RNA CONSTRUCT

Abstract

The invention relates to RNA constructs, and particularly, although not exclusively, to mRNA constructs and saRNA replicons and to nucleic acids and expression vectors encoding such RNA constructs. The invention extends to the use of such RNA constructs in therapy, for example in treating diseases and/or in vaccine delivery. The invention extends to pharmaceutical compositions comprising such RNA constructs, and methods and uses thereof.

Claims

1. An RNA construct encoding: (i) at least one therapeutic biomolecule; and (ii) at least one viral innate inhibitor protein (IIP).

2. The RNA construct according to claim 1, wherein the construct is a mRNA molecule.

3. The RNA construct according to claim 1, wherein the construct is a saRNA molecule.

4. The RNA construct according to claim 1, wherein the construct comprises or is derived from a positive stranded RNA virus selected from the group of genus consisting of: alphavirus; picornavirus; flavivirus; rubivirus; pestivirus; hepacivirus; calicivirus and coronavirus, preferably an alphavirus, optionally VEEV.

5. The RNA construct according to claim 1, wherein the at least one innate inhibitor protein (IIP) is: (i) HPV16 E6, or an orthologue thereof, (ii) HSV ICP34.5, or an orthologue thereof, (iii) HCV E2, or an orthologue thereof, (iv) HCV NS5a, or an orthologue thereof, (i) VACV E3L, or an orthologue thereof, (ii) VACV K3L, or an orthologue thereof, (iii) MERS ORF8B, or an orthologue thereof, (iv) KSHV ORF52, or an orthologue thereof, and/or (v) Ebola VP35, or an orthologue thereof.

6. The RNA construct according to claim 1, wherein the at least one innate inhibitor protein (IIP) is Vaccinia C6, or an orthologue thereof.

7. The RNA construct according to claim 1, wherein the at least one innate inhibitor protein (IIP) is EV71-2Apro, or an orthologue thereof.

8. The RNA construct according to claim 1, wherein the at least one innate inhibitor protein (IIP) is BVDV nPro, or an orthologue thereof.

9. The RNA construct according to claim 1, wherein the at least one innate inhibitor protein (IIP) is HSV Us1, or an orthologue thereof.

10. The RNA construct according to claim 1, wherein the at least one innate inhibitor protein (IIP) is Simian Virus 5 (PIV5 Non-structural protein V), or an orthologue thereof.

11. The RNA construct according to claim 1, wherein the at least one innate inhibitor protein (IIP) is (i) an ORF3b*57 variant of the wild type of SARS-CoV-2 ORF3b, or an orthologue thereof, or (ii) an ORF3b*57 Ecuador variant of the wild type of SARS-CoV-2 ORF3b, or an orthologue thereof.

12. The RNA construct according to claim 1, wherein the at least one innate inhibitor protein (IIP) is: (i) an ORF3b*57 Pangolin variant of the wild type of SARS-CoV-2 ORF3b, or an orthologue thereof, or (ii) an ORF3b*79 variant of the wild type of SARS-CoV-2 ORF3b, or an orthologue thereof.

13. The RNA construct according to claim 1, wherein the at least one innate inhibitor protein (IIP) is an ORF3b*79 Pangolin variant of the wild type of SARS-CoV-2 ORF3b, or an orthologue thereof.

14. The RNA construct according to claim 1, wherein the therapeutic biomolecule comprises a therapeutic protein, preferably the protein or peptide is an antigen, and more preferably a viral antigen.

15. A nucleic acid sequence encoding the RNA construct according to claim 1.

16-24. (canceled)

Description

[1109] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:—

[1110] FIG. 1 shows a schematic of various embodiments (denoted 1-7) of the RNA construct of the invention (e.g. a saRNA replicon on the left, or a mRNA construct). The saRNA replicon (1-4) is based on an alpha virus backbone. This so-called ‘Stealthicon’ vector includes a 5′ UTR followed by nucleic acid encoding Non-structural Proteins (NSP1-4) from an alphavirus, such as VEEV, a sub-genomic promoter (SGP), a GOI (Gene of Interest), such as a viral, bacterial, fungal or mammalian protein or antigen, a viral innate inhibitor protein (IIP), a 3′ UTR and a 3′ poly A tail. The mRNA construct (5-7) includes a 5′ UTR, a GOI (Gene of Interest), such as a viral, bacterial, fungal or mammalian protein or antigen, a viral innate inhibitor protein (IIP), a 3′ UTR and a 3′ poly A tail. The order of the IIP and GOI can be varied for both saRNA and mRNA as shown in the different illustrated embodiments;

[1111] FIG. 2 illustrates the immune response in a subject vaccinated (an initial primer jab followed by a subsequent boost jab) with a messenger RNA (mRNA) vaccine;

[1112] FIG. 3 illustrates the immune response in a subject vaccinated (an initial primer jab followed by a boost jab) with a standard self-amplifying (saRNA) vaccine;

