WO2014138792A1 - Double-stranded rna - Google Patents

Abstract

The present invention relates to nucleic acid molecules comprising a double stranded region complementary to a target gene of an influenza virus, in particular dsRNA fragments specific for the NSI and NEP (NS2) genes. The mvention further relates to expression vectors, cells, compositions and transgenic non-human mammals and avians comprising the polynucleotides, as well as methods of treating or preventing influenza in a subject by administering the polynucleotide, vector, cell or composition to the subject.

Description

DOUBLE-STRANDED UNA

FIELD OF THE INVENTION

The present invention relates to nucleic acid molecules comprising a double- stranded region complementary to a target gene of an influenza virus. The invention further relates lo expression vectors, cells and compositions comprising the polynucleotides, as well as methods of treating or preventing influenza in a subject by administering the polynucleotide, vector, cell or composition to the subject.

BACKGROUND OF THE INVENTION

Three types of influenza viruses, types A, B, and C are known and they belong to a family of single-stranded negative-sense enveloped RNA viruses called Orthomyxoviridae. The viral genome is approximately 12000 to 15000 nucleotides in length and comprises eight RNA segments (seven in Type C) that encode eleven proteins.

Influenza A virus infects many animals such as humans, pigs, horses, marine mammals, and birds. Its natural reservoir is in aquatic birds, and in avian species most influenza virus infections cause mild localized infections of the respiratory and intestinal tract. However, the virus can have a highly pathogenic effect in poultry, with sudden outbreaks causing high mortality rates in affected poultry populations.

Influenza A viruses can be classified into subtypes based on allelic variations in antigenic regions of two genes that encode surface glycoproteins, namely, hemagglutinin (HA) and neuraminidase (NA) which are required for viral attachment and cellular release. Other major viral proteins include the nucleoprotein, the nucleocapsid structural protein, matrix proteins (Ml and M2), polymerases (PA, PB1 and PB2), and non-structural proteins (NS1 and .NS2).

At least sixteen subtypes of HA (HI to H16) and nine NA (Nl lo N9) antigenic valiants are known in influenza A virus. Avian influenza strains can also be characterized as low pathogenic and highly pathogenic strains. Low pathogenic strains typically only have two basic amino acids at positions -1 and -3 of the cleavage site of the HA precursor, while highly pathogenic strains have a multi-basic cleavage site. Subtypes H5 and H7 can cause highly pathogenic infections in poultry and certain subtypes have been shown to cross the species barrier to humans. Highly pathogenic H5 and 117 viruses can also emerge from low pathogenic precursors in domestic poultry. Symptoms of avian influenza infection range from typical influenza type

symptoms (fever, cough, sore throat and muscle aches) to conjunctivitis, pneumonia, acute respiratory distress, and other life-threatening complications.

There is a need to develop ways of controlling influenza virus survival and/or replication in humans and animals such as poultry not only to improve productivity and welfare in the livestock industry, but to also reduce health risks to humans.

SUMMARY OF THE INVENTION

The present inventors have determined that a polynucleotide comprising a dsRNA region that is complementary to a pailicular region of the NSl gene segment in the influenza virus genome targets a sequence comprising two overlapping reading frames, one reading from encoding NSl and the second encoding the NEP polypeptide. Thus, the polynucleotide of the invention is able to target two message RNAs and is particularly effective at inhibiting the replication of influenza virus in a cell.

Accordingly, the present invention provides an isolated nucleic acid molecule comprising a double-stranded region, wherein the double-stranded region comprises a sequence of nucleotides complementary to a target sequence, and wherein the target sequence is at least 90% identical to any one of SEQ ID NOs:l to 6, 12 to 19. 25 or 26.

In one particular embodiment, the double-stranded region comprises a sequence of nucleotides at least 95% identical to any one of SEQ TD NOs:1 to 6, 12 to 19, 25 or 26.

In yet another embodiment, the double-stranded region comprises a sequence of nucleotides identical lo any one of SEQ ID NOs: 1 to 6, 12 to 19, 25 or 26.

In another embodiment, the double-stranded region comprises a sequence of nucleotides at least 90%, or at least 95%, identical to any one of SEQ ID NOs:2 to 6.

In yet another embodiment, the double-stranded region comprises a sequence of nucleotides identical to any one of SEQ ID NOs:2 to 6.

In another embodiment, the double-stranded region comprises a sequence of nucleotides at least 95% identical to SEQ ID NO:2. In one particular embodiment, the double-stranded regions comprises a sequence of nucleotides identical to SEQ ID NO:2.

While the double-stranded region may be of any length sufficient to induce

RNA interference in a cell, in one embodiment, the double-stranded region is 19 to 23 nucleotides in length. For example, the double-stranded region may be 19, 20, 21, 22 or 23 nucleotides in length.

In one embodiment, the isolated nucleic acid molecule comprises, or consist of, RNA or an analog thereof. In one particular embodiment, the isolated nucleic acid molecule is an siRNA, shRNA, eshRNA, or miRNA.

In an embodiment, the RNA comprises a sequence which is at least 90%, or at least 95%, identical to any one of SEQ ID NOs:13 to 19, 22 to 24, 26 or 27. In yet another embodiment, the RNA comprises, or consists of, a sequence of nucleotides identical to any one of SEQ ID NOs: 13 to 19, 22 to 24, 26 or 27. In a further embodiment, the RNA comprises, or consists of, a sequence which is at least 90%, or at least 95%, identical to any one of SEQ ID NOs:22 to 24. In yet another embodiment, the RNA comprises, or consists of, a sequence of nucleotides identical to any one of SEQ ID NOs: 22 to 24.

In another embodiment, the isolated nucleic acid molecule further comprises one or more double-stranded regions comprising a sequence of nucleotides complementary to a target sequence in an influenza A gene. In one particular embodiment, the one or more double-stranded regions comprises a sequence of nucleotides at least 90%; identical to SEQ ID NO:9 or 10.

In yet another embodiment, the isolated nucleic acid molecule reduces influenza A virus replication in an animal cell and/or reduces production of infectious influenza A virus particles in an animal cell and/or reduces the expression of an influenza A virus polypeptide in an influenza A virus infected animal cell when compared lo an isogenic influenza A virus infected animal cell lacking the RNA molecule.

The present invention further provides a nucleic acid construct encoding the nucleic acid molecule of die invention.

In one embodiment, the nucleic acid construct comprises a sequence of nucleotides at least 95% identical to any one of SEQ ID NOs:7, 8, 11, 20 or 21.

In another embodiment, the nucleic acid construct comprises a sequence of nucleotides identical to any one of SEQ ID NOs:7, 8, 11 , 20 or 21.

In yet another embodiment, the nucleic acid construct comprises one or more promoters operably linked to the open reading frame encoding a nucleic acid molecule of the invention, preferably an RNA molecule of the invention. In one embodiment, the one or more promoters is an RNA polymerase III promoter. Examples of RNA polymerase III promoters include U6 and HI promoters.

The present invention further provides a vector comprising the isolated nucleic acid molecule of the invention and/or the nucleic acid construct of the invention. Preferably, the vector is an expression vector.

The present invention further provides a cell comprising the isolated nucleic acid molecule of the invention, the nucleic acid construct of the invention, and/or the vector of the invention.

In one embodiment, the cell is an avian cell or a mammalian cell.

In another embodiment, the cell is a chicken, turkey or duck cell. In one particular embodiment the cell is a chicken primordial germ cell.

In another embodiment, the cell is a bacteria] cell. For example, the cell may be a Gram negative bacterial cell, and in one particular embodiment the cell is an E. coli cell.

The present invention further provides a composition comprising the isolated nucleic acid molecule of the invention, the nucleic acid construct of the invention, the vector of the invention, and/or the cell of the invention.

In one embodiment, the composition is a pharmaceutical and/or veterinary pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient.

In another embodiment, the composition is an animal feed composition.

The present invention further provides a method of treating or preventing influenza in a subject, the method comprising administering to the subject the isolated nucleic acid molecule of the invention, the nucleic acid construct of the invention, the vector of the invention, the cell of the invention, and/or the composition of the invention.

The subject may be human. Alternatively, the subject may be a non-human animal. In one embodiment, the non-human animal is an avian. In one particular embodiment, the subject is poultry. The poultry may be, for example, a chicken, turkey or duck.

