WO2010036391A2

WO2010036391A2 - A novel rna-based expression system

Info

Publication number: WO2010036391A2
Application number: PCT/US2009/040851
Authority: WO
Inventors: David Onyabe; David Hone
Original assignee: Bacilligen, Inc.
Priority date: 2008-04-16
Filing date: 2009-04-16
Publication date: 2010-04-01
Also published as: WO2010036391A3

Abstract

The present invention provides novel compositions and methods that enable rapid production of proteins and RNA. In particular, a novel system is provided comprised of Cystovirυs procapsids alone or with p8 and single-stranded (ss) RNA (ssRNA) encoding a gene of interest. The ssRNA is uploaded by the procapsids and converted to double-stranded (ds) RNA (dsRNA), thereby producing functional capsids. The resulting capsids are useful for expression of recombinant proteins, such as enzymes, hormones, cytokines, adjuvants and vaccine antigens, and RNA, such as catalytic RNA, antisense RNA, siRNA, hairpin loops and microRNA, in cell-free systems as well as in plant, insect and mammalian cells.

Description

A NOVEL RNA-BASED EXPRESSION SYSTEM

David Y. Onyabe and David M. Hone

Background

|00011 The elevated threat of bioterrorism and the renewed threat of emerging pathogens, such as H5N 1 influenza virus, have caused a change in government priorities, which now call for the development of an array of defense measures against this insidious form of weaponry. Although numerous toxins and viral and bacterial pathogens are considered high risk, it is possible to develop therapeutic and preventive pharmaceutical stockpiles as countermeasures to this subset of biologies. The shortfall of this approach is the high cost to tax payers to maintain such a stockpile of countermeasures, which have short shelf half-lives and require constant financial oversight for maintenance, as well as the fact that it is possible to make genetic modifications to these bioweapons that render them insensitive to conventional countermeasures. Moreover, the emergence of new serotypes of influenza and other viruses that cause high human mortality revealed another gap in the preparedness of the nation against biological threats.

[0002) These factors have engendered a need to develop novel strategies that enable a rapid response against existing and emerging biological threats that can be deployed as a countermeasure in the event of an unnatural or natural outbreak due to a bioweapon or emergent pathogen. One such strategy is to develop a highly flexible platform technology that enables the rapid production of bioactive proteins, protein antigens or monoclonal antibodies that afford prophylactic or therapeutic countermeasures against imminent biological threats. |0003| The prior art provides dsRNA phage and capsids derived from Cystovirus that are produced in bacteria and can be deployed via a bacterial vector or as a purified preparation. Although these compositions have many useful applications, the need to produce well-characterized and validated seed lots prior to production of clinical materials (i.e. US Food and Drug Regulations, CFR-21) creates an obligatory hurdle that expands the timeframe in which these biologies can be manufactured and released.

Summary of the Invention

[0004| The instant invention relates to a system for expressing a nucleic acid of interest. The invention relates to Cystovirus procapsids, a source of p8 and a single-stranded (ss) RNA (ssRN A) encoding the gene product of interest.

|0005| The invention also contemplates articles of manufacture, such as a kit, comprising the various reagents of interest to enable a user to express an ssRN A encoding an expressed product of interest, such as, an RNA or a polypeptide.

[0006] Other objects and features of the invention of interest are described below.

Description of the Invention

|0007| An embodiment of the present invention has been met by providing novel compositions comprised of Cystovirus procapsids and ssRNA encoding a gene of interest. The particular Cystovirus from which the procapsids are derived is not important to the present invention and include, but not limited to, previously documented dsRNA bacteriophage, such as, phi-6, phi-8, phi- 10, phi- 12 and phi- 13 (Mindich et al., Adv. Vir. Res., 35: 137 ( 1988); Mindich et al., J. Bacterid., 181 :4505, (1999); Hoogstraten et al. Virol. 272:218, (2000); Mindich et al., J. Virol., 62: 1 180, ( 1988) Qiao et al., Virol., 275:218, (2000)). Alternatively, dsRNA bacteriophage can be isolated from terrestrial sources using methods well documented in the prior art (Mindich et al., Adv. Vir. Res., 35: 137 ( 1988); Mindich et al., J. Bacteriol., 181 :4505, (1999); Silander et al., Proc. Natl. Acad. Sci., 102: 19009, (2005).

[0008] Procapsids of Cystovirus are typically comprised of four proteins, p 1 , p2, p4 and p7 (Mindich et al., J. Bacteriol., 181 :4505, ( 1999); Huiskonen et al., Structure, 14: 1039, (2006)), which are encoded by the genomic segment (gs)-L (gsL) of cystoviruses (Mindich et al., J. Bacteriol., 181 :4505, ( 1999)). (Cystoviruses also contain medium (gsM) and small (gsS) genomic segments.) The gsL can include 5 '-replication and 3 '-replication and transcription initiation sequences; however, in a preferred embodiment gsL lacking the 5 '-packaging sequence (herein referred to as "5'-pac") are utilized, thereby preventing uptake into the procapsids and creating additional space for cargo RNA sequences. Expression and assembly of these proteins can be achieved in commercially available cell-free transcription and translation systems, i.e. TNT* Quick Coupled Transcription/Translation System (Promega, Madison WI). Alternatively, expression and assembly of these proteins can be achieved in bacteria. The bacterial strain in which the procapsids are produced in the present invention is not critical thereto and include, but are not limited to: Escherichia spp.. Salmonella spp.. Shigella spp., Bacillus spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Mycobacterium spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp., Vibrio spp. etc. Detailed listing of strains useful to the present inventions can be found elsewhere (e.g. US Pat. No. 7,018,835). Extraction and purification of procapsids from the bacterial strains is achieved using methods well know in the art (Mindich et al., Adv. Vir. Res., 35: 137 ( 1988); Mindich et al., J. Bacterid., 181 :4505, (1999); Hoogstraten et al. Virol. 272:218, (2000); Mindich et al., J. Virol., 62: 1 180, ( 1988) Qiao et al., Virol., 275:218, (2000); Silander et al., Proc. Natl. Acad. Sci., 102: 19009, (2005)). |0009| Genes of interest can be any gene that encodes any protein or parts thereof that can be expressed in cell-free systems and in the target host cell, that is, the expressed product of interest. Genes of interest can also encode attenuated viruses and virus-like particles; the latter can be engineered to disseminate throughout the host and deliver either transient or stable phenotypic traits to host cells (e.g. Marillonnet et al., Proc. Natl. Acad. Sci (USA), 101 :6857; 2004). In some instances the gene of interest encodes vaccine antigens, can be any molecule that is expressed by any viral, bacterial, or parasitic pathogen prior to or during entry into, colonization of, or replication in their animal host; the Cystovirus capsids may express vaccine antigens or parts thereof that originate from viral, bacterial and parasitic pathogens and so on. These pathogens can be infectious in humans, domestic animals or wild animal hosts.

