WO2012079115A1 - Immunostimulatory oligonucleotides - Google Patents

Abstract

The present invention relates to isolated or synthetic RNA molecules comprising a double-stranded region and which also comprise an immunostimulatory sequence. The invention also relates to the treatment and prevention of diseases in a subject by administering an isolated or synthetic RNA molecule comprising a double-stranded region and an immunostimulatory sequence.

Description

IMMUNOSTIMULATORY OLIGONUCLEOTIDES

FIELD OF THE INVENTION

The present invention relates to RNA molecules comprising a double-stranded region and an immunostimulatory sequence. The invention also relates to the treatment and prevention of diseases by administering to a subject an RNA molecule comprising a double-stranded region and an immunostimulatory sequence.

BACKGROUND OF THE INVENTION

Viral infection remains an important health problem in both humans and animals with adverse economic and social consequences. Since the first reports of virus transmission from poultry to humans in 1997, highly pathogenic avian influenza (HPAI) H5N1 virus has spread throughout much of Asia, Europe, the Middle East and Africa. Cases of H5N1 in domestic poultry and water birds have been reported in over 40 countries since 2003, with the death or preventative culling of more than 200 million chickens causing devastation amongst poultry industries (Cyranoski, 2003), particularly in South-East Asia. During 2004, widespread outbreaks of H5 1 occurred throughout Asia, where the virus crossed the species barrier to infect humans. Since then there have been almost 500 confirmed human cases of H5N1 infection reported in 12 countries (Abbott, 2003). Furthermore, the 60 % human mortality rate of H5N1 is alarming when compared to the approximate 2 % mortality rate of seasonal influenza A (Layne et al, 2009).

In the past decade a number of new viral diseases have emerged (Mackenzie and Field, 2004; Mungall et al, 2008), including the highly pathogenic and zoonotic Hendra virus (Hev) and Nipah virus (NiV). While typically presenting as an acute respiratory infection in horses, Hev has also human deaths as a result of acute respiratory disease or encephalitis. The initial outbreak of Niv infecting pigs in Malaysia resulted in 265 human cases including 105 deaths. Attempts to control the outbreak resulted in the culling of 1.1 million pigs (Mungall et al., 2008).

In addition, there are a number of other viral pathogens that cause disease in economically important livestock animals such as chickens, pigs, fish, sheep and cattle. Other viral diseases of livestock animals include Newcastle Disease, Chicken Anaemia and Infectious Bursal Disease in chickens, Foot and Mouth Disease in cloven-hoofed animals, Porcine Reproductive and Respiratory Syndrome (PRRS) and Classical Swine Fever in pigs, Bluetongue and Akabane disease in sheep, and Infectious Salmon Anemia, Infectious Hematopoietic Necrosis Virus disease (IHNV), Viral Haemorrhagic Septicaemia and Infectious Pancreatic Necrosis in fish.

Novel therapies to combat pathogenic viruses are urgently needed. While vaccines against some viruses such as H5N1 are available, the lack of strain-specific vaccines during early-stage outbreaks creates a demand for alternative therapies. Compounding this is the shortcomings of existing antivirals and their loss of utility due to emerging viral resistance. RNA interference (RNAi), the natural cellular pathway in which dsRNA input sequence is used to degrade target mRNA, is the basis for many therapeutics currently being developed against major human diseases. However, it is well-established that select RNAi molecules can trigger off-target pro-inflammatory and antiviral cytokines which in many cases cause unwanted side effects (Schlee et al, 2006).

Accordingly, there remains a need for novel therapies to combat viral diseases in humans and animals.

SUMMARY OF THE INVENTION

The present inventors describe multifunctional RNAi molecules comprising an immunostimulatory sequence, and demonstrate that the positioning of the immunostimulatory sequence in the RNAi molecules advantageously determines the immunostimulatory ability of the molecule.

Accordingly, in one aspect, the present invention provides an isolated or synthetic RNA molecule comprising a double-stranded region, wherein the double- stranded region comprises a sense strand and an antisense strand, and wherein

the antisense strand comprises a first nucleotide sequence that is complementary to a target nucleic acid, and

the sense strand comprises a second nucleotide sequence complementary to the first nucleotide sequence, and an immunostimulatory sequence 5' and/or 3' of the second nucleotide sequence.

Preferably, the isolated or synthetic RNA molecule is capable of reducing the expression of the target gene and stimulating an immune response.

In one embodiment, there are preferably five or less nucleotides 5' of the immunostimulatory sequence that is 5' of the second nucleotide sequence, and/or five or less nucleotides 3' of the immunostimulatory sequence that is 3' of the second nucleotide sequence.

In a preferred embodiment, there are no nucleotides 5' of the immunostimulatory sequence that is 5' of the second nucleotide sequence, and/or no nucleotides 3' of the immunostimulatory sequence that is 3' of the second nucleotide sequence.

In yet another embodiment, the nucleotide sequence that is complementary to the target nucleic acid is about 19 to about 50 nucleotides in length.

In another embodiment, the nucleotide sequence that is complementary to the target nucleic acid comprises one or more mismatches with the target nucleic acid.

In yet another embodiment, the immunostimulatory sequence comprises one or more mismatches with the sense strand.

In one embodiment, the nucleotide sequence that is complementary to the target nucleic acid is perfectly complementary to the target nucleic acid.

While the isolated or synthetic RNA molecule of the invention may reduce the expression or level of transcript of any target nucleic acid of interest, in one embodiment, the target nucleic acid is a pathogen nucleic acid. The pathogen may be, for example, one that is capable of causing disease in a mammal, poultry or fish.

In one embodiment, the pathogen is a viral pathogen. The viral pathogen may be, for example, influenza virus, Hendra virus, SARS coronavirus, Ebolavirus, Newcastle Disease Virus, Chicken Anaemia Virus, Infectious Bursal Disease Virus, Foot and Mouth Disease Virus, Porcine Reproductive and Respiratory Syndrome (PRRS) Virus, Classical Swine Fever Virus, Bluetongue virus, Akabane virus, Infectious Salmon Anemia virus, Infectious Hematopoietic Necrosis Virus, Viral Haemorrhagic Septicaemia virus and Infectious Pancreatic Necrosis virus. In one particular embodiment, the viral pathogen is influenza virus or Hendra virus.

In another embodiment, the target nucleic acid is a host nucleic acid.

While the sense strand may comprise an immunostimulatory sequence 5' and/or 3' of the second nucleotide sequence, the present inventors have found that positioning the immunostimulatory sequence 5' of the second nucleotide sequence advantageously results in an increased level of immune response stimulation than compared to positioning of the immunostimulatory sequence 3' of the second nucleotide sequence. Thus, in a preferred embodiment, the immunostimulatory sequence is 5' of the second nucleotide sequence.

In one embodiment, the immunostimulatory sequence is 4 to 9 nucleotides in length.

In yet another embodiment, the immunostimulatory sequences is about 5 nucleotides in length. In another embodiment, the immunostimulatory sequence comprises a region at least four nucleotides in length consisting of nucleotides selected from uridine and/or guanos ine.

In yet another embodiment, the immunostimulatory sequence comprises a polyuridine and/or polyguanosine motif.

In one embodiment, the immunostimulatory sequences comprises a polyUG motif.

In one particular embodiment, the immunostimulatory sequence is at least 75% identical to a sequence selected from UGUGU, GGUU, UUGGUG, UUGGUU, UUUU and GUCCUUCAA.