The present invention further provides a non-human transgenic organism comprising the isolated nucleic acid molecule of the invention, the nucleic acid construct of the invention, the vector of the invention, the cell of the invention.

In one embodiment, the non-human transgenic organism is a plant.

In another embodiment, the non-human transgenic organism is a non-human transgenic animal.

In one embodiment, the non-human transgenic animal is an avian, for example poultry. In one particular embodiment, the non-human transgenic animal is a chicken, turkey or duck.

The present invention further provides the non-human transgenic organism of the invention for use in breeding. The present invention further provides the non-human transgenic organism of the invention for use in food production.

The present invention further provides a method of reducing the level of expression of influenza A vims NS1 and NEP genes in a cell, the method comprising introducing into the cell the isolated nuclei acid molecule of the invention, the isolated polynucleotide of the invention, the vector of the invention, and/or the composition of the invention.

In one embodiment, the level of NSl and/or NEP mRNA in the cell is reduced in comparison to an isogenic cell that does not comprise the cell the isolated nucleic acid molecule of the invention, the isolated polynucleotide of the invention, the vector of the invention, the cell of the invention, and/or the composition of the invention.

The present invention further provides an isolated nucleic acid molecule of the invention, the isolated polynucleotide oi^' the invention, the vector of the invention, the cell of the invention, and/or the composition of the invention for use in the treatment or prevention of influenza.

The present invention further provides use of the isolated nucleic acid molecule of the invention, the isolated polynucleotide of the invention, the vector of the invention, the cell of the invention, and/or the composition of the invention in the manufacture of a medicament for the treatment or prevention of influenza.

The present invention further provides a method of making a transgenic non- human animal, the method comprising:

(i) introducing a first nucleic acid comprising a transposon into a cell, wherein the nucleic acid encodes the isolated nucleic acid of the invention,

(ii) introducing a second nucleic acid encoding a transposase into the cell, (ii) selecting a transgenic cell comprising the first nucleic acid in the genome of the cell,

(iii) regenerating a transgenic non-human animal from the cell, and

(iv) breeding the transgenic non-human animal.

In one embodiment, the transposon is a Tol2 transposon and the transposase is Tol2 transposase.

In another embodiment, the cell is a chicken primordial germ cell.

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 FIGURES

Figure 1. Hemagglutination assay to measure inhibition of virus replication. MDCK cells were transfected with shRNA plasmids and control EGFP plasmid. Cells were infected 24 hrs post transfection with H1N1 (PR8) influenza vims. Supernatant was harvested from infected cells 48 hours post infection and a hemagglutination assay performed. Results are represented as percentage inhibition compared to EGFP control (set at 100% or no inhibition).

Figure 2. qPCR assay to measure inhibition of virus replication. MDCK cells were transfected with shRNA plasmids and control EGFP plasmid. Cells were infected 24 hrs post transfection with H1N1 (PR8) influenza virus. RNA was harvested from infected cells 48 hours post infection and a qPCR assay performed to measure viral RNA. Results were made relative to the EGFP control sample and are represented as a percentage of this control.

Figure 3. Tertiary structure of (A) miR30 and (B) NSl NEP miR30 structure with bulges and mismatches (NS l/NEP S+L).

Figure 4. Relative expression of different miRised NS1/NEP constructs in DFI cells. DFI cells were transfected with the miRised NSl/NEP and brummelkamp loop NSl/NEP constructs using lipofectamine 2000. At 48 hours post infection total RNA was extracted from the transfected cells and cDNA synthesised. A polyA tail step was included in the cDNA synthesis. Realtime PCR was performed on the cDNA using a forward primer specific for NS l/NEP and a universal reverse primer. The levels of 55 were also measured and results normalised to these levels. The graph shows relative expression of NS 1/NEP from the different constructs relative to a DFI control.

Figure 5. In vitro silencing of NSl/NEP constructs as measured by hemagglutination assay. MDCK cells were transfected with the shRNA plasmid and control EGFP plasmid. Cells were infected 24 hrs post transfection with ITINI (PR8) influenza virus at a MOl of 0.001. Supernatant was harvested from infected cells 48 hours post infection and a hemagglutination assay performed. Results are presented as a percentage inhibition compared to the eGFP negative control (set at 100% of no inhibition).

KEY TO THE SEQUENCE LISTING

SEQ ID NO: I - NS1 -NEP target region sequence

SEQ ID NO:2 - NS1 -NEP taiget sequence 1

SEQ ID NO:3 - NS 1 -NEP target sequence 2

SEQ ID NO:4 - NS1-NEP target sequence 3

SEQ ID NO:5 - NS1-NEP target sequence 4

SEQ ID NO:6 - NS1 -NEP target sequence 5

SEQ ID NO:7 -- NS1-NEP U6 promoter construct

SEQ ID NO:8 - NSl-NEP HI promoter construct

SEQ ID NO:9 - PB 1 -2257 siRNA target sequence

SEQ ID NO: 10 - PB-1498 siRNA target sequence

SEQ ID NO: 1 1 - Multi-warhead sequence

SEQ ID NO:! 2 - Common target sequence

SEQ ID NO: 13 - RNA version of NS 1 -NEP target region sequence

SEQ ID NO: 14 - RNA version of NS 1 -NEP target sequence 1

SEQ ID NO: 15 - RNA version of NSl-NEP taiget sequence 2

SEQ ID NO: 16 - RNA version of NSl-NEP target sequence 3

SEQ ID NO: 17 - RNA version of NSl-NEP target sequence 4

SEQ ID NO: 18 - RNA version of NS 1 -NEP target sequence 5

SEQ ID NO:19 - RNA version of common target sequence

SEQ ID NO:20 - NS 1/NEP miR30 L U64 promoter construct

SEQ ID NO-.21 - NSl/NEP miR30 S+L U64 promoter construct

SEQ ID NO:22 - NSl/NEP shRNA with Brummelkamp loop (BK)

SEQ ID NO:23 - NSl NEP miR30 structure without bulges and mismatches (NSl/NEP L)

SEQ ID NO:24■■ NS l/NEP miR30 structure with bulges and mismatches (NSl/NEP S+L)

SEQ ID NO:25 -- Extended NSl-NEP target sequence region

SEQ ID NO:26 - RNA version of extended NS I -NEP target sequence region SEQ ID NO:27 - Sense strand of modified miRNA version of taiget sequence I SEQ ID NO:28 - mir30

DETAILED DESCRIPTION OV THE INVENTION

General Techniques and Selected 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, molecular biology, RNA silencing, virology, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the molecular biology, microbiological and cell culture 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^Kl ed., Cold Spring Harbour Laboratory Press (2001), T.A. Brown (editor). Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, 1RL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, I L 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 other embodiments, the subject may be avian, for example poultry such as a chicken, turkey or a duck. In another embodiment, the subject is a pig.

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.

As used herein the terms "treating", "treat" or "treatment" include administering a therapeutically effective amount of a nucleic acid constnict, vector, cell and/or nucleic acid molecule of the invention sufficient to reduce or eliminate at least one symptom of an influenza A virus infection, especially avian influenza virus, infection. The term "preventing" refers to protecting a subject that is exposed to influenza A virus from developing at least one symptom of an influenza A vims infection, or reducing the severity of a symptom of infection in a subject exposed to influenza A vims.

The influenza virus may be an influenza A virus. The influenza A virus may be selected from influenza A viruses isolated from avian and mammalian organisms. In particular, the influenza A virus may be selected from H1 N1 , H1 N2, H1 N3, H1 4, H1 5, H1N6, ITI N7, H1N9, H2N1 , H2N2, H2N3, H2N4, H2N5. H2N7, H2 8, H2N9, Η3Ν , H3N2, H3N3, H3N4, H3 5, H3N6, H3N8, Η4ΝΊ , H4N2, H4N3, H4N4, H4N5, H4N6, H4N8, H4N9, H5N1. H5N2, H5N3, H5N6. H5N7, H5N8, H5N9, H6NI, H6N2, H6N3, H6N4, H6N5, H6N6, H6N7, H6N8, H6N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7 7, H7N8, H7N9, H9N1 , H9N2, H9N3, H9N5. H9N6, H9N7, H9N8, H10NI , H10N3, H10N4, H10N6, H10N7, H10NS, H10N9, H11N2, H11 3, H11N6, H11N9, H12N1, H12N4, H12N5, H12N9, H13N2, H13N6, H13N8, H13N9, H14N5, H15N2, H15N8, H 15N9 and H 16N3. In one embodiment, the influenza A virus is selected from H I N1 , H3N2, H7N7, and/or H5 1 .

