WO2004085645A1

WO2004085645A1 - Rna interference in fish

Info

Publication number: WO2004085645A1
Application number: PCT/EP2004/003078
Authority: WO
Inventors: Michael Horne; Andrew Cartner Barnes
Original assignee: Novartis Ag; Novartis Pharma Gmbh
Priority date: 2003-03-24
Filing date: 2004-03-23
Publication date: 2004-10-07
Also published as: GB0306715D0; TW200506054A

Abstract

Gene silencing through siRNA has been shown to function in aquatic organisms such as shrimp. This technique can be applied to prevent or treat infections in these organisms by intracellular pathogens such as viruses.

Description

RNA interference in fish

Field of the Invention

The present invention concerns a method of treating or preventing infectious pathogenic disease in aquatic organisms employing RNA silencing technology, and further relates to medicaments suitable for administration to aquatic organisms comprising double-stranded RNA or a precursor thereof.

Background of the Invention

Commercial aquaculture has been expanding rapidly in recent decades. Fueling the growth is the increasing demand for seafood products around the world, particularly in Asia and Europe, and static or falling supplies from wild stocks. However, the viability of the industry is increasingly vulnerable to disease epidemics associated with intensive farming, particularly those caused by external parasites, viruses, bacteria, protozoa, fungi, algae and the like.

in particular, the farming and ranching of shell-fish has seen major variations in yields due to disease. Some countries, such as Taiwan, have largely withdrawn from shrimp farming having previously held dominating positions in the early growth of the industry. Catastrophic outbreaks of disease have also occurred in China, Indonesia, India, Panama,' Honduras and Ecuador. By far the most serious cause of loss comes from viral disease conditions, which can result in very high levels of mortality. Whitespot virus, a member of the new genus Whispovirus in the Nimaviridae, Yellowhead virus (Rhabdoviridae) and Taura virus (Picornaviridae), in particular have been associated with losses which have virtually eliminated economic shrimp farming in some areas.

Nevertheless, shell-fish farming has the potential to become the most valuable sector of the aquaculture industry and has seen enormous expansion both in tonnage and in its geographical spread throughout temperate regions. The largest sector, currently, is the farming of shrimp, although many shellfish such as Abalone species, have joined the more traditional oyster, muscles and clam industries to create a valuable sub-sector of aquaculture. Thailand is now the leading shrimp farming country in the world followed, in the Eastern Hemisphere, by China, Indonesia and India. Ecuador, Mexico and Venezuela are the main producers in the Western Hemisphere. Virtually all farmed shrimp belong to the penaeidae, a family of decapod crustaceans, with the Great Tiger Shrimp, Penaeus monodon and White Shrimp Litopenaeus vannamei predominating. World production of shrimp in 2000 approximated 1.2 million metric tones.

There are currently no effective therapies for such viral infections and the immune systems of shellfish are not of a nature that can be used for vaccination in the sense in which the term is applied in higher animals. In simplistic terms they rely on non-specific, cellular immunity.

The present invention concerns the application of a revolutionary gene silencing technology in treating and preventing viral and other infections in aquatic organisms, including shellfish.

Summary of the Invention

In a first aspect, the invention provides the use of isolated double stranded RNA or an expression vector capable of directing transcription of double stranded RNA, in the manufacture of a medicament for the treatment or prevention of infectious diseases in fish. These isolated double stranded RNA sequences and expression vectors are defined as RNAi agents.

In another aspect, the invention provides an expression vector capable of transcribing complementary strands of a double stranded RNA molecule when transfected into fish cells. In one embodiment the vector comprises a DNA sequence to be transcribed, which sequence is flanked by two promoters functional in eukaryotic cells, the first promoter controlling transcription of one of the strands, and the second promoter controlling transcription of the complementary strand, wherein the transcript of one of the strands is complementary to an RNA molecule originating from an intracellular fish pathogen along at least a portion of the transcript. ln an alternative embodiment the vector comprises a first DNA sequence and a second DNA sequence, wherein said first sequence is operatively linked to a first promoter and said second sequence is operatively linked to a second promoter, wherein said promoters are functional in eukaryotic cells, and wherein the one of the transcripts is complementary to an RNA molecule originating from an intracellular fish pathogen along at least a portion of the transcript.

In another embodiment the vector comprises a DNA sequence which is operatively linked to a promoter functional in a eukaryotic cell, such that the sequence is transcribed in a fish cell to generate a transcript having substantially self-complementary sequences capable of forming a double-stranded hair-pin structure, wherein one strand of the double-stranded hair-pin structure is complementary to an RNA molecule originating from an intracellular fish pathogen along at least a portion of said strand.

In a further aspect the invention provides a double stranded RNA molecule in which one of the strands is complementary to at least a portion of an RNA sequence originating from an intracellular fish pathogen, especially a viral pathogen.

In a still further aspect the invention provides a pharmaceutical composition for prophylaxis or treatment of pathogenic diseases in fish, comprising an RNAi agent as defined according to the invention.

In another aspect of the invention there is provided a fish transformed with an RNAi agent as disclosed herein. Further, there is provided a live-feed organism (optionally itself a shellfish or finfish) transformed with an RNAi agent for feeding fish. The live-feed organism is preferably Artemia.

