Modified nodavirus rna for gene delivery

TECHNICAL FIELD

[0001] This invention is in the field of gene delivery, more particularly in the delivery of genes for expression using modified nodavirus RNA packaged in virus-like particles (VLPs).

BACKGROUND ART

[0002] The Nodaviridae have bipartite RNA genomes, that is to say their genomes consist of two separate single-stranded RNA molecules, designated RNA1 and RNA2. These are both packaged within the same virion. RNA1 encodes an RNA replicase, and RNA2 encodes the virion capsid protein. In flock house virus (FHV), RNA1 is 3.1 kb long and RNA2 is 1.4 kb.

[0003] The replicase product of FHV RNA1 is specific for the viral genome and this enables FHV to replicate autonomously [Ball et al. (1992) J.Virol. 66:2326-34: Gallagher et (l. (1983) J.Virol. 46:481-89]. In a natural situation, the replicase is highly template specific and replicates only viral RNA1 and RNA2, but self-replication also occurs in the absence of RNA2. Furthermore, even though FHV is an insect virus, it can self-replicate in many different cell types, including plants, vertebrates and yeasts.

[0004] Manipulation of RNA2 and the FHV capsid protein has been widely reported. The insertion of HIV epitopes into surface loops has been reported [e.g. Scodeller et al. (1995) Vaccine 13:1233-39; Buratti et al. (1996) J.Immunol.Methods 197:7-18; Schiappacassi et al. (1997) J.Virol.Methods 63:121-27]. More generally, the virion has been used as an epitope display system [Lorenzi & Burrone (1999) Immunotechnol. 4:267-72; see also WO96/05293].

[0005] In contrast, manipulation of FHV RNA1 and the replicase has not been pursued. In fact, the self-replication function of RNA1 has been shown to be very sensitive to manipulation of RNA1 and its ORF [Ball (1995) J. Virol 69:720-727].

[0006] During replication of RNA1, a small sub-genomic RNA called RNA3 is also transcribed from the 3′ end of RNA1, RNA3 encodes for two small proteins of unknown function: B 1 (in the same open reading frame and with the same translational stop codon as the replicase) and B2 (in the +1 open reading frame with respect to the replicase) [Ball (1992) J.Virol 66:2335-45; Johnson & Ball (1999) J.Virol. 73:7933-79421. Transcription of RNA3 seems to be controlled by an internal promoter which becomes active when a double stranded RNA+/RNA− intermediate is formed. It is an object of invention to permit modification and manipulation of the RNA1 molecule to exploit its ability to self-replicate, and to provide such modified RNA1 molecules.

DISCLOSURE OF THE INVENTION

[0007] The invention provides a modified nodavirus RNA1 molecule which includes a heterologous insertion downstream of the replicase ORFs of said RNA1. The insertion is preferably downstream of the replicase and B2 ORFs of said RNA 1.

[0008] The RNA1 Molecule

[0009] The invention is based around the RNA1 molecule of a nodavirus. RNA1 encodes an RNA replicase which specifically replicates the nodavirus genome, and sequences from several nodaviruses are available e.g. flock house virus (accession X77156), black beetle virus (accession X02396 & K02560), striped jack nervous necrosis virus (AB025018), pariacoto virus (AF171942), nodamura virus (AF174533), black beetle virus (NC—001411), and halibut nervous necrosis virus (AJ401165).

[0010] In relation to the present invention, a ‘nodavirus RNA1’ sequence includes a sequence found in nature, as well as fragments, variants and mutants thereof that encode replicases which retain the ability to specifically amplify their own genes.

[0011] It will be appreciated that the RNA1 molecule of the invention may be prepared by modification of a RNA1 obtained from a nodavirus by using standard molecular biology techniques, or may be assembled synthetically by enzymatic and/or chemical means.

[0012] ORFs Within the RNA1 Molecule

[0013] The available RNA1 sequences for nodaviruses (see above) indicate the location of the replicase (or ‘A’) and ‘B2’ ORFs. The insertion in the molecules of the present invention is downstream of the replicase ORF, and preferably also downstream of the B2 ORF.

[0014] In FHV, RNA1 is 3107 nucleotides long. The replicase ORF is encoded by nucleotides 40-3033, and the B2 ORF is encoded by nucleotides 2738-3055. According to the invention, therefore, the heterologous insertion for FHV is situated between nucleotides 3033 and 3107. and preferably between nucleotides 3055 and 3107.

[0015] Preferably, the heterologous insertion is more than 5 nucleotides downstream of the replicase ORF (i.e. downstream of nucleotide 3038 in FHV) e.g. more than 10, 15, 20, 25, 30, 35, 40, 45 or 50 nt downstream. More preferably, it is more than 5 nucleotides downstream of the B2 ORF (i.e. downstream of nucleotide 3060 in FHV) e.g. more than 10, 15, 20, 25, 30, 35, 40, 45 or 50 nt downstream of the ORFs. Similarly, the heterologous insertion is preferably more than 5 nucleotides upstream of 3′ end of RNA1 (i.e. upstream of nucleotide 3102 in FHV) e.g. more than 10, 15, 20, 25, 30, 35, 40, 45 or 50 nt upstream of the 3′ end. Retaining a length of native 3′ terminal sequence of a RNA1 helps ensure that the RNA1 retains its ability to self-replicate (Ball (1995) J.Virol 69:720-721.

[0016] If the heterologous insertion is within 3 nucleotides of the final codon in the replicase or B2 ORF (i.e. between nucleotides 3034-3036 or 3056-3058), it is preferred that the 5′ end of the insertion maintains a stop codon for the ORF (i.e. a stop codon at nucleotides 3034-3036 for the replicase or at 3056-3058 for B2).

[0017] The Heterologous Insertion

[0018] The modified RNA1 of the invention carries a heterologous insertion. This insertion preferably comprises one or more protein-coding regions, with their own start and stop codons.

[0019] As the insertion is downstream of one or more ORFs native to RNA1, to assist in its translation the 5′ portion of the heterologous insertion may also comprise a sequence that directs cap-independent translation. Typically, therefore, the heterologous insertion will comprise an internal ribosome entry site (IRES) functionally linked to a protein coding region. Any suitable IRES can be used, such as the hepatitis C virus or encephalomyocarditis virus IRES, or those from picornaviruses, foot-and-mouth disease, virus, echovirus 11′, classical swine fever virus, the c-myc proto-oncogene etc. [e.g. Martinez-Salas (1999) Curr. Opin. Biotechnol. 10:458-464].

[0020] The heterologous insertion does not destroy the ability of the RNA1 to self-replicate via its encoded replicase. Thus the modified RNA1 can deliver a gene of interest for expression, and is able to direct its own replication.

