Self-amplifying RNA and methods

Abstract

The technology described herein relates to compositions and methods for generating modified self-amplifying RNA (saRNA) with enhanced expression, and immunological properties. The disclosed saRNA constructs are designed to encode multiple proteins from a single transcript. Novel sequence elements and engineered vector mutations further augment protein output and control the vector tropism. Expression of immune-modulatory proteins mitigates adaptive immune responses against both the saRNA vector and its encoded transgenes. The incorporation of chemically modified nucleotides reduces innate immune activation, while improved synthesis methods minimize double-stranded RNA contaminants.

Description

[0001] SELF-AMPLIFYING RNA AND METHODS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003]

[0001] This application claims benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 63 / 640,069 filed April 29, 2024, U.S. Provisional Application No. 63 / 646,327 filed May 13, 2024, U.S. Provisional Application No. 63 / 647,984 filed May 15, 2024, U.S. Provisional Application No. 63 / 661,378 filed June 18, 2024, U.S. Provisional Application No. 63 / 696,428 filed September 19, 2024, and U.S. Provisional Application No. 63 / 717,421 filed November 7, 2024, the contents of each of which are incorporated herein by reference in their entireties.

[0004] GOVERNMENT SUPPORT

[0005]

[0002] This invention was made with government support under Grant Nos. 5U01CA265713 and R01EB029483 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007]

[0003] The instant application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on April 28, 2025, is named 701586-000136WOPT_SL.xml and is 169,969 bytes in size.

[0008] TECHNICAL FIELD

[0009]

[0004] Disclosed herein are self-amplifying RNAs (saRNAs), transfected cells and / or pharmaceutical compositions comprising the same. Also disclosed are methods of using and manufacturing such saRNAs.

[0010] BACKGROUND

[0011]

[0005] Self-amplifying RNAs (saRNAs) undergo replication and amplification, inside the cell, to afford robust and durable expression of an encoded protein cargo (Geall et al., 2023, PMID: 36384351; Minnaert et al., 2021, PMID: 34324884). saRNAs conventionally utilize alphavirus sequences, replacing the structural genes with the genes encoding the cargo(s) of interest (Bloom et al., 2021, PMID: 33093657). The resulting synthetic construct encodes an RNA- dependent RNA polymerase (RdRp) and the cargo(s) on the same RNA strand. Self-amplifying RNAs (saRNAs) can use RNA-dependent RNA polymerase (RdRp) from an alphavirus and conserved sequence elements (CSEs) at the 5’ and 3’ ends to enable replication, resulting in strong and sustained expression of the encoded protein cargo. Once expressed, the RdRp recognizes conserved sequence elements (CSEs) at the 5’ and 3’ ends to transcribe full-length copies. Subsequently, the RdRp recognizes a sub-genomic promoter (SGP) to initiate transcription of the cargo sequence(s). The sub-genomic transcript is capped by the RdRp and translated for production of the encoded cargo protein.

[0012]

[0006] Some of the advantages of saRNA include increased potency per mg RNA compared to non-replicating mRNA (nrRNA) and extended duration of action (Minnaert, K. Advanced Drug Delivery Reviews, 2021. PMID: 34324884)(Geall, A. Expert Opinion on Drug Discovery, 2022. PMID: 36384351). The RdRp encoded by saRNA recognizes conserved secondary structures and sequences, referred to as the conserved sequence elements and sub genomic promoter (SGP), which allow for transcription of both negative and positive strand saRNA as well as the mRNA encoding the cargo of interest. The current understanding of the saRNA field is that the incorporation of modified nucleotides into saRNA results in inactivation of replicase activity and the abrogation of downstream efficacy (Geall, A. Expert Opinion on Drug Discovery, 2022. PMID: 36384351)( Voigt, E. NPJ vaccines, 2022. PMID: 36323666)(Novartis AG, 12 / 83 l,252)(Kairuz, D. Frontiers in Immunology, 2022. PMID: 36353641)(Minnaert, K. Advanced Drug Delivery Reviews, 2021. PMID: 34324884). These works teach that incorporation of modified nucleotides at >25% substitution results in saRNA that does not produce sufficient antigen to be therapeutically effective.

[0013]

[0007] There have been 10+ clinical trials employing saRNA since 2015, and though preclinical evidence is promising, human data has demonstrated lower seroconversion, lower neutralizing antibodies levels, and decreased production of antibodies after booster compared to non-replicating mRNA (Geall, A. Expert Opinion on Drug Discovery, 2022. PMID: 36384351). One hypothesized reason for this, is the early and intense activation of the innate immune response by saRNA containing unmodified nucleotides, which hinders both saRNA replication and the launch of cargo from the saRNA SGP. In knockout mice deficient for type I IFN a and b receptor subunit 1, saRNA resulted in higher IgG specific antibody titers and seroconversion compared to WT mice (Pepini, T. The Journal of Immunology, 2017. PMID: 28416600)(Zhong, Z. Nano Today, 2018. DOI: 10.1016 / J.NANTOD.2018.10.005). Recently, corticosteroid immune suppression was explored as a co-therapy to minimize innate immune response, but ended up suppressing seroconversion. This same study did demonstrate that removal of dsRNA contaminants, which activate TLR3 and stimulate type I IFN expression, increased vaccine immunogenicity (Zhong, Z. Molecular Therapy, 2021. PMID: 33484964). A Type I IFN suppression of saRNAis further supported by the fact that IM is the optimal delivery route for saRNA, as intradermal injection has been shown to elicit higher type I IFN response than IM (Zhong, Z. Vaccines, 2019. PMID: 31450775). In sum, previous research points towards the potential for substantially increased saRNA efficacy if the early interferon response could be overcome. Part of what makes saRNA so potent as a vaccine candidate is that it acts as its own adjuvant. However, to achieve a balance between adjuvancy and IFN-mediated suppression of saRNA launch, controlling the early IFN response is essential (Zhong, Z. Nano Today, 23. (2018) DOI: 10.1016 / J.NANTOD.2018.10.005). Without a mechanism to evade early recognition during endosomal escape and preliminary expression, saRNA may continue to falter in the clinic.

[0014]

[0008] saRNA holds promise for a platform capable of addressing the shortcomings of mRNA by decreasing the dose of vaccines and administration frequency of protein-encoding therapeutics. Decreasing the required dose of both RNA and lipid nanoparticles (LNPs) will mitigate adverse events and reduce the occurrence of rare but serious adverse events (Ju et al., 2023, PMID: 36539526; Ndeupen et al., 2021, PMID: 34841223; Trougakos et al., 2022, PMID: 35537987). Additionally, order of magnitude reductions in dose requirements will significantly bolster the manufacturing capacity to enhance production speed, reduce vaccine production costs, and democratize the distribution of vaccines against emerging pathogens. However, early clinical evidence from saRNA vaccine trials shows decreased efficacy and reduced neutralizing antibody levels compared to mRNA (Low et al., 2022, PMID: 36513697). Recently, results from a late-stage clinical trial reveal comparable immunogenicity of a saRNA vaccine at l / 10th of the dose of an equivalent mRNA vaccine, however, side effects were similar between the treatment groups (Akahata et al., 2023, PMID: 37586325). Strategies to decrease the immunogenicity of saRNA are urgently needed to enable further clinical development and translation.

[0015]

[0009] The original discovery of how modified nucleotides enable mRNA to evade the immune system by Kariko and Weissman paved the way for the field of mRNA therapeutics (Kariko, K. Immunity, 2005. PMID: 16111635)(Karik6, K. Molecular Therapy, 2008. PMID: 18797453). mRNA containing chemically modified nucleotides are significantly less immunogenic (Kariko, K. Molecular Therapy, 2008. PMID: 18797453)(Kormann, M. Nature Biotechnology, 2011. PMID: 21217696)(Karik6, K. Immunity, 2005. PMID: 16111635). Currently, the clinical-gold standard in mRNA therapeutics is the complete substitution of uridine for N1 -methylpseudouridine (mlY) which has the greatest impact on the suppression of the type I interferon (IFN) response traditionally elicited by dsRNA and ssRNA (Kariko, K. Immunity, 2005. PMID: 16111635). On a molecular level, modified nucleotides alter the stability or accessibility of specific base pairs, change hydrogen bonding patterns, and shift RNA hydrophobicity. Along with altering RNA stability, some of these new interactions change RNA primary or secondary structure such that RNA-protein interactions are either stabilized or inhibited (Kierzek, E. Nature Communications, 2022. PMID: 35277476)( Harcourt, E. Nature, 2017. PMID: 28102265)(Davis, D. Nucleic Acids Research, 1995. PMID: 8559660). It is in part through these mechanisms that modified nucleotides imbue RNA with immune evasion properties. However, these same RNA-RNA and RNA-protein interactions are essential for self-amplifying RNA (saRNA) function.

[0016]

[0010] Previous studies have suggested that the level of substitution of saRNAs with modified nucleotides should be less than 25% otherwise the expression of transgenes encoded by saRNAs expression decreases relative to comparable saRNAs without such modifications (see, e.g., Voigt, E. NPJ vaccines, 7. (2022) (USSN 12 / 831,252) (WO2011005799)

[0017] (US10532067B2), (US11291682B2), (US20220054525A1), (US10487332B2),

[0018] (US20200048636A1), (US20220056449A1), (EP3964584A1), (WO2012006376), (US20220192997A1), (US11058762B2), (US20210290755A1), (US20140242152), (US20220313815A1), (US20210347828A1), (WO2022137128A2), (US20140271829A1).

[0019] For example, USSN 12 / 831,252 teaches that incorporation of 0.01% - 25% modified nucleotide is the optimal substitution ratio that maintains expression of a transgene cargo, either reporter construct or vaccine antigen. At higher substitution ratios, expression of the transgene cargo is decreased.

[0020] [Oil] By performing a screen of chemically modified nucleosides, specific modifications were identified that result in robust expression of a protein encoded by self-amplifying RNA while simultaneously reducing the innate immune response (McGee et al., 2023, PMID: 37745375).

[0021]

[0012] The ability to modify self-amplifying RNA to reduce immunogenicity while maintaining the ability to produce proteins at high levels for long duration enables its use for diverse vaccine and therapeutic applications. However, there remains a need to determine the optimal genetic architecture for expression of multiple proteins simultaneously from a single multivalent modified self-amplifying RNA and to identify other modified nucleotides that are compatible with self-amplifying RNA.

[0022]

[0013] The faithful expression of multiple proteins from a polycistronic RNA transcript is dependent on the strategy employed to facilitate separation of the encoded polypeptides. A common strategy is to employ viral-derived 2A peptides or ‘ribosomal skip’ sequences (Provost et al., 2007, PMID: 17941043). This strategy has been employed to express multiple proteins from the same open reading frame (ORF) including cytosolic, membrane, and secreted proteins. However, the proper localization or stability of the encoded proteins is not guaranteed due to the presence of scar sequences that are added, improper trafficking, or destabilization of the resulting polypeptide (de Felipe et al., 2010, PMID: 19946875; Reinhardt et al., 2020, PMID: 32109368; Yan et al., 2010, PMID: 19951694). As a result, internal ribosomal entry sites (IRES) sequences are often preferred for co-expression of multiple proteins from a polycistronic transcript. IRES sequences enable the faithful expression of multiple proteins in a scar-free manner without interfering with protein localization. However, the presence of modified nucleosides has been previously shown to inhibit IRES-mediated protein expression (Wesselhoeft et al., 2019, PMID: 30902547). The development of modified self-amplifying RNA containing IRES sequences is needed to ensure the faithful expression of multiple proteins at high levels and for long duration, to enable next generation of RNA vaccines and therapeutics.

[0023]

[0014] The inherent short half-life of mRNA necessitates a large dose to be effective, which increases the risk of adverse side effects, limits global accessibility, and restricts applications. The use of mRNA for the in situ production of therapeutic proteins requires multiple frequent injections to be effective. Modified self-amplifying can encode therapeutic proteins for the advancement of systemic therapies. In viral pandemic scenarios, a low dose modified saRNA can be used for expression of a broadly neutralizing monoclonal antibody. Furthermore, the prolonged production of therapeutic proteins in situ has the potential to improve the patient experience, potentially replacing time intensive intravenous infusions with simple outpatient injections.

[0024]

[0015] Messenger RNA (mRNA) has become an increasingly important therapeutic modality for the treatment of various diseases, where the mRNA encodes for a protein or antigen of interest. mRNA suffers from several limitations including low protein expression, a short period of protein expression, and non-targeted expression in all cells.

[0025]

[0016] saRNA has the potential to significantly enhance protein-encoding therapeutics by providing robust and prolonged expression of therapeutic proteins. This improvement is due to the saRNA’ s inherent capability for exponential amplification and replication within cells, resulting in sustained protein production over an extended period. The prolonged nature of protein production from saRNA can facilitate more effective protein replacement therapies, which are essential for addressing various protein deficiencies. By administering saRNA, it is possible to maintain therapeutic protein levels in patients for longer durations without the need for frequent dosing. Furthermore, saRNA technology holds promise for advancing the field of immunotherapies. The ability of saRNA to produce biologies in situ — that is, directly within the patient's body — presents a convenient and efficient solution. This method circumvents the complexities and costs associated with traditional biologic production and distribution. In situ production means that therapeutic proteins or other biologies can be generated precisely where they are needed, enhancing the treatment's effectiveness.

[0026]

[0017] Alphaviruses have evolved distinct cellular tropisms that result in context dependent enhancements in fitness and viral propagation. For example, Venezuela Equine Encephalitis (VEEV) and Eastern Equine Encephalitis (EEEV) have been found to differ in their tropism towards myeloid cells (Gardner et al., 2008, PMID: 18768986). (Trobaugh et al., 2019, PMID: 31658290). VEEV efficiently infects myeloid cells and EEEV does not. As a result, VEEV induces greater systemic expression of inflammatory cytokines than EEEV. Prior work has determined that the difference in tropism is a result of sequences encoded in the 3’ untranslated region (UTR) (Trobaugh et al., 2014, PMID: 24352241). The generation of chimeric selfamplifying RNA vectors containing UTRs that result in an alternative tropism may enhance the bioactivity of encoded proteins and diminish the inflammatory response to administration of the saRNA.

[0027]

[0018] The RdRp of saRNA is derived from RNA viruses. As a result, these proteins have the capacity to be recognized as foreign and produce an adaptive immune response. Small peptides derived from the nspl-4 proteins are presented on MHC I and MHC II complexes. Furthermore, the encoded cargo transgene(s) may contain genes that are not fully human in nature. As a result, an adaptive immune response can be generated against the saRNA vector and / or the transgene that is encoded. This may result in diminished transgene expression and / or undesirable side effects, especially in the case of chronic administration of the saRNA vector.

[0019] The early and intense type I interferon response triggered by saRNA detection hampers replication and antigen expression, similar to unmodified mRNA. The interferon response from the initial recognition of the RNA can be resolved by the inclusion of modified nucleotides (US12, 115,217). However, the double-stranded RNA intermediates generated during RNA replication can trigger a downstream inflammatory response. Chronic culture of RNA viruses in human cells under selection has been shown to result in the accrual of adaptive mutations. The identification of optimal mutations for self-amplifying RNA vectors can result in improved vectors conferring enhanced protein production and reduced inflammatory responses.

[0028]

[0020] There remains a need to determine the optimize saRNA for therapeutic use, including increased protein expression, reduced immunogenicity and increased stability.

[0021] Furthermore, there is a need to identify optimal pharmaceutical compositions for delivery of saRNA to diverse cell and tissue types.

[0029]

[0022] Additionally, there is a need to achieve high purity and low double-stranded RNA of saRNA to minimize inflammatory responses and maximize potency.

[0030] SUMMARY

[0031]

[0023] Disclosed herein are saRNA and modified saRNA molecules, transfected cells and pharmaceutical compositions comprising the same. Also disclosed are methods of using such molecules and compositions and methods of manufacturing the same. Advantageously, the saRNA and modified saRNA molecules, transfected cells and pharmaceutical compositions disclosed herein confer one or more improved properties selected from increased transfection efficiency, increased replication, increased protein expression, reduced immunogenicity, and increased stability. In certain embodiments, the modified saRNA permits production of multiple proteins from a single transcript. In certain embodiments, the modified saRNA has low double-stranded RNA content. The technology described herein relates to methods and compositions of modified nucleoside triphosphates for transcriptionally and translationally active self-replicating RNA. The technology described herein relates to methods of use and compositions of self-amplifying RNA that is highly or fully substituted with chemically modified nucleotides (e.g., which can be introduced post-transcriptionally).

[0032]

[0024] In some embodiments, multiple antibody polypeptide chains are encoded as cargos of interest.

[0033]

[0025] In some embodiments, multiple receptor polypeptide chains are encoded as the cargos of interest.

[0034]

[0026] In some embodiments, multiple heterodimerizing polypeptide chains are encoded as the cargos of interest.

[0035]

[0027] In some embodiments, a receptor and ligand pair are encoded as the cargos of interest.

[0028] The technology described herein is directed to self-amplifying RNA systems. In particular, described herein are methods and compositions for the preparation of modified nucleoside triphosphates and subsequent incorporation, via transcription, in self-amplifying RNA at a percentage greater than 25%. The resulting modified self-amplifying RNA is translationally active in cells and produces protein(s). The prepared modified nucleotides are compatible with the self-amplifying RNA machinery, even at high levels or complete substitution. Self-amplifying RNA is comprised of sequences derived from an RNA virus and additional cargo sequences. The resulting modified self-amplifying RNA can be delivered to cells or tissues of animals and humans by pharmaceutical compositions described herein.

[0036]

[0029] In one embodiment of any aspect herein, the modified self-amplifying RNA is generated by in vitro transcription (IVT). In other embodiments, the self-amplifying RNA is delivered to cells as plasmid DNA that is transcribed into RNA and contains the necessary sequences for replication.

[0037]

[0030] In one embodiment of any aspect herein, the RNA contains a 5’ cap structure. In a preferred embodiment of any aspect herein, the 5’ cap structure is directly upstream of an adenosine nucleotide or analog. In a preferred embodiment of any aspect herein, the 5’ cap structure is directly upstream of a guanosine nucleotide or analog.

[0038]

[0031] In one embodiment of any aspect herein, the RNA contains a 3’ poly-A tail. In one embodiment of any aspect herein, the RNA contains a 5’ UTR, a 3’ UTR, or a combination of both. In one embodiment of any aspect herein, the inclusion of sequence elements from the VEEV virus results in enhanced expression of a functional protein over a longer duration than conventional mRNA.

[0039]

[0032] The present technology also relates to methods and compositions involving selfamplifying RNA (saRNA) that are extensively or fully substituted with chemically modified nucleotides. These saRNA molecules are optimized for repeated dosing and long-term transgene expression by encoding one or more proteins that inhibit antigen presentation.

[0040]

[0033] The present technology also pertains to methods and compositions involving the generation and use of mutant self-amplifying ribonucleic acid (saRNA) vectors that confer enhanced protein production and compatibility with host cells. The vectors are transcribed into RNAs that are extensively or fully substituted with chemically modified nucleotides. These self-amplifying RNA (saRNA) molecules are optimized for expression of antigens or proteins for vaccines and therapeutics.

[0041]

[0034] In one aspect, a self-amplifying RNA (saRNA) is provided comprising a nucleotide sequence encoding (i) a mutated non- structural protein 2 (NSP2), wherein the mutation is D584N and encoded by a nucleotide substitution from GAC to AAC and (ii) at least one protein or fragment thereof, and wherein the saRNA is derived from VEEV.

[0042]

[0035] In one embodiment, the saRNA exhibits protein expression when introduced into a cell that is greater than in a comparable VEEV saRNA lacking the D584N mutation. In a particular embodiment, the increase in protein expression is at least about 2-fold, about 3-fold, about 4- fold, or about 5-fold greater or more compared to a comparable saRNA lacking the mutation.

[0036] In one embodiment, the saRNA exhibits protein expression that is specifically enhanced in immune cells when introduced into a subject (e.g., a human) as compared to a comparable saRNA lacking the D584N mutation. In a particular embodiment, the immune cell is selected from T cells, dendritic cells, macrophages, monocytes, B cells or a combination thereof. In one embodiment, protein expression is specifically enhanced by about 2-fold or more.

[0043]

[0037] In one embodiment, the saRNA enhances antigen-specific immune responses when introduced into a subject as comparable to a comparable saRNA lacking the D584N mutation. In a particular embodiment, the increase is at least 20%, at least about 60%, or at least about 80% or more.

[0044]

[0038] In one embodiment, activation of the innate immune response is decreased when the saRNA is introduced in a subject compared to a comparable saRNA lacking the D584N mutation. In a particular embodiment, the decrease is at least about 20%, at least about 60%, or at least about 80% or more.

[0045]

[0039] In one embodiment, the saRNA further comprises a chimeric 3’UTR sequence. In a particular embodiment, the chimeric 3’ UTR comprises nucleotides sequences from two or more RNA viruses (e.g., alphaviruses).

[0046]

[0040] In a particular embodiment, the saRNA exhibits protein expression when introduced into a cell that is synergistically enhanced by the presence of the chimeric 3’UTR and the D584N mutation.

[0047]

[0041] The protein or fragment thereof may be any suitable protein. In one embodiment, the protein or fragment thereof is an antigen (e.g., a viral antigen). In another embodiment, the protein or fragment thereof is a therapeutic protein (e.g., a coagulation factor, an antibody, a bi-specific antibody).

[0048]

[0042] In certain embodiments, the sequence encoding the antigen or therapeutic protein is operably linked to a sequence encoding an Fc domain or an albumin-binding domain by a peptide linker.

[0049]

[0043] The antigen may vary. In one embodiment, the antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a cancer antigen or a cancer-associated antigen.

[0050]

[0044] The therapeutic protein may vary. In one embodiment, the therapeutic protein selected from enzymes, anticoagulants, antibodies, bi-specific antibodies, cytokines, fusion proteins, transcription factors, receptors, or peptides.

[0051]

[0045] In one embodiment, the at least one protein or fragment thereof is a chimeric antigen receptor (CAR).

[0046] In certain embodiments, the saRNA comprises at least two proteins or fragments thereof. In one embodiment of any aspect herein, multiple proteins are expressed from the same modified self-amplifying RNA strand by inclusion of one or more 2A sequence between the coding sequences of each protein.

[0052]

[0047] In certain embodiments, a 2A sequence or IRES sequence operably links the nucleotide sequences encoding the at least two proteins or fragments thereof. In one embodiment, a furin cleavage site is placed in front of a 2A sequence to prevent the addition of extra amino acids to the N or C terminus of a protein. In one embodiment of any aspect herein, the expression level of multiple proteins expressed from the same modified self-amplifying RNA are equal to or greater than levels of expression resulting from other methods to establish constitutive expression of multiple proteins.

[0053]

[0048] In some embodiments, a saRNA described herein comprises sequences encoding at least two proteins or fragments thereof. In one embodiment, at least one of the at least two protein sequences is an adaptive immune response inhibitor protein.

[0054]

[0049] In one embodiment, the at least two proteins comprise at least one antigen and an adaptive immune response inhibitor protein. In certain embodiments, the adaptive immune response inhibitor protein transiently modulates the immune response to prolong antigen availability and / or reduce immunogenicity of the saRNA when introduced into a subject.

[0055]

[0050] In one embodiment, the saRNA comprises at least two antigens, i.e., a multivalent saRNA. In certain embodiments, the at least two antigens are from the same organism (e.g., virus), or more particularly different strains or serotypes of the same organism. In certain embodiments, the at least two antigens are from different organisms (e.g., different viruses).

[0051] In one embodiment, the saRNA comprises at least two CARs.

[0056]

[0052] In certain embodiments, the saRNA comprises at least three proteins or fragments thereof, or at least four proteins or fragments thereof, wherein each of the coding sequences for a protein or fragment are operably linked to the downstream sequence by an IRES sequence.

[0057]

[0053] In one embodiment, at least two of the at least three proteins are antigens. In certain embodiments, at least one of the at least three proteins is an adaptive immune response inhibitor protein.

[0058]

[0054] In one embodiment, at least two of the at least three proteins are CARs.

[0059]

[0055] In one embodiment, at least three of the at least four proteins are antigens. In certain embodiments, at least one of the at least four proteins is an adaptive immune response inhibitor protein,

[0060]

[0056] In one embodiment, at least three of the at least four proteins are CARs.

[0057] In one embodiment, the saRNA nucleotide sequence comprises modified nucleotides. In a particular embodiment, the nucleotide sequence comprises greater than about 25% modified nucleotides, more particularly, between 50% and 100% modified pyrimidines. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocyti dines substituted for cytidines.

[0061]

[0058] In certain embodiments, the increase in protein expression is synergistic in the presence of the D584N mutation and the adaptive immune response inhibitor and / or the 3’ UTR and / or the modified nucleotides.

[0062]

[0059] In a second aspect, a saRNA is provided comprising a nucleotide sequence encoding (i) a chimeric 3 ’UTR sequence; and (ii) at least one protein or fragment thereof.

[0063]

[0060] In one embodiment, the chimeric 3 ’UTR comprises sequences from at least two RNA viruses. In a particular embodiment, the RNA viruses are alphaviruses are selected from Venezuela Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV), Sindbis Virus (SIN), Chikungunya Virus (CHIKV), Eastern Equine Encephalitis Virus (EEEV), Mayaro Virus (MAYV), Getah Virus (GETV), Ross River Virus (RRV), Una Virus (UNAV), Middleburg Virus (MIDV), O'nyong nyong virus (ONNV), Barmah Forest Virus (BFV), Mucambo Virus (MUCV), Tonate Virus (TONV), Everglades Virus (EVEV), Rio Negro Virus (RNV), Turnip Rosette Virus (TROV), Highlands J Virus (HJV), Western Equine Encephalitis Virus (WEEV), Fig Mosaic Emaravirus (FMV), Aura Virus (AURAV), Kunjin Virus (KUN).

