementary to each other to be specifically hybridizable.
As used herein “fragment” and similar words based on this, relate to portions of a nucleic acid, polynucleotide or oligonucleotide shorter than the full sequence of a reference. The sequence of bases in a fragment is unaltered from the sequence of the corresponding portion of the molecule from which it arose; there are no insertions or deletions in a fragment in comparison with the corresponding portion of the molecule from which it arose. As contemplated herein, a fragment of a nucleic acid or polynucleotide, such as 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 or more bases in length, up to a length that is one base shorter than the full length sequence. Oligonucleotides may be chemically synthesized and may be used as siRNAs, PCR primers, or probes.
Detection and Labeling. A targeting polynucleotide, such as a polynucleotide that includes an siRNA sequence, as well as a viral polynucleotide target, may be detected in many ways. Detecting may include any one or more processes that result in the ability to observe the presence and or the amount of a targeting polynucleotide. In one embodiment a sample nucleic acid containing a targeting polynucleotide or a viral target may be detected prior to expansion. In an alternative embodiment a targeting polynucleotide in a sample may be expanded to provide an expanded targeting polynucleotide, or an expanded viral target, and the expanded polynucleotide is detected or quantitated. Physical, chemical or biological methods may be used to detect and quantitate a targeting polynucleotide. Physical methods include, by way of nonlimiting example, surface plasmon resonance (SPR) detection such as binding a probe to a surface and using SPR to detect binding of a targeting polynucleotide to the immobilized probe, or having a probe in a chromatographic medium and detecting binding of a targeting polynucleotide in the chromatographic medium. Physical methods further include a gel electrophoresis or capillary electrophoresis format in which targeting polynucleotides are resolved from other polynucleotides, and the resolved targeting polynucleotides are detected. Chemical methods include polymerase chain reaction (PCR) methods, and hybridization methods generally in which a targeting polynucleotide hybridizes to a probe. Biological methods include causing a targeting polynucleotide or a target polynucleotide to exert a biological effect on a cell, and detecting the effect. The present invention discloses examples of biological effects which may be used as a biological assay. These include enumeration of virions by particle counting, plaque assays, evaluation of cytopathic effects on infected cells, and the like. In many embodiments, the polynucleotides may be labeled as described below to assist in detection and quantitation. For example, in embodiments not including expansion, a sample nucleic acid may be labeled by chemical or enzymatic addition of a labeled moiety such as a labeled nucleotide or a labeled oligonucleotide linker.
Expanded polynucleotides may be detected and/or quantitated directly. For example, an expanded polynucleotide may be subjected to electrophoresis in a gel that resolves by size, and stained with a dye that reveals its presence and amount. Alternatively an expanded targeting polynucleotide may be detected upon exposure to a probe nucleic acid under hybridizing conditions (see below) and binding by hybridization is detected and/or quantitated. Detection is accomplished in any way that permits determining that a targeting polynucleotide has bound to the probe. This can be achieved by detecting the change in a physical property of the probe brought about by hybridizing a fragment. A nonlimiting example of such a physical detection method is SPR.
An alternative way of accomplishing detection is to use a labeled form of the expanded polynucleotide, and to detect the bound label. A label may be a radioisotopic label, such as 125I, 35S, 32P, 14C, or 3H, for example, that is detectable by its radioactivity. Alternatively, a label may be selected such that it can be detected using a spectroscopic method, for example by fluorescence, phosphorescence, or chemiluminescence. Thus a label that fluoresces, or that phosphoresces, or that induces a chemiluminescent reaction, may be employed. A label may still further be a ligand in a specific ligand-receptor pair; the presence of the ligand is then detected by the secondary binding of the specific receptor, which commonly is itself labeled for detection.
Interfering RNA
According to the invention, gene expression of respiratory viral targets is attenuated by RNA interference. Expression products of a viral gene are targeted by specific double stranded siRNA nucleotide sequences that are complementary to at least a segment of the viral target that contains any number of nucleotides between 15 and 30, or in many cases, contains anywhere between 21 and 25 nucleotides. The target may occur in the 5′ untranslated (UT) region, in a coding sequence, or in the 3′ UT region. See, e.g., PCT applications WO00/44895, WO99/32619, WO01/75164, WO01/92513, WO 01/29058, WO01/89304, WO02/16620, and WO02/29858, each incorporated by reference herein in their entirety.