"Vims replication^" as used herein refers to the amplification of the viral genome in a host cell.

By "reduces the expression of, "reducing the expression of^", or "reducing the level of expression" a polypeptide, polynucleotide or gene is meant that the translation of a polypeptide sequence and/or transcription of a polynucleotide sequence in a host cell is down-regulated or inhibited. The degree of down-regulation or inhibition will vary with the nature and quantity of the nucleic acid construct or nucleic acid molecule provided to the host cell, the identity, nature, and level of nucleic acid molecule(s) of the invention expressed from the construct, the time after administration, etc., but will be evident e.g., as a detectable decrease in target gene/sequence protein 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% as compared to a ceil not treated according to the present invention will be achieved.

As used herein, the term "transposon" refers to a genetic element that can move (transpose) from one position to another within the genome of an organism by processes which do not require extensive DNA sequence homology between the transposon and the site of insertion nor the recombination enzymes need for classical homologous crossing over.

As used herein, the term "introducing" as it relates to a nucleic acid construct or nucleic acid molecule is to be taken in the broadest possible sense and include any method resulting in the nucleic acid construct or nucleic acid molecule being present in a cell or organism. For example, the nucleic acid construct or nucleic acid molecule may be delivered to a cell as naked DNA via any suitable transl^'ection or transformation technique such as, for example, electroporation. Alternatively, the nucleic acid construct or nucleic acid molecule may be inserted into the genome and/or be expressed by a transgene in a cell.

As used herein, "isogenic" refers to organisms or cells that are characterised by essentially identical genomic DNA, for example the genomic DNA is at least about 92%, preferably at least about 98%, and most preferably at least about 99%, identical to the genomic DNA of a corresponding organism or cell (for example an organism or cell which is genetically the same except for lacking a nucleic acid construct of the invention).

As used herein, the term "and/or" towards the end of a list of a number of options means the same as any one or more or all of the listed alternatives.

Nucleic Acids and RNA Interference

The terms "RNA interference", "RNAi" or "gene silencing" are well known in the art and 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 RN.A molecule shares substantial or total sequence identity. It has been shown that RNA interference can be achieved using non-RNA double stranded molecules (see, for example. US 20070004667).

The present invention includes nucleic acid molecules comprising and/or encoding double-stranded regions for RNA interference. The nucleic acid molecules are typically RNA but may comprise chemically-modified nucleotides and non- nucleotides.

The double-stranded regions should be at least 1 contiguous nucleotides, for example about 19 to 23 nucleotides, or may be longer, for example 30 or 50 nucleotides, or 100 nucleotides or more. The full-length sequence corresponding to the entire gene transcript may be used.

The degree of identity of a double-stranded region of a nucleic acid molecule to the targeted U'anscript 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. The nucleic acid molecules of the present invention may be siRNA, shRNA, miRNA, short interfering nucleic acid (si A), short interfering modified oligonucleotide, chemically-modified siRNA, posl-transcriptional gene silencing RNA (ptgsRNA), and others.

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 1 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.

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' L'UCAAGAGA 3'. In one embodiment, the nucleic acid molecule is an extended shRNA ("eshRNA") that can be processed by the RNAi machinery into multiple siRNA duplexes (Liu et al.. 2007). An eshRNA construct typically comprises a single promoter, two or three sequences encoding siRNA sequences targeting a gene of interest and a loop sequence.

Included shRNAs are dual or bi-finger and multi-finger hairpin dsRNAs, in which the RNA molecule comprises two or more of such stem-loop stractures separated by single-stranded spacer regions.

MicroRNAs (miRNAs) are small single-stranded non-coding RNAs that play critical roles in the regulation of biological processes. MicroRNAs are initially transcribed as a long, single-stranded miRNA precursor known as a primary-miRNA (pri-miRNA), which may contain one or several miRNAs. These pri-miRNAs typically contain regions of localized stem-loop hairpin stractures that contain tlie mature miRNA sequences. Pri-miRNAs are processed into 70-100 nucleotide pre-miRNAs in the nucleus by the double-stranded RNA-specil^'ic nuclease Drosha. These 70- 100 nucleotide pre-miRNAs are transported to the cytoplasm, where they are processed by the enzyme Dicer into single-stranded mature miRNAs of about 1 -25 nucleotides. As known in the art, naturally-occurring or synthetic miRNAs may be modified to comprise a sequence of nucleotides complementary to one or more target gene sequences of interest.

ieroRNAs can be produced by modifying naturally occurring molecules to include a double stranded portion comprising the target sequence. Examples of such miRNAs that can be modified for use in molecules of the invention include, but are not limited to, miR30. miR19b, miR17 and miR107.

Several factors need to be taken into account when designing miRNAs such as the size of the miR, position (5' or 3' ), loop sequence, bulges and other sequence. Once the appropriate length is identified, the bulges and mismatches are identified and the final structure checked using, for example, m-fold. Guidelines for the design of miRNAs are described in Griffiths-Jones (2004), Griffiths- Jones (2006 and 2008), Kozomara and Griffiths-Jones (201 1 and 2014), and Zuker (2003).

Generally to "miRise" a shRNA structure its length needs to be extended to the length of the mature miRNA being mimicked. Mismatches then need to be matched to their position in the miRNA hairpin structure that is being mimicked. Such mismatches may be, for example, the sense strand being extended needs by one or a few nucleotides, for example two nucleotides. In an embodiment, is some instances there is a mismatch (when compared to the target) at the 5' end of the sense strand. When extending sequence to make it the appropriate size for the microRNA of choice the changes need to be made to the sense sequence and the new sequence need to be examined by the Taxman algorithm (Taxman et al., 2006) and free energy of the structure calculated to ensure it is optimal.

When the RNA is against a viral molecule it is important to look at the consensus sequences of the target to ensure the extensions being made are still targeting conserved sequence. Alterations in sequences to introduce mismatches seen in the microRNA structures should preferably be done in the sense strand only which gives rise to the non-active siRNA 'passenger' strand. The anti-sense strand of the RNA stem (designed to give rise to the active siRNA 'guide' strand) should preferably remain the same.

As known in the art, and as indicated above, RNAi molecules such as siRNA, shRNA and miRNAs may contain a "bulge" in the antisense strand in order to reduce or eliminate off-target silencing (Dua et al.. 201 1). For example, a nucleic acid molecule of the invention comprising a double-stranded region of 19 nucleotides in length may comprise a single non-matching nucleotide at the 5' end of the molecule. Thus, despite the antisense strand of the double-stranded region comprising 20 nucleotides and the sense strand comprising 19 nucleotides, such nucleic acid molecules of the invention are still be considered to comprise a double-stranded of 1 nucleotides.

Once designed, the nucleic acid molecules comprising a double-stranded region can be generated by any method known in the art, for example, by in vitro transcription, recombinantly (such as by expression of a nucleic acid construct of the invention in a cell of the invention), or by synthetic means.

Modifications or analogs of nucleotides can be introduced to improve the properties of the nucleic acid molecules of the invention. Improved properties include increased nuclease resistance and/or increased ability to permeate cell membranes. Accordingly, the terms "nucleic acid molecule" and "double-stranded RNA molecule" includes synthetically modified bases such as, but not limited to, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl-, 2-propyl- and other alkyl- adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiuracil, 8- halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substiluted adenines, 8-halo guanines, 8-atnino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other substituted guanines, other aza and deaza adenines, other aza and deaza guanines, 5-trifluoromethyl uracil and 5- trifltioro cytosine.

Nucleic Acid Molecules

By "isolated nucleic acid molecule" we mean a nucleic acid 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. In some, if not all embodiments, the nucleic acid molecules of the invention do not exist in nature. Thus, in these circumstances the "isolated nucleic acid molecule" can also be considered as an "exogenous nucleic acid molecule" when present, for example, in a cell of the invention. Furthermore, the term "nucleic acid molecule" is used interchangeably herein with the term "polynucleotide".