In another aspect the invention provides a method of treating or preventing infectious disease in fish, comprising administering to the fish an RNAi agent or pharmaceutical composition as defined herein. Further, the invention provides a method of reducing the viral load of an RNA virus in a fish comprising administering to the fish an RNAi agent or pharmaceutical composition according to the invention.

Detailed description of the Invention The phenomenon of post transcriptional gene silencing (PTGS) was first seen by Jorgensen et al who, in introducing a gene for the amplification of purple colour in petunias, "co- suppressed" the resident gene thereby achieving a mottled or white colour rather than the deepened purple colour expected. Following this initial discovery a similar phenomenon of transgene-induced silencing was found in many other plants, in fungi (Neurospora crassa, "gene quelling") and eventually it has been shown to exist in mammals including humans.

The first evidence that gene silencing could be triggered by double stranded RNA (dsRNA) came from work in nematodes (Caenorhabditis elegans) using antisense RNA. It was later found that the injection of a small amount of dsRNA into the gut silenced function of the homologous gene throughout the whole worm and also in its first generation off-spring.

Further evidence for RNA silencing has been provided by studies based on feeding C. elegans with bacteria engineered to produce dsRNA, by immersion of worms in buffer containing dsRNA, by injecting Drosophila embryos with a gene gun and by engineering flies to contain an inverted repeat of the gene to be silenced.

The RNA silencing mechanism appears to be achieved by a similar, stepwise process in each case:

(i) dsRNA, on entering a cell, is digested into 21-23 nucleotide pieces referred to as small interfering RNA (siRNA) or "Guide RNA". The size is important and consistent. This cleavage is achieved by the RNAIII polymerase, "Dicer" (originally the DCR-1 enzyme from C. elegans). Dicer is an RNA-specific ribonuclease and its function is ATP dependent. The cleavage results in 19-21 base pair duplexes each with 2-nucleotide, 3' overhangs, (ii) The siRNA duplexes bind to a nuclease complex to form an RNA-induced, silencing complex (RISC) which is activated by the unwinding of the siRNA duplex. Each RISC contains a single siRNA and a different RNase which together target homologous transcripts of mRNA by base pairing with and cleaving the mRNA, 12 nucleotides from the 3' end, (iii) Cleavage of the mRNA of the homologous gene therefore destroys its function by preventing translation into protein. Sequence specificity is crucial as a single base-pair mismatch between the siRNA and the homologous gene dramatically reduces silencing While RNA interference has been shown to occur in a handful of well-studied organisms, it is not clear whether it is a general phenomenon exhibited by all taxonomic groupings. In particular, it was unclear until now whether all aquatic organisms share this gene silencing mechanism.

We have now succeeded in showing that RNA interference (RNAi) or RNA silencing not only functions in aquatic organisms including crustaceans, molluscs and finfish, but that it is capable of being applied to counteract pathogenic infections in these organisms, and thereby to combat diseases which commonly ravage the aquaculture industry. Where RNAi is used to target key pathogen-originating RNA molecules, the pathogen is incapacitated, disabled or killed and is unable to reproduce or proceed through its destructive parasitic life- cycle.

The target organisms of the invention are aquatic organisms defined principally as all species of finfish and shellfish. Shellfish are broadly divided into two main categories: molluscs and crustaceans. Molluscs can be further divided into three categories: univalves, bivalves and cephalopods. The preferred fish to which the inventive treatment is applied are invertebrate fish.

In the context of the present invention by "RNA" is meant a sequence of ribonucleotides linked by phosphodiester bonds (as in naturally-occurring RNA molecules), or (in the case of synthetic molecules), phosphorothioate, phosphoramidate, or phosphotriester bonds, or any other chemical linkage known in the art of synthesis of oligonucleotides. Oligoribonucleotides incorporating chemically modified bases or sugars are also encompassed by the term "RNA". Optionally the bases in the ribonucleotides are exclusively those which are naturally occurring: cytosine, uracil, guanine and adenosine.

RNA silencing uses double stranded RNA molecules in which at least a portion of one strand (the antisense strand) is capable of hybridizing specifically with a region of an RNA molecule originating from a pathogenic organism (the "target RNA") within the target cell of an aquatic organism. The pathogen may be bacterial, viral, protozoan or fungal. In general the target RNA is a messenger RNA molecule produced by transcription from the genome or plasmids of the pathogen, although it can be ribosomal RNA, tRNA, etc. In the case of viral RNA, the target RNA can be genomic RNA (of single or double-stranded RNA viruses), or it may be messenger RNA which is transcribed from genomic DNA (of single or double-stranded DNA viruses) or genomic RNA. The target RNA may be protein-coding or non-encoding.

