Molecules triggering the ribosome quality control (RQC) mechanism

Abstract

Novel hybrid polynucleotide mRNA (or DNA that encodes the mRNA) has one or more antigens and a sequence that can trigger the cellular Ribosome Quality Control (RQC) mechanism in a cell. The polynucleotide will thus have a triggering sequence, usually a sequence of AAA codons (lysine residues) which stalls translation by the ribosome after the synthesis of the antigenic peptide. This can result in the encoded antigen being presented on MHC-I to enhance an immune response.

Description

[0001] MOLECULES TRIGGERING THE RIBOSOME QUALITY CONTROL (RQC) MECHANISM

[0002] Introduction

[0003] The present invention relates to (usually synthetic, or non-natural) polynucleotides. These can be used for triggering the Ribosome Quality Control (RQC) mechanism in cells and may result in stalling of the protein translation. As a result they can be used in vaccines.

[0004] Despite their remarkable recent success, there is considerable variability in efficacy of mRNA vaccines for different antigens, mainly due to issues with their presentation on major histocompatibility complexes (MHCs). For instance, highly structured proteins with stable 3D conformations can be resistant to degradation and presentation on MHCs.

[0005] The RQC mechanism exists in most eukaryotic species and is a natural mechanism to deal with errors in the translation process, for example when aberrant proteins or incomplete proteins are produced as a result of interrupted translation. As its name suggests, it is a Quality Control mechanism that is a cell’s way of recycling unwanted or mutant proteins resulting from errors in the translation process.

[0006] It has been postulated that the RQC may play a role in sampling cellular proteins toto detect possible viral infections (see, for example, Trentini et al, PNAS, Vol 117 (8), 4099-4108, February 2020). However, no-one in the art has suggested that the natural RQC could be harnessed to assist antigen presentation for the purpose of enhancing vaccine efficacy.

[0007] Summary of the invention

[0008] The invention aims to enhance the immune response to vaccines, such as by harnessing the Ribosome Quality Control (RQC) mechanism, so as to increase the efficacy of antigen presentation on MHCs. This is counter-intuitive as the RQC mechanism deals with errors that occur in the translation process and so it is surprising that this mechanism could be used in a positive way, such as to enhance vaccines.

[0009] The invention, in a first aspect, relates to a synthetic, man-made, non-natural (or non- naturally occurring) polynucleotide (sequence). It may comprise an RQC triggering means, such an RQC triggering sequence element or structure. The polynucleotide thus comprises (one or more) means for triggering the RQC mechanism. Hence the polynucleotides of the invention are designed to deliberately stall the RQC, contrary to conventional wisdom which would try to avoid stalling mRNA translation and protein synthesis.

[0010] The polynucleotide can comprise either mRNA or DNA(if the latter, then DNA encoding mRNA).

[0011] Suitably the polynucleotide will encode a (e.g. specific or desired) protein, such as one or multiple antigenic proteins, or antigens. Thus, the polynucleotide may comprise an Open Reading Frame (ORF) which may encode a desired protein or antigen (such as a viral or cancer antigen).

[0012] Preferably the polynucleotide has the structure:

[0013] Start codon - ORF - optional linker - stop codon.

[0014] The RQC triggering means may comprise one or more of the following elements or features: a. A stalling sequence, or a sequence which results in triggering the RQC mechanism, such as (a stretch of) AAA codon(s), namely codons that encode lysine(s); b. A structured or structural element, such as a stem loop, optionally present in the ORF, and suitably after the antigen coding sequence; c. The absence of a stop codon, for example there being no stop codon in the polynucleotide; d. A truncated or shortened ORF, for example wherein there is no stop codon after the coding sequence; and / or e. One or more modified polynucleotide(s), for example one that may trigger RQC, for example one that blocks translation or causes ribosome frameshift, suitably located after the antigen coding sequence.

[0015] The RQC triggering means may comprise a stalling sequence, and this is preferably one or more, or a series of, lysine codons (the codon for lysine being AAA). Thus, preferably the triggering means is a series of AAA codons. The triggering means may thus comprise a plurality of lysine codons and / or a poly A sequence, for example, suitably located after the ORF but before the stop codon.

[0016] The number of lysine codons is preferably from 4, or up to 100, such as from 6 or 12 to 24 or 36, optimally from 6 to 24 codons. Suitably there are from 6 or 9 or 12 or 15 codons and / or up to 15 or 18 or 21 or 24 codons. Preferably there are from 10 to 14 or 14 to 30 codons.