According to the methods of the present invention, respiratory viral gene expression, and thereby respiratory viral replication, is suppressed using siRNA. A targeting polynucleotide according to the invention includes an siRNA oligonucleotide. Such an siRNA can also be prepared by chemical synthesis of nucleotide sequences identical or similar to a viral sequence. See, e.g., Tuschl, Zamore, Lehmann, Bartel and Sharp (1999), Genes & Dev. 13: 3191-3197, incorporated herein by reference in its entirety. Alternatively, a targeting siRNA can be obtained using a targeting polynucleotide sequence, for example, by digesting a respiratory viral ribopolynucleotide sequence in a cell-free system, such as but not limited to a Drosophila extract, or by transcription of recombinant double stranded viral cRNA.
Efficient silencing is generally observed with siRNA duplexes composed of a 16-30 nt sense strand and a 16-30 nt antisense strand of the same length. In many embodiments each strand of an siRNA paired duplex has in addition a 2-nt overhang at the 3′ end. The sequence of the 2-nt 3′ overhang makes an additional small contribution to the specificity of siRNA target recognition. In one embodiment, the nucleotides in the 3′ overhang are ribonucleotides. In an alternative embodiment, the nucleotides in the 3′ overhang are deoxyribonucleotides. Use of 3′ deoxynucleotides provides enhanced intracellular stability.
A recombinant expression vector of the invention, when introduced within a cell, is processed to provide an RNA that includes an siRNA sequence targeting a respiratory virus. Such a vector is a DNA molecule cloned into an expression vector comprising operatively-linked regulatory sequences flanking the viral targeting sequence in a manner that allows for expression. From the vector, an RNA molecule that is antisense to viral RNA is transcribed by a first promoter (e.g., a promoter sequence 3′ of the cloned DNA) and an RNA molecule that is the sense strand for the viral RNA target is transcribed by a second promoter (e.g., a promoter sequence 5′ of the cloned DNA). The sense and antisense strands then hybridize in vivo to generate siRNA constructs targeting the respiratory virus for silencing of the viral gene. Alternatively, two constructs can be utilized to create the sense and anti-sense strands of a siRNA construct. Further, cloned DNA can encode a transcript having secondary structure, wherein a single transcript has both the sense and complementary antisense sequences from the target gene or genes. In an example of this embodiment, a hairpin RNAi product is similar to all or a portion of the target gene. In another example, a hairpin RNAi product is a siRNA. The regulatory sequences flanking the viral sequence may be identical or may be different, such that their expression may be modulated independently, or in a temporal or spatial manner.
In certain embodiments, siRNAs are transcribed intracellularly by cloning the viral gene templates into a vector containing, e.g., a RNA pol III transcription unit from the smaller nuclear RNA (snRNA) U6 or the human RNase P RNA H1. One example of a vector system is the GeneSuppressor™ RNA Interference kit (commercially available from Imgenex). The U6 and H1 promoters are members of the type III class of Pol III promoters. The +1 nucleotide of the U6-like promoters is always guanosine, whereas the +1 for H1 promoters is adenosine. The termination signal for these promoters is defined by five consecutive thymidines. The transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3′ UU overhang in the expressed siRNA, which is similar to the 3′ overhangs of synthetic siRNAs. Any sequence less than 400 nucleotides in length can be transcribed by these promoter, therefore they are ideally suited for the expression of around 21-nucleotide siRNAs in, e.g., an approximately 50-nucleotide RNA stem-loop transcript. The characteristics of RNAi and of factors affecting siRNA efficacy have been studied (See, e.g., Elbashir, Lendeckel and Tuschl (2001). Genes & Dev. 15: 188-200).
An initial BLAST homology search for the selected siRNA sequence is done against an available nucleotide sequence library to ensure that only a viral gene, but no host gene, is targeted. See, Elbashir et al. 2001 EMBO J. 20(23):6877-88.
Synthesis of Polynucleotides
Oligonucleotides corresponding to targeting nucleotide sequences, and polynucleotides that include targeting sequences, can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer. Methods for synthesizing oligonucleotides include well-known chemical processes, including, but not limited to, sequential addition of nucleotide phosphoramidites onto surface-derivatized particles, as described by T. Brown and Dorcas J. S. Brown in Oligonucleotides and Analogues A Practical Approach, F. Eckstein, editor, Oxford University Press, Oxford, pp. 1-24 (1991), and incorporated herein by reference.