For the purposes of describing the present invention, the term "double stranded RNA" ("dsRNA") is used to refer. to duplex RNA/RNA or RNA/DNA oligomers in the size-range preferably 15-30 bp, usually 20-25 bp, more preferably 21-23 bp (siRNAs or guide RNAs), and in particular 21, 22 or 23 bp long. Preferably dsRNA molecules have 2, 3, 4 or 5 nt 3' overhangs on each strand of the duplex. The term dsRNA also encompasses any larger precursor duplex RNA molecule which can be digested in a cell to generate siRNAs, and any single oligoribonucleotide with self-complementary sequences which assumes a hairpin formation to produce a duplex structure. The length of the hybridized portion of the hairpin is typically the same as that provided above for the siRNA type of agent or longer by 4-8 nucleotides. Precursor duplex RNA molecules may be up to 1500bp in length (e.g. in the range of 30-1500bp, optionally 50-1000bp, optionally 100-500bp) or even longer, and the region of duplex formation may be over a portion or over the entirety of the length of the precursor. As used throughout the present specification "portion" means a part of a molecule, or the complete molecule, unless otherwise specified. Optionally the dsRNA has regions of complementarity to portions of two or more different target RNAs, wherein the target RNAs may be different RNAs from a single pathogen species, or different target RNAs from two or more different pathogen species.

The sense and antisense strands of the double-stranded RNA are substantially complementary so that hybridization occurs in vivo. Theses strands are optimally, but not necessarily perfectly complementary. Throughout the present specification the term "complementary" has the conventional meaning in the field, i.e. it is meant that a nucleic acid sequence can form hydrogen bond(s) with another nucleic acid sequence, e.g. by Watson- Crick base-pairing. In the case of RNA molecules, for instance, there is complementarity at a given position when adenosine is paired (adjacent) with uracil and cytosine is paired (adjacent) with guanine, and thereby aligned for optimal hydrogen bonding. The percent complementarity of two nucleic acid sequences indicates the percentage of contiguous residues in the first sequence which can form hydrogen bonds with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, and 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. The sense and antisense strands of the dsRNA are optimally at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% complementary.

One of the strands of the double stranded RNA is capable of hybridizing to the target RNA to be destroyed within the cell, i.e. these molecules are substantially complementary. This strand is preferably perfectly complementary to a portion or the entirety of the target RNA, at least along part of its length. However a certain degree of mismatch can be tolerated in the RISC completes, so insertions, deletions and single point mutations in the dsRNA are possible. Nevertheless, it is preferred that there is complementarity of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%.

RNA silencing can be induced by synthesizing double stranded RNA (e.g. siRNA) molecules in vitro and transfecting these molecules into living host cells. In vitro synthesis methods include chemical synthesis and in vitro transcription methods, which are well known in the art. "Isolated" dsRNA refers to a dsRNA molecule that has been prepared in vitro. In vitro transcription may rely on the RNA polymerases of phages T7, T3 or SP6, for instance. RNA prepared in this way may be purified prior to being introduced into a target organism, for instance by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. The RNA may be dried for storage or dissolved in an aqueous solution. Following RNA preparation, duplex formation may be initiated in vitro before the double stranded RNA is introduced into the target organism, such as by annealing through heating and subsequent cooling. The sense and antisense strands may be covalently crosslinked. Alternatively, complementary sense and antisense strands may be introduced separately or together into the target organism, allowing hybridization to occur in vivo.

Alternatively, double-stranded RNA molecules can be synthesized in vivo in host cells by transcription from a vector template. In vivo synthesis from a vector is preferred due to continuous expression in stably transfected cells. In accordance with one embodiment of the invention siRNA is administered to aquatic organisms by means of a vector which is capable of transcribing double stranded RNAs in vivo.

Expression vectors can be constructed by techniques well known in the art and referred to, for instance, in Sambrook et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989. Such vectors are preferably plasmid DNA vectors or (retro-)viral vectors, which are relatively stable and therefore capable of being administered to aquatic organisms by a variety of routes, such as immersion in water, or in feed. Preferably the vector can be replicated in prokaryotic cells. If sustained effects are desired, the vector may be designed to replicate in eukaryotic cells. RNA is transcribed in vivo within the target cell from a vector equipped with appropriate transcription regulatory elements, including a suitable promoter and optionally terminator, enhancer or silencer sequences. The vectors of the invention can remain episomal (extrachromosomal) or become chromosomally integrated, for example by incorporating retroviral long-terminal repeat (LTR) sequences and a sequence encoding the corresponding retroviral integrase.

The dsRNA coding sequence(s) is operatively linked to a promoter. Within an expression vector, "operatively linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. The promoter may be constitutive or inducible. It may be an RNA polymerase II or RNA polymerase III promoter. The promoter may be selected to be one which functions in a wide variety of eukaryotic cells, such as the CMV promoter. Alternatively it may be a promoter sequence specific to the aquatic organism which is to be transfected with the DNA template, for instance, endogenous fish cytokine, heat shock or actin promoters. The vector may or may not incorporate other transcriptional regulatory elements such as enhancers, termination sequences, polyadenylation sequences (such as the BGH polyadenylation signal), and so on. Preferably, the dsRNA encoded by the vector is incapable of being translated into protein in prokaryotic or eukaryotic cells (e.g. the dsRNA lacks an IRES sequence and/or a start codon and/or a Shine-Dalgarno sequence, and/or is not polyadenylated and/or is too short to be translated).