In a preferred embodiment, the immunostimulatory sequence comprises a sequence selected from UGUGU, GGUU, UUGGUG, UUGGUU, UUUU and GUCCUUCAA

In one embodiment, the target nucleic acid may be an influenza virus transcript. Thus, the double-stranded region may comprise a sequence of nucleotides at least 90% identical to any one of SEQ ID NOs: 1 to 7.

Preferably, the double-stranded region comprises a sequence of nucleotides selected from any one of SEQ ID NOs: 1 to 7.

In yet another embodiment, the double-stranded region comprises a sequence of nucleotides at least 90% identical, more preferably 100% identical, to SEQ ID NO: l .

In another embodiment, the RNA molecule comprises a sequence of nucleotides at least 90% identical, more preferably 95%, more preferably 100% identical to any one of SEQ ID NOs: 8 to 10.

The target nucleic acid may be a Hendra virus transcript. Thus, in one embodiment, the double-stranded region of the RNA molecule may comprise a sequence of nucleotides at least 90% identical to SEQ ID NO: 13 or SEQ ID NO: 14. Preferably the double-stranded region comprises a sequence of nucleotides selected from SEQ ID NO: 13 or SEQ ID NO: 14.

In another aspect, the present invention provides a composition comprising the isolated or synthetic RNA molecule of the invention.

In one embodiment, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

In another aspect, the present invention provides food and/or drink comprising the isolated or synthetic RNA molecule of the invention.

In another aspect, the present invention provides a cell comprising the isolated or synthetic RNA molecule of the invention and/or the composition of the invention. In yet another aspect, the present invention provides a vector comprising a nucleic acid molecule encoding the isolated or synthetic RNA molecule of the invention or the sense strand thereof.

In another aspect, the present invention provides a method of preparing an immunostimulatory RNA molecule, the method comprising synthesizing an RNA molecule according to the invention.

In one embodiment, synthesizing the RNA molecule comprises chemically synthesizing the molecule or transcribing the molecule in vitro.

In a preferred embodiment, synthesizing the RNA molecule comprises chemically synthesizing the molecule.

In yet another aspect, the present invention provides a method of treating or preventing a disease in a subject, the method comprising administering the isolated or synthetic RNA molecule of the invention and/or the composition of the invention to the subject.

In one embodiment, the disease is caused by a pathogen.

In one embodiment, the pathogen is a virus.

In one particular embodiment, the virus is influenza virus or Hendra virus.

In one embodiment, the subject that is administered the isolated or synthetic RNA molecule of the invention and/or the composition of the invention is a mammal, poultry or fish.

The mammal may be, for example, a human, pig, horse, bovine or sheep.

The poultry may be for example, a chicken, duck, turkey or goose.

In one embodiment, the poultry is a chicken.

In one embodiment, the fish is a salmonid.

In one embodiment, the method comprises administering the RNA molecule and/or the composition in food, drinking water and/or in an aerosol.

In another aspect, the present invention provides a method of reducing the expression of one or more genes and stimulating an immune response in a subject, the method comprising administering to the subject the isolated or synthetic RNA molecule of the invention and/or the composition of the invention.

In one embodiment, the immune response that is stimulated is an increase in production of IFN-β, IFN-a, IFN-λ, IL-Ιβ, TNF-a and/or IL-18.

In yet another aspect, the present invention provides a method of inhibiting viral replication in a subject, the method comprising administering to the subject the isolated or synthetic RNA molecule of the invention and/or the composition of the invention. In another aspect, the present invention provides use of the isolated or synthetic RNA molecule of the invention and/or the composition of the invention in the manufacture of a medicament for the treatment or prevention of disease.

In another aspect, the present invention provides the isolated or synthetic RNA molecule of the invention and/or the composition of the invention for use in the treatment or prevention of disease.

In yet another aspect, there is provided use of the isolated or synthetic RNA molecule of the invention and/or the composition of the invention in the manufacture of a medicament for reducing the expression of one or more genes and stimulating an immune response in a subject.

In one aspect, the present invention provides the isolated or synthetic RNA molecule of the invention and/or the composition of the invention for use in reducing the expression of one or more genes and stimulating an immune response in a subject.

In yet another aspect, the present invention provides use of the isolated or synthetic RNA molecule of the invention and/or the composition of the invention in the manufacture of a medicament for inhibiting viral replication in a subject.

In another aspect, the present invention provides the isolated or synthetic RNA molecule of the invention and/or the composition of the invention for use in inhibiting viral replication in a subject.

As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1. Induction of IFN-β in chicken DF-1 cells by siRNAs. IFN-β message levels induced in DF-1 cells by siRNAs (20 pmol) or polyLC (0.8 μg). Each value is the mean + s.d (3 replicates) and representative of data from 3 experiments, a = /XO.001 between MP-592 and PA-2087 after 8 h. b = / 0.001 between PA-2087 and PB2-2240 after 8 h.

Figure 2. Increased immunostimulatory properties of siRNAs containing 5'- UGUGU-3'. Message levels of cytokines in DF-1 cells induced by PB1-2257, isPB l- 2257 and Scramble (20 pmol) after 8 h. Each value is the mean + s.d. (3 replicates) and representative of data from 3 experiments a = <0.001 between isPB l-2257 and both PB 1-2257 and Scramble.

Figure 3. Immunostimulatory properties of siRNAs depend on the mode of synthesis. IFN-β mRNA levels were measured in DF-1 cells transfected with T7- siRNAs (white bars, 20 pmol) or c-siRNAs (black bars, 20 pmol) at (A) 8 h or (B) 24 h. a = ><0.05 between paired white bars (T7-siRNA) and black bars (c-siRNA). b = p<0.05 between c-PB l-2257 and c-isPBl-2257 at 24 h (C) IFN-β message levels in DF-1 cells induced by isPB l-2557 (chemically synthesized, 20 pmol) or 2Me-isPB l- 2257 (chemically-synthesized, 20 pmol). a. = <0.001 between isPBl-2257 and 2Me- isPB l-2257 at 24 h. (D) IFN-β message levels in DF-1 cells induced by PB1-2257, isPB 1-2557 or Scramble expressed as T7-siRNAs (20 pmol, white bars) or plasmid driven shRNAs (0.8 ug, black bars) a. = p<0.05 between paired white bars (T7- siRNAs) and black bars (shRNAs).

Figure 4. Immunostimulatory siRNAs require TLR3 but not Mda5 for type I

IFN induction in DF-1 cells. Control (white bars), Mda5 -knockdown (striped bars) and TLR3 -knockdown (black bars) DF-1 cells were transfected with PB1-2257 (T7-siRNA, 20 pmol). Levels of IFN-β were measured by QRT-PCR 24 h after PB 1-2257 stimulation, a = <0.001 between IFN-β levels induced by PB 1-2257 in control cells (white bars) and TLR3 KD cells (black bars).

Figure 5. Attaching 5'-UGUGU-3' to siRNAs increases induction of type I IFN. Message levels of IFN-β in DF-1 cells transfected with chemically-synthesized PB1-2257, uPB l-2257 or PBl-2257u (all 20 pmol) for 8 h or 24 h. a = p<0.05 between levels of IFN-β induced by uPB l-2257 and PB1-2257. Each value is the mean + s.d (3 replicates) and representative of data from 3 experiments.

Figure 6. Inhibition of H1N1 influenza virus replication in MDCK cells by siRNAs. MDCK cells were transfected with T7 -siRNAs (20 pmol) or polyLC (0.8 μg) and infected 24 h later with influenza A/WSN/1933 (H1N1) virus at moi of 10^"3. Virus titers were measured 48 h post-infection TCID₅₀ assay. Each value is the mean + s.d (3 replicates) and representative of data from 2 experiments. Levels of virus inhibition are relative to cells treated with media alone, a = p<0.05 between virus levels in cell supernatants of cells treated with PB 1-2257 or uPB 1-2257.