[0064]

[0061] The at least one protein or fragment thereof can be any suitable protein or fragment thereof. In one embodiment, the at least one protein or fragment thereof is an antigen (e.g., a viral antigen) or therapeutic protein (e.g., IL2, an antibody, a bispecific antibody).

[0065]

[0062] In certain embodiments, the sequence encoding the therapeutic protein is operably linked to a sequence encoding an Fc domain or an albumin-binding domain by a peptide linker.

[0063] In one embodiment, the protein or fragment thereof is an antigen selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a cancer antigen or a cancer-associated antigen.

[0066]

[0064] In one embodiment, the at least one protein is a therapeutic protein selected from enzymes, anticoagulants, antibodies, bi-specific antibodies, cytokines, fusion proteins, transcription factors, receptors, or peptides.

[0067]

[0065] In one embodiment, the at least one protein is a chimeric antigen receptor (CAR).

[0068]

[0066] In one embodiment, the saRNA reduces off-target immune activation when introduced into a subject (e.g., a human) compared to a comparable saRNA lacking the chimeric 3’ UTR. In a particular embodiment, the reduction is at least about 2-fold.

[0067] In one embodiment, the saRNA enhances protein expression levels when introduced into a cell in comparison a comparable saRNA lacking the chimeric 3’ UTR. In a particular embodiment, protein expression increased by about 3 -fold or greater compared to a comparable saRNA lacking a chimeric 3’ UTR sequence.

[0069]

[0068] In one embodiment, the saRNA exhibits altered or restricted cellular tropism when introduced into a subject (e.g., a human) compared to a comparable saRNA lacking the chimeric 3’ UTR. In certain embodiments, the saRNA exhibits enhanced tropism for immune cells or cancer cells.

[0070]

[0069] In one embodiment, the saRNA comprises at least two proteins or fragments thereof.

[0071]

[0070] In one embodiment, the nucleotide sequence encoding the at least two proteins are operably linked by a 2A sequence or an IRES sequence. In a particular embodiment, a furin cleavage site is placed in front of a 2A sequence to prevent the addition of extra amino acids to the N or C terminus of a protein.

[0072]

[0071] In one embodiment, the at least two proteins comprise at least one antigen and at least one adaptive immune responsive inhibitor protein.

[0073]

[0072] In one embodiment, the saRNA comprises at least two antigens, i.e., a multivalent saRNA. The at least two antigens may be from the same organism (e.g., the same virus), or different strains or serotypes thereof. Alternatively, the at least two antigens may be from different organisms (e.g., different viruses).

[0074]

[0073] In certain embodiments, at least one of the at least two proteins is a CAR.

[0075]

[0074] In certain embodiment, the saRNA comprises at least three proteins or fragments thereof, or at least four proteins or fragments thereof, wherein each of the coding sequences for a protein or fragment are operably linked to the downstream sequence by an IRES sequence.

[0076]

[0075] In one embodiment, at least two of the at least three proteins are antigens. In certain embodiments, at least one of the at least three proteins is an adaptive immune response inhibitor protein.

[0077]

[0076] In certain embodiments, at least two of the at least three proteins are CARs.

[0078]

[0077] In one embodiment, at least three of the at least four proteins are antigens. In certain embodiments, at least one of the at least four proteins is an adaptive immune response inhibitor protein.

[0079]

[0078] In one embodiment, at least three of the at least four proteins are CARs.

[0080]

[0079] In one embodiment, the saRNA nucleotide sequence comprises modified nucleotides. In a particular embodiment, the nucleotide sequence comprises greater than about 25% modified nucleotides, more particularly, between 50% and 100% modified pyrimidines. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocyti dines substituted for cytidines.

[0081]

[0080] In certain embodiments, the increase in protein expression is synergistic in the presence of the 3’UTR and the adaptive immune response inhibitor and / or the modified nucleotides.

[0082]

[0081] In a third aspect, a saRNA is provided comprising a nucleotide sequence comprising greater than about 25% of cytidines substituted with 5-flourocytidine and encoding at least one protein or a fragment thereof.

[0083]

[0082] In one embodiment, the nucleotide sequence comprises between about 50% and about 100% of cytidines substituted with 5-flourocytidine.

[0084]

[0083] In one embodiment, the nucleotide sequence comprises between about 50% and about 100% of cytidines substituted with 5-flourocytidine.

[0085]

[0084] In one embodiment, the saRNA expresses the at least one protein or fragment thereof when introduced in a cell at a level equal to or greater than a comparable saRNA lacking 5- flourocytidine substitution or having a reduced percentage of substitution.

[0086]

[0085] In one embodiment, the saRNA reduces innate immune response activation when introduced into a subject (e.g., a human) compared to a comparable saRNA lacking 5’flourocytidine substitution or having a reduced percentage of substitution.

[0087]

[0086] In one embodiment, the at least one protein or fragment thereof is an antigen (e.g., a viral antigen) or a therapeutic protein (e.g., IL2, an antibody, a bispecific antibody).

[0088]

[0087] In one embodiment, the saRNA comprises at least two proteins or fragments thereof.

[0089]

[0088] In one embodiment, the nucleotide sequence encoding the at least two proteins are operably linked by a 2A sequence or an IRES sequence. In a particular embodiment, a furin cleavage site is placed in front of a 2A sequence to prevent the addition of extra amino acids to the N or C terminus of a protein.

[0090]

[0089] In one embodiment, the at least at least two proteins comprise an antigen and an adaptive immune responsive inhibitor protein.

[0091]

[0090] In certain embodiments, the at least two protein sequences are at least two antigens, i.e., a multivalent saRNA. In one embodiment, the at least two antigens are from the same organism, or different strains or serotypes thereof. In another embodiment, the at least two antigens are from different organisms.

[0092]

[0091] In certain embodiment, the saRNA comprises at least three proteins or fragments thereof, or at least four proteins or fragments thereof, wherein each of the coding sequences for a protein or fragment are operably linked to the downstream sequence by an IRES sequence.

[0092] In certain embodiments, at least two of the at least three proteins are antigens. In one embodiment, at least one of the at least three proteins is an adaptive immune response inhibitor protein.

[0093]

[0093] In certain embodiments, at least two of the at least three proteins are CARs.

[0094]

[0094] In one embodiment, at least three of the at least four proteins are antigens. In a particular embodiment, at least one of the at least four proteins is an adaptive immune response inhibitor proteins.

[0095]

[0095] In one embodiment, at least three of the at least three proteins are CARs.

[0096]

[0096] In one embodiment, the saRNA further comprises additional modifications selected from a D584N mutation, a chimeric 3’ UTR or a combination thereof. In certain embodiments, the one or more additional modifications results in a synergistic increase in protein expression when the saRNA in introduced into a cell in comparison to a comparable saRNA comprising about 100% of cytidines substituted with 5’flourocytidine.

[0097]

[0097] In a fourth aspect, a saRNA is provided comprising a nucleotide sequence encoding (i) at first nucleotide sequence encoding a first protein or fragment thereof; (ii) a second nucleotide sequence encoding a second protein of fragment thereof; and (ii) an IRES which operably links the first and second nucleotide sequence, and wherein the nucleotide sequence comprises greater than 25% modified pyrimidines.

[0098]

[0098] In one embodiment, the saRNA further comprises (iii) a third nucleotide sequence encoding a third protein or fragment thereof and (iv) a second IRES sequence operably linking the second nucleotide sequence to the third nucleotide sequence.

[0099]

[0099] In one embodiment, the saRNA further comprises (v) a fourth nucleotide sequence encoding a fourth protein or fragment thereof and (vi) a third IRES sequence operably linking the third nucleotide sequence to the fourth nucleotide sequence.

[0100]

[0100] In certain embodiments, the nucleotide sequence comprises between 26% and 100% modified pyrimidines.

[0101]

[0101] In one embodiment, the nucleotide sequence comprises between about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% modified pyrimidines.

[0102]

[0102] In one embodiment, the nucleotide sequence comprises greater than about 25% modified pyrimidines selected from 5-fluorocytidine, 5-methylcytidine, 5- hydroxymethylcytidine, 5-methyluridine, 5-fluorouridine, or combinations thereof.

[0103] In one embodiment, the nucleotide sequence comprises between about 50% and about 100% 5-fluorocytidine. In a particular embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine.

[0103]

[0104] In certain embodiments, the expression of the protein downstream of the IRES sequence is enhanced relative to a comparable saRNA having about 25% or less modified pyrimidines. In one embodiment, expression of the downstream protein is enhanced by least about 2-fold, about 3-fold, about 4-fold, or about 5-fold greater or more compared to a comparable saRNA having about 25% or less modified pyrimidines.

[0104]

[0105] In one embodiment, the at least two proteins are selected from antigens (e.g., viral antigens) and therapeutic proteins (e.g., IL2, antibodies, bispecific antibodies, enzymes, coagulation proteins).

[0105]

[0106] In certain embodiments, the at least two proteins are at least two antigens, i.e., a multivalent saRNA. In one embodiment, the at least two antigens are from the same organism, or different strains or serotypes thereof. In another embodiment, the at least two antigens are from different organisms.

[0106]

[0107] In one embodiment, at least one of the at least two proteins is a functional protein. In one embodiment, the activity of the functional protein is modulated by the presence of at least one stimuli. The stimuli may be endogenous or exogenous. In certain embodiments, the at least one stimuli is an agent (e.g., a cell, protein, or small molecule or a combination thereof). In one embodiment, the concentration of the agent modulates the functional protein. In certain embodiments, the stimuli is selected from light, temperature, magnetic field, or a combination thereof. In one embodiment, the wavelength of the light modulates the functional protein. In certain embodiments, the specific temperature modulates the functional protein.

[0107]

[0108] In one embodiment, at least one of the at least two proteins is an adaptive immune response inhibitor protein. The adaptive immune response inhibitor protein may be, for example, a viral immune evasion protein. In a particular embodiment, the immune evasion protein is selected from BNLF2A or UL49.5.

[0108]

[0109] In one embodiment, at least one of the at least two proteins or fragments thereof is an antigen. In another embodiment, at least two of the at least three proteins or fragments thereof is an antigen. In a further embodiment, at least three of the at least three proteins or fragments thereof is an antigen.

[0109] [HO] The antigen may vary. In one embodiment, the antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a cancer antigen or a cancer-associated antigen. [Hl] The at least two proteins or fragments thereof may vary. In one embodiment, at least one of the at least two proteins is a therapeutic protein.

[0110]

[0112] The therapeutic protein may vary. In one embodiment, the therapeutic protein is selected from the group consisting of therapeutic proteins include enzymes, antibodies, bispecific antibodies, cytokines, transcription factors, receptors, or peptides.

[0111]

[0113] In certain embodiments, the least one of the at least two proteins is a chimeric antigen receptor (CAR).

[0112]

[0114] In any of the aspects above, the saRNA may comprise a unique barcode sequence inserted into the 5’ or 3’ untranslated region (UTR) of the saRNA.

[0113]

[0115] In a fifth aspect, a lipid nanoparticle encapsulating the saRNA or modified saRNA disclosed herein.

[0114]

[0116] The composition of the lipid nanoparticle may vary. In one embodiment, the lipid nanoparticle comprises an ionizable lipid and a helper lipid. The helper lipid may be, for example, cholesterol, a polyethylene glycol (PEG)-modified lipid or a combination thereof.

[0115]

[0117] In one embodiment, the lipid nanoparticle contains l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE). In a particular embodiment, the lipid nanoparticle has a greater transfection efficiency than a lipid nanoparticle comprising DSPC.

[0116]

[0118] In certain embodiments, the lipid nanoparticle further comprises a targeting moiety. Representative, non-limiting examples of targeting moieties include antibodies, aptamers and peptides.

[0117]

[0119] In a sixth aspect, a cell is provided comprising the saRNA, modified saRNA or lipid nanoparticle disclosed herein.

[0118]

[0120] The cell may vary. In one embodiment, the cell is in vitro. In other embodiments, the cell is in in vivo.

[0119]

[0121] In one embodiment, the cell is selected from an immune cell, a cancer cell, a muscle cell or a lung cell.

[0120]

[0122] In one embodiment, the cell is a human cell, and more particularly, a human immune cell, cancer cell, muscle cell or lung cell.

[0121]

[0123] In a seventh aspect, a pharmaceutical composition comprising (i) a pharmaceutically acceptable excipient and (ii) the saRNA, modified saRNA or polymeric carrier, polycationic protein or peptide, or lipid nanoparticle (LNP) associated with the same (e.g., encapsulating the same).

[0122]

[0124] In one embodiment, the pharmaceutical composition comprises lipid nanoparticles containing l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE). In certain embodiments, the lipid nanoparticle comprising DOPE has a greater transfection efficiency than a lipid nanoparticle comprising DSPC.

[0123]

[0125] In one embodiment, the pharmaceutical composition comprises a lipid nanoparticle comprising a sterol, such as cholesterol. In certain embodiments, the molar ratio of cholesterol is less than about 60%, less than about 50%, less than about 40%, less than about 30% or less about that about 20%. In one embodiment, the transfection efficiency of the lipid nanoparticle comprising cholesterol is improved relative to a comparable lipid nanoparticle containing more or less cholesterol.

[0124]

[0126] In an eighth aspect, a method of increasing expressing at least one protein, comprising administering an saRNA, modified saRNA or lipid nanoparticle encapsulating the same to a cell, wherein the saRNA comprises a nucleotide sequence encoding one or more proteins or fragments thereof and one or more features selected from (i) a non-structural protein 2 (nsp2) encoding a D584N mutation, (ii) a chimeric 3’ UTR, (ii) at least 25% of the cytidines are substituted by 5-flourocytidines, wherein administration results in increased expression of at least one protein in comparison saRNA lacking one or more of the features.

[0125]

[0127] Optionally, the saRNA encodes two or more proteins or fragments thereof, for example, two, three or four or more proteins or fragments thereof. In one embodiment, at least one of the two or more proteins is an adaptive immune response inhibitor protein. In certain embodiments, the at least two or more proteins are operably linked by a 2A sequence or an IRES sequence.

[0126]

[0128] Optionally, the one or more proteins are linked to a Fc or albumin domain by a peptide linker.

[0127]

[0129] In one embodiment, the one or more proteins are antigens (e.g., viral antigens) or therapeutic proteins (e.g., IL2, antibodies, bispecific antibodies).

[0128]

[0130] In one embodiment, the cell in in vitro. In alternative embodiments, the cell is in vivo.

[0129]

[0131] In one embodiment, the cell is selected from an immune cell, a cancer cell, a muscle cell or a lung cell.

[0130]

[0132] In one embodiment, the cell is a human cell, and more particularly, a human immune cell, cancer cell, muscle cell or lung cell.

[0131]

[0133] In one embodiment, the protein expression is increased by about is enhanced by about by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 2-fold, about 3-fold, about 4-fold or about 5-fold or more.

[0134] In a ninth aspect, a method of enhancing serum protein levels of at least one protein or fragment thereof is provided, comprising administering a saRNA, modified saRNA or lipid nanoparticle encapsulating the same to a subject (e.g., a human), wherein the saRNA or modified saRNA encodes at least one protein or fragment thereof is provided, wherein the at least one protein or fragment thereof linked to a Fc or albumin domain by a peptide linker disclosed herein to subject, and wherein administration results in enhanced serum protein levels relative to a comparable saRNA encoding an antigen or protein lacking an Fc or albumin domain.

[0132]

[0135] Optionally, the saRNA encodes two or more proteins or fragments thereof, for example, two, three or four or more proteins or fragments thereof. In one embodiment, at least one of the two or more proteins is an adaptive immune response inhibitor protein. In certain embodiments, the at least two or more proteins are operably linked by a 2A sequence or an IRES sequence.

[0133]

[0136] Optionally, the one or more proteins are linked to a Fc or albumin domain by a peptide linker.

[0134]

[0137] In one embodiment, the one or more proteins are antigens (e.g., viral antigens) or therapeutic proteins (e.g., IL2, antibodies, bispecific antibodies).

[0135]

[0138] In certain embodiments, the administration is local, and expression is increased by about at least 5-fold compared to a comparable saRNA encoding an antigen protein lacking the Fc-domain or albumin domain.

[0136]

[0139] In one embodiment, administration is to a subject via intramuscular (IM) or subcutaneous (SC) injection. In a particular embodiment, the serum protein levels following IM or SC are increased by at least about 2-fold, about 5-fold, about 10-fold, about 20-fold, or about 100-fold.

[0137]

[0140] In a tenth aspect, a method of reducing immunogenicity of a saRNA is provided, comprising administering a saRNA, modified saRNA or lipid nanoparticle to a subject (e.g., a human), wherein the saRNA comprises a nucleotide sequence encoding one or more proteins or fragments thereof and one or more features selected from (i) a non- structural protein 2 (nsp2) encoding a D584N mutation, (ii) a chimeric 3’ UTR, (ii) at least 25% of cytidines substituted by 5-flourocytidines, and wherein administration results in reduced immunogenicity.

[0138]

[0141] Optionally, the saRNA encodes two or more proteins or fragments thereof, for example, two, three or four or more proteins or fragments thereof. In one embodiment, at least one of the two or more proteins is an adaptive immune response inhibitor protein. In certain embodiments, the at least two or more proteins are operably linked by a 2A sequence or an IRES sequence.

[0142] Optionally, the one or more proteins are linked to a Fc or albumin domain by a peptide linker.

[0139]

[0143] The reduction in immunogenicity may vary. In one embodiment, the immunogenicity is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold or about 5- fold or more.

[0140]

[0144] In an eleventh aspect, a method of reducing an adaptive immune response to a saRNA is provided, comprising administering the saRNA, modified saRNA or lipid nanoparticle disclosed herein to a subject (e.g., a human), wherein the saRNA comprises a nucleotide sequence encoding one or more proteins or fragments thereof and one or more features selected from (i) a non-structural protein 2 (nsp2) encoding a D584N mutation, (ii) a chimeric 3’ UTR, (ii) at least 25% of cytidines substituted by 5-flourocytidines, and wherein administration results in a reduced adaptive immune response.

[0141]

[0145] Optionally, the saRNA encodes two or more proteins or fragments thereof, for example, two, three or four or more proteins or fragments thereof. In one embodiment, at least one of the two or more proteins is an adaptive immune response inhibitor protein. In certain embodiments, the at least two or more proteins are operably linked by a 2A sequence or an IRES sequence.

[0142]

[0146] Optionally, the one or more proteins are linked to a Fc or albumin domain by a peptide linker.

[0143]

[0147] In one embodiment, the one or more proteins are antigens (e.g., viral antigens) or therapeutic proteins (e.g., IL2, antibodies, bispecific antibodies).

[0144]

[0148] The reduction in adaptive immune response may vary. In one embodiment, the adaptive immune response is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold or about 5-fold or more.

[0145]

[0149] In a twelfth aspect, a method of treating a disease or disorder is provided, comprising administering an saRNA to a cell, wherein the saRNA comprises a nucleotide sequence encoding one or more features selected from (i) a D584N mutation in non-structural protein 2 (NSP2), (ii) a chimeric 3’ UTR, (ii) at least two proteins or fragments thereof, wherein at least one protein is an adaptive immune response protein, (iv) at least two or more proteins operably linked by an IRES sequence, wherein greater than about 25% of the nucleotide sequence comprises modified pyrimidines, wherein administration results treatment of the disease or disorder.

[0150] In one embodiment, the disease or disorder is an infectious disease or disorder (e.g., a viral disease).

[0146]

[0151] The one embodiment, the disease or disorder is cancer, a cardiovascular disorder, a neurological disorder, an autoimmune disorder, a metabolic disorder or a respiratory disorder.

[0147]

[0152] In a thirteenth aspect, a method of manufacturing a saRNA is provided, comprising: (i) preparing a linearized plasmid DNA template; (ii) performing in vitro transcription using a mutant T7 RNA polymerase that generates reduced double-stranded RNA (dsRNA) impurities; and (iii) purifying the saRNA, wherein reduced dsRNA impurities result in simplified downstream purification and decreased innate immune responses when administered to a subject and simplified downstream purification.

[0148]

[0153] In one embodiment, the mutant T7 RNA polymerase reduces dsRNA impurities by at least 50% compared to wild-type T7 RNA polymerase.

[0149]

[0154] In a fourteenth aspect, a method of high-throughput screening lipid nanoparticle (LNP) formulations is provided comprising: (i) encapsulating multiple barcoded saRNAs into different LNP formulations, wherein each saRNA is associated with a unique barcode; (ii) administering the LNP formulations to a biological model; (iii) isolating saRNA from target tissues or cells; and (iv) quantifying barcode abundance by next-generation sequencing (NGS) to identify LNP formulations with optimized functional saRNA delivery.

[0150]

[0155] In one embodiment, the method further comprises (v) screening different modified nucleotides within the same LNP formulation or screening combinations of different LNP formulations and modified nucleotides to identify optimized formulations.

[0151]

[0156] In one embodiment, the optimized formulations exhibit one or more properties selected from increased cellular uptake, endosomal escape, or translation efficiency relative to nonoptimized formulations.

[0152] BRIEF DESCRIPTION OF THE DRAWINGS

[0153]

[0157] FIG. 1 shows a schematic detailing the effect of nucleoside modification on an IRES containing messenger RNA (mRNA) or self-amplifying RNA (saRNA). In mRNA, the presence of the modified nucleosides inhibits the activity of the IRES sequence, resulting in no expression of the protein downstream of the IRES. Conversely, modified nucleosides are removed during replication of the self-amplifying RNA and the protein downstream of the IRES sequence is expressed.

[0154]

[0158] FIG. 2 shows a schematic depicting an mRNA encoding GFP and iRFP, the latter after an IRES sequence. The expression of GFP indicates cap-dependent translation. The expression of iRFP indicates cap-independent translation. When the reporter mRNA is synthesized with wildtype nucleotides, the expression of both proteins occurs. When synthesized with 100% Nl- methylpseudouridine or 5-methylcytidine substitution, the IRES -dependent expression of the iRFP protein is completely inhibited.

[0155]

[0159] FIG. 3 shows the results of a flow cytometry screen performed to assess the impact of modified nucleosides substituted into the reporter construct depicting in FIG. 2. As the percentage of substitution is increased, the percentage of cells expressing iRFP (iRFP+ %) is decreased, indicating reduced IRES-mediated protein expression.

[0156]

[0160] FIG. 4 shows the results of a flow cytometry experiment performed to assess the impact of modified nucleosides on IRES mediated expression in a mRNA or saRNA. When synthesized with wildtype nucleotides, the expression of both proteins occurs in the case of mRNA. When synthesized with 100% 5-methylcytidine substitution, the IRES-dependent expression of the BFP protein from the mRNA is completely inhibited. In the case of saRNA containing the same IRES sequence, the expression of both reporter proteins occurs with high efficiency.

[0157]

[0161] FIG. 5 shows a schematic detailing the expression of two antigens from a single modified self-amplifying RNA. After replication, modified nucleosides are removed from the self-amplifying RNA and the antigen downstream of the IRES sequence is expressed in addition to the antigen expressed through cap-dependent translation.

[0158]

[0162] FIG. 6 shows a schematic of the results of a flow cytometry experiment performed to assess the surface expression of Spike protein and HA protein in C2C12 transfected with a modified self-amplifying RNA encoding both antigens separated by a P2A sequence (top) or IRES sequence (bottom).

[0159]

[0163] FIG. 7 shows a depiction of self-amplifying RNA constructs encoding antigens from multiple serotypes of dengue virus as well as western blot results showing the correct expression of these encoded antigens in HEK293T cells.

[0160]

[0164] FIG. 8 shows a schematic depicting various self-amplifying RNA constructs encoding one or multiple antigens derived from one or multiple serotypes of dengue virus.

[0161]

[0165] FIG. 9 shows a schematic depicting a self-amplifying RNA encoding four distinct proteins.

[0162]

[0166] FIG. 10 shows flow cytometry results depicting the co-expression of multiple proteins in cells transfected with unmodified or modified self-amplifying RNA constructs.

[0163]

[0167] FIG. 11 shows a schematic detailing the approach for screening lipid nanoparticles containing a modified self-amplifying RNA reporter in C2C12 and Jurkat cell lines.

[0168] FIG. 12 shows the properties (size, PDI, encapsulation efficiency) of lipid nanoparticles containing a 5-hydroxymethylcytidine modified self-amplifying RNA synthesized with 50 mol% ionizable lipid, 10 mol% DSPC or DOPE, 38.5 mol% cholesterol, 1.5 mol% DMG-PEG2K.

[0164]

[0169] FIG. 13 shows the results of a flow cytometry experiment performed to measure the transfection efficiency LNPs synthesized with DSPC or DOPE containing a modified selfamplifying RNA encoding a fluorescent reporter in C2C12 cells.

[0165]

[0170] FIG. 14 shows the results of a flow cytometry experiment performed to measure the transfection efficiency LNPs synthesized with DSPC or DOPE containing a modified selfamplifying RNA encoding a fluorescent reporter in Jurkat cells.

[0166]

[0171] FIG. 15 shows the results of In-vivo transfection of a 20% cholesterol (soft) and a 60% cholesterol (stiff) LNP formulation using FLuc mRNA analyzed via BLI. Mice were injected with 1 ug of mRNA in the respective LNP formulation or a PBS mock injection and imaged at 24 and 48 hours post LNP treatment.