If desired, the DNA vector may carrier a reporter or marker gene which enables cells, tissues or organisms which have been successfully transfected to be identified. Examples of such marker genes are green fluorescent protein (GFP), firefly luciferase, and beta-galactosidase. Transfection can also be verified by techniques such as PCR and Southern Blotting.

A vector can be designed to achieve transcription of double stranded RNA in vivo in a target cell in a multitude of ways. In one type of construct the DNA sequence to be transcribed is flanked by two promoters, one controlling transcription of one of the strands, and the other that of the complementary strand. These two promoters may be the same or different. These promoters may be termed opposing promoters. In vivo, the complementary RNA strands anneal to create dsRNA molecules.

In another type of construct, vectors may be engineered to express from a single vector cassette a small, stem-loop or hairpin RNA (shRNA) which is processed in vivo to siRNAs. One side of the stem encodes a sequence, preferably of at least 18 nucleotides, which is complementary to a portion of a target RNA, and the other side of the stem is sufficiently complementary to the first side of the stem to hydridize with the first side to form a duplex stem. The intervening loop portion is preferably 4, 7, 11 or more nucleotides in length. Optimally, the duplex stem includes the preferred.21-23 nucleotide sequences of the siRNA desired to be produced in vivo. In one specific embodiment the shRNA sequence is situated between a polymerase III promoter and a 4-5 thymidine transcription termination site. Further description of such vectors can be found in WO 03/006477, which is incorporated herein by reference.

Alternatively an expression vector may incorporate two separate promoters, each of which directs transcription of either the sense or the antisense strand of a dsRNA. These two promoters may be the of the same type or may be different. In vivo, the complementary RNA strands anneal to create dsRNA molecules. It is also possible for the sense and antisense strands of the dsRNA to be encoded by separate vectors, which are co-transfected into the target organism.

If it is desired for an expression vector to be eliminated rapidly from the cells of a target organism, it is possible to introduce into the vector a so-called "suicide" gene. This gene encodes a polypeptide capable of inducing programmed cell death (PCD). Exemplary polypeptides capable of introducing PCD in animal cells include matrix proteins of rhabdoviruses, such as the IHNV matrix protein (IHNV M), the IPNV VP2 protein, adenovirus 5 E1A protein and hepatitis B virus X protein.

Plasmids are commercially available for gene-silencing purposes, such as the Genesuppressor™ system distributed by Biocarta. Ambion's Silencer™ siRNA kit enables th e production of siRNA by in vitro transcription. Libraries of siRNA molecules and custom- made siRNA molecules are also readily available from commercial providers.

As referred to generically herein, an "RNAi agent" is either a double stranded RNA or an expression vector engineered to be capable of transcribing a double stranded RNA within a target cell, which double stranded RNA is at least partially complementary to a portion of an RNA molecule derived from an intracellular pathogen of fish.

Concurrent downregulation or degradation of multiple target RNAs is possible by concurrent administration of a plurality of RNAi agents. Alternatively, a plurality of RNAi agents can be administered, each RNAi agent having complementarity to a different portion of a single target RNA.

As used herein, the term "transfection" is interchangeable with "transformation" and refers to the introduction of foreign DNA or RNA into eukaryotic cells. RNAi agents can be administered to the target aquatic organisms of the invention by techniques including, but not limited to: electroporation, injection, microinjection, jet injection, immersion, ingestion (feeding), calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, poly brene-mediated transfection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics (particle bombardment, e.g. using a gene gun). Viral vectors transfect cells directly. For shellfish such as shrimps an immersion, feeding or transdermal transfection approach is preferred. The RNAi agent can be introduced into the target organism in naked form. By "naked" is meant the RNAi agent is free from any delivery vehicle that can act to facilitate entry into the target cell, such as liposomal formulations, charged lipids, or precipitating agents. If preferred, a delivery agent may be used. The RNAi agent may be delivered in a composition which further comprises a nucleic acid condensing agent such as spermidine, protamine sulphate, poly-lysine, or others known in the art.

An RNAi agent can be administered to a fish (shellfish or finfish) at any stage in the life- cycle, including to sperm ("milt"), unfertilized or fertilized eggs, or embryos, to the immature or larval phases (e.g. shrimp nauplii), or to the mature phases. For practical reasons, if the RNAi agent is to be delivered by injection or microinjection to a subject, it is preferred if the subject is visible to the naked eye. In the case of shrimp it is most beneficial to administer the RNAi agent prior to or during the 100-150 day grow-out period of the post larval stages. One option is to microinject finfish or shellfish gonads, so that any progeny inherit the RNAi . agent. Methods of transfecting finfish are known in the art, for instance through research done into the DNA vaccines field (e.g. in US 5,780,448, which is incorporated herein by reference). A specific method of transfection of mollusks by microinjection is described in EP1163844.