lOOOlOJ The viral pathogens from which the viral antigens are derived, include, but are not limited to, Orthomyxoviruses, such as influenza virus (Taxonomy ID: 59771 ); Retroviruses, such as RSV, HTLV-I (Taxonomy ID: 39015) and HTLV-II (Taxonomy ID: 1 1909); Papillomaviridae, such as HPV (Taxonomy ID: 337043); Herpesviruses, such as EBV (Taxonomy ID: 10295); CMV (Taxonomy ID: 10358) or herpes simplex virus (ATCC No.: VR- 1487); Lentiviruses, such as HIV-I (Taxonomy ID: 12721 ) and HIV-2 (Taxonomy ID: 1 1709); Rhabdoviruses, such as rabies; Picornoviruses, such as Poliovirus (Taxonomy ID: 12080); Poxviruses, such as vaccinia (Taxonomy ID: 10245); Rotavirus (Taxonomy ID: 10912); and Parvoviruses, such as adeno-associated virus 1 (Taxonomy ID: 85106).

|000111 Examples of viral antigens can be found in the group including but not limited to the human immunodeficiency virus antigens, Nef (National Institute of Allergy and Infectious Disease HIV Repository Cat. No. 183; Genbank Accession No. AF238278), Gag, Env (National Institute of Allergy and Infectious Disease HIV Repositoiy Cat. No. 2433; Genbank Accession No. U39362), Tat (National Institute of Allergy and Infectious Disease HIV Repository Cat. No. 827; Genbank Accession No. M l 13137), mutant derivatives of Tat, such as Tat-Δ3 l -45 (Agwale et al., Proc. Natl. Acad. Sci. USA 99: 10037; 2002), Rev (National Institute of Allergy and Infectious Disease HIV Repository Cat. No. 2088; Genbank Accession No. L14572), and Pol (National Institute of Allergy and Infectious Disease HIV Repository Cat. No. 238; Genbank Accession No. AJ237568); and T and B cell epitopes of gpl20 (Hanke and McMichael, AIDS Immunol Lett., 66: 177; 1999; Hanke et al., Vaccine, 17:589; 1999; Palker et al., J. Immunol., 142:3612-3619; 1989); as well as chimeric derivatives of HIV-I Env and gpl20, such as, but not restricted to, fusion between gp l 20 and CD4 (Fouts et al., J. Virol. 2000, 74: 1 1427- 1 1436; 2000), truncated or modified derivatives of HlV-I env, such as, but not restricted to gpl40 (Stamatos et al., J Virol, 72:9656-9667; 1998) or derivatives of HIV-I Env and/or gpI40 thereof (Binley et al., J Virol, 76:2606-2616; 2002; Sanders, et al., J Virol, 74:5091 -5100, 2000; Binley et al., J Virol, 74:627-643; 2000), the hepatitis B surface antigen (Genbank Accession No. AF043578; Wu et al., Proc. Natl. Acad. Sci., USA, 86:4726-4730; 1989); rotavirus antigens, such as VP4 (Genbank Accession No. AJ293721 ; Mackow et al., Proc. Natl. Acad. Sci., USA, 87:518-522; 1990) and VP7 (GenBank Accession No. AY003871 ; Green et al., J. Virol., 62: 1819- 1823; 1988), influenza virus antigens, such as hemagglutinin or (GenBank Accession No. AJ404627; Pertmer and Robinson, Virology, 257:406; 1999); nucleoprotein (GenBank Accession No. AJ289872; Lin et al., Proc. Natl. Acad. Sci., 97: 9654-9658; 2000); and herpes simplex virus antigens, such as thymidine kinase (Genbank Accession No. AB047378; Whitley et al., In: New Generation Vaccines, pages 825-854).

[00012| The bacterial pathogens, from which the bacterial antigens are derived, include, but are not limited to: Mycobacterium spp., Helicobacter spp., such as, Helicobacter pylori, Salmonella spp., Shigella spp., Escherichia spp., such as, E. coli, Rickettsia spp., Listeria spp., Legionella spp., such as, Legionella pneumoniae, Pseudomonas spp., Vibrio spp., Bacilli, such as. Bacillus antliracis and Borellia spp., such as, Borellia burgdorferi.

[00013| Examples of protective antigens of bacterial pathogens include the somatic antigens of enterotoxigenic E. coli, such as the CFA/I fimbria! antigen (Yamamoto et al., Infect. Immun., 50:925-928; 1985) and the nontoxic B-subunit of the heat-labile toxin (Klipstein et al., Infect. Immun., 40:888-893; 1983); pertactin of Bordetella pertussis (Roberts et al., Vacc, 10:43-48; 1992), adenylate cyclase-hemolysin of B. pertussis (Guiso et al., Micro. Path., 1 1 :423-431 ; 1991 ), fragment C of tetanus toxin of Clostridium tetani (Fairweather et al., Infect. Immun., 58: 1323- 1326; 1990), OspA of Borellia burgdorferi (Sikand et al., Pediatrics, 108: 123- 128; 2001 ; Wallich et al., Infect Immun, 69:2130-2136; 2001 ), protective paracrystalline-surface-layer proteins of Rickettsia prowazekii and Rickettsia typhi (Carl ct al., Proc Natl Acad Sci USA, 87:8237-8241 ; 1990), listeriolysin (also known as "LIo" and "HIy") and/or the superoxide dismutase (also know as "SOD" and "p60") of Listeria monocytogenes (Hess et al., Infect. Immun. 65: 1286-92; 1997; Hess, J., et al., Proc. Natl. Acad. Sci. 93: 1458-1463; 1996; Bouwer et al., J. Exp. Med. 175: 1467-71 ; 1992), the urease of Helicobacter pylori (Gomez-Duarte et al., Vaccine 16, 460-71 ; 1998; Corthesy-Theulaz et al., Infection & Immunity 66, 581-6; 1998), and the Bacillus anthracis protective antigen and lethal factor receptor-binding domain (Price et al., Infect. Immun. 69, 4509-4515; 2001).

[00014] The parasitic pathogens, from which the parasitic antigens are derived, include, but are not limited to: Plasmodium spp., such as Plasmodium falciparum (ATCC No.: 30145); Trypanosome spp., such as Trypanosoma cruzi (ATCC No.: 50797); Giardia spp., such as Giardia intestinalis (ATCC No.: 30888D); Boophilus spp., Babesia spp., such as Babesia microti (ATCC No.: 30221 ); Entamoeba spp., such as Entamoeba histolytica (ATCC No.. 30015); Eimeria spp., such as Eimeria maxima (ATCC No. 40357); Leishmania spp. (Taxonomy ID: 38568); Schistosome spp., Brugia spp., Fascida spp., Dirofilaria spp., Wuchereria spp., and Onchocerea spp.