[0167]

[0172] FIG. 16 shows a schematic depicting a self-amplifying RNA system encoding a protein tagged with an Fc domain that results in improved biodistribution and higher serum concentration than a protein lacking the Fc domain.

[0168]

[0173] FIG. 17 shows a schematic depicting mRNA and self-amplifying RNA (saRNA) constructs encoding nanoLuciferase (nanoLuc) with or without fusion to additional IgGl Fc and albumin domains (top) and the in vitro transfection results of the depicted constructs in C2C12 after 100 ng or 10 ng LNP transfection (bottom).

[0169]

[0174] FIG. 18 shows the systemic levels of nanoLuc expression in C57BL / 6 mice after intramuscular administration of 1 microgram of mRNA or saRNA in SM102 LNPs.

[0170]

[0175] FIG. 19 shows the systemic levels of nanoLuc murine IgGl Fc fusion expression in C57BL / 6 mice after intramuscular administration of 1 microgram of mRNA or saRNA in SMI 02 LNPs.

[0171]

[0176] FIG. 20 shows the systemic levels of nanoLuc murine albumin fusion expression in C57BL / 6 mice after intramuscular administration of 1 microgram of mRNA or saRNA in SMI 02 LNPs.

[0172]

[0177] FIG. 21 Shows systemic levels following administration of 10 ng saRNA encoding either nanoLuc-human IgGl Fc fusion, nanoLuc-murine IgGl FcyR-fusion, nanoLuc-murine IgGl Fc neonatal receptor-null fusion, nanoLuc-murine IgGl fusion, or 10 ng mRNA encoding nanoLuc-murine IgGl Fc fusion.

[0178] FIG. 22 shows the results of a microscopy experiment performed to assess the compatibility of saRNA with 100% substitution of cytidine for 5-fluorocytidine with lipid nanoparticle (LNP) delivery into C2C12 cells (top). Following LNP delivery of saRNA or mRNA encoding a mCherry fluorescent reporter, saRNA with 100% substitution of cytidine with 5-fluorocytidine showed compatibility with LNP delivery as determined by appreciable levels of construct expression. C2C12 cells were electroporated with an mCherry saRNA synthesized with 100% substitution of 5-fluorocytidine and expression was verified by flow cytometry (bottom).

[0173]

[0179] FIG. 23 shows a synthetic scheme to afford modified cytidine and uridine triphosphates.

[0174]

[0180] FIG. 24 is a schematic depicting the advantage of removing double-stranded RNA for modified self-amplifying RNA encoding an exemplary mCherry cargo. The removal of doublestranded RNA from modified self-amplifying RNA results in a minimized inflammatory immune response.

[0175]

[0181] FIG. 25 shows the results of a blot analysis of the double-stranded RNA content conducted on synthesized messenger RNA, unmodified self-amplifying RNA, and 5- hydroxymethylcytidine or 5-methylcytidine modified self-amplifying RNA (top) and the results from an experiment to transfect Jurkat T cells with modified messenger RNA, unmodified self-amplifying RNA, or modified self-amplifying RNA with or without doublestranded RNA depletion (bottom).

[0176]

[0182] FIG. 26 shows the levels of IL-6 in the serum of mice intramuscularly administered LNPs containing purified modified mRNA, unmodified self-amplifying RNA, or modified self-amplifying RNA. The mice administered modified self-amplifying RNA that was depleted of double-stranded RNA had the lowest levels of IL-6.

[0177]

[0183] FIG. 27 shows the levels of expression of a luciferase reporter in C57BL / 6 mice after intramuscular administration.

[0178]

[0184] FIG. 28 shows a denaturing gel of unmodified or modified self-amplifying RNAs or mRNA encoding firefly luciferase synthesized with a wildtype T7 polymerase (Megascript) or mutant T7 polymerase (Yeasen) (top), a dot-blot analysis and quantification of the doublestranded RNA content of unmodified or modified self-amplifying RNAs encoding firefly luciferase synthesized by a wildtype T7 polymerase (MegaScript) or a mutant T7 polymerase (Yeasen) (bottom).

[0179]

[0185] FIG. 29 shows a denaturing gel of unmodified or modified self-amplifying RNAs encoding mCherry synthesized with a wildtype T7 polymerase (MegaScript) or mutant T7 polymerase (Yeasen) (top), shows a dot-blot analysis and quantification of the double-stranded RNA content of unmodified or modified self-amplifying RNAs encoding mCherry synthesized by a wildtype T7 polymerase (MegaScript) or a mutant T7 polymerase (Yeasen) (bottom).

[0180]

[0186] FIG. 30 shows a schematic drawing depicting an exemplary structure for a barcoded self-amplifying RNA used for high-throughput, in vivo screening. saRNA is synthesized encoding an RdRp, a firefly luciferase reporter protein, and a 10 base pair barcode located between the 3’ end of the cargo protein and upstream of the VEEV 3’ UTR. Upon successful localization to the cytosol of a cell and translation of the RdRp, replication of saRNA will result in amplification of the barcode sequence, which can be extracted for NGS and quantification of functional saRNA delivery.

[0181]

[0187] FIG. 31 depicts the ability to reproducibly isolate and amplify the barcoded region from cells transfected with barcoded saRNA. RNA extracts from cells transfected with saRNA were used to produce cDNA and then amplified using primers specific to regions of saRNA encompassing the barcode-containing region. For five RNA extracts performed in parallel, all samples produced distinct, single bands with the size anticipated for correct PCR products.

[0182]

[0188] FIG. 32 shows a schematic detailing the ability for high-throughput screening with barcoded saRNA to detect functional transfection of RNA cargo into the cytosol of translationally active cells. Following treatment with a library of barcoded LNPs, saRNA barcode amplification and relative abundance is measured via NGS is used to determine LNP efficacy. In contrast to barcoded nucleotide screening methods, this provides an output that can distinguish between target tissue localization, uptake, and functional cytosolic delivery and translation.

[0183]

[0189] FIG. 33 shows a schematic depicting the recognition of a presented antigen by an immune cell capable of triggering an adaptive immune response. The presented antigen can be a foreign RdRp peptide or a transgene peptide. The expression of an adaptive immune response prevention protein (AIP protein) interrupts antigen presentation.

[0184]

[0190] FIG. 34 shows a schematic depicting exemplary designs of a self-amplifying RNA encoding a cargo protein and an additional protein that suppresses the adaptive immune response.

[0185]

[0191] FIG. 35 shows the results of an experiment where Jurkat T cells were electroporated with mCherry encoding saRNA, AIP-2A-mCherry encoding saRNA, or buffer (mock). The expression of mCherry (x axis) and HLA A / B / C (y axis) was detected by flow cytometry. The expression of the AIP protein resulted in a decrease in HLA A / B / C expression in the mCherry + population.

[0192] FIG. 36 shows the results four days after transfection of Jurkat T cells with mCherry encoding saRNA, AIP-2A-mCherry encoding saRNA, or buffer (mock). The expression of mCherry (x axis) and HLA A / B / C (y axis) was detected by flow cytometry. The expression of the AIP protein resulted in a decrease in HLA A / B / C expression in the mCherry + population.

[0186]

[0193] FIG. 37 shows the results of an experiment where Jurkat T cells were electroporated with mCherry encoding saRNA, BNLF2A-2A-mCherry encoding saRNA, UL49.5-2A- mCherry encoding saRNA, UL49.5-IRES-mCherry encoding saRNA, or buffer (mock). The expression of mCherry (x axis) and HLA A / B / C (y axis) was detected by flow cytometry. The expression of the AIP proteins resulted in a decrease in HLA A / B / C expression in the mCherry + population. The mean fluorescence intensity (MFI) of the mCherry- and mCherry+ populations are graphed in the bar chart.

[0187]

[0194] FIG. 38 depicts a VEEV saRNA in comparison to a chimeric saRNA comprising chimeric sequence elements in the 3’ untranslated region along with sequence elements from the original virus. The chimeric construct is generated by taking a base vector, for example VEEV, and replacing sequences in the 3’ untranslated region with a chimeric sequence derived from another RNA virus. The VEEV vector transfects undesired cell types, innate immune cells for example that may result in excessive generation of inflammatory cytokines in addition to the expression of the encoded protein by desired cell types. By comparison, the chimeric saRNA vector transfects only the desired cell type.

[0188]

[0195] FIG. 39 shows the percentage of GFP+ human PBMCs that were transfected with the VEEV or chimeric saRNA constructs.

[0189]

[0196] FIG. 40 shows the amount of GFP expressing HEK293 cells transfected with VEEV or chimeric saRNA constructs synthesized with or without various nucleoside modifications.

[0190]

[0197] FIG. 41 shows the amount of GFP expressing THP1 cells transfected with VEEV or chimeric saRNA constructs synthesized with or without various nucleoside modifications.

[0191]

[0198] FIG. 42 shows the amount of GFP expressing C2C12 cells transfected with VEEV or chimeric saRNA constructs synthesized with or without various nucleoside modifications.

[0192]

[0199] FIG. 43 shows longitudinal luciferase expression in BALB / c mice, measured by In Vivo Imaging System (IVIS), following intramuscular injection of LNPs containing 1 microgram of either VEEV saRNA (saRNA), saRNA containing a chimeric 3’ UTR (Chimeric saRNA), mRNA, or PBS (top) and images of biological replicate mice expressing luciferase on day 7 post administration (bottom).

[0200] FIG. 44 and FIG. 45 show representative images of mice on day 3 post administration of VEEV saRNA (saRNA) or chimeric saRNA illustrating a difference in the expression coming from a region where draining lymph nodes are located.

[0193]

[0201] FIG. 46 shows the change in weight after administration of LNPs containing 1 microgram of either VEEV saRNA (saRNA), Chimeric saRNA, mRNA, or PBS to BALB / C mice.

[0194]

[0202] FIG. 47 depicts the workflow utilized for identifying adaptive mutations that enhance the performance of self-amplifying RNA vector.

[0195]

[0203] FIG. 48 shows the results of a denaturing electrophoresis gel to assess the length and integrity of self-amplifying RNA vectors synthesized with or without complete substitution of 5-methylcytidine. The RNAs analyzed in the gel were used in downstream example 41.

[0196]

[0204] FIG. 49 shows the luminescent signal at 24 hours resulting from transfection of Jurkat cells with LNPs containing self-amplifying RNAs synthesized with or without 5- methylcytidine (m5C) generated from VEEV, nsp3 mutant (Q48P) or nsp2 mutant (D584N) templates. The performance is greatly improved by the inclusion of the mutation in nsp2 relative to the original VEEV or previously reported nsp3 mutant.

[0197]

[0205] FIG. 50 shows the luminescent signal at 24 hours resulting from transfection of THP1 cells with LNPs containing self-amplifying RNAs synthesized with or without 5- methylcytidine (m5C) generated from VEEV, nsp3 mutant (Q48P) or nsp2 mutant (D584N) templates. The performance is greatly improved by the inclusion of the mutation in nsp2 relative to the original VEEV or previously reported nsp3 mutant.

[0198]

[0206] FIG. 51 shows the luminescent signal at 72 hours resulting from transfection of THP1 cells with LNPs containing self-amplifying RNAs synthesized with or without 5- methylcytidine (m5C) generated from VEEV, nsp3 mutant (Q48P) or nsp2 mutant (D584N) templates. The performance is greatly improved by the inclusion of the mutation in nsp2 relative to the original VEEV or previously reported nsp3 mutant.

[0199]

[0207] FIG. 52 shows the luminescent signal at 24 hours resulting from transfection of HEK293 cells with MessengerMax™ containing self-amplifying RNAs synthesized with or without the nsp2 mutation (D584N), a chimeric 3’ UTR, and a combination of the nsp2 mutation and the chimeric 3’ UTR.

[0200]

[0208] FIG. 53 shows the luminescent signal at 24 hours resulting from transfection of THP1 cells with MessengerMax™ containing self-amplifying RNAs synthesized with or without the nsp2 mutation (D584N), a chimeric 3’ UTR, and a combination of the nsp2 mutation and the chimeric 3’ UTR.

[0209] FIG. 54 shows the expression of luciferase measured by IVIS imaging of BALB / C mice 24 hours after intramuscular administration of m5C modified VEEV saRNA or VEEV saRNA containing the D584N mutation in the nsp2 protein.

[0201]

[0210] FIG. 55 shows increased expression of luciferase in the popliteal lymph node region at 24 hours following administration of m5C modified VEEV saRNA or VEEV saRNA containing the D584N mutation in the nsp2 protein.

[0202]

[0211] FIG. 56 shows the expression of luciferase in the popliteal lymph node region at 72 hours following administration of m5C modified VEEV saRNA or VEEV saRNA containing the D584N mutation in the nsp2 protein.

[0203]

[0212] FIG. 57 shows the levels of IFNal in the serum of mice 24 hours after administration of PBS, m5C VEEV saRNA, m5C VEEV saRNA with D584N mutation in the nsp2 protein, m5C VEEV saRNA with a chimeric 3’ UTR, m5C VEEV saRNA with D584N mutation in the nsp2 protein and a chimeric 3’ UTR.

[0204]

[0213] FIG. 58 shows a schematic detailing the expression of two chimeric antigen receptors from a single modified self-amplifying RNA. After replication, modified nucleosides are removed from the self-amplifying RNA and the CAR downstream of the IRES sequence is expressed in addition to the CAR expressed through cap-dependent translation.

[0205]

[0214] FIG. 59 shows the results of a flow cytometry experiment performed to assess the expression of two chimeric antigen receptors encoded on the same self-amplifying RNA. A self-amplifying RNA encoding a HER2 targeting inhibitory CAR and an IRES sequence followed by a CD 19 activating CAR was synthesized with 100% 5-methylcytidine substitution and transfected into Jurkat cells by electroporation. The presence of both receptors on the surface of the cells was confirmed by antibody staining.

[0206]

[0215] FIG. 60 shows the expression of secreted nano-luciferase in mouse serum after intramuscular administration of LNPs containing 5 -hydroxymethylcytidine modified selfamplifying RNA encoding nanoLuc, nanoLuc-albumin fusion, or nanoLuc-Fc fusion.

[0207]

[0216] FIG. 61 shows the expression at multiple time points of secreted nano-luciferase in mouse serum after intramuscular administration LNPs containing 5 -hydroxymethylcytidine modified self-amplifying RNA encoding nanoLuc-Fc fusion.

[0208]

[0217] FIG. 62 depicts a comparison between the mean fluorescence intensity in HEK293 cells transfected with GFP encoding VEEV or chimeric saRNAs.

[0209]

[0218] FIG. 63 shows the amount of transfected HEK293 or THP1 cells that were transfected with the VEEV or chimeric saRNA constructs,

[0219] FIG. 64 depicts a comparison between GFP encoding VEEV saRNA and a GFP encoding chimeric saRNA described in examples 1 and 2. HEK293 and THP1 cells were transfected with a VEEV saRNA and the chimeric saRNA by lipofection (HEK293) or electroporation (THP1). The VEEV saRNA resulted in protein expression in both HEK293 and THP1 cells. In contrast, the chimeric saRNA only resulted in protein expression in HEK293 cells.

[0210]

[0220] FIG. 65 shows the results of a flow cytometry experiment to measure the levels of mCherry expression in Jurkat cells transfected with mCherry encoding saRNA or AIP-2A- mCherry encoding saRNA.

[0211]

[0221] FIG. 66 shows the results of a flow cytometry experiment to measure the levels of MHC expression (HLA A / B / C) on the surface of Jurkat cells transfected with mCherry encoding saRNA or AIP-2A-mCherry encoding saRNA.

[0212] DETAILED DESCRIPTION

[0213]

[0222] The summary of the present invention detailed herein is not intended to describe each disclosed embodiment or every implementation of the present invention. The description and examples that herein exemplify illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0214]

[0223] Described herein are transcriptionally and translationally active saRNA comprising a nucleotide sequence encoding at least one protein or fragment thereof and one or more features selected from (i) a non-structural protein 2 (nsp2) encoding a D584N mutation, (ii) a chimeric 3’ UTR, (iii) at least 25% of cytidines substituted by 5-flourocyti dines. In certain embodiments, the saRNA encodes two or more (e.g., three or more, four or more) proteins or fragments thereof. In certain embodiments, the nucleic acid sequences encoding the proteins or fragments are operably linked by 2A sequences or IRES sequences. In a particular embodiment, the saRNA encodes two or more proteins, wherein the coding sequences for the proteins are separated by an IRES sequence and the cytidines or uridines are substituted with modified nucleotides in an amount greater than about 25%, more particularly, between 30 and 100%, more particularly, between 50% and 100% and even more particularly, 100%. The modified nucleotides can be any suitable modified nucleotides, including but not limited to those disclosed herein.

[0224] In some embodiments, these saRNA molecules are useful for therapeutic purposes and advantageously confer one or more of the following compared to a compared to a comparable self-amplifying RNA lacking one of the features disclosed herein: increased transfection efficiency, increased replication, increased protein expression, increased serum concentration, increased compatibility with the encoded RNA-dependent RNA polymerase, reduced immunogenicity and increased stability. In certain embodiments, the saRNAs described herein permit increased expression of cargo protein(s) and reduced expression of inflammatory cytokines. The modified saRNA molecules are comprised of sequences from one or more (e.g., two or more) RNA viruses (e.g., alphaviruses) that confer replication in addition to specific sequences encoding one or more (e.g., two or more, three or more, four or more) cargos such as therapeutic proteins or antigens (e.g., viral antigens). Optionally, the sequences that confer replication may be mutated. The resulting modified saRNAs can be delivered to cells to provide transfected cells, for example for therapeutic use, or to cells or tissues by pharmaceutical compositions described herein.

[0215]

[0225] The modifications introduced into the saRNA are removed during the first round of RNA replication. This allows for restoration of the functionality of encoded RNA structures such as IRES elements that are otherwise altered or inhibited by the presence of chemically modified nucleosides. Such structures are often utilized for enabling or controlling protein expression. Described herein are modified saRNAs that facilitate the functional expression of multiple proteins (e.g., cytosolic, membrane or secreted proteins). Additionally, the technology can be combined with RNA delivery strategies (e.g., lipid nanoparticles) to transfect cells in vivo. Described herein are formulations that confer efficient transfection of cells with modified saRNA.

[0216]

[0226] Described herein are methods of utilizing barcoded modified self-amplifying RNA to efficiently identify lipid nanoparticle formulations conferring high efficiency delivery in vivo via high throughput screening approaches.

[0217]

[0227] The invention is based, in part, upon the discovery that certain modified nucleotides via a modified nucleoside triphosphate, can be incorporated into saRNA at levels above 25%, including greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%-100% or even 100%, and that the resulting saRNA can still express cargo or cargos of interested encoded on the saRNA.

[0218]

[0228] Furthermore, under certain circumstances, the level of expression of transgenes encoded by the saRNA can even be higher than the level of expression of comparable saRNA without such modifications. These specific modifications allow for maintenance of self- amplifying function of the saRNA as well as maintaining expression of the cargo protein encoded by one or more cargos.

[0219]

[0229] Described herein are general frameworks for expressing cargo from self-amplifying RNA that comprises modified nucleotides at greater than 25% substitution and is able to maintain or increase expression capabilities compared to unmodified saRNA. This enables expressing vaccine antigen, protein replacement therapies, antibodies, enzymes, or modifying cells and equipping them with controllable behavior via modified self-amplifying RNA: (1) expression of cargo protein(s) in cells; (2) external control of cell behavior by administration of external factors that increase or decrease activity of cargo protein(s); (3) internal control of cell behavior by logic computation circuits that sense inputs and perform logic computation.

[0220]

[0230] Disclosed herein are self-amplifying RNAs containing greater than 25% substitution of one or more given nucleotide with modified nucleotides. These highly-substituted saRNA are not only capable of maintaining saRNA functionality, but confer suppression of innate immune activation.

[0221]

[0231] Additionally, these highly-substituted saRNA are more efficacious, increasing cargo expression and transfection efficiency. The saRNA described herein have the potential to unlock the next frontier in RNA therapeutics that has been hampered by the inherent immunogenicity of the constructs; this work provides a blueprint for the modification and potentiation of saRNA therapeutics as vaccines, long-lasting cell therapies, protein replacement therapies, and any other conceived embodiment of RNA therapeutics.

[0222]

[0232] In some embodiments, self-amplifying RNA is created by utilizing sequences derived from the viral elements capable of copying and generating additional RNA. Exemplary viruses include alphaviruses, flaviviruses, measles viruses, coronaviruses and rhabdoviruses. In particular embodiments, self-amplifying RNA is created by utilizing sequences derived from Venezuela Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV), Sindbis Virus (SIN), Kunjin Virus (KUN), Measles virus (MV), Rabies virus (RABV), and Vesicular Stomatitis virus (VSV). In other embodiments, modified self-amplifying RNA is created by utilizing sequences derived from Venezuela Equine Encephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV), Western Equine Encephalitis Virus (WEEV), Chikungunya Virus (CHIKV), Mayaro Virus (MAYV), Ross River Virus (RRV), Semliki Forest Virus (SFV), Sindbis Virus (SIN), Kunjin Virus (KUN), Measles virus (MV), Rabies virus (RABV), and Vesicular Stomatitis virus (VSV) or another virus evolutionarily related to the aforementioned virus species. In a specific embodiment, the replication can be the result of inclusion of conserved noncoding sequence elements on the 5’ and 3’ ends of the RNA strand in addition to the inclusion of coding sequences for the proteins nspl, nsp2, nsp3, and nsp4. The expression of any genetically encodable protein is enabled by placing the coding sequence after a subgenomic promoter sequence or an internal ribosome entry site (IRES) sequence. In addition, noncoding RNA can be transcribed by placement of a microRNA or siRNA sequence after the subgenomic promoter.

[0223]

[0233] In some embodiments of the aspects, an saRNA as described herein comprises at least one nucleotide modification, as described further herein. In some embodiments of the aspects, an saRNA as described herein does not comprises nucleotide modifications.

[0224]

[0234] In some embodiments, a self-amplifying RNA (saRNA) comprises at least 25% modified nucleotides, wherein the modified nucleotides comprise a pyrimidine nucleoside phosphate with a moiety on carbon 5 of the pyrimidine, wherein the moiety is selected from the group consisting of methyl, ethyl, propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, and hydroxypropyl functional groups, at least one internal ribosomal entry site (IRES) and at least two cargos of interest.

[0225]

[0235] In some embodiments, a self-amplifying RNA (saRNA) comprises at least 25% modified nucleotides, wherein the modified nucleotides comprise a pyrimidine nucleoside phosphate with a moiety on carbon 5 of the pyrimidine, wherein the moiety is selected from the group consisting of methyl, ethyl, propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, and hydroxypropyl functional groups, at least one adaptive immune response inhibitory protein and at least one cargo of interest.

[0226]

[0236] In some embodiments the modified nucleotides comprise a modified cytidine selected from the group of 5-methylcytidine, 5-hydroxymethylcytidine, and / or a modified uridine selected from the group of 5-methyluridine, 5-hydroxymethyluridine.

[0227]

[0237] In some embodiments, a self-amplifying RNA (saRNA) comprises at least 25% modified nucleotides, wherein the modified nucleotides comprise a pyrimidine nucleoside phosphate with a moiety on carbon 5 of the pyrimidine, wherein the moiety is selected from the group consisting of fluoro, bromo, iodo, azide, functional groups, at least one cargo of interest.

[0228]

[0238] In some embodiments the modified nucleotides of the saRNA comprise a modified cytidine or uridine selected from the group of 5-methylcytidine, 5-fluorocytidine, 5- bromocytidine, 5-iodocytidine, 5-azidocytidine, 5-trifluoromethylcytidine, 5- difluoromethylcytidine, 5-fluorouridine, 5-bromouridine, 5-iodouridine, 5-azidouridine, 5- trifluoromethyluridine, 5-difluoromethyluridine, 5-methyluridine, 5-hydroxymethyluridine, 5- hydroxymethylcytidine, 5-ethyluridine, 5-ethylcytidine, 5-propyluridine, 5-propylcytidine, 5- nitrouridine, 5-nitrocytidine, 5-difluorohydroxymethyluridine, 5- difluorohydroxymethylcytidine, 5-monofluorohydroxymethyluridine, 5- monofluorohydroxymethylcytidine, 5-hydroxyethylcytidine, 5-hydroxyethyluridine, 5- hydroxypropylcytidine, 5 -hydroxypropyluridine, or any combination thereof.

[0229]

[0239] In some embodiments the modified nucleotides of the saRNA comprise one more modified nucleotides.

[0230]

[0240] The technology described herein is directed to modified self-amplifying RNA systems. Described herein are methods and compositions utilized nucleoside modified self-amplifying RNA for therapeutic applications. Modified self-amplifying RNA incorporates chemically modified nucleotides that confer reduced immunogenicity and compatibility with the encoded RNA dependent RNA polymerase. Modified self-amplifying RNA is comprised of sequences from RNA viruses that confer replication in addition to specific sequences encoding therapeutic proteins or viral antigens. In some embodiments, the co-expression of adaptive immune response inhibiting proteins confer reduced antigen presentation, which results in diminished immunogenicity upon re-dosing saRNA and can prolong transgene expression. In some embodiments, the identification of beneficial mutations in the RNA-dependent RNA polymerase coding sequence results in enhanced performance relative to the original unoptimized sequence; the identified mutations do not interfere with the compatibility of previously identified modified nucleotides. The resulting modified self-amplifying RNA can be delivered to cells or tissues by pharmaceutical compositions described herein. The modifications introduced into the self-amplifying RNA are removed during the first round of RNA replication. This allows for restoration of the functionality of encoded RNA structures that are otherwise altered or inhibited by the presence of chemically modified nucleosides. Such structures are often utilized for controlling protein expression. Described herein are modified self-amplifying RNAs depleted of double-stranded RNA which enables higher expression and reduced expression of inflammatory cytokines. Described herein are methods of utilizing barcoded modified self-amplifying RNA to efficiently identify lipid nanoparticle formulations conferring high efficiency delivery in vivo via high throughput screening approaches.