In one approach in a method of administering an RNAi agent to a target organism a live-feed organism carrying an RNAi agent is used. For instance, shrimp and other aquatic organisms consume single-celled and multicellular food sources such as plankton, plankton-like filter feeders, and algae. These food sources can also be employed in aquaculture. It may be desired to transform these food sources with an RNAi agent, in order that the target organism ultimately receives the RNAi agent by ingesting the food source. Artemia, Spirulina, Daphnia, Gammarus, Rotifera, bloodworms and tubifex worms are example of live- feed sources used to nourish farmed shrimp, and which can be genetically engineered to deliver RNAi agents to the shrimp (or to other aquatic organisms). It is suspected in certain cases that commonly-used food source organisms are themselves involved in the chain of transmission of infectious diseases to the target organisms. It is therefore possible to achieve a dual effect by effectively treating pathogenic infections in the live-feed organisms and in the target organisms which consume the live-feed organisms. A live-feed organism is not necessarily alive when administered to the target organism. For example, live-feed organisms may be prepared in freeze-dried or frozen form.

The invention relates in one aspect to pharmaceutical or "vaccine" compositions comprising an RNAi agent comprising, or capable of directing transcription of, dsRNA in which a portion of one RNA strand is complementary to a portion of a target RNA of a pathogen of shellfish or finfish.

The pharmaceutical compositions of the invention may be in solid, semi-solid or liquid form. Depending on the mode of administration and on the nature of the RNAi agent, pharmaceutical compositions may be formulated appropriately for delivery to the target aquatic organism of choice. In one embodiment of the invention, a pharmaceutical composition comprises at least one RNAi. agent, optionally multiple RNAi agents, and further comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers or vehicles with which the RNAi agent can be admixed include conventional excipients, and may be, for example, aqueous solvents such as water, saline or PBS, oil, dextrose, glycerol, wetting or emulsifying agents, bulking agents, stabilizers, anti-oxidants, preservatives, coatings, binders, fillers, disintegrants, diluents, lubricants, pH buffering agents, and the like. For transmucosal 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, detergents, bile salts, and fusidic derivatives.

In certain embodiments of the invention the RNAi agent is administered in a pharmaceutical composition in conjunction^'with an adjuvant. The adjuvant may be selected from any substance known in the art for this purpose, including Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, saponins (such as QuilA), muramyl dipeptides, avridine, aluminium hydroxide, aluminium phosphate, oils, oil emulsions, dextran sulphate, glucans, CpG oligomers, cytokines, and block co-polymers. The amount of adjuvant added depends on the nature of the adjuvant itself.

Where the RNAi agent is dsRNA, care should be taken to ensure that the carrier or excipient is sterile and RNase free. In one embodiment the dsRNA is administered in conjunction with an RNase inibitor (such as RNasin™). The preferred amount of the RNase inhibitor per unit dose is from about 4 to 4000 units, usually from about 400 to 4000 units and more usually from about 400 to 1500 units. In addition or as an alternative, competitor RNA may be provided in the pharmaceutical compositions of the invention, to serve as a competitive inhibitor of RNase activity. The precise sequence of the competitor RNA is irrelevant to its competitor activity, but it should be provided in excess over the amount of RNAi agent in the composition.

In one embodiment the invention encompasses the use of an RNAi agent in the manufacture of a medicament for inhibiting the function of a target RNA in a finfish or shellfish cell.

The invention also encompasses the use of an RNAi agent in the manufacture of a medicament for the therapeutic or prophylactic treatment of infections or infestations by bacteria, viruses, protozoa, fungi, and other pathogens, especially intracellular pathogens. The treatment may achieve eradication of the infection or infestation, or it may reduce the impact, limit the spread of the infectious agent, and/or ameliorate associated symptoms. The pathogen may be killed, incapacitated, prevented from reproducing, replicating or multiplying, or rendered non-pathogenic by the treatment.

Crustaceans are a commercially important subphylum of shellfish which includes but is not limited to shellfish selected from the group consisting of shrimp, prawns, lobsters, crayfish and crabs. The prophylactic and treatment method of the invention is particularly applicable to the families Penaeidae, Sergestidae, Palaemonidae, Nephropidae, Astacidae, Cambaridae, Parastacidae, Palinuridae, Portunidae and Potamidae among the crustaceans.

Notable examples of Penaeidae include: Penaeus monodon, Penaeus chinensis, Penaeus indicus, Penaeus stylirostris, Penaeus merguiensis, Penaeus vannamei, Penaeus setiferus, Penaeus japonis, Penaeus aztecus, Penaeus duorarum, Penaeus semisulcatus, Penaeus teraoi, Penaeus orientalis, Penaeus plebejus, Penaeus esculentus, Penaeus paulensis, Penaeus penicillatus, Penaeus schmitti, Penaeus subtilis, Penaeus kerathurus. Metapenaeus ensis, Metapenaeus dobsoni, Metapenaeus endeavouri, Metapenaeus monoceros, and Xiphopenaeus kroyeπ. Palaemonidae include Macrobrachium rosenbergii, Macrobrachium malcolmsonii, and Palaemon serratus. Lobsters from the families Homaridae, Nephropidae and Palinuridae include the Homarus spp. (e.g. Homarus americanus, Homarus gammarus), Palinarus spp (e.g. Palinarus elephas, Palinarus interruptus, Palinarus argus), Panuliris spp, Calinectes spp and Nephrops spp (e.g. Nephrops norvegicus). Crayfish from the families Astacidae, Cambaridae and Parastacidae include Astacus astacus, Astacus leptodactylus, Pacifastacus leniusculus, Procambarus clarkia, Cherax destructor, Cherax quadricarinatus, and Cherax tenuimanus. Cultured crabs include Cancer spp, Callinectes spp, Carcinus spp, Portunus spp (e.g. Portunus trituberculatus) other members of the family Portunidae (e.g. Scylla serrata), and Eriocheir sinensis in the family Potamidae.