Figure 7. Inhibition of H5N1 influenza virus growth in HD-11 cells by UGUGU-tagged siRNAs. HD-1 1 cells transfected with T7-siRNAs (5 pmol) or polyLC (0.1 μg) were infected 24 h later with H5N1 at moi of 10^_1to 10^"7. Virus titers were measured 72 h post-infection by TCID₅₀ assay. Each value is the mean + s.d (4 replicates). Levels of virus inhibition are relative to cells treated with media alone, a = p<0.05 between virus levels in cell supernatants of cells treated with PB 1-2257 or uPB 1-2257.

KEY TO THE SEQUENCE LISTING SEQ ID NO: l - PB 1-2257 siRNA.

SEQ ID NO:2 - PA-2087 siRNA.

SEQ ID NO:3 - NP-1484 siRNA.

SEQ ID NO:4 - PB1-129 siRNA.

SEQ ID NO:5 - PB2-2240 siRNA.

SEQ ID NO: 6 - NP-1496 siRNA.

SEQ ID NO:7 - MP-592.

SEQ ID NO:8 - isPBl-2257.

SEQ ID NO:9 - uPB 1-2257

SEQ ID NO: 10 - PB l-2257u

SEQ ID NO : 11 - Scramble

SEQ ID NO : 12 - 2Me-isPB 1-2257

SEQ ID NO: 13 - siN121p siRNA

SEQ ID NO: 14 - siN600 siRNA

DETAILED DESCRIPTION

General techniques and definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, microbiology especially virology, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the microbiological, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al, Molecular Cloning: A Laboratory Manual, 3^rd ed., Cold Spring Harbour Laboratory Press (2001), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al, (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al, (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

As used herein, the term "subject" refers to an animal, e.g., a bird or mammal. In one embodiment, the subject is a human. In another embodiment, the subject may be a mammal such as, for example, a pig or a horse. In another embodiment, the subject may be an avian, for example poultry such as a chicken, turkey or a duck.

The term "avian" as used herein refers to any species, subspecies or race of organism of the taxonomic Class Aves, such as, but not limited to, such organisms as chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and ratites including ostrich, emu and cassowary. The term includes the various known strains of Gallus gallus (chickens), for example, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Australorp, Cornish, Minorca, Amrox, California Gray, Italian Partidge-coloured, as well as strains of turkeys, pheasants, quails, duck, ostriches and other poultry commonly bred in commercial quantities.

The term "poultry" includes all avians kept, harvested, or domesticated for meat or eggs, for example chicken, turkey, ostrich, game hen, squab, guinea fowl, pheasant, quail, duck, goose, and emu.

The term "salmonid" as used herein refers to fish of the Salmonidae family and includes salmon, trout, char and whitefish. Non-limiting examples of salmon include Atlantic salmon, Chinook salmon, pink salmon, coho salmon, cherry salmon, sockeye salmon and chum salmon. Non-limiting examples of trout include rainbow trout, brown trout, brook trout and lake trout.

"Administering" as used herein is to be construed broadly and includes administering an isolated or synthetic RNA molecule as described herein to a subject as well as providing an isolated or synthetic RNA molecule as described herein to a cell.

As used herein the terms "treating", "treat" or "treatment" include administering a therapeutically effective amount of an isolated or synthetic RNA molecule of the invention sufficient to reduce or eliminate at least one symptom of disease.

The term "preventing" refers to protecting a subject from developing at least one symptom of disease, or reducing the severity of a symptom of disease in a subject.

The term "about" as used herein refers to a range of +1-5% of the specified value. Immunostimulatory RNAi molecules

The isolated or synthetic RNA molecules of the present invention comprise a double stranded region comprising an antisense strand complementary to a target nucleic acid, as well as an additional immunostimulatory sequence located at the 5' or 3' of the molecule. Thus, the isolated or synthetic RNA molecules of the invention are able to reduce the expression of a target nucleic acid by RNA interference (RNAi) and additionally possess immunostimulatory activity.

Immunostimulatory Sequences

As used herein, the term "immunostimulatory sequence" refers to a nucleotide sequence that has "immunostimulatory activity", i.e. a nucleotide sequence that is capable of stimulating an immune response in a cell or an animal. By way of non- limiting example, an immunostimulatory sequence may involve the induction of cytokine or other responses due to signalling through receptors such as TLR7 and/or TLR8 or through alternatives such as the RIG-I pathway and/or may stimulate the production or increased production of cytokines, such as, but not limited to, IFN-a, IFN-β, IFN-λ, IL-Ι β, IL-18, IFN-γ, TNF-a, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17 and/or may involve the recruitment or regulation of immune cells such as dendritic cells, macrophages and/or natural killer (NK) cells.

Typically, an immunostimulatory sequence is a short nucleotide sequence, for example of about 2 to about 15 nucleotides in length, preferably about 4 to 9 nucleotides in length. Immunostimulatory sequences include uridine and guanosine rich sequences (Sioud, 2005), and sequences containing GU motifs (Heil et al, 2004). Preferred immunostimulatory sequences for inclusion in the isolated or synthetic RNA molecule of the invention include sequences at least 75%, more preferably 90%, more preferably 90%, more preferably 100% identical to UGUGU, GGUU, UUGGUG, UUGGUU, UUUU or GUCCUUCAA. The skilled person will understand that it may be possible to alter a limited number of nucleotides within an immunostimulatory sequence and maintain the immunostimulatory ability of the molecule. Preferably no more than two nucleotides are altered, more preferably only one nucleotide is altered in the immunostimulatory sequence. It is also preferred that any alteration to the immunostimulatory sequence comprise a substitution which does not result in the loss of a uridine or guanosine from the immunostimulatory sequence.

The person skilled in the art will appreciate that a limited number of nucleotides may be added 5' of the immunostimulatory sequence located 5' of the second nucleotide sequence and/or 3' of the immunostimulatory sequence located 3' of the second nucleotide sequence and still maintain the ability of the RNA molecule to stimulate an immune response and reduce expression of a target nucleic acid. Preferably, there are 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, more preferably 1, more preferably no nucleotides 5' of the immunostimulatory sequence located 5' of the second nucleotide sequence and/or 3' of the immunostimulatory sequence located 3' of the second nucleotide sequence.

RNA Interference

The terms "RNA interference", "RNAi" or "gene silencing" refer generally to a process in which a double-stranded RNA molecule reduces the expression of a target nucleic acid sequence with which the double-stranded RNA molecule shares substantial or total identity. However, it has more recently been shown that RNA interference can be achieved using non-RNA double stranded molecules (see, for example, US 20070004667).

By "reduces the expression of or "reducing the expression of a target nucleic acid or gene or polypeptide is meant that the transcription of a nucleotide sequence and/or translation of a polypeptide sequence in a cell is down-regulated or inhibited. The degree of down-regulation or inhibition will vary with the nature and quantity of the isolated or synthetic RNA molecule administered to the host cell, but will be evident e.g., as a detectable decrease in target gene expression and/or related target or cellular function, or e.g., decrease in level of viral replication, etc.; desirably a degree of inhibition greater than 10%, 33%, 50%, 75%, 90%, 95% or 99% or more as compared to a cell not treated according to the present invention will be achieved.

A "target nucleic acid" as used herein refers to a nucleic acid or gene of which the level of expression, or level of transcript, is to be reduced or inhibited. Accordingly, the term "target nucleic acid" as used herein may refer to an mRNA transcript of a gene of interest. In one embodiment, the target nucleic acid is, for example, one or more gene or gene transcripts encoding a polypeptide involved in virus production, virus replication, virus infection, and/or transcription of viral RNA. In other embodiments, the target nucleic acid may be a host nucleic acid, such as for example, genes associated with cancer and cell transformation, angiogenic genes, immunomodulator genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.