[1113] FIG. 4 illustrates the immune response in a subject vaccinated (an initial primer jab followed by a boost jab) with one embodiment of the RNA construct of the invention, for example the Stealthicon vector shown in FIG. 1;

[1114] FIG. 5 illustrates the antigen expression level in a subject vaccinated (an initial primer jab followed by a boost jab) with one embodiment of the RNA construct of the invention, i.e. the Stealthicon vector shown in FIG. 1;

[1115] FIG. 6 shows f-Luc expression in HeLa cells following transfection with VEEV replicons containing selected IIP in F-T2A configuration relative to expression in HEK293T/17 cells. HEK293T/17 and HeLa cells were transfected with saRNA (100 ng) containing luciferase as a reporter protein and assessed for protein expression after 24 hr;

[1116] FIG. 7 shows f-Luc expression in HeLa cells following transfection with VEEV replicons containing selected IIP in F-T2A configuration relative to expression in HEK293T/17 cells. HEK293T/17 and HeLa cells were transfected with saRNA (100 ng) containing luciferase as a reporter protein and assessed for protein expression after 24 hr;

[1117] FIG. 8 shows f-Luc expression in HeLa cells following transfection with selected VEEV replicons containing IP in F-T2A configuration relative to expression in HEK293T/17 cells. HEK293T/17 and HeLa cells were transfected with saRNA (100 ng) containing luciferase as a reporter protein and assessed for protein expression after 24 hr;

[1118] FIG. 9 shows f-Luc expression in HeLa cells following transfection with VEEV replicons containing IIP in a double sub-genomic promoter (DSGP) configuration relative to expression in HEK293T/17 cells. HEK293T/17 and HeLa cells were transfected with saRNA (100 ng) containing luciferase as a reporter protein and assessed for protein expression after 24 hr;

[1119] FIG. 10 shows the increase in VEGF-A expression in HeLa cells following transfection with saRNA containing the IIP HSV ICP34.5 in a F-T2A configuration compared to saRNA without IIP and relative to expression in HEK293T/17 cells. HEK293T/17 and HeLa cells were transfected with saRNA (100 ng) containing VEGF-A as a secreted reporter protein and assessed for protein expression in the culture media after 48 hr by ELISA;

[1120] FIG. 11 compares f-Luc expression in HeLa cells following transfection with saRNA containing f-Luc as the GOI (construct 1) and with the IIP MERS ORF4a in F-T2A configuration (constructs 2a and 2b), IRES configuration (construct 3b) and DSGP configurations (constructs 4a and 4b) shown in FIG. 1. HeLa cells were transfected with RNA (100 ng) containing luciferase as a reporter protein and assessed for protein expression after 24 hr, and

[1121] FIG. 12 shows n-Luc expression f-Luc expression in HeLa cells following transfection with mRNA containing IIP in F-T2A configuration relative to expression in HEK293T/17 cells. HEK293T/17 and HeLa cells were transfected with RNA (100 ng) containing luciferase as a reporter protein and assessed for protein expression after 24 hr.

EXAMPLES

[1122] The inventors hypothesized that cis encoding proteins from non-viral sources, such as humans and other mammals, that are known to inhibit the innate recognition of saRNA or mRNA, would dampen the innate sensing in the host cell, and enhance both the protein expression and immunogenicity of RNA vaccines. Thus, the inventors designed and tested a range of RNA constructs (saRNA and mRNA) containing viral innate immune inhibitor proteins (IIPs) and a gene of interest (GOI), and then characterized whether these constructs enhance both intracellular and secreted protein expression (encoded by the gene of interest).

[1123] Materials and Methods

[1124] Cloning of saRNA Replicon Plasmids Containing IIPs

[1125] SaRNA encoding firefly luciferase (fLuc) and replicase derived from the Venezuelan equine encephalitis virus (VEEV) were cloned into a plasmid vector, as previously described (1). Replicon plasmids containing reporter gene followed by IIP (firefly luciferase f-Luc; Uniprot: Q27758) were generated with Furin-T2A or double sub-genomic promoters. Double sub-genomic (DSG) constructs are designed to initiate transcription of separate RNA molecules encoding the fLuc and IIP and were produced by cloning into a base double sub-genomic vector using Gibson assembly and a nucleotide base overlap. Briefly, plasmid DNA was restriction digested for 2 h at 37° C. and used in a NEB Builder HiFi DNA assembly reaction with gene fragment strings synthesised by GeneArt (Regensburg, Germany) or Integrated DNA Technologies (IDT) (Iowa, USA) according to manufacturer's protocol (New England BioLabs, UK). Furin-T2A (F-T2A) constructs designed to generate a single RNA transcript from the VEEV primary sub-genomic promoter with no stop codon for fLuc translation were produced by cloning IIP with F-T2A sequence into restriction enzyme sites of the corresponding DSG plasmid vector. After incubation at 50° C. for 30 min, 2 uL of the NEB Builder HiFi assembly reaction was used to transform NEB 10-alpha bacteria and the transformants plated onto LB agar plates and incubated overnight. Colonies were selected, expanded overnight and recombinant plasmid purified using Qiagen plasmid miniprep kits (Qiagen, UK). Purified clonal plasmids were analysed using a diagnostic restriction enzyme digest and those which exhibited the correct digestion pattern were fully sequenced to confirm nucleotide identity (Eurofins, Germany).