[0017] The polynucleotide according to the invention may thus be able to: a. Stall, halt or interrupt translation, or a translating ribosome; and / or b. May cause ribosome collision, dissociation of ribosomal subunits, and / or degradation of a nascent polypeptide (via the proteosome).

[0018] Preferably the polypeptide results in presentation (of the peptide or antigen) on (the surface of) MHC-1 / 2.

[0019] Molecules of the invention (such a polynucleotide) may be isolated, non-natural, manmade and / or purified.

[0020] The ORF suitably encodes a protein (polypeptide) to be used as a vaccine, or as an antigen.

[0021] The invention can thus be thought of as e.g. hybrid (mRNA), that may increase or maximise expression of an (e.g. encoded antigen), such as on an MHC. It may comprise a sequence that can selectively trigger the RQC mechanism.

[0022] A second aspect of the invention relates to the use of a polynucleotide (e.g. of the first aspect) for: a. Stalling, halting, or interrupting translation, or of a translating ribosome; b. Causing ribosome collision, dissociation of ribosomal subunits and / or degradation of a nascent peptide; c. Presenting or increasing (presentation) of an antigen on an MHC; d. Maximising and / or increasing expression of an antigen, or increasing the efficiency of antigen presentation of an MHC; e. Activation of an immune response; and / or f. Preparation of a vaccine.

[0023] A third aspect of the present invention relates to a method of triggering (or activating) the RQC (such as in a cell) the method comprising: a. Inserting or locating a polynucleotide of the first aspect into a cell; b. Causing said polynucleotide to be (at least partially) translated (for example in a cell), optionally in a ribosome.

[0024] A fourth aspect of the invention relates to a vector comprising a polynucleotide of the first aspect.

[0025] A fifth aspect of the invention relates to a cell (e.g. MHC 1 or 2) comprising a polynucleotide of the first invention or a vector of the fourth aspect.

[0026] The cell may present, or has on its cell surface, a protein (such as an antigen) as a result of the RQC mechanism (in that cell).

[0027] A sixth aspect relates to the use of the RQC mechanism in a cell to present an (heterogenous or foreign) protein (such as an antigen) on the surface of said cell.

[0028] Details of the invention

[0029] Nascent peptides, produced by mRNAs that trigger the RQC, can be presented on MHC-I. This is often independent of their intrinsic folding properties. Therefore, any antigen, irrespective of its structure and / or amino acid sequence, could be efficiently designated for presentation on MHC-I, and may thus activate the immune system.

[0030] The invention aims to create novel hybrid polynucleotides, such as mRNA, that can (maximise) expression of (encoded) antigens (e.g., viral proteins or cancer antigens), This may use a sequence that may (selectively) trigger the RQC mechanism. This may designate the encoding antigen(s) to MHC-I and / or augment activation of the immune response. This antigen presentation boosting technology may thus create a versatile and broadspectrum platform, such as for increased efficacy of existing and novel vaccines, suitably across a range of antigens. It may have high translational and commercial potential. This technology could also be used for DNA vaccines, e.g. by designing DNAs that encode mRNAs, suitably with an open reading frame that may harbour the antigen and / or the RQC-trigger sequence. mRNA and DNA vaccines

[0031] Current vaccines mainly rely on delivery of peptides or inactivated pathogens. This can be costly and may require complex processes, e.g. that involve purification of proteins and / or ensuring that the proteins contain the typical features of the antigen (such as the correct 3D structure and / or post-translational modifications).

[0032] An mRNA vaccine may alleviate such problems. The vaccine antigen can be encoded by the host cell and / or may undergo all the necessary modifications (as the typical antigen would in natural case). However, there is considerable variability in efficacy of different antigens, often due to issues with their presentation on MHCs. For instance, highly structured proteins that have stable 3D structures can be highly resistant to degradation and targeting to MHC.

[0033] The industry currently uses mRNA modifications, sequence optimisation, and propriety UTR sequences to enhance or modulate the stability and I or translation efficiency of the mRNAs, usually in order to produce more proteins. In principle, producing more proteins could result in more presentation on MHCs, and thus a bigger or increased immune response. However, our innovation may result in (e.g. exponential) antigen presentation from the same number of mRNAs, often regardless of the type of mRNA modifications.