The nucleotide sequence complementary to a target nucleic acid should be at least about 19 contiguous nucleotides, for example about 19 to 30 nucleotides, or may be longer, for example 35 or 50 nucleotides, or 100 nucleotides or more. The full- length sequence corresponding to the entire gene transcript may be used. Preferably, they are about 19 to about 30 nucleotides in length, more preferably about 19 to about 21 nucleotides in length.

The degree of identity of a double-stranded region of a nucleic acid molecule to the targeted transcript should be at least 90% and more preferably 95-100%. The nucleic acid molecule may of course comprise unrelated sequences which may function to stabilize the molecule.

An isolated or synthetic RNA molecule of the invention may be blunt-ended, or alternatively the RNA molecule may have a 3 ' overhang at one or both ends of the molecule. An overhang may be one, two, three or more nucleotides in length. In one embodiment, the RNA molecule of the invention comprises a blunt end at the 5' end of the antisense strand and a two-nucleotide overhang at the 3 ' end of the antisense strand. An RNA molecule comprising an overhang may be generated by chemical synthesis or by in vitro transcription. In the case of in vitro transcription, the transcript tail may be included in the overhang, so that the overhang may comprise a plurality of U residues, for example, between 1 and 5 U residues. It is noted that synthetic siRNAs that have been studied in mammalian systems often have 2 overhanging U residues. In one embodiment, only unmodified ribonucleotides are used in the anti-sense and/or sense strand of the double-stranded region of the RNA molecule, while the overhang(s) of the antisense and/or sense strand may include modified ribonucleotides and/or deoxyribonucleotides.

The term "short interfering RNA" or "siRNA" as used herein refers to a nucleic acid molecule which comprises ribonucleotides capable of inhibiting or down regulating gene expression, for example by mediating RNAi in a sequence-specific manner, wherein the double stranded portion is less than 50 nucleotides in length, preferably about 19 to about 23 nucleotides in length. For example the siRNA can be a nucleic acid molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siRNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary.

As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid (siNA), short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, RNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by the isolated or synthetic RNA molecules of the invention can result from siRNA mediated modification of chromatin structure to alter gene expression.

By "shRNA" or "short-hairpin RNA" is meant an RNA molecule where less than about 50 nucleotides, preferably about 19 to about 23 nucleotides, is base paired with a complementary sequence located on the same RNA molecule, and where said sequence and complementary sequence are separated by an unpaired region of at least about 4 to about 15 nucleotides which forms a single-stranded loop above the stem structure created by the two regions of base complementarity. An Example of a sequence of a single-stranded loop includes: 5' UUCAAGAGA 3'.

Nucleic Acids

By "isolated" RNA molecule it is meant an RNA molecule which has generally been separated from the nucleotide sequences with which it is associated or linked in its native state (if it exists in nature at all). Preferably, the isolated nucleic acid molecule is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. Furthermore, the terms "RNA molecule" and "nucleic acid molecule" are used interchangeably herein with the term "polynucleotide".

As used herein "synthetic RNA" means a ribonucleic acid that is assembled in vitro. Any chemical system known in the art for ribonucleotide polymer synthesis is contemplated, including the chemical synthesis methods described in Verma and Eckstein (1998) and in US 6,989,442. The synthetic RNAs can also be prepared by enzymatic transcription from synthetic DNA templates or from DNA plasmids isolated from recombinant bacteria. Typically, phage RNA polymerases are used such as T7, T3 or SP6 RNA polymerase (Milligan and Uhlenbeck, 1989). In a preferred embodiment, the isolated or synthetic RNA molecules of the invention are chemically synthesized.

The term "complementary" as used herein refers to the relationship between two strands of a double-stranded nucleic acid molecule, wherein the two strands hybridise to each other via Watson-Crick base pairing bonds. In the context of the isolated or synthetic RNA molecule of the invention, the antisense strand that is complementary to the target nucleic acid is sufficiently complementary so as to be capable of inducing RNA interference. Thus, the antisense strand of the isolated or synthetic RNA molecule of the invention may comprise non-complementary nucleotides to the target nucleic acid as long as it is capable of inducing RNA interference.

In addition, the isolated or synthetic RNA molecule of the invention may comprise one or more non-pairing mismatches (i.e. non-complementary nucleotides) between the sense and antisense strands. The introduction of a non-pairing nucleotide mismatch into an isolated or synthetic RNA molecule of the invention results in one or more bulges in the secondary structure of the molecule and increases its immunostimulatory activity. The mismatch typically comprises one or more, for example, about 2 to 8, more preferably about 2 to 5 nucleotides located in the antisense strand of the isolated or synthetic RNA molecule of the invention that are non- complementary to the sense strand. In one embodiment, the mismatches are non- complementary to the immunostimulatory sequence. The RNA molecule of the invention will maintain sufficient complementarity to the target nucleic acid for the RNA molecule to direct cleavage of the target nucleic acid via RNA interference.

The % identity of a nucleic acid molecule is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 19 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 19 nucleotides. Alternatively, the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. Alternatively, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Preferably, the two sequences are aligned over their entire length.

With regard to the defined nucleic acid molecules, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the nucleic acid molecule comprises a nucleotide sequence which is at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.

An isolated or synthetic RNA molecule of the present invention may comprise a nucleotide sequence that selectively hybridises to a target nucleic acid under stringent conditions. As used herein, under stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodS04 at 50°C; (2) employ during hybridisation a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42°C in 0.2 x SSC and 0.1% SDS.

The nucleic acid molecules are typically RNA but may comprise chemically- modified nucleotides and non-nucleotides. Thus "RNA" as used herein refers to ribonuocleotides as well as modified nucleotides and nucleotide analogs. For example, it may be desirable in certain embodiments to utilize unmodified ribonucleotides in the antisense strand while employing modified ribonucleotides and/or modified or unmodified deoxyribonucleotides at some or all positions in the sense strand. According to certain embodiments of the invention, only unmodified ribonucleotides are used in the double-stranded portion of the antisense and/or the sense strand of the RNA molecule, while the overhang(s) of the antisense and/or sense strand may include modified ribonucleotides and/or deoxyribonucleotides.

Numerous nucleotide analogs and modified nucleotides are known in the art, and their effect on properties such as hybridization and nuclease resistance has been explored. For example, various modifications to the base, sugar and internucleoside linkage have been introduced into oligonucleotides at selected positions, and the resultant effect relative to the unmodified oligonucleotide compared. A number of modifications have been shown to alter one or more aspects of the oligonucleotide such as its ability to hybridize to a complementary nucleic acid or its stability. For example, useful 2'-modifications include halo, alkoxy and allyloxy groups. US patent numbers 6,403,779; 6,399,754; 6,225,460; 6, 127,533; 6,031,086; 6,005,087; 5,977,089, and references therein disclose a wide variety of nucleotide analogs and modifications that may be of use in the practice of the present invention. See also Crooke, S. (ed.) "Antisense Drug Technology: Principles, Strategies, and Applications" Marcel Dekker; ISBN: 0824705661 ; 1st edition (2001) and references therein. As will be appreciated by one of ordinary skill in the art, analogs and modifications may be tested using, for appropriate assays in order to select those that effectively reduce expression of a target nucleic acid and stimulate an immune response.