[1126] Plasmids that had IIP followed by the reporter gene in the F-T2A or DSG form as well as constructs that utilized the ECMV IRES internal ribosomal entry sequence (which initiates protein translation from the IRES element at a site internal to a messenger RNA transcript; Bochkov and Palmenburg, Biotechniques 41(3):283-4, 2006) were generated by VectorBuilder (VectorBuilder, Germany) using standard molecular techniques.

[1127] The incorporated interferon inhibiting proteins (IIP) can be found with the following database identifiers/accession numbers: EBOV VP35 (Ebola virus VP35; NP_066244.1; Accession Number—NCBI Reference Sequence: NC_002549.1; UniProtKB—Q05127 (VP35_EBOZM); EV71-2Apro (Enterovirus 71 2A pro; Accession Numbers—GenBank KC875402.1 and AG028195.1; UniProtKB—Q66478 (POLG_HE71B); HCV E2 (hepatitis C virus E2; NS1 Protein from polyprotein AAA45534.1; Accession Number—Genomic RNA Translation AAA45534.1; UniProtKB—P27958 (384-746) (POLG_HCV77)); HCV NS5a (hepatitis C virus NS5a; isolate H—Genomic RNA translation: AAA45534.1; UniProtKB—P27958 (POLG_HCV77)); HPV E6 (Human papillomavirus E6; NP_041325.1; Accession Number—NCBI Reference Sequence: NC_001526.4; UniProtKB—P03126 (VE6_HPV16)); HSV ICP34.5 (Herpes simplex virus ICP34.5; YP_009137073.1; Accession Number—NCBI Reference Sequence: NC_001806.2; UniProtKB—P36313 (ICP34_HHV11)); KSHV ORF52 (Kaposi's sarcoma-associated herpesvirus ORF52; Accession Number—Genomic DNA Translation: ACY00451.1; UniProtKB—F5HBL8 (F5HBL8_HHV8)); MERS ORF8b (Middle East Respiratory Syndrome virus ORF8b; Accession Number—Genomic RNA Translation ANF29170.1; UniProtKB—A0A1W5LGP6 (A0A1W5LGP6_MERS)); VACV C6 Vaccinia C6 (vaccinia virus C6; Accession Number—Genomic DNA Translation: AAA69602.1; UniProtKB—P17362 (C6_VACCW)); VACV K3L (vaccinia virus K3L; Accession Number—Genomic DNA Translation: AAA48009.1; UniProtKB—P20639 (K3 VACCC)); PIV 5 V (Parainfluenza virus 5 V; ENA protein ID: AAA47882.1; GenBank Accession Number J03142.1; UniProtKB—P11207; V_PIV5)); SARS ORF3b*57 variant (Severe acute respiratory syndrome coronavirus 2 (2019-nCoV) (SARS-CoV-2) ORF3b protein-mutated stop codon at AA 23; Genomic RNA Translation QTT40181.1; UniProtKB—PoDTF1 (ORF3B_SARS2)); SARS ORF3b*79 variant (Severe acute respiratory syndrome coronavirus 2 (2019-nCoV) (SARS-CoV-2) ORF3b protein-mutated stop codons at AA 23 and AA 57; Genomic RNA Translation QTT40181.1; UniProtKB—PoDTF1 (ORF3B_SARS2)); SARS ORF3b*57 Ecuador variant (Severe acute respiratory syndrome coronavirus 2 (2019-nCoV) (SARS-CoV-2) ORF3b protein-mutated stop codon at AA 23; Ecuador mutation at AA 24 (L24M); Genomic RNA Translation QTT40181.1; UniProtKB—PoDTF1 (ORF3B_SARS2)); Pangolin ORF3b *57 (Pangolin Coronavirus—Genomic RNA Translation: QIG55946.1; ORF3b protein-mutated stop codon at AA 23; UniProtKB—A0A6M3G7Q4 (A0A6M3G7Q4_9BETC)); Pangolin ORF3b *79 (Pangolin Coronavirus—Genomic RNA Translation: QIG55946.1; ORF3b protein-mutated stop codons at AA 23 and AA 57; UniProtKB—A0A6M3G7Q4 (A0A6M3G7Q4_9BETC)); MERS ORF4a (Middle East respiratory syndrome-related coronavirus (MERS-CoV) NS4A protein—Genomic RNA Translation: AGV08457.1; UniProtKB: T2BBG6 (T2BBG6_MERS)); BVDV nPro (Bovine viral diarrhea virus (BVDV) (Mucosal disease virus) N-terminal protease (aa 1-168)—Genomic RNA Translation: AAA42854.1; UniProtKB: P19711 (POLG_BVDVN)); HSV US1 (Human herpesvirus 2 (strain HG52) (HHV-2) (Human herpes simplex virus 2) E3 ubiquitin ligase ICP22 US1—Genomic DNA Translation: CAB06708.1; UniProtKB: P89474 (ICP22_HHV2H)); MERS CoV M (Middle East respiratory syndrome-related coronavirus (MERS-CoV) Membrane protein (M)-Genomic RNA Translation: AGV08396.1; UniProtKB: T2BB40 (T2BB40_MERS)). [1128] (1) A. K. Blakney, P. F. McKay, R. J. Shattock, Structural Components for Amplification of Positive and Negative Strand VEEV Splitzicons. Frontiers in Molecular Biosciences 5, 71 (2018).