[00015| Examples of protective antigens of parasitic pathogens include the circumsporozoite antigens of Plasmodium spp. (Sadoff et al., Science, 240:336-337; 1988), such as the circumsporozoite antigen of P. bergerii or the circumsporozoite antigen of P. falciparum; the merozoite surface antigen of Plasmodium spp. (Spetzler et al., Int. J. Pept. Prot. Res., 43:351-358; 1994); the galactose specific lectin of Entamoeba histolytica (Mann et al., Proc. Natl. Acad. Sci., USA, 88:3248-3252; 1991 ), gp63 of Leishmania spp. (Russell et al., J. Immunol., 140: 1274-1278; 1988; Xu and Liew, Immunol., 84: 173-176; 1995), gp46 of Leishmania major (Handman et al., Vaccine, 18:301 1 -3017; 2000) paramyosin of Brugia malayi (Li et al., MoI. Biochem. Parasitol., 49:315-323; 1991 ), the triose-phosphate isomcrase of Schistosoma mansoni (Shoemaker et al., Proc. Natl. Acad. Sci., USA, 89: 1842- 1846; 1992); the secreted globin-like protein of Trichostrongylus colubriformis (Frenkel et al., MoI Biochem. Parasitol., 50:27-36; 1992); the glutathione-S-transferase of Frasciola hepatica (Hillyer et al., Exp. Parasitol., 75: 176-186; 1992), Schistosoma bovis and S. japonicum (Bashir et al., Trop. Geog. Med., 46:255-258; 1994); and vaccine antigens such as KLH of Schistosoma bovis and S. japonicum (Bashir et al., supra, 1994).

|00016| As mentioned earlier, the gene of interest may encode an endogenous immunogen, which may be any cellular protein, immunoregulatory agent, or therapeutic agent, or parts thereof, that may be expressed in the recipient cell, including, but not limited to tumor, transplantation, and autoimmune immunogens, or fragments and derivatives of tumor, transplantation, and autoimmune immunogens thereof. Thus, in the present invention, the gene of interest may encode tumor, transplant, or autoimmune immunogens, or parts or derivatives thereof. Alternatively, the gene of interest may encode synthetic genes (made, for example, as described herein), which encode tumor-specific, transplant, or autoimmune antigens or parts thereof, for example.

|00017| Examples of tumor specific antigens include prostate specific antigen (Gattuso et al., Human Pathol., 26: 123- 126; 1995), TAG-72 and CEA (Guadagni et al., Int. J. Biol. Markers, 9:53-60; 1994), MAGE- I and tyrosinase (Coulie et al., J. Immunothera., 14: 104-109; 1993). It has been shown in mice that immunization with non-malignant cells expressing a tumor antigen provides a vaccine effect, and also helps the animal mount an immune response to clear malignant tumor cells displaying the same antigen (Koeppen et al., Anal. N.Y. Acad. Sci., 690:244-255; 1993).

[00018] Examples of transplant antigens include the CD3 molecule on T cells (Alegre et al., Digest. Dis. Sci., 40:58-64; 1995). Treatment with an antibody to CD3 receptor has been shown to rapidly clear circulating T cells and reverse cell-mediated transplant rejection (Alegre et al., supra, 1995).

[00019] Examples of autoimmune antigens include IASb chain (Topham et al., Proc. Natl. Acad. Sci., USA, 91 :8005-8009; 1994). Vaccination of mice with an 18 amino acid peptide from the IASb chain has been demonstrated to provide protection and treatment to mice with experimental autoimmune encephalomyelitis (Topham et al., supra, 1994).

|00020| In addition, the gene of interest can encode an adjuvant, and can be used to increase host immune responses to immunogens. The particular adjuvant encoded by the gene of interest is not critical to the present invention and may be the A subunit of cholera toxin (i.e. CtxA; GenBank Accession Nos. XOOl 71 , AFl 75708, D3OO53, D30052), or parts and/or mutant derivatives thereof (e.g. the A l domain of the A subunit of Ctx (i.e. CtxA l ; GenBank Accession No. K02679), from any classical Vibrio cholerae (e.g. V. cholerae strain 395, ATCC No. 39541 ) or El Tor V. cholerae (e.g. V. cholerae strain 2125, ATCC No. 39050) strain. Alternatively, any bacterial toxin that is a member of the family of bacterial adenosine diphosphate-ribosylating exotoxins (Krueger and Barbier, Clin. Microbiol. Rev., 8:34; 1995) may be used in place of QxA; for example, the A subunit of heat-labile toxin (referred to herein as EItA) of enterotoxigenic Escherichia coli (GenBank Accession No. M35581), pertussis toxin Sl subunit (e3.g. ptxSl, GenBank Accession Nos. AJ007364, AJ007363, AJ006159, AJ006157 etc.); as a further alternative, the adjuvant may be one of the adenylate cyclase-hemolysins of Bordetella pertussis (ATCC No. 8467), Bordetella bronchi septica (ATCC No. 7773) or Bordetella parapertussis (ATCC No. 15237), e.g. the cyaΛ genes of B. pertussis (GenBank Accession No. X 14199), B. parapertussis (GenBank Accession No. AJ249835) or B. bronchiseptica (GenBank Accession No. Z371 12).

|000211 In addition, or alternatively, the gene of interest can encode a cytokine, which is useful as an adjuvant or for production of the recombinant proteins as therapeutics. The particular cytokine encoded by the gene of interest is not critical to the present invention includes, but not limited to, interleukin-4 (herein referred to as "IL-4"; Genbank Accession No. AF352783 (murine IL-4) or NM_000589 (human IL-4), IL-5 (Genbank Accession No. NM_O1O558 (murine IL-5) or NM_000879 (human IL-5)), IL-6 (Genbank Accession No. M20572 (murine 1L-6) or M29150 (human IL-6)), IL- I O (Genbank Accession No. NM_010548 (murine IL- 10) or AF418271 (human IL-IO)), IL-12-p40 (Genbank Accession No. NM 008352 (murine IL- 12 p40) or AY008847 (human IL-12 p40)), IL-12-p70 (Genbank Accession No. NM_O08351/NM_O08352 (murine IL-12 p35/40) or AF093065/AY008847 (human IL- 12 p35/40)), TGFb (Genbank Accession No. NMJ)1 1577 (murine TGFbI) or M603 I 6 (human TGFbI)), and TNFa (Genbank Accession No. X0261 1 (murine TNFa) or M26331 (human TNFa)).

[00022| Recombinant DNA and RNA procedures for the introduction of a functional gene of interest capable of expressing a transgene of interest, such as, an immunoregulatory agent in target host cells or tissues are described herein. [00023| As exemplary vaccine constructs to be encoded by the gene of interest, virus-like particles (herein referred to as "VLP") can be constructed to induce protective immune responses against viral pathogens. Influenza VLP's have been shown to self assemble following plasmid expression of gene sequences encoding the hemagglutinin (HA), neuraminidase (NA), and the matrix proteins (M 1 and M2) (Latham et al., J. Virol, 75:6154-6165; 2001 ). VLP's so constructed are further capable of membrane fusion and budding to further potentiate antibody-producing immune responses and protective immunity in animal models (Pushko et al., Vaccine 23:5751 ; 2005). HIV VLP's can be similarly assembled from minimal sequences encoding amino acids 146-231 of the capsid protein, a six amino acid myristylation sequence, the sequence encoding the P2 peptide, a GCN4 leucine zipper domain, and the gpl όO envelope precursor (Accola et al., J. Virol, 74:5395-5402; 2000). The major protein LI of HPV has been shown to self-assemble into VLP's in a variety of cell lines and produces humoral and cellular immunity, making the gene encoding this protein an attractive immunogen (Shi et al., J. Virol., 75(21): 10139-10148; 2001 ).