[0231]

[0241] The modification of one or more untranslated region to include chimeric elements derived from other viral species confers an alternative or specific tropism that may reduce inflammatory cytokine expression or improve bioactivity.

[0242] The technology described herein is directed to circular RNA, modified messenger RNA and self-amplifying RNA systems. In particular, described herein are methods and compositions utilizing unmodified and nucleoside modified self-amplifying RNA for therapeutic applications. Modified self-amplifying RNA incorporates chemically modified nucleotides that confer reduced immunogenicity and compatibility with the encoded RNA- dependent RNA polymerase. Modified messenger RNA incorporates chemically modified nucleotides to confer reduced immunogenicity. Circular RNA results in more durable protein expression due to its increased stability. Modified self-amplifying RNA is comprised of sequences from RNA viruses that confer replication in addition to specific sequences encoding therapeutic proteins. The inclusion of chimeric sequences imparts an alternative or specific cellular tropism that may reduce off-target expression that can result in inflammatory or detrimental responses. In some embodiments, the inclusion of mutations identified as imbuing enhanced performance are incorporated into the RNA-dependent RNA polymerase. The resulting modified messenger RNA, circular RNA or self-amplifying RNA (e.g., in some embodiments harboring at least one mutant sequence) can be delivered to cells or tissues by pharmaceutical compositions described herein.

[0232]

[0243] The technology described herein is also directed to modified self-amplifying RNA systems wherein modified nucleoside triphosphates are included within the RNA at >25%. Described herein are methods and compositions utilized nucleoside modified self-amplifying RNA for therapeutic and vaccine applications. Modified self-amplifying RNA incorporates chemically modified nucleotides that confer reduced immunogenicity and compatibility with the encoded RNA-dependent RNA polymerase. Modified self-amplifying RNA is comprised of sequences from RNA viruses that confer replication in addition to specific sequences encoding therapeutic proteins or viral antigens. The resulting modified self-amplifying RNA can be delivered to cells or tissues by pharmaceutical compositions described herein. Described herein are modified nucleoside triphosphates and self-amplifying RNAs containing such structures that facilitate the functional expression of proteins. Additionally, the technology can be combined with RNA delivery strategies (e.g., lipid nanoparticles) to transfect cells in vitro or in vivo.

[0233] I. Definitions

[0234]

[0244] The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description and examples that follow exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0235]

[0245] “2A” or “2A sequence”, as used herein, refers to a peptide, or a sequence encoding such a peptide that induces ribosomal skipping during translation. 2A sequences are typically 18- 22 amino acids long and exhibit a core motif of DXEXNPGP (SEQ ID NO: 74).

[0236]

[0246] “About” as used herein, has its ordinary meaning of approximately. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. Where ranges are provided, they are inclusive of the boundary values.

[0237]

[0247] “Adaptive immune response” as used herein refers an antigen-specific response. Antigen specificity allows for the generation of responses that are tailored to specific antigens, pathogens or pathogen -infected cells.

[0238]

[0248] "Administering” or “introducing to a subject”, as used herein refers to the placement of a saRNA, or a nucleic acid or cell or composition comprising an saRNA, as disclosed herein, into a subject by a method or route which results in at least partial delivery of the saRNA, or a nucleic acid or cell or composition comprising an saRNA, at a desired site. Pharmaceutical compositions comprising the saRNA, or a nucleic acid or cell or composition comprising an saRNA, as disclosed herein, can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and / or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and / or the subject being treated.

[0239]

[0249] “Analog” as used herein refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance.

[0240]

[0250] “Antibody” is used herein in the broadest sense unless clearly indicated otherwise. Therefore, an “antibody” can be naturally occurring or man-made, such as monoclonal antibodies produced by conventional hybridoma technology. Antibodies include monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and / or one or more complementarity determining regions of these antibodies. “Antibody” refers to any form of antibody or antigen binding fragment thereof and includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies), and antibody fragments.

[0241]

[0251] “Antibody fragment” as used herein refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments, diabodies, and multi-specific antibodies formed from antibody fragments.

[0242]

[0252] “Antigen” as used herein refers to an agent or moiety that elicits an immune response; and / or that is specifically bound by an antibody or to a T cell receptor (e.g., when presented by an MHC molecule). In some embodiments, an antigen elicits a humoral response (e.g., which may involve or include production of antigen-specific antibodies); in some embodiments, an antigen elicits a cellular response (e.g., which may involve or include T-cells whose receptors specifically interact with the antigen). In some embodiments, an antigen binds to an antibody and may or may not induce a particular physiological response in an organism. In some embodiments, an antigen is or comprises a polypeptide or epitope thereof. In some embodiments, an antigen is a recombinant antigen. In one embodiment, the antigen is an infectious agent (e.g., viral, bacterial, fungal) antigen, allergenic antigen or a tumor antigen. In certain embodiments, the antigen is an auto-antigen, i.e., any self-antigen which is mistakenly recognized by the immune system as being foreign.

[0243]

[0253] “Binding” as used herein refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts — including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell). “Specific binding” refers to a chemical or physical interaction between two molecules, compounds, cells and / or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third non- target entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.

[0244]

[0254] “Cancer” as used herein refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells.

[0245]

[0255] “Cargo” as used herein refers to nucleic acid cargos or protein cargos, which can be encoded by conventional or self-replicating RNA. The terms “protein”, “cargo”, or “protein of interest” refers to any genetically encodable polypeptide or nucleic acid sequence of known or unknown function. Representative non limiting examples of nucleic acid cargos include noncoding RNA, long non-coding RNA, microRNA, and siRNA. Representative non-limiting examples of protein cargos include receptors, ligands, enzymes, transmembrane receptors, ligands, reporter proteins (e.g., fluorescent proteins), enzymes, transcription factors, antibodies, antibody fragments, antigens, viral components, etc. In some embodiments, at least one of the cargos is selected from the group consisting of: receptor, transcription factor, enzyme, cytokine, reporter protein, antigen, antibody, hormone protein, contractile protein, structural protein, transport protein, peptide, etc. In some embodiments, at least one of the cargos is selected from the group consisting of: receptor, transcription factor, enzyme, cytokine, reporter protein, antigen, antibody, hormone protein, contractile protein, structural protein, transport protein, peptide, a fusion protein etc. These are examples of cargo proteins with known function and should not be interpreted as an exclusive list. Cargo proteins with unknown function can also be genetically encoded

[0246]

[0256] “ Chimera” as used herein refers to an entity having two or more incongruous or heterogeneous parts or regions. Disclosed herein, for example, are chimeric nucleotide sequences such as chimeric UTR sequences comprising sequences derived from different viral species.

[0247]

[0257] “ Chimeric antigen receptor” as used herein refers to engineered receptors, which graft an antigen specificity onto cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combination thereof). Chimeric antigen receptors are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors. In some embodiments, Chimeric antigen receptors comprise an antigen-specific targeting regions, an extracellular domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain.

[0248]

[0258] ““ Co-administration” and “co-administering” or “combination therapy” refer to both concurrent administration (administration of two or more therapeutic agents at the same time) and time-varied administration (administration of one or more therapeutic agents at a time different from that of the administration of an additional therapeutic agent or agents), as long as the two or more therapeutic agents are present in the patient to some extent, preferably at effective amounts, at the same time. In certain embodiments, the modified saRNA or circular RNA disclosed herein is co-administered with at least one additional therapeutic agent, e.g., an infective agent or anticancer agent. In particularly preferred aspects, the co-administration of both agents results in synergistic activity and / or therapy.

[0249]

[0259] “Comparable” as used herein refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In certain embodiments, the modified saRNA disclosed herein is compared to a comparable saRNA having fewer modifications, e.g., fewer than 25% modified cytosines or uracils. In certain embodiments, the modified saRNA disclosed herein is compared to a comparable mRNA, wherein each molecule has an IRES sequence.

[0250]

[0260] “ Conserved sequence element” or “CSE” as used herein refers to “nucleotide sequence found in alphavirus RNA. These sequence elements are termed “conserved” because orthologs are present in the genome of different alphaviruses, and orthologous CSEs of different alphaviruses preferably share a high percentage of sequence identity and / or a similar secondary or tertiary structure. The term CSE includes CSE 1, CSE 2, CSE 3 and CSE 4.

[0251]

[0261] “Contacting" as used herein refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, transfection, transduction, perfusion, injection, or other delivery method known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.

[0252]

[0262] “ Control element” as used herein means elements that provide (collectively) for the replication, transcription, post-transcriptional processing, and translation of a coding sequence in a recipient cell, for example, promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES), enhancers, and splice junctions.

[0253]

[0263] “ Conventional mRNA” as used herein refers to messenger RNA that does not have selfreplicating functions. In some embodiments, conventional mRNA is generated by in vitro transcription (IVT). In some embodiments, conventional mRNA contains a 5’ cap structure and a poly- A tail. In some embodiments, conventional mRNA contains 5’ and 3’ untranslated regions.

[0254]

[0264] “Cytokine” as used herein refers to a secreted, low-molecular-weight (about 5 to 20 kDa) protein expressed by cells that regulate the nature, intensity, and duration of an immune response by exerting a biological effect on immune cells that express receptors that bind the cytokine. Cytokines may possess different biological effects when bound by different cell types and can modulate the balance between the humoral and the cell-based (innate) immune response. Cytokines play an important role in activating and stimulating cells of the immune system. Cytokines are typically divided into four structural families including the hematopoietin family, interferon (IFN) family, chemokine family, and tumor necrosis factor (TNF) family. The term cytokine encompasses interleukins, lymphokines, monokines, interferons, colony stimulating factors, and chemokines.

[0255]

[0265] “Corresponding” as used herein refers two or more nucleic acids, nucleic acid sequences, or portions thereof, etc., that have the same base sequence, where a base sequence uses four options at each position - namely 1) adenosine, 2) guanosine, 3) uridine (and analogs thereof), and 4) cytidine (and analogs thereof). Modified nucleotides can be classified as analogs of the unmodified nucleotide (e.g., 5-methylcytidine and 5 hydroxymethylcytidine are classified as cytidine analogs; e.g., 5-methyluridine and 5 hydroxymethyluridine are classified as uridine analogs).

[0256]

[0266] “Decreased”, “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal, e.g., for an individual without a given disorder.

[0257]

[0267] “Derived from” as used herein refers to an origin or source, and may include naturally occurring, recombinant, unpurified or purified molecules. In some embodiments of any of the aspects, a sequence which is derived from a reference, origin, or source sequence comprises at least 80% sequence identity to the reference, origin, or source sequence. In some embodiments of any of the aspects, a sequence which is derived from a reference, origin, or source sequence comprises at least 85% sequence identity to the reference, origin, or source sequence. In some embodiments of any of the aspects, a sequence which is derived from a reference, origin, or source sequence comprises at least 90% sequence identity to the reference, origin, or source sequence. In some embodiments of any of the aspects, a sequence which is derived from a reference, origin, or source sequence comprises at least 95% sequence identity to the reference, origin, or source sequence. In some embodiments of any of the aspects, a sequence which is derived from a reference, origin, or source sequence comprises at least 98% sequence identity to the reference, origin, or source sequence. In some embodiments of any of the aspects, a sequence which is derived from a reference, origin, or source sequence comprises at least 99% sequence identity to the reference, origin, or source sequence.

[0258]

[0268] “Disease” as used herein refers to a state of health of a subject in which the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate. In comparison, a “disorder” in aa subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health.

[0259]

[0269] “Domain” is used herein to refer to a section or portion of an entity. In one embodiment, a domain is a section of a polypeptide; in some such embodiments, a domain is characterized by a particular structural element (e.g., a particular amino acid sequence or sequence motif, a- helix character, P-sheet character, coiled-coil character, random coil character, etc.), and / or by a particular functional feature (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0260]

[0270] “Encode” as used herein refers to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first (e.g., an saRNA encodes a protein). The second molecule may have a chemical structure that is different from the chemical nature of the first molecule.

[0261]

[0271] “Expressed” or “expression” as used herein refers to the translation from an saRNA nucleic acid molecule to give a protein or polypeptide or a portion thereof. In some embodiments, “expression” may refer to the production of protein to elicit the intended therapeutic effect. To examine the extent of protein expression, a test sample (e.g., a sample of cells in culture expressing the desired protein) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g. mouse) or a non-human primate (e.g., monkey) model) is contacted with a nucleic acid (e.g., nucleic acid in combination with a lipid of the present invention). In some embodiments, “expression” may refer to the percentage of a cell population that expresses the cargo. In a preferred embodiment, “high expression” refers to over-production of the cargo of interest with respect to total amount. In another preferred embodiment, “high expression” refers to a percentage of a cell population that is equal to or greater than the control that is expressing the cargo of interest. In another embodiment, “high expression” may refer to the production of a sufficient or large amount of protein. The level of expression desired is dependent on the application of interest. The terms “high expression” and “low expression” may be used in embodiments to compare cargo expression with respect to total amount or percentage of cell expression, between cells of interest.

[0262]

[0272] The term "expression" can refer to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (e.g., saRNA, mRNA) or antisense RNA derived from a nucleic acid fragment or fragments and / or to the translation of saRNA or mRNA into a polypeptide.

[0263]

[0273] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following a coding region, e.g. 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0264]

[0274] “Helper lipid” as used herein refers to a lipid that contributes to the structural integrity and fluidity of the LNP, facilitating efficient encapsulation and release of the therapeutic agent (e.g., modified saRNA). Non-limiting helper lipids include phospholipids, cholesterol lipids, polymers, cationic lipids, neutral lipids, charged lipids, steroids, steroid analogues, polymer conjugated lipids, stabilizing lipids, or any combination thereof.

[0265]

[0275] “Highly substituted” or “high substitution” as used herein refers to the replacement of the natural nucleotide (e.g., a pyrimidine, and more particularly, cytosine, uracil or a combination thereof) with a high percentage of the corresponding analog. Where the high percentage is greater than 25%; where the percentage is greater than 30%, where the percentage is greater than 35%; where the percentage is greater than 40%; where the percentage is greater than 45%; where the percentage is greater than 50%; where the percentage is greater than 55%; where the percentage is greater than 60%; where the percentage is greater than 65%; where the percentage is greater than 70%; where the percentage is greater than 75%; where the percentage is greater than 80%; where the percentage is greater than 85%; where the percentage is greater than 90%; where the percentage is greater than 95%; where the percentage is 100%. Additionally, the terms “100% substitution”, “100% replaced”, “full substitution”, “fully substituted”, and “completely replaced” may be used interchangeably throughout the disclosure of this invention. Full substitution means that there is no presence of the given nonmodified nucleotide in the synthesis or final product of the self-amplifying RNA. A given percentage of substitution means the fractional mixture of a given nucleotide that is comprised of analog and naturally occurring either cytidine, adenosine, guanosine, or uridine. In one embodiment, between about 50 and 100%, of one of a combination of 5-methylcytidine, 5- fhiorocytidine, 5-bromocytidine, 5-iodocytidine, 5-azidocytidine, 5-trifluoromethylcytidine, 5- difluoromethylcytidine, 5-hydroxymethylcytidine, 5-ethylcytidine, 5-propylcytidine, 5- nitrocytidine, 5-difluorohydroxymethylcytidine, 5-monofluorohydroxymethylcytidine, 5- hydroxyethylcytidine, 5-hydroxypropylcytidine is substituted into the saRNA.

[0266]

[0276] In one embodiment of any aspect herein, multiple cell types are modified with highly substituted self-replicating RNA.

[0267]

[0277] “Hormone” as used herein refers to a peptide or protein hormone regulates various bodily functions such as growth, metabolism, reproduction, and mood. Non-limiting examples include insulin and glucagon.

[0268]

[0278] “ Innate immune system” as used herein refers to the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that the cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host

[0269]

[0279] “Increased” or “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10- 100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

[0280] The term “input” or “inputs” refers to a stimuli that interacts with the cargo of the selfreplicating RNA. Input or inputs include, but are not limited to, a cell, a protein, an enzyme, a small molecule, a specific wavelength or wavelengths of light, a thermal stimuli, or application of a magnetic field. Input or inputs include, but are not limited to, a cell, a protein, an enzyme, a small molecule, DNA, RNA, a specific wavelength or wavelengths of light, athermal stimuli, or application of a magnetic field.

[0270]

[0281] “In vitro" as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe)

[0271]

[0282] “In vivo" as used herein refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0272]

[0283] “Immune response” refers to a response elicited in a subject. An immune response may refer to cellular immunity, humoral immunity or may involve both. An immune response may also be limited to a part of the immune system. For example, in certain embodiments, an immunogenic composition may induce an increased IFNy response. In certain embodiments, an immunogenic composition may induce a mucosal IgA response (e.g., as measured in nasal and / or rectal washes). In certain embodiments, an immunogenic composition may induce a systemic IgG response (e.g., as measured in serum). In certain embodiments, an immunogenic composition may induce virus-neutralizing antibodies or a neutralizing antibody response. In certain embodiments, an immunogenic composition may induce a cytolytic (CTL) response by T cells.

[0273]

[0284] “Internal ribosome entry site” or “IRES” as used herein refers to an RNA sequence or structural element ranging in size from 10 nt to 1000 nt or more, capable of initiating translation of a polypeptide in the absence of a typical RNA cap structure, e.g., in the middle of an RNA sequence. An IRES is typically about 500 nt to about 700 nt in length. For example, an IRES sequence can permit production of multiple proteins from a single mRNA transcript. In certain embodiments, two or more polypeptides encoded by the modified saRNA disclosed herein are separated by an IRES sequence.

[0274]

[0285] In one embodiment of any aspect herein, the modified self-amplifying RNA contains an

[0275] IRES sequence that is inhibited by the presence of modified nucleosides in a messenger RNA.

[0276]

[0286] “Ionizable lipid” as used herein refers to a pH-sensitive lipid. These lipids are designed to show net neutrality at physiological pH but become positively charged inside the acidic endosomes An ionizable lipid may be a cationic lipid or an anionic lipid. In addition to an ionizable moiety, an ionizable lipid may contain an alkyl or alkenyl group, e.g., greater than six carbon atoms in length (e.g., greater than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16 carbons, 18 carbons, 20 carbons or more in length). Additional ionizable lipids that may be included in an LNP described herein are disclosed in Jayaraman et al. (Angew. Chem. Int. Ed. 51 :8529-8533 (2012)), Semple et al. Nature Biotechnol. 28: 172-176 (2010)), and U.S. Pat. Nos. 8,710,200 and 8,754,062, each of which is incorporated herein by reference in its entirety.

[0277]

[0287] “Lipid encapsulated” as used herein refers to a lipid nanoparticle that provides an active agent or therapeutic agent, as the saRNA disclosed herein, with full encapsulation, partial encapsulation, or both. In an embodiment, the saRNA disclosed herein is fully encapsulated in the lipid nanoparticle.

[0278]

[0288] “Lipid nanoparticle” as used herein refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which include one or more lipids (e.g., cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids). In some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as the saRNA disclosed herein, to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some embodiments, the lipid nanoparticles of the invention comprise a nucleic acid. Such lipid nanoparticles typically comprise a cationic lipid and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.

[0279]

[0289] “Modified nucleotide” as used herein refers to any analog of cytidine, adenosine, guanosine, uridine, or pseudouridine. These analogs can include isomers of the nitrogenous base, as well as inclusion or exclusion of chemical groups, both natural occurring and synthetically introduced, on any aspect of the nitrogenous base. It is explicitly stated herein that modifications to the sugar-phosphate backbone are not included in the definition of the term “modified nucleotide.” This exception is not meant to exclude the methylation at the 2’0 position of the first and second initiating nucleotides, also referred to as Cap-1 and cap-2 structures. In multiple aspects, described herein are saRNAs comprising modified nucleotides.

[0280]

[0290] “Modulate” or “modulating” as used herein refers to mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0281]

[0291] “Mutant” or “mutation” as used herein with reference to a polypeptide or a gene encoding a polypeptide means a polypeptide or gene encoding a polypeptide having one or more allelic variants, splice variants, derivative variants, substitution variants, deletion variants, and / or insertion variants, fusion polypeptides, orthologs, and / or interspecies homologs.

[0282]

[0292] “Non- structural protein” or “non structural protein” or “nsp” as used herein refers to a protein of viral origin which is not part of the viral particle. In some embodiments of any of the aspects, the nsp is nspl, nsp2, nsp3, or nsp4. In some embodiments of any of the aspects, the nsp is nsp2. In some embodiments of any of the aspects, the nsp is VEEV nspl, VEEV nsp2, VEEV nsp3, or VEEV nsp4. In some embodiments of any of the aspects, the nsp is VEEV-derived nspl, VEEV-derived nsp2, VEEV-derived nsp3, or VEEV-derived nsp4. In some embodiments of any of the aspects, the nsp is VEEV nsp2. In some embodiments of any of the aspects, the nsp is VEEV-derived nsp2. Exemplary sequences of nsps are known in the art. By way of non-limiting example, wild-type VEEV nspl has the sequence of amino acids 1-535 of SEQ ID NO: 3, wild-type VEEV nsp2 has the sequence of amino acids 536-1329 of SEQ ID NO: 3, wild-type VEEV nsp3 has the sequence of amino acids 1330-1879 of SEQ ID NO: 3, and wild-type VEEV nsp4 has the sequence of amino acids 78-613 of SEQ ID NO: 4. When mutations of a nsp are referred to, the numbering refers to the sequence of the nsp itself and not to the entirety of SEQ ID NO: 3 or 4.

[0283]

[0293] In some embodiments of any of the aspects, a nspl comprises the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nspl consists essentially of the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nspl consists of the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nspl comprises the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nspl consists essentially of the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nspl consists of the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nspl comprises a sequence with at least 80% sequence identity to the sequence of amino acids 1- 535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nspl consists essentially of a sequence with at least 80% sequence identity to the sequence of amino acids 1- 535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nspl consists of a sequence with at least 80% sequence identity to the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nspl comprises a sequence with at least 95% sequence identity to the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nspl consists essentially of a sequence with at least 95% sequence identity to the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nspl consists of a sequence with at least 95% sequence identity to the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nspl comprises a base sequence with at least 80% sequence identity to the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nspl consists essentially of a base sequence with at least 80% sequence identity to the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nspl consists of a base sequence with at least 80% sequence identity to the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nspl comprises a base sequence with at least 95% sequence identity to the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nspl consists essentially of a base sequence with at least 95% sequence identity to the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nspl consists of a base sequence with at least 95% sequence identity to the sequence of amino acids 1-535 of SEQ ID NO: 3.

[0284]

[0294] In some embodiments of any of the aspects, a nspl comprises the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a nspl consists essentially of the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a nspl consists of the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a wild-type nspl comprises the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a wild-type nspl consists essentially of the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a wild-type nspl consists of the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a wild-type nspl comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a wildtype nspl consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a wild-type nspl consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a wild-type nspl comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a wild-type nspl consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a wild-type nspl consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a nspl comprises a base sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a nspl consists essentially of a base sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a nspl consists of a base sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a nspl comprises a base sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a nspl consists essentially of a base sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments of any of the aspects, a nspl consists of a base sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 75.

[0285]

[0295] In some embodiments of any of the aspects, a nsp2 comprises the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp2 consists essentially of the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp2 consists of the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp2 comprises the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wildtype nsp2 consists essentially of the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp2 consists of the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp2 comprises a sequence with at least 80% sequence identity to the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp2 consists essentially of a sequence with at least 80% sequence identity to the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp2 consists of a sequence with at least 80% sequence identity to the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp2 comprises a sequence with at least 95% sequence identity to the sequence of amino acids 536- 1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp2 consists essentially of a sequence with at least 95% sequence identity to the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp2 consists of a sequence with at least 95% sequence identity to the sequence of amino acids 536- 1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp2 comprises a base sequence with at least 80% sequence identity to the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp2 consists essentially of a base sequence with at least 80% sequence identity to the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp2 consists of a base sequence with at least 80% sequence identity to the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp2 comprises a base sequence with at least 95% sequence identity to the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp2 consists essentially of a base sequence with at least 95% sequence identity to the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp2 consists of a base sequence with at least 95% sequence identity to the sequence of amino acids 536-1329 of SEQ ID NO: 3.

[0286]

[0296] In some embodiments of any of the aspects, a nsp2 comprises the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a nsp2 consists essentially of the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a nsp2 consists of the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a wild-type nsp2 comprises the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a wild-type nsp2 consists essentially of the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a wild-type nsp2 consists of the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a wild-type nsp2 comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a wildtype nsp2 consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a wild-type nsp2 consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a wild-type nsp2 comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a wild-type nsp2 consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a wild-type nsp2 consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a nsp2 comprises a base sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a nsp2 consists essentially of a base sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a nsp2 consists of a base sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a nsp2 comprises a base sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a nsp2 consists essentially of a base sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 76. In some embodiments of any of the aspects, a nsp2 consists of a base sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 76.