[1129] Cloning of Plasmids Containing IIPs for RNA Transcription

[1130] IIP were inserted into a base plasmid using restriction digestion followed by Gibson assembly with a nucleotide base overlap region and included a F-T2A sequence to allow for a single transcript expression of the n-Luc followed by an IIP. The base plasmid consisted of an mRNA encoding a luminous shrimp nanoluciferase (n-Luc) expression cassette with a T7 promoter, an alpha-globin 5′ UTR and a beta-globin 3′ UTR. Briefly, the n-Luc plasmid construct was linearized with restriction enzymes for 2 h at 37° C. and then used in a NEB Builder HiFi DNA assembly reaction essentially as described in the NEB Builder HiFi assembly protocol (New England BioLabs, UK). After incubation at 50° C. for 30 min, 2 uL of the assembly reaction was used to transform NEB 10-alpha bacteria as per protocol and the transformants plated onto LB agar plates and incubated overnight for colony growth. Colonies were selected and expanded overnight, the recombinant plasmid purified from the bacteria using Qiagen plasmid miniprep kit (Qiagen, UK) and purified clonal plasmids were analysed initially using a diagnostic restriction enzyme digest and those which exhibited the correct digestion pattern were fully sequenced to confirm nucleotide identity (Eurofins, Germany).

[1131] Plasmids that had IIP followed by the n-Luc in the F-T2A or DSG form as well as constructs that utilized the ECMV IRES internal ribosomal entry sequence (which initiates protein translation from the IRES element at a site internal to a messenger RNA transcript; Bochkov and Palmenburg, Biotechniques 41(3):283-4, 2006) for both the saRNA replicons and the plasmids used for mRNA transcription were generated by VectorBuilder (VectorBuilder, Germany) using standard molecular techniques.

[1132] In Vitro Transcription of saRNA

[1133] Plasmid DNA (pDNA) was transformed into Escherichia coli (E. coli) (New England BioLabs, UK) and cultured in 100 mL of Luria Broth (LB) with 100 μg/mL of carbenicillin (Sigma Aldrich, UK). pDNA was isolated using a Plasmid Plus MaxiPrep kit (QIAGEN, UK) and the final concentration measured on a NanoDrop One (ThermoFisher, UK). saRNA was transcribed from the pDNA template using CleanCap Reagent AG (Tebu-bio, France) to produce an RNA transcript with a naturally occurring Cap 1 structure. Briefly, the pDNA template was linearized for 3 h at 37° C., then 1 μg of the linearized pDNA template used in the standard CleanCap Transcription protocol (Tebu-bio, France) according to the manufacturer's protocol. Transcripts were purified by LiCl precipitation at −20° C. for at least 30 min, centrifuged at 20,000 g for 20 min at 4° C. to pellet the RNA, rinsed once with 70% EtOH, centrifuged again at 20,000 g for 5 min at 4° C. and resuspended in UltraPure H.sub.2O (Ambion, UK) and stored at −80° C. until further use.

[1134] In Vitro Transcription of RNA

[1135] pDNA was transformed into E. coli (New England BioLabs, UK), cultured in 100 mL of Luria Broth (LB) with 100 μg/mL of carbenicillin (Sigma Aldrich, UK). Plasmid was purified using a Plasmid Plus MaxiPrep kit (QIAGEN, UK) and the concentration and purity measured on a NanoDrop One (ThermoFisher, UK). RNA was transcribed from the plasmid DNA template using the MEGAscript™ T7 Transcription protocol (ThermoFisher, UK) followed by a ScriptCap™ m7G Capping System post translation (Cambio, UK). Briefly, pDNA was linearized for 3 h at 37° C., and 1 μg of the linearized pDNA template used in the standard reaction protocol. After the MEGAscript™ T7 Transcription the transcripts were purified by LiCl precipitation at −20° C. for at least 30 min, then centrifuged at 20,000 g for 20 min at 4° C. to pellet the RNA, rinsed once with 70% EtOH, centrifuged again at 20,000 g for 5 min at 4° C. and resuspended in UltraPure H.sub.2O (Ambion, UK). The transcripts were then post-transcriptionally capped using the ScriptCap™ m7G Capping System standard protocol and finally LiCl precipitated as described above. Purified and Cap 1 capped RNA was then resuspended in UltraPure H.sub.2O (Ambion, UK) and stored at −80° C. until further use.