Among the molluscs the families Halotidae, Littorinidae, Strombidae, Mytilidae, Arcidae, Pteridae, Pectinidae, Ostreidae, Cardiidae, Tridacnidae, Mactridae, Solecurtidae, Corbiculidae, Veneridae, Myidae and Hiatellidae all comprise species which are currently cultured. These include various species of abalone, conch, mussel, cockle, oyster, scallop and clam. Within the Haliotidae, cultured species include Haliotis discus, Haliotis diversicolor, Haliotis midae, Haliotis rufescens and Haliotis tuberculata. Examples of Mytilidae include Mytilus chilensis, Mytilus coruscus, Mytilus edulis, Mytilus galloprovincialis, Perna canaliculus and Perna viridis. The Pectinidae include Argopecten purpuratus and Patinopecten yessoensis. The Ostreidae include Crassostrea gigas, Crassostrea iredalei, Crassostrea virginica, Ostrea edulis, Ostrea chilensis and Saccostrea commercialis. Pearl oysters include Pinctada fucata Martensii, Pinctada maxima and Pinctada margaritifera. The clams include Corbicula japonica of the Corbiculidae family, and Mercenaria mercenaria, Ruditapes decussates and Ruditapes philippinarum of the Veneridae.

Finfish may be cartilaginous fish (e.g. sharks or rays) or bony (vertebrate) fish. Finfish may be freshwater or saltwater species. The preferred finfish to be treated with the "vaccination" method of the invention are those being commercially farmed, including, but not limited to: sturgeon, eel, bream, carp, koi, catfish, salmonid fish including salmon and trout, char, cod, perch, seabass, grouper, snapper, jack, tuna, tilapia, mullet, turbot, halibut and sole.

The "vaccination" technique of the invention is useful against any intracellular pathogens of aquatic organisms, especially pathogens of invertebrate fish, particularly viruses but also bacteria and protozoans. The-viruses may be double- or single-stranded RNA viruses or DNA viruses. In one embodiment RNA viruses are the preferred pathogens to be treated ' with an RNAi agent, since the genomic RNA can be degraded by this method. The lists of pathogens provided herewith include examples of particular pathogens, but these lists are non-exhaustive and non-limiting.

Shrimps are susceptible to a large and growing range of viruses, including: Yellow-head virus (YHV), Taura Syndrome Virus (TSV), White Spot Syndrome Virus (WSSV - which also infects swimming crabs), Monodon baculovirus (MBV), Baculovirus penaei (BP), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Reo-like virus, Hepatopancreatic parvovirus (HPV), and the virus causing Baculoviral midgut gland necrosis. Representative examples of the numerous viral conditions identified in mollusks include: Oyster Velar Virus Disease (OWD), Iridoviruses, Herpes-Type Virus disease of Oysters, Haemocytic Infection Virus Disease of Oysters, Virus-like disease of Mussels, and Viral infection of clams. Other intracellular pathogens cause the diseases: Bonamiosis (Bonamia exitiosus, Bonamia ostreae, Mikrocytos roughley), MSX disease (Haplosporidium nelsoni), Perkinsosi (Perkinsus marinus, Perkinsus olseni/atlanticus), Marteilosis (Marteilia refringens, Marteilia Sydney), and Mikrocytosis (Mikrocytos mackini). Examples of viruses which infect fin-fish are: Infectious Salmon Anaemia Virus (ISAV), Infectious Pancreatic Necrosis Virus (IPNV), Infectious Hematopoietic Necrosis Virus (IHNV), Iridovirus, Viral Nervous Necrosis Virus (VNNV), Salmon Pancreas Disease Virus (SPDV), Spring Viremia of Carp Virus (SVCV), Viral Hemorrhagic Septϊcemia Virus (VHSV), Lymphocystis Virus, and Cardiomyopathy Virus. Intracellular bacterial pathogens of fish include: Renibacterium salmoninarum (causative agent of Bacterial Kidney Disease), Piscirickettsia salmonis (causative agent of Salmonid Rickettsial Septicemia), Yersinia ruckerii, Photobacterium damselae, Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium chelonei, Nocardia asteroids etc.

Target RNA molecules may originate from any of the above pathogens. Preferred target RNAs are those from viruses that infect shrimps and prawns, especially YHV, TSV (both single-stranded RNA viruses), IHHNV (a single-stranded DNA virus) and WSSV (a double- stranded DNA virus). The sequences of target RNAs from all these viruses have been published and are available in the literature and in public databases. Structural or non- structural protein RNA sequences may be targets. For WSSV, target RNAs may be identified for instance in PCT applications WO 01/38351, WO 03/000900 or WO 01/09340 (including those encoding VP19, VP24, VP26 and VP28) and in Genbank entries AF440570, AY422230, AY422229, AY422228, AY422227, and AY422226, to name just a few. TSV sequences are available in Genbank entry AF277675, and in Mari, J. et al. 2002, J. Gen. Virol. 83: 915 — 26. YHV sequences are available in Genbank under record numbers AF540644, AY052786, AF102829, and AF148846.