[00024] Recombinant DNA and RNA procedures to generate RNA capable of expressing a VLP in target host cells or tissues are described herein.

[00025| In another embodiment, the gene of interest can express recombinant proteins useful as therapeutics and laboratory reagents. Examples of recombinant proteins include but are not limited to: calcitonin, CTLA-Ig fusion protein, glucagon, hyaluronidase, insulin, insulin-like growth factor- 1 , interferon alpha-2a, interferon alpha-2b, parathyroid hormone, somatropin, somatropin antagonist, P53, platelet-derived growth factor, urate oxidase, Factor VIII, Factor Vila, Arylsulfatase B, bone morphogenic protein-2, bone morphogenic protein-7, DNase, erythropoietin, Factor IX, follicle stimulating hormone, β-galactosidase, glucocerebrosidase, glucosidase, human cariogenic hormone, iduronidase, iduronate-2 sulfatase, luteinizing hormone, tumor necrosis factor receptor-IgG Fc fusion protein, tissue plasminogen activator (tPA), thyroid stimulating hormone etc. [00026| Recombinant DNA and RNA procedures to produce RNA capable of expressing therapeutics and laboratory reagent proteins in cell-free systems and in target host cells or tissues are described herein.

[00027| In a further embodiment, the gene of interest can encode a molecule with a binding activity, such as a ligand which binds another entity or one member of a binding pair. Examples of such include a receptor and the ligand which binds and activates that receptor, antibodies and antigens, lectins and the cognate sugars and so on. Thus, in the case of antibodies, the expressed protein can comprise an antibody, one chain of an antibody, a part of the heavy chain and the light chain, a complementarity-determining region, or any polypeptide that can bind an epitope or a determinant. Thus, monoclonal antibodies (Mabs) or epitope-binding parts thereof can be expressed as taught herein wherein the antibody can have a specific binding activity, such as, but not limited to anti-CD 1 Ia Mab, anti-CD20 Mab, anti-CD52 Mab, anti-HER2 receptor Mab, anti-immunoglobulin E Mab, anti-TNF Mab, anti-interleukin-2 receptor Mab, anti-platelet Mab, anti-respiratory syncytial virus Mab, anti-EGF-receptor Mab etc. Recombinant DNA and RNA procedures to produce RNA capable of expressing Mabs in cell-free systems or in target host cells or tissues are described herein.

Initiation of protein expression

|00028| As mentioned, the gene of interest is capable of expressing proteins in target yeast, plant and mammalian cells; preferably, expression in each host background utilizes specific translation initiation sequences to increase the efficiency of expression. For efficient expression of the recombinant protein of interest, yeast expression cassettes are comprised of translation loop formation sequences (herein referred to as "TLFS") in the 5' and 3' ends of the rdsRNA segment. The particular TLFS useful for expression of recombinant proteins in yeast is not important to the present invention and include, but are not limited to, the TLFS located at the 5' and 3' ends of the YAP l and p i 50 genes of Saccharomyces cerevisiae (Zhou et al., Proc Natl Acad Sci USA 98: 1531 - 1536 (2001 )), hepatitis C virus (Rosenfeld and Racaniello, J Virol 79: 10126-10137 (2005)), and cricket paralysis virus cricket paralysis virus (Thompson et al., Proc Natl Acad Sci USA 98: 12972-12977 (2001 )).

|00029| For efficient expression of a recombinant protein of interest, plant expression cassettes are comprised of TLFS in the 5' and 3' ends of the recombinant dsRNA (rdsRNA) segment (reviewed in Dreher and Miller, Virol. 344: 185; 2006). For example, the 5'-end of the 3'-UTR of barley yellow dwarf luteovirus (BYDV) RNA carries about 100 base sequence that facilitates translation initiation at the 5'-proximal AUG of the mRNA (Guo et al., RNA, 6: 1808; 2000; Wang et al., EMBO, 16:4107; 1997). This BYDV-like TLFS is conserved in all luteovirυses and in the dianthovirus (Mizumoto ct al., J. Virol., 77: 121 13; 2003) and nccrovirus (Meulewaeter et al., Nucl. Acid Res., 32: 1721 ; 2004; Shen and Miller, J. Virol., 78:4655; 2004) genera of the Tombusviridae. The cap independent translation element (BTE) is defined by a 17-nιιcleotide conserved sequence, GGAUCCUGGGAAACAGG (SEQ ID NO: 1), that forms a stem-loop structure and by at least one additional stem-loop, whose loop base-pairs to the 5'-UTR (Guo et al., MoI. Cell 7: 1 103; 2001). A tract in this element also has potential to base-pair near the 3 '-end of 18S rRNA (Wang et al., supra, 1997). This may contribute to recruitment and recycling of the ribosomes to and within the TLFS. However, the particular TLFS useful for expression of recombinant proteins in plants is not important to the present invention and may include, but are not limited to, the TLFS located in the cmcifer-infecting tobamovirus (Ivanov et al., Virology 232: 32; 1997; Skulachev et al., Virology 263: 139; 1999; Dorokhov et al., J. Gen. Virol., 87: 2693; 2006)), tomato bushy stunt virus (Monkewich et al., J. Virol., 79:4848; 2005), maize Hsp 101 gene (Dinkova et al., Plant J., 41 :722; 2005), potato leafroll polerovirus (Jaag et al., Proc Natl Acad Sci USA 100:8939; 2003), and Rhopalosiphum padi virus (Domicr ct al, Virol., 268: 264; 2000; Woolaway ct al., J Virol 75: 10244; 2001 ). |00030| Efficient expression of a recombinant protein of interest in mammalian cells entails the utilization of expression cassettes containing TLFS in the 5' and 3' ends of the gene of interest (Dreher and Miller, Virol. 344: 185; 2006). The particular TLFS is not important to the present invention and can include, but is not limited to, the 5'-UTR and 3'-UTR of the hepatitis C virus (herein referred to as "HCV"). The 3' UTR of the TLFS in HCV is comprised of a polypyrimidine sequence that is recognized by the polypyrimidine tract binding protein (herein referred to as "PTBP" or "PTB") and does not contain a polyadenosine (also know as "poly-A") sequence. The use of polyadenosine, as described in (US Pat. No. 7,018,835 and US Patent Appl. No. 200601 15493), impedes the binding of PTB in mammalian cells.

[00031 ] In addition, RNA in the present invention can contain "Kozak" sequences adjacent to the translation start codon (e.g. Kozak, J. MoI. Biol. 196:947-950; 1987). Preferably, expression cassettes contain a 5'-TLFS and 3'-TLFS, and a Kozak sequence functionally linked to the 5'-end UTR. This configuration is designed to optimize expression of genes of interest in mammalian cells.

1000321 As mentioned above, the RNA can also encode small inhibitory RNA (siRNA), hairpin loops, antisense RNA or ribozymes, which are capable of silencing and/or regulating host genes. Examples of siRNA sequences or targets include, but are not limited to: rapamycin siRNA (Iwamaru et al., Oncogene, 26:2435-44,2007); conserved regions in HIV- I gag, pol, int and vpu genes (Chang et al., Gene Ther. 12: 1 133-1 144 (2005)); epidermal growth factor receptor (Vollmann et al.; Int J Oncol. 28: 1531 -1542 (2006)); surface antigen region of Hepatitis B virus (Giladi et al., MoI Ther. 8:769-776 (2003)); NS3 and NS5B regions of Hepatitis C virus (Takigawa et al., Microbiol Immunol. 48:591-598 (2004)).

