Sarna backbones and methods of use

Abstract

Self-amplifying RNA (saRNA) vectors are disclosed. The described saRNA vectors are adapted from genotypically diverse alphaviruses. The described saRNA vectors are used in the expression of heterologous genes. Efficacious saRNA vaccines and therapies may be produced from such saRNA vectors.

Description

saRNA BACKBONES AND METHODS OF USECross-Reference to Related Applications

[0001] This application claims priority from US application No. 63 / 734,908 filed 17 December 2024 and entitled saRNA BACKBONES AND METHODS OF USE which is hereby incorporated herein by reference for all purposes. For purposes of the United States of America, this application claims the benefit under 35 U.S.C. §119 of US application No. 63 / 734,908 filed 17 December 2024 and entitled saRNA BACKBONES AND METHODS OF USE which is hereby incorporated herein by reference for all purposes.Field

[0002] The invention pertains to self-amplifying RNA (saRNA) vectors, in particular, those that are adapted from alphavriruses.Background

[0003] The synthetic messenger ribonucleic acid (mRNA) platform has received significant attention after the successful global production and distribution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines by Moderna and Pfizer-BioNTech. With the approved 30 - 100 pg mRNA dosage of these vaccines, individuals had to receive multiple booster shots to confer lasting immunity against SARS-CoV-2. The transient expression of the encoded gene of interest (GOI) and short half-life of mRNA are a double-edged sword by contributing to the safety profile but lead to low production of the encoded protein. To generate long-lasting immunity at a lower dose, synthetic mRNA may be redesigned to contain an alphavirus RNA-dependent RNA polymerase and conserved sequence elements to make a self-amplifying RNA (saRNA). Due to the replication properties that result in higher and longer encoded gene expression, saRNA is a suitable next-generation alternative to non-replicating mRNA. As such, various SARS-CoV-2 saRNA vaccines have been clinically approved to be administered at a 6 to 20-fold lower dose than their mRNA counterparts.

[0004] Alphaviral genomes consist of about 10-12 kb single-stranded, positive sense RNA segmented into two cistrons. The first cistron encodes for the non-structural proteins 1 through 4 (nsP1-4) that comprise the replication machinery. The second cistron, the subgenomic RNA, encodes for the structural proteins. In saRNA vectors derived from alphaviruses, the structural proteins are entirely removed and replaced by a gene of interest (or GOI(s)). Upon delivery into the cytosol, the positive sense genomic saRNA transcript is translated by host machinery to produce the non- structural polyproteins that are processed to form the replicase complex. Utilizing promoter sequences in the 3’ untranslated region (UTR), the replicase transcribes the full genomic minus strand. Subsequently, the replicase makes the full genomic positive strands using conserved sequence elements in the 5’ region of the minus strand. The replicase utilizes the subgenomic promoter to make the positive sense subgenomic RNA at a higher rate than the genomic strand. The subgenomic RNA is translated to produce the therapeutic GOI.

[0005] The design space of saRNA vectors is promising but is relatively unexplored. In a head-to-head comparison of Venezuelan Equine Encephalitis Virus (VEEV) and Semliki Forest Virus (SFV) RNA replicons encapsulated in modified dendrimer nanoparticles, expression of the HIV-1 Gag and Ebola glycoproteins was higher in the VEEV group. Similarly, 20-50% proliferation of T cells expressing an immunodominant epitope of cytoplasmic ovalbumin (cOVA) was observed in transgenic mice fourteen days after immunization with VEEV replicons expressing cOVA. In contrast, no proliferation was observed when using an SFV replicon. A prima facie assessment of this study shows that they compared replicons derived from the old- and new-world alphaviral families. Using a similar framework, another group (Dominguez et al) compared four virus-like replicons (VRPs) in different cell lines and noted that heterologous gene expression was cell-specific and influenced by the parental strain used. Notably, the use of VRPs for delivery may partially explain the difference in infectivity of the VRPs and is limited in in vivo application due to antivector immunity. Therefore, it is evident that the alphaviral source impacts the functionality of its cognate saRNA. Additionally, it is unknown to what extent the alphavirus properties as an infectious clone can be inferred to the functionality of its derived saRNA.

[0006] The inventors have recognised a general need for adapting different alphaviruses including new- and old-world viruses, into saRNA vectors, and in particular, those that are suitable for protein expression and vaccine applications.Summary

[0007] This application has a number of aspects. These include, without limitation:• Genetically diverse alphaviral saRNA vectors for use in the expression of heterologous genes.• Use of the described saRNA vectors in the manufacturing of a medicament for treating, inhibiting and / or ameliorating a disorder and / or disease in a subject.

[0008] Some aspects of the invention pertain to a self-amplifying RNA (saRNA) vector. The saRNA vector comprises a nsPs sequence which encodes non-structural proteins (nsPs), a 5’-UTR sequence located upstream of the nsPs sequence, a 3- UTR sequence located downstream of the nsPs sequence, a subgenomic promoter (SGP) sequence between the nsPs sequence and the 3’-UTR, a gene of interest (GOI) sequence between the subgenomic promoter sequence and the 3’-UTR, wherein the gene of interest sequence encodes at least one heterologous protein, a 5’-cap upstream of the 5’-UTR sequence, and a poly(A) tail downstream of the 3’- UTR sequence. The non-structural proteins comprise nsP1 , nsP2, nsP3 and nsP4 proteins.

[0009] In some embodiments, the 5’-UTR sequence comprises a nucleotide sequence of one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45, or a nucleotide sequence having at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

[0010] In some embodiments, the subgenomic promoter sequence comprises a nucleotide sequence of one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59, or a nucleotide sequence having at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% sequence identity to any one ofSEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

[0011] In some embodiments, the 3’-UTR sequence comprises a nucleotide sequence of one of SEQ ID NO: 19-20, 26-29, 36-38, and 46-52, or a nucleotide sequence having at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

[0012] In some embodiments, the nsPs sequence and / or 5’-UTR and / or 3’-UTR, and / or subgenomic promoter sequence comprises or is derived from a species of alphaviruses selected from the group consisting of Tonate virus, Chikungunya virus, O’nyong-nyong virus, Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus, Barmah Forest virus, Mayaro virus, Ross River virus, Semliki Forest virus, and Sindbis virus.

[0013] In some embodiments, the gene of interest (GOI) sequence encodes a therapeutic protein. In some embodiments, the gene of interest (GOI) sequence encodes a chimeric antigen receptor (CAR) protein.

[0014] Some aspects of the invention pertain to use of the described saRNA vector in the manufacture of a medicament for treating, inhibiting and / or ameliorating a disorder and / or disease in a subject. The disorder and / or disease may comprise a cancer or a viral infection. In some embodiments, the medicament comprises a vaccine.

[0015] Further aspects and example embodiments are illustrated in the accompanying drawings and / or described in the following description.

[0016] It is emphasized that the invention relates to all combinations of the above features, even if these are recited in different claims.Brief Description of the Drawings

[0017] The accompanying drawings illustrate non-limiting example embodiments of the invention.

[0018] Figure 1 is a schematic diagram illustrating a saRNA vector according to an example embodiment of the invention.

[0019] Figure 2 are results from the transfection of saRNA-eGFP BHK-21 cells and innate sensing in A549-Dual reporter cells. Figure 2a is a schematic illustrating the process of designing saRNA vectors. Figure 2b illustrates a rooted phylogenetic tree based on amino acid sequence of the alphaviral replicases used in this study. Bolded virus names resulted in competent saRNA vectors. Figure 2c show the results from an XTT assay showing viability of BHK cells after transfection with saRNA relative to a lipofectamine only control. Data shown are mean (SD) from n = 3 compared to VEEV_TrD using Two-way ANOVA followed by Dunnett’s multiple comparison test. Figure 2d is a plot showing the percentage of viable cells as a function of the tested saRNA vectors. Figure 2e is a plot showing the percentage of eGFP+ cells as a function of the tested saRNA vectors. Figure 2f is a plot showing the corresponding geometric mean fluorescence intensity 24 hours after transfection with 500 ng of saRNA as a function of the tested saRNA vectors. Data shown are mean (SD) from n = 3 compared to VEEV_TrD using One-way ANOVA. Figure 2g is a plot showing fold change of eGFP transcripts in total RNA of BHK cells at different time points after transfection as a function of the tested saRNA vectors. Data shown are mean (SD) from n = 3 compared to 4 h using Two-way ANOVA followed by Dunnett’s multiple comparison test. Figure 2h is a plot showing NF-KB induction relative to lipofectamine only control as determined using a secreted alkaline phosphatase assay at 4 and 24 h post-transfection as a function of the tested saRNA vectors. Figure 2i is a plot showing relative luminescence indicating levels of IRF3 / 7 induction 4 h (left) and 24 h (right) post-transfection as a function of the tested saRNA vectors. Data shown are mean (SD) from n = 3 compared to VEEV_TrD using One-way ANOVA followed by T ukey’s multiple comparison test. *P < 0.05; **P < 0.01 ; ***P < 0.001 ; ****p < 0.0001 .

[0020] Figure 3 are results showing saRNA-Fluc expression and subsequent cytokine response in BALB / cJ mice. Figure 3a are representative bioluminescence images of mice after bilateral intramuscular injection with 5 pg of saRNA-Fluc in LNPs. Figure 3b is a plot of total flux obtained from mice as a function of days post-injection. The data shown are mean (SD) from n = 6 of biologically independent animals from twostudies. Figures 3c, 3d, and 3e are plots of the area under the curve (AUC) of total flux curves from 2-6 or 8-33 days or for the duration of the study, respectively for each of the tested saRNA vectors. The data shown are mean (SEM) compared using Brown-Forsythe ANOVA followed by Dunnett’s multiple comparison test. Figure 3f is a plot showing fold change in systemic cytokines relative the PBS controls, 4 hours after injections. Data shown are mean (SD) from n = 3 and compared to VEEV_TrD using One-way ANOVA followed by Dunnett’s multiple comparison test. Figure 4g is a plot showing the concentration of cytokines whose relative levels could not be calculated as PBS controls were below the lower detection limit. *P < 0.05; **P < 0.01.