[1136] Measurement of IIP Activity

[1137] In order to establish the ability of saRNA containing viral IIP to increase saRNA f-luc expression relative to saRNA without IIP; the ability of mRNA containing IIP to increase mRNA n-luc expression relative to mRNA without IIP and the ability of mRNA containing IIP to increase f-luc expression from saRNA without IIP, constructs were tested in interferon competent HeLa cells and expression compared to that obtained in HEK293T/17 cells which do not have a functional antiviral signalling pathway. Both cell lines were cultured in high glucose Dulbecco's Modified Eagle's Medium (cDMEM) (Sigma-Aldrich, Merck, UK) containing 10% (v/v) fetal bovine serum (FBS), 5 mg/mL L-glutamine (Gibco, ThermoFisher, UK) and 5 mg/mL penicillin/streptomycin (Sigma-Aldrich, Merck, UK).

[1138] Assessment of IIP on saRNA Firefly Luciferase (f-Luc) Expression

[1139] HEK293T/17 cells were plated at a density of 25000 cells per well and HeLa cells at a density of 10000 cells per well into flat clear bottom 96-well plates (Corning Costar) and incubated for 24 hr. 10 uL of OptiMEM (ThermoFisher, UK) containing 0.15 μL lipofectamine MessengerMAX (ThermoFisher, UK) and 100 ng of saRNA IIP constructs or saRNA control (no IIP) was added to triplicate wells and after a further 24 hr, plates were centrifuged at 630 g for 5 min at room temperature, 50 μL of medium removed from each well and 50 μL of ONE-Glo™ Ex Reagent D-luciferin reagent (Promega, UK) added and mixed by pipetting. The total volume from each well was then transferred to a flat bottom opaque white 96-well plate (Corning Costar) and fluorescence measured on a FLUOstar OMEGA plate reader within 10 min (BMG LABTECH, UK). Background fluorescence from control wells containing no saRNA was subtracted from the signal for each well containing saRNA. Then the signal obtained for saRNA containing IIP in HeLa cells was expressed as a fold change from signal obtained with control saRNA and to that obtained in HEK293T/17 cells.

[1140] Assessment of IIP on saRNA VEGF-A Expression

[1141] HEK293T/17 or Hela cells were transfected with 100 ng saRNA containing the VEGF-A gene using the same methods as described for testing of constructs expressing f-Luc. After 48 hr the VEGF-A in the cell culture media was measured using a human VEGF-A ELISA kit (Invitrogen, UK). Briefly, assay plate wells were washed twice with 400 uL wash buffer before addition of test samples or VEGF-A standard (15.6 μg/ml to 1000 μg/ml). Plates were then incubated at room temperature for 2 hr in a microplate shaker (300 rpm; Jencons Scientific Ltd, UK) before washing six times with 400 uL wash buffer 100 uL of Biotin-conjugate detection antibody (1:100 dilution) was added to each well and plates incubated in a microplate shaker (1 hr RT, 300 rpm). After six washes with 400 uL of wash buffer, the streptavidin-HRP (1:100 dilution) second layer conjugate (100 uL) was added and after a further 1 hr incubation and six further washes, 100 uL of TMB substrate was added to each well. After incubation in the dark for 30 min at RT in the dark, 100 uL of the Stop solution was added and the absorbance of each well read at 450 nm in a VersaMax microplate spectrophotometer (Molecular Devices, UK). VEGF-A levels in the samples were determined by interpolation to the standard curve.

[1142] Assessment of IIP on RNA Nano-Luciferase (n-Luc) Expression

[1143] HEK293T/17 cells were plated at a density of 25000 cells per well and HeLa cells at a density of 10000 cells per well into flat clear bottom 96-well plates (Corning Costar) and incubated for 24 hr. 10 uL of OptiMEM (ThermoFisher, UK) containing 0.15 μL lipofectamine MessengerMAX (ThermoFisher, UK) and 100 ng of saRNA IIP constructs or saRNA control (no IIP) was added to triplicate wells and after a further 24 hr, plates were centrifuged at 630 g for 5 min at room temperature, 50 μL of medium removed from each well and 50 μL of NanoDLR™ Stop & Glo® Reagent (Promega, UK) added and mixed by pipetting. The total volume from each well was then transferred to a flat bottom opaque white 96-well plate (Corning Costar) and fluorescence measured on a FLUOstar® OMEGA plate reader within 10 min (BMG LABTECH, UK). Background fluorescence from control wells containing no RNA was subtracted from the signal for each well containing RNA. Then the signal obtained for RNA containing IIP in HeLa cells was expressed as a fold change from signal obtained with control RNA and to that obtained in HEK293T/17 cells.