The terms "nucleic acid molecule" or "polynucleotide" refer to an oligonucleotide, polynucleotide or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, and combined with carbohydrate, lipids, protein, or other materials to perform a particular activity defined herein. In a preferred embodiment, the nucleic acid molecule is an RNA molecule.

As used herein, the term "nucleic acid molecule comprising a double-stranded region" means that the molecule can be fully double stranded or have a combination of single stranded and double stranded portions such as one or more loops or bulges, and/or one or more (such as two, three or four) additional 5' and/or 3' nucleotides overhanging the double stranded region.

As used herein, the term "target sequence'^" refers to a region of the influenza virus NS1 gene, and NEP gene, or mRNAs encoded by said genes. When present in a cell infected with the influenza virus, a nucleic acid molecule of the invention is processed into single stranded nucleic acids which hybridize the mRNAs, and initiate gene silencing. As used herein, the term "complementary to a target sequence" refers to the reverse complement of the mRNAs encoded by said genes such that the processed single stranded nucleic acids which hybridize the mRNAs. The single stranded nucleic acids which hybridize the mRNAs do not necessarily need to be a perfect complement (although it is preferred), but must be sufficiently complementary to initiate gene silencing.

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 die 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.

A nucleic acid molecule of the present invention may selectively hybridise to a polynucleotide that encodes an influenza A virus polypeptide 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 forraamide, for example, 50% (vol/vol) formamide with 0.1 bovine serum albumin, 0.1 % Ficoll, 0.1 % polyvinylpyrrolidone, 50 mM sodiiun 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% dexlran sulfate at 42°C in 0.2 x SSC and 0.1 % SDS.

Usually, monomers of a nucleic acid are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a relatively short monomeric units, e.g., 19-25, to several hundreds of monomeric units. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodi!hioate, phosphoroselenoate, phospliorodiselenoatc, phosphoroanilothioate, phosphoranilidate, phosphoramidate.

Nucleic Acid Constructs

As used herein, "nucleic acid construct" refers to any nucleic acid molecule that encodes a double-stranded RNA molecule as defined herein and includes the nucleic acid molecule in a vector, the nucleic acid molecule when present in a cell as an extracliromosomal nucleic acid molecule, and a nucleic acid molecule that is integrated into the genome. Typically, the nucleic acid construct will be double stranded DNA or double-stranded RNA, or a combination thereof. Furthermore, the nucleic acid construct will typically comprise a suitable promoter operably linked to the open reading frame encoding the double-stranded RNA. The nucleic acid construct may comprise a first open reading frame encoding a first single strand of the double- stranded RNA molecule, with the complementary (second) strand being encoded by a second open reading frame by a different, or preferably the same, nucleic acid construct. The nucleic acid construct may be a linear fragment or a circular molecule and it may or may not be capable of replication. The skilled person will understand that the nucleic acid construct of the invention may be included within a suitable vector. Transfection or transformation of die nucleic acid construct into a recipient cell allows the cell to express an RNA molecule encoded by the nucleic acid construct.

The nucleic acid construct of the invention may express multiple copies of the same, and/or one or more (e.g. 1 , 2, 3, 4, 5, or more) including multiple different, RNA molecules comprising a double-stranded region, for example a short hairpin RNA. RNA molecules considered to be the "same" as each other are those that comprise only the same double-stranded sequence, and RNA molecules considered to be "different" from each other will comprise different double-stranded sequences, regardless of whether the sequences to be targeted by each different double-stranded sequence are within the same, or a different gene, or sequences of two different genes. Examples of additional dsRNA molecules which may be encoded by a nucleic acid construct of the invention are described in WO 2008/138072.

The nucleic acid construct also may contain additional genetic elements. The types of elements that may be included in the construct are not limited in any way and may be chosen by one widi skill in the art. In some embodiments, the nucleic acid construct is inserted into a host cell as a transgene. In such instances it may be desirable to include "stuffer" fragments in the construct which are designed to protect the sequences encoding the RNA molecule from the transgene insertion process and to reduce die risk of external transcription read through. Stuffer fragments may also be included in the construct to increase the distance between, e.g., a promoter and a coding sequence and/or terminator component. The stuffer fragment can be any length from 5- 5000 or more nucleotides. There can be one or more stuffer fragments between promoters. In the case of multiple stuffer fragments, they can be the same or different lengths. The stuffer DNA fragments are preferably different sequences. Preferably, the stuffer sequences comprise a sequence identical to that found within a cell, or progeny thereof, in which they have been inserted. In a further embodiment, the nucleic acid construct comprises stuffer regions flanking the open reading frame(s) encoding the double stranded RNA(s).

Alternatively, the nucleic acid construct may include a transposable element, for example a transposon characterized by terminal inverted repeat sequences flanking die open reading frames encoding the double stranded RNA(s). Examples of suitable tiansposons include Tol2, mini-Tol, Sleeping Beauty, Mariner and Galluhop.

Other examples of an additional genetic element which may be included in the nucleic acid construct include a reporter gene, such as one or more genes for a fluorescent marker protein such as GFP or RFP; an easily assayed enzyme such as beta- galactosidase, luciferase, beta-glucuronidase, chloramphenicol acetyl Iransferase or secreted embryonic alkaline phosphatase; or proteins for which immunoassays are readily available such as hormones or cytokines. Other genetic elements that may find use in embodiments of the present invention include those coding for proteins which confer a selective growth advantage on cells such as adenosine deaminase, aminoglycodic phosphotransferase, dihydrofolate reductase, hygromycin-B- phosphotransferase, or drug resistance.

Where the nucleic acid construct is to be transfected into an animal, it is desirable that the promoter and any additional genetic elements consist of nucleotide sequences that naturally occur in the animal's genome. It is further desirable that the sequences encoding RNA molecules consist of influenza A virus sequences.

Vectors and Host Cells

In some instances it may be desirable to insert the nucleic acid construct and/or nucleic acid molecule of the invention into a vector. The vector may be, e.g., 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 diose 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 D A viral vector.

The vector into which the nucleic acid construct is inserted may also include a transposable element, for example a transposoii 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.

The present invention also provides a host cell into which the nucleic acid construct, nucleic acid molecule 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 dsRNA molecule. For in vitro production, eukaryotic cells or prokaryotic cells can be used.

Useful eukaryotic host cells may be animal, plant, or fungal cells. As animal cells, mammalian cells such as CHO, COS. 3T3, DPI, CEF, MDCK myeloma, baby hamster kidney (BHK), HeLa, or Vero cells, amphibian cells such as Xenopus oocytes, or bisect cells such as Sf9, Sf21 , or Tn5 cells can be used. CHO cells lacking DHFR gene (dhfr-CHO) or CHO -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.

Useful prokaryolic cells include bacteria] cells, such as E. oli, for example, JM109, DH5a, and HB101, or Bacillus subtilis.

Culture medium such as DM EM, MEM, RPM1 1640, or 1MDM 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.

Transgenic Non-Human Organisms

A "transgenic non-human organism" refers to a plant or an animal, other than a human, that contains a nucleic acid construct ("transgene") not found in a wild-type plant or animal of the same species or breed. A "transgene" as referred to herein has the normal meaning in the art of biotechnology and includes a genetic sequence which has been produced or altered by recombinant DNA or RNA technology and which has been introduced into a plant or an animal, preferably avian, cell. The transgene may include genetic sequences derived from an animal or plant cell. Typically, the transgene has been introduced into an animal or plant by human manipulation such as, for example, by transformation but any method can be used as one of skill in the art recognizes. A tra sgene includes genetic sequences that are introduced into a chromosome as well as those that are extrachromosomal.

Techniques for producing transgenic animals are well known in the art. A useful general textbook on this subject is Houdebine, Transgenic animals - Generation and Use (Harwood Academic, 1997).

Heterologous DNA can be introduced, for example, into fertilized ova. For instance, totipotent or pluripotent stem cells can be transformed by microinjection, calcium phosphate mediated precipitation, liposome fusion, retroviral infection or other means, the transformed cells are then introduced into the embryo, and the embryo then develops into a transgenic animal. In one method, developing embryos are infected with a retrovirus containing the desired DNA, and transgenic animals produced from the infected embryo. In an alternative method, however, the appropriate DNAs are coinjected into the pronucleus or cytoplasm of embryos, preferably at the single cell stage, and the embryos allowed to develop into mature transgenic animals.