[0021] Packaging the Modified RNA1

[0022] In order to deliver the modified RNA1 to a cell, it is preferred to package it e.g. within a virus-like particle (VLP) or pseudovirion.

[0023] A nodavirus particle may be used, comprising protein expressed from RNA2 (optionally modified). If this approach is used, the RNA2 is preferably from the same nodavirus as the modified RNA1 which is packaged.

[0024] As nodaviruses are native to insect cells, however, where delivery to a mammalian cell is desired, it is preferred to use a different package. A preferred package is a papillomavirus VLP (e.g. Touze & Coursaget (1998) Nucl.Acid.Res. 26:1317-1323; Kawana et al. (1998) J.Virol. 72:10298-10300; Müller et al. (1997) Virol. 234:93-111; Zhao et al. (1998) Virol. 243:482-491: Unckell et al. (1997) J. Virol. 71:2934- 2939; WO97/46693; WO98/02548 etc.], which is able to bind to receptors on mammalian cells.

[0025] Using HPV VLPs offers several advantages. Firstly, HPV VLPs bind to a wide range of cell types, with the highest level of binding being observed with epithelial and mesenchymal cells, and only low levels of anti-VLP antibodies have been observed in humans [Le Cann et al. (1995) J Clin.Microbiol. 33:1380-1382]. Secondly, L1 proteins of different HPV type can be expressed so, as immunity induced by the use of VLPs is predominantly type specific, the presence of pre-existing anti-HPV antibodies may be eluded and multiple immunisations may be feasible. Thirdly, as nodavirus RNA1 is small, it can overcome a known limitation with HPV VLPs, namely the upper limit of 7-8 kbp on encapsidated nucleic acid. Nodavirus RNA thus offers significant advantages over other nucleic acid.

[0026] The papillomavirus is preferably a human papillomavirus, such as HPV-6. The VLP capsid may comprise proteins L1 and L2, or may be made solely of L1.

[0027] In order to facilitate packaging of the modified RNA 1, the proteins which form the package (e.g. the capsid proteins in a VLP) may be modified to include a sequence or structure which can interact specifically with the modified RNA1. The in vivo specificity of RNA/protein interactions is a further advantage of using nodavirus RNA, compared with problems encountered using DNA plasmids.

[0028] A particularly useful packaging system of this type relies on the tat/TAR interaction of immunodeficiency viruses (e.g. HIV, BIV, etc.). In this system, the package includes a motif from the tat protein (e.g. a minimal tat sequence, such as amino acids 48-59 of HIV tat: see also Derse et al. (1991) J.Virol 65:7012-15; Chen & Frankel (1994) Biochemistry 33: 2708-2715: Puglisi et al. (1995) Science 270: 1200-1202) that specifically interacts with a TAR motif (e.g. a minimal TAR sequence, such as the minimal 59mer, or the motifs described in WO92/02228) in the modified RNA1, Thus the modified RNA1 of the invention will additionally comprise a TAR motif. This may be upstream or downstream of a gene within the heterologous insertion, but is preferably downstream. As an alternative, the TAR motif may be placed upstream of the replicase ORF (i.e. before nucleotide 40 in FHV). In a papillomavirus-based packaging system, a tat sequence may be inserted in the L1 protein, preferably at or near the C-terminal. Although this region is not essential for capsid formation [Paintsil et al (1996) Virology 223:238-244], the sequence must not disrupt the ability of the VLP to assemble [Miller et al. (1997) Virology 234:93-111]. The tat and TAR interaction results in packaging of the modified RNA1 within the package. Use of the tat/TAR interaction from BIV is preferred, as this is a strong interaction that requires no cellular factors.

[0029] It will be appreciated that the use of the tat/TAR interaction for packaging nucleic acids within a VLP is not restricted to the modified RNA1 of the invention.

[0030] Further Aspects of the Invention

[0031] The modified RNA1 of the invention is typically positive-sense and single-stranded. The invention also provides: (i) single-stranded nucleic acid complementary to RNA1 of the invention. (ii) single-stranded nucleic acid complementary to ti): (iii) single-stranded nucleic acid comprising a sequence complementary to RNA1 of the invention: (iv) single-stranded nucleic acid comprising a sequence complementary to (i): and (v) double-stranded nucleic acid in which one of the strands is (i), (ii), (iii) or (iv). The term ‘nucleic acid’ encompasses RNA, DNA, as well as analogs such as PNA (peptide nucleic acid) and backbone-modified DNA or RNA (e.g. phosphorothioates etc.). It will be appreciated that nucleic acid can be transcribed by RNA polymerase in vitro or in vivo to produce the modified RNA1 of the invention.

[0032] The invention also provides a VLP containing nucleic acid of the invention (e.g. containing a modified RNA1). The VLP is preferably a papillomavirus VLP.

[0033] The invention also provides a papillomavirus L1 protein modified to include a motif from an immunodeficiency virus tat protein.

[0034] The invention also provides a process for producing a modified RNA1 molecule of the invention, comprising the steps of: (a) obtaining nucleic acid comprising or encoding a nodavirus RNA1 sequence: and (b) inserting a heterologous sequence downstream of the replicase ORF and B2 ORF within said RNA1 sequence. Where FHV is used, it should be noted that its replicase shows maximal activity below 28° C.

[0035] The invention also provides a process for producing a VLP of the invention, comprising the steps of: transfecting a cell with nucleic acid according to the invention: transfecting a cell with nucleic acid encoding a VLP capsid protein, optionally modified to include a motif specific for modified RNA1: and purifying VLPs from the cell. This process preferably takes place in yeast.

[0036] In order to introduce VLP-encoding and RNA1-encoding nucleic acid into the same yeast, it is possible to mate haploid strains (e.g. a haploid strain expressing a capsid protein can be mated with a haploid strain of opposite mating type expressing RNA1).

[0037] Nucleic acid (especially modified RNA1) and VLPs of the invention are also provided for use as medicaments. They are particularly useful for gene delivery eg. in gene therapy.

[0038] The invention also provides a process for delivering a nucleic acid sequence to a cell, comprising the step of introducing a VLP of the invention into said cell. This process man; be carried out in vitro (cells in culture) or in vivo.

[0039] Nodaviruses

[0040] Nodaviruses are divided into the alphanodaviruses and the betanodaviruses, and include Nodamura virus, black beetle virus, Boolarra virus, flock house virus (FHV). gypsy moth virus. Manawatu virus, atlantic halibut virus, tiger puffer nervoti necrosis virus. barfin flounder nervous necrosis virus, Japanese flounder nervous necrosis virus. Dicentrarchus labrax encephalitis virus, pariacoto virus, dragon nervous necrosis virus, halibut nervous necrosis virus, malabaricus nervous necrosis virus, redspotted grouper nervous necrosis virus, umbrina cirrosa nodavirus, and striped Jack nervous necrosis virus. RNA1 molecules from any of these viruses may be used according to the invention. As the best-studied nodaviruses at the molecular level, however, FHV and Nodamura virus are preferred. Further details of nodaviruses can be found in Garzon & Charpentier [pages 351-370 of Atlas of invertebrate viruses (eds. Adams & Bonami). CRC press (1992)] and in Hendry [pages 227-276 of Viruses of invertebrates (ed. Kurstak), Marcel Deker ( 1991)].