Example 1—Structural Design of Viral Innate Inhibitor Protein (IIP) Constructs

[1144] Viral innate inhibitor proteins (IIPs) can be incorporated into an RNA construct of the invention, which can be a self-amplifying RNA (saRNA) or messenger RNA (mRNA), in order to reduce or ablate the innate recognition and response that may modify or reduce protein expression and translation, i.e. the protein encoded by a Gene of Interest (GOI), which can be any therapeutic biomolecule.

[1145] Various embodiments of design configurations for the RNA construct of the invention are shown in FIG. 1. SaRNA expression constructs are based on an alphavirus backbone where the non-structural proteins are maintained, but the gene of interest (GOI) is inserted downstream of a subgenomic promotor (SGP) replacing the structural genes of the virus (see Embodiment “1” in FIG. 1). The GOI can be any protein at all, and may include viral, bacterial, fungal or mammalian protein, i.e. a biotherapeutic protein. However, the inventors envisage that the RNA construct of the invention will demonstrate significant utility in the vaccine space, and so the GOI would encode a vaccine antigen, such as a viral, bacterial or fungal protein, such as a coat protein.

[1146] saRNA Constructs (Left Hand of FIG. 1)

[1147] Any IIP can be encoded within the saRNA using the following design approaches: [1148] Embodiment “2a” in FIG. 1 shows a saRNA construct encoding a fusion protein including a peptide cleavage motif (e.g. furin-T2a), such that the protein encoded by the GOI (e.g. the antigen of interest) and the IIP are cleaved into separate proteins on translation in the host cell; [1149] In Embodiment “2b” in FIG. 1, the order of the GOI and IIP have been reversed, such that the IIP is 5′ of the GOI, again with a peptide cleavage motif between the IIP and the GOI so that two separate proteins are produced in the host cell following translation of the saRNA construct; [1150] In Embodiment “3a”, the IIP has been inserted downstream of the GOI stop codon. The subgenomic promoter drives translation of the GOI, and expression/translation of the IIP is driven by the inclusion of an internal ribosomal entry site (IRES); [1151] In Embodiment “3b”, the order of the GOI and IIP has been reversed such that translation of the IIP is promoted by the subgenomic promotor and of the GOI by the IRES; [1152] In Embodiment “4a”, the IIP has been inserted downstream of the GOI stop codon. Translation of the GOI is promoted by the first subgenomic promoter and translation of the IIP is driven by the inclusion of a second subgenomic promotor; [1153] In Embodiment “4b”, the position of the IIP and GOI have been swapped around, i.e. with the IIP placed before the GOI.

[1154] mRNA Constructs (Right Hand of FIG. 1)

[1155] Referring to FIG. 1, any IIP can also be encoded within mRNA (see Embodiment “5”) using the following design approaches: [1156] In embodiment “6a”, the mRNA construct encodes a fusion protein including a peptide cleavage motif (e.g. F-T2a) such that the GOI and IIP are cleaved into separate proteins on translation; [1157] In Embodiment “6b”, the order of the GOI and IIP have been reversed such that the IIP is 5′ of the GOI; [1158] In Embodiment “7a”, the IIP has been inserted downstream of the GOI stop codon where translation is driven by the inclusion of an internal ribosomal entry site (IRES); [1159] In Embodiment “7b”, the order of the GOI and IIP has been reversed such that translation is promoted by the subgenomic promotor and the GOI by the IRES.

[1160] The inventors have tested a large number of viral IIPs in the various embodiments of RNA constructs illustrated in FIG. 1, and believe that they each have potential to modify expression and response to saRNA and RNA.

Example 2—Construction and Testing of saRNA Constructs Comprising a Viral Innate

[1161] Inhibitor Protein (IIP) The inventors designed, constructed and then tested a series of diverse viral IIPs in different replicon configurations on expression of the reporter gene, f-Luc or VEGF-A, and the results of the expression studies are shown in FIGS. 6-11.

[1162] Referring to FIG. 6, there is shown the fold increase in f-Luc expression in HeLa cells following transfection with VEEV replicons containing HPV E6, HSV ICP34.5, HCV E2, VACV E3L, MERS ORF8b or VACV K3L in an F-T2A configuration. HEK293T/17 and HeLa cells were transfected with saRNA (100 ng) containing luciferase as a reporter protein and assessed for protein expression after 24 hr. HeLa cells are known to have more intact IFN expression pathways compared to HEK293T/17 and therefore increased expression (fold increase) relative to a control (saRNA containing luciferase as reporter protein and no IIP) indicates that the IIP is increasing saRNA expression. Of these IIP, HSV ICP34.5 produced the greatest increase in f-Luc expression. Data shown are constructs providing a greater than −2-fold increase in luciferase expression in HeLa cells relative to expression in HEK293T/17 cells and are mean f SEM of data obtained in 3 independent experiments using 3 separate batches of saRNA.