[0034] The invention may utilise the powerful cellular RQC process that induces degradation of proteins, and present the peptides on MHCs, often regardless of their 3D structure. Therefore, our invention may combine the advantages of the mRNA vaccine technology and the concept of RQC. This may enable safe and efficient delivery of antigen coding mRNAs to the RQC process which then target the resultant antigenic peptides to MHCs. This invention may not be limited to any specific antigen and may have high translational and commercial potential.

[0035] The invention will now be described with reference to the accompany Examples, which are not to be construed as being limiting.

[0036] Brief Description Of The Drawings

[0037] The invention will now be described by reference to the drawings, in which:

[0038] Figure 1 : Schematic overview of the Ribosome Quality Control (RQC) mechanism. If a translating ribosome stalls, due to reasons such as presence of Poly(A) within the Open Reading Frame, the trailing ribosomes collide into the stalled ribosome. These collision events are detected by the RNA-binding protein, ZNF598, which triggers the RQC process. Activation of RQC results in dissociation of ribosomal subunits, degradation of the nascent peptide via proteasome and presentation on MHC-I.

[0039] Figure 2. A) Schematic representation of the design of the antigen-expressing mRNAs. These mRNAs are designed to encode the Ovalbumin (OVA) antigen. 12K denotes the 12x (AAA) codons that would trigger RQC on this mRNA construct. 6TS is the control sequence that does not trigger RQC. B) Western blot analysis of the expression of OVA protein encoded by the 6TS or 12K mRNA prototypes in the indicated cells. Different amounts of mRNAs (300 and 600 ng) were used in parallel to assess the linearity of the expression of the protein. MG132 proteasome inhibitor was used to assess stabilization of the 12K encoded proteins that otherwise are degraded by ubiquitination-induced proteasome-mediated degradation.

[0040] Figure 3: Schematic overview of the FACS assay for detection of the SIINFEKL antigen presentation on the cell surface. The 25D1 .16 monoclonal antibody reacts with the ovalbumin-derived peptide SIINFEKL bound to H-2Kb isoform of MHC class I (MHC-I), but not with unbound H-2Kb, or H-2Kb bound with an unrelated peptide.

[0041] Figure 4: Representative figure demonstrating the effectiveness of the Antigen-to- Surface Enhancement Technology (ASET) platform in enhancing antigen presentation on the cell surface. NIH3T3 mouse fibroblast cells that stably express the H2KB MHC- I isoform were transfected with 600 ng of OVA- 6TS or OVA-12K coding mRNAs. 24 h after transfection cells were analysed by FACS for measurement of the presence of the MHC-I / SIINFEKL complex on the surface using the 25D1.16 monoclonal antibody. A) representative graph that shows enhanced percentage of cells with positive MHC- I / SIINFEKL on the cell surface in 0VA-12K mRNA transfected, compared with the OVA-6TS or no-RNA controls detected by FACS using the 25D1.16 monoclonal antibody. B) Quantitation of 6 independent experiment as shown in A. The 0VA-12K mRNA results in ~2-fold increased antigen presentation (23.39 ± 1 .22 % for 0VA-12K compared with 11.86± 0.92% for OVA-6TS control; t-test p-value>0.001 ), indicating the effectiveness of the RQC-trigger sequence in augmenting the presentation of antigen on cell surface.

[0042] Figure 5: Schematic design of the antigen presentation boosting platform which contains an optimised number of AAA codons that triggers RQC in-frame with one or multiple antigens within the Open Reading Frame. The context optimised linker between antigen(s) and RQC-trigger sequence determines the number of times that the antigens are translated by the ribosome before RQC is triggered.

[0043] Figure 6: Schematic representation of different embodiments of the invention, and five different ways of triggering the RQC mechanism using a polynucleotide of the invention.

[0044] Figure 7: Schematic representation of mice immunisation.

[0045] Figure 8: Quantification and characterization of antigen-specific CD8+ T cells.

[0046] Figure 9: Quantification and characterization of antigen-specific IFN-y+CD8+T cells.

[0047] Figure 10: Quantification and characterization of antigen-specific CD8+T cells.

[0048] Figure 11 : Quantification and characterization of antigen-specific IFN-y+CD8+T cells.

[0049] Figure 12: Quantification of antigen-specific IFN-y.

[0050] Figure 13: ASET mRNA reduces tumour growth in “immune hot” MC38-OVA and “immune cold” B16-OVA tumours. ASET mRNA works synergistically with anti-PD1 therapy.