BRIEF DESCRIPTION OF DRAWINGS

[0041]
FIG. 1 shows CDNA, from FHV RNA1. The start codons for the replicase (40), B1 (2728) and B2 (2738) are boxed. The reading frames for (a) replicase/B1 and (b) B2 are both shown for the complete genome. The start of the RapApep fragment expressed in E.coli is underlined.

[0042]
FIG. 2 is a schematic representation of the pFHV[1.0] plasmild. The HDV ribozyme is hatched, and the transcription terminator is black.

[0043]
FIG. 3 shows restriction sites with the RNA1 region of pFHV[1.0]

[0044]
FIG. 4 shows the removal of a BssHII site in RNA1.

[0045]
FIG. 5 shows pFHV-BsshIIΔ.

[0046]
FIGS. 6 and 7 show the construction of pFHV-MutΔ and pSK+-ADH2-RNA1/SpeI, respectively.

[0047]
FIG. 8 shows pBS24.1-6L1.

[0048]
FIG. 9 shows pSL+-ADH2-RNA1-MutΔ.

[0049]
FIG. 10 shows the construction of pEGFP-1/TAR.

[0050]
FIG. 11 shows pFHV-EGFP-1/TAR, with the TAR shown as horizontal dashes and the GFP coding region dotted.

[0051]
FIG. 12 shows pFHV-EGFP-1-TARΔ.

[0052]
FIG. 13 shows the construction of pFHV-IRES-EGFP-1, with the IRES shown as vertical dashes.

[0053]
FIG. 14 shows the construction of pFHV-IRES-EGFP-1-b-TAR.

[0054]
FIG. 15 shows pBS-ADH2-RNA1-EGFP-1 TARΔ(19.6 kb).

[0055]
FIG. 16 shows pBS-ADH2-RNA1-MutΔ(18.1 kb).

[0056]
FIG. 17 shows pBS-ADH2-RNA1-MutΔ(−) (18.1 kb).

[0057]
FIG. 18 shows pBS-ADH2-RNA1-IRES-EGFP-1/b-TAR 20.2 kb).

[0058]
FIG. 19 shows a Northern blot of total RNA from yeasts transformed with the vectors of FIGS. 16-18. Lane 1 shows a negative control (total RNA from AB110): lane 2 is RepA(+) clone 5; lane 3 is RepA(+) clone 7; lane 4 is RepA(−) clone 3: lane 5 is RepA(−) clone 4: lane 6 is RepA(+)-GFP. Yeast rRNA 26S (2250bp) and 18S (1650bp) are also shown.

[0059]
FIG. 20 shows a Northern blot for EGFP-1 of total RNA from yeasts transformed with the vector of FIG. 18. Lane 1 shows RNA extracted from a 24 hour induced culture: lane 2 is after 48 hours: lane 3 is after 72 hours: lane 4 is a negative control (total RNA from AB110).

[0060]
FIG. 21 shows constructs for His-tagged E. coli expression of replicase and B2. and

[0061]
FIG. 22 shows SDS-PAGE of proteins expressed from these constructs. Lane 1 of 22(A) is a MW market plus fraction 14; lanes 2 to 9 are fractions 15 to 22: lane 10 is column flow-through. Lane 1 of 22(B) is the soluble fraction of cellular lysis: lane 2 is column flow-through: lanes 3 to 11 are fractions 21 to 29: lane 12 is a MW marker.

[0062]
FIG. 23 shows mass spectrometry of RepApep.

[0063]
FIG. 24 is a western blot of total protein extracts from yeasts transformed with the sector of FIG. 15. Lanes 1 to 5 are extracts immunoreacted with sera from five immunised mice at 1:5000 dilution: lane 6 is a negative control, comprising a mix of pre-immunisation sera.

[0064]
FIG. 25 is a western blot of total protein extracts from various yeast strains over a 72 hour period. Lane 1 is a negative control; lane 2 is a total protein extract from pBD-R1-G;TΔ clone 5 at 48 hours: lanes 3 to 5 are total protein extracts of RepA(+)-GFP strains, clone number 7, at 24, 28 and 72 hours: lanes 6 to 8 show the same for clone number 9.

[0065]
FIG. 26 is a western blot of total protein extracts from RepA(+)-GFP strains. Lane 1 is a negative control (total protein extract from AB110). Lane 2 shows protein from a 24 hour induced culture; lane 3 is after 48 hours; lane 4 is after 72 hours.

[0066]
FIG. 27 shows the construction of pBS-ADH2/GAP-6L1Δ4-Tat.

[0067]
FIG. 28 shows a western blot on CsCl-purified 6L1Δ4-tat protein under non-reducing conditions using anti-6L1 (1:5000).

[0068]
FIG. 29 illustrates the expression and packaging of modified RNA1 in VLPs in yeast.

[0069]
FIG. 30 shows a Northern blot of total RNA from yeast strains RepA (−) lane 2 grown 24 hr. lane 3 grown 48 hr), two different RepΔ(+) strains (lanes 4 and 5 grown 24 hr. lanes 6 and 7 grown 48 hr) and strain RepΔ(+)-IRES-EGFP/bTar (lane 8 grown 24 hr. lane 9 grown 48 hr). A negative control RNA (lane 1) and RNA molecular weight markers are also shown. Arrows indicate the position of the signals corresponding to RNA3.

[0070]
FIG. 31 shows a Northern blot of the RNAs from (1) lane 3 and (2) lane 6 of FIG. 30.

[0071]
FIG. 32 shows a Northern blot as described for FIG. 30. but using a different probe. Arrows indicate the position of the signals corresponding to RNA1 minus strand.

[0072]
FIG. 33 shows FIG. 4 shows a primer extension experiment on total RNA from 48 hr grown yeast strains RepA (−) (lane 2), RepA(+) (lane 3), RepA(+)-EGFP/hTar (lane 4) and RepA(+)-IRES-EGFP/bTar (lane 5). A negative control RNA (lane 1) and a positive control on in vitro transcribed RNA1 (lane 6) are also reported. Letters a, b, c and d in lane 4 identify bands (three of them also visible in lanes 3 and 5) which correspond to the nucleotides indicated on the RNA1 5′ sequence.