The treatment method of the invention is not only useful for therapeutic applications - to ameliorate or cure disease conditions - but also for prophylaxis, since in many cases the pathogen will be present in a target organism which is asymptomatic, and targeted RNA destruction can prevent an infection taking hold.

The pharmaceutical compositions of the invention comprise a therapeutically effective amount of an RNAi agent. The desired therapeutic effect is achieved by an amount of RNAi agent that reduces the amount of target RNA by at least 30 percent, at times preferably by 40, 50, 60, 70, 80, or 90 percent or higher. Exemplary doses include milligram, microgram, or nanogram amounts of the molecule per kilogram of subject. The optimum dose varies according to the route of administration, and nature of the pathogen, the subject and the target RNA, but can be determined by the skilled person without undue burden. Similarly the frequency and timing of administration of the RNAi agent can best be determined by simple experimentation.

In the case of DNA vectors, a desired dosage is at least one plasmid copy per cell, and optionally multiple copies per cell. The appropriate dosage for in vitro-generated dsRNA may be for instance of the order of at least a 10 fold excess of dsRNA molecules per cell. However, substoichiometric doses may also be sufficient, since RNAi is thought to have a catalytic mechanism. The precise dosage can easily be determined by the skilled person for any particular target gene in a specific target organism, by measuring an appropriate end- point (e.g. presence or absence of symptoms of pathogenic disease, survival of the target organism upon challenge by the pathogen etc.). The concentration of DNA vector in an injectable pharmaceutical solution is preferably 2 to 200 μg/ml, more preferably about 5 to 10 μg/ml. The amount of injected solution is preferably in the range 1 to 500 μl, more preferably 5 to 100 μl, more preferably 10 to 50 μl. The precise volume and concentration of solution depends on the age and species of the target organism, and can be determined by experimentation.

In accordance with the invention, an RNAi agent may be combined with one or more antigens or DNA vaccines in a single composition, in order to treat or prevent multiple diseases in a target organism, or to combat a single disease with multiple approaches. A kit- of-parts may be provided in which an RNAi agent is combined with other immunogenic components (e.g. antigens or DNA vaccines) for separate, sequential or simultaneous administration.

Examples

Demonstration of RNA interference and its therapeutic efficacy in trout

Viral haemorrhagic septicaemia (VHS) is a disease to which rainbow trout (Onchorhynchus mykiss) are susceptible. It is known that the glycoprotein (G protein) is essential for infectivity of the virus and therefore for its pathogenicity. A DNA vector is constructed from which dsRNA is transcribed in vivo in the fish. One strand of the dsRNA is designed to be complementary to the VHS G protein RNA. The DNA vector is transfected into the fish by intramuscular injection.

In a challenge experiment, "vaccinated" trout are exposed to virulent VHSV, and the response of the treated fish is compared to the response in the control group.

Demonstration of RNA interference and its therapeutic efficacy in crustaceans

The WSSV VP28 coding sequence is amplified by PCR, cloned, and sequenced. To establish the detection system for RNAi silencing of VP28 in vitro, the VP28 fragment is subcloned into the pCMV-LUC vector and transfected into CHO cells or Hela cells. The silencing effect is assessed by measuring the LUC activity. To construct VP28 RNAi libraries, five DNA fragments spanning the whole VP28 coding region are amplified by PCR and cloned into RNAi vector pSilencer 1.0-U6. The RNAi libraries are created and transfected into the CHO or Hela cells for screening of RNAi suppressing VP28 expression using the established detection system. Multiple rounds of screening are performed to single out the individual RNAi clone(s) that inhibit VP28 gene expression and subsequently the RNAi sequence(s) obtained.

If more than one RNAi clone is obtained, combinational use of the RNAi clones to silence VP28 is tested in the CHO or Hela cell system. In addition, primary cultures of swimming crab are tested for transient infection of WSSV. Delivery of RNAi into the primary cultures is achieved by electroporation or adding RNAi into the culture medium and the inhibition effect of WSSV investigated, assuming successful viral infection of the primary cultured cells.

To facilitate RNAi expression in shrimp, an endogenous RNAi vector is constructed using shrimp U6 promoter. The shrimp U6 promoter is sequenced using PCR homology cjoning and gene walking approaches and its ability to synthesise RNAi assessed in the primary cultured cells of the swimming crab. The desirable U6 construct is selected for in vivo delivery of RNAi into swimming crab.

In vivo delivery routes of DNA into Crustacea (swimming crab or scampi) including intramuscular injection or direct injection into the haemolymph are determined using the gene construct containing a marker, LacZ gene. The RNAi fragments previously selected are inserted into the endogenous shrimp U6 plasmid for in vivo delivery using the method determined above. Inhibition of WSSV and protective effect in swimming crabs is measured in the presence of WSSV in tissues detected by PCR/in situ hybridisation and animal mortalities during viral challenge, respectively. Optimal doses of RNAi plasmid DNA and a time course of RNAi administration are investigated.