[0051] EXAMPLE 1

[0052] The mRNA translation process has three phases: initiation, elongation, and termination. Apart from the strict requirement to regulate the rate of translation at the initiation phase, the rate of elongation ( / .e., the speed at which the ribosome translates the Open Reading Frame) is also subject to regulation. In addition, an elongating ribosome can be stalled for reasons such as presence of damaged nucleotide or presence of a stretch of >18 nucleotide poly(A) sequence within the Open Reading Frame.

[0053] When a ribosome stalls, the trailing ribosomes collide with the stalled ribosome. We (Garzia & Jafarnejad, et al, Nature Communications 2017) and other showed that these collision events are detected by proteins such as ZNF598, which then trigger a process called the Ribosome Quality Control (RQC). Once activated, RQC induces the ubiquitinoylation of the nascent peptide, which leads to their degradation via the proteasomal pathway (Figure 1).

[0054] Recently, it was demonstrated that the proteasomal degradation of the nascent proteins generated by mRNAs that trigger RQC leads to the MHC-I display of the resultant peptides, including highly structured proteins that otherwise evade MHC-I presentation (Trentini et al, PNAS 2020). Therefore, any peptide that is encoded by an mRNA with a RQC-triggering sequence such as poly(A) within the Open Reading Frame could be displayed on MHC-I, irrespective of its amino acid sequence or folding structure (Figure 1).

[0055] Recent evidence also indicate that highly efficiently translated mRNAs, such as those that encode ribosomal proteins and translation factors) are more frequently targeted by RQC, likely due to higher chance of presence of trailing ribosomes that col lied with the stalled ribosome (Juszkiewicz and Hegde, Mol Cell 2017; Park and Subramaniam, PLoS Biol 2019; Goldman, et al, Mol Cell 2021 ; Jafarnejad, unpublished data). Therefore, increasing the translation efficiency of an RQC-targeted, antigen-coding mRNA, through inclusion of sequences that enhance the mRNA translation efficiency, would likely augment the rate of RQC and antigen presentation.

[0056] Therefore, we developed the Antigen-to-Surface Enhancement Technology (ASET) platform, to harness the cellular RQC process for enhancing the presentation of antigens on MHC. We tested the effect of the ASET platform on presentation of the Ovalbumin (OVA) antigen on MHCs. We first tested the expression of the OVA protein by two mRNAs: 1 - a mRNA where a RQC-trigger 12K sequence (K=Lysine, which is encoded by AAA codon. Therefore, this sequence has 36 Adenosines that encode 12 lysine amino acids) is present at the 3' end of the Open-Reading Frame of the OVA. 2- A control mRNA wherein the 12K sequence is replaced by 6 Tyrosine and 6 Serine amino acids (6TS) (Figure 2A).

[0057] Western blot analysis showed that while the 6TS mRNA encodes the OVA antigen proportional to the number of transfected mRNAs, the 12K mRNA results in less protein expression. However, upon inhibition of the proteasome pathway by MG132, expression of the 12K-OVA is restored (Figure 2B). This clearly indicates that presence of 12K sequence rapidly directs the antigen towards proteasomal degradation. We next tested the presentation of the OVA antigen on MHCs by using Fluorescent Activated Cell Sorting (FACS) assay. In this assay, we utilise an antibody that detects the complex between MHC and SIINFEKL, an epitope within the OVA antigen (Figure 3). Our data showed that presence of 12K sequence enhances SIINFEKL antigen presentation on MHCs by up to 2-fold (Figure 4).

[0058] The invention aims to use or harness the Ribosome Quality Control (RQC) mechanism to enhance the effectiveness of mRNA and DNA vaccines, for example by increasing the efficacy of antigen presentation, suitably on major histocompatibility complexes (MHCs). This approach may enhance the immune response to vaccines against various antigens, including cancer, viruses, and / or other pathogens.

[0059] The invention thus provides novel hybrid mRNAs that may (maximise or increase) the expression of an encoded antigen (e.g. a viral protein or cancer antigen). These may be modified (in a number of ways) so that they are selectively targeted by the RQC mechanism, such as to designate the encoding antigen(s) to MHC-I, and thus may augment (activation of) immune response.