1000331 Examples of antisense RNA sequences include but are not limited to: human papilloma virus E6 and E7 genes (Schiffelers et al., Expert Opin Biol Ther. 5:359 (2005)); human Jun N-terminal kinase 1 (Betigeri et al., MoI Pharm. 3:424-430 (2006)); human c-Met, a receptor tyrosine kinase, (Chu et al., Surg Neurol. 65:533-538 (2006)); HIV- I gag (Ramezani et al., Front Biosci. 1 1 :2940-2948 (2006)),or microRNA (e.g. Ying et al., Methods MoI. Biol;342: l , (2006) and so on.

|00034| Examples of ribozyme sequences include, but are not limited to: human CPEB3 gene (Salehi-Ashtiani et al., Science, 313: 1788-1792 (2006)); hepatitis delta virus (Been, Curr Top Microbiol Immunol. 307:47-65 (2006)); glmS genes of many Gram positive bacteria (Urban et al., J. MoI. Biol., 373:521, (2007)); the universal ribozyme, ribonuclease P (Torres-Larios et al., Curr Opin Struct Biol.16:327-335 (2006)); Neurospora Varkud satellite (VS) ribozyme (Hoffmann et al., Proc Natl Acad Sci U S A. 100:7003-7008 (2003)); multimeric hammerhead ribozymes against various HIV-I genes (Ramezani et al., supra); and ribozymes targeting Hepatitis C virus 5' and 3' untranslated regions (Jarczak et al., FEBS J. 272:5910-5922 (2005)).

[00035| Said resulting capsids that express small inhibitory RNA, antisense RNA or ribozymes express and silence or regulate host genes are useful as immunosuppressants, immunoregulatory agents, and anticancer, viral and gene therapeutics.

Production of capsid coat protein p8

|00036| In a preferred embodiment, the present invention provides compositions comprised of Cystovirus procapsids, Cystovirus capsid coat protein p8 and ssRNA encoding a gene of interest. Capsids that bear a complete p8 shell are capable of spontaneous entry into target cells (Jaalino et al., Structure, 15: 15, (2007). This property can be used, therefore, as a means to transfect cells of interest and is useful for the use of capsids produced by the current invention as expression vectors in yeast, plant and mammalian cells.

|00037| The particular p8 used in the present invention is not important and can be, but no limited to, the capsid coat protein designated p8 of phi-6 (GenBank Accession No. NP 620340), phi-8 (GenBank Accession No. NP_524570), phi 12 (GenBank Accession No.NP_690826) and phi- 13 (GenBank Accession No.NP_690807). Alternatively, capsid coat protein p8 can be derived from any dsRNA bacteriophage isolated from terrestrial sources using methods well documented in the prior art (Silander et al., Proc. Natl. Acad. Sci., 102:19009, (2005)). Purification of p8 can be achieved using previously documented procedures for the purification of p8 (Olkkonen J MoI Biol. 1991 Apr 5;218(3):569, (1991)). Alternatively, combinations standard protein purification procedures, such as, size fractionation, ion exchange and affinity purification can be used to purify p8. The identity of p8 can be demonstrated by SDS-PAGE gel electrophoresis, immunoblot N-terminal sequencing, and mass spectrometry procedures well known in the art (Olkkonen J MoI Biol. 1991 Apr 5;218(3):569, ( 199 I )).

Recombinant DNA, RNA and protein methods

|00038| Expression cassettes can be made using known methods, and can be made synthetically by any qualified commercial vendor, such as DNA 2.0 Inc. (Menlo Park, CA, USA), Blue Heron Biotechnology (Bothell, WA, USA), Geneart Inc. (Toronto Ont, CA) and Genscript Inc. (Piscataway, NJ, USA). To synthesize expression cassettes a series of short sequences of 100-200 base pairs in length are generated and ligated to form the full-length sequence using procedures well know in the art (Ausubel et al., Short Protocols in Molecular Biology, John Wiley and Sons, New York, N. Y. ( 1992)). The synthetic DNA can be produced using an Applied Biosystems International ABI ^{I M} 3900 High-Throughput DNA Synthesizer (Foster City, Calif.) and procedures provided by the manufacturer.

|00039| Single stranded RNA (herein referred to as "ssRNA") can be prepared by in vitro transcription of cDNA encoding the gene of interest as the template for the in vitro transcription reactions. To ensure precise ends are produced encoding the appropriate Cystovirus 5' and 3' replication and transcription initiation sequences, ssRNA employed in the present study is synthesized in vitro using PCR-generated DNA fragments encoding the expressed sequences of interest. PCR fragments are preferable as compared to restriction enzyme linearized plasm ids, as the 5' and 3' ends encode the precise replication and transcription initiation sequences. The primers used are designed such that the forward primer carries, for example, an SP6 promoter sequence and places the start of transcription at the 5' guanidine. PCR is performed using Accuprime pfx polymerase (Invitrogen, Carlsbad, CA), not Taq, to create blunt ends. Successful amplification of sequences can be verified by agarose gel electrophoresis followed by purification of the PCR reaction products using QlAquick PCR purification kit (Qiagen, Valencia, CA, Cat. No. 28104). The concentration of the PCR-generated fragments encoding RNA can be determined by spectrophotometry.

|00040| Preparations of ssRNA are synthesized using, for example, DuraScribe SP6 polymerase kit (Epicentre, Madison, WI) according to the manufacturer's instructions using the PCR-generated DNA. The transcription reactions are treated with DNase I to remove the template DNA and the reaction is purified using MEGAclear^R kit according to the manufacturer's instructions (Ambion, Austin, TX, Cat. No. 1909). The synthetic ssRN A transcripts encoding genes of interest are verified for size, for example, in a 1% denaturing gel and the RNA concentrations are determined by spectrophotometry.

Nucleic acid methods

|00041 | The recombinant DNA procedures used in the present invention, including, but not limited to, PCR, restriction endonuclease (herein referred to as "RE") digestions, DNA ligation, agarose gel electrophoresis, DNA purification, and dideoxynucleotide sequencing, are described elsewhere (Miller, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.; 1992); (Bothwell et al.. Methods for Cloning and Analysis of Eukaryotic Genes, Eds., Jones and Bartlett Publishers Inc., Boston, Mass. ( 1990)); and (Ausubel et al., supra), bacteriophage-mediated transduction (de Boer, infra); (Miller, supra, 1992) and (Ausubel et al., supra), or chemical (Bothwell et al., supra); (Ausubel et al., supra); (Feigner et al, Proc. Natl. Acad. Sci., USA, 84:7413 7417 ( 1987); and Farhood, Annal. N. Y. Acad. Sci., 716:23 34 (1994)) electroporation (Bothwell et al., supra); (Ausubel et al., supra); (Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.; 1992) and physical transformation techniques (Johnston et al, Meth. Cell Biol, 43(Pt A):353 365 ( 1994); (Bothwell et al., supra). The genes can be incorporated on phage (de Boer et al., Cell, 56:641-649; 1989), plasmids vectors (Wu et al., AIDS Res Hum Retroviruses., 13: 1 187, ( 1997)) or spliced into the chromosome (Hone et al., Microb. Pathog., 5:407, ( 1988)) of the target strain.