Claims

1. Use of an RNAi agent in the preparation of a medicament for the prophylactic or therapeutic treatment of infectious disease in fish.

2. Use according to claim 1 wherein the fish are farmed fish for human or animal consumption.

3. Use according to claim 1 or claim 2 wherein the fish are finfish or shellfish.

4. Use according to claim 3 wherein the fish are finfish other than zebrafish.

5. Use according to claim 1 or claim 2 wherein the fish are invertebrate fish.

6. Use according to any of claims 1 to 5 wherein the RNAi agent is an isolated double stranded RNA molecule.

7. Use according to any of claims 1 to 5 wherein the RNAi agent is a vector capable of directing transcription of double stranded RNA in a fish cell.

8. Use according to claim 7 wherein the vector is an episomal vector which is incapable of integrating into the chromosomes of the fish cell.

9. Use according to any qf claims 1 to 8 wherein the infectious disease is caused by an intracellular pathogen.

10. Use according to claim 9 wherein the intracellular pathogen is an RNA virus.

11. Use according to claim 9 wherein the intracellular pathogen is selected from Whitespot virus (WSSV), Taura syndrome virus (TSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Yellow-head virus (YHV).

12. An isolated double stranded RNA sequence comprising substantially complementary sense and antisense RNA strands, wherein said sense or said antisense RNA strand is substantially complementary to an RNA sequence originating from an intracellular fish pathogen along at least a portion of said sense or antisense RNA strand.

13. An isolated double stranded RNA sequence according to claim 12 wherein said sense or said antisense RNA strand is at least 70% complementary to said RNA sequence originating from an intracellular pathogen along a portion of at least 20 nucleotides of said sense or said antisense RNA strand.

14. An isolated double stranded RNA sequence according to claim 12 or claim 13 which is 20-25 bp in length.

15. An isolated double stranded RNA sequence according to any of claims 12 to 14 wherein said sense and antisense RNA strands are at least 70% complementary.

16. An expression vector capable of directing transcription of substantially complementary sense and antisense strands of a double stranded RNA molecule when transfected into fish cells, wherein said sense or said antisense RNA strand is substantially complementary to an RNA molecule originating from an intracellular fish pathogen along at least a portion of said strand.

17. An expression vector according to claim 16 wherein said sense or said antisense strand is at least 70% complementary to said RNA molecule originating from an intracellular fish pathogen over a portion of at least 20 nucleotides of said strand.

18. An expression vector according to claim 17 where said sense and antisense RNA strands are at least 70% complementary.

19. An expression vector according to any of claims 16 to 18 comprising a DNA sequence to be transcribed, which sequence is flanked by two promoters functional in eukaryotic cells, the first promoter directing transcription of either the sense or the antisense RNA strand, and the second promoter directing transcription of the complementary RNA strand.

20. An expression vector according to any of claims 16 to 18 comprising a first DNA sequence and a second DNA sequence, wherein said first sequence is operatively linked to a first promoter and said second sequence is operatively linked to a second promoter, wherein said promoters are functional in eukaryotic cells, wherein the transcripts from said first sequence and said second sequence are said sense and said antisense RNA strands, respectively.

21. An expression vector comprising a DNA sequence operatively linked to a promoter functional in a eukaryotic cell, such that the sequence is transcribed in a fish cell to generate a transcript having substantially self-complementary sequences capable of forming a double- stranded hair-pin structure, wherein one strand of the double-stranded hair-pin structure is substantially complementary to an RNA molecule originating from an intracellular fish pathogen along at least a portion of said strand.

22. An expression vector according to claim 21 wherein said strand is at least 70% complementary to said RNA molecule originating from an intracellular fish pathogen over a portion of at least 20 nucleotides of said strand.

23._^ An expression vector according to any of claims 16 to 22 wherein said promoter or promoters are derived from the genome of a fish.

24. An expression vector according to any of claims 16 to 23 which further carries a gene encoding a polypeptide capable of inducing programmed cell death.

25. A pharmaceutical composition comprising at least one RNAi agent selected from the group consisting of double stranded RNA sequences according to any of claims 12 to 15, and expression vectors according to any of claims 16-24, and a pharmaceutically acceptable carrier. ^• -

26. A pharmaceutical composition according to claim 25 comprising at least two of said RNAi agents.

27. A fish transfected with the expression vector of any of claims 16 to 24.

28. A live-feed organism for feeding fish, transfected with an RNAi agent.

29. A live-feed organism according to claim 28 which is Artemia.

30. A method of treating or preventing infectious disease in a fish, comprising administering to said fish a pharmaceutical composition according to claim 25 or claim 26.

31. A method of reducing viral load of an RNA virus in a fish, comprising administering to said fish a pharmaceutical composition according to claim 25 or claim 26.

32. A kit-of-parts for simultaneous, separate or sequential administration, comprising at least two different expression vectors, one expression vector being capable of transcribing the sense strand of a double stranded RNA molecule in vivo in a eukaryotic cell, and a second expression vector being capable of transcribing the antisense strand of that double stranded RNA molecule in vivo in a eukaryotic cell, wherein said sense or said antisense strand is substantially complementary to an RNA molecule originating from an intracellular fish pathogen along at least a portion of said RNA strand.