[0073]
FIG. 34 shows a S1 mapping experiment using the same total RNAs reported in FIG. 33. The results of S1 reactions using a negative control RNA (lane 1) and a positive control on in vitro transcribed RNA1 (lane 6) are also reported.

[0074]
FIG. 35 shows the results obtained by RT-PCR using nucleic acids extracted from diploid HPV-6VLP co-expressing RNA1-IRES-GFP/hTar and HPV-6 L1/hTAT. 35A shows yeast control RNA (lanes 1 and 3) and VLP-derived nucleic acids (lanes 2 and 4); 35B shows yeast RNA (lanes 1) and nucleic acids derived from VLPs either untreated (lane 3) or treated with Benzonase (lane 2); 35C shows yeast RNA (lanes 1) and nucleic acids derived from VLPs either untreated (lane 3) or treated with RNAse A (lane 2) in the presence of mouse RNA as internal control.

MODES FOR CARRYING OUT THE INVENTION

[0075] The RNA1 of FHV

[0076] The sequence of RNA1 from the FHV genome is given in GenBank as accession number X77156. A double-stranded cDNA (3107bp) corresponding to the RNA1 is shown in FIG. 1.

[0077] This cDNA is present in plasmid pFHV[1,0], described by Ball [J.Virol. (1995) 69:720-727], which was used in subsequent experiments. pFHV[1,0] also includes: an 18bp T7 promoter upstream from the RNA1 sequence, with a single G between the promoter and the RNA1 5′ end; a hepatitis delta virus ribozyme (89bp) positioned downstream of the RNA1 sequence, such that cleavage of the ribozyme generates the native 3′ end of RNA1; and a 136bp T7 transcriptional terminator which seems to assist ribozyme activity. This is shown in FIG. 2.

[0078] Modification of RNA1 for Cloning Purposes

[0079] To facilitate cloning, various changes were made to RNA1.

[0080] Removal of a BssHII Site

[0081] As shown in FIG. 3, the native RNA1 sequence includes two nearby BssHII restriction sites (nucleotides 1270 and 1278). One of these was removed by introducing a silent mutation that did not affect the replicase amino acid sequence.

[0082] The SphI/BsshII fragment of RNA1 was replaced by a PCR fragment generated using primers RI900-f and RI1272M-r (Table I). The first primer included the SphI site necessary for cloning, while the sequence of the reverse primer included a G to A mutation at position 1271, thereby eliminating one of the two BsshII restriction sites (FIG. 4) without altering the replicase ORF. The resulting plasmid was called pFHV-BsshIIΔ (FIG. 5).

[0083] Introduction of Additional Sites

[0084] In order to insert heterologous sequences into RNA1, two additional restriction sites were introduced: a SacII site at position 3062. a NotI site at position 3072 (downstream from the translational stop codons for the replicase and B2 ORFs). In addition, a Unique SalI site was inserted into pFHV[0,1] downstream from the T7 transcriptional terminator (nucleotide 3337). These sites were introduced by PCR amplification, as follows (FIG. 6):

[0085] primers ApaI-f and DRS-r (Table I) amplified a fragment of RNA1 between the ApaI site (position 2349) and position 3101. Introduction of the SacII and NotI restriction sites was achieved by introducing the two sequences in the DRS-r primer (FIG. 6a).

[0086] primers DRS-f (complementary to DRS-r) and RI-KpnI-r amplified a fragment spanning the region from nucleotide 3039 to the KpnI site (position 3368) (FIG. 6a).

[0087] The fusion of the two DNA fragments was obtained by denaturing and mixing equal amounts of the two amplification products which annealed by means of a 62 bp complementary region present in the two PCR fragments. Following elongation cycles with Taq polymerase in the only presence of dNTPs and amplification cycles in the presence of Taq and primers ApaI-f and RI-KpnI-r, a blunt ended DNA fusion fragment was obtained (FIG. 6b) which was then cloned in pCR2. I vector to check the DNA sequence. The ApaI-KpnI fragment was excised from pCR2.1 and used to replace the homologous fragment in pFHV[1,0] and pFHV-BsshIIΔ. obtaining a plasmid named pFHV-Mut (not shown) and a plasmid named pFHV-MutΔ (FIG. 6c), containing the entire RNA1 cDNA with only one BsshII site and the new SacII. NotI and SalI restriction sites.

[0088] Cloning of RNA1 into Yeast Vector

[0089] In order to express RNA1 (and modified versions of it) in yeast, the cDNA was cloned into a yeast vector, downstream of a yeast-specific promoter. This vector directs the transcription of RNA1 molecules that encode the replicase and (after ribozyme cleavage) have 5′ and 3′ ends compatible with self-replication. The overall approach is shown in FIG. 7.

[0090] Cloning of ADH2/GAP in pBluescript SK+

[0091] The promoter used for expression was the ADH2/GAP promoter. As a source of this promoter, yeast expression vector pBS24.1-6L1 (FIG. 8; see also WO00/09699) was used. The DNA fragment ADH2/GAP-6L1 was extracted from plasmid PBS24.1-6L1 by digestion with SacI and SalI, and was cloned in pBluescript-SK+(Stratagene) digested with the same enzymes. The resulting construct was named pSK+-ADH-6L1.

[0092] PCR Amplification of the Yeast ADH2/GAP promoter

[0093] A portion of the yeast promoter was amplified by PCR carried out on pSK+-ADH-6L1 using primer ADH/Sac (Table I), which anneals on the yeast promoter at the SacI site, and reverse primer P-R1rev, which anneals on the promoter at the fusion with the 6L1 gene, which additionally included 13 nucleotides from RNA1 (FIG. 7a).

[0094] PCR Amplification of RNA1/Sph

[0095] A portion of RNA1 cDNA from pFHV[1,0] was amplified using primer RNA1-f, which anneals at the 5′ end of RNA1 (+1 position). and reverse primer RNA1-r, which anneals at the unique SphI restriction site at nucleotide 1021 (FIG. 7b).

[0096] ADH2-RNA1/Sph Fusion

[0097] The fusion was obtained by denaturing and mixing equal amounts of the two amplification products of FIGS. 7a & b, thus favouring the annealing of the 13 bp complementary region present in both PCR fragments (FIG. 7c). Following elongation cycles with Taq only in the presence of dNTPs and amplification cycles in the presence of Taq and the primers ADH/SAC and RNA1-r, a fusion blunt ended DNA fragment was obtained.

[0098] Cloning of the ADH2-RNA1/Sph in pCR2.1

[0099] This blunt ended PCR product was cloned in pCR2.1 (Invitrogen), giving plasmid pCR-ADH2/RNA1 (FIG. 7d). Its DNA sequence was checked, and confirmed the expected promoter/RNA1 fusion sequence TAAATCTA GTTTCGAAA, although some mutations were present in other regions of the amplified product. The only clone which had a correct amplified sequence, however, had a mutation which eliminated the SphI site necessary for the further steps (FIG. 7d).