[00042J RE's (New England Biolabs, Beverly, Mass.), T4 DNA ligase (New England Biolabs, Beverly, MA) and Taq polymerase (Life Technologies, Gaithersburg, MD) were used according to the manufacturers' protocols; plasmid DNA was prepared using small-scale (Qiagen Miniprep^IM kit, Santa Clarita, CA) or large-scale (Qiagen Midiprep^{1 M} kit, Santa Clarita, CA) plasmid DNA purification kits according to the manufacturer's protocols (Qiagen, Santa Clarita, CA); nuclease-free, molecular biology grade deionized water, Tris-HCl (pH 7.5), EDTA pH 8.0, 1 M MgCh, 100% (v/v) ethanol, ultra-pure agarose, and agarose gel electrophoresis buffer were purchased from Life Technologies, Gaithersburg, MD. RE digestions, PCRs, DNA ligation reactions and agarose gel electrophoresis were conducted according to well-known procedures (Sambrook et al., supra, 1989; Ausubel et al., supra, 1990). Nucleotide sequencing to verify the DNA sequence of each recombinant plasmid described in the following examples was accomplished by conventional automated DNA sequencing techniques using an Applied Biosystems automated sequencer, model 373A. |000431 PCR primers can be purchased from Integrated DNA Technologies (Coralville, IA) or the University of Maryland Biopolymer Facility (Baltimore, MD) and were synthesized using, for example, an Applied Biosystems DNA synthesizer (model 373A). PCR primers were used at a concentration of 200 μM and annealing temperatures for the PCR reactions were determined using Clone manager software version 4.1 (Scientific and Educational Software Inc., Durham, NC) or OLIGO primer analysis software version 4.0. PCRs can be conducted in a Bio Rad iCycler, (Hercules, CA). The PCR primers for the amplifications can be designed using Clone Manager¹* software version 4.1 (Scientific and Educational Software Inc., Durham, NC) and OLIGO primer analysis software version 4.0. The software enables the design of PCR primers and RE sites that are compatible with the specific DNA fragments being manipulated. Primer annealing, elongation and denaturation times in the PCRs were set according to standard procedures (Ausubel et al., supra). The RE digestions and the PCRs were subsequently analyzed by agarose gel electrophoresis using standard procedures (Ausubel et al., supra; Sambrook, supra). A positive clone was defined as one that displays the appropriate RE pattern and/or PCR pattern. Plasmids identified through this procedure can be further evaluated using standard DNA sequencing procedures, as described herein.

|00044| Escherichia coli strains Topl O and DH5α are purchased from Invitrogen (Carlsbad, CA) and strain SCS I 10 is purchased from Stratagene (La Jolla, CA). Recombinant plasmids are introduced into E. coli, for example, by electroporation using a Gene Pulser (BioRad Laboratories, Hercules, CA) set at , for example, 200 Ω, 25 μF and 1.8 kV, or chemical transformation, as described (Ausubel et al., supra).

[00045| Bacterial strains can be grown on tryptic soy agar (Difco, Detroit, MI) or in tryptic soy broth (Difco, Detroit, MI), unless otherwise stated, at an appropriate temperature. Medium can be supplemented with 100 μg/ml ampicillin, 50 μg/ml kanamycin, and/or chloramphenicol 20 μg g/ml (Sigma, St. Louis, MO) as needed. Bacterial strains were stored at -80° C in tryptic soy broth (Difco) containing 30% (v/v) glycerol (Sigma, St Louis, MO) at about 10⁹ colony-forming units (herein referred to as "cfu") per ml.

Reagent List

|00046| Kpnl (New England Biolabs, Beverly, MA, Cat. No. ROl 42S), Pstl (New England Biolabs, Beverly, MA, Cat. No. RO 140S), tryptic soy broth (Difco, Detroit, MI, Cat. No. 21 1822), tryptic soy agar (Difco, Detroit, MI., Cat. No. 236920), Miniprep⁰⁰ plasmid DNA purification kit (Qiagen, Valencia, CA, Cat. No. 27106), glycerol (Sigma, St. Louis, MO, Cat. No. G5516), Hpal (New England Biolabs, Beverly, MA, Cat. No. RO 105S), calf intestinal alkaline phosphatase (New England Biolabs, Beverly, MA, Cat. No. M0290S), Vent^R DNA polymerase (New England Biolabs, Cat. No. M0254S), QIAquick PCR purification kit (Qiagen, Cat. No. 28106, Valencia, CA.), diaminopimelic acid (Sigma-Aldrich, St. Louis, MO, Cat. No. Dl 377), BgIII (New England Biolabs, Beverly, MA, Cat. No. ROl 44S), IPTG (Invitrogen, Carlsbad, CA, Cat. No. 15529-019), cell culture lysis reagent (Promega, Madison, WI, Cat. No. E 1531 ), lysozyme (Sigma, St. Louis, MO, Cat. No. L6876), potassium phosphate (Sigma, St. Louis, MO, Cat. No. P5379), magnesium chloride (Sigma, St. Louis, MO, Cat. No. M 1028) Dralll (New England Biolabs, Beverly, MA, Cat. No. R0510S), Psil (New England Biolabs, Beverly, MA, Cat. No. V0279S), Proteinase K (Ambion, Austin, TX, Cat. No. 2542-2548), Durascribe T7 transcription kit (Epicentre, Madison, Wl), Durascribe SP6 transcription kit (Epicentre, Madison, WI), MEGAscript^® T7 transcription kit (Ambion, Austin, TX, Cat. No. 1334), MEGAscript^R SP6 transcription kit (Ambion, Austin, TX, Cat. No. 1330), MEGAclear columns (Ambion, Austin, TX, Cat. No. 1908), BrightStar biotinylated RNA millennium marker (Ambion, Austin, TX, Cat. No. 7170), BrightStar nylon membrane (Ambion, Austin, TX, Cat. No. 10102), BrightStar Biodetect kit (Ambion, Austin, TX, Cat. No. 1930), Tris-HCI buffer (Quality Biological, Gaithersburg, MD, Cat. No. 351 -007- 100), magnesium chloride (Sigma-Aldrich, St. Louis, MO, Cat. No M 1787), ammonium acetate (Sigma-Aldrich, St. Louis, MO, Cat. No. A2706), sodium chloride (Sigma-Aldrich, St. Louis, MO, Cat. No. S7653), potassium chloride (Sigma-Aldrich, St. Louis, MO, Cat. No. P391 1 ), dithiothreitol (Sigma-Aldrich, St. Louis, MO, Cat. No. D9779), EDTA (Sigma-Aldrich, St. Louis, MO, Cat. No. E8008), polyethylene glycol 4000 (Fluka, Buchs, CH, Cat. No. 95904), SUPERase RNase inhibitor (Ambion, Austin, TX, Cat. No 2694,), biotin- 14-CTP (Invitrogen, Carlsbad, CA, Cat. No. 19519-016) and RNase ONE ribonuclease (Promega, Madison, WI, Cat. No. M4261).