[0297] In some embodiments of any of the aspects, a nsp3 comprises the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp3 consists essentially of the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp3 consists of the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp3 comprises the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp3 consists essentially of the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp3 consists of the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wildtype nsp3 comprises a sequence with at least 80% sequence identity to the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp3 consists essentially of a sequence with at least 80% sequence identity to the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wildtype nsp3 consists of a sequence with at least 80% sequence identity to the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp3 comprises a sequence with at least 95% sequence identity to the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp3 consists essentially of a sequence with at least 95% sequence identity to the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a wild-type nsp3 consists of a sequence with at least 95% sequence identity to the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp3 comprises a base sequence with at least 80% sequence identity to the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp3 consists essentially of a base sequence with at least 80% sequence identity to the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp3 consists of a base sequence with at least 80% sequence identity to the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp3 comprises a base sequence with at least 95% sequence identity to the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp3 consists essentially of a base sequence with at least 95% sequence identity to the sequence of amino acids 1330-1879 of SEQ ID NO: 3. In some embodiments of any of the aspects, a nsp3 consists of a base sequence with at least 95% sequence identity to the sequence of amino acids 1330-1879 of SEQ ID NO: 3.

[0287]

[0298] In some embodiments of any of the aspects, a nsp3 comprises the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a nsp3 consists essentially of the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a nsp3 consists of the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a wild-type nsp3 comprises the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a wild-type nsp3 consists essentially of the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a wild-type nsp3 consists of the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a wild-type nsp3 comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a wildtype nsp3 consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a wild-type nsp3 consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a wild-type nsp3 comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a wild-type nsp3 consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a wild-type nsp3 consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a nsp3 comprises a base sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a nsp3 consists essentially of a base sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a nsp3 consists of a base sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a nsp3 comprises a base sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a nsp3 consists essentially of a base sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 77. In some embodiments of any of the aspects, a nsp3 consists of a base sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 77.

[0288]

[0299] In some embodiments of any of the aspects, a nsp4 comprises the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a nsp4 consists essentially of the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a nsp4 consists of the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a wild-type nsp4 comprises the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a wild-type nsp4 consists essentially of the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a wild-type nsp4 consists of the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a wild-type nsp4 comprises a sequence with at least 80% sequence identity to the sequence of amino acids 78- 613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a wild-type nsp4 consists essentially of a sequence with at least 80% sequence identity to the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a wild-type nsp4 consists of a sequence with at least 80% sequence identity to the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a wild-type nsp4 comprises a sequence with at least 95% sequence identity to the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a wild-type nsp4 consists essentially of a sequence with at least 95% sequence identity to the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a wild-type nsp4 consists of a sequence with at least 95% sequence identity to the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a nsp4 comprises a base sequence with at least 80% sequence identity to the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a nsp4 consists essentially of a base sequence with at least 80% sequence identity to the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a nsp4 consists of a base sequence with at least 80% sequence identity to the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a nsp4 comprises a base sequence with at least 95% sequence identity to the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a nsp4 consists essentially of a base sequence with at least 95% sequence identity to the sequence of amino acids 78-613 of SEQ ID NO: 4. In some embodiments of any of the aspects, a nsp4 consists of a base sequence with at least 95% sequence identity to the sequence of amino acids 78-613 of SEQ ID NO: 4.

[0289]

[0300] In some embodiments of any of the aspects, a nsp4 comprises the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a nsp4 consists essentially of the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a nsp4 consists of the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a wild-type nsp4 comprises the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a wild-type nsp4 consists essentially of the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a wild-type nsp4 consists of the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a wild-type nsp4 comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a wildtype nsp4 consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a wild-type nsp4 consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a wild-type nsp4 comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a wild-type nsp4 consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a wild-type nsp4 consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a nsp4 comprises a base sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a nsp4 consists essentially of a base sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a nsp4 consists of a base sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a nsp4 comprises a base sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a nsp4 consists essentially of a base sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 78. In some embodiments of any of the aspects, a nsp4 consists of a base sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 78.

[0290]

[0301] In some embodiments of any of the aspects, wild-type VEEV nspl has the sequence of amino acids 1-535 of SEQ ID NO: 3. In some embodiments of any of the aspects, wild-type VEEV nsp2 has the sequence of amino acids 536-1329 of SEQ ID NO: 3. In some embodiments of any of the aspects, wild-type VEEV nsp3 has the sequence of amino acids 1330-1886 of SEQ ID NO: 3 and amino acids 1-6 of SEQ ID NO: 4. In some embodiments of any of the aspects, wild-type VEEV nsp4 has the sequence of amino acids 218-275 of SEQ ID NO: 4.

[0291]

[0302] “Nucleic acid” or “nucleic acid sequence” as used herein refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any doublestranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., template DNA, plasmid DNA, vector DNA. Suitable RNA can include, e.g., saRNA, mRNA.

[0292]

[0303] “Open Reading Frame” or “ORF” as used herein means a portion of a DNA molecule that, when translated into amino acids, contains no stop codons. The saRNA disclosed herein may have one or more ORFs (e.g., two, three, four or five ORFs). Bi- / multicistronic mRNA: mRNA, that typically may have two (bicistronic) or more (multi ci stronic) open reading frames (ORF) (coding regions or coding sequences).

[0293]

[0304] “Operably linked” as used herein refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0294]

[0305] “Pharmaceutical composition” as used herein refers to the active agent in combination with a pharmaceutically acceptable carrier e.g., a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in or within nature.

[0295]

[0306] “Protein" and “polypeptide" are used herein interchangeably to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0296]

[0307] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0297]

[0308] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested to confirm that a desired activity, e.g., activity and specificity of a native or reference polypeptide is retained.

[0298]

[0309] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into lie or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into lie; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu.

[0310] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a polypeptide which retains at least 50% of the wild-type reference polypeptide’s activity. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0299]

[0311] In some embodiments, the polypeptide described herein can be a variant of a polypeptide sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a protein or fragment thereof that retains activity of the native or reference polypeptide. A wide variety of, for example, PCR-based, site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan to generate and test artificial variants.

[0300]

[0312] A variant amino acid or DNA sequence can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).

[0301]

[0313] A variant amino acid sequence can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to a native or reference sequence. As used herein, “similarity” refers to an identical amino acid or a conservatively substituted amino acid, as described herein. Accordingly, the percentage of “sequence similarity” is the percentage of amino acids which is either identical or conservatively changed, e.g., “sequence similarity” = (% sequence identity)+(% conservative changes). It should be understood that a sequence that has a specified percent similarity to a reference sequence necessarily encompasses a sequence with the same specified percent identity to that reference sequence. The skilled person will be aware of various computer programs, using different mathematical algorithms, which are available to determine the identity or similarity between two sequences. For instance, use can be made of a computer program employing the Needleman and Wunsch algorithm (Needleman et al. (1970)); the GAP program in the Accelrys GCG software package (Accelerys Inc., San Diego U.S.A.); the algorithm of E. Meyers and W. Miller (Meyers et al. (1989)) which has been incorporated into the ALIGN program (version 2.0); or more preferably the BLAST (Basic Local Alignment Tool using default parameters); see e.g., US Patent 10,023,890, the content of which is incorporated by reference herein in its entirety. As used herein, the phrase “maintains the same function”, when used in reference to an enzyme, catalyzes the same reaction as a reference enzyme.

[0302]

[0314] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragment of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. A wide variety of, site-specific mutagenesis approaches, e.g., Kunkel’s method, cassette mutagenesis, PCR site- directed mutagenesis (e.g., traditional PCR, primer extension, or inverse PCR), whole plasmid mutagenesis, in vivo site-directed mutagenesis, CRISPR / Cas-guided mutagenesis, are known in the art and can be applied by the ordinarily skilled artisan to introduce mutations into specific nucleic acid loci. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Braman, Jeff, ed. (2002) In Vitro Mutagenesis Protocols, Methods in Molecular Biology, Vol. 182 (2nd ed.); Khudyakov and Fields (2002), Artificial DNA: Methods and Applications, CRC Press; Hsu et al. (2014), Cell 157 (6): 1262- 78; Cerchione et al. (2020) PLOS ONE 15 (4): e0231716; and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0315] “Prevent” as used herein with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined time. In certain embodiments herein, the saRNA molecules or modified saRNA molecules serve as vaccines for the prevention of a disease or disorder.

[0303]

[0316] “Promoter” as used herein nucleic acid sequence sufficient to direct transcription of an operably linked nucleic acid molecule. Also included in this definition are those transcription control elements (for example, enhancers) that are sufficient to render promoter-dependent gene expression controllable in a cell type-specific, tissue-specific, or temporal-specific manner, or that are inducible by external signals or agents; such elements, which are well- known to skilled artisans, may be found in a 5' or 3' region of a gene or within an intron.

[0304]

[0317] “Self-amplifying RNA” or “saRNA” or “self-replicating RNA” or “srRNA” or “selfamplifying messenger RNA” or “smRNA” are used interchangeably and refer to an RNA strand capable of undergoing replication activity that results in replicate strands from an original strand. In addition, the term “self-amplifying RNA” can refer to a polymer of nucleic acids that is capable of replication of the entire nucleic acid polymer in both the negative-strand and positive-strand conformations. It is capable of synthesizing additional self-amplifying RNA in part through production of various non- structural proteins of viral origin capable of acting as, among other activities, an RNA dependent RNA polymerase. An integral aspect of saRNA is the ability for the non- structural proteins to generation both new full length saRNA strands, as well as RNA produced from a sub-genomic promoter. Self-replicating RNA is known to exist in the form of RNA viral genomes, and artificial self-replicating RNA can be created by utilizing components from RNA viruses along with sequences of interest. The components from RNA viruses can be used in combination or from a particular RNA virus exclusively. In therapeutic saRNA, the RNA produced by transcription from the sub-genomic protomer encodes for the cargo(s) of interest. The modified saRNA disclosed herein can encode one or more biologically active peptides, polypeptides, or proteins. The modified saRNA can comprise one or more modifications as compared to wild type mRNA and in certain embodiments, is highly or completely modified. Modifications of the saRNA may be located in any region of the molecule, including a coding region, an untranslated region, or a cap or tail region. In some embodiments a modified saRNA comprises at least one nucleotide which is modified, e.g., is not cytidine, adenosine, uridine, or guanosine.

[0318] “Sequence” as used herein with respect to saRNA refers to the particular and individual order, i.e. the succession of its nucleotides.

[0305]

[0319] “ Stable” See as used herein with reference to the saRNA compositions disclosed herein, means that the compositions maintain one or more aspects of their physical structure and / or activity over a period of time under a designated set of conditions. In some embodiments, the period of time is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, weeks or more, including for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more; in some embodiments, the designated set of conditions is or comprises a temperature above a low temperature threshold. In some embodiments, a low temperature threshold is above about -80° C., -70° C., -50° C., -30° C., -20° C., 0° C., 2° C., 4° C., 8° C., 15°, 20° C., 30° C., 40° C. or higher. In some embodiments, a composition is considered to be stable based on maintenance of colloidal content comprising lipid nanoparticles (LNPs). In some embodiments, a composition is stable based on maintenance of one or more of LNP characteristics (including, e.g., but not limited to its Z- average and / or poly dispersity index (PDI)). In some embodiments, a composition is considered to be stable based on maintenance of nucleic acid integrity, degree (e.g., percent) of nucleic acid encapsulation, and / or nucleic acid expressibility (e.g., level of expression of an encoded polypeptide, as may be expressed for example as percent of a relevant reference level). In some embodiments, compositions described herein are considered stable if lipid nanoparticles within such compositions exhibit less than about 20 nm change in Z-average (including, e.g., less than 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, l l nm, or less change in Z-average) over a certain period of time under a designated set of conditions compared to a relevant reference level. In some embodiments, compositions described herein are considered stable if lipid nanoparticles within such compositions exhibit less than about 10 nm change in Z-average (including, e.g., less than 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, or less change in Z-average) over a certain period of time under a designated set of conditions compared to a relevant reference level. In some embodiments, compositions described herein are considered stable if at least 50% (including e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more) nucleic acid encapsulation is maintained in such compositions over a certain period of time under a designated set of conditions compared to a relevant reference level. In some embodiments, compositions described herein are considered stable if at least 50% (including e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more) of expression level of an encoded polypeptide is maintained over a certain period of time under a designated set of conditions compared to a relevant reference level.

[0320] “Statistically significant” or “significantly" refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference.

[0306]

[0321] “Subgenomic promoter” as used herein refers to a promoter of a subgenomic mRNA of a viral nucleic acid.

[0307]

[0322] “Subject” as used herein refers to a human or animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0308]

[0323] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a disease or disorder. In some embodiments of any of the aspects, the subject is a human. In some embodiments of any of the aspects, the subject is a livestock animal. In some embodiments of any of the aspects, the subject is a domestic or tamed animal, such as a pet, including but not limited to a dog, cat, guinea pig, rabbit, rat, mouse, or hamster. In some embodiments of any of the aspects, the subject is a fish or a bird or a lizard or a snake. A subject can be male or female.

[0309]

[0324] A subject can be one who has been previously diagnosed with or identified as suffering from or having such a condition in need of treatment or one or more complications related to such a condition, and optionally, have already undergone treatment for such a disease or disorder or the one or more complications related to that disease or disorder. Alternatively, a subject can also be one who has not been previously diagnosed as having that disease or disorder or one or more complications related to the disease or disorder. For example, a subject can be one who exhibits one or more risk factors for the disease or disorder or one or more complications related to the disease or disorder or a subject who does not exhibit risk factors. A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0310]

[0325] “Target tissue” as used herein refers to any one or more tissue types of interest in which the delivery of the modified saRNA disclosed herein would result in a desired biological and / or pharmacological effect. Examples of target tissues of interest include specific tissues, organs, and systems or groups thereof. In certain embodiments, a target tissue may be a the brain, muscle (e.g., skeletal muscle), kidney, pancreas, lung, spleen, vascular endothelium in vessels (e.g., intra-coronary or intra-femoral), or tumor tissue (e.g., via intratumoral injection). An “off- target tissue” refers to any one or more tissue types in which the expression of the encoded protein does not result in a desired biological and / or pharmacological effect. In certain embodiments, the off-target tissues may include the liver and the spleen.

[0311]

[0326] “ T7 polymerase” as used herein refers to an enzyme derived from the T7 bacteriophage. It is highly specific for the T7 promoter sequence and catalyzes the synthesis of RNA from a DNA template in the 5' to 3' direction. See, e.g., Maslak et al, Biochemistry 1994, 33: 6918- 6924; Martin et al Prog. Nucleic Acid Res. Mol. Biol. 2005 80: 323-47; and Sousa et al Prog. Nucleic Acid Res. Mol. Biol. 2003 73: 1-41, both incorporated herein by reference.

[0312]

[0327] “Therapeutically effective amount” as used herein refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.

[0313]

[0328] “Transcription factor” as used herein refers to a protein that possesses a biological function including regulation of transcription of genes. Transcription factors possesses a DNA- binding domain (DBD) that allows the protein to bind a specific sequence of DNA (an enhancer element or promoter sequence). Upon binding the enhancer or promoter element, the transcription factor's presence can aide in initiation of transcription by stabilizing transcription initiation complex formation and / or activity, for example. Transcription factors also bind to regulatory DNA sequences, such as enhancer sequences, which can be many hundreds of base pairs downstream or upstream from the transcribed gene.

[0314]

[0329] “ Transfected” or “transduced” as used herein refers to a process by which exogenous nucleic acid such as the modified saRNA disclosed herein is transferred or introduced into the host cell (e.g., into the intracellular space of cells). A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny. The exogenous nucleic acid may be introduced stably or transiently into the host cell. Transfection can be achieved any number of known methods, for example, treating the cells with high concentrations of salt, an electric field, or detergent, to render the host cell outer membrane or wall permeable to nucleic acid molecules of interest, microinjection, PEG-fusion, and the like. In some embodiments, transfection or delivery occurs by electroporation. In some embodiments, transfection or delivery is by lipid nanoparticles. In some embodiments, transfection or delivery occurs directly, without modification of the RNA or vehicle to carry the RNA. In some embodiments, transfection or delivery occurs through conjugation of cell-reactive or targeting moieties to the RNA

[0315]

[0330] “ Transfection efficiency” as used herein refers to the percentage of cells that successfully take up and express foreign genetic material (such as the disclosed saRNA) during the process of transfection.

[0316]

[0331] “ Translation” as used herein refers to the process by which a polypeptide is synthesized by a ribosome ‘reading’ the sequence of a polynucleotide.

[0317]

[0332] “ Translation efficiency” as used herein refers to a rate or amount of protein or peptide production from a ribonucleotide transcript. In certain embodiments, translation efficiency can be expressed as amount of protein or peptide produced per given amount of transcript that codes for the protein or peptide.

[0318]

[0333] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0334] “Tropism” as used herein refers to “tropism” preferential entry of the modified saRNA into certain target cell or target tissue types, optionally and preferably followed by expression (e.g., transcription and, optionally, translation) within the cell of sequences encoded by the modified saRNA.

[0319]

[0335] “Vaccine” as used herein refers to a prophylactic or therapeutic material providing at least one antigen, preferably an immunogen. The antigen or antigenic function may stimulate the body's adaptive immune system to provide an adaptive immune response.

[0320]

[0336] “Viral species” as used herein refers viruses that are group based on several criteria, including genetic similarity, host range, and ecological niche. As one example, alphaviruses belong to the family Togaviridae and include at least 29 recognized species. Weaver SC et al. 2005. Togaviridae, p 999-1008. (In Fauquet CM et al. (ed), Virus taxonomy: Vlllth Report of the International Committee on Taxonomy of Viruses. Elsevier / Academic Press, London, England.)

[0321]

[0337] “Wild-type” or “naturally occurring” are used herein with reference a polypeptide or polynucleotide sequence refers to a polypeptide or polynucleotide sequence that occurs in a native population without genetic modification

[0322] II. Compositions

[0323]

[0338] Disclosed herein are saRNA molecules, as well as transfected cells and pharmaceutical compositions comprising the same.

[0324] Self-Amplifying RNAs

[0325]

[0339] Disclosed herein is a saRNA comprising a nucleotide sequence encoding (e.g., from 5 to 3) (i) sequences from at least one RNA virus (e.g., an alphavirus) that confer replication; and (ii) at least one cargo of interest (e.g., a protein).

[0326]

[0340] In one embodiment, the saRNA comprises sequences from at least two viral species (e.g., two alphavirus species) that confer replication.

[0327]

[0341] In one embodiment, described herein is a self-amplifying RNA (saRNA) comprising a nucleotide sequence encoding (i) a mutated non- structural protein 2 (nsp2), wherein the mutation is D584N and (ii) at least one protein or fragment thereof. In one embodiment, described herein is a self-amplifying RNA (saRNA) comprising a nucleotide sequence encoding (i) a mutated non- structural protein 2 (nsp2), wherein the mutation is D584N and encoded by a nucleotide substitution from GAC to AAC and (ii) at least one protein or fragment thereof.

[0342] In some embodiments, the saRNAis derived from VEEV. In some embodiments of any of the aspects, the saRNAis at least 80%, at least 85%, at least 90%, at least 95%, or greater in sequence identity to VEEV.

[0328]

[0343] In some embodiments, the sequence of the saRNA, excluding the at least one protein or fragment thereof, is derived from VEEV In some embodiments, the sequence of the saRNA, excluding the at least one protein or fragment thereof, is at least 80%, at least 85%, at least 90%, at least 95%, or greater in sequence identity to VEEV.

[0329]

[0344] In some embodiments, the mutated nsp2 is derived from VEEV. In some embodiments of any of the aspects, the mutated nsp2 is at least 80%, at least 85%, at least 90%, at least 95%, or greater in sequence identity to VEEV. In some embodiments, the mutated nsp2 is mutated relative to wild-type VEEV nsp2. In some embodiments, the mutated nsp2 is mutated relative to amino acids 536-1329 of SEQ ID NO: 3. In some embodiments, wild-type or VEEV nsp2 is a polypeptide having the sequence of amino acids 536-1329 of SEQ ID NO: 3. In referring to “D584N”, in some embodiments, the numbering refers to the sequence of 536-1329 of SEQ ID NO: 3 and not to the entirety of SEQ ID NO: 3.

[0330]

[0345] In some embodiments, a saRNA comprising a sequence encoding D584N nsp2 exhibits an increase in protein expression when introduced into a cell compared to a comparable saRNA encoding a nsp2 lacking the D584N mutation. In some embodiments, a saRNA comprising a sequence encoding D584N nsp2 exhibits an increase in protein expression when introduced into a cell compared to a comparable saRNA encoding a wild-type nsp2. In some embodiments, the increase in expression is at least about 2-fold greater. In some embodiments, the increase in expression is at least about 3-fold greater. In some embodiments, the increase in expression is at least about 4-fold greater. In some embodiments, the increase in expression is at least about 5 -fold greater.

[0331]

[0346] In some embodiments, a saRNA comprising a sequence encoding D584N nsp2 exhibits protein expression that is specifically enhanced in immune cells compared to a comparable saRNA encoding a nsp2 lacking the D584N mutation, when introduced in a subject. In some embodiments, a saRNA comprising a sequence encoding D584N nsp2 exhibits protein expression that is specifically enhanced in immune cells compared to a comparable saRNA encoding a wild-type nsp2, when introduced in a subject. In some embodiments, the immune cell is selected from T cells, dendritic cells, macrophages, monocytes, B cells or a combination thereof. In some embodiments, the enhanced protein expression is at least about 2-fold greater. In some embodiments, the enhanced protein expression is at least about 3 -fold greater. In some embodiments, the enhanced protein expression is at least about 4-fold greater. In some embodiments, the enhanced protein expression is at least about 5-fold greater.

[0332]

[0347] In one embodiment, a saRNA is provided comprising a nucleotide sequence encoding (e.g., from 5’ to 3)’ : (a) at least one non-structural protein (nsp) derived from at least one virus (e.g., from at least one alphavirus; optionally, comprising one or more mutations); (b) a subgenomic promoter (SGP) derived from at least one virus (e.g., from at least one alphavirus); and (c) at least one cargo of interest (e.g., a protein).

[0333]

[0348] In a particular embodiment, the replication-conferring sequence is non-structural protein 2 (nsp2) and the mutation is D584N, encoded by a nucleotide substitution of GAC to A AC.

[0334]

[0349] In a particular embodiment, the nucleotide sequence comprises at least 25% modified pyrimidines, as discussed further herein. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine substitution for cytidine.

[0335]

[0350] In one embodiment, a modified saRNA is provided comprising a nucleotide sequence encoding (e.g., from 5’ to 3’) (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus (e.g., from at least one alphavirus: optionally, at least one of the non-structural proteins comprises at least one mutation); (c) a subgenomic promoter (SGP) derived from at least one virus (e.g., from at least one alphavirus); (d) 5’ untranslated region (UTR) derived from at least one virus (e.g., from at least one alphavirus); (e) the at least one cargo of interest (e.g., a protein or fragment thereof); and (f) a 3’ untranslated region (UTR) derived from at least one virus (e.g., from at least one alphavirus); and (g) a poly-Atail.

[0336]

[0351] In a particular embodiment, the nsp2 comprises the mutation D584N, encoded by a nucleotide substitution of GAC to AAC. In one embodiment, described herein is an saRNA comprising a nucleotide sequence encoding (i) a chimeric 3 ’UTR sequence; and (ii) at least one protein or fragment thereof. In one embodiment, an saRNA described herein further comprises a chimeric 3 ’UTR sequence. In a particular embodiment, one or more of the UTRs (3’ UTR or 5’ UTR) are chimeric, i.e., comprised of sequences from more than one virus, such as a DNA or RNA virus. In one embodiment, the saRNA comprises a chimeric 3 ’UTR. In one embodiment, the chimeric 3 ’UTR comprises sequences from at least two RNA viruses. In one embodiment, the chimeric 3 ’UTR comprises sequences from at least two alphaviruses.

[0337]

[0352] In some embodiments, the alphavirus is Venezuela Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV), Sindbis Virus (SIN), Chikungunya Virus (CHIKV), Eastern Equine Encephalitis Virus (EEEV), Mayaro Virus (MAYV), Getah Virus (GETV), Ross River Virus (RRV), Una Virus (UNAV), Middleburg Virus (MIDV), O'nyong nyong virus (ONNV), Barmah Forest Virus (BFV), Mucambo Virus (MUCV), Tonate Virus (TONV), Everglades Virus (EVEV), Rio Negro Virus (RNV), Turnip Rosette Virus (TROV), Highlands J Virus (HJV), Western Equine Encephalitis Virus (WEEV), Fig Mosaic Emaravirus (FMV), Aura Virus (AURAV), or Kunjin Virus (KUN). In some embodiments, the at least two alphaviruses are selected from the group consisting of: Venezuela Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV), Sindbis Virus (SIN), Chikungunya Virus (CHIKV), Eastern Equine Encephalitis Virus (EEEV), Mayaro Virus (MAYV), Getah Virus (GETV), Ross River Virus (RRV), Una Virus (UNAV), Middleburg Virus (MIDV), O'nyong nyong virus (ONNV), Barmah Forest Virus (BFV), Mucambo Virus (MUCV), Tonate Virus (TONV), Everglades Virus (EVEV), Rio Negro Virus (RNV), Turnip Rosette Virus (TROV), Highlands J Virus (HJV), Western Equine Encephalitis Virus (WEEV), Fig Mosaic Emaravirus (FMV), Aura Virus (AURAV), and Kunjin Virus (KUN).

[0338]

[0353] In some embodiments, a saRNA comprising sequences encoding both a modified / mutated non- structural protein (e.g., D584N nsp2) and a chimeric 3’UTR exhibits protein expression that is enhanced compared to the absence of one of the modification / mutation of the non- structural protein (e.g., D584N nsp2) or the chimeric 3’UTR. In some embodiments, a saRNA comprising sequences encoding both a modified / mutated non- structural protein (e.g., D584N nsp2) and a chimeric 3’UTR exhibits protein expression that is synergistically enhanced compared to the absence of one of the modification / mutation of the non- structural protein (e.g., D584N nsp2) or the chimeric 3’UTR.