An example of a synthetic procedure uses Expedite RNA phosphoramidites and thymidine phosphoramidite (Proligo, Germany). Synthetic oligonucleotides are deprotected and gel-purified (Elbashir et al. (2001) Genes & Dev. 15, 188-200), followed by Sep-Pak C18 cartridge (Waters, Milford, Mass., USA) purification (Tuschl et al. (1993) Biochemistry, 32:11658-11668). Complementary ssRNAs are incubated in an annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90° C. followed by 1 h at 37° C. to hybridize to the corresponding ds-siRNAs.
Other methods of oligonucleotide synthesis include, but are not limited to solid-phase oligonucleotide synthesis according to the phosphotriester and phosphodiester methods (Narang, et al., (1979) Meth. Enzymol. 68:90), and to the H-phosphonate method (Garegg, P. J., et al., (1985) “Formation of internucleotidic bonds via phosphonate intermediates”, Chem. Scripta 25, 280-282; and Froehler, B. C., et al., (1986a) “Synthesis of DNA via deoxynucleoside H-phosphonate intermediates”, Nucleic Acid Res., 14, 5399-5407, among others) and synthesis on a support (Beaucage, et al. (1981) Tetrahedron Letters 22:1859-1862) as well as phosphoramidate techniques (Caruthers, M. H., et al., “Methods in Enzymology,” Vol. 154, pp. 287-314 (1988), U.S. 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 others described in “Synthesis and Applications of DNA and RNA,” S. A. Narang, editor, Academic Press, New York, 1987, and the references contained therein, and nonphosphoramidite techniques. Solid phase synthesis helps isolate the oligonucleotide from impurities and excess reagents. Once cleaved from the solid support the oligonucleotide may be further isolated by known techniques.
Inhibitory Polynucleotides of the Invention
The invention provides broadly for oligonucleotides intended to provoke an RNA interference phenomenon upon entry into a cell infected with a respiratory viral pathogen. The present invention, while not restricted in the nature of a respiratory virus target, emphasizes oligonucleotides targeting RSV and several strains of influenza A. RNA interference is engendered within the cell by appropriate double stranded RNAs one of whose strands is identical to or highly similar to a sequence in a target gene of the virus. In general, an oligonucleotide that targets a respiratory virus may be a DNA or an RNA, or it may contain a mixture of ribonucleotides and deoxyribonucleotides. An example of the latter is an RNA sequence terminated at the 3′ end with a deoxydinucleotide sequence, such as d(TT), d(UU), d(TU), d(UT), as well as other possible dinucleotides. In additional embodiments the 3′ overhang may be constituted of ribonucleotides having the bases specified above, or others. Furthermore, the oligonucleotide pharmaceutical agent may be single stranded or double stranded. Several embodiments of the therapeutic oligonucleotides of the invention are envisioned to be oligoribonucleotides, or oligoribonucleotides with 3′ d(TT) terminals. A single stranded targeting polynucleotide, upon entry into a mammalian cell, is readily converted to a double stranded molecule by endogenous enzyme activity resident in the cell.
Most generally the invention provides oligonucleotides or polynucleotides that may range in length anywhere from 15 nucleotides to as long as 200 nucleotides. The polynucleotides include a first nucleotide sequence that targets the genome of a respiratory syncytial virus or an influenza A virus. The first nucleotide sequence consists of either a) a sequence whose length is any number of nucleotides from 15 to 30, or b) a complement thereof. Such a polynucleotide is termed a linear polynucleotide herein.
The invention additionally provides that, in the targeting polynucleotide, a sequence chosen from SEQ ID NOS:1-263 may be part of a longer targeting sequence such that the targeting polynucleotide targets a sequence in the viral genome that is longer than the first nucleotide sequence represented by SEQ. This is illustrated in
The invention further provides a sequence that is a fragment of any of SEQ ID NOS:1-263 that is at least 15 nucleotides in length (and at most 1 base shorter than the reference SEQ ID NO:; illustrated in
Still further the invention provides a sequence that is a complement to any of the above-described sequences (shown in
Thus the polynucleotide may be single stranded, or it may be double stranded. In still further embodiments, the polynucleotide contains only deoxyribonucleotides, or it contains only ribonucleotides, or it contains both deoxyribonucleotides and ribonucleotides. In important embodiments of the polynucleotides described herein the target sequence consists of a sequence that may be either 15 nucleotides (nt), or 16 nt, or 17 nt, or 18 nt, or 19 nt, or 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 length. In still additional advantageous embodiments the targeting sequence may differ by up to 5 bases from a target sequence in the viral pathogen genome.