[1163] Referring to FIG. 7, there is shown f-Luc expression in HeLa cells following transfection with VEEV replicons containing KHSV ORF52, EBOV VP35, SARS ORF3b* 57 variant, SARS ORF3b*79 variant, SARS ORF3b*57 Equador variant or Pangolin ORF3b* 57 in an F-T2A configuration relative to expression in HEK293T/17 cells. Details of experimental methods are provided in FIG. 6. Of these EBOV VP35 and SARS ORF3b*79 variant produced the greatest increase in f-Luc expression. Data shown are constructs providing a greater than 2-fold increase in luciferase expression in HeLa cells relative to expression in HEK293T/17 cells and are mean f SEM of data obtained in 3 independent experiments using 3 separate batches of saRNA.

[1164] Referring to FIG. 8, there is shown f-Luc expression in HeLa cells following transfection with selected VEEV replicons containing IIP in F-T2A configuration relative to expression in HEK293T/17 cells. Details of experimental methods are provided in FIG. 6. PIV V5 and MERS ORF4a produced the greatest increase inf-Luc expression. Data shown are constructs providing a greater than 2-fold increase in luciferase expression in HeLa cells relative to expression in HEK293T/17 cells and are mean f SEM of data obtained in 3 independent experiments using 3 separate batches of saRNA.

[1165] Referring to FIG. 9, there is shown f-Luc expression in HeLa cells following transfection with VEEV replicons containing IIP in a double sub-genomic promoter (DSGP) configuration relative to expression in HEK293T/17 cells. Details of experimental methods are provided in FIG. 6. HCV E2, VACV E3L and PIV 5V produced similar increases in f-Luc expression. Data shown are constructs providing a greater than 2-fold increase in luciferase expression in HeLa cells relative to expression in HEK293T/17 cells and are mean f SEM of data obtained in 3 independent experiments using 3 separate batches of saRNA.

[1166] Referring to FIG. 10, there is shown the increase in VEGF-A secretion from HeLa cells following transfection with saRNA containing the IIP HSV ICP34.5 in a F-T2A configuration compared to saRNA without IIP and relative to expression in HEK293T/17 cells. HEK293T/17 and HeLa cells were transfected with RNA (100 ng) containing VEGF-A as a reporter protein and assessed for protein expression and secretion into the culture media after 48 hr by ELISA. HeLa cells are known to have more intact IFN expression pathways compared to HEK293T/17 and therefore increased expression relative to a control (RNA containing VEGF-A as GOI and no IIP) indicates that HSV ICP34.5 increases saRNA GOI expression. Data are from one experiment and represent the mean f SEM of three replicate measurements.

[1167] Referring to FIG. 11, there is shown f-Luc expression in HeLa cells following transfection with saRNA containing f-Luc as the GOI (construct 1) and with the IIP MERS ORF4a in F-T2A configuration (constructs 2a and 2b), IRES configuration (construct 3b) and DSGP configurations (constructs 4a and 4b) shown in FIG. 1. Details of experimental methods are provided in FIG. 6. Data are mean±SEM of data obtained in 3 independent experiments using 3 separate batches of saRNA. P<0.05 repeated measures ANOVA compared to construct with no IIP.

Example 3—Construction and Testing RNA Constructs Comprising a Viral Innate Inhibitor Protein (IIP)

[1168] The inventors designed, constructed and then tested a series of diverse viral IIPs, and the results of the expression studies are shown in FIG. 12.

[1169] Referring to FIG. 12, there is shown n-Luc expression in HeLa cells following transfection with RNA containing IIP in F-T2A configuration relative to expression in HEK293T/17 cells. Details of experimental methods are provided in FIG. 6. Data shown are constructs providing a greater than 2-fold increase in luciferase expression and are mean±SEM of data obtained in 3 independent experiments using 3 separate batches of RNA.

CONCLUSIONS

[1170] The inventors believe that the constructs described herein display many advantages over those described in the prior art, including: [1171] i) insertion of nucleotide sequences encoding any of the innate modulatory proteins directly into the RNA construct, such as mRNA or saRNA, enabling dual protein expression of the IIP protein and the biotherapeutic molecule encoded by the gene of interest; [1172] ii) as opposed to delivering two different and separate strands of RNA, one encoding the gene of interest (GOI), i.e. the therapeutic biomolecule, and one encoding the IIP, a single strand is required to be delivered; [1173] iii) the IIP inhibits innate sensing of RNA, thus enabling higher protein expression; [1174] iv) when the RNA construct is a saRNA, the IIP expression itself is self-amplified by virtue of being co-expressed on the sub-genome strand with the GOI; and/or [1175] v) an increase in both the magnitude and duration of protein expression compared to conventional VEEV RNA replicon constructs.