[0100] Cloning of the ADH2/RNA1 Fusion Product in pSK+-ADH2-6L1

[0101] To overcome this problem, the fusion product was excised from pCR2.1 as a SacI-Spe fragment and cloned into pSK+-ADH2-6L1 (FIG. 7e) digested with the same restriction enzymes, thus obtaining the final construct pSK+-ADH2-RNA1/SpeI (FIG. 7f).

[0102] Introducing Modified RNA1 into the Yeast Vector

[0103] The EagI-KpnI fragment from pFHV-BsshIIΔ (FIG. 5) was inserted in place of the corresponding fragment in pSK+-ADH2-RNA1/SpeI (FIG. 7f), generating a construct named pSK+-ADH2-RNA1-BsshIIΔ. The BsshII-KpnI fragment in this construct was replaced with the corresponding fragment from pFHV-MutΔ (FIG. 6c). generating a plasmid named pSK+-ADH2-RNA1-MutΔ (FIG. 9).

[0104] Insertion of Heterologous Sequences

[0105] A heterologous insertion was made inside the RNA1 sequence. A gene for expressing green fluorescent protein (GFP) [Chalfie et al. (1994) Science 263:802-806] was inserted downstream from the replicase and B2 open reading frames, together with a TAR sequence from HIV.

[0106] To make the heterologous insert, the starting point was plasmid PEGFP-1 (Clontech) digested with BsrgI and NotI. A dsDNA sequence with BsrgI (5′) and NotI (3′) cohesive ends and including the HIV-1 TAR sequence (nucleotides 454-520 HIV-1 genome, accession K03455) was obtained by annealing two complementary oligonucleotides, TAR-f and TAR-r, and this was inserted into the digested pEGFP-1 to form pEGFP-1/TAR (FIG. 10). The TAR oligo maintained the EGFP ORF and also included a translational stop codon just downstream from the BsrgI site, immediately followed by new BstEII and NheI restriction sites. A TAR sequence has also been successfully inserted upstream of the replicase ORF.

[0107] The SacII-NotI fragment from pEGFP-1/TAR, including the GFP coding sequence followed by the HIV-1 TAR sequence, was cloned in pFHV-Mut digested with the same restriction enzymes, obtaining a plasmid named pFHV-EGFP-1/TAR (FIG. 11). The SphI/SacII fragment of this plasmid was replaced with the corresponding fragment from pFHV-MutΔ (FIG. 6c). to generate pFHV-EGFP-1/TARΔ (FIG. 12) with only one BsshII restriction site.

[0108] Facilitating Expression of the Heterologous Insert

[0109] Polycistronic RNAs can be translated in mammalian cells by insertion of an internal ribosomal entry site (IRES) [Parks et al. (1986) J.Virol. 60:376-384] between the two genes of interest, thereby permitting cap-independent translation of the second (downstream) gene. To facilitate expression from the heterologous insertion, the IRES from encephalomyocarditis virus was thus inserted in front of the EGFP-1 gene. This IRES will typically provide translation of the EGFP protein only in mammalian cells, as IRES-mediated translation is selectively inhibited in yeast [Venkatesan et al. (1999) Nucl. Acids Res. 27:562-572.].

[0110] Plasmid pCMV-IRES/EGFP-1 (FIG. 13) was digested with EcoRI, treated with klenow enzyme to blunt-end the site and, following inactivation of the enzymes, was digested with NotI. generating a 5′ bluntended-3′ NotI fragment containing the EMCV FRES fused to the EGFP-1 gene. Similarly the plasmid pFHV-MutΔ (FIG. 6c) was digested with SacII, treated with klenow enzyme and then digested with NotI. The IRES-EGFP-1 fragment was cloned into the digested plasmid, giving pFHV-IRES-EGFP-1 (FIG. 13).

[0111] To assist in packaging RNA expressed from this construct, the TAR from BIV (nucleotides 5-30 BIV genome, accession M32690) was inserted downstream from the EGFP gene. The TAR sequence was constructed by annealing two complementary oligonucleotides (b-TAR-f and b-TAR-r) with NotI compatible cohesive ends. This also introduced two new restriction sites, BstEII (next to the 5′ NotI site) and XhoI (next to the 3′ NotI site). The double-stranded NotI fragment was cloned into NotI-digested pFHV-IRES-EGFP-1, to give pFHV-IRES-EGFP-1/b-TAR (FIG. 14). The correct orientation of the NotI fragment was confirmed.

[0112] Cloning into Yeast Expression Vectors

[0113] Four yeast expression plasmids were constructed with the following basic 5′ to 3′ layout: yeast specific promoter: modified RNA1; HDV ribozyme: T7 terminator, yeast polyA site.

[0114] Initially, the SacI-BsshII fragment pBS24.1-6L1 (FIG. 8) was replaced with the corresponding fragment from pSK+-ADH2-RNA1/BsshIIΔ, to give plasmid pBS-ADH2-RNA1/BsshIIΔ-6L1, in which part of the RNA1 cDNA was fused to the 3′ end of L1 gene sequence (not shown). This was manipulated as follows:

[0115] 1. The BsshII-SalI L1 fragment was replaced with the BsshII-SalI fragment from pFHV-EGFP-1/TARΔ, giving pBS-ADH2-RNA1-EGFP-1/TARΔ (FIG. 15).

[0116] 2. The BsshII-SalI L1 fragment was replaced with the BsshII-SalI fragment from pFHV-MutΔ. giving pBS-ADH2-RNA1-MutΔ (FIG. 16).

[0117] 3. Filling in the unique BsshII site (1273) in pBS-ADH2-RNA1-MutΔ. with interruption of the replicase ORF, generated pBS-ADH2-RNA1-MutΔ (−) (FIG. 17).

[0118] 4. Replacement of the BsshII-SalI fragment (including part of the modified RNA1 cDNA sequence) in pBS-ADH2-RNA1-MutΔ with the corresponding BsshII-SalI fragment (including IRES-EGFP-1/b-TAR) from pFHV-IRES-EGFP-1/b-TAR generated a plasmid named pBS-ADH2-RNA1-IRES-EGFP-1/b-TAR (FIG. 18).

[0119] Yeast Transformation

[0120] Plasmids pBS-ADH2-RNA1-EGFP-1 TARΔ. pBS-ADH2-RNA1-MutΔ. pBS-ADH2-RNA1-MutΔ(−) and pBS-ADH2-RNA1-IRES-EGFP-1/b-TAR (FIGS. 15-18), were introduced by transformation into a AB110 strain previously obtained in the laboratory (WO00/09699) and which expresses HPV-16L2 protein. The resulting strains were named pBS-RI-G/TΔ (clones 4 and 5 selected). pBS-RI-MutΔ (clones 1 and 3 selected). pBS-RI-MutΔ (-) (clones 5 and 7 selected) and pBS-RI-IRES-G/b-TAR (clones 5 and 9 selected), respectively.