[00047| The cDNA sequences encoding the recombinant dsRNA (rdsRNA) segments can be generated using convention recombinant DNA techniques involving PCR and assembly of desired elements synthetically by ligation. Alternatively, recombinant segments can be generated synthetically using an Applied Biosystems ABI™ 3900 High-Throughput DNA Synthesizer (Foster City, CA) and procedures provided by the manufacturer. To synthesize the cDNA copies of segment L and the recombinant segments, a series of segments of the full-length sequence is generated by PCR and ligated together to form the full-length segment using procedures well know in the art (Haas et al., Curr Biol 6:315; 1996; Andre et al., J Virol 72: 1497; 1998; Fouts et al., J Virol 74: 1 1427; 2000). Briefly, synthetic oligonucleotides 100-200 nucleotides in length (i.e. preferably with sequences at the 5' end and 3 'end that match at the 5' and 3' end of the oligonucleotides that encode the adjacent sequence) are produced using, for example, an automated DNA synthesizer (e.g. Applied Biosystems ABI™ 3900 High-Throughput DNA Synthesizer (Foster City, CA)). Using the same approach, the complement oligonucleotides are synthesized and annealed with the complementary partners to form double stranded oligonucleotides. Pairs of double stranded oligonucleotides (i.e. those that encode adjacent sequences) are joined by ligation to form a larger fragment. These larger fragments are purified by agarose gel electrophoresis and isolated using a gel purification kit (e.g. QIAEX® II GcI Extraction System from Qiagcn, Santa Cruz, CA, Cat. No. 12385). This procedure is repeated until the full-length DNA molecule is created. After each round of ligation the fragments can be amplified by PCR to increase the yield. Procedures for de novo synthetic gene construction are well known in the art, and are described elsewhere (Andre et al., supra, ( 1998); et al., Haas infra, (1996)); alternatively synthetic genes can be purchased commercially, e.g. from the Midland Certified Reagent Co. (Midland, TX).

[00048| The invention is amenable for the in vitro expression of a foreign gene product. Thus, the various components of an expression system as described herein are combined under conditions conducive and which would enable packaging and expression of a foreign gene or transgene carried by a capsid-like structure as taught herein.

[00049] The invention also contemplates articles of manufacture comprising components of interest that will enable a user to use the packaging system and the expression system of interest to express of foreign gene of interest, which, for example, is introduced as a single-stranded RNA encoding the gene of interest. For example, a kit can comprise a dsRNA phage procapsid composition along with purified p8 protein. In another embodiment, the kit can comprise a vector for expressing the p8 protein. In yet another embodiment, a kit can comprise an expression system, with the necessary components, for expressing the procapsid. In another embodiment, the kit can comprise a capsid carrying a known ssRNA carrying the gene to be expressed. The reagents can be in liquid form, or a liquid that is frozen, or desiccated for reconstitution by the user. Such a kit can include a vial containing a fluid for reconstituting the dry form. The kit can include instructions for use.

|00050| The reagents can be suspended in suitable medium that maintains the reagents in a stable form. Thus, the regents can be carried in a suitable buffer, such as a phosphate buffer, a citrate buffer, a bicarbonate buffer and so on, as known in the art, and as a design choice. The liquid formulation can carry various additives as known in the art for obtaining desired properties of the liquid, lyophilized or desiccated composition of interest. Thus, a liquid composition can include, for example, various carriers, diluents and excipients as known in the art. Suitable such additives include salts, sugars, emulsifiers, bulking agents, chelating agents, buffers, stabilizers, isotonifϊers, preservatives, detergents, surfactants, antioxidants and so on as known in the art and as a design choice.

|000511 Liquid formulations can be lyophilized or desiccated as known in the art. A suitable stabilizer, such as glycerol or other sugar can be included for lyophilization as known in the art.

[000521 The instant invention encompasses formulations, such as, liquid formulations or solid compositions having stability at temperatures found in a laboratory, including a commercial refrigerator and freezer, such as a device that maintains a temperature of from about -20° C to about 5° C, or other freezers that attain lower temperatures, such as a -80° C freezer. The compositions preferably are stable for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, greater than 1 week, greater than 2 weeks and so on. Lyophilized or desiccated forms may be stable for longer periods of time, such as, for about IO days, for about 20 days, for about 30 days, for about 40 days, for about 50 days, for about 60 days, for about 120 days, for about 180 days, for about a year, for about 2 years or more. The stability can be assessed, for example, by determining procapsid formation, capsid formation or foreign gene expression as compared to a baseline. The degree of accepted stability is a design choice.

|00053| An article of manufacture can comprise a containing means, a container or containers and a label. Suitable containing means include, for example, bottles, vials, syringes and test tubes. The containing means may be formed from a variety of materials such as glass or plastic. The containing means holds a composition that contributes to packaging or expression as taught herein. The containing means may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label on or associated with the one or more containing means indicates that the composition is used for the methods taught herein, namely expression of an ssRNA of interest. The article of manufacture may further comprise additional containing means comprising a suitable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes and package inserts with instructions for use. '

|00054| The invention now will be exemplified in the following non-limiting examples demonstrating certain aspects of the invention of interest.

Example 1

Preparation of Cvstovirus RNA packaging mixture

|00055| The procapsids of cystoviruses, such as dsRNA phage phi-6, package RNA that contain the appropriate recognition sequences to produce dsRNA within the procapsids; completion of this process produces a capsid, which is capable of synthesizing and secreting RNA in cell-free systems as well as in target host cells. To produce procapsids, Escherichia coli strain Bl-gsL (expresses phi-6 procapsids) is grown in 1 liter liquid cultures to an optical density at 600 nm of 1.2. The bacteria are harvested by centrifugation at 5000 x g for 15 min and washed 3 x in 100 ml Tris-buffered saline, pH7.3. To lyse cells, the bacteria are resuspended in 15 ml of 1 mg/ml lysozyme reconstituted in 10 mM potassium phosphate, pH 7.5, 1 mM magnesium chloride. The mixture is digested with gentle shaking at 25" C for 1 hour. The lysate is then digested with RNase-free DNase 1 (New England BioLabs, Beverly, MA, Cat. No. M0303L) at 37 ° C for 30 min. Next, the lysate is subjected to centrifugation at 12,000 rpm for 15 min, and the procapsids are purified by tangential flow that has a 500,000 molecular weight cutoff (Millipore, Billerica, MA). The presence of procapsids in the retentate is verified using standard electrophoresis procedures (Ausubel et al, supra, (1990)) that employs 15.5 % (w/v) polyacrylamide gels containing 20 % SDS (w/v) followed by immunoblot. Immunodetection is achieved with rabbit polyclonal antibodies against proteins Pl and P4.

[00056| In parallel, phi-6 capsid coat protein p8 is purified using strain BI-p8, which carries plasmid pBACIL-p8 and overexpresses p8.