[0021] Figure 4 are results showing saRNA expression in 1 x 1 cm2human skin explants. Figure 4a are bioluminescence images of skin explants after injection of 2.5 pg of saRNA-Fluc vectors encapsulated in LNPs. Figure 4b is a plot showing total flux obtained from the image in Figure 4a as a function of 2, 4 and 7 days post-injection. Dotted line represent the background flux from skin explants injected with PBS.Figure 4c is a plot showing total Flue expression presented as area under the curve of total flux from day 2 to 7 as a function of the tested saRNA vectors. Figure 4d is a plot showing relative eGFP transcript levels present in total RNA extracted from skin explants on the 4thday after injection with saRNA-eGFP vectors encapsulated in LNPs. Data shown are mean ± SD from n = 3 compared using One-way ANOVA followed by Tukey’s multiple comparison test. *P< 0.05; ****p < 0.0001 .

[0022] Figure 5 are results illustrating humoral and cellular immunity of saRNA-spike vaccines in C57BL / 6J mice with n = 5 per group. Figure 5a is a schematic representation of the vaccination study in which mice were intramuscularly injected with 0.5 or 5 pg of saRNA-spike encapsulated in LNPs. Four weeks after the priming injection, mice were bled and boosted with the same dosage received at priming. At six weeks, the study was terminated, and spleens and blood were obtained from all groups. Figures 5b and 5c are plots showing concentration of anti-spike IgG in blood sera at 4 and 6 weeks, respectively, as determined by ELISA for all groups immunized with 0.5 and 5 pg. Dotted line indicates the PBS control levels. Figure 5d is a plot showing fold change in anti-spike IgG titres from 4 to 6 weeks after thebooster injection. Figure 5e is a plot showing ratio of lgG2c to I gG 1 of all groups. Dotted line indicates lgG2c / lgG1 = 1. Figure 5f is a plot showing percent (%) neutralization of virus reporter particles pseudotyped with SARS-CoV-2 spike protein. % neutralization obtained as a percentage decrease Renilla luciferase signal compared to PBS groups. Figure 5g is a plot showing quantification of spike-specific splenocytes producing IFN-y. Black and blue dotted lines indicate results from PBS- control splenocytes stimulated with spike peptides and Concanavalin A, respectively. All data shown are mean (SD) from n = 5 independent animals and compared using Two-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05; **P < 0.01 ; ***P < 0.001 ; ****P < 0.0001 .

[0023] Figure 6 are results from comparing heterologous and homologous vaccination of C57BI mice with VEEV_TC83 (VE) and CHIKV (CH). Figure 6a is a schematic illustrating the design of the vaccination study in which fifteen mice received a priming injection of VEEV_TC83 or CHIKV encoding SARS-Cov-2 spike on Day 0. On Day 28, 5 mice from each vaccination group was randomly assigned to either no boost or a homologous or heterologous booster injection. Figure 6b is a plot showing concentration of anti-spike IgG in blood sera on Day 42 as determined by ELISA. Figure 6c is a plot showing terminal anti-spike IgG titres relative to Day 28 titres before boosting to demonstrate boosting benefits. Figure 6d is a plot showing ratio of lgG2c to I gG 1 of all groups. Figure 6e is a plot showing percent neutralization of virus reporter particles pseudotyped with SARS-CoV-2 spike protein. Figure 6f is a plot showing the number of spike-specific splenocytes producing IFN-y. Black dotted line indicates results from PBS-control splenocytes stimulated with spike peptides. All data shown are mean (SD) from n = 5 independent animals and compared using Two-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05; **P < 0.01 ; ***P < 0.001 ; ****P < 0.0001 .

[0024] Figure 7 illustrates a correlation summary of platforms that were assessed in the experiments. Figure 7a shows a summary of platforms used to assess saRNA vectors in this study. Figure 7b is a heat map of Pearson’s correlation coefficient between mean fluorescence intensity of EGFP-positive BHK cells (MFI), Balb / c mice total luminescence (Flux-M), C57BL / 6 terminal anti-spike IgG titres (IgG) and humanskin explants total luminescence (Flux-S).

[0025] Figure 8 is a schematic diagram of a BFV-eGFP-saRNA plasmid construct according to an example embodiment.

[0026] Figure 9 is a schematic diagram of a CHlKV-eGFP-saRNA plasmid construct according to an example embodiment.

[0027] Figure 10 is a schematic diagram of an EEEV-eGFP-saRNA plasmid construct according to an example embodiment.

[0028] Figure 11 is a schematic diagram of a MAYV-eGFP-saRNA plasmid construct according to an example embodiment.

[0029] Figure 12 is a schematic diagram of a ONNV-eGFP-saRNA plasmid construct according to an example embodiment.

[0030] Figure 13 is a schematic diagram of a RRV-eGFP-saRNA plasmid construct according to an example embodiment.

[0031] Figure 14 is a schematic diagram of a SFV-eGFP-saRNA plasmid construct according to an example embodiment.

[0032] Figure 15 is a schematic diagram of a SINV-eGFP-saRNA plasmid construct according to an example embodiment.

[0033] Figure 16 is a schematic diagram of a TONV-eGFP-saRNA plasmid construct according to an example embodiment.

[0034] Figure 17 is a schematic diagram of a WEEV-eGFP-saRNA plasmid construct according to an example embodiment.

[0035] Figure 18 is a schematic diagram of a VEEV_TC83-eGFP-saRNA plasmid construct according to an example embodiment.

[0036] Figures 19a, 19b and 19c are plots illustrating the results of co-transfection of different saRNA-eGFP constructs with VEEV_TC83 mCherry. Figure 19a is a plotshowing the percentage of eGFP positive cells. Figure 19b is a plot showing the percentage of TC83 mCherry positive cells. Figure 19c is a plot showing the percentage of double-positive cells (mCherry+; eGFP+) in the co-transfected samples.

[0037] Figures 20a and 20b are plots illustrating the geometric mean fluorescence intensity of (a) eGFP or (b) mCherry in single or double positive cells after cotransfection of different saRNA-eGFP with VEEV_TC83 mCherry.

[0038] Figure 21 illustrates PCR efficiencies of the NSP1 , eGFP barcode region of different saRNA constructs.

[0039] Figure 22 illustrates results from the validation of using qPCR to compare the different yields from a reaction containing pooled saRNA constructs.

[0040] Figure 23 illustrates flow cytometry results from experiments comparing CAR- saRNA constructs.Detailed Description

[0041] Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive sense.Definitions

[0042] The term “strain” refers to a genetic variant or subtype of a microorganism such as a bacterial strain or a specific strain of a virus.

[0043] The term “upstream” means the direction towards the 5’ end of a nucleic acid sequence.

[0044] The term “downstream” means the direction towards the 3’ end of a nucleicacid sequence.

[0045] A “5’-cap” refers to a cap structure that is present at the 5’ end of the saRNA molecule. The 5’-cap may include a modified guanine (G) nucleotide, which is attached to the saRNA by linkage to an unusual triphosphate at the 5' end.Guanosine can be methylated at position 7, and is, for example, m 7 G or 3'-O-Me-m 7 G. A 5’-cap may include a naturally occurring RNA 5' cap, e.g., 7-methylguanosine (m7G), or 5' end cap analogs that resemble RNA end cap structures and are attached to RNA and, for example, modified to have the ability to stabilize RNA in vivo and / or in cells.

[0046] A "polyadenylation sequence" or "poly(A) tail" means an mRNA region downstream of the 3'-UTR that includes multiple consecutive adenosine monophosphates, e.g., directly downstream of the 3'-UTR. The poly (A) tail can include 10 to 300 adenosine monophosphates.

[0047] The term “5'-untranslated region (5'-UTR)" means the saRNA region that does not encode a polypeptide, directly upstream of the first codon of the RNA transcript to be translated by ribosomes (i.e. , 5'), i.e. , the initiation codon.

[0048] The term "3'-untranslated region (3'-UTR)" means the saRNA region that does not encode a polypeptide, directly downstream (i.e., 3') of the codon that signals the termination of translation of the RNA transcript, i.e., a stop codon.

[0049] The term "derived from" as used in the context of a nucleic acid, i.e. for a nucleic acid "derived from" (another) nucleic acid, means that the nucleic acid, which is derived from (another) nucleic acid, shares e.g. at least 40%, 50%, 60%, 70%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid from which it is derived.

[0050] The term “sequence identity” means determining the relationship between at least two nucleotide sequences by comparing the sequences. Identity indicates the level of sequence relatedness as determined by the number of matches between lists of amino acid residues or nucleotide residues. The identity of related polypeptides orpolynucleotides can be calculated by known methods. When applied to polynucleotides, “% identity” or "percent identity" is defined as the difference between a candidate sequence of nucleotides after aligning it with a second sequence for maximum percent identity. The percentage of residues identical to those of the second sequence, with gaps introduced where necessary. Methods and programs for alignment are known in the art. The program can be, for example, BLAST, the Smith- Waterman algorithm, or the Needleman-Wunsch algorithm, etc.

[0051] A “chimeric antigen receptor” or “CAR” is an artificial engineered receptor comprising parts of antigen receptors. Antigen receptors are surface receptors that are able to bind antigens or epitopes. B-cell and T-cell receptors are examples of antigen receptors. A non-limiting example of a chimeric antigen receptor is a hybrid receptor that comprise domains from T-cell or B-cell receptors fused to major histocompatibility complex (MHC) domains and / or antigenic peptide sequences. A chimeric antigen receptor comprises a target binding moiety that specifically binds to a desired target peptide or antigen.

[0052] As used herein, unless the position of the nucleotide sequence is indicated otherwise, the nucleotide sequence is linked from the 5' end to the 3' end or located from the 5' end to the 3' end.Example Embodiments

[0053] Aspects of the present invention pertain to saRNA vectors for use in the expression of heterologous genes. The saRNA vector comprises or is derived from a positive stranded RNA virus. In some embodiments, the positive stranded RNA virus is an alphavirus. The saRNA vectors of the present invention are produced by adapting genetically diverse alphaviruses into vectors. In some embodiments, the saRNA vector comprises or is derived from one of the following species of alphaviruses: Tonate virus, Chikungunya virus, O’nyong-nyong virus, Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus, Barmah Forest virus, Mayaro virus, Ross River virus, Semliki Forest virus, Sindbis virus, Aura, Bebaru virus, Cabassou, Fort Morgan, Getah virus, Kyzylagach, Middleburg,Mucambo virus, Ndumu, Pina virus, virus Triniti, Una, Whataroa, and Y-62-33.

[0054] Proof-of-concept experiments have demonstrated that efficacious saRNA vaccines and therapies, as supported by high transgene expression and / or low immunogenicity, can be produced using the described saRNA vectors.