[0339]

[0354] In some embodiments, a saRNA comprising sequences encoding both a modified / mutated non- structural protein (e.g., D584N nsp2) and a chimeric 3’UTR exhibits enhanced protein expression levels compared to the absence of the chimeric 3’UTR. In some embodiments, the enhancement is at least about 2-fold. In some embodiments, the enhancement is at least about 3 -fold.

[0340]

[0355] In some embodiments, a saRNA comprising sequences encoding both a modified / mutated non- structural protein (e.g., D584N nsp2) and a chimeric 3’UTR exhibits reduced off-target immune activation compared to the absence of the chimeric 3’UTR. In some embodiments, the reduction is at least about 2-fold.

[0341]

[0356] In some embodiments, a saRNA comprising sequences encoding both a modified / mutated non- structural protein (e.g., D584N nsp2) and a chimeric 3’UTR exhibits altered or restricted cellular tropism compared to the absence of the chimeric 3’UTR.

[0357] In some embodiments, a saRNA comprising sequences encoding both 1) a modified / mutated non- structural protein (e.g., D584N nsp2) and 2) a chimeric 3’UTR and / or a sequence encoding an adaptive immune inhibitor protein exhibits protein expression that is enhanced compared to the absence of one of 1) the modification / mutation of the non- structural protein (e.g., D584N nsp2) or 2) either or both of the chimeric 3 ’UTR and the adaptive immune inhibitor protein. In some embodiments, a saRNA comprising sequences encoding both 1) a modified / mutated non- structural protein (e.g., D584N nsp2) and 2) a chimeric 3’UTR and / or a sequence encoding an adaptive immune inhibitor protein exhibits protein expression that is synergistically enhanced compared to the absence of one of 1) the modification / mutation of the non- structural protein (e.g., D584N nsp2) or 2) either or both of the chimeric 3’UTR and the adaptive immune inhibitor protein.

[0342]

[0358] In some embodiments, a saRNA comprising sequences encoding a modified / mutated non- structural protein (e.g., D584N nsp2), a chimeric 3’UTR, and a sequence encoding an adaptive immune inhibitor protein exhibits protein expression that is enhanced compared to the absence of the adaptive immune inhibitor protein. In some embodiments, a saRNA comprising sequences encoding a modified / mutated non-structural protein (e.g., D584N nsp2), a chimeric 3’UTR, and a sequence encoding an adaptive immune inhibitor protein exhibits protein expression that is synergistically enhanced compared to the absence of the adaptive immune inhibitor protein.

[0343]

[0359] In some embodiments, a saRNA comprising sequences encoding a modified / mutated non-structural protein (e.g., D584N nsp2), a chimeric 3’UTR, and a sequence encoding an adaptive immune inhibitor protein exhibits decreased activation of the innate immune response compared to the absence of the adaptive immune inhibitor protein. In some embodiments, the decrease is at least about 20%. In some embodiments, the decrease is at least about 60%. In some embodiments, the decrease is at least about 80%.

[0344]

[0360] In a particular embodiment, the nucleotide sequence comprises at least 25% modified pyrimidines, as discussed further herein. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine substitution for cytidine.

[0345]

[0361] In one embodiment, a saRNA is provided comprising a nucleotide sequence encoding (e.g., from 5’ to 3’): (a) at least one non-structural protein (nsp) derived from at least one virus (optionally, comprising one or more mutations); (b) a subgenomic promoter (SGP) derived from at least one virus; and (c) at least two cargoes of interest (e.g., at least two proteins or protein fragments).

[0362] In a particular embodiment, the replication-conferring sequence is non- structural protein 2 (nsp2) and the mutation is D584N, encoded by a nucleotide substitution of GAC to A AC.

[0346]

[0363] In a particular embodiment, one or more of the UTRs (3’ UTR or 5’ UTR) are chimeric, i.e., comprised of sequences from more than one virus, such as a DNA or RNA virus. In one embodiment, the saRNA comprises a chimeric 3 ’UTR.

[0347]

[0364] In a particular embodiment, the nucleotide sequence comprises at least 25% modified pyrimidines, as discussed further herein. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine substitution for cytidine.

[0348]

[0365] In one embodiment, a modified saRNA is provided comprising a nucleotide sequence encoding (e.g., from 5’ to 3’): (a) a 5’ cap; (b) non-structural protein 1 (nspl), non- structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus (optionally, at least one of the non-structural proteins comprises at least one mutation); (c) a subgenomic promoter (SGP) derived from at least one virus; (d) 5’ untranslated region (UTR) derived from at least one virus; (e) the at least two cargos of interest (e.g., at least proteins or fragments thereof); and (f) a 3’ untranslated region (UTR) derived from at least one virus; and (g) a poly-Atail.

[0349]

[0366] In a particular embodiment, nsp2 comprises the mutation D584N, encoded by a nucleotide substitution of GAC to A AC.

[0350]

[0367] In a particular embodiment, one or more of the UTRs (3’ UTR or 5’ UTR) are chimeric, i.e., comprised of sequences from more than one virus, such as a DNA or RNA virus. In one embodiment, the saRNA comprises a chimeric 3 ’UTR.

[0351]

[0368] In a particular embodiment, the nucleotide sequence comprises at least 25% modified pyrimidines, as discussed further herein. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine substitution for cytidine.

[0352]

[0369] In certain embodiments, the coding sequence for at least two proteins are separated by a 2A sequence or an IRES sequence.

[0353]

[0370] In one embodiment of any aspect herein, a 2A sequence is used to separate two proteins encoded in the same open reading frame. In one embodiment, a furin cleavage site is placed in front of a 2A sequence to prevent the addition of extra amino acids to the N or C terminus of a protein.

[0354]

[0371] In a saRNA molecule comprising a IRES sequence, a first coding sequence (e.g., protein coding sequence) can be translated by a cap-dependent, ribosome scanning, mechanism with its own 5'-UTR, whereas translation of a subsequent coding sequence (e.g., protein sequence) be accomplished by direct recruitment of a ribosome to an IRES in a capindependent manner. An IRES sequence can allow eukaryotic ribosomes to bind and begin translation without binding to a 5' capped end.

[0355]

[0372] In one embodiment, a saRNA is provided comprising a nucleotide sequence encoding (e.g., from 5’ to 3’): (a) at least one non- structural protein derived from at least one virus (optionally, comprising at least one mutation); (b) a subgenomic promoter (SGP) derived from at least one virus; and (c) at least two cargos of interest (e.g., two proteins or fragments thereof) operably linked by an internal ribosome entry site (IRES) sequence.

[0356]

[0373] In a particular embodiment, the at least one non-structural protein is non- structural protein 2 (nsp2) and the mutation is D584N, encoded by a nucleotide substitution of GAC to A AC.

[0357]

[0374] In a particular embodiment, the nucleotide sequence comprises at least 25% modified pyrimidines, as discussed further herein. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine substitution for cytidine.

[0358]

[0375] In one embodiment, a saRNA is provided comprising a nucleotide sequence encoding (e.g., from 5’ to 3’) (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus (optionally, at least one of the non-structural proteins comprises at least one mutation); (c) a subgenomic promoter (SGP) derived from at least one virus; (d) 5’ untranslated region (UTR) derived from at least one virus; (e) the at least two cargos of interest (e.g., two proteins) operably linked by an internal ribosome entry site (IRES) sequence; (f) a 3’ untranslated region (UTR) derived from at least one virus; and (g) a poly-Atail, wherein the saRNA comprises at least 25% modified nucleotides.

[0359]

[0376] In a particular embodiment, nsp2 comprises the mutation D584N, encoded by a nucleotide substitution of GAC to A AC.

[0360]

[0377] In a particular embodiment, one or more of the UTRs (3’ UTR or 5’ UTR) are chimeric, i.e., comprised of sequences from more than one virus, such as a DNA or RNA virus. In one embodiment, the saRNA comprises a chimeric 3 ’UTR.

[0361]

[0378] In a particular embodiment, the nucleotide sequence comprises at least 25% modified pyrimidines, as discussed further herein. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine substitution for cytidine.

[0362]

[0379] In one embodiment, a saRNA is provided comprising a nucleotide sequence encoding (e.g., from 5’ to 3’): (a) at least one non-structural protein derived from at least one virus (optionally, comprising at least one mutation); (b) a subgenomic promoter (SGP) derived from at least one virus; and (c) at least two cargos of interest (e.g., two proteins or fragments thereof) operably linked by a 2A sequence.

[0363]

[0380] In a particular embodiment, the at least one non-structural protein is non- structural protein 2 (nsp2) and the mutation is D584N, encoded by a nucleotide substitution of GAC to A AC.

[0364]

[0381] In a particular embodiment, the nucleotide sequence comprises at least 25% modified pyrimidines, as discussed further herein. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine substitution for cytidine.

[0365]

[0382] In one embodiment, a saRNA is provided comprising a nucleotide sequence encoding (e.g., from 5’ to 3’): (a) at least one non-structural protein derived from at least one virus (optionally, comprising at least one mutation); (b) a subgenomic promoter (SGP) derived from at least one virus; (c) at least one cargo of interest (e.g., a protein or fragment thereof; (d) an IRES sequence or 2A sequence; and (e) a sequence encoding at least one an adaptive immune response inhibitor protein.

[0366]

[0383] The adaptive immune response inhibitor protein may be any suitable adaptive immune response inhibitor protein. In one embodiment, the adaptive immune response inhibitor protein is a viral immune evasion protein. In a particular embodiment, the viral immune evasion protein is selected from BNLF2, UL49.5 or combinations thereof.

[0367]

[0384] In a particular embodiment, the at least one non-structural protein is non-structural protein 2 (nsp2) and the mutation is D584N, encoded by a nucleotide substitution of GAC to A AC.

[0368]

[0385] In a particular embodiment, the nucleotide sequence comprises at least 25% modified pyrimidines, as discussed further herein. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine substitution for cytidine.

[0369]

[0386] In some embodiments, a saRNA comprising sequences encoding 1) a modified / mutated non-structural protein (e.g., D584N nsp2) and 2) a chimeric 3’UTR, a sequence encoding an adaptive immune inhibitor protein, and / or one or more modified nucleotides as described herein exhibits protein expression that is enhanced compared to the absence of one of 1) the modification / mutation of the non-structural protein (e.g., D584N nsp2) or 2) one or more of the chimeric 3’UTR, the adaptive immune inhibitor protein, and the one or more modified nucleotides. In some embodiments, a saRNA comprising sequences encoding 1) a modified / mutated non-structural protein (e.g., D584N nsp2) and 2) a chimeric 3’UTR, a sequence encoding an adaptive immune inhibitor protein, and / or one or more modified nucleotides as described herein exhibits protein expression that is synergistically enhanced compared to the absence of one of 1) the modification / mutation of the non- structural protein (e.g., D584N nsp2) or 2) one or more of the chimeric 3’UTR, the adaptive immune inhibitor protein, and the one or more modified nucleotides.

[0370]

[0387] In some embodiments, a saRNA comprising sequences encoding a modified / mutated non- structural protein (e.g., D584N nsp2), a chimeric 3’UTR, a sequence encoding an adaptive immune inhibitor protein, and / or one or more modified nucleotides as described herein exhibits protein expression that is enhanced compared to the absence of the one or more modified nucleotides. In some embodiments, a saRNA comprising sequences encoding a modified / mutated non- structural protein (e.g., D584N nsp2), a chimeric 3’UTR, a sequence encoding an adaptive immune inhibitor protein, and / or one or more modified nucleotides as described herein exhibits protein expression that is synergistically enhanced compared to the absence of the one or more modified nucleotides.

[0371]

[0388] In some embodiments, a saRNA comprising sequences encoding 1) a modified / mutated non- structural protein (e.g., D584N nsp2) and 2) a chimeric 3’UTR and / or one or more modified nucleotides as described herein exhibits protein expression that is enhanced compared to the absence of one of 1) the modification / mutation of the non- structural protein (e.g., D584N nsp2) or 2) one or more of the chimeric 3’UTR and the one or more modified nucleotides. In some embodiments, a saRNA comprising sequences encoding 1) a modified / mutated non- structural protein (e.g., D584N nsp2) and 2) a chimeric 3’UTR and / or one or more modified nucleotides as described herein exhibits protein expression that is synergistically enhanced compared to the absence of one of 1) the modification / mutation of the non- structural protein (e.g., D584N nsp2) or 2) one or more of the chimeric 3’UTR and the one or more modified nucleotides.

[0372]

[0389] In some embodiments, a saRNA comprising sequences encoding a modified / mutated non- structural protein (e.g., D584N nsp2), a chimeric 3’UTR, and one or more modified nucleotides as described herein exhibits protein expression that is enhanced compared to the absence of the one or more modified nucleotides. In some embodiments, a saRNA comprising sequences encoding a modified / mutated non-structural protein (e.g., D584N nsp2), a chimeric 3’UTR, and one or more modified nucleotides as described herein exhibits protein expression that is synergistically enhanced compared to the absence of the one or more modified nucleotides.

[0373]

[0390] In other aspects described herein is an saRNA comprising (e.g., from 5’ to 3’): (a) at least one non-structural protein derived from at least one virus (e.g., at least one alphavirus); (b) a subgenomic promoter (SGP) derived from at least one virus (e.g., at least one alphavirus); and (c) least one cargo of interest. In additional aspects described herein is an saRNA comprising from 5’ to 3’: (a) at least one non- structural protein derived from at least one virus (e.g., at least one alphavirus); (b) a subgenomic promoter (SGP) derived from at least one virus (e.g., at least one alphavirus); (c) at least one adaptive immune response inhibitory protein (AIP); (d) an IRES or 2A sequence; and (e) at least one cargo of interest.

[0374]

[0391] In another embodiment, the modified saRNA comprises (e.g., from 5’ to 3’): (a) a 5’ cap; (b) non-structural protein 1 (nspl), non- structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus (e.g., at least one alphavirus; optionally, at least one of the non-structural proteins comprises at least one mutation); (c) a subgenomic promoter (SGP) derived from at least one virus (e.g., at least one alphavirus); (d) 5’ untranslated region (UTR) derived from at least one virus (e.g., at least one alphavirus); (e) the at least one cargo of interest; (f) at least one adaptive immune response inhibitory protein (AIP); (g) a 3’ untranslated region (UTR) derived from at least one virus (e.g., at least one alphavirus); and (h) a poly-Atail.

[0375]

[0392] In one aspect described herein is an saRNA comprising (e.g., from 5’ to 3’): (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus (e.g., from at least one alphavirus), wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one virus (e.g., from at least one alphavirus); (d) 5’ untranslated region (UTR) derived from at least one virus (e.g., from at least one alphavirus); (e) the at least one cargo of interest; (f) at least one adaptive immune response inhibitory protein (AIP); (g) a 3’ untranslated region (UTR) derived from at least one virus (e.g., from at least one alphavirus), wherein the 3’ UTR comprises at least one 3'- conserved sequence element (3' CSE); and (h) a poly-Atail.

[0376]

[0393] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising (e.g., from 5’ to 3’): (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus; (c) a subgenomic promoter (SGP) derived from at least one virus; (d) a 5’ untranslated region (UTR) derived from at least one virus; (e) at least one adaptive immune response inhibitory protein; (f) at least one cargo of interest; (g) a 3’ untranslated region (UTR) derived from at least one virus; and (h) a poly-A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5- hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNAis methylated at the 2'0 position of the ribose (Capl).

[0377]

[0394] In a particular embodiment, nsp2 comprises the mutation D584N, encoded by a nucleotide substitution of GAC to A AC.

[0378]

[0395] In a particular embodiment, one or more of the UTRs (3’ UTR or 5’ UTR) are chimeric, i.e., comprised of sequences from more than one virus, such as a DNA or RNA virus. In one embodiment, the saRNA comprises a chimeric 3 ’UTR.

[0379]

[0396] In a particular embodiment, the nucleotide sequence comprises at least 25% modified pyrimidines, as discussed further herein. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine substitution for cytidine.

[0380]

[0397] In one embodiment, the saRNA comprises (e.g., from 5’ to 3’) (a) a 5’ cap; (b) non- structural protein 1 (nspl), non- structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non- structural protein 4 (nsp4), each derived from at least one virus (e.g., from at least one alphavirus; optionally, at least one of the non- structural proteins comprises at least one mutation), wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one virus (e.g., from at least one alphavirus); (d) 5’ untranslated region (UTR) derived from at least one virus (e.g., from at least one alphavirus); (e) the at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one virus (e.g., from at least one alphavirus), wherein the 3’ UTR comprises at least one 3'- conserved sequence element (3' CSE); and (g) a poly-Atail.

[0381]

[0398] In a particular embodiment, nsp2 comprises the mutation D584N, encoded by a nucleotide substitution of GAC to A AC.

[0382]

[0399] In a particular embodiment, one or more of the UTRs (3’ UTR or 5’ UTR) are chimeric, i.e., comprised of sequences from more than one virus, such as a DNA or RNA virus. In one embodiment, the saRNA comprises a chimeric 3 ’UTR.

[0383]

[0400] In a particular embodiment, the nucleotide sequence comprises at least 25% modified pyrimidines, as discussed further herein. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine substitution for cytidine

[0384]

[0401] In one embodiment, the modified saRNA comprises a nucleotide sequence encoding (e.g., from 5’ to 3’): (a) a 5’ cap; (b) 5’ untranslated region (UTR) derived from at least one virus; (c) non- structural protein 1 (nspl), non- structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus (optionally, at least one of the non-structural proteins comprises at least one mutation), wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (d) a subgenomic promoter (SGP) derived from at least one virus; (e) the at least one cargo of interest; (f) at least one adaptive immune response inhibitory protein (AIP); a 3’ untranslated region (UTR) derived from at least one virus, wherein the 3’ UTR comprises at least one 3'- conserved sequence element (3' CSE); and (g) a poly- A tail.

[0385]

[0402] In a particular embodiment, nsp2 comprises the mutation D584N, encoded by a nucleotide substitution of GAC to A AC.

[0386]

[0403] In a particular embodiment, one or more of the UTRs (3’ UTR or 5’ UTR) are chimeric, i.e., comprised of sequences from more than one virus, such as a DNA or RNA virus. In one embodiment, the saRNA comprises a chimeric 3 ’UTR.

[0387]

[0404] In a particular embodiment, the nucleotide sequence comprises at least 25% modified pyrimidines, as discussed further herein. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine substitution for cytidine

[0388]

[0405] In one aspect described herein is a plasmid vector comprising: (a) a cytomegalovirus (CMV) promoter positioned at the 5’ end for driving expression of the saRNA; (b) a 5’ untranslated region (UTR) derived from at least one alphavirus, containing at least one 5’- conserved sequence element (5’ CSE); (c) a coding sequence for self-amplifying RNA (saRNA) comprising: (i) non-structural proteins 1 (nspl), 2 (nsp2), 3 (nsp3), and 4 (nsp4), wherein nsp2 comprises a substitution of the codon at amino acid position 584 from GAC to AAC; (ii) a subgenomic promoter (SGP) derived from at least one alphavirus; (iii) the at least one cargo of interest; (d) a poly(A) tail positioned at the 3’ end of the saRNA coding sequence; (e) a 3’ untranslated region (UTR) derived from at least one alphavirus, containing at least one 3’- conserved sequence element (3’ CSE); (f) a self-cleaving ribozyme positioned downstream of the poly(A) tail, facilitating precise cleavage of the transcript to produce a functional saRNA molecule; (g) an antibiotic resistance marker for selection in bacterial cultures; (h) a multiple cloning site (MCS) for the insertion of additional sequences; and (i) a bacterial origin of replication (ori) for maintenance and amplification of the plasmid in bacterial host cells.

[0389]

[0406] In a particular embodiment, one or more of the UTRs (3’ UTR or 5’ UTR) are chimeric, i.e., comprised of sequences from more than one virus, such as a DNA or RNA virus. In one embodiment, the saRNA comprises a chimeric 3 ’UTR.

[0390]

[0407] In a particular embodiment, the nucleotide sequence comprises at least 25% modified pyrimidines, as discussed further herein. In one embodiment, the nucleotide sequence comprises about 100% 5-flourocytidine substitution for cytidine

[0391]

[0408] The virus may be any suitable virus. In one embodiment, the virus selected alphaviruses, flaviviruses, measles viruses, and rhabdoviruses or combinations thereof.

[0409] In one embodiment, the virus is an alphavirus. The alphavirus replication machinery is composed of four nonstructural proteins (nsPl to -4), which are expressed as one of two polyproteins (P123 or P1234). P1234 is expressed as a read-through of an opal termination codon at the end of nsP3. These precursor polyproteins are cleaved by a protease within nsP2.

[0392]

[0410] In one embodiment, the alphavirus is selected from the group consisting of: Venezuela Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV), Sindbis Virus (SIN), Chikungunya Virus (CHIKV), Eastern Equine Encephalitis Virus (EEEV), Mayaro Virus (MAYV), Getah Virus (GETV), Ross River Virus (RRV), Una Virus (UNAV), Middleburg Virus (MIDV), O'nyong nyong virus (ONNV), Barmah Forest Virus (BFV), Mucambo Virus (MUCV), Tonate Virus (TONV), Everglades Virus (EVEV), Rio Negro Virus (RNV), Highlands J Virus (HJV), Western Equine Encephalitis Virus (WEEV), and Aura Virus (AURAV).

[0393]

[0411] In one embodiment, the virus is selected from Turnip Rosette Virus (TROV), Highlands J Virus (HJV), Fig Mosaic Emaravirus (FMV), Kunjin Virus (KUN), Measles virus (MV), Coronavirus (CoV), Rabies virus (RABV), Vesicular Stomatitis virus (VSV), or combinations thereof.

[0394]

[0412] The virus from which the non- structural protein(s), SGP, 5 ’ UTR and 3 ’UTR are derived may be the same or different.

[0395]

[0413] In one embodiment, the 5 ’UTR, the 3 ’UTR or both are derived from different viral species. Alphaviruses, for example, have evolved distinct cellular tropisms that result in context dependent enhancements in fitness and viral propagation. For example, Venezuela Equine Encephalitis (VEEV) and Eastern Equine Encephalitis (EEEV) have been found to differ in their tropism towards myeloid cells (Gardner et al., 2008, PMID: 18768986). (Trobaugh et al., 2019, PMID: 31658290). VEEV efficiently infects myeloid cells and EEEV does not. As a result, VEEV induces greater systemic expression of inflammatory cytokines than EEEV. Prior work has determined that the difference in tropism is a result of sequences encoded in the 3’ untranslated region (UTR) (Trobaugh et al., 2014, PMID: 24352241). The generation of chimeric self-amplifying RNA vectors containing UTRs that result in an alternative tropism may enhance the bioactivity of encoded proteins and diminish the inflammatory response to administration of the saRNA.

[0396]

[0414] In a particular embodiment, the 5 ’UTR sequence is chimeric and comprises sequence from at least two viral species (e.g., two alphavirus species). In a particular embodiment, the 3 ’UTR is chimeric and comprises sequences from at least two viral species (e.g., two alphavirus species). In certain embodiments, the modified saRNA disclosed herein exhibits enhanced tropism toward a target cell or tissue in comparison to a comparable saRNA not comprising a chimeric 3’UTR. In one embodiment, tropism is enhanced by about at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 125%, about 150%, about 175%, or about 200% or more in comparison to a suitable control.

[0397]

[0415] In one embodiment, the target is the brain, lung, skeletal muscle, heart, liver, kidney, spleen, gastrointestinal tract, nervous tissue or pancreas.

[0398]

[0416] In another embodiment, the target cell is an immune cell, cancer cell, muscle cell, liver cell or lung cell.

[0399]

[0417] The saRNAs described herein can self-replicate due to inclusion of conserved sequence elements (CSEs) located on the 5' and 3' ends of the RNA in combination with protein machinery (RNA-dependent RNA polymerase or RdRp) encoded by the coding sequences for nspl-4. The saRNA can also include amplification of a sub-genomic RNA, e.g., encoding the cargo of interest, from a subgenomic promoter (SGP) that is recognized by the RdRp. As such, the exemplary saRNAs described herein can comprise at least the following domains: nspl-4 (see e.g., nucleotides 62-5701 of SEQ ID NO: 1 and nucleotides 5702-7543 of SEQ ID NO: 1, which translate to SEQ ID NO: 3 and SEQ ID NO: 4, respectively); 3' CSE (e.g., SEQ ID NO: 8); 5' CSE (e g., SEQ ID NO: 9); and / or a SGP (e g., SEQ ID NO: 10).

[0400]

[0418] The conserved 3'- conserved sequence element (3' CSE) is encoded in the 3' UTR and is dependent on the viral machinery (e.g., nsPl-4) used in the saRNA. For VEEV, the 3' CSE is in the 3' UTR sequence and can comprise SEQ ID NO: 8 (see e.g., nucleotides 7673-7742 of SEQ ID NO: 1).

[0401]

[0419] The conserved 5' sequence element (5' CSE) is contained in the nspl coding sequence and is 51 nt. The 5' CSE is also dependent on viral machinery (e.g., nsPl-4) used to generate the saRNA. For VEEV, the 5' CSE can comprise SEQ ID NO: 9 (see e.g., nucleotides 149-199 of SEQ ID NO: 1).