Another method used to produce a transgenic animal involves microinjecting a nucleic acid into pro-nuclear stage eggs by standard methods. Injected eggs are then cultured before transfer into the oviducts of pseudopregnant recipients.

Transgenic animals may also be produced by nuclear transfer technology. Using this method, fibroblasts from donor animals are stably transfected with lasmid incorporating the coding sequences for a binding domain or binding partner of interest under the control of regulatory sequences. Stable transfectants are then fused to enucleated oocytes, cultured and transferred into female recipients.

Sperm-mediated gene transfer (SMGT) is another method that may be used to generate transgenic animals. This method was first described by Lavitrano et al. (1989).

Another method of producing transgenic animals is linker based sperm-mediated gene transfer technology (LB-SMGT). This procedure is described in US 7,067,308. Briefly, freshly harvested semen is washed and incubated with murine monoclonal antibody mAbC (secreted by the hybridotna assigned ATCC accession number PTA- 6723) and then the construct DNA. The monoclonal antibody aids in the binding of the DNA to the semen. The sperm/DNA complex is then artificially inseminated into a female.

Germline transgenic chickens may be produced by injecting replication- defective retrovirus into the subgerminal cavity of chick blastoderms in freshly laid eggs (US 5,162,215; Bosselman et al., 1989; Thoraval et al.. 1995). The retroviral nucleic acid carrying a foreign gene randomly inserts into a chromosome of the embryonic cells, generating transgenic animals, some of which bear the transgene in their germ line. Use of insulator elements inserted at the 5' or 3' region of the fused gene construct to overcome position effects at the site of insertion has been described (Chim et al, 1993).

Another method for generating germline transgenic animals is by using a transposon, for example the Tol2 transposon, to integrate a nucleic acid construct of the invention into tire genome of an animal. The Tol2 transposon which was first isolated from the medaka fish Oryzias latipes and belongs to the hAT family of transposons is described in Koga et al. (1996) and Kawakami et al. (2000). Mini-Tol2 is a variant of Tol2 and is described in Balciunas et al. (2006). The Tol2 and Mini-Tol2 transposons facilitate integration of a transgene into the genome of an organism when co-acting with the Tol2 transposase. By delivering the Tol2 transposase on a separate non- replicating plasmid, only the Tol2 or Mini-Tol2 ransposon and transgerie is integrated into the genome and the plasmid containing the Tol2 transposase is lost within a limited number of cell divisions. Tims, an integrated Tol2 or Mini-Tol2 transposon will no longer have the ability to undergo a subsequent transposition event. Additionally, as Tol2 is not known to be a naturally occurring avian transposon, there is no endogenous transposase activity in an avian cell, for example a chicken cell, to cause further transposition events.

Any other suitable transposon system may be used in the methods of the present invention. For example, the transposon system may be a Sleeping Beauty, Frog Prince or Mosl transposon system, or any transposon belonging to the tcl/mariner or hAT family of transposons may be used.

The injection of avian embryonic stem cells into recipient embryos to yield chimeric birds is described in US 7.145,057. Breeding the resulting chimera yields transgenic birds whose genome is comprised of exogenous DNA.

Methods of obtaining transgenic chickens from long-term cultures of avian primordial germ cells (PGCs) are described in US 20060206952. When combined with a host avian embryo by known procedures, those modified PGCs are transmitted through the germline to yield transgenic offspring.

In one embodiment, transfection reagents can be mixed with an isolated nucleic acid molecule, polynucleotide or nucleic acid constnict as described herein and injected directly into the blood of developing avian embryos. This method is referred to herein as "direct injection" (see WO 2013/155572). Using such a method the isolated nucleic acid molecule, polynucleotide or nucleic acid construct of the invention is introduced into primordial germ cells (PGCs) in the embryo and inserted into the genome of the avian.

Accordingly, in the methods of the present invention an isolated nucleic acid, polynucleotide or nucleic acid construct is eomplexed or mixed with a suitable transfection reagent. The term "transfection reagent" as used herein refers to a composition added to the polynucleotide for enhancing the uptake of the polynucleotide into a eukaryotic cell including, but not limited to, an avian cell such as a primordial germ cell. While any transfection reagent known in the art to be suitable for transfecting eukaryotic cells may be used, the present inventors have found that transfection reagents comprising a cationic lipid are particularly useful in the methods of the present invention. Non-limiting examples of suitable commercially available transfection reagents comprising cationic lipids include Lipofectamine (Life Technologies) and Lipofectamine 2000 (Life Technologies). The polynucleotide may be mixed (or "complexed") with the transfection reagent according to the manufacturer's instructions or known protocols. By way of example, when trarisfecling plasmid DNA with Lipofectatnine 2000 transfection reagent (Invitrogen, Life Technologies), DNA may be diluted in 50 μΐ Opit-MEM medium and mixed gently. The Lipofectamine 2000 reagent is mixed gently and an appropriate amount diluted in 50 μΐ Opti-MEM medium. After a 5 minute incubation, the diluted DNA and transfection reagent are combined and mixed gently at room temperature for 20 minutes.

A suitable volume of the transfection mixture may then be directly injected into an avian embryo in accordance with the method of the invention. Typically, a suitable volume for injection into an avian embryo is about 1 μΐ to about 3 μΙ, although suitable volumes may be determined by factors such as the stage of the embryo and species of avian being injected. The person skilled in the art will appreciate that the protocols for mixing the transfection reagent and DNA, as well as the volume to be injected into the avian embryo, may be optimised in light of the teachings of the present specification.

Prior to injection, eggs are incubated at a suitable temperature for embryonic development, for example around 37.5 to 38°C, with the pointy end (taglion) upward for approximately 2.5 days (Stages 12-17), or until such time as the blood vessels in the embryo are of sufficient size to allow injection. The optimal lime for injection of the transfection mixture is the time of PGC migration that typically occurs around Stages 12-17, but more preferably Stages 13-14. As the person skilled in the art will appreciate, broiler line chickens typically have faster growing embryos, and so injection should preferably occur early in Stages 13-14 so as to introduce the transfection mixture into the bloodstream at the time of PGC migration.

To access a blood vessel of the avian embryo, a hole is made in the egg shell.

For example, an approximately 10 mm hole may be made in the pointy end of the egg using a suitable implement such as forceps. The section of shell and associated membranes are carefully removed while avoiding injury to the embryo and it's membranes.

Following injection of the transfection mixture into the blood vessel of the avian embryo, the egg is sealed using a sufficient quantity of parafilm, or other suitable sealant film as known in the art. For example, where a 10 mm hole has been made in the shell, an approximately 20 mm square piece of parafilm may be used to cover the hole. A warm scalpel blade may then be used to affix the parafilm to the outer egg surface. Eggs are then turned over to the pointy-end down position and incubated at a

temperature sufficient for the embryo to develop, such as until later analysis or hatch. The direct injection technique is further described in WO 2013/155572.

A viral delivery system based on any appropriate virus may be used to deliver the nucleic acid constructs of the present invention to a cell. In addition, hybrid viral systems may be of use. The choice of viral delivery system will depend on various parameters, such as efficiency of delivery into the cell, tissue, or organ of interest, transduction efficiency of the system, pathogenicity, immunological and toxicity concerns, and the like. It is clear that there is no single viral system that is suitable for all applications. When selecting a viral delivery system to use in the present invention, it is important to choose a system where nucleic acid constnict-containing viral particles are preferably: 1 ) reproducibly and stably propagated; 2) able to be purified to high titers; and 3) able to mediate targeted delivery (delivery of the nucleic acid expression construct to the cell, tissue, or organ of interest, without widespread dissemination).

The non-human transgenic animals of the invention have use in breeding and food production. Once a non-human transgenic animal with an increased resistance to influenza has been produced using the method of the invention, it can be bred to select for disease resistant progeny. The disease resistant progeny, for example, disease resistant poultry, are then suitable for distribution to producers for further breeding an food production. Methods for the production of food from livestock animals, such as poultry, are well known in the art.

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 nucleic acid construct(s) and/or nucleic acid 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/liposornic 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 01. 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 belween 0.8 and 1.2, inclusive, such as between 0.8 and 0.9, inclusive.

Administration of a nucleic acid construct, nucleic acid molecule and/or composition may conveniently be achieved 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 nucleic acid construct, nucleic acid 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 earner is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by tire 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.