In several embodiments of the invention, the polynucleotide is an siRNA consisting of the targeting sequence with optional inclusion of a 3′ dinucleotide overhang described herein.
Alternatively, in recognition of the need for a double stranded RNA in RNA interference, the oligonucleotide or polynucleotide may be prepared to form an intramolecular hairpin looped double stranded molecule. Such a molecule is formed of a first sequence described in any of the embodiments of the preceding paragraphs followed by a short loop sequence, which is then followed in turn by a second sequence that is complementary to the first sequence. Such a structure forms the desired intramolecular hairpin. Furthermore, this polynucleotide is disclosed as also having a maximum length of 200 nucleotides, such that the three required structures enumerated may be constituted in any oligonucleotide or polynucleotide having any overall length of up to 200 nucleotides. A hairpin loop polynucleotide is illustrated in
RSV Strains as Targets
For RSV, although any portion of the viral genome may be targeted, it is reasonable that targeting genes that code for viral-specific enzymatic functions should provide potent siRNA candidates. These include the L, F, G and P genes. The L gene, located at the 5′ end of the viral genome, is expressed in only small amounts, so that effective RNAi silencing may require only small amounts of siRNA. The structural genes may also be targeted.
Sequences of the following representative strains of RSV subgroups A and B were used to select target sequences that are either common between subgroups A and B (Table 1) or specific for subgroup A (Table 2) or B (Table 3):
The viral gene sequences were aligned to seek common or unique regions. For each targeted gene or region at least two targets were chosen unless not applicable (NA). It was more difficult to find target sequences common to both subgroups than to find targets unique for either of them, since there are very few homologous or identical sequences available. In
this case (Table 1), it was sometimes necessary to introduce a mismatch into the 5′ base of one end of the RNA duplex.
In order provide siRNA agents directed to the 5′-end of the viral genome, trail-targets unique to either strain B1 or strain 9320 individually are shown below (Table 4). The 5′ terminus of the (+) strand (anti-genomic RNA) is targeted, i.e., the nascent leader sequence produced when the viral genome begins to replicate using the positive RNA strand (the antigenomic RNA) as template.
represent mismatches.
Based upon the targets listed above in Table 1-Table 4, siRNAs may be created as follows: a) Each strand of a double stranded siRNA are provided with a 3′-dTdT overhang b) In case a “mismatch” is needed, G:C are changed to G:T or G:A; A:T will be changed to A:C or A:G; and C:G will be changed to C:T or C:A.
Influenza a Strains as Targets
siRNA target sequences of lengths of 25 and 19 nucleotides within the (+) stranded mRNA sequences have been identified in all eight segments. The sequences are presented in Table 5-Table 12. siRNA sequences s from each of the eight segments. The entry for each sequence also shows the starting nucleotide location in the mRNA from the 5′ end.
Another approach for selection of the potent siRNA candidate sequences targeting influenza A is to focus on the subtype-specificity of the two major viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The subtypes that have been reported to cause human infection are H5N1, H7N7, and H9N2. The following are examples of the subtype-specific HA and NA targets.
The sequences in Table 13 are shared by six H5N1 strains isolated from chicken infected by HPAI viruses in China between year 2000 and 2003. These H5N1 strains are listed in the Table 14.
The above sequences are shared by six H5N1 strains isolated from chicken or swine infected by in China between year 2001 and 2003. These H5N1 strains are listed in Table 16 below.
All these above sequences are shared by six H7N7 strains isolated from Mallard in Sweden in year 2002. These H7N7 strains are listed in Table 18 below.
By comparison of the six HA sequences, it was found the HA1 related domain has higher frequency of point mutations, therefore, 10 targets are chosen from the HA2 domain.
Additional siRNA duplexes against several influenza A H5N1 mRNA sequences were identified. These are shown in Table 19. Their positions are illustrated in
Combinations of siRNA
Several embodiments of the invention provide pharmaceutical compositions containing two or more oligonucleotides or polynucleotides each of which includes a sequence targeting genes in the genome of a respiratory virus. Related embodiments provide methods of treating cells, and methods of treating respiratory viral infections, using the combinations, as well as uses of such combination compositions in the manufacture of pharmaceutical compositions intended to treat respiratory viral infections. The individual polynucleotide components of the combination may target different portions of the same gene, or different genes, or several portions of one gene as well as more than one gene, in the genome of the viral pathogen. An advantage of using a combination of oligonucleotides or polynucleotides is that the benefits of inhibiting expression of a given gene are multiplied in the combination. Greater efficacy is achieved in knocking down a gene or silencing a viral genome by use of multiple targeting sequences. Enhanced efficiency in inhibiting viral replication is achieved by targeting more than one gene in the viral genome.