[0055] Figure 1 illustrates a saRNA vector according to an example embodiment of the invention. The saRNA vector comprises a sequence which encodes non-structural proteins (nsPs). The non-structural proteins (nsPs) comprise nsP1 , nsP2, nsP3 and nsP4 proteins. Such a sequence may be referred to herein as a “nsPs Sequence”. nsP1 protein is a viral capping enzyme and is believed to facilitate the anchoring of the replication complex to the host membrane. nsP2 protein is believed to provide helicase, triphosphatase, and protease activity. nsP3 protein is believed to facilitate with host cells factors during replication. nsP4 protein is believed to be a RNA- dependent RNA polymerase (RdRP). The nsP1 , nsP2, nsP3 and nsP4 proteins form a replicative complex.

[0056] In some embodiments, the saRNA vector comprises a nsPs Sequence which comprises or is derived from a sequence from a genome of one or more species or strains of an alphavirus which encodes the nsPs. The nucleotide sequence may be a wild-type alphavirus sequence, and / or a variant of the wild-type alphavirus sequence. In some embodiments, the nsPs Sequence is derived from one alphavirus species or strain. In some embodiments, the nsPs Sequence is derived from a plurality of alphaviruses variants to form a chimera or recombinant nsPs protein. The sequences which encode the nsP1 , nsP2, nsP3, and nsP4 proteins may comprise or may be derived from more than one alphavirus strain. For example, the nsPs sequence may encode a chimera or recombination nsPs protein. The chimera or recombinant nsPs protein comprises a first sequence which encodes the nsP1 and nsP2 proteins derived from one alphavirus species or strain, and a second sequence which encodes the nsP3 and nsP4 proteins derived from another alphavirus species or strain. However, this is only an example as an illustration. Other combinations of sequences from different alphavirus species or strains to form a chimera or recombinant nsPs protein are within the scope of the invention. In some example embodiments, thensPs Sequence comprises or is derived from one or more alphavirus species or strains listed in Table 1.Table 1. Examples of alphaviruses from which the saRNA vector may comprise or may be derived.

[0057] The saRNA vector comprises a 5’-UTR sequence located upstream of the nsPs Sequence. The 5’ untranslated region (5’-UTR) sequence may be located directly upstream of the sequence which encodes the nsP1 protein. In some embodiments, the 5’-UTR sequence of the saRNA vector comprises or is derived from a 5’-UTR sequence from a genome of a species or strains of an alphavirus. The nucleotide sequence may be a wild-type alphavirus sequence, and / or a variant of the wild-type alphavirus sequence. In some embodiments, the 5’-UTR sequence of the saRNA vector comprises a nucleotide sequence listed in Table 2, i.e. , any one of SEQ ID NO: 13-15, 21-23, 30-32, 39-45 or a nucleotide sequence having at least 40%, or at least 50%, or at least 60% or at least 70% sequence identity, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.Table 2. Example 5’-UTR Sequences in the saRNA Vector

[0058] In some example embodiments, the 5’-UTR sequence of the saRNA vector is a sequence which comprises or is derived from the 5’-UTR sequence from the genome of one of the alphavirus species or strains listed in Table 1. In some exampleembodiments, the 5’-UTR sequence of the saRNA vector comprises a nucleotide sequence which has at least 40%, at least 50%, or at least 60% or at least 70% sequence identity, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to the 5’-UTR sequence from the genome of one of the alphavirus species or strains listed in Table 1 .

[0059] The saRNA vector comprises a 3’ untranslated region (3’-UTR) sequence located downstream of the nsPs Sequence. In some embodiments, the 3’-UTR sequence of the saRNA vector comprises or is derived from a 3’-UTR sequence from a genome of a species or strains of an alphavirus. The nucleotide sequence may be a wild-type alphavirus sequence, and / or a variant of the wild-type alphavirus sequence. In some embodiments, the 3’-UTR sequence of the saRNA vector comprises a nucleotide sequence listed in Table 3, i.e., any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52, or a nucleotide sequence having at least 40%, at least 50%, or at least 60% or at least 70% sequence identity, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.Table 3. Example 3’-UTR Sequences in the saRNA Vector

[0060] In some example embodiments, the 3’-UTR sequence of the saRNA vector is a sequence which comprises or is derived from the 3’-UTR sequence from the genome of one of the alphavirus species or strains listed in Table 1. In some example embodiments, the 3’-UTR sequence of the saRNA vector comprises a nucleotide sequence which has at least 40%, at least 50%, or at least 60% or at least 70% sequence identity, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to the 5’-UTR sequence from the genome of one of the alphavirus species or strains listed in Table 1 .

[0061] The saRNA vector comprises a subgenomic promoter (SGP) sequence located downstream of the nsPs Sequence, between the nsPs Sequence and the 3’- UTR sequence. In some embodiments, the subgenomic promoter sequence is located directly downstream of the sequence which encodes the nsP4 protein. Insome embodiments, the subgenomic promoter sequence of the saRNA vector comprises or is derived from a subgenomic promoter sequence from a genome of a species or strains of an alphavirus. The nucleotide sequence may be a wild-type alphavirus sequence, and / or a variant of the wild-type alphavirus sequence. In some embodiments, the subgenomic promoter sequence of the saRNA vector comprises a nucleotide sequence listed in Table 4, i.e., any one of SEQ ID NO: 16-18, 24-25, 33- 35, and 53-59 or a nucleotide sequence having at least 40%, at least 50%, or at least 60% or at least 70% sequence identity, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.Table 4. Example Subgenomic promoter sequences in the saRNA Vector

[0062] In some example embodiments, the subgenomic promoter sequence of the saRNA vector is a sequence which comprises or is derived from the subgenomic promoter sequence from the genome of one of the alphavirus species or strains listed in Table 1. In some example embodiments, the subgenomic promoter sequence of the saRNA vector comprises a nucleotide sequence which has at least 40%, at least 50%, or at least 60% or at least 70% sequence identity, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to the subgenomic promoter sequence from the genome of one of the alphavirus species or strains listed in Table 1.

[0063] In some embodiments, the saRNA vector comprises a gene of interest (GOI) sequence which encodes at least one heterologous protein. The gene of interest sequence may be located downstream of the subgenomic promoter sequence, and upstream of the 3’-UTR. In some embodiments, the gene of interest sequence is located between the subgenomic promoter sequence and the 3’-UTR.

[0064] In some embodiments, the heterologous protein comprises a therapeutic protein. A "therapeutic protein" is a protein that has a therapeutic effect when introduced into a eukaryote (e.g., a mammal such as a human). Exemplary therapeutic proteins may include proteins and peptides derived from pathogens, such as bacteria, viruses, fungi, protozoa / or parasites. In some embodiments, theheterologous protein comprises an antigen such as a viral antigen that is derived from a virus, or a bacterial antigen that is derived from bacteria, or a fungal antigen that is derived from a fungus, or a protozoan antigen that is derived from a protozoa, or a plant antigen that is derived from a plant, etc. The therapeutic protein may be an immunogen or an antigen such as a tumour immunogen or antigen or cancer immunogen or antigen. The therapeutic protein may be a eukaryotic polypeptide, such as a mammalian polypeptide. The mammalian polypeptide may for example be an enzyme, an enzyme inhibitor, a hormone, an immune system protein, a receptor, a binding protein, a transcription or translation factor, tumour growth supressing protein, a structural protein, a blood protein, etc.

[0065] In some embodiments, the gene of interest sequence comprises a CAR sequence which encodes a chimeric antigen receptor (CAR) protein.

[0066] The saRNA vector additionally comprises a 5’-cap located directly upstream of the 5’-UTR, and a poly-A tail located directly downstream of the 3’-UTR.

[0067] The sequence of the saRNA vector or a portion thereof may be chemically modified to optimize RNA structural stability and / or translation efficiency and / or reducing innate response, etc. with the goal for example, of improving therapeutic properties. Examples of suitable modification techniques include RNA methylation modification, such as 5-methylcytidine (5mC) modification; however, it will be known that other suitable types of nucleotide modifications may also be used.

[0068] The invention is further described with reference to the following specific examples, which are not meant to limit the invention, but rather to further illustrate it.EXAMPLESExample 1

[0069] The twelve alphaviruses that are listed in Table 1 above were adapted into the described saRNA vectors. All sequences for the alphavirus genus (taxid: 11019) were obtained from the National Center for Biotechnology Information - virus database. For down selection, the inventors filtered out viruses with incomplete nucleotidesequences, excluded those that exclusively infect insects and fish and prioritized those that have been isolated in humans, pigs and hosts. Additionally, the inventors intentionally avoided isolates that have previously been used as viral replicons or saRNA to express heterologous proteins except for the control saRNA. Of the remaining viruses, exemplar variants that are well-characterized isolates as determined by the International Committee on Taxonomy of viruses were chosen except for the Venezuelan equine encephalitis virus (VEEV) groups. For the control vector, the inventors chose the Trinidad donkey isolate of the VEEV species due to its wide application in pre-clinical and clinal studies. Additionally, the inventors included the TC-83 vaccine strain of the VEEV virus. VEEV_TC83 is an attenuated variant derived from VEEV_TrD by serial dilution in guinea-pig heart cells. Phylogenetic analysis on the amino acid sequence of the non-structural proteins (Figure 2b) was performed using a neighbour-joining algorithm, thus showing the diverse range of vectors that were assessed in baby hamster kidney (BHK) cells. All other components of each virus such as the untranslated region and the non-structural proteins were unmodified. The saRNA vectors were then cloned into a plasmid designed for in vitro transcription. Figures 8 to 18 are schematic diagrams of exemplary saRNA plasmid constructs designed for use in the experiments. The gene of interest (GOI) sequence in each of these illustrated saRNA constructs comprise a reporter gene sequence which encodes Enhanced Green Fluorescent Protein (EGFP).Screening of alphaviruses in BHK cells

[0070] BHK cells (2 x 104cells / well) were transfected with different amounts of saRNA, and their viability determined using XTT assay after 24 hours (Figure 2h). The XTT assay indicated a reduction of viable cells relative to the un-transfected control at the highest saRNA concentration (250 and 500 nanograms). Compared to the VEEV_TrD control, all vectors showed relatively similar levels of viability after transfection with saRNA (Figure 2c). For further evaluation on the performance of the saRNA-eGFP vectors, equimolar amounts (~500 nanograms) were transfected into 2.5 x 105BHK-21 cells using lipofectamine and flow cytometry was performed after 24 h. Flow cytometry showed that all saRNA transfections resulted in a drop in viability (< 95%) except for the EEEV, BFV, MAYV, RRV, and SFV constructs (Figure 2d).VEEV_TC83, TONV, CHIKV and ONNV resulted in more GFP+ cells compared to the VEEV_TrD control by 22%, 46%, 28% and 15%, respectively (Figure 2e). The GFP+ cells transfected with VEEV_TC83 and TONV showed 1.5- and 2.7-fold higher mean fluorescence intensity (MFI) relative to VEEV_TrD. GFP+ cells transfected with CHIKV and ONNV had similar MFI levels as VEEV_TrD (Figure 2f). Surprisingly, saRNA vectors derived from the selected EEEV, WEEV, SFV, SI NV, BFV, MAYV and RRV variants resulted into little to no eGFP-positive cells. For the vectors that showed less than 2% GFP+ cells, a high amount of saRNA was transfected into BHK cells, but in each case did not result in an increase GFP-positive cells. Considering that all vectors were derived from alphaviruses with the capacity to replicate in mammalian cells, more so in BHK cells that are permissive to viral infections, it was suspected that the replicases were defective. Relative to the 4thh after transfection, qPCR analysis confirmed that no amplification of the saRNA occurred by the 24thh but rather a significant decrease in transcripts detected. Henceforth, all experiments were performed using the VEEV_TrD, VEEV_TC83, TONV, CHIKV and ONNV- derived saRNA vectors.