A nucleic acid construct, nucleic acid 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.

Therapeutic and Prophylactic Methods

The present invention utilises the isolated nucleic acid molecules and nucleic acid constructs of the invention for the treatment and prevention of influenza in a subject. In one embodiment, the therapeutic and/or prophylactic methods of the invention comprise administering the isolated nucleic acid molecule of the invention, the nucleic acid construct of the invention, the vector of the invention, the ceil of the invention, or the composition of the invention to the subject.

In some instances it may be desirable to combine the administration of the inventive nucleic acid molecules, constructs, vectors or compositions ("the compounds of the invention") described herein with one or more other anti-viral agents in order to inhibit, reduce, or prevent one or more symptoms or characteristics of infection. In certain embodiments of the invention, the compounds of the invention are combined with one or more other antiviral agents such as NA inhibitors, M inhibitors, etc. Examples include amantadine or rimantadine and/or zanamivir, oseltamivir, peramivir (BCX-1812, RWJ-270201) Ro64-0796 (GS 4104) or RWJ-270201. However, the administration of the compounds of the invention may also be combined with one or more of any of a variety of agents including, for example, influenza vaccines (e.g., conventional vaccines employing influenza viruses or viral antigens as well as D A vaccines) of which a variety are known.

The compounds of the invention may be present in the same mixture as the other agent(s) or the treatment regimen for an individual includes both the compounds of tire invention and the other agent(s), not necessarily delivered in the same mixture or at the same time. Thus, as used herein, the term "combination" is not intended to indicate that compounds must be present in, or administered to a subject as, a single composition of matter, e.g., as part of the same dosage unit (e.g., in the same aerosol formulation, particle composition, tablet, capsule, pill, solution, etc.) although they may be. Instead, in certain embodiments of the invention the agents are administered individually but concurrently. As used herein the term "coadministration" or "concurrent administration" of two or more compounds is not intended to indicate that the compounds must be administered at precisely the same time. In general, compounds are coadministered or administered concurrently if they are present within the body at the same time in less than de minimis quantities.

The inventive therapeutic protocols described herein involve administering an effective amount of compound of the invention, simultaneously with, or after exposure to influenza virus. For example, uninfected individuals may be treated with an inventive composition prior to exposure to influenza; at risk individuals (e.g., the elderly, immunocompromised individuals, persons who have recently been in contact with someone who is suspected, likely, or known to be infected with influenza virus, etc.) can b treated substantially contemporaneously with exposure, e.g., within 2 hours or less following exposure. In other embodiments a subject is treated at a later time, e.g., within 2-12, 12-24, 24-36, or 36-48 hours, following a suspected or known exposure. The subject may be symptomatic or asymptomatic. In certain embodiments the subject is protected by administration of a compound of the invention up to 48 hours, up to 24 hours, up to 12 hours, up to 3 hours, etc., before an exposure. Of course

individuals suspected or known to be infected may receive inventive treatment at any time.

Influenza viruses infect a wide variety of species in addition to humans. The present invention includes the use of the inventive compositions for the treatment of nonhuman species, particularly species such as chickens, ducks, turkeys, swine, and horses. For the treatment and prevention of influenza in livestock animals, it may be desirable to formulate the inventive nucleic acid molecules, constructs, vectors or compositions into an animal feed or in drinking water.

EXAMPLES

Example 1. Identification of S1- EP target region

The present inventors have identified a region of the influenza genome which overlaps the NS1 and NEP open reading frames in genome segment 8. Within this region, the inventors have advantageously identified a 23 nucleotide sequence that is conserved across many influenza virus isolates, thus making it an excellent target for molecules that induce RNA interference in a cell. The sequence of the 23 nucleotide conserved region is provided as SEQ ID NO:l.

Provided in SEQ ID NOs:2 to 6 are 19-mer oligonucleotides complementary to target sequences within the conserved 23 nucleotide region of NSl-NEP. Nucleic acid molecules comprising a double-stranded region complementary to a target sequence comprising any one of SEQ ID NOs:2 to 6 share the common structural and functional capability of being able to inhibit the expression of a target sequence comprising SEQ ID NO: 12. Expression constructs comprising an shRNA targeting the NSl-NEP conserved region under control of the U64 promoter (SEQ ID NO:7) and HI promoter (SEQ ID NO:8) were also prepared, along with a multi-warhead construct expressing shRNA targeting NSl -NEP, PB I -2257 and NP-1498 (SEQ ID NO:l l ).

Example 2. In vitro silencing of Influenza PR8 in MDCK cells by shRNA

The inventors tested the ability of an shRNA comprising a sequence complementary to SEQ ID NO:2 to induce RNA interference and inhibition of viral replication in MDCK cells infected with the PR8 strain of influenza virus. In this experiment, the shRNA sequence was expressed from the U64 promoter (SEQ ID NO:7).

MDCK cells were freshly passed on the day prior to experiments so that the cells were healthy and not over confluent. The cells were trypsinised to remove them

from the flask, counted and aliquoted into 1.5 ml microcentrifuge tubes at 1 x 10⁶ cells per tube. Cells were pelleted at 4000 rpm for 3 minutes in a bench-top microcentrifuge.

MDCK cells were cultured in Earls Modified Eagle's Medium containing 10% Foetal Calf Serum (FCS), 10 mM Hepes, 2 mM glutamine, supplemented with penicillin (100 U/ml) and streptomycin (100 μ«/ιτ>1) at 37°C in a humidified atmosphere containing 5% CO₂ . Logarithmic phase cells were trypsinized, counted and aliquoted into 1.5 million cells for each electroporation. Cells were resuspended in 100 μΐ of electroporation solution T (Amaxa), mixed with 2.5 pg of plasmid DNA and eleciroporated using program T20 of the Amaxa Nucleofector. Cells were plated out in a 24 well plate at 250 000 cells per well in duplicate for each infection.

After incubation for 31 hours, medium was removed from the cells and 200 μ] of influenza PR8 virus diluted in Viral Growth Media was added. The Viral Growth Media comprised EMEM, 0.3% bovine serum albumen, HEPES, glutamine, penicillin/streptomycin and 5 pg/ml trypsin. Infections were performed in duplicate. The cells were incubated at 37°C for 1 hour. Virus was removed from wells and 500 μΐ of Viral Growth Medium added to each well. After 66 hours incubation, the supematanls were removed and hemagglutination assays were performed. Cells were removed in RLT buffer and used in real time PCR assays. The results of the hemagglutination assays are provided in Table 1 and Figure 1.

Table 1. Results of Hemaglutination Assays

Example 3. Quantitative PCR

Total RNA was isolated from cells described above in Example 2 using a

Qiagen RNeasy kit and viral mRNA was quantified by real time PCR with specific primers for Influenza A M gene (Heine et al., 2007) using an ABI 7700 sequence detection system (Applied Biosystems). A primer/probe mix was prepared by mixing together equal volumes of primers IVA-D161M (18 μΜ) and IVA-D162M (18 μΜ) and probe lVA-Ma(FAM) (5 μΜ) (Heine et al., 2007). Real-time reverse transcription- PCR (RRT-PCR) reactions were set up in 25 μΐ volumes comprising 5.75 μΐ nuclease- free water; 12.5 μΐ TaqMan 2x Universal PCR master mix no AmpErase UNG; 0.625 μΐ 40x Multiscribe and RNase inhibitor mix; 4.125 μΐ test primer probe mix; and 2 μΐ of total RNA. Reactions were run in a real-time PCR theraiocycler with the following parameters; 30 min at 48^CC (reverse transcription), 10 min at 95°C (hot-start Taq polymerase activation), and 45 cycles of 15 sec at 95°C and 1 min at 60°C (target amplification). PCR results were analysed were made relative to the EGFP control sample and represented as a percentage of this control. Results in Figure 2 show that NS1/NEP sliRNA reduced viral RNA levels by greater than 90% compared to the BGFP control plasmid.

Example 4. MiRised hairpins

Methods

miRising hairpin design

A miR30 structure (Figure 3A) was chosen as previous work in the inventors lab had shown this is a good structure. To miRise a shRNA structure the length of the sense strand needs to be extended.