[0121] Confirmation of Self-Replication

[0122] Confirmation of RNA Replication

[0123] Total RNAs were extracted from strains pBS-RI-MutΔ. pBS- RI-MutΔ(−) and pBS-RI-IRES-G/b-TAR and labelled as RepA(+). RepA(−) and RepA(+)-GFP respectively. These RNAs, together with a negative control RNA from the AB110 strain labelled as c-, were analysed by Northern blot using a non radioactive (BrighStar Psoralen-Biotin labelling kit, Ambion) DNA probe (600 bp) obtained by PCR carried out on pFHV[1,0] using primers ApaI-f and repA-Rev.

[0124]
FIG. 19 shows the results, indicating that high hybridization signals are only detected with RNAs extracted from yeast clones designed to express the replicase ORF, both when the RNA1 sequence has no insertions (lanes 2 and 3) and when the IRES-EGFP/bTAR sequence is introduced downstream from the replicase and B2 ORFs (lane 6). A much weaker signal is detected with RNAs from yeast clones transformed with a plasmid where the replicase ORF is interrupted (lanes 4 and 5), while no signal is detected with the negative control RNA.

[0125] These results confirm that the replicase can sustain self-replication of RNA1 even when a heterologous insert (foreign gene) is introduced in the wild type RNA1 sequence.

[0126] To confirm that the self-replicating modified RNA1 did contain the IRES-EGFP/b-TAR sequence, a Northern blot was performed using a non radioactive DNA probe of the entire EGFP region. The experiment confirmed expression of the recombinant RNA1 at 24. 48 and 72 hours (FIG. 20).

[0127] Confirmation of Protein Expression

[0128] As well as confirming self-replication at the RNA level, protein expression was also assayed using anti-replicase antibodies.

[0129] To obtain anti-replicase antibodies, the C-terminal portion of the replicase (DVWEK . . . SNNRK, referred to as RepApep) was expressed in E. coli. This protein includes the complete B1 protein so, advantageously, anti-RepApep antibodies should detect B1 expression as well as replicase. The same region was also expressed with a +1 frameshift to obtain bacterially expressed B2 protein. Detection of B1 or B2 proteins would confirm self-replication.

[0130] The RepApep portion of the replicase ORF was PCR amplified using primers RepA-dir and RepA-rev containing NheI (5′) and XhoI (3′) restriction sites. The PCR blunt-ended product was cloned into pCR-BluntII-TOPO (Invitrogen) and the sequence of the inserts from different clones was checked. The NheI(5′)-XhoI(3′) fragment was extracted from one of the correct clones by digestion with the two restriction enzymes and was cloned into pTrc-HisA (Invitrogen), generating a plasmid named pTrc-RepApep-His6 containing an ORF where the RepApep was in frame with a hexahistidine tag (FIG. 21a).

[0131] The entire B2 ORF was amplified by PCR using primers B2-dir and B2-rev containing the same NheI and XhoI sites. The PCR product was digested with the two enzymes and directly cloned into pTrc-HisA (Invitrogen), generating a plasmid named pTrc-B2-His6 containing an ORF where the B2 was in frame with a hexahistidine tag (FIG. 21 b).

[0132] Total soluble protein extracts from bacterial clones expressing either of the two recombinant proteins were subjected to IMAC using Ni-NTA resin (Hoffmann-La Roche) and fractions enriched for RepApep and B2 protein were collected (FIG. 22). The RepApep protein showed an unexpected migration profile in SDS-PAGE, so the purified protein was subjected to mass spectrometry (FIG. 23) which confirmed the expected 15-16 kDa MW.

[0133] Fractions containing the enriched protein were pooled, dialysed against PBS, and protein content was measured with a BCA test (Pierce). The proteins were used to immunise groups of 5 mice either 3 times (repApep, 50 μg/dose) or 4 times (protein B2, 25 μg/dose) at two week intervals. Vaccines were administered intraperitoneally (IP) using 200 μl of antigen and the same volume of MF59 [Ott et al. (1995) pages 277-296 of Vaccine design. The subunit and adjuvant approach (eds. Powell & Newman) Plenum Press]. Pre-immune sera were collected prior the immunization and serum samples were taken two weeks after each immunization.

[0134] Serum samples of the 5 mice immunised with RepApep were tested at 1:5000 dilution on total protein extracts prepared from the yeast strain pBS-RI-G/TΔ (clone 4), and detection of a ˜110 kDa protein was observed in all of them, in agreement with the existence of yeast expressed replicase (FIG. 24).

[0135] The same dilution of the serum used in lane 4 of FIG. 24 was used for a Western blot analysis on total protein extracts from different yeast strains grown under inducing conditions for variable time periods. The experiment confirmed that the RNA1 replicase is present at 24, 48 and 72 hours in strains expressing the modified RNA1 (FIG. 25).

[0136] The serum used in lane 4 of FIG. 24 was used for a Western blot analysis on total protein extract prepared at 24, 48 and 72 hours induction from the yeast strain carrying pBS-RI-G/TΔ (FIG. 26). A protein band (lanes 2 and 3) was detected between the 20 kDa and the 7 kDa MW markers which is not present in the negative control extract (lane 1) and in the extract prepared after a 72 hour induction (lane 4). This is in agreement with the synthesis of B1 protein in yeast, again confirming self-replication of the modified RNA1. The absence of B1 protein at 72 hours is in agreement with the B1 and B2 expression data previously obtained in mammalian cells [Johnson & Ball (1999) J.Virol. 73:7933-7942].

[0137] Confirmation of RNA3 Expression

[0138] Total RNAs were extracted from yeast strains expressing the plasmids pBS-ADH2-RNA1MutΔ(−). pBS-ADH2-RNA1MutΔ and pBS-ADH2-RNA1-IRES-EGFP-1/b-Tar, grown for 24 and 48 hours and labelled as RepA(−), RepA(+) and RepA(+) IGFP/b-Tar, respectively. These RNAs were analysed by Northern blot analysis using the non radioactive double stranded DNA probe as for FIG. 19. FIG. 30 shows that overexposing the film reveals a weak signal corresponding to a RNA species migrating as expected for RNA3 is detected both at 24 and 48 in the strain RepA(+) (lanes 4 to 7). A Northern blot was performed using the RNA from lane 6 over a longer time period on agarose gel and the filter was cut before hybridisation to include only the area were RNA3 was present (FIG. 31). This confirms the existence of RNA3. In the case of RNA derived from the strain RepA(+) IGFP/b-Tar (FIG. 30, lanes 8 and 9), the corresponding RNA3, which would migrate slower due to the insertion, could not be observed, presumably masked by the intense RNA1 hybridisation signal.