[0071] Differences in GFP+ cells and their respective MFI after transfection of equal saRNA copy numbers pointed towards different rates of saRNA replication, translation, and interaction with other host cellular mechanisms. Therefore, the inventors sought to characterize the relative replication of the genomic and subgenomic mRNA using amplicons in the eGFP region. At 4 h post-transfection, similar levels of eGFP were detected for all saRNA constructs (Figure 2g). Afterwards at 12 and 24 h, the eGFP transcripts increased significantly thus indicating high levels of saRNA amplification in all transcripts. To decouple the genomic and subgenomic replication, the inventors sought to quantify the levels of nsP1-containing transcripts. However, a direct comparison between the constructs could not be performed due to use of different primer pairs.

[0072] Given the differences in saRNA genomic and subgenomic replication as determined by qPCR, the inventors sought to characterize the innate immune activation through the NF-KB and interferon regulatory factor (IRF) 3 / 7 pathways using A549-Dual reporter cell line. A549-Dual cells stably express secretedembryonic alkaline phosphatase (SEAP) under the control of the IFN-p minimal promoter fused to five NF-KB binding sites. Additionally, the cells stably express secreted Lucia luciferase under the control of an ISG54 minimal promoter in conjunction with five IFN-stimulated response elements. Considering that the saRNA generated via in vitro transcription and purified by lithium chloride precipitation, the inventors quantified the relative amount of double-stranded RNA due to their characterization as pathogen-associated molecular patterns. Dot blot analysis indicated that all saRNA tested had similar levels of dsRNA content. At 4 h posttransfection of the A549-Dual cells, NF-KB or IRF3 / 7 induction by the different saRNA constructs was similar and close to the lower detection limit of the assay (Figures 2h- i). 24 h later, NF-KB induction was highest in cells transfected with VEEV_TrD and VEEV_TC83. ONNV resulted in the highest induction of IRF3 / 7. TONV had substantially lower levels of NF-KB and IRF3 / 7 induction in A549 Dual cells despite having higher levels of genomic and subgenomic RNA than the VEEV_TrD. Similarly unexpected, lower NF-KB and same IRF induction levels were observed in the CHIKV group when compared to VEEV_TrD considering that CHIKV had 2-3 higher saRNA replication. These results indicated that saRNA vectors activated the innate immunity via multiple axes at different levels as dictated by the parental alphavirus properties. saRNA vector design modulates luciferase expression BALB / c mice

[0073] Having established significant difference in transgene expression in vitro from VEEV_TrD, VEEV_TC83, TONV, CHIKV and ONNV, the inventors sought to compare their expression profiles of firefly luciferase (Flue) in BALB / cJ mice in vivo. A lipid nanoparticle (LNP) formulation was used to deliver all saRNA-Fluc vectors. After bilateral injection of 5 pg LNPs and phosphate buffer saline (PBS) as a control, bioluminescence images were obtained for all groups (Figure 3a) and quantified as total flux (Figure 3b). saRNA-Fluc vectors from VEEV_TrD, VEEV_TC83, TONV, CHIKV and ONNV had similar global maxima of bioluminescence on the first day of in vivo imaging and declined till day 6. Similarly, the area under the curve (AUC) of total flux for the first 6 days showed no difference in luciferase expression between the saRNA vectors (Figure 3c). However, the recovery pattern of luminescence diverged afterwards. ONNV-Fluc continuously declined and reached background levels by theeighth day. VEEV_TrD, VEEV_TC83, TONV and CHIKV saRNA-Fluc had local maxima occurring after the 8thpost-injection. VEEV_TrD and CHIKV saRNA Flue declined to background levels around day 22. VEEV_TC83 declined but steadied from day 18 to the end of the study. TONV-Fluc showed expression throughout the study but declined steadily at a lower rate compared to the other vectors. The difference in kinetics after the 8thwas also evident by a significant difference in AUC by VEEV_TC83 and TONV vectors when compared to the control saRNA (Figure 3d). Ranking of total Flue expression as presented by total area under the total flux curve for the duration of the study was VEEV_TC83 - CHIKV > TONV > ONNV > VEEV_TrD with only TONV being significant (Figure 3e).

[0074] The inventors further explored the impact of LNP-saRNA administration on systemic pro-inflammatory cytokines. Assessment of the physical properties of the LNPs indicated that the particles had relatively similar sizes, polydispersity indices and zeta potential. As particle characteristics were nearly identical, the inventors assumed that differences observed could be attributed to the encapsulated saRNA vector. 4 h after bilateral intramuscular injection of LNPs containing 5 pg of saRNA, mice were bled, and the serum used to quantify the levels of systemic pro- inflammatory cytokines. Administration of the different saRNA vectors encapsulated in LNPs resulted in elevated levels of pro-inflammatory cytokines relative to the PBS- injected controls (Figures 3f-g). TONV- and ONNV-Fluc resulted in significantly lower IP-10, MIP-ip and MCP-1 levels compared to the VEEV_TrD control.Vector design modulates saRNA expression in human skin tissue explants

[0075] After observing that VEEV_TrD, VEEV_TC83, TONV and ONNV constructs resulted in high eGFP expression of in mammalian cells and similar trends observed in bioluminescence studies in mice, the inventors assessed Flue expression in human skin explants. Results generated using preclinical murine models often fail to translate into efficient therapies in humans. Human skin explants provide a proxy for the tissue architecture and immune landscape in humans compared to small animals, hence their suitability for ex vivo surveys of saRNA therapeutics. Similarly, the inventors injected LNPs containing 2.5 pg of saRNA-Fluc intradermally into the skin explants.Bioluminescence imaging indicated that all vectors resulted in Flue expression in human skin explants. By the seventh day, all saRNA-Fluc vectors had returned to baseline except for VEEV_TC83 (Figure 4b). The total protein expression over the course of the experiment showed that only VEEV_TC83 resulted in significantly higher expression than the control saRNA (Figure 4c). Additionally, qPCR on total RNA from skin explants showed no significant difference in the relative quantity of genomic and subgenomic transcripts on the 4thday after saRNA-LNP intradermal injections (Figure 4d).Standard prime-boost vaccination with saRNA-spike vectors modulates humoral and cellular immunity in a preclinical murine model

[0076] Having demonstrated that the Flue expression kinetics in mice vary with the vector design, the inventors sought to determine the immunogenicity of the saRNA vectors encoding the SARS-CoV-2 spike antigen. The vaccine study was performed in C57BL / 6J mice by priming with LNPs containing 0.5 or 5 pg saRNA-spike, boosted at week 4 after primary vaccination and terminated by week 6 (Figure 5a). Serum samples were collected before boosting and at endpoint to assess Anti-spike immunoglobulin G (IgG) was assessed on serum samples collected at 4 weeks after priming (Figure 5b) and 2 weeks after booster (Figure 5c) immunization.

[0077] Primary and booster vaccination with 0.5 pg of VEEV_TrD generated detectable but lower anti-spike IgG antibodies. However, primary vaccination with 5 pg VEEV_TrD resulted in higher IgG titres which did not increase after booster injections. Vaccination with VEEV_TC83 resulted in a clear dose response between the 0.5 and 5 pg immunizations with both primary and booster injections leading to high anti-spike IgG titres. CHlKV-spike vaccination resulted in high levels of anti-spike IgG titres before and after booster injections. No dose response was observed in mice vaccinated with CHIKV vectors. Vaccination with 0.5 pg of TONV-spike resulted in detectable but low anti-spike IgG titres before and after boosting. However, 5 pg vaccination TONV resulted in high IgG titres that increased significantly after boosting. Primary vaccination with 0.5 or 5 pg of ONNV-spike generated low antispike IgG at week 4. However, booster injections of 0.5 and 5 pg of ONNV-spikeresulted in > 100-fold increase of IgG titres (Figure 5d).

[0078] Further assessment of anti-spike lgG1 and lgG2c subclasses recapitulated the trends observed in endpoint IgG titres. However, the lgG2c to lgG1 ratios indicated varied biases towards Th1- or Th2- immunity (Figure 5e). It was expected that saRNA vaccination would result in a Th1-biased immunity as shown in previous pre-clinical and clinical studies. At 0.5 pg dose, VEEV_TrD vaccination showed generated equivalent levels of lgG1 and lgG2c thus inferring to a balanced Th1-Th2 immunity while 5 pg dosage generated a Th1-biased immunity. Vaccination with VEEV_TC83 and CHIKV resulted in a Th1-biased immunity at both doses. 0.5 pg vaccination with TONV-spike resulted in Th1-biased immunity while 5 pg generated a balanced Th1- Th2 immunity. Vaccination of mice with ONNV-spike at both doses resulted in a balanced Th1-Th2 immunity.

[0079] To assess the neutralization capacity of the anti-spike antibodies generated after vaccination, endpoint serum samples were used to neutralize infection of SARS- CoV-2 pseudotyped viral reporter particles into HEK-293T cells expressing human ACE2 receptor. VEEV_TrD, VEEV_TC83 and TONV vaccinations showed a significant difference in pseudovirus inhibition at the two doses (Figure 5f). Meanwhile, 0.5 and 5 pg of CHIKV and ONNV resulted in relatively similar levels of pseudovirus neutralization. It was observed that VEEV_TrD vaccination at 0.5 and 5 pg had resulted in similar titres of anti-spike IgG, but this did not register in the neutralization assay as 0.5 pg generated fewer neutralizing antibodies than the 5 pg dose. Contrary to the VEEV_TrD observation, ONNV-spike vaccination at 0.5 pg resulted in ~100-fold lower IgG titres than the 5 pg dose at endpoint but generated similar levels of neutralizing antibodies as the 5 pg vaccination.