Pharmaceutical Compositions
The targeting polynucleotides of the invention are designated “active compounds” or “therapeutics” herein. These therapeutics can be incorporated into pharmaceutical compositions suitable for administration to a subject.
As used herein, “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
Suitable carriers are described in textbooks such as Remington's Pharmaceutical Sciences, Gennaro A R (Ed.) 20th edition (2000) Williams & Wilkins P A, USA, and Wilson and Gisvold's Textbook of Organic Medicinal and Pharmaceutical Chemistry, by Delgado and Remers, Lippincott-Raven., which are incorporated herein by reference. Preferred examples of components that may be used in such carriers or diluents include, but are not limited to, water, saline, phosphate salts, carboxylate salts, amino acid solutions, Ringer's solutions, dextrose (a synonym for glucose) solution, and 5% human serum albumin. By way of nonlimiting example, dextrose may used as 5% or 10% aqueous solutions. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral, nasal, inhalation, transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intravenous, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., 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 elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release pharmaceutical active agents over shorter time periods. Advantageous polymers are biodegradable, or biocompatible. Liposomal suspensions (including liposomes targeted to 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 U.S. Pat. No. 4,522,811. Sustained-release preparations having advantageous forms, such as microspheres, can be prepared from materials such as those 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, e.g., as described in U.S. Pat. No. 5,703,055. Delivery can thus also include, e.g., intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a kit, e.g., in a container, pack, or dispenser together with instructions for administration.
Also within the invention is the use of a therapeutic in the manufacture of a pharmaceutical composition or medicament for treating a respiratory viral infection in a subject.
Delivery
In several embodiments the siRNA polynucleotides of the invention are delivered into cells in culture by liposome-mediated transfection, for example by using commercially available reagents or techniques, e.g., Oligofectamine™, LipofectAmine™ reagent, LipofectAmine 2000™ (Invitrogen), as well as by electroporation, and similar techniques. Additionally siRNA polynucleotides are, is delivered to animal models, such as rodents or non-human primates, through inhalation and instillation into the respiratory tract. Additional routes for use with animal models include intravenous (IV), subcutaneous (SC), and related routes of administration. The pharmaceutical compositions containing the siRNAs include additional components that protect the stability of siRNA, prolong siRNA lifetime, potentiate siRNA function, or target siRNA to specific tissues/cells. These include a variety of biodegradable polymers, cationic polymers (such as polyethyleneimine), cationic copolypeptides such as histidine-lysine (HK) polypeptides see, for example, PCT publications WO 01/47496 to Mixson et al., WO 02/096941 to Biomerieux, and WO 99/42091 to Massachusetts Institute of Technology), PEGylated cationic polypeptides, and ligand-incorporated polymers, etc. positively charged polypeptides, PolyTran polymers (natural polysaccharides, also known as scleroglucan), a nano-particle consists of conjugated polymers with targeting ligand (TargeTran variants), surfactants (Infasurf; Forest Laboratories, Inc.; ONY Inc.), and cationic polymers (such as polyethyleneimine). Infasurf® (calfactant) is a natural lung surfactant isolated from calf lung for use in intratracheal instillation; it contains phospholipids, neutral lipids, and hydrophobic surfactant-associated proteins B and C. The polymers can either be uni-dimensional or multi-dimensional, and also could be microparticles or nanoparticles with diameters less than 20 microns, between 20 and 100 microns, or above 100 micron. The said polymers could carry ligand molecules specific for receptors or molecules of special tissues or cells, thus be used for targeted delivery of siRNAs. The siRNA polynucleotides are also delivered by cationic liposome based carriers, such as DOTAP, DOTAP/Cholesterol (Qbiogene, Inc.) and other types of lipid aqueous solutions. In addition, low percentage (5-10%) glucose aqueous solution, and Infasurf are effective carriers for airway delivery of siRNA30.