[0139] Confirmation of RNA Minus Strand

[0140] RNAs extracted from strains RepA(−), RepA(+) and RepA(+) IGFP/b-Tar grown at 24 and 48 hours were analysed by Northern blot using a strand specific non-radioactive probe(BrighStar Psoralen-Biotin labelling kit. Ambion to detect the RNA minus strand. The strand specific probe was an in vitro transcript obtained by using the plasmid pFHV-MutΔ linearised with SacII. FIG. 32 shows that a strong hybridisation signal corresponding to RNA of the expected size is detected only in clones expressing the replicase protein, both when the RNA1 sequence has no insertions (lanes 4 and 5) and when the IRES-EGFP/b-Tar sequence is introduced downstream from the B2 ORF (lanes 6 and 7). No signal is detected in control RNA (lane 1) and when the replicase ORF is interrupted (lanes 2 and 3).

[0141] Primer Extension and S1 Mapping Analysis of RNA1

[0142] Standard primer extension analysis was carried out on total RNAs from strains RepA(−). RepA(+) and RepA(+) IGFP/b-Tar using AMV reverse transcriptase and the T4 kinase labelled oligonucleotide R1-120

[0143] The extension products were run on a 8% denaturing polyacrylamide/urea gel with a control sequence carried out on plasmid pSK+-ADH2-RNA1/SpeI (FIG. 7f) by using the same primer. FIG. 33 shows that shorter extension products can be detected.

[0144] S1 mapping analysis was carried out using the same RNAs. The probe was a double-stranded PCR fragment obtained from plasmid pSV40-FHV [0,0] using oligos AZ15-for (nt 6285 in the plasmid) and R1-120. The PCR fragment was labelled with T4 kinase, digested with NcoI to remove the radioactive label from the strand which had the same polarity of the mRNA to be detected, and gel purified. Aliquots of the radioactive probe were hybridised to total RNAs and digestion with S1 nuclease was carried out under standard conditions. As a control, a T7 in vitro transcribed RNA1 from pFHV [1,0] was also hybridised to the probe and S1 digested. The S1 treated products were run on a 8% denaturing polyacrylamide/urea gel along with a DNA sequence obtained by using the primer R1-90rev and complementary to RNA1.

[0145] As shown in FIG. 34, bands corresponding to full-length RNA can be detected in samples expressing the replicase A without or with GFP and IREs-GFP insertions (lanes 3, 4 and 5).

[0146] Cloning and Sequencing of the 5′ End of RNA1

[0147] Total RNA derived from the strain RepA(+)-IGFP/b-Tar was used to clone the 5′ end of the RNA1 species. Cloning was carried out by using the Ambion FirstChoice™ RLM-RACE Kit, which allows selective cloning of capped mRNA molecules. The primers used were:

[0148] 1) Reverse transcription with R1-rev

[0149] 2) Outer PCR with the outer RNA adapter primer supplied with the kit and reverse primer AZ-9rev (nt 426 on RNA 1)

[0150] 3) Inner PCR with the inner RNA adapter primer supplied with the kit and R1-120.

[0151] Cloning of the final PCR- product was carried out into pZero blunt vector (Invitrogen). Sequencing of 10 clones revealed that the cloned sequence of all of them began AAAACAG (nucleotide 16 of RNA1 sequence). This corresponds to product c shown in FIG. 33. Band d in FIG. 33 may thus represent a degradation product of c, and band b may be a capped form.

[0152] Cloning and Sequencing of the 5′ End of RNA3

[0153] Using the same experimental procedures, the 5′ region of RNA3′ was cloned from the same yeast clones. The primers used were:

[0154] 1) Reverse transcription with AZ-5rev (nt 3101 of RNA1)

[0155] 2) Outer PCR with the outer RNA adapter primer supplied with the kit and reverse primer AZ-5rev

[0156] 3) Inner PCR with the inner RNA adapter primer supplied with the kit and R3-2809rev (nt 2830 on RNA1).

[0157] The final PCR products were digested with NcoI (nt 2801 on RNA1) and BamHI (within inner adapter primer) and cloned in pTRC-HisB (Invitrogen) digested with the same enzymes.

[0158] Sequencing of 10 clones from RT-PCR carried out with RNA from the RepA(+) strain revealed that same sequence in each case, beginning at nucleotide 2721 of RNA1 (GTTACCAA . . . ). This corresponds to the RNA3 start site already reported in the literature.

[0159] Sequencing of 18 clones from RT-PCR carried out with RNA from the RepA(+)-IGFP/b-Tar strain revealed that none corresponds to the RNA3 transcriptional start site, but all of them initiate with upstream sequences belonging to RNA1.

[0160] Packaging in HPV VLPs

[0161] To promote packaging of the modified RNA1 carrying a TAR insert, the L1 coat protein of HPV-6 was modified to include a complementary tat motif at its C-terminal.

[0162] The construction of the yeast expression vector to achieve this included several steps (FIG. 27). Firstly, plasmid pBS24.1-6L1 (FIG. 8) was digested with BsshI and SalI, thus eliminating the last 4 amino acids of the L1 protein. This plasmid expresses HPV-6 L1 capsid protein in yeast. A short BsshII-SalI dsDNA, obtained by annealing complementary oligonucleotides 6L1Δ4 and 6L1Δ4inv, was inserted, thereby reconstituting the L1 ORF, giving it a translational stop codon and a new NotI restriction site. The resulting yeast expression plasmid was named pBS24.1-6L1Δ4. PCR amplification of the HIV-1 TAR-binding domain of tat (amino acids 36 to 72) was carried out on a plasmid containing the cDNA of HIV-1 strain IIIB by using the primers Tat-dir and Tat-rev, which also included a BsshII site at the 5′ end and NotI and SalI sites at the 3′ end. The resulting blunt-ended fragment was cloned into pCR2.1 plasmid and sequenced. The BsshII-SalI fragment was extracted from pCR2.1 and ligated with pBS24.1-6L1Δ4 digested with the same restriction enzymes. The resulting plasmid was named pBS24.1-6L1Δ4-Tat.

[0163] pBS24.1-6L1Δ4-Tat was introduced by transformation into a JSC310 strain previously obtained in the laboratory (WO00/09699) and which expresses HPV-6L2 protein. Different clones from the transformation experiments were analyzed for 6L1Δ4-Tat expression and clone 2 was selected for further experiments.