[00057] The Cystovirus packaging mixture is produced by admixing the purified procapsids and p8, as described (Olkkonen J. MoI. Biol. 1991 Apr 5;2 I 8(3):569, (1991 )). The procapsids and p8 are suspended in 50 mM Tris-HCl (pH 8.2), 3 mM MgCIa, 100 mM ammonium acetate, 20 mM NaCl, 5 mM KCl, 5 mM dithiothreitol (DTT), 1 mM ATP, 1 mM CTP, 1 mM GTP, 1 mM UTP, 5% polyethylene glycol 4000, and 2 units Superase RNase Inhibitor and frozen at -80° C. Aliquots of this suspension can be used to package RNA sequences containing sequences homologous to the 5' and 3' ends of phi-6 genomic RNA segments.

Example 2

Packaging RNA encoding an adjuvant

|00058| To utilize this system for the expression of an adjuvant, a synthetic DNA sequence encoding adjuvants are constructed:

BI-IL2: 5'-phi-6 gsS-Pac-»TLFS->IL-2->TLFS-»Phi-6 gsS-3'UTR, BI-CTLA-4: 5'-phi-6 gsS-Pac->TLFS-»CTLA-4 antagonist-* TLFS-Phi- 6->gsM-3'UTR,

BI-NSP3: 5'-phi-6 gsL-Pac-TLFS-NSP3-TLFS-Phi-6 gsL-3'UTR

|00059| The cDNA sequences encoding the rdsRNA segments are generated synthetically using an Applied Biosystems ABI™ 3900 High-Throughput DNA Synthesizer (Foster City, CA) and procedures provided by the manufacturer. To synthesize die cDNA copies of a series of sub-segments are synthesized and ligated together to form the full-length segment using procedures well know in the art (Haas et al., Curr Biol 6:315; 1996; Andre et al., J Virol 72: 1497; 1998; Fouts et al., J Virol 74: M 427; 2000). Briefly, synthetic oligonucleotides 100-200 nucleotides in length (i.e. preferably with sequences at the 5' end and the 3 'end that match at the 5' and 3 ' ends of the oligonucleotides that encodes the adjacent sequence) are produced using, for example, an automated DNA synthesizer (e.g. Applied Biosystems ABI™ 3900 High-Throughput DNA Synthesizer (Foster City, CA)). Using the same approach, the complement oligonucleotides are synthesized and annealed with the complementary partners to form double stranded oligonucleotides. Pairs of double stranded oligonucleotides (i.e. those that encode adjacent sequences) and joined by ligation to form a larger fragment. These larger fragments are purified by agarose gel electrophoresis and isolated using a gel purification kit (e.g. QIAEX^® II Gel Extraction System, from Qiagen, Santa Cruz, CA, Cat. No. 12385). This procedure is repeated until the full-length DNA molecule is created. After each round of ligation the fragments can be amplified by PCR to increase the yield. Procedures for de novo synthetic gene construction are well known in the art, and are described elsewhere (Andre et al., supra, ( 1998); et al., Haas supra, ( 1996)).

(00060| These synthetic DNA sequences are then used as templates to generate the ssRNA using DuraScribe SP6 polymerase kit (Epicentre, Madison, WI) according to the manufacturer's instructions. The transcription reactions are treated with DNase I to remove the template DNA and the reaction is purified using a MEGAclear* kit according to manufacturer's instructions (Ambion, Austin, TX, Cat. No. 1909). The synthetic ssRNA transcripts encoding BI-IL2, BI-CTLA-4 and BI-NSP3 are verified for size, for example, in a 1% denaturing gel and the RNA concentrations are determined by spectrophotometry.

1000611 To produce capsids, an admixture comprised of an aliquot of the Cystovirus packaging mixture produced in Example 1 and 1 μg of each synthetic ssRN A transcript encoding BI-IL2, BI-CTLA-4 and BI-NSP3 is prepared. This admixture is incubated at room temperature for 4 hours.

1000621 Following incubation the presence of dsRNA, the capsids are purified by tangential flow and examined by agarose gel electrophoresis and SDS-PAGE. The results show that the above admixture produced capsids that are capable of expressing and adjuvant in mammalian cells.

Example 3

Expression of an adjuvant

1000631 To evaluate the adjuvant produced in Example 2, an animal study is performed to test the efficacy of a 100 μg DNA vaccine encoding tumor antigen MUC I under control of the CMV promoter, which is suspended in 100 μl of endotoxin-free phosphate-buffered saline (PBS) injected alone or in the presence of a capsid adjuvant in mice transgenic for human MUC l , using methods well known in the art. To identify an appropriate dose of capsid adjuvant, a range of capsid doses are mixed with the MUCl DNA vaccine in the amounts show below.

4 lOO ng 100 μg

5 300 ng 100 μg

6 i μg 100 μg

7 3 μg 100 μg

8 _.10 μg 100 μg

9 30 μg 100 μg

10 LQP.μg 100 μg _

[000641 Groups of ten mice are vaccinated intramuscularly with the DNA vaccine alone or with one of the admixtures, three times, at weeks 0, 2 and 6. Then 8 weeks after the final dose, the mice are challenged with 10⁶ human small cell lung carcinoma cells expressing MUC l , and deaths are recorded daily. The results of this study show that MUC l vaccine is more effective at controlling MUC l -positive cancers when administered with the capsid adjuvant.

|00065| The instant application claims benefit to U.S. Ser. No. 61/045,289 filed 16 April 2008, the contents of which are incorporated herein by reference in entirety.

|00066| All references cited herein are herein incorporated by reference in entirety.

|00067| It will be evident to one of skill in the art that various modifications can be made without departing from the scope and spirit of the teachings herein.

Claims

We claim:

1. A composition comprising a dsRNA phage procapsid, components thereof or a nucleic acid which expresses said components; and an isolated p8 protein or a nucleic acid which expresses said p8 protein.

2. The composition of claim 1 , wherein said dsRNA phage comprises a Cystoviais.

3. The composition of claim 2, wherein said Cystovirus is phi-6, phi-8, phi-10, phi-12 or phi-13.

4. The composition of claim 2, wherein said Cystovirus is phi-6.

5. The composition of claim 2, wherein said components of said Cystovirus procapsid comprise pi , p2, p4 and p7.

6. The composition of claim 5, wherein said components are expressed using an in vitro expression system.

7. The composition of claim 5, wherein said components are expressed by a recombinant organism.

8. The composition of claim 1 , wherein said p8 is obtained from a Cystovirus.

9. The composition of claim 8, wherein said Cystovirus is phi-6, phi-8, phi-10, phi- 12 or phi- 13.

10. The composition of claim 8, wherein said Cystovirus is phi-6.

1 1. The composition of claim 1 , further comprising an ssRN A encoding an expressed product of interest.

12. The composition of claim 1 1 , wherein said product is an RNA.

13. The composition of claim 1 1 , wherein said product is a polypeptide.

14. The composition of claim 1 1 , wherein said product is an adjuvant.

15. The composition of claim 1 1, wherein said product is an antigen.

16. The composition of claim 1 1 , wherein said product is a cytokine.

17. The composition of claim 1 1 , wherein said product is a ligand.

18. The composition of claim 17, wherein said ligand comprises an epitope-binding site.