Using fluorescence-labeled siRNA suspended in an oral-tracheal delivery solution of 5% glucose and Infasurf examined by fluorescence microscopy, it has been shown that after siRNA is delivered to mice via the nostril or via the oral-tracheal route, and washing the lung tissues the siRNA is widely distributed in the lung (see co-owned WO 2005/01940, incorporated by reference herein in its entirety). The delivery of siRNA into the nasal passage and lung (upper and deeper respiratory tract) of mice was shown to successfully silence the indicator genes (GFP or luciferase) delivered simultaneously with the siRNA in a plasmid harboring a fusion of the indicator gene and the siRNA target (see co-owned WO 2005/01940). In addition, experiments reported by the inventors, working with others, have demonstrated that siRNA species inhibit the replication of SARS coronavirus, thus relieving the lung pathology, in the SARS-infected rhesus monkeys30.
siRNA Recombinant Vectors
Another aspect of the invention pertains to vectors, preferably expression vectors, containing an siRNA polynucleotide of the 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, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel (1990) G
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type such as a cell of the respiratory tract. Tissue-specific regulatory elements are known in the art. The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector. The DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that includes an siRNA targeting a viral RNA. Regulatory sequences operatively linked to a nucleic acid can be chosen that direct the continuous expression of the RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.
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 intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, 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.
Methods of Assaying for Virus Titer or Amount
In practicing the present invention assaying for the virus may be carried out by several procedures. Among these are, by way of nonlimiting example, immunoblot (Western blot), immunoprecipitation (I.P.), and combined reverse transcription-polymerase chain reaction (RT-PCR) assay, and the like. Such procedures measure the reduction of the synthesis of the targeted mRNAs or their protein products that may present in the lysate or supernatant of the transfected tissue cultures. Likewise, these assays may be used as diagnostic procedures, to measure the reduction of the synthesis of the targeted mRNAs or their products that may present in homogenized tissue samples, nasopharyngeal washes, secretions, obtained from infected animals or human subjects.
In the RT-PCR assay methods the synthesis of viral genomic RNA is detected using primers complementary to the sequence across the junction between NS1 and NS2 ORFs. The result of this RT-PCR using NS1/NS2 primers will reflect the synthesis of the whole genomic RNA.
The methods further include assays wherein siRNA-induced interference of viral replication in tissue culture is measured by quantitative real-time PCR (RTQ-PCR), TCID50, viral plaque assay, immunofluorescence assay, and immunohistochemistry, and the like, as known to a worker of skill in the field of the invention. In addition the methods further include assaying for the presence of virus, employing the said TCID50 method to monitor the inhibition of viral replication in tissue culture, wherein the viral titer is measured by any kind of cell pathogenic endpoints, including by way of nonlimiting example, cell fusion, cytopathic effect (CPE), cell adsorption, and the like.
The methods further include assays wherein the said siRNA-mediated viral replication in tested animals is measured by RTQ-PCR, pathology, immunohistochemistry, and re-isolation of virus, and the like.
Primers are designed for RT-PCR detection that is used to measure the reduction of mRNA synthesis by RNAi. The RT reaction is initiated by hexamer or poly-dT primers; and PCR is performed by using upstream and downstream primers specific for each gene targeted by siRNA.
For the detection of genomic RNA synthesis, a pair of primers (up and down) is designed correspondent to sequences within NS1 and NS2 ORFs. The purpose of using “joint-crossing” primers, instead of primers complementary to either end of the viral genome, is to avoid the big size of whole RNA transcripts (around 15K-nt of length) that is not easy to handle. The product of this RT-PCR, by the design, will reflect the synthesis of the whole genomic RNA.
Primers that have been identified, and the sizes of the RT-PCR products, are listed below in Tables 20, 21, and 22.
This Example reports evaluation of the inhibitory effects of siRNAs on H5N1 infected cells in culture.
Design and Synthesis of siRNAs
Two siRNAs respectively targeting the NP(NP-1 & NP-2; Table 19, SEQ ID NOS:) and M2 (M2-1 & M2-2; Table 19, SEQ ID NOS:) genes of H5N1 were developed. These siRNAs target sequences conserved in several strains of H5N1 virus (e.g. gi|47834945—10-1506, gi|8452827—1-1497, gi|13925158—46-1542, gi|61927237—46-1542, gi|59940391—44->1535, gi|9802277—46-1542). The ds-siRNA, fluorescein-labeled at the 5′-sense strand, were chemically synthesized by Proligo BioTech Ltd (Paris, France). Their anti-H5N1 effects were detected in siRNA-transfected MDCK cells (Madin-Darby canine kidney; ATCC® Number: CCL-34, Manassas, Va.) infected with H5N1 virus. The inhibition of H5N1 was determined by cytopathic effects (CPE), back titration of the released virus in culture media by standard TCID50 (50% tissue culture infectious dose) protocol and quantification of intracellular viral RNA using real-time RT-PCR as described below.