[0080] The inventors stimulated splenocytes with overlapping peptides of SARS-CoV- 2 spike glycoprotein and performed an IFN-y ELISpot assay. A clear dose response was observed in all groups except the VEEV_TC83 group (Figure 5g). All vaccinated groups resulted in higher IFN-y-producing splenocytes than the control group thus indicating the capacity of all vectors to induce a robust cellular immunity in a vaccine regimen. The results show that ONNV induced higher cellular responses than theother groups. This is notable since it was relatively lower initially with the antibody response. This may suggest that ONNV may be a more useful vector for vaccines where a robust T cell response, like a personalized cancer vaccine is needed.Heterologous vector vaccination using CHIKV-VEEV saRNA-spike results in higher humoral immunity compared to other vaccination regimens

[0081] Having established that homologous prime-boost vaccination against the spike antigen using VEEV_TC83 (VE) and CHIKV (CH) vectors resulted in the highest humoral immunity at the low and high dose, the inventors sought to characterize the performance of heterologous boosting regimens. Therefore, C57BL / 6 mice were primed with 2.5 pg of VEEV_TC83 or CHIKV saRNA-spike. Four weeks after the prime injection, the mice were boosted with PBS, VEEV_TC83 or CHIKV (Figure 6a). Anti-spike IgG levels were elevated two and four weeks after the priming injection with VE and CH, with the later resulting in a significant increase. At the 6thweek, total anti-spike IgG levels were quantified (Figure 6b). Homologous vaccination of VE-VE resulted in higher IgG titres relative to the single primed VE groups. CH-CH homologous vaccination did not provide any boosting benefits compared to its prime- only counterpart. The heterologous CH-VE groups had significantly higher anti-spike IgG titers compared to the VE-CH groups as well as its homologous CH-CH counterpart by 10- and 6-folds. CH-VE vaccination resulted in higher IgG titers than the standard VE-VE groups. Compared to the titres observed before priming, it was evident that boosting with VEEV_TC83 in a homologous or heterologous regimen resulted in the highest boosting benefit (Figure 6c).

[0082] Further assessment of IgG 1 and lgG2c subclasses indicated that both homologous and heterologous vaccinated groups led to a Th1-biased immunity, although no significant differences were observed between the groups (Figure 6d). Similar trends as the terminal IgG titers were observed in an assay to determine the neutralization capacity of the anti-spike antibodies. Higher IFN-y secreting splenocytes as determined via ELISpot analysis was observed for the prime-only, homologous and heterologous vaccinated groups. However, the single-primed CHIKV groups had significantly lower levels that all other vaccination regimen.Summary

[0083] In this experiment, the performance of some examples of the described saRNA vectors (VEEV_TrD, VEEV_TC83, TONV, CHIKV, ONNV) was examined in multiple platforms. Such platforms include eGFP expression in BHK cells, bioluminescence studies in human skin explants and Balb / c mice as well immunogenicity of saRNA-spike vaccines in C57BL / 6 mice (Figure 7). The highest correlation (r = 0.91) was observed between the total luminescence and terminal antispike IgG titres after immunization with 5 pg of saRNA. Moderate correlation was observed between human skin bioluminescence studies and mice imaging and immunogenicity studies (r = 0.39 and 0.47, respectively). Little to no correlation was observed between in vitro BHK eGFP expression, immunogenicity in a preclinical murine model, and Flue expression in human skin explants.

[0084] The exploratory nature of the study relied on sequences deposited on NCBI but not exclusively on reference sequences. Five out of 12 vectors showed high protein expression in BHK cells including the VEEV_TrD and VEEV_TC83 vectors that were used as controls. Due to the permissiveness of BHK cells to alphavirus infection, it can be inferred that failure to launch as saRNA could be attributed to erroneous sequences that result in non-functional replicases or conserved sequence elements. Besides cost-limitations on the synthesis of plasmid DNA to launch multiple variants from the same alphavirus species, there is a lack of high throughput methods to enable in vitro and in vivo assessment of saRNA. Notably, ranking of total antispike IgG for CHIKV, VEEV_TC83 and VEEV_TrD was similar to Flue total expression while TONV had higher Flue expression than ONNV, but their ranking was reversed in terms of IgG generation. Furthermore, the inventors demonstrated that the platform chosen to assess saRNA vectors plays an integral role (Figure 7). Though bioluminescence and immunogenicity were assessed in different mice strains, the results were highly correlated unlike other studies that have indicated that levels of antigen expression do not predict immunogenicity of saRNA vectors or recombinant virus particles. The human skin explant bioluminescence studies had lowest correlations with mice in vivo results followed by in vitro results. The observed low correlation of luminescence between human skin explants and Balb / c could beattributed to differences in cellular uptake of nanoparticles and subsequent response to the synthetic saRNA. The multi-faceted approach taken to measuring expression and immunogenicity underscores the complexity in the diverse functionality of saRNA modalities.

[0085] The results of these experiments support that alphaviruses, other than and in addition to VEEV, can be adapted into saRNA vectors which perform differently in transgene expression and vaccination efficacy in an infectious disease model.Materials and methods usedCell lines

[0086] All cell lines were cultured at 37 °C and 5% CO2 in base media supplemented with 10% Fetal bovine serum (FBS), 1 % Glutamax, 1 % Pen-Strep (100 U / mL-100 pg / mL) and other selective antibiotics as required. BHK-21 (ATCC #CCL-10) were cultured in Minimum Essential Medium (ThermoFisher Scientific™ #11095072, USA); HEK 293T Lenti-X (Takara™ #632180, USA) in in Dulbecco’s Modified Eagle’s Medium (DMEM, ThermoFisher Scientific #11960069); A549 Dual (Invivogen™ #a549d-nfis, USA) in DMEM with 100 pg / mL Normocin, 100 pg / mL Zeocin and 10 pg / mL Blasticidin; 293T-hsACE2 (Integral Molecular™ #C-HA102, USA) in DMEM with 10 mM HEPES (Fisher Scientific™ #BP299, USA) and 0.5 pg / mL puromycin.Plasmids

[0087] Viral RNA sequences obtained from NCBI were modified by replacing the structural proteins with enhanced green fluorescent protein (eGFP) sequences to make the various saRNA vectors (Table 1). saRNA sequences were placed downstream of the T7 promoter, followed by poly-adenine tail of 30 nucleotides and Asci or Sapl restriction enzyme sites for linearization. All plasmids for saRNA-eGFP were synthesized by VectorBuilder™ (USA). Gene fragments of firefly luciferase (Flue), or the prefusion-stabilized SARS-CoV-2 spike glycoprotein (GenelD: 4740568) were obtained from pAKB001-VEEV_TrD-Fluc and pAKB018-VEEV_TrD-CoV-S, respectively, and cloned into saRNA plasmids using the NEBuilder HIFI DNAassembly kit (New England Biolabs™ #E5520, USA) according to the manufacturer’s instructions. All plasmids were transformed into E. coli (NEB), cultured in Luria Broth with 100 pg / mL carbenicillin and extracted using a plasmid maxiprep kit (Qiagen™, USA).In vitro transcription of saRNA

[0088] 125 pg / mL of pDNA was linearized with Asci or Sapl (NEB™, USA) at 37°C for 2 h and enzyme-inactivated for 20 min at manufacturer’s recommended temperature. RNA for cell culture experiments were generated using the Mmessage™ machine kits (ThermoFisher Scientific™ #AM1344, USA). saRNA for ex- and in vivo studies in human skin and mice, respectively, were generated in an IVT rection mixture of Tris- HCI, magnesium acetate, spermidine, dithiothreitol, unmodified nucleotide triphosphates, pyrophosphatase, RNase inhibitor, T7 RNA polymerase (NEB #M0251 L) at 30 °C for 2 h, as previously described and further capped using the CellScript Cap-1 system (CellScript #C-SCCS1710, USA). All RNA was purified using lithium chloride precipitation and dissolved in nuclease-free water. RNA concentration and purity was determined using the Nanodrop One system (ThermoFisher Scientific™). RNA was aliquoted and stored at -70 °C before use.Cell culture transfection

[0089] BHK, HEK, A549-dual cells were seeded overnight. Transfection of saRNA into the cells was done using Lipofectamine 3000 reagent (Invitrogen™ #L3000001) according to the manufacturer’s manual. Before transfection, complete media was replaced with OptiMEM™ (ThermoFisher Scientific™ #11058021). Downstream assays were performed 24 h post-transfection unless stated otherwise.Flow cytometry

[0090] All cells were dissociated using TrypLE reagent according to manufacturer’s instructions. Cells were washed twice in FACS buffer (PBS + 2% FBS), stained with the far red Live / Dead dye (ThermoFisher Scientific™) for 30 min at 4 °C and washed twice again before data acquisition using the Cytoflex S (Beckman™, USA). Dataanalysis was done using the FlowJo™ software.