[0402]

[0420] The subgenomic promoter (SGP) is located at the end of nsp4 and contains sequences within and after the coding sequence of nsp4. This is also dependent on viral machinery (e.g., nsPl-4) used to generate the saRNA. The sequence for the SGP in VEEV can comprise: SEQ ID NO: 10 (see e.g., nucleotides 7308-7578 of SEQ ID NO: 1).

[0403]

[0421] In one embodiment, the saRNA further comprises at least one 5’ conserved sequence element (CSE) and / or 3’ at least one conserved sequence element (CSE). In one embodiment, the at least one 5’ conserved sequence element (CSE) and / or 3’ at least one conserved sequence element (CSE) derived from at least one virus.

[0422] In one embodiment, the nspl, nsp2, nsp3, nsp4, SGP, 5’ UTR, 3’ UTR, 5’ CSE, and / or 3’ CSE are derived from the same virus. Optionally, one or more of the nsps comprise at least one mutation. In a particular embodiment, the nsp2 is mutated and more particularly, the mutation is D584N.

[0404]

[0423] In another embodiment, at least one of the nspl, nsp2, nsp3, nsp4, SGP, 5’ UTR, 3’ UTR, 5’ CSE, and / or 3’ CSE are derived from different viruses. Optionally, one or more of the nsps are mutation. In a particular embodiment, the nsp2 is mutated and more particularly, the mutation is D584N.

[0405]

[0424] In one embodiment, the nspl, nsp2, nsp3, and nsp4 proteins encoded by the saRNA comprise SEQ ID NO: 3 and / or SEQ ID NO: 4 or at least one amino acid sequence that is at least 90% identical to SEQ ID NO: 3 and / or at least 90% identical to SEQ ID NO: 4, that maintains the same function.

[0406]

[0425] In some embodiments, the RNA dependent RNA polymerase is mutated relative to the original sequence. In some embodiments, beneficial mutations for the RNA-dependent RNA polymerase are generated by chronic positive selection in the presence of an antibiotic such as puromycin. In some embodiments, the mutation(s) in the RNA-dependent RNA polymerase provide enhanced protein expression and / or reduced activation of the innate immune system. In some embodiments, the mutation is in nsp2 of the VEEV RNA-dependent RNA polymerase.

[0407]

[0426] In certain embodiments, the mutation(s) in the RNA-dependent RNA polymerase (the one or more non- structural proteins) provide enhanced protein expression and / or reduced activation of the innate immune system or response. In a particular embodiment, protein expression is enhanced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% or more relative to a comparable saRNA lacking a mutation in the at least one non- structural protein. In a particular embodiment, upon administration, the activation of the innate immune system or response is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% or more relative to a comparable saRNA lacking a mutation in the at least one non-structural protein. In a particular embodiment, upon administration, the activation of the innate immune system or response is reduced by at least about 20% or more relative to a comparable saRNA lacking a mutation in the at least one non-structural protein. In a particular embodiment, upon administration, the activation of the innate immune system or response is reduced by at least about 60% or more relative to a comparable saRNA lacking a mutation in the at least one non-structural protein. In a particular embodiment, upon administration, the activation of the innate immune system or response is reduced by at least about 80% or more relative to a comparable saRNA lacking a mutation in the at least one non- structural protein. In some embodiments, the mutation is in nsp2 of the VEEV RNA-dependent RNA polymerase.

[0408]

[0427] In some embodiments, the amino acid at position 584 is mutated. In some embodiments, the mutation at position 584 of nsp2 enhances protein production. In some embodiments, the mutation at position 584 of nsp2 decreases the activation of the innate immune system. In some embodiments, the nucleotide G at position 3398 is replaced with A.

[0409]

[0428] In some embodiments, the mutation is a C to T mutation at position 3399 resulting in a D to N amino acid change at amino acid position 584 in nsp2. n some embodiments, the amino acid at position 584 is substituted from D to N. In a particular embodiment, 584 nsp2 is mutated from GAC to AAC.

[0410]

[0429] In multiple aspects, described herein are saRNAs comprising a 5’ cap, which is found on the 5’ end of the saRNA molecule. In some embodiments of any of the aspects, the 5’ cap is derived from at least one virus (e.g., at least one alphavirus). In some embodiments of any of the aspects, the 5’ cap is selected from the group consisting of cap-0, cap-1, and cap-2. The 5’ cap can be involved in translation, nucleocytoplasmic transport, splicing, and / or stabilization of saRNA against 5' exonucleolytic degradation.

[0411]

[0430] In eukaryotes, the 5' cap, referred to as cap-0, is found on the 5' end of an RNA molecule. Cap-0 consists of a guanine nucleotide connected to mRNA via a 5' to 5' triphosphate linkage. This guanosine is methylated on the 7 position directly after capping in vivo by a methyltransferase. Cap-0 can also be referred to as a 7-m ethylguanylate cap, abbreviated m7G. In some embodiments of any of the aspects, an saRNA as described herein comprises cap-0. In some embodiments of any of the aspects, an saRNA as described herein comprises a m7G 5’ cap. ARCA (Anti -Reverse Cap Analog) consisting of 3'-O-Me-m7G(5')ppp(5')G is a nonlimiting example of a reagent for producing an saRNA comprising a 5’ CapO.

[0412]

[0431] In multicellular eukaryotes and some viruses, further 5’ cap modifications exist, including the methylation of the 2' hydroxy-groups of the first 2 ribose sugars of the 5' end of the RNA. cap-1 has a methylated 2'-hydroxy group on the first ribose sugar, while cap-2 has methylated 2'-hydroxy groups on the first two ribose sugars. In some embodiments of any of the aspects, the initiating nucleoside of an saRNA as described herein is methylated at the 2'0 position of the ribose (Capl). In some embodiments of any of the aspects, Capl has the following chemical structure: m7GpppNm. CLEANCAP AU consisting of m7G(5')ppp(5')(2'OMeA)pU is a non-limiting example of a reagent for producing an saRNA comprising a 5’ Capl. In some embodiments of any of the aspects, the initiating nucleotide and the subsequent nucleotide of an saRNA as described herein are both methylated at the 2'0 position of the ribose (Cap2). In some embodiments of any of the aspects, Cap2 has the following chemical structure: m7GpppNmNm.

[0413]

[0432] In some embodiments of any of the aspects, the initiating nucleotide directly proximal to the 5’ cap in an saRNA as described herein comprises an adenosine or adenosine analog. In some embodiments of any of the aspects, the initiating nucleotide directly proximal to the 5’ cap in an saRNA as described herein comprises a guanosine or guanosine analog. In some embodiments of any of the aspects, the first two initiating nucleotides directly proximal to the 5’ cap in an saRNA as described herein comprises: in position 1 an adenosine or adenosine analog and in position 2 a uridine or a uridine analog. In some embodiments of any of the aspects, the first three initiating nucleotides directly proximal to the 5’ cap in an saRNA as described herein comprises: in position 1 a guanosine or guanosine analog, in position 2 an adenosine or adenosine analog, and in position 3 a uridine or a uridine analog.

[0414]

[0433] In some embodiments of any of the aspects, the initiating nucleotide of an saRNA as described herein comprises an adenosine or adenosine analog, and the initiating nucleotide of the saRNA is methylated at the 2'0 position of the ribose (Capl).

[0415]

[0434] In some embodiments of any of the aspects, the initiating nucleotide of an saRNA as described herein comprises an adenosine or adenosine analog, and the initiating nucleotide and the subsequent nucleotide of the saRNA are both methylated at the 2'0 position of the ribose (Cap2).

[0416]

[0435] In some embodiments of any of the aspects, the initiating nucleotide of an saRNA as described herein comprises a guanosine or guanosine analog, and wherein the initiating nucleotide of the saRNA is methylated at the 2'0 position of the ribose (Capl).

[0417]

[0436] In some embodiments of any of the aspects, the initiating nucleotide of an saRNA as described herein comprises a guanosine or guanosine analog, and wherein the initiating nucleotide and the subsequent nucleotide of the saRNA are both methylated at the 2'0 position of the ribose (Cap2).

[0418]

[0437] In some embodiments of any of the aspects, the initiating nucleotide of an saRNA as described herein comprises an adenosine or adenosine analog, and the 5’ cap is a m7G 5’ cap- 0. In some embodiments of any of the aspects, the initiating nucleotide of an saRNA as described herein comprises a guanosine or guanosine analog, and the 5’ cap is a m7G 5’ cap-0. In one embodiment, a modified saRNA is provided comprising from 5’ to 3’ : (a) at least one non- structural protein derived from at least one virus (optionally, comprising at least one mutation); (b) a subgenomic promoter (SGP) derived from at least one virus; and (c) at least one cargo of interest; herein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-fluorocytidine, 5- fluorouridine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2'0 position of the ribose (Capl).

[0419]

[0438] In a particular embodiment, the mutation is a codon change at amino acid 584 of nsp2 from GAC to AAC.

[0420]

[0439] In one embodiment, a self-amplifying RNA (saRNA) is provided comprising from 5’ to 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non- structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus (optionally, at least one of the nsps comprises at least one mutation); (c) a subgenomic promoter (SGP) derived from at least one virus; (d) a 5’ untranslated region (UTR) derived from at least one virus; (e) at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one virus; and (g) a poly-Atail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-fluorocytidine, 5-fluorouridine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2'0 position of the ribose (Capl).

[0421]

[0440] In a particular embodiment, the 5’ UTR, the ‘3UTR or both are chimeric, i.e., comprises of sequences from two or more viral species.

[0422]

[0441] In a particular embodiment, the mutation is 584 nsp2 GAC to AAC.

[0423]

[0442] In certain embodiments, the modified saRNA or herein contains a mutation at the location that corresponds to amino acid 33 of nsp2, specifically a C to U mutation in the RNA sequence or C to T mutation in the DNA sequence, leading to a synonymous mutation.

[0424]

[0443] In one embodiment, the IRES sequence(s) drive cap-independent translation from a viral genome. In certain embodiments, an IRES sequence can be derived from a cell or virus. In some embodiments, the IRES sequence drives cap-independent translation and is derived from a viral genome. In one embodiment, the IRES sequence is derived from the RNA genome selected from the group consisting of: poliovirus, coxsackievirus, enterovirus, rhinovirus, encephalomyocarditis virus (EMCV), aphthovirus (FMDV), hepatitis C virus (HCV), hepatitis A virus, swine fever virus, Moloney murine leukemia (MMLV), rous sarcoma virus, human immunodeficiency virus.

[0425]

[0444] In one embodiment, the IRES sequence is derived from a human transcript. In a particular embodiment, the human transcript is selected from the group consisting of: fibroblast growth factor (FGF1 / FGF2), platelet-derived growth factor B (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), eukaryotic initiation factor 4G, c-myc, L-myc, Piml, p53, Bcl-Xl, Bcl-2.

[0426]

[0445] Also disclosed herein are circular RNAs. As used herein, the term circular RNA or circRNA refers to an RNA molecule that forms a covalently closed continuous loop, which is capable of being translated to produce a protein or antigen. Circular RNAs lack free 5’ and 3’ ends, making them resistant to exonuclease degradation, thus offering enhanced stability over linear RNAs. Circular RNAs may be synthesized by intron-mediated circularization, or ribozyme-mediated self-circularization.

[0427]

[0446] In one embodiment, a circRNA is provided comprising: (a) a 5’ untranslated region (UTR) that may be derived from at least one virus or other eukaryotic or prokaryotic source; (b) at least one cargo of interest, such as a sequence encoding a therapeutic protein, antigen, or enzyme; (c) at least one chimeric 3’ UTR sequence derived in part from one or more viruses, which may include sequences imparting enhanced stability, translational efficiency, or alternative or specific tropism; and (d) a regulatory element for enhanced ribosomal binding and translation initiation. The circRNA may optionally include internal ribosome entry sites (IRES) to facilitate translation independent of a 5’ cap structure.

[0428]

[0447] In another embodiment, a circRNA is provided comprising: (a) a 5’ and 3’ untranslated region (UTR) derived from at least one virus; (b) the at least one cargo of interest; (c) at least one chimeric sequence derived in part from at least one virus; and (d) at least one additional element selected from RNA aptamers, miRNA binding sites, or other regulatory RNA sequences designed to control translation, degradation, or cellular localization of the circRNA.

[0429]

[0448] In one embodiment, a circRNA is provided comprises at least one modified nucleotide, as further described herein. In another embodiment, a circRNA is provided comprising no modified nucleotides. Modified nucleotides may include, but are not limited to, modifications that reduce immunogenicity, enhance stability, or improve translation efficiency and include, but are not limited to, those modified described herein.

[0430]

[0449] In one embodiment, a circRNA is provided chimeric sequences partly derived from multiple sources, including but not limited to viral, bacterial, and eukaryotic RNA sequences. These chimeric sequences may impart specific or alternative cellular tropism that is desirable for therapeutic and vaccine applications, potentially reducing off-target effects and improving the efficiency of transfection and protein production.

[0431]

[0450] In a particular embodiment, a circRNA is provided comprising: (a) at least one cargo of interest, wherein the circRNA comprises at least 5% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5- methyluridine (5mU), 5-hydroxymethylcytidine, 5-hydroxymethyluridine (50HmU), Nl- methylpseudouridine, pseudouridine, 2-thiouridine, 5-methoxyuridine, 5-formylcytidine, N6- methyladenosine, and other modifications that enhance circRNA functionality or stability; and (b) regulatory elements that include, but are not limited to, IRES elements, miRNA target sites, or aptamers to facilitate specific cellular interactions and translation control.

[0432]

[0451] In a particular embodiment, a circRNA is provided comprising: (a) an untranslated region (UTR) that enhances translation efficiency; (b) the at least one cargo of interest; (c) at least one chimeric 3’ UTR sequence derived from viral RNAthat improves stability or cellular targeting; (d) additional regulatory elements, such as ribosome-binding sites or RNA binding protein interaction sites; wherein the circRNA comprises at least 5% modified nucleotides, selected from the group consisting of 5-methylcytidine, 5 -methyluridine, 5- hydroxymethylcytidine, 5-hydroxymethyluridine, N1 -methylpseudouridine, pseudouridine, 2- thiouridine, 5-methoxyuridine, 5-formylcytidine, N6-methyladenosine.

[0433]

[0452] In one aspect, described herein is a messenger RNA (mRNA) comprising from 5’ to 3’: (a) a 5’ cap: (b) a 5’ untranslated region (UTR) derived from at least one virus; (c) at least one of cargo of interest; (d) a 3’ untranslated region (UTR) derived from at least one virus; and (e) a poly- A tail.

[0434]

[0453] In some embodiments, a messenger RNA (mRNA) comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the following group: 5- methylcytidine, 5-methyluridine, 5-hydroxymethylcytidine, 5-hydroxymethyluridine, Nl- methylpseudouridine, pseudouridine, 2-thiouridine, 5-methoxyuridine, 5-formylcytidine, N6- methyladenosine.

[0435]

[0454] In some embodiments, a circular RNA comprises an internal ribosomal entry site (IRES) sequence upstream of a coding sequence and a chimeric 3 ’ UTR.

[0436]

[0455] In some embodiments, the adaptive immune response inhibitory protein reduces the expression of molecules associated with antigen presentation including MHC I or MHC II complexes.

[0437]

[0456] In some embodiments, the vector contains chimeric sequences that confer an alternative cellular tropism compared to an equivalent vector without the chimeric sequences.

[0438]

[0457] In some embodiments, the chimeric tropism altering sequence is derived from an RNA or DNA virus.

[0439]

[0458] In some embodiments, the chimeric sequence is from a 3’ untranslated region (UTR).

[0459] In one aspect described herein is an mRNA comprising from 5’ to 3’ : (a) a 5’ cap;(b) 5’ untranslated region (UTR) derived from at least one virus; (c) the at least one cargo of interest; (d) at least one chimeric sequence derived in part from at least one virus; (e) a chimeric 3’ untranslated region (UTR) derived from at least one virus; and (f) a poly-A tail.

[0440]

[0460] In one aspect described herein is an mRNA comprising from 5’ to 3’ : (a) a 5’ cap;(b) 5’ untranslated region (UTR) derived from at least one virus; (c) the at least one cargo of interest; (d) at least one chimeric sequence derived in part from at least one virus; (e) a chimeric 5’ untranslated region (UTR) derived from at least one virus; and (f) a poly-A tail.

[0441]

[0461] In some embodiments of the aspects, an mRNA as described herein comprises at least one nucleoside modification, as described further herein. In some embodiments of the aspects, an mRNA as described herein does not comprises nucleotide modifications.

[0442]

[0462] In some embodiments of the aspects, an mRNA is constructed from chimeric sequences partly derived from multiple viruses. The chimeric sequences may impart an alternative or specific cellular tropism that is desirable for therapeutics and vaccines by reducing off-target transfection and protein production.

[0443]

[0463] In one aspect, described herein is a messenger RNA (mRNA) comprising: (a) at least one cargo of interest; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5 -hydroxymethylcytidine, 5-hydroxymethyluridine, Nl- methylpseudouridine, pseudouridine, 2-thiouridine, 5-methoxyuridine, 5-formylcytidine, N6- methyladenosine; (b) wherein the initiating nucleotide of the mRNA is methylated at the 2’0 position of the ribose (Capl).

[0444]

[0464] In one aspect, described herein is a messenger RNA (saRNA) comprising: (a) a 5’ cap; (b) a 5’ untranslated region (UTR); (c) least one cargo of interest; (d) a chimeric 3’ untranslated region (UTR) derived from at least one virus; and (e) a poly-A tail; wherein the mRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethylcytidine, 5- hydroxymethyluridine, N1 -methylpseudouridine, pseudouridine, 2-thiouridine, 5 methoxyuridine, 5-formylcytidine, N6-methyladenosine; and wherein the initiating nucleotide of the mRNA is methylated at the 2’0 position of the ribose (Capl).

[0445]

[0465] Described herein in multiple aspects are circular RNAs. As used herein, the term circular RNA or circRNA refers to an RNA molecule that forms a covalently closed continuous loop, which is capable of being translated to produce a protein or antigen. Circular RNAs lack free 5’ and 3’ ends, making them resistant to exonuclease degradation, thus offering enhanced stability over linear RNAs. Circular RNAs may be synthesized by intron-mediated circularization, or ribozyme-mediated self-circularization.

[0446]

[0466] In one aspect, described herein is a circRNA comprising: (a) a 5’ untranslated region (UTR) that may be derived from at least one virus or other eukaryotic or prokaryotic source; (b) at least one cargo of interest, such as a sequence encoding a therapeutic protein, antigen, or enzyme; (c) at least one chimeric 3’ UTR sequence derived in part from one or more viruses, which may include sequences imparting enhanced stability, translational efficiency, or alternative or specific tropism; and (d) a regulatory element for enhanced ribosomal binding and translation initiation. The circRNA may optionally include internal ribosome entry sites (IRES) to facilitate translation independent of a 5’ cap structure.

[0447]

[0467] In another aspect, described herein is a circRNA comprising: (a) a 5’ and 3’ untranslated region (UTR) derived from at least one virus; (b) the at least one cargo of interest; (c) at least one chimeric sequence derived in part from at least one virus; and (d) at least one additional element selected from RNA aptamers, miRNA binding sites, or other regulatory RNA sequences designed to control translation, degradation, or cellular localization of the circRNA.

[0448]

[0468] In some embodiments of the aspects, the circRNA as described herein comprises at least one modified nucleotide, as further described herein. In some embodiments of the aspects, the circRNA comprises no modified nucleotides. Modified nucleotides may include, but are not limited to, modifications that reduce immunogenicity, enhance stability, or improve translation efficiency.

[0449]

[0469] In some embodiments of the aspects, a circRNA as described herein is constructed from chimeric sequences partly derived from multiple sources, including but not limited to viral, bacterial, and eukaryotic RNA sequences. These chimeric sequences may impart specific or alternative cellular tropism that is desirable for therapeutic and vaccine applications, potentially reducing off-target effects and improving the efficiency of transfection and protein production.

[0450]

[0470] In one aspect, described herein is a circRNA comprising: (a) at least one cargo of interest, wherein the circRNA comprises at least 5% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5- methyluridine, 5-hydroxymethylcytidine, 5-hydroxymethyluridine, N1 -methylpseudouridine, pseudouridine, 2-thiouridine, 5-methoxyuridine, 5-formylcytidine, N6-methyladenosine, and other modifications that enhance circRNA functionality or stability; and (b) regulatory elements that include, but are not limited to, IRES elements, miRNA target sites, or aptamers to facilitate specific cellular interactions and translation control.

[0471] In another aspect, described herein is a circRNA comprising: (a) an untranslated region (UTR) that enhances translation efficiency; (b) the at least one cargo of interest; (c) at least one chimeric 3’ UTR sequence derived from viral RNA that improves stability or cellular targeting; (d) additional regulatory elements, such as ribosome-binding sites or RNA binding protein interaction sites; wherein the circRNA comprises at least 5% modified nucleotides, selected from the group consisting of 5 -methylcytidine, 5-methyluridine, 5-hydroxymethylcytidine, 5- hydroxymethyluridine, N1 -methylpseudouridine, pseudouridine, 2-thiouridine, 5- methoxyuridine, 5-formylcytidine, N6-methyladenosine.

[0451]

[0472] In multiple aspects described herein is an saRNA comprising: (a) at least one non- structural protein derived from at least one virus; (b) a subgenomic promoter (SGP) derived from at least one virus; and (c) at least one cargo of interest. In additional aspects described herein is an saRNA comprising from 5’ to 3’ : (a) at least one non-structural protein derived from at least one virus; (b) a subgenomic promoter (SGP) derived from at least one virus; (c) at least one chimeric sequence derived in part from at least one virus; (d) an IRES or 2A sequence; and (e) at least one cargo of interest.

[0452]

[0473] In other aspects described herein is an saRNA comprising: (a) at least one non-structural protein derived from at least one alphavirus; (b) a subgenomic promoter (SGP) derived from at least one alphavirus; and (c) least one cargo of interest. In additional aspects described herein is an saRNA comprising from 5’ to 3’: (a) at least one non-structural protein derived from at least one alphavirus; (b) a subgenomic promoter (SGP) derived from at least one virus; (c) at least one chimeric sequence derived in part from at least one virus; (d) an IRES or 2A sequence; and (e) at least one cargo of interest.

[0453]

[0474] In one aspect described herein is an saRNA comprising: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non structural protein 4 (nsp4), each derived from at least one virus; (c) a subgenomic promoter (SGP) derived from at least one virus; (d) 5’ untranslated region (UTR) derived from at least one virus; (e) the at least one cargo of interest; (f) at least one chimeric sequence derived in part from at least one virus; (g) a chimeric 3’ untranslated region (UTR) derived from at least one virus; and (h) a poly- A tail.

[0454]

[0475] In one aspect described herein is an saRNA comprising from 5’ to 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus; (c) a subgenomic promoter (SGP) derived from at least one virus; (d) 5’ untranslated region (UTR) derived from at least one virus; (e) the at least one cargo of interest; (f) at least one chimeric sequence derived in part from at least one virus; (g) a chimeric 3’ untranslated region (UTR) derived from at least one virus; and (h) a poly- A tail.

[0455]

[0476] In one aspect described herein is an saRNA comprising: (a) a 5’ cap; (b) non- structural protein 1 (nspl), non- structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non structural protein 4 (nsp4), each derived from at least one alphavirus; (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) 5’ untranslated region (UTR) derived from at least one alphavirus; (e) the at least one cargo of interest; (f) at least one chimeric sequence derived in part from at least one virus; (g) a chimeric 3’ untranslated region (UTR) derived from at least one alphavirus; and (h) a poly-Atail.

[0456]

[0477] In one aspect described herein is an saRNA comprising from 5’ to 3’: (a) a 5’ cap; (b) non- structural protein 1 (nspl), non- structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one alphavirus; (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) 5’ untranslated region (UTR) derived from at least one alphavirus; (e) the at least one cargo of interest; (f) at least one chimeric sequence derived in part from at least one virus; (g) a chimeric 3’ untranslated region (UTR) derived from at least one alphavirus; and (h) a poly-Atail.

[0457]

[0478] In one aspect described herein is an saRNA comprising: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non structural protein 4 (nsp4), each derived from at least one virus, wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one virus; (d) 5’ untranslated region (UTR) derived from at least one virus; (e) the at least one cargo of interest; (f) at least one chimeric sequence derived in part from at least one virus; (g) a chimeric 3’ untranslated region (UTR) derived from at least one virus, wherein the 3’ UTR comprises at least one 3’- conserved sequence element (3'CSE); and (h) a poly-Atail.

[0458]

[0479] In one aspect described herein is an saRNA comprising from 5’ to 3’: (a) a 5’ cap; (b) 5’ untranslated region (UTR) derived from at least one virus; (c) non-structural protein 1 (nspl), non structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus, wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (d) a subgenomic promoter (SGP) derived from at least one virus; (e) the at least one cargo of interest; (f) at least one chimeric sequence derived in part from at least one virus; a chimeric 3’ untranslated region (UTR) derived from at least one virus, wherein the 3’ UTR comprises at least one 3’- conserved sequence element (3'CSE); and (g) a poly-Atail.

[0480] In one aspect described herein is an saRNA comprising from 5’ to 3’: (a) a 5’ cap; (b) non- structural protein 1 (nspl), non- structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one alphavirus, wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) 5’ untranslated region (UTR) derived from at least one alphavirus; (e) the at least one cargo of interest; (f) a chimeric 3’ untranslated region (UTR) derived from at least one alphavirus, wherein the 3’ UTR comprises at least one 3’- conserved sequence element (3’CSE); and (g) a poly-Atail.