Cell Culture, siRNA Transfection and H5N1 Virus Infection
MDCK cells were cultured and maintained in MEM medium with 10% fetal bovine serum (FBS, Invitrogen). Around 5000 cells were set in each well of a 96-well plate for viral infection and replication assay. The cells were transected with 100, 50, 25 and 12.5 nM siRNA mixing with 0.5 μl of Lipofectamine 2000 (Invitrogen, CA). Unrelated siRNA targeting luciferase (GL21) or siRNAs targeting SARS coronavirus (C-1) and transfectant Lipofectamine 2000 alone (C-2) were included in the experiments as negative controls. Six hours after transfection, the culture medium was removed and the cells were washed twice with PBS before H5N1 virus infection. One hundred microliters of 100 TCID50H5N1 virus (strain: A/Hong Kong/486/97)7 diluted in MEM with 1% FBS was added to the transfected cells. Culture supernatant and the cells were respectively collected at 12, 16 and 24 hours post-infection for detection of released virus titers and intracellular viral RNA copies. Twenty-four hours after infection, the CPEs were observed and recorded under phase-contrast microscopy. The experiments were performed in triplicate and repeated at least three times.
Quantitative RT-PCR
Q-RT-PCR is carried out as follows. Total intracellular RNA was isolated using RNeasy Mini kit (Qiagen, Germany), in accordance with the manufacturer's instructions. The reverse-transcription experiments were performed by oligo-dT priming using ThermoScript RT-PCR systems (Invitrogen, CA). Real-time PCR was then performed using the primers shown in Table 23.
Five μl of the RT product (template), 1 μl of each forward and reverse primers (final concentration 500 nM), 2 μl of 255 mM MgCl2, and 9 μl of H2O were mixed with 2 μl of SYBR Green I Master Mix (Roche, USA) Master Mix and real-time quantification was carried out using an ABI7900 Sequence Detection System. The PCR conditions were: 50° C. for 5 min, 95° C. for 10 min, then 40 cycles of 95° C. for 10 sec, 61° C. for 5 sec and 72° C. for 5 sec. Copies of β-actin were also measured as an internal control. The copy numbers of intracellular viral RNA per 1000 copies of β-actin were calculated and expressed as relative viral RNA copies as compared to the untreated control (copies of treated/copies of untreated×100%).
Measurement of Viral Titers
The virus titers in cultures with or without siRNA treatment were tested as follows. Briefly, the conditioned medium from infected cells was diluted in 10-fold serial steps in MEM with 1% FBS. Each dilution was used for infecting cells according to the standard TCID50 protocol. Briefly, cells were set in 96-well dishes sixteen hours before infection. Seventy-two hours post-infection, CPEs were observed under phase-contrast microscopy and CPE-positive (CPE+) cells determined. TCID50 is then evaluated as follows:
where h=an interpolated log10 value of a dilution step, which is added to the log10 step above the 50% value. The infectious viral titer was calculated and expressed as relative virus yielded as compared to the untreated control (titer of treated/titer of untreated×100%).
Results
The siRNA NP-1 (Table 19) treatment reduced H5N1 virus production in culture media (
Treatment with siRNA directed against M2-1 (Table 19) also exhibited about 80 to 94% of inhibitory effects to the virus growth (
In a second experiment, the cells in culture were treated with various siRNAs at 50 nM. The virus produced in the culture supernatant media was collected at three time points. The samples were titrated by determining the tissue culture infection dose required to infect 50% of the cells in the culture (TCID50) (
The results in this Example demonstrate that various siRNAs directed against different genes in the H5N1 genome are highly effective in inhibiting viral replication in infected cells grown in culture.
Combinations of siRNAs directed against different genes in the H5N1 genome were identified in order to provide effective anti-H5N1 activity. This Example presents two such combinations.
In this Example optimally effective siRNAs, and optimally effective combinations of siRNAs, targeting different respiratory viral genes in their ability to silence the cognate target gene(s) or inhibiting viral replication, are identified by experiments in cell culture. The siRNAs effective in vitro are to be candidates to be further tested and used in vivo.