In certain embodiments of the invention, the analog or modification results in an RNA molecule with increased absorbability (for example, increased absorbability across a mucus layer or increased oral absorption), increased stability in the blood stream or within cells or increased ability to cross cell membranes. As will be appreciated by one of ordinary skill in the art, analogs or modifications may result in altered Tm, which may result in increased tolerance of mismatches between the RNA molecule sequence and the target while still resulting in effective suppression or may result in increased or decreased specificity for desired target transcripts.

Usually, monomers of a nucleic acid are linked by phosphodiester bonds or analogs thereof to form oligonucleotides. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate.

The present inventors have found that masking the immunostimulatory sequence in the RNA molecule of the invention with a methyl group reduces cytokine induction in cultured cells. Thus, in a preferred embodiment, the immunostimulatory sequence in the isolated or synthetic RNA molecule of the invention is not methylated.

Vectors and Host Cells

In some instances it may be desirable to insert a nucleic acid encoding the isolated or synthetic RNA molecule of the invention, or a strand thereof, into a vector. The vector may be, for example, a plasmid, virus or artificial chromosome derived from, for example, a bacteriophage, adenovirus, adeno-associated virus, retrovirus, poxvirus or herpesvirus. Such vectors include chromosomal, episomal and virus- derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, and viruses, vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, cosmids and phagemids. Thus, one exemplary vector is a double-stranded DNA phage vector. Another exemplary vector is a double-stranded DNA viral vector.

The vector into which the nucleic acid encoding the RNA molecule is inserted may also include a transposable element, for example a transposon characterized by terminal inverted repeat sequences flanking the open reading frames encoding the double stranded RNA(s). Examples of suitable transposons include Tol2, Mini-Tol2, Sleeping Beauty, Mariner and Galluhop. Reference to a Tol2 tansposon herein includes a transposon derived from Tol2 such as Mini-Tol2.

It will be appreciated by the person skilled in the art that a vector may encode a single strand of an RNA molecule of the invention. Thus, the sense and antisense strands may be encoded by separate vectors and then hybridise to form an RNA molecule comprising a double-stranded region. In some embodiments, the sense strand is connected to the antisense strand via a linker molecule. Examples of linker molecules include, but are not limited to, a polynucleotide linker and a non-nucleotide linker. Alternatively, a vector may express a single RNA strand that self-hybridises to form the RNA molecule of the invention comprising a double-stranded region.

The present invention also provides a host cell into which the isolated or synthetic RNA and/or the vector of the present invention has been introduced. The host cell of this invention can be used as, for example, a production system for producing or expressing the RNA molecule. For in vitro production, eukaryotic cells or prokaryotic cells can be used.

Useful prokaryotic cells include bacterial cells, such as E. coli, for example, JM109, DH5a, and HB 101, ox Bacillus subtilis.

Useful eukaryotic host cells may be animal, plant, or fungal cells. As animal cells, mammalian cells such as CHO, COS, 3T3, MDCK myeloma, baby hamster kidney (BHK), HeLa, or Vero cells, amphibian cells such as Xenopus oocytes, or insect cells such as Sf9, Sf21, or Tn5 cells or avian cells such as DF1 or CEF cells can be used. CHO cells lacking DHFR gene (dhfr-CHO) or CHO K-l may also be used. The vector can be introduced into the host cell by, for example, the calcium phosphate method, the DEAE-dextran method, cationic liposome DOTAP (Boehringer Mannheim) method, electroporation, lipofection, etc.

Culture medium such as DMEM, MEM, RPM11640, or IMDM may be used for animal cells. The culture medium can be used with or without serum supplement such as fetal calf serum (FCS). The pH of the culture medium is preferably between about 6 and 8. Cells are typically cultured at about 30° to 40° C for about 15 to 200 hr, and the culture medium may be replaced, aerated, or stirred if necessary.

Therapeutic Methods

The present invention provides a method of treating or preventing a disease in a subject, the method comprising administering an isolated or synthetic RNA molecule of the invention to a subject. The disease to be treated may be any which may benefit from an enhanced immune response in the subject. The disease may be selected from, for example, a disease caused by an infectious pathogen including viral pathogens such as, but not limited to influenza virus, Hendra virus, SARS coronavirus, Ebolavirus, Newcastle disease virus, chicken anaemia virus, infectious bursal disease virus, foot and mouth disease virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, bluetongue virus, a herpes virus, Nipah virus, Infectious Salmon Anemia virus, Infectious Hematopoietic Necrosis Virus, Viral Haemorrhagic Septicaemia virus and Infectious Pancreatic Necrosis virus. Thus, the isolated or synthetic RNA molecules of the invention, or compositions comprising the molecules can be used to reduce or inhibit pathogen infection or replication in a subject.

In one embodiment, the target nucleic acid is a pathogen nucleic acid. For example, the target nucleic acid may be a viral gene involved in infection, survival and/or replication of the virus, or a gene encoding a protein associated with bacterial invasion, infection, replication and/or survival.

While particularly suited for use in the treatment or prevention of diseases caused by pathogenic agents, the isolated or synthetic RNA molecule of the invention is useful for the treatment or prevention of other diseases in which it is desirable to reduce the expression of a gene and simultaneously stimulate an immune response. Thus, in an alternative embodiment, the target nucleic acid may be a host nucleic acid. Non-limiting examples of target host nucleic acids include genes in tumor cells that have been shown to code for proteins associated with tumor growth and survival, such as growth factors and receptors, anti-apoptotic proteins, downstream signal transduction proteins, proteins associated with angiogenesis, and proteins that may be involved with metastasis.

In yet another embodiment, the target nucleic acid may be a host nucleic acid associated with an undesirable or excessive host immune response. Thus, the isolated or synthetic RNA molecule of the invention may stimulate a desirable immune response and simultaneously reduce the expression of a host target nucleic acid associated with an undesirable or excessive host immune response.

In one embodiment of the invention, an effective amount of an isolated or synthetic RNA molecule is delivered to a cell or organism prior to, simultaneously with, or after exposure to a pathogen such as for example influenza virus or Hendra virus. Preferably, the amount of the isolated or synthetic RNA molecule is sufficient to reduce or delay one or more symptoms of infection.

An "effective amount" or "therapeutically effective amount" of an RNA molecule is an amount sufficient to produce a desired effect, for example, inhibition of expression of a target nucleic acid in comparison to the normal expression level detected in the absence of the RNA molecule and/or stimulation of an immune response. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with the RNA molecule relative to the control is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of a target nucleic acid include, for example examination of protein or mRNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art. Similarly, the stimulation of an immune response can be determined using methods known to the person skilled in the art. For example, immunostimulation may be measured by detecting an increase in recruitment of immune cells such as dendritic cells, macrophages or other immune cells such as, but not limited to B cells, T-cells, and natural killer cells. Alternatively, immunostimulation may be measured by detecting an increase in cytokine level or production using known techniques.

A composition of the invention may comprise a single RNA molecule species, targeted to a single site in a single target nucleic acid, or may comprise a plurality of different RNA molecule species, targeted to one or more sites in one or more target nucleic acids. In addition, the isolated or synthetic RNA molecules in the composition may comprise the same immunostimulatory sequence, or alternatively the composition may comprise different species of RNA molecules comprising different immunostimulatory sequences.

In some embodiments of the invention, it will be desirable to utilize compositions containing collections of different RNA molecule species targeted to different genes. For example, it may be desirable to attack a virus at multiple points in the viral life cycle using a variety of isolated or synthetic RNA molecules of the invention directed against different viral transcripts. In one embodiment, the compositions of the invention contains an isolated or synthetic RNA molecule targeted to each segment of a viral genome.