Sense strand:

AAIJGGAAUGAUAACACAGU (SEQ ID NO: 14) - 19 bases extend by 3 to make 22

gAAUGGAAUGAUAACACAGUuc (SEQ ID NO:27) - 22 base pairs

When extending sequence to make it the appropriate size for the microRNA of choice the changes need to be made to the sense sequence and the new sequence need to be examined by the Taxman algorithm (Taxman et al., 2006) and free energy of the structure calculated to ensure it is optimal.

When the RNA is against a viral molecule it is important to look at the consensus sequences of the target to ensure the extensions being made are still targeting conserved sequence. Alterations in sequences to introduce mismatches seen in the microRNA structures should be done in the sense strand only which gives rise to the non-active siRNA 'passenger' strand. The anti-sense strand of the RNA stem (designed to give rise to the active siRNA 'guide' strand) should remain the same.

Modelling a shRNA on a miR

Several factors need to be taken into account such as the size of the miR, position (5 or 3'), loop sequence, bulges and other sequence (see, for example, Griffiths-Jones, 2004; Griffiths-Jones, 2006 and 2008; Kozomara and Griffiths-Jones, 201 1 and 2014). Once the appropriate length is identified, the bulges and mismatches are identified and the final structure checked using m-fold (Zuker, 2003).

Constructs encoding the following three RNA molecules were prepared where the open reading frame encoding the RNA molecule are operably linked to the full strength U6 (FS U6) promoter:

• shRNA with Brummelkamp loop (BK)

AAUGGAAUGAUAACACAGUuucaagagaACUGUGUUAUCAUUCCAUU (SEQ ID NO: 22)

(Sense or Passenger) Loop (Guide or Antisense) (construct encoding the shRNA provided as SEQ ID NO:7)

NS1/NEP miR30 structure without bulges and mismatches (NS1/NEP L) GAAUGGAAUGAUAACACAGUUCcugugaagcagcagauggggGAACUGUGUUAUC AUUCCAUUC (SEQ ID NO:23)

(Sense or Passenger) Loop (Guide or

Antisense) (construct encoding SEQ ID NO:23 provided as SEQ ID NO:20)

NS1/NEP miR30 structure with bulges and mismatches (NSI/NEP S+L) (tertiary structure shown in figure 3B)

gcgaAAUGGAAUGAUAUCACACAGUUCcugugaagcagcagauggggGAACUGUGUU AUG AUUCC AUUCu gc

(Sense or Passenger) Loop (Guide or

Antisense) (SEQ ID NO:24) (construct encoding SEQ ID NO:24 provided as SEQ ID NO:21)

Testing antiviral activity in vitro

MDCK cells were transfected with the NSI NEP constructs and EGFP control 24 h prior to infection with HlNl PRS influenza virus. Cell supernatant were harvested at 48 hrs for HA assay.

MDCK cells were freshly passed on the day prior to experiments so that the cells were healthy and not over confluent. The cells were trypsinised to remove them from the flask, counted and aliquoted into 1.5 ml microcentrifuge tubes at 1 x 106 cells per tube. Cells were pelleted at 4000 rpm for 3 minutes in a bench-top microcentrifuge.

MDCK cells were cultured in Earls Modified Eagle's Medium containing 10%

Foetal Calf Serum (PCS), 10 mM Hepes, 2 niM glutamine, supplemented with penicillin (100 U/rnl) and streptomycin (100 ,ug/ml) at 37°C in a humidified atmosphere containing 5% C02 . Logarithmic phase cells were trypsinized, counted and aliquoted into 1.5 million cells for each electroporation. Cells were resuspended in 100 μΐ of electroporation solution T (Amaxa), mixed with 2.5 pg of plasmid DNA and electroporated using program T20 of the Amaxa Nucleofector. Cells were plated out in a 24 well plate at 250 000 cells per well in duplicate for each infection.

After incubation for 3 1 hours, medium was removed from the cells and 200 μ| of influenza PR8 virus diluted in Viral Growth Media was added. The Viral Growth Media comprised EMEM, 0.3% bovine serum albumen, HEPES, glutamine, penicillin/streptomycin and 5 pg/ml trypsin. Infections were performed in duplicate. The cells were incubated at 37°C for 1 hour. Virus was removed from wells and 500 μΐ of Viral Growth Medium added to each w'ell. Supematants were harvested after 48 hrs and vires measured by HA. Expression of NS1/NEP from different constructs

Expression of the different miRised constructs was tested in the chicken cell line

DF1.

DFI cells (DFI : Atcc No. CRL- 12203) were grown in DME supplemented with 10% foetal bovine serum, 2 mM glutamine, 10 mM Hepes, 1.5 g/1 sodium bicarb, 4.5g/l glucose, 0.01 % (w/v) penicillin and 0.01% (w/v) streptomycin at 37°C with 5% (v/v) C02 and subctiltured twice weekly.

DFI cells were transfected with the different NS1/NEP constructs using lipofectamine2000 according to the manufacturers instructions.

48 hours post transfection RNA was extracted as described below.

RNA extractions

All RNA extractions were carried out using Trizol Reagent (Invitrogen) according to the manufactures instructions. Glycogen (10 g; Invitrogen) was added to the aqueous phase and 80% ethanoJ was used for the wash step to enhance the precipitation of small RNAs. If RNA extraction was not performed straight away cell pellets were frozen at -80°C. RNA pellets were resuspended in 20 μΐ of NF water. Prior to reverse transcription, RNA samples were treated with RQl DNase (Promega) according to the raanufactuer' s instructions. RNA and cDNA concentrations were determined using a NanoDrop 1000 spectrophotometer (Thermo-Scientific). All RNA sample were stored at -80"C.

Realtime PCR

RT-PCR was carried out using die following conditions unless odrerwise stated 94°C 2 min; 94°C, 30 sec, 55°C, 30 sec and 72°C, 1 min for 30 cycles followed by a final extension of 72°C for 5 min.

Analysis of S1 NEP expression was carried out using qRT-PCR polyadenylation method ( Shi and Chiang, 2005). Polyadenylation reactions contained 1 ng of total RNA (8 μΙ), 0.25 μΐ (Ί 50 U) of yeast poly(a) polymerase (PAP: catalog no. 74225; USB corporation), 4 μΐ 5x PAP buffer and 1 μΐ of 10 nM rATP (ambion) and NF water (Promega) in a final volume of 20 μΙ. Reaction were incubated at 37°C for 30 min and the 95°C for 5 min and stored at -20"C. First strand cDNA synthesis was performed using Superscript M (superscript HI first strand synthesis supermix, Invitrogen). Annealing reactions contained 4 μΐ of polyadenylated total RNA, 3 μΐ of modified ikugi-dT primer (miR-PTA) at 25 niM and 1 μιΐ of annealing buffer mix, incubated at 65°C for 5 min and cooled on ice. First-strand cDNA synthesis reaction was carried out according to the manufacturer instructions for oligo-dt primed RNA. Complementary DNA samples were stored at -20°C. All PAP and cDNA synthesis incubations were carried out in a thermal cycler (MasterCycler S; Eppendorf).

Quantatative analysis was performed using StepOneP!us real-time PCR system (Machine ans software; Applied Biosvstems), with universal reverse primer PAM-URP to recognize miR-PTA sequence and control or NS1/NEP1 specific forward primers. Chicken U6 small-nuclear RNA (U6) was used as the reference control. Reactions mixes contained 2 μΐ cDNA diluted 1 :50 in NF water, 10 μΐ SYBR Green PCR Master Mix (Applied Biosvstems), 0.8 μΐ of each primer (final concentration 200 nM) and NF water to 20 μΐ final volume. All samples were analysed in triplicate in 96 well MicroAmp PCR plates (Applied Biosvstems). Cycle settings were 94°C. 10 min; 94°C 15 sec and 60°C, 1 min (40 cycles). Melt curve was 95T. 15 sec; 60°C, 1 min; ramp 0.3°C/sec: 95°C, 15 sec (one cycle).

Construct expression assay

The inventors used the "direct injection" method (WO 2013/155572) where transfection reagents are mixed with an isolated nucleic acid molecule, polynucleotide or nucleic acid construct and injected directly into the blood of developing avian embryos. Using such a method the isolated nucleic acid molecule, polynucleotide or nucleic acid constract of the invention is introduced into primordial genu cells (PGCs) in the embryo and inserted into the genome of the avian.