[0060] This antigen presentation boosting platform (Figure 5) may augment production and / or MHC-display of peptide(s), suitably encoded in engineered mRNAs. This may be achieved through unique optimised combination of (one or more of): - A stalling sequence, or a modification which results in triggering the RQC mechanism: (e.g. a Poly(A) sequence of optimised length, a structured or structural element, such as a stem loop, absence of a stop codon, a truncated or shortened ORF, one or more modified polynucleotide(s), for example one that may block translation: Figure 5) within the Open Reading Frame of an mRNA encoding antigenic peptide sequence(s), which acts as an RQC-trigger to selectively target encoded peptide for presentation on MHC-I. - Context optimised linker between antigen or antigens that are encoded by ORF. This linker will determine the number of times that the antigens are translated by the ribosome before RQC is triggered.

[0061] This proprietary antigen presentation boosting technology creates a versatile and broad-spectrum platform for increased efficacy of existing and novel vaccines across a range of antigens. It is particularly suitable for development based on mRNA vaccine platforms, the excellent efficiency of which has recently been demonstrated for the SARS-COV2 vaccines generated by Pfizer, Moderna, and other pharmaceutical companies, making this approach very attractive to the pharmaceutical industry. However, this technology could also be used for DNA vaccines, as they encode mRNAs that could harbour the RQC-trigger.

[0062] EXAMPLE 2

[0063] Material and methods

[0064] Mice

[0065] Eight to twelve weeks old female wild type (WT) on a C57BL / 6J background were provided by The Jackson Laboratory (USA). Animals were bred in-house by the Comparative Medicine Unit (CMU) and maintained according to the regulations of the European Union and the Health Products Regulatory Authority (HPRA). They were kept in individual ventilated cages (IVC), specific-pathogen free (SPF) conditions with 12 hours light cycle, 21 °C and unrestrictive food and water consumption. All procedures were conducted under animal license number AE19136 / 1582 and were approved by TCD Animal Research Ethics Committee (Ethical Approval Number 091210).

[0066] Cell culture conditions

[0067] All cells were cultured in a steri-cycle CO2 incubator HEPA class 100 (Thermo Scientific) with 5% CO2, 95% humidity, 37°C and darkness. Base medium used for cell culture RPMI 1640 GlutaMAX (Gibco) was supplemented with 50U / ml penicillin and 50pg / ml streptomycin (Gibco)', 10% heat-inactivated, filter-sterilized Foetal Bovine Serum (FBS) (Biosera) and 45.5pM [3-mercaptoethanol (Gibco) to make complete medium (cRPMI). T cell medium was made by supplementing cRPMI with 0.88mM sodium pyruvate (Gibco), 10mM MEM Non-Essential Amino Acids (Gibco) and 0.35% (v / v) 100X MEM Vitamins (Gibco).

[0068] Splenocytes

[0069] Spleens were collected from euthanized mice and single cell suspension were obtained after homogenising by mechanical dissociation with a sterile plunger and filtering cell suspension in cRPMI through a 40pm cell strainer (Fisher Scientific). Cells were centrifuged at 1200rpm for 5m in to remove all the remaining tissue debris. Supernatants were decanted and cell pellets resuspended in 1 ml of 0.88% ammonium chloride (NH4CI) for 2min to lyse erythrocytes with subsequent quenching of reaction by cRPMI and centrifugation. Cell pellets were resuspended in 5ml of T cell medium and live cells counted with trypan blue. For ex vivo antigen restimulation assays, cells were plated in sterile Il-bottom 96 well plates in either 10OpI of T cell media at 10x106cells / mL for intracellular staining studies or 200pl of T cell media at 2x106cells / ml for cytokine ELISAs. Cells were then restimulated with appropriate antigen concentrations and controls as detailed in the methods and figure legends for each experiment.