In some instances it will be desirable to combine the administration of the isolated or synthetic RNA molecules of the invention with one or more other anti -viral agents in order to treat or prevent disease caused by infection with the virus. For example, the isolated or synthetic RNA molecules of the invention may be combined with anti-viral agents such as, but not limited to, amantadine, ramantadine, zanamavir, oseltamavir, peramavir and/or famciclovir. The isolated or synthetic RNA molecule of the invention may also be combined with any one or more of a variety of agents such as vaccines, for example, influenza vaccines. The RNA molecule of the invention may be present in the same mixture or composition as the other therapeutic agent or may be delivered separately to the other therapeutic agent.

Compositions and Administration

In a preferred embodiment, a composition of the invention is a pharmaceutical composition comprising a suitable carrier. Suitable pharmaceutical carriers, excipients and/or diluents include, but are not limited to, lactose, sucrose, starch powder, talc powder, cellulose esters of alkonoic acids, magnesium stearate, magnesium oxide, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, gelatin, glycerin, sodium alginate, antibacterial agents, antifungal agents, gum arabic, acacia gum, sodium and calcium salts of phosphoric and sulfuric acids, polyvinylpyrrolidone and/or polyvinyl alcohol, saline, and water.

In some embodiments, the isolated or synthetic RNA molecules of the invention are complexed with one or more cationic lipids or cationic amphiphiles, such as the compositions disclosed in US 4,897,355; US 5,264,618; or US 5,459, 127. In other embodiments, they are complexed with a liposome/liposomic composition that includes a cationic lipid and optionally includes another component, such as a neutral lipid (see, for example, US 5,279,833; US 5,283, 185; and US 5,932,241). In other embodiments, they are complexed with the multifunctional molecular complexes of US 5,837,533; 6, 127, 170; and 6,379,965 or, desirably, the multifunctional molecular complexes or oil/water cationic amphiphile emulsions of WO 03/093449. The latter application teaches a composition that includes a nucleic acid, an endosomolytic spermine that includes a cholesterol or fatty acid, and a targeting spermine that includes a ligand for a cell surface molecule. The ratio of positive to negative charge of the composition is between 0.1 to 2.0, preferably 0.5 and 1.5, inclusive; the endosomolytic spermine constitutes at least 20% of the spermine-containing molecules in the composition; and the targeting spermine constitutes at least 10% of the spermine-containing molecules in the composition. Desirably, the ratio of positive to negative charge is between 0.8 and 1.2, inclusive, such as between 0.8 and 0.9, inclusive.

A 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, intramuscular, intraperitoneal, intrathecal), mucosal (e.g., oral, rectal, intranasal, buccal, vaginal, respiratory), enteral (e.g., orally, such as by tablets, capsules or drops, rectally) and transdermal (topical, e.g., epicutaneous, inhalational, intranasal, eyedrops, vaginal). Solutions or suspensions used for parenteral, intradermal, enteral or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, 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. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms is achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions is brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the isolated or synthetic RNA molecule of the invention in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the RNA molecule into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the RNA molecule is incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions are also prepared using a fluid carrier for use as a mouthwash, wherein the RNA molecule in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the RNA molecules 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. Systemic administration can also be by mucosal or transdermal means. For mucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for mucosal administration, detergents, bile salts, and fusidic acid derivatives. Mucosal administration is accomplished through the use of nasal sprays or suppositories. For transdermal administration, the RNA molecules are formulated into ointments, salves, gels, or creams as generally known in the art.

A pharmaceutically acceptable vehicle is understood to designate a compound or a combination of compounds entering into a pharmaceutical composition which does not cause side effects and which makes it possible, for example, to facilitate the administration of the RNA molecule, to increase its life and/or its efficacy in the body, to increase its solubility in solution or alternatively to enhance its preservation. These pharmaceutically acceptable vehicles are well known and will be adapted by persons skilled in the art according to the nature and the mode of administration of the isolated or synthetic RNA molecule chosen. Administration of an RNA molecule and/or composition may conveniently be performed by injection into an avian egg, and generally injection into the air sac. Notwithstanding that the air sac is the preferred route of in ovo administration, other regions such as the yolk sac or chorion allantoic fluid may also be inoculated by injection. The hatchability rate might decrease slightly when the air sac is not the target for the administration although not necessarily at commercially unacceptable levels. The mechanism of injection is not critical to the practice of the present invention, although it is preferred that the needle does not cause undue damage to the egg or to the tissues and organs of the developing embryo or the extra-embryonic membranes surrounding the embryo.

Generally, a hypodermic syringe fitted with an approximately 22 gauge needle is suitable for avian in ovo administration. The method of the present invention is particularly well adapted for use with an automated injection system, such as those described in US 4,903,635, US 5,056,464, US 5, 136,979 and US 20060075973.

In another embodiment, the isolated or synthetic RNA molecule and/or composition of the invention is administered via pulmonary delivery, such as by inhalation of an aerosol or spray dried formulation. For example, the aerosol may be administered by an inhalation device or nebulizer (see for example US 4,501,729), providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues. Solid particulate compositions containing respirable dry particles of micronized nucleic acid compositions can be prepared by grinding dried or lyophilized nucleic acid compositions, and then passing the micronized composition through, for example, a 400 mesh screen to break up or separate out large agglomerates. A solid particulate composition comprising the nucleic acid compositions of the invention can optionally contain a dispersant which serves to facilitate the formation of an aerosol as well as other therapeutic compounds. A suitable dispersant is lactose, which can be blended with the nucleic acid compound in any suitable ratio, such as a 1 to 1 ratio by weight.

Nebulizers are commercially available devices which transform solutions or suspensions of an active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers comprise the active ingredient in a liquid carrier in an amount of up to 40% w/w preferably less than 20% w/w of the formulation. The carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride or other suitable salts. Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxybenzoate, anti-oxidants, flavorings, volatile oils, buffering agents and emulsifiers and other formulation surfactants. The aerosols of solid particles comprising the active composition and surfactant can likewise be produced with any solid particulate aerosol generator. Aerosol generators for administering solid particulate therapeutics to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a therapeutic composition at a rate suitable for human administration.

An isolated or synthetic RNA molecule and/or composition of the invention can also be added to animal feed or drinking water. It can be convenient to formulate the feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.

EXAMPLES

Example 1. Materials and methods

siRNAs and shRNAs

siRNAs were synthesized by T7 RNA polymerase using the Ambion Silencer® siRNA Construction Kit according to manufacturer's instructions (Ambion inc, Austin, TX) with template oligonucleotides purchased from Geneworks (Adelaide, Australia). Chemically-synthesized siRNAs were purchased from Sigma (St Louis, MO). The synthesis of plasmid-driven short hairpin (sh)RNAs under control of the chicken U6-4 promoter was performed as described previously (Hinton and Doran, 2008).

Cell culture

The continuous chicken fibroblast cell line DF-1 (American Type Culture Collection (ATCC), CRL-12203) was maintained in DMEM supplemented with 10 % (v/v) heat-inactivated foetal calf serum (FCS), 2 mM 1-glutamine, 1.5 % (w/v) sodium bicarbonate, 100 U/mL penicillin and 100 μg/mL streptomycin. Madin-Darby canine kidney (MDCK) (ATCC CCL-34) cells were maintained in DMEM containing 10 % heat- inactivated FCS, 2 mM 1-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Vero 76 cells (ATCC CRL-1587) were maintained in DMEM supplemented with 10 % (v/v) FCS, 100 U/mL penicillin and 100 μg/mL streptomycin. The continuous chicken macrophage cell line HD-1 1 was maintained in RPMI media supplemented with 10 % (v/v) FCS, 10 mM HEPES, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μ£ξ/ι ί streptomycin. All cells were incubated at 37 °C under a 5 % CO₂/95 % air atmosphere.