Cultured permissive cell lines, such as, by way of nonlimiting example, A549 (ATCC® Number: CCL-185™, a type II alveolar epithelial lung carcinoma cell line) are infected with an RSV viral strain or an influenza A strain such as H5N1, and then are transfected with various siRNAs either individually or in combination. In the case of combinations, two siRNAs targeting one single gene, or a combination of siRNAs targeting two or more genes are to be employed. The total siRNA dosage of single or combination of siRNAs is kept the same. In different experimental protocols, the siRNA transfection is performed several hours prior to the RSV or influenza A infection, or simultaneously with viral infection, or several hours post-infection. These different procedures provide information on whether the tested siRNAs exhibit prophylactic and/or therapeutic effects at the cellular level.
The extent of inhibition of target gene(s), or the inhibition of viral replication is assayed in a variety of ways. Nonlimiting examples of assays include the following procedures:
1) Immunoblot (Western) is performed using cell lysates and RSV or influenza A specific antibody against a given viral antigen.
2) Immunoprecipitation (IP) is performed using cell lysates and RSV or influenza A specific antibody against single gene products as above.
3) rvtr-PCR is performed to demonstrate the inhibition of mRNA transcription or the inhibition of viral RNA replication. Special primers are designed to detect transcripts of targeted gene or detect whether a tested siRNA oligo(s) can also target the genomic RNA.
4) The measurement of cell-fusion-based TCID50 could be used to compared cell cultures treated with specific siRNA of unrelated control siRNA, to monitor the inhibition of viral replication.
5) Immunofluorescence or immunohistochemistry also can be used to titrate virus titers, thus to monitor the siRNA-mediated inhibition of viral replication.
In this Example various siRNAs, individually or their combinations, are examined to determine their efficacy in treating RSV or avian influenza H5N1 in animal models. An RSV or influenza H5N1 strain is used to infect the test animals through the airway, by inhalation or instillation. The siRNAs are delivered through the same route, and are applied prior to, simultaneously with, or after RSV or influenza H5N1 infection. By varying siRNA delivering times, it is possible to get information of the efficacy of siRNAs in inhibiting RSV or influenza H5N1 replication in the test animal, reducing RSV or influenza H5N1-induced pathology, and relieving the RSV or influenza H5N1-like symptoms, as well as the information on whether RSV or influenza H5N1-specific siRNAs demonstrate prophylactic or therapeutic effect on experimental RSV or influenza H5N1 infection in animals.
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. Infection of rodents usually results in a low to moderate level of viral replication that peaks at 4 days and is quickly clear. These animals do not show overt respiratory tract disease, but show lung pathology. At high doses of virus infection they show weigh loss, changes in pulmonary function, and ruffled fur that is indicative of disease.
Same diagnostic assays as described above in Example 3 are used to monitor the siRNA-mediated gene-silencing and inhibition of RSV or influenza H5N1 infection on samples taken from the animals, such as nasal, buccal or pharyngeal swabs. Additionally, lung pathology, lung immunohistochemistry, and symptom observation are carried out to determine the efficacy of siRNAs on RSV or influenza H5N1 infection in vivo.
This Example describes studies of siRNA-mediated inhibition of RSV or influenza H5N1 viral replication in non-human primates. The efficacy and safety of dose administration are major goals of this study.
Based upon our protocol used on rhesus monkey model for another respiratory disease, SARS, the dose of siRNA of up to 30 mg/kg weigh is tolerable, showing no signs of toxicity to the animals(22). The respiratory delivery (by inhalation and instillation) of siRNAs that had been pre-screened by in non-primate mammals showed substantial effect on inhibition of viral replication, and reduction of virus-induced pathology and disease-like symptoms. For the present RSV or influenza H5N1 study, the same delivery route is used, and similar dosages of siRNA are delivered.
In addition to the experiments described for small animal study (Example 4), the following assays are readily performed in primates and contribute to a more direct assessment of therapeutic efficacy.
1) Virus shedding: Viral shedding is measured using nasopharynx wash samples of infected monkeys. The virus yield is titrated by either TCID50 (cell-fusion based) and/or immunofluorescence assay.
2) RTQ-PCR of nasopharynx wash samples: to monitor changes of viral genome copy numbers.
3) Symptom monitoring: it is likely to find bronchiolitis symptoms, as observed in human infant patients.
Although the invention has been described and illustrated with respect to various exemplary embodiments thereof, equivalent embodiments and various other alterations, additions and omissions may be made therein and thereto without departing from the spirit and scope of the present invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2005/040048 | 11/4/2005 | WO | 00 | 6/1/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60625677 | Nov 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2005/003858 | Feb 2005 | US |
| Child | 11792179 | US |