[0070] Mice immunization

[0071] Immune Response study: Intramuscular (i.m.) vaccination was performed on days 0 and 14 using 27G / 0.5in tuberculin syringes (BDTM) in both hind limbs (biceps femoris muscles) by injecting 50pl / leg. Animals received 10pg apyrogenic ovalbumin (OVA) (Lionex GmbH (Germany)) or 10pg of mRNA-LNPs coding for OVA (RIBOPRO). 1 mg 50nm polystyrene particles (Phosphorex, Inc® (USA)) in combination with 10pg OVA per mouse was used as positive control (Figure 7A). Immunotherapeutic Study: 5x105MC38-OVA or B16F10-OVA cancer cells in exponential growth phase were administered by subcutaneous (s.c.) injection in the lateral flank of mice in a volume of 50 pl. Mice were monitored daily and tumour growth was measured using a calliper and volumes (mm3) recorded. When tumours reached 10-20mm3daily, (day 0) and one week after the first vaccination (day 7) OVA mRNA-LNPs (10 pg) by i.m. vaccination. Mice were humanely euthanised when the lesion reached 1500 mm3or if necrosis was noted. Some mice also received anti-PD1 checkpoint inhibitor (Assay Genie) (200 pg) in a volume of 200 pl by systemic (i.p.) vaccination on day 5, 10 and 15 after the first immunization. Tumour growth over time and survival rates were analysed upon finalisation of the study (Figure 7B).

[0072] Quantification and characterization of antigen-specific CD8+T cells

[0073] One week after booster (day 21 after injections), mice were culled by a 5-minute exposure to CO2. Afterwards, spleens were collected and splenocytes were isolated as previously explained. 20x106viable splenocytes were added to Flow Cytometry tubes with lids (ThermoFisher) in T cell medium, to analyse antigen-specific CD8+T cells by tetramer staining. To do so, splenocytes were stained with PE-labelled H-2K(b) Chicken OVA257-264 SIINFEKL tetramers, provided by the National Institutes of Health Tetramer Core Facility (NIH) (Emory University) at 1 :40 dilution in 10OpI T cell medium per sample for 2h in a cell culture incubator (5% CO2, 95% humidity, darkness, and 37°C) and with orbital shaking (TITRAMAX 100 - Heidolph Instruments) (250 rpm). During the last 40 minutes of the staining, anti-CD16 / CD32 monoclonal antibodies (mAbs) (BD Biosciences) to block FcyRII / lll (0.5pl per sample in 50pl of 1xPBS+2% FBS (FACS buffer)) were added. After 10 minutes of incubation, antibody staining of specific extracellular molecules to characterize different cell populations was performed to study OVA-specific CD8+T cells (Table 1) (in 100 pl / sample of FACS buffer + Brilliant stain buffer (BD Biosciences)), with an incubation time of 30 minutes. Finally, cells were washed in 2ml 1xPBS and pellets were resuspended in 1 ml live- dead stain Aqua 510 (BV510) at 1 :500 dilution in 1xPBS to stain live cells. After 30 minutes, cells were washed twice in 2 ml 1xPBS (by centrifugation at 1200rpm for 5 min) and fixed for 15 minutes at RT in 2% paraformaldehyde. Finally, cells were washed in 2 ml 1xPBS, and pellets were resuspended in 250pl of FACS buffer. Samples were kept at 4°C until acquisition in a 4 laser / 16 fluorescent parameter LSR- Fortessa analyser until reaching 1x104CD8+T cells. Analysis was carried out in FlowJo (V10X) (Figure 8).

[0074] Single stain (SS) samples were used to compensate the wavelength of the different fluorochromes to avoid spectral overlaps. This was done by diluting two drops of OneComp eBeads Compensation Beads (Invitrogen™) in 1 ml of 1xPBS. Then, 200 pl of the mix were added to Flow Cytometry tubes and 10pl / tube of the corresponding antibody dilution were added to finally have all the required SS samples. In the case, of the live-dead SS, unstimulated cells were single stained with 1 ml of the live-dead stain Aqua 510 (BV510) dilution.

[0075] Furthermore, unstained cells were used to set the forward scatter area (FSC-A) and the side scatter area (SSC-A) voltages. Finally, FMOs were used to set the boundaries of negative / positive cell populations for each of the antibodies.

[0076] Table 1. Panel for the quantification and characterization of antigen-specific CD8+T cells.