Transfections

DF-1 and MDCK cells were cultured in 12-well plates (1 x 10⁵ cells per well) for 24 h before transfection in medium lacking penicillin and streptomycin. Cells were transfected using Lipofectamine 2000 (Invitrogen) according to manufacturer's guidelines. After 4 h, the transfection media was changed to growth media containing penicillin (100 U/mL) and streptomycin (100 μg/mL) and 10 % FCS. HD-11 cells were transfected in 96 well plates using Lipofectamine 2000 according to manufacturer's guidelines.

RNA isolation, reverse transcription and quantitative real-time PCR

RNA was harvested using Tri-reagent (Sigma) according to manufacturer's instructions. One microgram of extracted RNA was treated with DNase (Sigma) according to manufacturer's instructions and reverse-transcribed to complementary DNA (cDNA) using a Reverse Transcription kit (Promega). Quantitative real-time PCR (QRT-PCR) experiments were conducted on an ABI Prism 7700 Sequence Detection System (Applied Biosystems). The comparative threshold cycle (Ct) method was used to derive fold change gene expression. Primers and probes were designed using Primer Express software (Applied Biosystems). Where possible, the probe sets were designed across intron:exon boundaries. PCR cycling was performed as follows: 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 sec, 61 °C for 30 sec and 68 °C for 30 sec.

Viruses

Influenza A/WSN/1933 (H1N1) and influenza A/Vietnam/1203/2004 (H5N1) were passaged in the allantoic fluid of 10-day embryonated specific pathogen-free chicken eggs. H5N1 virus was handled in enhanced BSL3 conditions at the CSIRO Australian Animal Health Laboratory. Allantoic fluid was harvested, aliquoted and stored at -80 °C for inoculations. Virus titer was measured by plaque assays, whereby serial 10-fold dilutions of supernatants of MDCK cells (H1N1) or Vero cells (H5N1) infected with virus were added onto a monolayer of 1% semisolid agar. Two days after infection, plaques were visualized by staining with crystal violet. TCID50

H1N1 virus work

10-fold dilutions of supernatants containing virus were added to a 96-well tissue culture plate. MDCK cells were added to all wells and the plates were incubated for 1 h at 37 °C, 5 % CO2. The medium was discarded and replaced with virus growth media (EMEM, 0.3% bovine serum albumin, 1 mM Hepes, 5 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 5 μg/mL trypsin). The plates were incubated for 4 days. The infectious titer was calculated by the method of Reed and Muench (1938).

H5N1 virus work

10-fold dilutions of H5N1 virus were made in PBS and added to a 96-well tissue culture plate containing Vero 76 cells in growth media. Plates were incubated for 5 days at 37 °C, 5 % CO2 and scored for cytopathic effect. The infectious titer was calculated by the method of Reed and Muench (1938).

Statistics

The difference between two groups was statistically analysed by Student's i-test.

A -value of O.05 was considered significant.

Example 2. Results

siRNAs designed against three different conserved regions of the influenza A genome were selected based on their silencing ability (Ge et al., 2003) (Table 1, SEQ ID NOs: l, 2, 5 and 7). To test the ability of these siRNAs to induce cytokine production in chicken cells, these three siRNAs were synthesized using T7 RNA polymerase and transfected into the immortalized chicken fibroblast cell line, DF-1. Since DF-1 cells have previously been shown to produce high levels of IFN-β in response to the dsRNA mimetic polyLC (Karpala et al, 2008), they provide a model cell line for assessing the type I IFN response to siRNA. MP-592, PA-2087 and PB2- 2240 induced IFN-β to differing levels, effectively giving low, moderate and high induction of IFN-β, respectively, relative to induction by polyLC, with maximum levels observed after 24 h (Figure 1). Table 1. siRNA sequences.

The ability of distinct sequences to dictate the immunostimulatory properties of siRNAs in mammalian cells was illustrated recently (Judge et al, 2005) where the induction of pro-inflammatory cytokines was directly related the presence of a 5 bp motif, 5'-UGUGU-3'. The inventors conducted a bioinformatics search of RNAi sequences targeting influenza A and found that PB l-2257 (SEQ ID NO:5), a 19 bp siRNA targeting the PB 1 segment of influenza A contains a motif from base pairs 3-7, 5'-UCUGU-3', remarkably similar to 5'-UGUGU-3' (Table 1). Previous studies have shown PBl-2257 to be an effective silencer of influenza A subtype HlNl replication (Ge et al., 2003). A modified version of PB l-2257 (immunostimulatory (is)PBl-2257; SEQ ID NO:8) was created where a single substitution at position 4 (C to G) was made, hence creating a 5'-UGUGU-3' motif (Table 1). An irrelevant siRNA (Scramble; SEQ ID NO: 11) was also synthesized as a negative control by random shuffling of the PBl-2257 sequence (Table 1). isPB l-2257 induced IFN-a, IFN-β, IFN-λ and IL-Ι β to a greater extent than PBl-2257 and Scramble after transfection into DF-1 cells (Figure 2). Interestingly, PBl-2257, isPB l-2257 and Scramble induced IL-Ι β, but not the related cytokine, IL-18. These results demonstrate that siRNAs featuring the 5'- UGUGU-3' motif are particularly inflammatory in chicken cells and that a single nucleoside substitution within an siRNA sequence can greatly effect that siRNAs immunostimulatory profile.

It has been reported that mode of synthesis can impact the immunostimulatory properties of siRNAs (Schlee et al, 2006). To test this parameter in our study, chemically-synthesized siRNAs (c-siRNAs) and siRNAs synthesized using T7 RNA polymerase (T7-siRNAs) were transfected into DF-1 cells at identical concentrations and the induced changes in IFN-β message levels were measured. By 8 h post- transfection (Figure 3A), the T7-isPB 1-2257 had induced a large increase in IFN-β however, this increase was not observed for the c-siRNA variant, nor the PB 1-2257 or Scramble. At 24 h (Figure 3B), PB 1-2257 and isPBl-2257 induced significant increases in IFN- β up to 10-fold higher for c-siRNAs than T7-siRNAs. Scramble induced moderate IFN- β expression at 24 h as a T7-siRNA, but not a c-siRNA. Together, these results demonstrated that nucleoside sequence and mode of synthesis both impact on the level of siRNA- induced cytokine production in chicken cells.

To further probe the role of 5'-UGUGU-3' in IFN stimulation, a chemically- synthesized derivative of isPB 1-2257 was synthesized with a methyl group masking the 5'-UGUGU-3' motif (Table 1; SEQ ID NO: 12). Masking of the 5'-UGUGU-3' motif caused an almost total abolition of IFN- β induction by isPBl-2257 at 24 h (Figure 3C), confirming the importance of this motif to the inflammatory properties of isPB 1-2257. To further test how the mode of RNAi expression influences immunostimulation, PB 1- 2257, isPB 1-2257 and Scramble were transfected into DF-1 cells as c-siRNA, or as plasmid-expressed short hairpin (sh)RNAs driven by a chicken U6 promoter and linked by the Brummelkamp sequence, a method previously used to synthesize and express antiviral shRNAs in chicken cells (Hinton and Doran, 2008). While isPB 1-2257, and to a lesser extent, PB 1-2257 and Scramble, induced IFN- β in DF-1 cells at 24 h, the same sequences expressed as plasmid-expressed shRNAs were not stimulatory (Figure 3D).