More specifically, we used the miniTol2 plasmid system containing the different constructs. The plasmid DNA was diluted in 50 μΐ Opit-MEM medium and mixed gently. The Lipoi^'ectamine 2000 reagent was mixed gently and an appropriate amount diluted in 50 μΐ Opti-MEM medium. After a 5 minute incubation, the diluted DNA and transfection reagent were combined and mixed gently at room temperature for 20 minutes.

A suitable volume (typically 1 μΐ to about 3 μΐ) of the transfection mixture was then directly injected into an avian embryo in accordance with the method of the invention. Prior to injection, eggs were incubated at 38°C, with the pointy end (taglion) upward for approximately 2.5 days (Stages 12-17), or until such time as the blood vessels in the embryo were of sufficient size to allow injection. The optimal time for injection of the transfection mixture is the time of PGC migration that typically occurs around Stages 12-17, but more preferably Stages 13-14.

To access a blood vessel of the avian embryo, an approximately 10 mm hole was made in the pointy end of the egg using forceps. The section of shell and associated membranes were carefully removed while avoiding injury to the embryo and its membranes.

Following injection of the transfection mixture into the blood vessel of the avian embryo, the egg was sealed using a sufficient quantity of paral lm using a warm scalpel blade to affix the parafilm to the outer egg surface. Eggs were then turned over to the pointy-end down position and incubated at a temperature sufficient for the embryo to develop, such as until later analysis

Results

In ovo construct expression assay

Table 2 shows the results of the in ovo expression assay. This result shows the miRising the constructs may be a very useful step to allow high expression.

Table 2. In ovo Expression Assay Results

Relative expression levels

5 Expression levels of the NS1-NEP hairpin with the traditional Brummelkamp loop (BK) and miR30 loop and stem structure (S+L) structure behind full strength U6 (FS U6) was measured in DF1 cells. DF1 cells were transfected with the constructs and RNA extracted from the cells 48 h post transfection.

Realtime PCR analysis of expression levels (Figure 4) showed higher levels of 10 NS1/NEP hairpin expression with the miR30 loop and stem construct than the brummelkamp loop construct.

Comparison of in vitro silencing of influenza PR8 in MDCK cells by different NS I /NEP shRNA structures

15 In vitro silencing of the NS1 -NEP hairpin with the traditional Brummelkamp loop (BK) and miR30 loop and stem structure (S+L) structure behind full strength U6 (FS U6) was measured in MDCK cells. MDCK cells were transfected 24 h prior to infection with H1N1 PR8 influenza virus. Cell supematants were harvested at 48 hrs for hemagglutination assay.

20 Figure 5 shows the silencing results as measured by a hemagglutination assay.

Silencing was demonstrated with all NSl EP constructs.

The present application claims priority from US 61/783,764 filed 14 March 25 2014, the entire contents of which are incorporated herein by reference.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. 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.

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.

REFERENCES

Balciunas et al. (2006) PLoS Genet., 10:el69.

Bosselman et al. (1989) Science. 243:533-534.

Chim et al. (1993) Cell, 74:504-514.

Dua et al. (201 1 ) Mol Thcr, 19: 1676- 1687.

Griffiths-Jones (2004) Nucl Acids Res., 32: 109-1 11.

Griffiths-Jones et al. (2006) Nucl Acids Res., 34:140-144.

Griffiths-Jones et al. (2008) Nucl Acids Res., 36:154-158 Heine et al. (2007) Avian Dis„ 51:370-372.

Kawakami et al. (2000) Proc Natl Acad Sci USA, 97: 1 1403-11408. Koga et al. (1996) Nature, 383:30.

Kozomara and Griffiths-Jones (2011) Nucl Acids Res., 39:152-157. Kozomara and Griffiths-Jones (2014) Nucl Acids Res., 42:68-73. Lavitrano et ai. (1989) Cell. 57:717-723.

Liu et al. (2007) Nucl Acids Res., 35:5683-5693.

Shi et al. (2005) Biotechniques, 39:519-525.

Taxman et al. (2006) BMC Biotechnol., 24:6:7.

Thoraval et al. ( 1995) Transgenic Research, 4:369-36.

Zuker (2003) Nucl Acids Res., 3.1:3406-3415.

Claims

1. An. isolated nucleic acid molecule comprising a double-stranded region, the double-stranded region comprising a sequence of nucleotides complementary to a target sequence, wherein the target sequence is at least 90% identical to any one of SEQ ID NOs: 1 to 6, 12 to 19, 25 or 26.

2. The isolated nucleic acid molecule of claim 1, wherein the target sequence is identical to any one of SEQ ID NOs: 1 to 6, 12 to 1 , 25 or 26.

3. The isolated nucleic acid molecule of claim 1 or claim 2, wherein the double- stranded region is 19 to 23 nucleotides in length.

4. The isolated nucleic acid molecule of any one of claims 1 to 3 which comprises, or consists of, RNA.

5. The isolated nucleic acid molecule of claim 4 which is an siRNA, sh NA, eshRNA, or miRNA.

6. A nucleic acid construct encoding the isolated nucleic acid molecule of any one of claims 1 to 5.

7. A vector comprising the isolated nucleic molecule of any one of claims 1 to 5 and/or the nucleic acid construct of claim 6.

8. The vector of claim 7, wherein the vector is an expression vector.

9. A cell comprising the isolated nucleic acid molecule of any one of claims 1 to 5, the nucleic acid construct of claim 6, and/or the vector of claim 7 or claim 8.

10. The cell of claim 9, wherein the cell is a mammalian cell.

11. The cell of claim 9, wherein the cell is a bacterial cell.

1.2. A composition comprising the isolated nucleic acid molecule of any one of claims 1 to 5, the nucleic acid construct of claim 6, the vector of claim 7 or claim 8, and/or the cell of any one of claims 9 to 11.

5 13. A method of treating or preventing influenza in a subject, the method comprising administering to the subject the isolated nucleic acid molecule of any one of claims 1 to 5, the nucleic acid construct of claim 6, the vector of claim 7 or claim 8, the cell of any one of claims 9 to 11 , and/or the composition of claim 12.

10 14. The method of claim 13, wherein the subject is a non-human animal.

15. The method of claim 14, wherein the non-human animal is an avian.

16. The method of claim 15, wherein the avian is poultry.

15

17. A non-human transgenic organism comprising the isolated nucleic acid molecule of any one of claims I to 5, the nucleic acid construct of claim 6, the vector of claim 7 or claim 8, the cell of any one of claims 9 to 1 1 .

20 18. The non-human transgenic organism of claim 17 which is a plant.

19. The non-human transgenic organism of claim 17 which is a non-human transgenic animal.

25 20. The non-human transgenic animal of claim 1 which is an avian.

21. The non-human transgenic animal of claim 20 which is poultry.

22. The non-human transgenic organism of any one of claims 17 to 21 for use in 30 breeding and/or for use in food production.

23. A method of reducing the level of expression of influenza A virus NS1 and NEP genes in a cell, the method comprising introducing into the cell the isolated nucleic acid molecule of any one of claims 1 to 5, the nucleic acid construct of claim 6, the vector of

35 claim 7 or claim 8, and/or the composition of claim 12.

24. The isolated nucleic acid molecule of any one of claims 1 to 5, the nucleic acid construct of claim 6, the vector of claim 7 or claim 8, the cell of any one of claims 9 to 1 1 , and/or the composition of claim 12 for use in the treatment or prevention of influenza.

5

25. Use of the isolated nucleic acid molecule of any one of claims 1 to 5, the nucleic acid construct of claim 6, the vector of claim 7 or claim 8, the cell of any one of claims 9 to 11, and/or the composition of claim 12 in the manufacture of a medicament for the treatment or prevention of influenza.

10

26. A method of making a transgenic non-human animal, the method comprising:

(i) introducing a first nucleic acid comprising a transposon into a cell, wherein the nucleic acid encodes the isolated nucleic acid of any one of claims 1 to 5,

(ii) introducing a second nucleic acid encoding a transposase into the cell,

15 (ii) selecting a transgenic cell comprising the first nucleic acid in the genome of the cell,

(iii) regenerating a transgenic non-human animal from the cell, and

(iv) breeding the transgenic non-human animal.

20 27. The method of claim 26, wherein the transposon is a Tol2 transposon and the transposase is Tol2 transposase.

28. The method of claim 26 or claim 27, wherein the cell is a chicken primordial germ cell.

25