[0091] Flow cytometry experiments were performed to determine CAR expression followed a modified staining protocol. The cells were dissociated using Trypsin-EDTA (0.25%) (Gibco™, #25200056), washed twice with PBS, and stained with Fixable Viability Stain 575V (BD Biosciences, # 565694) at room temperature for 15 min.After the incubation, the cells were washed twice with FACS buffer and stained with an antibody cocktail consisting of anti-G4S rabbit antibody Alexa Fluor 488 (Cell Signalling, #50515S) and anti-Myc mouse antibody PE (Cell Signalling, #3739S) at 4°C for 30 min. The cells were then washed twice with FACS buffer before data acquisition using the FACSymphony A1 (BD Biosciences, USA). Data analysis was done using the FlowJo™ software.Isolation of RNA and quantitative real time PCR

[0092] Total RNA from cell cultures was isolated using the Monarch Total RNA™ miniprep kit (NEB #T2010) including the DNase treatment step. The Luna one-step RT-qPCR kit (NEB #E3005L) was utilized according to the manufacturer’s instructions. 100 ng of total RNA was used as template.NF-KB-SEAP and IRF-Luc reporter assays

[0093] Detection of SEAP in cell culture supernatant by QUANTI-Blue™ (absorbance recorded at 655 nm) and / or detection of Lucia in cell supernatant by QUANTI-Luc™ (recorded using luminescence) were performed according to the manufacturer's recommendations (InvivoGen™). Absorbance and luminescence readings were obtained using the Tecan Infinite microplate reader.Lipid nanoparticle (LNP) formulation

[0094] LNPs were produced through rapid T tube mixing of RNA in aqueous phase (pH 4.52, sodium acetate buffer) and lipid mixture in ethanol phase using the Pump 33 DDS Syringe pump (Harvard Apparatus™) at a total flow rate of 20 mL / min and flow rate ratio of 3:1 (RNA: lipid). A molar percentage of 45:15.9:37.85:1.25 lipid mixture of ALC-0315 ((4-hydroxybutyl)azanediyl)di(hexane-6,1-diyl) bis(2-hexyldecanoate)) (BroadPharm™), DSPC (1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine) (Avanti Polar Lipids), plant-derived cholesterol (Avanti Polar Lipids™), and DMG-PEG 2000 (1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) (Avanti Polar Lipids™) was used. LNPs were diluted 10-fold with PBS and purified using sterilized MWCO 10 kDa centrifugal filters (Amicon™, Millipore Sigma™) at 4 °C and 2000 x g. Size, polydispersity index, and zeta potential of the LNPs were measured using the Zetasizer Nano (Malvern Instruments) and Zetasizer 7.12 software (Malvern™). The Quant-iT Ribogreen RNA assay kit (ThermoFisher Scientific™) was used to determine the percentages of encapsulated RNA and RNA concentrations, as previously described.Mice in vivo luciferase imaging

[0095] All animal studies were approved by the University of British Columba Animal Care Committee and complied to all ethical regulations for animal testing and research. Female BALB / cJ mice (Jackson Laboratories™, USA), 6-8 weeks of age, were placed into groups of n = 3. Mice were injected intramuscularly in both hind legs with LNPs containing 5 pg of saRNA-fluc. On specific days, as indicated in the results section, mice were injected intraperitoneally with 100 pL of 30 mg / mL D-Luciferin Substrate (PerkinElmer™, USA) and allowed to rest for 7 min. Mice were then anesthetized using isoflurane and imaged on the I VIS Lumina LT™ in vivo (PerkinElmer™) using a 3 min exposure. Luminescence from each injection site was quantified using an equal detection area using the Live Imaging Software and expressed total flux (p / s). 4 h after injection of saRNA-fluc LNPs, mice were bled through the saphenous vein to obtain serum for cytokine assessment.Ex vivo luciferase expression in human skin explants

[0096] Studies using human skin explants were done in accordance with protocols approved by the UBC Clinical Research Ethics Board. Specimen was obtained from patients undergoing elective abdominoplasty. Human skin tissue was processed as previously described. Briefly, excess subcutaneous fat layer was removed, excised into 1 cm2section and washed with PBS. Skin explants were cultured at 37 °C with 5% CO2 in DMEM supplemented with 10% FBS, 1 % Glutamax™, 1 % Pen-Strep™.LNPs containing 2.5 g of saRNA-fluc were injected intradermally into the explants using a 30-g insulin syringe. At 2, 4 and 7 days after injection, skin explants were inverted, and fresh media was added supplement with 1.5 mg / mL of D-Luciferin Substrate. Plates were incubated at 37 °C for 20 min and imaged on the MS Lumina LT in vivo (PerkinElmer™) using a 5 min exposure. Luminescence was quantified as mentioned above.Immunogenicity of saRNA-spike

[0097] Female C57BL / 6L mice (n=5 per group) aged 6-8 weeks received 5 and 0.5 pg of saRNA-spike LNPs via a single intramuscular injection for the standard primeboost vaccination. For the heterologous vaccination study, mice were immunized with 2.5 pg of saRNA-LNPs. Booster shots were provided on day 28. Blood samples were collected on days 27 and 42 (before and after boosting, respectively) to obtain serum for spike-specific ELISA and pseudovirus neutralization assay, unless otherwise specified. On day 42, spleens were collected from immunized mice for IFN-y ELISpot assay.Serum collection

[0098] Blood obtained via the saphenous vein on days indicated above was left to clot at room temperature for 1 h and then centrifuged at 1 ,500 x g and 4 °C for 10 min. Serum was collected and stored at -70 °C until use.Pro-inflammatory cytokine levels in mice

[0099] 4 h after injection of LNPs containing saRNA-Fluc, mice were bled, and sera obtained. After a 1 :12 serum dilution in diluent provided, the following cytokine levels were assessed using a U-PLEX kit (Meso Scale Diagnostics™ #K15069M, USA): GM-CSF, IFN-a, IFN-p, IFN-y, IL-5, IL-6, IP-10, KC, MCP-1 , MIP-1a, MIP-1p, MIP-2, TNF-a.SARS-CoV-2 Spike-specific ELISA.

[0100] MaxiSorp high-binding ELISA plates (Thermo Scientific™ # 442404, USA)were coated with 100 p.L of 1 jag / mL recombinant SARS-CoV-2 spike protein (R&D™ #10549-CV, USA) or a mixture of unlabelled goat anti-mouse Kappa (Southern Biotech™ #1050-01 , USA) and Lambda (Southern Biotech™ #1060-01) light chains in PBS overnight at 4 °C. The plates were washed three times with wash buffer (PBS + 0.05% (v / v) Tween-20), blocked with assay buffer (1 % BSA (w / v) in wash buffer) for 1 h at 37 °C and then washed again. A 5-fold serial dilution of standard IgG (Southern Biotech™ #0107-01) and a 1 :250 dilution of mouse serum samples was done in assay buffer. 50 pL of standard and samples were added to the wells and incubated for 1 h at 37 °C. After incubation, the plates were washed. 100 pL of secondary antibody was added to the plates after a 1 :4000 dilution of anti-mouse IgG-HRP (Southern Biotech™ #1030-05), lgG1-HRP (Southern Biotech™ #1070-05) and lgG2c-HRP (Southern Biotech™ #1079-05) and incubated at 37°C for 1 h. After incubation, the plates were washed and then developed by adding 100 pL TMB ELISA substrate (Thermo Scientific™ #34028). After 5 min, the reaction was stopped by adding 100 pL of 0.18 M sulfuric acid. Absorbance was measured at 450 nm using a Tecan™ plate reader.SARS-CoV-2 pseudovirus neutralization assay

[0101] Serum collected at the end of the vaccine study was heat-inactivated at 56 °C for 30 min. a 1 :5 dilution of serum was done in cell culture medium. 50 pL of the diluted sample was added to a 96-well plate. 50 pL of 5 pg / mL of SARS-CoV-2 Renilla™ luciferase reporter virus particles (RVP) (Integral™ #RVP-701 L, USA) diluted in cell culture medium was added to the serum containing wells. The serum- RVP mixture was incubated at 37 °C for 1 h. Thereafter, 100 pL containing 4 x 104293T-hsACE2 cells was added to the serum-RVP mixture and incubated a 37 °C and 5% CO2 for 48 h. Relative luciferase activity was determined using the Dual-Glo™ luciferase assay system (Promega™ #E16110). % inhibition of SARS-CoV-2 pseudovirus was determined by fold change between vaccinated groups and PBS controls.I FN-Y ELISpots

[0102] 6 weeks after immunization, excised spleens in PBS were manually minced using the flat end of a syringe plunger. The cell suspension was passed through a 70 pm strainer and centrifuged at 300 x g and 4 °C for 10 min. After removal of supernatant, the cells were resuspended in RPMI™ 1640 supplemented with 10% FBS and 1 % glutamax and counted. The Mouse IFN-y enzyme-linked immunosorbent spot (ELISpot™) kit (R&D systems™ #EL485, USA) was used to quantify the IFN-y T cell response according to the manufacturer’s instructions. Briefly, the precoated plate was blocked using resuspension media at room temperature for 20 min. 100 pL of 2.5 x 106splenocytes / mL was added to the plate. 100 pL of media only (negative control) or 2 pg / mL of SARS-CoV-2 spike peptides (JPT # PM-WCPV-S-1 , Germany) or 2.5 pg / mL of Concanavalin A (ThermoFisher Scientific™ #00497893) was added to the wells and incubated for 48 h at 37 °C and 5% CO2. Plates were developed according to manufacturer’s instructions and read using the AID ELISpot™ Reader (AID GmbH, Germany).Detection of dsRNA by Dot Blot Analysis

[0103] saRNA constructs of interest were first diluted in STE buffer (0.1 M NaCI, 1 mM EDTA, 50 mM Tris-HCI, pH 7.0) to a final concentration of 250 ng / pL. Each construct (500 ng total) was then dotted onto a dry, positively charged nylon membrane (GVS #1226556) and allowed to dry at room temperature for 30 minutes. The membrane was blocked in EveryBlot Blocking Buffer™ (Bio-Rad™ #12010020) at room temperature for 15 minutes with agitation. After blocking, the membrane was incubated in J2 anti-dsRNA murine antibody (diluted in RPMI and 5% FBS, prepared by Exalpha Biologicals, Inc. #10613002) at 4 °C overnight with agitation. The membrane was then washed three times with 15 mL PBS-T (0.5% Tween-20, 10 mM phosphate buffer saline, pH 7.2) at room temperature for 15 minutes per wash with agitation. Subsequently, the membrane was incubated with Alexa Fluor™ 488- conjugated anti-mouse goat antibody (Invitrogen™ #A-11001), diluted 1 :10,000 in PBS-T, at room temperature for 1 hour with agitation. Prior to imaging, the membrane was washed three times in PBS-T as previously described. Fluorescence detection was performed using the Sapphire Biomolecular Imager (Azure Biosystems™, USA).The resulting image was processed in Imaged™, where rolling ball background subtraction was applied, and the signal intensities of each dot was quantified.Statistical analysis

[0104] Graphs were prepared in GraphPad Prism™, version 8. Statistical differences were analyzed as noted in the figure legends. Figures 2a, 5a and 7a were created with BioRender.com™.Example 2Co-transfection experiments to determine compatibility of non-structural proteins

[0105] The inventors sought to determine if co-transfection of different saRNAs would impact each other’s performance. In the co-transfection experiments, human embryonic kidney (HEK) cells were seeded overnight. saRNA was mixed with Lipofectamine™ 3000. Single transfection experiments involved transfecting the cells with 1 pg of TC83 eGFP, TONV eGFP, CHIKV eGFP, ONNV eGFP or TC83 mCherry. Co-transfection experiments involved transfecting the cells with 1 pg of TC83 mCherry + 1 pg of each of the saRNA eGFP. saRNA-lipoplexes were added onto the cells, and were then left for 24 hours in an incubator. The samples were prepared for flow cytometry by staining the cells with Far Red viability dye. The data was then analyzed by FlowJo™.