[0164] Clone 2 was grown under inducing conditions and cesium chloride (CsCl) gradient purification of VLPs was carried out as described in WO00/09699. The CsCl fraction corresponding to a density of 1.28-1.29 g/cm3 was run under non reducing conditions on SDS-PAGE and analysed by Western blot using an anti-6L1 antibody (FIG. 28). The detection of a band which migrated slower than the 115 kDa Mw protein marker indicates that disulphide bonds among different L1/Tat monomers are formed, which is a known requirement for efficient self-assembly of HPV VLPs [Sapp et al. (1998) J. Virol. 72:6186-89].

[0165] The expression of replicase, self-replication of modified RNA1 carrying a TAR insert, expression of L1-Tat, and packaging of modified RNA1 is illustrated in FIG. 29. All steps take place in the same yeast cell, resulting in infectious VLPs for delivering a gene of interest to mammalian cells.

[0166] Yeast strain JSC310 containing the plasmid pBS24.1-6L1Δ4-Tat. clone 2, was mated with strain AB110 containing the plasmid pBS-ADH2-RNA1-EGFP-1/TARΔ to obtain a diploid clone co-expressing the recombinant RNA1 derivative (containing the GFP gene and the human TAR sequence) and the recombinant HPV6 L1/hTAT protein. Different clones from the mating experiment were tested for L1 and replicase A protein expression and for RNA1-EGFP-1/TAR transcript levels. One diploid clone was chosen and grown under inducing conditions. CsCl gradient purification of VLPs was carried out as described in WO/00/09699.

[0167] The CsCl fractions corresponding to a density of 1.28-1.29 g/cm3 were pooled and dialysed against PBS. The dialyzed VLPs were used for different experiments carried out following different procedures and reported in FIG. 35.

[0168] In FIG. 35A, the VLPs were treated with phenol, phenol-chloroform, chloroform and ethanol precipitated to obtain a material devoid of proteins. Aliquots of the final nucleic acids pellet suspended in water were subjected to RT-PCR without any additional treatment by using the OneStep RT-PCR kit (Qiagen) following the kit instructions. The primer used were R1-235 for (nt 235 on RNA1) and R1-1272M-r, resulting in the expected 1037 nt band (lanes 2 and 4, which are the same sample repeated in two different RT-PCR analysis). Unrelated yeast RNA was included as a control.

[0169] In FIG. 35B, a 100 μl aliquot of the pooled VLPs was subjected to a 24 hr treatment at 37° C. with 25 units of Benzonase (Merck) before the fenol/chlorophorm extraction. The final nucleic acids pellet was subjected to RT-PCR using the same primers as before. The 1037 nt band is observed, both when the VLPs had been previously treated with Benzonase (lane 2) and without any Benzonase treatment.

[0170] In FIG. 35C, a 100 μl aliquot of the pooled VLPs was subjected to a 1 hr treatment at 37° C. with 1 μg RNAse A before the phenol/chloroform extraction. Total mouse RNA was included in the sample as internal control of the RNAse treatment. Following phenol/chloroform extraction, the final nucleic acids pellet was subjected to RT-PCR by using two different sets of primers, the two RNA1 primers used for FIG. 35A and two primers amplifying an ˜600 nt long band corresponding to mouse GAPDH mRNA. While the RNA1 band (empty arrow) is visible both when the VLPs had been previously treated with RNAse (lane 2) and without any RNAse treatment (lane 3), GAPDH mRNA can be amplified (black arrow) from the untreated sample (lane 3) and it is barely visible after RNAse treatment.

[0171] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

1TABLE IprimersNAMESEQUENCE (5′-3′)ADH-SACCCCAATTCGTCTTCAGAGCTCATTGTTTG (Sacl)P-R1-revTTGTTTCGAAAACTAGATTTACAGAATTACAATCAATACCTACCGTC(underlined sequence corresponds to RNA1)RNA1-tGTTTTCGAAACAAATAAAACAGAAAAGCGAAACRNA1-rTGAGCATGCTCCCCTTCTGGACC (Sphl)TAR-fGTACAAGTAAGGTTACCGCTAGCGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAGC (BstEll and Nhel)TAR-rGGCCGCTAAGCAGTGGGTTCCCTAGTTAGCCAGAGAGCTCCCAGGCTCAGATCTGGTCTAACCAGAGAGACCCGCTAGCGGTAACCTTACTT (Bstell and Nhel)DRS-fCCCACCCGCAAAACTGTAGGTGCCGCGGAGGAGCGGCCGCACCCGTTCTAGCCCGAAAGGGC (Sacll and Notl)DRS-rGCCCTTTCGGGCTAGAACGGGTGCGGCCGCTCCTCCGCGGCACCTACAGTTTTGCGGGTGGG (Sacll and Notl)Apal-fCCGTGTCGAAGGCTATCTCTGTACRl-Kpnl-rACGGAATTAATTCGAGCTCGGTACCCGTCGACCTCGATCCGGATATAGTTCCTCCTT (Kpnl and Sall)Rl900-fGGATTGATACCGAACTACACGTGCGRl1272M-rTATTGGCGCGCACTCACTTCTGGTA (Bsshll and point mutation)RepA-DirACGTTAGCTAGCGATGTCTGGGAGAAAATTACACATGACAGC (Nhel)RepA-RevTAACGTCTCGAGTCACTTCCGGTTGTTGGAAGGCTG (Xhol)b-TAR-fGGCCGCACTCGAGGCTCGTGTAGCTCATTAGCTCCGAGCGGTCACCGC(Xhol and Bstell)b-TAR-rGGCCGCGGTGACCGCTCGGAGCTAATGAGCTACACGAGCCTCGAGTGC(Xhol and Bstell)B2-DirACGTTAGCTAGCCCAAGCAAACTCGCGCTAATCCAG (Nhel)B2-RevTAACGTCTCGAGCTACAGTTTTGCGGGTGGGGG (Xhol)6L1Δ4CGCGCCTAAGCGGCCGCG (Notl)6L1Δ4-invGGATTCGCCGGCGCAGCT (Notl)Tat-dirACTGCGCGCCGTTTGTTTCATGACAAAAGCC (Bsshll)Tat-revAGTGCGGCCGCTTACTGCTTTGATAGAGAAGCTTG (Notl)R1-120GCACCCAGATACGGTTGCAATCCCGACAZ15-forGCATGCATCTCAATTAGTCAGCAACCAGGATCR1-90revCAATTCAGTTCGGGTGATCTGGTGTTCTCCAZ-9revTTCGCAGTCCAGTGCTTGAGTTTGGAZ-5revGCCCTTTCGGGCTAGAACGGGR3-2809revGGTGCGTCTTGGTAGCTCATTCCCATGR1-234forCGAAGACCCCGATAGAGACACGTTTC

[0172]

Modified nodavirus rna for gene delivery

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