[0077] Quantification and characterization of antigen-specific IFN-y+CD8+T cells

[0078] Following the same protocol as above, splenocytes from vaccinated mice were collected on day 21 . Then, 1x106splenocytes from each mouse were plated in T cell medium and in triplicate in sterile 96 well plates round bottom. Afterwards, they were stimulated for 2h with cognate antigen OVA at 200pg / ml and SIINFEKL peptide 1 pg / ml followed by 12h of treatment with the protein transport inhibitor GolgiPlug containing Brefeldin A (BD Biosciences) (1 pg / ml). After this, splenocytes were washed with 10OpI 1xPBS per well and stained with live-dead stain Aqua 510 (BV510) 1 :1000 dilution in PBS+1 % FBS (25pl / well) for 15min on ice. Thereafter, cells were washed and incubated for 10min with anti-CD16 / CD32 monoclonal antibodies (mAbs) to block FcyRII / lll (1 :100 dilution) in 25pl / well of PBS+1 % FBS. After this, antibody staining of specific extracellular molecules was performed to characterize T cell populations and study activated CD8+T cells (Table 2) (in 25pl / well of FACS buffer+Brilliant stain buffer), with an incubation time of 15 minutes on ice. After washing the cells in 1xPBS, these were fixed and permeabilized using the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen). Then, staining of intracellular IFN-y was performed overnight a 4°C to analyse the activated IFN-y+CD8+T cell population in the samples (Table 2) (in 50pl / well of IxPermeabilization buffer). Finally, cells were washed in 200pl 1x Permeabilization buffer, transferred into Flow Cytometry tubes and washed again to resuspended pellets in 250pl of FACS buffer. Samples were kept at 4°C until acquisition in a 4 laser / 16 fluorescent parameter LSR-Fortessa analyser (as described above) until reaching 1 x104CD8+T cells. Analysis was carried out in FlowJo (V10X) (Figure 9).

[0079] Table 2. Panel for the quantification and characterization of antigen-specific IFN- Y+CD8+T cells.

[0080] Results

[0081] Quantification and characterization of antigen-specific CD8+T cells. Figure 10A shows a representative dot plot from each of the groups of mice immunized with either 10pg OVA, 10pg of mRNA-LNPs or 1 mg of 50nm polystyrene (PS) particles with 10pg OVA (n=5 per group) indicating the percentage of CD8+T cells specific to H-2Kb OVA epitope SIINFEKL and that were activated (CD44hiTmer+CD8+T cells). This was determined by FACS, staining with tetramers 7 days after i.m. booster. Figure 10B shows scatter plots with individual values (n=5 per group) and mean with SEM of the percentage or total number of activated CD8+T cells specific to H-2Kb OVA epitope SIINFEKL (CD44hiTmer+CD8+T cells) in each group, either over the total number of CD8+T cells or over the total number of splenocytes. One-way ANOVA with Tukey correction for multiple comparisons was applied for the statistical analysis. Statistical significance was set at p<0.05. p values were represented as **p<0.005, ***p<0.0005, and ****p<0.0001. Quantification and characterization of antigen-specific IFN-y+CD8+T cells. Figure HAshows a representative dot plot from each of the groups of mice immunized with either 10pg OVA, 1 Opg of mRNA-LNPs or 1 mg of 50nm polystyrene (PS) particles with 10pg OVA (n=5 per group) indicating the percentage of IFN-y+activated (CD44+) CD8+T cells after re-stimulation of splenocytes with OVA antigen (200pg / ml) and SIINFEKL peptide (1 pg / ml) for 2h and a further 12h in the presence of Golgi Plug (containing Brefeldin A). This was determined by FACS 7 days after i.m. booster. Figure 11 B shows scatter plots with individual values (n=5 per group) and mean with SEM of the percentage and total number of activated IFN-y+CD8+T cells in each group, either over the total number of CD44+CD8+T cells or over the total number of splenocytes. One-way ANOVA with Tukey correction for multiple comparisons was applied for the statistical analysis. Statistical significance was set at p<0.05. p values were represented as *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001 .

[0082] Quantification of antigen-specific IFN-y. Splenocytes from immunized mice with either 10pg OVA, 10pg of mRNA-LNPs or 1 mg of 50nm polystyrene (PS) particles with 10pg OVA (n=5 per group) were collected at day 21 after the initial injection and restimulated with OVA antigen (200 pg / ml) and SIINFEKL peptide (1 pg / ml) for 72h (Figure 12). T cell medium was used as negative control and anti-CD3 (0.2pg / ml) with anti-CD28 (0.2pg / ml) were used as positive control. Antigen-specific release of IFN-y was determined by ELISA. Bar graphs show mean with SEM of the concentration of IFN-y (pg / ml) in each of the groups. One-way ANOVA with Tukey correction for multiple comparisons was applied for the statistical analysis.