[1176] Numbered Paragraphs

[1177] The following paragraphs form part of the description and not the claims [1178] 1. An RNA construct encoding: (i) at least one therapeutic biomolecule; and (ii) at least one viral innate inhibitor protein (IIP). [1179] 2. The RNA construct according to paragraph 1, wherein the construct is an mRNA, saRNA or a trans-replicon system, most preferably saRNA. [1180] 3. The RNA construct according to either paragraph 1 or paragraph 2, wherein the construct comprises or is derived from a positive stranded RNA virus selected from the group of genus consisting of: alphavirus; picornavirus; flavivirus; rubivirus; pestivirus; hepacivirus; calicivirus and coronavirus, preferably an alphavirus, optionally VEEV. [1181] 4. The RNA construct according to any preceding paragraph, wherein the at least one innate inhibitor protein (IIP) is HPV E6 or HSV ICP34.5, or an orthologue thereof. [1182] 5. The RNA construct according to any one of paragraphs 1-3, wherein the at least one innate inhibitor protein (IIP) is HCV E2 or HCV NS5a, or an orthologue thereof. [1183] 6. The RNA construct according to any one of paragraphs 1-3, wherein the at least one innate inhibitor protein (IIP) is VACV E3L or VACV K3L, or an orthologue thereof. [1184] 7. The RNA construct according to any one of paragraphs 1-3, wherein the at least one innate inhibitor protein (IIP) is MERS ORF8B, or an orthologue thereof. [1185] 8. The RNA construct according to any one of paragraphs 1-3, wherein the at least one innate inhibitor protein (IIP) is KSHV ORF52, or an orthologue thereof. [1186] 9. The RNA construct according to any one of paragraphs 1-3, wherein the at least one innate inhibitor protein (IIP) is Ebola VP35, or an orthologue thereof. [1187] 10. The RNA construct according to any one of paragraphs 1-3, wherein the at least one innate inhibitor protein (IIP) is Vaccinia C6, or an orthologue thereof. [1188] 11. The RNA construct according to any one of paragraphs 1-3, wherein the at least one innate inhibitor protein (IIP) is an ORF3b*57 variant of the wild type of SARS-CoV-2 ORF3b, or an orthologue thereof. [1189] 12. The RNA construct according to any one of paragraphs 1-3, wherein the at least one innate inhibitor protein (IIP) is an ORF3b*79 variant of the wild type of SARS-CoV-2 ORF3b, or an orthologue thereof. [1190] 13. The RNA construct according to any one of paragraphs 1-3, wherein the at least one innate inhibitor protein (IIP) is an ORF3b*57 Ecuador variant of the wild type of SARS-CoV-2 ORF3b, or an orthologue thereof. [1191] 14. The RNA construct according to any preceding paragraph, wherein the therapeutic biomolecule comprises a therapeutic protein, preferably the protein or peptide is an antigen, and more preferably a viral antigen. [1192] 15. A nucleic acid sequence encoding the RNA construct according to any preceding paragraph. [1193] 16. An expression cassette comprising a nucleic acid sequence according to paragraph 15. [1194] 17 A recombinant vector comprising the expression cassette according to paragraph 16. [1195] 18. A pharmaceutical composition comprising the RNA construct according to any one of paragraphs 1 to 14, the nucleic acid sequence according to paragraph 15, the expression cassette according to paragraph 16 or the vector according to paragraph 17, and a pharmaceutically acceptable vehicle. [1196] 19. A method of preparing the RNA construct according to any one of paragraphs 1 to 14, the method comprising: [1197] a) i) introducing, into a host cell, the vector according to paragraph 17; and [1198] ii) culturing the host cell under conditions to result in the production of the RNA construct according to any one of paragraphs 1 to 14; or [1199] b) transcribing the RNA construct from the vector according to paragraph 17. [1200] 20. The RNA construct according to any one of paragraphs 1 to 14, the nucleic acid sequence according to paragraph 15, the expression cassette according to paragraph 16 or the vector according to paragraph 17 or the pharmaceutical composition according to paragraph 18, for use as a medicament or in therapy. [1201] 21. The RNA construct according to any one of paragraphs 1 to 14, the nucleic acid sequence according to paragraph 15, the expression cassette according to paragraph 16 or the vector according to paragraph 17 or the pharmaceutical composition according to paragraph 18, for use in the prevention, amelioration or treatment of a protozoan, fungal, bacterial or viral infection. [1202] 22. The RNA construct according to any one of paragraphs 1 to 14, the nucleic acid sequence according to paragraph 15, the expression cassette according to paragraph 16, the vector according to paragraph 17 or the pharmaceutical composition according to paragraph 18, for use in the prevention, amelioration or treatment of cancer. [1203] 23. A vaccine comprising the RNA construct according to any one of paragraphs 1 to 14, the nucleic acid sequence according to paragraph 15, the expression cassette according to paragraph 16, the vector according to paragraph 17 or the pharmaceutical composition according to paragraph 18. [1204] 24. The RNA construct according to any one of paragraphs 1 to 14, the nucleic acid sequence according to paragraph 15, the expression cassette according to paragraph 16, the vector according to paragraph 17 or the pharmaceutical composition according to paragraph 18, for use in stimulating an immune response in a subject.