[0106] Co-transfection of different saRNA-eGFP constructs with VEEV_Tc83 mCherry resulted in a decrease in eGFP positive cells except for TONV when compared to the singly transfected samples (Figure 19a). In the co-transfected samples, the percentage of TC83 mCherry positive cells reduced significantly when compared to the single TC83 mCherry transfection (grey region in Figure 19b). The percentage of mCherry positive cells in the co-transfected cells was similar.Co-transfection of different saRNA-eGFP constructs with VEEV_TC83 mCherry

[0107] In the co-transfected samples, the percentage of double-positive cells(mCherry+; eGFP+) was lower for all saRNAs compared to the TC83 eGFP - TC83 mCherry control sample (Figure 19c). Co-transfection of TC83 mCherry with CHIKV- and ONNV eGFP significantly reduced the occurrence of double positive cells to < 1 % while TC83 mCherry only cells (mCherry +; eGFP-) dominated.

[0108] The geometric mean fluorescence intensity of eGFP and / or mCherry indicated interference between the different saRNA eGFP and TC83 mCherry. The eGFP and mCherry MFI for the control samples (TC83 eGFP co-transfected with TC83 mCherry) was similar for double and single positive cells (Figures 20a, 20b).However, the EGFP MFI for single positive samples in the co-transfected samples of CHIKV, ONNV and TONV was higher than the eGFP MFI in double positive cells (Figure 20a). In contrast, the mCherry MFI for the double positive cells of CHIKV- eGFP or ONNV-eGFP and TC83 mCherry was higher than single mCherry positive cells in the co-transfected samples (Figure 20b). This indicates some compatibility in replication between co-transfected VEEV_TC83 and TONV. In the case of CHIKV and ONNV co-transfection with VEEV_TC83, the latter seems to exert some dominance or exclusion against the other saRNAs.

[0109] These experiments show that chimeras or recombinants in the NSP regions of different alphavirus strains (e.g., VEEV_TC83 and TONV) may be compatible and used as the basis of novel compositions of replication competent chimeric saRNA vectors with enhanced protein expression.Massively parallel reporter assay to identify highly efficient synthetic saRNA constructs.

[0110] All constructs have unique barcodes between the eGFP and 3’UTR sequences (Table 5). A common capture sequence is present downstream of the barcode.Table 5. Barcode sequences used in saRNA constructs.

[0111] The inventors sought to determine if the RNA yield for each construct was comparable to each other when the in vitro transcription (IVT) is done using individual plasmids or when the plasmids are pooled together. Additionally, the inventors investigated if the barcode may be used to quantify relative amounts of each construct individually as well as its quantity being additive in the case of pooled IVT. The slopes of the non-structural region probed are not significantly different hence the performance of the PCR can be compared. This is also supported by the high correlations as shown by the heatmap.

[0112] To validate that qPCR can be used to compare the different yields from a pooled reaction, linearized DNA of three constructs were pooled, with 0.2 ug of one construct, 0.4 ug of each of the other constructs to a total of 1 ug. Triplicate IVT’s were performed, purified, then 5 pg was used for rt-qPCR analysis. Results are shown in Figure 22. The plot in Figure 22 shows that at lower concentrations of DNA (200 ng), a similar amount of NSP1 translation was seen, but at higher concentrations VEEV outperformed other constructs. Overall, the total amount of replication of the GFP and BC region were not significantly different.Example 3 - Evaluation of CAR expression using novel saRNA constructs

[0113] This experiment compared the level of expression between TC83-saRNA and TONV-saRNA constructs which encode a CAR, referred to as TC83-CAR-saRNA and TONV-CAR-saRNA. Additionally, it evaluated the effect of nucleotide substitutions on CAR expression by comparing constructs comprising non-modified RNA and constructs comprising 100% 5mC-substituted RNA for both TC83 and TONV backbones.

[0114] To determine expression, BHK cells were transfected with TC83-CAR-saRNA and TONV-CAR-saRNA constructs prepared with or without 100% 5mC substitution. Briefly, BHK cells were seeded overnight at 60-80% confluency. Each saRNA construct was mixed with Lipofectamine Messenger Max™ according to the manufacturer’s instructions. The saRNA-lipoplexes were then added to the cells at a working concentration of 1 nM and incubated for 24 hours at 37°C. The CAR sequence in each construct has a G4S linker and Myc tag to determine expression using flow cytometry. The samples were prepared by staining with Fixable Viability Stain 575V, followed by an anti-G4S Alexa Fluor 488 and anti-Myc PE antibody. The results were then analyzed using FlowJo™ software.

[0115] Flow cytometry showed that while the CAR was detected from both the TONV- CAR-saRNA and TC83-CAR-saRNA, the TONV construct showed stronger expression. The data showed that 16.4% Myc+ G4S+ positive cells were detected when the cells were transfected with TC83-CAR-saRNA versus 35.5% when the cells transfected with TONV-CAR-saRNA (no nucleotide modifications). The flow cytometry data also showed that 5mC-substituted RNA performed better in both cases. The data showed that 24.7% Myc+ G4S+ positive cells were detected when the cells were transfected with TC83-CAR-saRNA versus 50.1 % when the cells were transfected with TONV-CAR-saRNA (5mC substitution).Interpretation of Terms

[0116] Unless the context clearly requires otherwise, throughout the description and the claims:• “comprise”, “comprising”, and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”;“herein”, “above”, “below”, and words of similar import, when used to describe this specification, shall refer to this specification as a whole, and not to any particular portions of this specification;• “or”, in reference to a list of two or more items, covers all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list;• the singular forms “a”, “an”, and “the” also include the meaning of any appropriate plural forms. These terms (“a”, “an”, and “the”) mean one or more unless stated otherwise;• “and / or” is used to indicate one or both stated cases may occur, for example A and / or B includes both (A and B) and (A or B);• “approximately” when applied to a numerical value means the numerical value ± 10%;• where a feature is described as being “optional” or “optionally” present or described as being present “in some embodiments” it is intended that the present disclosure encompasses embodiments where that feature is present and other embodiments where that feature is not necessarily present and other embodiments where that feature is excluded. Further, where any combination of features is described in this application this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely," "only" and the like in relation to the combination of features as well as the use of "negative" limitation(s)” to exclude the presence of other features; and

[0117] Where a range for a value is stated, the stated range includes all sub-ranges of the range. It is intended that the statement of a range supports the value being at an endpoint of the range as well as at any intervening value to the tenth of the unit of the lower limit of the range, as well as any subrange or sets of sub ranges of the range unless the context clearly dictates otherwise or any portion(s) of the stated range is specifically excluded. Where the stated range includes one or both endpoints of the range, ranges excluding either or both of those included endpoints are also included in the invention.

[0118] Certain numerical values described herein are preceded by "about". In this context, "about" provides literal support for the exact numerical value that it precedes, the exact numerical value ±5%, as well as all other numerical values that are near to or approximately equal to that numerical value. Unless otherwise indicated a particular numerical value is included in “about” a specifically recited numerical value where the particular numerical value provides the substantial equivalent of the specifically recited numerical value in the context in which the specifically recited numerical value is presented. For example, a statement that something has the numerical value of “about 10” is to be interpreted as: the set of statements:• in some embodiments the numerical value is 10;• in some embodiments the numerical value is in the range of 9.5 to 10.5; and if from the context the person of ordinary skill in the art would understand that values within a certain range are substantially equivalent to 10 because the values with the range would be understood to provide substantially the same result as the value 10 then “about 10” also includes:• in some embodiments the numerical value is in the range of C to D where C and D are respectively lower and upper endpoints of the range that encompasses all of those values that provide a substantial equivalent to the value 10.

[0119] Specific examples of systems, methods and apparatus have been described herein for purposes of illustration. These are only examples. The technology provided herein can be applied to systems other than the example systems described above. Many alterations, modifications, additions, omissions, and permutations are possible within the practice of this invention. This invention includes variations on described embodiments that would be apparent to the skilled addressee, including variations obtained by: replacing features, elements and / or acts with equivalent features, elements and / or acts; mixing and matching of features, elements and / or acts from different embodiments; combining features, elements and / or acts from embodiments as described herein with features, elements and / or acts of other technology; and / oromitting combining features, elements and / or acts from described embodiments.

[0120] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any other described embodiment(s) without departing from the scope of the present invention.

[0121] Any aspects described above in reference to apparatus may also apply to methods and vice versa.

[0122] Any recited method can be carried out in the order of events recited or in any other order which is logically possible. For example, while processes or blocks are presented in a given order, alternative examples may perform routines having steps, or employ systems having blocks, in a different order, and some processes or blocks may be deleted, moved, added, subdivided, combined, and / or modified to provide alternative or subcombinations. Each of these processes or blocks may be implemented in a variety of different ways. Also, while processes or blocks are at times shown as being performed in series, these processes or blocks may instead be performed in parallel, simultaneously or at different times.

[0123] Various features are described herein as being present in “some embodiments”. Such features are not mandatory and may not be present in all embodiments. Embodiments of the invention may include zero, any one or any combination of two or more of such features. All possible combinations of such features are contemplated by this disclosure even where such features are shown in different drawings and / or described in different sections or paragraphs. This is limited only to the extent that certain ones of such features are incompatible with other ones of such features in the sense that it would be impossible for a person of ordinary skill in the art to construct a practical embodiment that combines such incompatible features. Consequently, the description that “some embodiments” possess feature A and “some embodiments” possess feature B should be interpreted as an express indication that the inventors also contemplate embodiments which combine features A and B (unless the description states otherwise or features A and B arefundamentally incompatible). This is the case even if features A and B are illustrated in different drawings and / or mentioned in different paragraphs, sections or sentences.