The inventors next investigated the receptor responsible for inducing type I IFN in response to immunostimulatory siRNAs in chicken cells. Immunostimulatory siRNAs are reported to act via Toll-like receptor 7 (TLR7) in mammals (Hornung et al, 2005). However, QRT-PCR demonstrated that DF-1 cells express extremely low levels of TLR7 (not shown), suggesting that other dsRNA-sensing molecules may recognise PB 1-2257. siRNAs were transfected into DF-1 cells to create cell populations knocked down for melanoma-differentiation-associated gene 5 (Mda5) and TLR3. QRT-PCR results confirmed that siRNAs reduced gene expression levels by >75 % and did not induce significant increases in type I IFN, (data not shown). siRNAs used to silence chicken TLR3 (Karpala et al, 2008) and MDA5 (Karpala et al, 2011) have been described previously. A strong induction of IFN-β in control DF-1 cells was observed 24 h after stimulation with PBl-2257 (Figure 4). This response was reduced by 90 % in TLR3 knock down DF-1 cells (pO.001). In contrast, cells with reduced levels of lacking Mda5 did not show a significant reduction in IFN-β expression.

To create a multi-functional antiviral with both immunostimulatory and silencing properties, the inventors attached 5'-UGUGU-3' to either the 5' or the 3' ends of c-PB 1-2257 without disrupting the silencing sequence itself (Table 1 : SEQ ID NOs:9 and 10). This modification resulted in a 3-fold enhancement of the immunostimulatory profile for the PB l-2257 variant tagged with the 5'-UGUGU-3' motif at the 5' end (Figure 5). In contrast, tagging at the 3' end appeared to have little enhancing effect.

In order to test siRNA-mediated inhibition of influenza virus replication we measured the replication of influenza A/WSN/1933 (H1N1) as a low-pathology model of H5 1. Due to the fact that DF-1 cells are poor at supporting virus replication (data not shown), we used MDCK cells for this experiment. The PBl segment sequence of influenza A/WSN/1933 (H1N1) (Genbank: CY034138.1) is identical that of the consensus sequence for H5N1 (avian, Asia 2003-2009) in the nucleoside region targeted by PB l-2257 (data not shown). When measured by TCID₅₀ assay (Figure 6), PBl-2257 tagged with the 5'-UGUGU-3 ' motif inhibited virus growth 2-3 fold more effectively than PB l-2257. Scramble was largely ineffective as an antiviral, while the positive control in this experiment, the long dsRNA mimetic polyLC, inhibited virus growth effectively.

The inventors tested the effect of UGUGU-tagged siRNAs on H5N1 growth in the immortalised chicken macrophage cell line, HD-1 1. H5N1 grew poorly in DF-1 cells. When measured by TCID₅₀ assay (Figure 7), PBl-2257 inhibited H5N1 replication in HD-1 1 cells by 40 % relative to cells treated with media alone. PBl- 2257 tagged with 5'-UGUGU-3' at the 5' end was the most effective treatment inhibiting H5N1 growth by 98 %. Interestingly, PBl-2257 tagged with 5'-UGUGU-3 ' at the 3' had little impact on the inhibition of H5 1 replication.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All publications discussed and/or referenced herein are incorporated herein in their entirety.

The present application claims priority from US 61/422,994, the entire contents of which are incorporated herein by reference.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

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Claims

CLAIMS;

1. An isolated or synthetic RNA molecule comprising a double-stranded region, wherein the double-stranded region comprises a sense strand and an antisense strand, and wherein

the antisense strand comprises a first nucleotide sequence that is complementary to a target nucleic acid, and

the sense strand comprises a second nucleotide sequence complementary to the first nucleotide sequence, and an immunostimulatory sequence 5' and/or 3' of the second nucleotide sequence.

2. The isolated or synthetic RNA molecule of claim 1, wherein the nucleotide sequence that is complementary to the target nucleic acid is about 19 to about 50 nucleotides in length.

3. The isolated or synthetic RNA molecule of claim 1 or claim 2, wherein the target nucleic acid is a pathogen nucleic acid.

4. The isolated or synthetic RNA molecule of claim 3, wherein the pathogen is a viral pathogen.

5. The isolated or synthetic RNA molecule of claim 1 or claim 2, wherein the target nucleic acid is a host nucleic acid.

6. The isolated or synthetic RNA molecule of any one of claims 1 to 5, wherein the immunostimulatory sequence is 4 to 9 nucleotides in length.

7. The isolated or synthetic RNA molecule of any one of claims 1 to 6, wherein the immunostimulatory sequence comprises a region at least four nucleotides in length consisting of nucleotides selected from uridine and/or guanosine.

8. The isolated or synthetic RNA molecule of claim 7, wherein the immunostimulatory sequence comprises a polyuridine and/or polyguanosine motif.

9. The isolated or synthetic RNA molecule of any one of claims 1 to 8, wherein the immunostimulatory sequence is at least 75% identical to a sequence selected from UGUGU, GGUU, UUGGUG, UUGGUU, UUUU and GUCCUUCAA.

10. A composition comprising the isolated or synthetic RNA molecule of any one of claims 1 to 9.

11. The composition of claim 10, which is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

12. A cell comprising the isolated or synthetic RNA molecule of any one of claims 1 to 9 and/or the composition of claim 10 or claim 1 1.

13. A vector comprising a nucleic acid molecule encoding the isolated or synthetic RNA molecule of any one of claims 1 to 9 or the sense strand thereof.

14. A method of preparing an immunostimulatory RNA molecule, the method comprising synthesizing an RNA molecule according to any one of claims 1 to 9.

15. The method of claim 14, wherein synthesizing the RNA molecule comprises chemically synthesizing the molecule or transcribing the molecule in vitro.

16. A method of treating or preventing a disease in a subject, the method comprising administering the isolated or synthetic RNA molecule of any one of claims 1 to 9 and/or the composition of claim 10 or claim 1 1 to the subject.

17. The method of claim 16, wherein the diseases is caused by a pathogen.

18. The method of claim 17, wherein the pathogen is a virus.

19. A method of reducing the expression of one or more genes and stimulating an immune response in a subject, the method comprising administering to the subject the isolated or synthetic RNA molecule of any one of claims 1 to 9 and/or the composition of claim 10 or claim 11.

20. The method of claim 19, wherein the immune response that is stimulated is an increase in production of IFN-β, IFN-a, IFN-λ, IL-Ι β, TNF-a and/or IL-18.

21. A method of inhibiting viral replication in a subject, the method comprising administering to the subject the isolated or synthetic RNA molecule of any one of claims 1 to 9 and/or the composition of claim 10 or claim 11.

22. Use of the isolated or synthetic RNA molecule of any one of claims 1 to 9 and/or the composition of claim 10 or claim 11 in the manufacture of a medicament for the treatment or prevention of disease.

23. The isolated or synthetic RNA molecule of any one of claims 1 to 9 and/or the composition of claim 10 or claim 11 for use in the treatment or prevention of disease.

24. Use of the isolated or synthetic RNA molecule of any one of claims 1 to 9 and/or the composition of claim 10 or claim 11 in the manufacture of a medicament for reducing the expression of one or more genes and stimulating an immune response in a subject.

25. The isolated or synthetic RNA molecule of any one of claims 1 to 9 and/or the composition of claim 10 or claim 11 for use in reducing the expression of one or more genes and stimulating an immune response in a subject.

26. Use of the isolated or synthetic RNA molecule of any one of claims 1 to 9 and/or the composition of claim 10 or claim 11 in the manufacture of a medicament for inhibiting viral replication in a subject.

27. The isolated or synthetic RNA molecule of any one of claims 1 to 9 and/or the composition of claim 10 or claim 11 for use in inhibiting viral replication in a subject.