[0083] ASET mRNA reduces tumour growth in “immune hot” MC38-OVA and “immune cold” B16-OVA tumours. ASET mRNA works synergistically with anti-PD1 therapy. MC38-OVA or B16F10-Ova cells were implanted in mice, where tumour volume was monitored following implantation. Once tumours reached 10-20 mm3(day 0), and one week after the first vaccination (day 7), mice received Ova ASET mRNA- LNPs (10 ug) by i.m. injection. In addition, the Ova ASET mRNA-LNPs ± anti-PD1 cohort received an anti-PD-1 check point inhibitor by i.p. injection on days 5, 10 and 15 following the first immunization (day 0). Mice were humanely euthanised when tumour volume reached 1500 mm3or if necrosis was noted. Tumour growth was monitored over time, and survival rates analysed upon study completion (Figure 13). Two-way ANOVA with Tukey correction for multiple comparisons was applied for the statistical analysis (*p<0.03, **p<0.002, ***p<0.0002, and ****p<0.0001) alongside simple survival analysis (Kaplan-Myer curves).

Claims

Claims1 . A polynucleotide comprising means for triggering the Ribosome Quality Control (RQC) mechanism.

2. A polynucleotide according to claim 1 which is synthetic, man-made, nonnatural (or non-naturally occurring) and / or wherein the RQC triggering means comprises a section, sequence, element or structure in the polynucleotide.

3. A polynucleotide according to claim 1 or 2 which is isolated or purified and / or comprises mRNA or DNA (encoding mRNA).

4. A polynucleotide according to any preceding claim which encodes one or several (e.g. specific or desired) antigen(s) or a protein (that is antigenic).

5. A polynucleotide according to any preceding claim which comprises an Open Reading Frame (ORF) which encodes an antigen (such as a viral or cancer antigen).

6. A polynucleotide according to any preceding claim which has the structure:Start codon - ORF - optional linker - stop codon7. A polynucleotide according to any preceding claim wherein the RQC triggering means comprises: a. A stalling sequence, or a sequence which results in the activation of the RQC mechanism, or stalling; b. A (highly) structured or structural element, such as a step loop, optionally in the ORF, after the antigen coding sequence; c. The absence of a stop codon (such as no stop codon in the polynucleotide); d. A truncated or shortened ORF (optionally wherein there is no stop codon after the coding sequence);e. One or more modified polynucleotide(s), optionally one that blocks translation, or causes ribosome frameshift, suitably after the antigen coding sequence.

8. A polynucleotide according to any preceding claim wherein the RQC triggering means comprises a stalling sequence, preferably a series of AAA (or lysine) codons.

9. A polynucleotide according to claim 8 wherein the stalling sequence comprises a plurality of lysine codons and / or a poly A tail (suitably at the end of the ORF).

10. A polynucleotide according to claim 8 or 9 wherein the number of lysine codons is from 4 to 100, such as from 6 or 9 or 12 up to 18, 24 or 36.11 . A polynucleotide according to any preceding claim which: a. Stalls, halts or interrupts translation, or a translating ribosome; and / or b. Causes ribosome collision, dissociation of ribosomal subunits and / or degradation of a nascent peptide (via the proteasome); or can enhance, activate or trigger an immune response.

12. A polynucleotide according to any preceding claim which results in, or can effect, presentation (of an antigen) on a cell, such as an MHC (e.g. -1 ).

13. The use of a polynucleotide according to any preceding claim for: a. Stalling, halting, or interrupting translation, or of a translating ribosome; b. Causing ribosome collision, dissociation of ribosomal subunits and / or degradation of a nascent peptide; c. Presenting or increasing (presentation) of an antigen on an MHC; d. Maximising and / or increasing expression of an antigen, or increasing the efficiency of antigen presentation of an MHC; e. Activation of an immune response; and / or f. Preparation of a vaccine.

14. A method of triggering (or activating) the RQC (in a cell), the method comprising: a. Inserting a polynucleotide according to any preceding claim into a cell; b. Causing the polynucleotide according to any preceding claim to be (at least partially) translated (such as in a cell), optionally in a ribosome.

15. A vector comprising a polynucleotide according to claims 1 to 12.

16. A cell comprising a vector according to claim 15 or a polynucleotide according to any of claims 1 to 12.

17. A cell according to claim 16 which presents, or has on its cell surface, a protein (such as an antigen) as a result of the RQC mechanism (in that cell).

18. The use of the RQC mechanism in a cell to present an (heterogenous or foreign) protein (such as an antigen) on the surface of said cell.

19. A vaccine comprising a polynucleotide according to any of claims 1 to 12 or a vector according to claim 15.

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