[0124] It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions, omissions, and sub-combinations as may reasonably be inferred. The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

WHAT IS CLAIMED IS:1 . A self-amplifying RNA (saRNA) vector comprising: a nsPs sequence which encodes non-structural proteins (nsPs), the non- structural proteins comprising nsP1 , nsP2, nsP3 and nsP4 proteins; a 5’-UTR sequence located upstream of the nsPs sequence; a 3-UTR sequence located downstream of the nsPs sequence; a subgenomic promoter (SGP) sequence between the nsPs sequence and the 3’-UTR; a gene of interest (GOI) sequence between the subgenomic promoter sequence and the 3’-UTR, wherein the gene of interest sequence encodes at least one heterologous protein; a 5’-cap upstream of the 5’-UTR sequence; and a poly(A) tail downstream of the 3’-UTR sequence, wherein the 5’-UTR sequence comprises a nucleotide sequence having at least 40% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

2. The saRNA vector according to claim 1 , wherein the 5’-UTR sequence comprises a nucleotide sequence having at least 50% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

3. The saRNA vector according to claim 1 , wherein the 5’-UTR sequence comprises a nucleotide sequence having at least 60% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

4. The saRNA vector according to claim 1 , wherein the 5’-UTR sequence comprises a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

5. The saRNA vector according to claim 1 , wherein the 5’-UTR sequence comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

6. The saRNA vector according to claim 1 , wherein the 5’-UTR sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

7. The saRNA vector according to claim 1 , wherein the 5’-UTR sequence comprises a nucleotide sequence set forth in any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

8. The saRNA vector according to any one of claims 1 to 7, wherein the 3’-UTR sequence comprises a nucleotide sequence having at least 40% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

9. The saRNA vector according to any one of claims 1 to 7, wherein the 3’-UTR sequence comprises a nucleotide sequence having at least 50% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

10. The saRNA vector according to any one of claims 1 to 7, wherein the 3’-UTR sequence comprises a nucleotide sequence having at least 60% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.11 . The saRNA vector according to any one of claims 1 to 7, wherein the 3’-UTR sequence comprises a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

12. The saRNA vector according to any one of claims 1 to 7, wherein the 3’-UTR sequence comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

13. The saRNA vector according to any one of claims 1 to 7, wherein the 3’-UTR sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

14. The saRNA vector according to any one of claims 1 to 7, wherein the 3’-UTR sequence comprises a nucleotide sequence set forth in any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

15. The saRNA vector according to any one of claims 1 to 14, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 40% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

16. The saRNA vector according to any one of claims 1 to 14, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 50% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

17. The saRNA vector according to any one of claims 1 to 14, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 60% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

18. The saRNA vector according to any one of claims 1 to 14, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

19. The saRNA vector according to any one of claims 1 to 14, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

20. The saRNA vector according to any one of claims 1 to 14, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.21 . The saRNA vector according to any one of claims 1 to 14, wherein thesubgenomic promoter sequence comprises a nucleotide sequence set forth in any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

22. A self-amplifying RNA (saRNA) vector comprising: a nsPs sequence which encodes non-structural proteins (nsPs), the non- structural proteins comprising nsP1 , nsP2, nsP3 and nsP4 proteins; a 5’-UTR sequence located upstream of the nsPs sequence; a 3-UTR sequence located downstream of the nsPs sequence; a subgenomic promoter (SGP) sequence between the nsPs sequence and the 3’-UTR; a gene of interest (GOI) sequence between the subgenomic promoter sequence and the 3’-UTR, wherein the gene of interest sequence encodes at least one heterologous protein; a 5’-cap upstream of the 5’-UTR sequence; and a poly(A) tail downstream of the 3’-UTR sequence, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 40% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

23. The saRNA vector according to claim 22, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 50% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

24. The saRNA vector according to claim 22, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 60% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

25. The saRNA vector according to claim 22, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 70% sequenceidentity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

26. The saRNA vector according to claim 22, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

27. The saRNA vector according to claim 22, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

28. The saRNA vector according to claim 22, wherein the subgenomic promoter sequence comprises a nucleotide sequence set forth in any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

29. The saRNA vector according to any one of claims 22 to 28, wherein the 5’- UTR sequence comprises a nucleotide sequence having at least 40% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

30. The saRNA vector according to any one of claims 22 to 28, wherein the 5’- UTR sequence comprises a nucleotide sequence having at least 50% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.31 . The saRNA vector according to any one of claims 22 to 28, wherein the 5’- UTR sequence comprises a nucleotide sequence having at least 60% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

32. The saRNA vector according to any one of claims 22 to 28, wherein the 5’- UTR sequence comprises a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

33. The saRNA vector according to any one of claims 22 to 28, wherein the 5’- UTR sequence comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

34. The saRNA vector according to any one of claims 22 to 28, wherein the 5’-UTR sequence comprises a nucleotide sequence having at least 90%sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

35. The saRNA vector according to any one of claims 22 to 28, wherein the 5’- UTR sequence comprises a nucleotide sequence set forth in any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

36. The saRNA vector according to any one of claims 22 to 35, wherein the 3’- UTR sequence comprises a nucleotide sequence having at least 40% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46- 52.

37. The saRNA vector according to any one of claims 22 to 35, wherein the 3’- UTR sequence comprises a nucleotide sequence having at least 50% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46- 52.

38. The saRNA vector according to any one of claims 22 to 35, wherein the 3’- UTR sequence comprises a nucleotide sequence having at least 60% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46- 52.

39. The saRNA vector according to any one of claims 22 to 35, wherein the 3’- UTR sequence comprises a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46- 52.

40. The saRNA vector according to any one of claims 22 to 35, wherein the 3’- UTR sequence comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46- 52.41 . The saRNA vector according to any one of claims 22 to 35, wherein the 3’- UTR sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-42. The saRNA vector according to any one of claims 22 to 35, wherein the 3’- UTR sequence comprises a nucleotide sequence set forth in any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

43. A self-amplifying RNA (saRNA) vector comprising: a nsPs sequence which encodes non-structural proteins (nsPs), the non- structural proteins comprising nsP1 , nsP2, nsP3 and nsP4 proteins; a 5’-UTR sequence located upstream of the nsPs sequence; a 3-UTR sequence located downstream of the nsPs sequence; a subgenomic promoter (SGP) sequence between the nsPs sequence and the 3’-UTR; a gene of interest (GOI) sequence between the subgenomic promoter sequence and the 3’-UTR, wherein the gene of interest sequence encodes at least one heterologous protein; a 5’-cap upstream of the 5’-UTR sequence; and a poly(A) tail downstream of the 3’-UTR sequence, wherein the 3’-UTR sequence comprises a nucleotide sequence having at least 40% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

44. The saRNA vector according to claim 43, wherein the 3’-UTR sequence comprises a nucleotide sequence having at least 50% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

45. The saRNA vector according to claim 43, wherein the 3’-UTR sequence comprises a nucleotide sequence having at least 60% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

46. The saRNA vector according to claim 43, wherein the 3’-UTR sequencecomprises a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

47. The saRNA vector according to claim 43, wherein the 3’-UTR sequence comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

48. The saRNA vector according to claim 43, wherein the 3’-UTR sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

49. The saRNA vector according to claim 43, wherein the 3’-UTR sequence comprises a nucleotide sequence set forth in any one of SEQ ID NO: 19-20, 26-29, and 36-38, and 46-52.

50. The saRNA vector according to any one of claims 43 to 49, wherein the 5’- UTR sequence comprises a nucleotide sequence having at least 40% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.51 . The saRNA vector according to any one of claims 43 to 49, wherein the 5’- UTR sequence comprises a nucleotide sequence having at least 50% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

52. The saRNA vector according to any one of claims 43 to 49, wherein the 5’- UTR sequence comprises a nucleotide sequence having at least 60% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

53. The saRNA vector according to any one of claims 43 to 49, wherein the 5’- UTR sequence comprises a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

54. The saRNA vector according to any one of claims 43 to 49, wherein the 5’- UTR sequence comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

55. The saRNA vector according to any one of claims 43 to 49, wherein the 5’-UTR sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

56. The saRNA vector according to any one of claims 43 to 49, wherein the 5’- UTR sequence comprises a nucleotide sequence set forth in any one of SEQ ID NO: 13-15, 21-23, 30-32, and 39-45.

57. The saRNA vector according to any one of claims 43 to 56, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 40% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

58. The saRNA vector according to any one of claims 43 to 56, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 50% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

59. The saRNA vector according to any one of claims 43 to 56, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 60% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

60. The saRNA vector according to any one of claims 43 to 56, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.61 . The saRNA vector according to any one of claims 43 to 56, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

62. The saRNA vector according to any one of claims 43 to 56, wherein the subgenomic promoter sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 16-18, 24-25, 33-35,and 53-59.

63. The saRNA vector according to any one of claims 43 to 56, wherein the subgenomic promoter sequence comprises a nucleotide sequence set forth in any one of SEQ ID NO: 16-18, 24-25, 33-35, and 53-59.

64. The saRNA vector according to any one of claims 1 to 63, wherein the nsPs sequence comprises or is derived from a species of alphaviruses selected from the group consisting of Tonate virus, Chikungunya virus, O’nyong-nyong virus, Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus, Barmah Forest virus, Mayaro virus, Ross River virus, Semliki Forest virus, and Sindbis virus.

65. The saRNA vector according to any one of claims 1 to 64, wherein the 5’-UTR sequence comprises or is derived from a species of alphaviruses selected from the group consisting of Tonate virus, Chikungunya virus, O’nyong-nyong virus, Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus, Barmah Forest virus, Mayaro virus, Ross River virus, Semliki Forest virus, and Sindbis virus.

66. The saRNA vector according to any one of claims 1 to 65, wherein the 3’-UTR sequence comprises or is derived from a species of alphaviruses selected from the group consisting of Tonate virus, Chikungunya virus, O’nyong-nyong virus, Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus, Barmah Forest virus, Mayaro virus, Ross River virus, Semliki Forest virus, and Sindbis virus.

67. The saRNA vector according to any one of claims 1 to 66, wherein the subgenomic promoter sequence comprises or is derived from a species of alphaviruses selected from the group consisting of Tonate virus, Chikungunya virus, O’nyong-nyong virus, Eastern equine encephalomyelitis virus, Western equine encephalomyelitis virus, Barmah Forest virus, Mayaro virus, Ross River virus, Semliki Forest virus, and Sindbis virus.

68. The saRNA vector according to any one of claims 1 to 67, wherein the gene ofinterest (GOI) sequence encodes a therapeutic protein.

69. The saRNA vector according to any one of claims 1 to 67, wherein the gene of interest (GOI) sequence encodes a chimeric antigen receptor (CAR) protein.

70. Use of the saRNA vector according to any one of claims 1 to 69 in the manufacture of a medicament for treating, inhibiting and / or ameliorating a disorder and / or disease in a subject.71 . The use of the saRNA vector according to claim 70, wherein the disorder and / or disease comprises a cancer or a viral infection.

72. The use of the saRNA vector according to claim 70 or 71 , wherein the medicament comprises a vaccine.

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