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Wo 2009/130479 A2 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 29 October 2009 (29.10.2009) WO 2009/130479 A2 (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 15/11 (2006.01) C12N 15/861 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, PCT/GB2009/001056 EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (22) International Filing Date: HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, 23 April 2009 (23.04.2009) KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, (25) Filing Language: English NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, (26) Publication Language: English SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 0807424.7 23 April 2008 (23.04.2008) GB (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant (for all designated States except US): ISIS IN¬ GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, NOVATION LIMITED [GB/GB]; Ewert House, Ewert ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, Place, Summertown, Oxford OX2 7SG (GB). TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (72) Inventors; and MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, TR), (75) Inventors/ Applicants (for US only): SEYMOUR, OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, Leonard, William [GB/GB]; Department Of Clinical MR, NE, SN, TD, TG). Pharmacology, University of Oxford, Old Road Campus, Headington, Oxford OX3 7DQ (GB). CAWOOD, Ryan Published: [GB/GB]; Department Of Clinical Pharmacology, Univer — without international search report and to be republished sity of Oxford, Old Road Campus, Headington, Oxford upon receipt of that report (Rule 48.2(gf) OX3 7DQ (GB). (74) Agents: ALI, Suleman et al; J.A. KEMP & CO., 14 South Square, Gray's Inn, London WClR 5JJ (GB). (54) Title: VIRUS (57) Abstract: The invention provides an attenuated virus. VIRUS Field of the Invention The present invention relates to attenuated viruses for use in vaccination. Background to the Invention The different tissues within an organism regularly exhibit varied gene expression and this variation is crucial to tissue and cell identity. The level of protein produced in a tissue from any given gene can be controlled at multiple points prior to translation in both the nucleus and cytoplasm. The recent discovery that an entire network of small non-coding RNA molecules (typically 21-23 nucleotides in length) called microRNAs exists within cells added another level of control to gene regulation. MicroRNAs have been shown to negatively regulate gene expression post-transcription through a number of mechanisms which all involve binding of the microRNA to complementary regions within a messenger RNA (mRNA). Such binding sites usually reside within the 3' un-translated region (UTR) of a transcript; however, they have also been discovered within exons and the 5' UTR. Following microRNA binding, translation is inhibited either by direct interactions between microRNA machinery and translation machinery, mRNA sequestration to P bodies or by an increase in mRNA turnover due to increased degradation. Efficient repression is probably a consequence of more than one of these mechanisms. Some microRNA genes show distinct tissue or cell type expression and their transcripts cannot be found in other tissue lineages. This allows efficient down- regulation in specific cell types and can be integral to their function and identity. This property has previously been exploited to efficiently regulate the expression of a factor IX transgene cassette. Summary of the Invention The invention concerns the use of microRNA binding sites to prevent virus infection and/or replication in vulnerable cells and tissues, thereby improving safety and efficacy of vaccination. By incorporating binding sites to microRNAs that are expressed in disease-associated tissues, viral replication can be decreased, restricted, or entirely ablated, to allow safer vaccination. The invention is of particular utility where viral infections compromise or kill cells that are normally involved in producing an immune response, since protection of these cells while allowing infection to proceed in less important cells should enable effective vaccination against viral pathogens that previously could not be subject to prophylactic vaccination. Accordingly, the present invention provides a virus for use in a method of vaccination of a host, which virus is attenuated by means of a microRNA binding sequence which is present within the genome of the virus, wherein attenuation is achieved by the microRNA binding sequence causing a reduction in the level of virus replication in host cells which express a microRNA that binds to the microRNA binding sequence of the virus. hi one embodiment the virus is one which when present in a cell in which it is able to replicate, expresses at least one (for example at least 2, 3, 4 or more) mRNA molecule that comprises a microRNA binding site. Detailed Description of the Invention The inventors have found that limiting the infectious tropism of viruses may be used to produce safer and more effective attenuated vaccines. The viruses are modified to substantially reduce their rate of replication in a cell which is important in causing disease in the host. Preferably the virus is able to replicate in other cells, for example at rate which is similar to (or the same as) the wild-type virus, and this replication leads to an immune response against the virus. It is to be understood that the term "binding sequence" mentioned herein includes a sequence which can directly bind to a microRNA (for example when the relevant sequence is present in an mRNA). However, unless the context requires otherwise, the term also includes the complement of such a binding sequence or any sequence which when expressed as RNA would lead to the generation of a sequence which is capable of binding microRNA, i.e. the term includes sense and antisense sequences in the genomes of viruses (in positive or negative strands) which correspond to sequences capable of binding microRNA. Vaccination/Stimulation of an Immune Response The invention relates to attenuating a virus for use in vaccination of a host. The vaccination may be prophylactic or therapeutic, and typically causes the host to have: (i) decreased susceptibility to infection by the wild-type form of the virus and/or (ii) decreased susceptibility to disease caused by the wild-type form of the virus and/or (iii) decreased disease symptoms when infected by the wild-type form of the virus. The vaccination may thus be protective against infection and/or disease. The invention also relates to use of the attenuated virus of the invention for stimulating an immune response in a host, for example stimulating an antibody and/or T cell response directed against the virus. The virus of the invention is typically administered in a form in which at least some of the viruses that are administered are capable of normal or attenuated replication in at least one cell type of the host (i.e. a live vaccine). However the invention is also applicable to a "killed vaccine" where the virus preparation has been subject to a treatment which should render all of the viruses incapable of replicating in any cell of the host. Introduction of microRNA binding sites into viruses for use in a killed vaccine will enhance the safety of such vaccines. The Host Human or Animal The host is an animal (including birds), preferably a mammal. The host may be a human or any of any of the groups or species mentioned below: Ungulates - Family: Suidae, Genus: Sue (Pigs) Family Bovidae, Sub family: Bovinae, Genus: Bos (Cows) Family: Bovidae, Sub family: Carpinae, Genus: Ovis (Sheep) Family: Equidae, Genus: Equus (Horses) Primates: Order: Primates, Sub order: Haplorrhini (Including Simian Monkeys), Sub family: Homininae (Gorillas, Chimpanzees) Tribe: Hominini (Humans) Other: Family: Canidae, Genus: Canis. (Dogs) Family: Felidae, Genus: Felis (Cats) Non-tetrapod chordates: Class: Actinopterygii (Fish) Class: Aves, Order: Galliformes (Land fowl), and Order: Anseriformes (Water Fowl) Family: Muridae (Rats, Gerbils, Mice, Hamsters) The host may be at (increased) risk of infection by the virus, or at (increased) risk of disease caused by the virus. The host may or may not inhabit a region for which the virus is endemic. The host may live in a population which includes individuals infected with (or carrying) the wild-type virus. The host may visit regions where the wild type virus is endemic and other individuals are carrying the virus or infected with it. The host may be an infant (for example less than 5, 3 or 1 year old) or may be old (for example more than 60, 70 or 80) years old. The host may be immunocompromised. The host typically has more than one cell type, for example at least 2, 3, 4, 5 or more cell types in which the virus is able to replicate. The Virus of the Invention This invention is applicable to any virus which in its wild-type form can act as a pathogen, i.e. which will typically be capable of infecting a host in its wild-type form.
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