[0459]

[0481] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising (e.g., from 5’ to 3’): (a) at least one non-structural protein derived from at least one virus; (b) a subgenomic promoter (SGP) derived from at least one virus; (c) at least one adaptive immune response inhibitory protein and (d) at least one cargo of interest; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5 methyluridine, 5-hydroxymethyluridine, and 5- hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0460]

[0482] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising (e.g., from 5’ to 3’): (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus; (c) a subgenomic promoter (SGP) derived from at least one virus; (d) a 5’ untranslated region (UTR) derived from at least one virus; (e) at least one chimeric sequence derived in part from at least one virus; (f) at least one cargo of interest; (g) a chimeric 3’ untranslated region (UTR) derived from at least one virus; and (h) a poly-A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5 -methyluridine, 5- hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0461]

[0483] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) at least one non-structural protein derived from at least one virus; (b) a subgenomic promoter (SGP) derived from at least one virus; (c) at least one chimeric sequence derived in part from at least one virus and (d) at least one cargo of interest; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5- hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0462]

[0484] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non- structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus; (c) a subgenomic promoter (SGP) derived from at least one virus; (d) a 5’ untranslated region (UTR) derived from at least one virus; (e) at least one cargo of interest; (f) a chimeric 3’ untranslated region (UTR) derived from at least one virus; and (g) a poly-A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5- hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0463]

[0485] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus, wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one virus; (d) a 5’ untranslated region (UTR) derived from at least one virus; (e) at least one cargo of interest; (f) a chimeric 3’ untranslated region (UTR) derived from at least one virus, wherein the 3’ UTR comprises at least one 3’- conserved sequence element (3'CSE); and (g) a poly-A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0464]

[0486] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising: (a) at least one non-structural protein derived from at least one alphavirus; (b) a subgenomic promoter (SGP) derived from at least one alphavirus; and (c) at least one cargo of interest; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5- hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0465]

[0487] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one alphavirus; (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) a 5’ untranslated region (UTR) derived from at least one alphavirus; (e) at least one cargo of interest; (f) a chimeric 3 ’ untranslated region (UTR) derived from at least one alphavirus; and (g) a poly-A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5 -methyluridine, 5- hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0466]

[0488] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) at least one non-structural protein derived from at least one alphavirus; (b) a subgenomic promoter (SGP) derived from at least one alphavirus; and (c) at least one cargo of interest; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5- methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0467]

[0489] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one alphavirus; (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) a 5’ untranslated region (UTR) derived from at least one alphavirus; (e) at least one cargo of interest; (f) a chimeric 3’ untranslated region (UTR) derived from at least one alphavirus; and (g) a poly-A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5- methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0468]

[0490] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non- structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one alphavirus, wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) a 5’ untranslated region (UTR) derived from at least one alphavirus; (e) at least one cargo of interest; (f) a chimeric 3’ untranslated region (UTR) derived from at least one alphavirus, wherein the 3’ UTR comprises at least one 3’ conserved sequence element (3'CSE); and (g) a poly-A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5- methyluridine, 5-hydroxymethyluridine, and 5 hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0469]

[0491] In other aspects described herein is an saRNA comprising: (a) at least one non-structural protein derived from at least one alphavirus; (b) a subgenomic promoter (SGP) derived from at least one alphavirus; and (c) least one cargo of interest. In additional aspects described herein is an saRNA comprising from 5’ to 3’: (a) at least one non-structural protein derived from at least one alphavirus; (b) a subgenomic promoter (SGP) derived from at least one alphavirus; and (c) least one cargo of interest.

[0470]

[0492] In one aspect described herein is an saRNA comprising: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non structural protein 4 (nsp4), each derived from at least one virus; (c) a subgenomic promoter (SGP) derived from at least one virus; (d) 5’ untranslated region (UTR) derived from at least one virus; (e) the at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one virus; and (g) a poly-A tail.

[0471]

[0493] In one aspect described herein is an saRNA comprising from 5’ to 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus; (c) a subgenomic promoter (SGP) derived from at least one virus; (d) 5’ untranslated region (UTR) derived from at least one virus; (e) the at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one virus; and (g) a poly-A tail.

[0472]

[0494] In one aspect described herein is an saRNA comprising: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non structural protein 4 (nsp4), each derived from at least one alphavirus; (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) 5’ untranslated region (UTR) derived from at least one alphavirus; (e) the at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one alphavirus; and (g) a poly- A tail.

[0473]

[0495] In one aspect described herein is an saRNA comprising from 5’ to 3’: (a) a 5’ cap; (b) non- structural protein 1 (nspl), non- structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one alphavirus; (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) 5’ untranslated region (UTR) derived from at least one alphavirus; (e) the at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one alphavirus; and (g) a poly- A tail.

[0474]

[0496] In one aspect described herein is an saRNA comprising: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non structural protein 4 (nsp4), each derived from at least one virus, wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one virus; (d) 5’ untranslated region (UTR) derived from at least one virus; (e) the at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one virus, wherein the 3’ UTR comprises at least one 3 ’-conserved sequence element (3 ’CSE); and (g) a poly- A tail.

[0475]

[0497] In one aspect described herein is an saRNA comprising from 5’ to 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus, wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one virus; (d) 5’ untranslated region (UTR) derived from at least one virus; (e) the at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one virus, wherein the 3’ UTR comprises at least one 3 ’-conserved sequence element (3 ’CSE); and (g) a poly- A tail.

[0476]

[0498] In one aspect described herein is an saRNA comprising: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non- structural protein 4 (nsp4), each derived from at least one alphavirus, wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) 5’ untranslated region (UTR) derived from at least one alphavirus; (e) the at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one alphavirus, wherein the 3’ UTR comprises at least one 3 ’-conserved sequence element (3 ’CSE); and (g) a poly-A tail.

[0477]

[0499] In one aspect described herein is an saRNA comprising from 5’ to 3’: (a) a 5’ cap; (b) non- structural protein 1 (nspl), non- structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one alphavirus, wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) 5’ untranslated region (UTR) derived from at least one alphavirus; (e) the at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one alphavirus, wherein the 3’ UTR comprises at least one 3’- conserved sequence element (3 ’CSE); and (g) a poly- A tail.

[0478]

[0500] In some embodiments of the aspects, an saRNA as described herein comprises at least one nucleotide modification, as described further herein. In some embodiments of the aspects, an saRNA as described herein does not comprises nucleotide modifications.

[0479]

[0501] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising: (a) at least one non-structural protein derived from at least one virus; (b) a subgenomic promoter (SGP) derived from at least one virus; and (c) at least one cargo of interest; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog, and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose.

[0480]

[0502] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus; (c) a subgenomic promoter (SGP) derived from at least one virus; (d) a 5’ untranslated region (UTR) derived from at least one virus; (e) at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one virus; and (g) a poly-A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose.

[0481]

[0503] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) at least one non-structural protein derived from at least one virus; (b) a subgenomic promoter (SGP) derived from at least one virus; and (c) at least one cargo of interest; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5- hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0482]

[0504] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non- structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus; (c) a subgenomic promoter (SGP) derived from at least one virus; (d) a 5’ untranslated region (UTR) derived from at least one virus; (e) at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one virus; and (g) a poly-A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5 -methyluridine, 5- hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0483]

[0505] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus, wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one virus; (d) a 5’ untranslated region (UTR) derived from at least one virus; (e) at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one virus, wherein the 3’ UTR comprises at least one 3’- conserved sequence element (3’ CSE); and (g) a poly-A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5- hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2'0 position of the ribose (Capl).

[0484]

[0506] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising: (a) at least one non-structural protein derived from at least one alphavirus; (b) a subgenomic promoter (SGP) derived from at least one alphavirus; and (c) at least one cargo of interest; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5- hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0507] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non- structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one alphavirus; (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) a 5’ untranslated region (UTR) derived from at least one alphavirus; (e) at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one alphavirus; and (g) a poly-A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5 -methyluridine, 5- hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0485]

[0508] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) at least one non-structural protein derived from at least one alphavirus; (b) a subgenomic promoter (SGP) derived from at least one alphavirus; and (c) at least one cargo of interest; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5- methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0486]

[0509] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one alphavirus; (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) a 5’ untranslated region (UTR) derived from at least one alphavirus; (e) at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one alphavirus; and (g) a poly- A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5 -methyluridine, 5- hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0487]

[0510] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one alphavirus, wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one alphavirus; (d) a 5’ untranslated region (UTR) derived from at least one alphavirus; (e) at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one alphavirus, wherein the 3’ UTR comprises at least one 3’ conserved sequence element (3’CSE); and (g) a poly-A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5 -methyluridine, 5- hydroxymethyluridine, and 5-hydroxymethylcytidine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2’0 position of the ribose (Capl).

[0488]

[0511] In one aspect, described herein is a modified self-amplifying RNA (saRNA) encapsulated or contained within a polymer nanoparticle. Said positively charged polymer nanoparticle possesses a diameter between 10 and 1000 nm, or more specifically 100 and 500 nm, or more specifically 100 and 200 nm. A preferred aspect is the use of a synthetic or natural polymer which contains an overall positive charge. Cationic polymers of choice include those discussed in the Han's review, where he discussed most of the common cationic polymer systems including PLL, poly(L-lysine); PEI, polyethyleneimine; pDMEAMA, poly(2- dimethylamino)ethyl-methacrylate; PLGA, poly(D,L-lactide-co-glycolide) and PVP (polyvinylpyrrolidone). See Garnett, M. C. Crit. Rev. Ther. Drug Carrier Sys. 1999, 16, 147- 207; Han, S.; Mahato, R. I.; Sung, Y. K.; Kim, S. W. Molecular Therapy 2000, 2, 302-317; Zauner, W.; Ogris, M.; Wagner, E. Adv. Drug. Del. Rev. 1998, 30, 97-113; Kabanov, A. V.; Kabanov, V. A. Bioconj. Chem. 1995, 6, 7-20; Lynn, D. M.; Anderson, D. G.; Putman, D.; Langer, R. J. Am. Chem. Soc. 2001, 123, 8155-8156; Boussif, O.; Lezoualc'h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. USA 1995, 92, 7297-7301; Choi, J. S.; Joo, D. K.; Kim, C. H.; Kim, K.; Park, J. S. J. Am. Chem. Soc. 2000, 122, 474-480; Putnam, D.; Langer, R. Macromolecules 1999, 32, 3658-3662; Gonzalez, M. F.; Ruseckaite, R. A.; Cuadrado, T. R. Journal of Applied Polymer Science 1999, 71, 1223-1230; Tang, M. X.; Redemann, C. T.; Szoka, F. C. In Vitro Gene Delivery by Degraded Polyamidoamine Dendrimers Bioconjugate Chem. 1996, 7, 703-714; Kukowska-latallo, J. F.; Bielinska, A. U.; Johnson, J.; Spinder, R.; Tomalia, D. A.; Baker, J. R. Proc. Nat. Acad. Sci. 1996, 93, 4897-4902; and Lim, Y.; Kim, S.; Lee, Y.; Lee, W.; Yang, T.; Lee, M.; Suh, M.; Park, J. J. Am. Chem. Soc. 2001, 123, 2460-2461.

[0489]

[0512] Some representative examples of further cationic polymers for use are described below. For example, poly(P-amino esters) have been explored and shown to condense plasmid DNA into soluble DNA / polymer particles for gene delivery. To accelerate the discovery of synthetic transfection vectors parallel synthesis and screening of a cationic polymer library was reported by Langer. Wolfert describes cationic vectors for gene therapy formed by self-assembly of DNA with synthetic block cationic co-polymers. Haensler and Szoka describe the use of cationic dendrimer polymers (polyamidoamine (PAMAM) dendrimers) for gene delivery. Wang describes a cationic polyphosphoester for gene delivery. Putnam describes a cationic polymer containing imidazole for the delivery of DNA. See Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000, 722, 10761-10768; Wolfert, M. A.; Schacht, E. H.; Toncheva, V.; Ulbrich, K.; Nazarova, O.; Seymour, L. W. Hum. Gene Ther. 1996, 7, 2123-2133; Haensler, J.; Szoka, F. Bioconj. Chem. 1993, 4, 372; and Wang, J.; Mao, H. Q.; Leong, K. W. J. Am. Chem. Soc. 2001; Putnam, D.; Gentry, C. A.; Pack, D. W .; Langer, R. Proc. Nat. Acad. Set. 2001, 98, 1200- 1205.

[0490]

[0513] In some embodiments, the 3’ UTR is derived from an alphavirus, e.g., selected from the group consisting of: Venezuela Equine Encephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV), Western Equine Encephalitis Virus (WEEV), Chikungunya Virus (CHIKV), Mayaro Virus (MAYV), Ross River Virus (RRV), Semliki Forest Virus (SFV), Sindbis Virus (SIN), Kunjin Virus (KUN).

[0491]

[0514] In some embodiments, the chimeric 3’ UTR confers a specific cellular tropism.

[0492]

[0515] In some embodiments, the chimeric 3’ UTR results in restricted protein expression in the liver.

[0493]

[0516] In some embodiments, the chimeric 3’ UTR results in restricted protein expression in innate immune cells.

[0494]

[0517] In some embodiments, the chimeric 3’ UTR confers an alternate cellular tropism.

[0495]

[0518] In some embodiments, the chimeric 3’ UTR does not interfere with replication in desirable cell types.

[0496]

[0519] In some embodiments, the chimeric 3’ UTR restricts replication in myeloid cells.

[0497]

[0520] In some embodiments, the chimeric 3’ UTR results in a reduced inflammatory response upon administration.

[0498]

[0521] In some embodiments, the chimeric 3’ UTR results in a reduced interferon response upon administration.

[0499] Modified Self-Amplifying RNAs

[0500]

[0522] Described herein are modified self-amplifying RNAs, i.e., saRNA comprising modified nucleotides.

[0523] The degree of substitution of the saRNA may vary. In one embodiment, the degree of modification is greater than about 25%, for example, greater than about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%-100% or even 100%. In some embodiments of any of the aspects, an saRNA as described herein comprises at least 25% modified nucleotides and at most 100% modified nucleotides. In one embodiment, an saRNA as described herein comprises at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% modified nucleotides.

[0501]

[0524] In some embodiments of any of the aspects, an saRNA as described herein comprises at least 25% modified nucleotides and at most 100% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine; e.g., cytidine and / or uridine).

[0502]

[0525] In some embodiments of any of the aspects, described herein is an saRNA comprising at least 25% modified nucleotides and at most 100% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine; e.g., cytidine and / or uridine). In some embodiments of any of the aspects, described herein is an saRNA comprising at least 25% modified cytidines. In some embodiments of any of the aspects, described herein is an saRNA comprising at least 25% cytidines modified with 5-flourocytidine.

[0503]

[0526] In some embodiments of any of the aspects, described herein is an saRNA comprising at least 25% modified nucleotides and at most 100% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine; e.g., cytidine and / or uridine) and encoding at least one protein or fragment thereof. In some embodiments of any of the aspects, described herein is an saRNA comprising at least 25% modified cytidines and encoding at least one protein or fragment thereof. In some embodiments of any of the aspects, described herein is an saRNA comprising at least 25% cytidines modified with 5-flourocytidine and encoding at least one protein or fragment thereof.

[0504]

[0527] In one embodiment, the modified saRNA comprises at least 25%, at least 26%, at least

[0505] 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least

[0506] 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least

[0507] 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least

[0508] 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least

[0509] 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least

[0510] 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least

[0511] 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least

[0512] 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least

[0513] 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

[0514] 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least

[0515] 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine, such as cytidine and / or uridine).

[0516]

[0528] In some embodiments of any of the aspects, an saRNA as described herein comprises at most 25%, at most 26%, at most 27%, at most 28%, at most 29%, at most 30%, at most 31%, at most 32%, at most 33%, at most 34%, at most 35%, at most 36%, at most 37%, at most 38%, at most 39%, at most 40%, at most 41%, at most 42%, at most 43%, at most 44%, at most 45%, at most 46%, at most 47%, at most 48%, at most 49%, at most 50%, at most 51%, at most 52%, at most 53%, at most 54%, at most 55%, at most 56%, at most 57%, at most 58%, at most 59%, at most 60%, at most 61%, at most 62%, at most 63%, at most 64%, at most 65%, at most 66%, at most 67%, at most 68%, at most 69%, at most 70%, at most 71%, at most 72%, at most 73%, at most 74%, at most 75%, at most 76%, at most 77%, at most 78%, at most 79%, at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, at most 99.1%, at most 99.2%, at most 99.3%, at most 99.4%, at most 99.5%, at most 99.6%, at most 99.7%, at most 99.8%, at most 99.9%, or at most 100% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine; e.g., cytidine and / or uridine).

[0517]

[0529] In one embodiment, the degree of modification is between about 25 and about 50%, between about 51 and about 75%, between about 75 and about 99%, between about 91.1 and about 99.9% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine, such as cytidine and / or uridine).

[0518]

[0530] In one embodiment, the degree of substitution comprises about 25% to about 35%, about 30% to about 40%, about 35% to about 45%, about 40% to about 50%, about 45% to about 55%, about 50% to about 60%, about 55% to about 65%, about 60% to about 70%, about 65% to about 75%, about 70% to 80%, about 75% to 85%, about 80% to about 90%, about 85% to about 95%, about 90% to about 100%, about 95% to about 100%, or about 99% to about 100% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine; e.g., cytidine and / or uridine).

[0519]

[0531] In some embodiments of any of the aspects, an saRNA as described herein comprises 25%-50% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine; e.g., cytidine and / or uridine). In some embodiments of any of the aspects, an saRNA as described herein comprises 51%-75% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine; e.g., cytidine and / or uridine). In some embodiments of any of the aspects, an saRNA as described herein comprises 75%-99% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine; e.g., cytidine and / or uridine). In some embodiments of any of the aspects, an saRNA as described herein comprises 100% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine; e.g., cytidine and / or uridine).

[0520]

[0532] In one embodiment, the modified saRNA comprises about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, or about 100% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine, such as cytidine and / or uridine).

[0521]

[0533] In in one embodiment, the modified saRNA comprises 0%, at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 11%, at most 12%, at most 13%, at most 14%, at most 15%, at most 16%, at most 17%, at most 18%, at most 19%, at most 20%, at most 21%, at most 22%, at most 23%, at most 24%, at most 25% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine, such as cytidine and / or uridine). In one embodiment, saRNA as described herein does not comprise any modified nucleotides.

[0522]

[0534] In some embodiments of any of the aspects, an saRNA as described herein comprises at least 25% modified pyrimidines and at most 100% modified pyrimidines. In some embodiments of any of the aspects, an saRNA as described herein comprises between 26% modified pyrimidines and 100% modified pyrimidines. In some embodiments of any of the aspects, an saRNA as described herein comprises greater than 25% modified pyrimidines. In some embodiments of any of the aspects, an saRNA as described herein comprises at least 25% modified pyrimidines. In one embodiment, an saRNA as described herein comprises at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% modified pyrimidines. In some embodiments, the pyrimidines are cytidine, uridine, or a combination thereof. In some embodiments, the pyrimidines are cytidine and uridine.

[0523]

[0535] In some embodiments, the modified pyrimidines are selected from the group consisting of 5-fluorocytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-methyluridine, 5- fluorouridine and combinations thereof. In some embodiments, each modified pyrimidines is 5-fluorocytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-methyluridine, or 5- fluorouridine.

[0536] In some embodiments, the saRNA comprises between about 50% and 100% 5- fluorocytidine. In some embodiments, the saRNA comprises at least 25% 5 -fluorocytidine. In some embodiments, the saRNA comprises 100% 5-fluorocytidine.

[0524]

[0537] In some embodiments, the saRNA comprises modifications of between about 50% and 100% of cysteines with 5-fluorocytidine. In some embodiments, the saRNA comprises modifications of at least 25% of cysteines with 5-fluorocytidine. In some embodiments, the saRNA comprises modifications of 100% of cysteines with 5-fluorocytidine.

[0525]

[0538] In some embodiments, the saRNA comprises modifications of between about 50% and 100% of pyrimidines with 5-fluorocytidine. In some embodiments, the saRNA comprises modifications of at least 25% of pyrimidines with 5-fluorocytidine. In some embodiments, the saRNA comprises modifications of 100% of pyrimidines with 5-fluorocytidine.

[0526]

[0539] In one embodiment, the modified saRNA comprises about 25% to about 50% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine, e.g., cytidine and / or uridine). In one embodiment, the modified saRNA comprises between about 51% and about 75% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine, e.g., cytidine and / or uridine). In one embodiment, the modified saRNA is about 75% to about 99% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine, e.g., cytidine and / or uridine). In one embodiment, the modified saRNA comprises 100% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine, e.g., cytidine and / or uridine). The modified nucleotide (nucleotide analog) may vary. In one embodiment, the modified nucleotide is a cytidine, adenosine, guanosine, uridine, or pseudouridine. These analogs can include isomers of the nitrogenous base, as well as inclusion or exclusion of chemical groups, both natural occurring and synthetically introduced, on any aspect of the nitrogenous base. It is explicitly stated herein that modifications to the sugar-phosphate backbone are not included in the definition of the term “modified nucleotide.” This exception is not meant to exclude the methylation at the 2’0 position of the first and second initiating nucleotides, also referred to as Cap-1 and cap-2 structures.

[0527]

[0540] In one embodiment, the modified nucleotide is a pyrimidine and particularly, cytosine (C), uracil (U) or a combination that of. In a particular embodiment, the modified nucleotide is a pyrimidine with a moiety on carbon 5 is selected from the group consisting of methyl, ethyl, propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxypropylfluoro, iodo, bromo, azido, monofluorohydroxymethyl, nitro, difluoromethyl, monofluoromethyl, and difluorohydroxymethyl functional groups.

[0541] In a particular embodiment, the modified nucleotide is a modified cytidine selected from the group of 5-methylcytidine, 5-hydroxymethylcytidine, and / or a modified uridine selected from the group of 5-methyluridine, 5-hydroxymethyluridine, 5-fluorocytidine, 5- fluorouridine, 5-bromocytidine, 5-iodocytidine, 5-azidocytidine, 5-bromouridine, 5- iodouridine, 5-azidouridine, 5-difluoromethyluridine, 5-nitrouridine, 5-nitrocytidine, 5- difluorohydroxymethyluridine, 5-difluorohydroxymethylcytidine, 5- monofluorohydroxymethyluridine, 5-monofluorohydroxymethylcytidine, 5- triflouromethylcytidine, 5-triflouromethylcytidine, 5- hydroxymethylcytidine, 5-ethylcytidine, 5-propylcytidine, 5-ethyluridine, 5-propyluridine, 5-nitrouridine, 5-hydroxyehtyluridine, 5- hydroxy ethylpropyluridine or a combination thereof.

[0528]

[0542] In a particular embodiment, the modified saRNA comprises 5-methyluridine or 5- hydroxymethyluridine, and one or both of 5-methylcytidine and 5-hydroxymethylcytidine

[0529]

[0543] The names and common abbreviations used for the modified nucleotides include such as 5-methylcytidine (5mC; m5C), 5-methyluridine (5mU; m5U), 5-hydroxymethyluridine (50HmU; hm5mU), 5-fluorocytidine (5fC, f5C), 5-bromocytidine (5BrC, Br5C), 5- iodocytidine (5IC, I5C), 5-azidocytidine (5azC, az5C), 5-fluorouridine (5fU, f5U), 5- bromouridine (5BrU, Br5U), 5-iodoouridine (5IU, I5U), 5-azidouridine (5azU, az5U),and 5- hydroxymethylcytidine (50HmC; hm5C) etc.

[0530]

[0544] In one embodiment, the modified saRNA comprises 100% 5-methylcytidine (5mC) substituted for cytidine. In one embodiment, the modified saRNA comprises 100% 5- fluorocytidine (5fC) substituted for cytidine. In one embodiment, the modified saRNA comprises 100% 5-bromocytidine substituted for cytidine. In one embodiment, the modified saRNA comprises 100% 5-iodocytidine substituted for cytidine. In one embodiment, the modified saRNA comprises 100% 5-azidocytidine substituted for cytidine. In one embodiment, the modified saRNA comprises 100% 5 -trifluoromethyl substituted for cytidine. In one embodiment, the modified saRNA comprises 100% 5-difluoromethylcytidine substituted for cytidine. In one embodiment, the modified saRNA comprises 100% 5-hydroxymethylcytidine substituted for cytidine. In one embodiment, the modified RNA comprises 100% 5- ethylcytidine substituted for cytidine. In one embodiment, the modified saRNA comprises 100% 5-propylcytidine substituted for cytidine. In one embodiment, the modified saRNA comprises 100% 5-nitrocytidine substituted for cytidine. In one embodiment, the modified saRNA comprises 100% 5-difluorohydroxymethylcytidine substituted for cytidine. In one embodiment, the modified saRNA comprises 100% 5-monofluorohydroxymethylcytidine substituted for cytidine. In one embodiment, the modified saRNA comprises 100% 5- hydroxy ethylcytidine substituted for cytidine. In one embodiment, the saRNA comprises 100% 5-hydroxypropylcytidine substituted for cytidine.

[0531]

[0545] In one embodiment, the modified saRNA comprises 100% 5 -methyluridine (5mU) substituted for uridine. In one embodiment, the saRNA comprises 100% 5 -fluorouridine (5fU) substituted for uridine. In one embodiment, the saRNA comprises 100% 5-bromouridine substituted for uridine. In one embodiment, the saRNA comprises 100% 5-iodouridine substituted for uridine. In one embodiment, the saRNA comprises 100% 5-azidouridine substituted for uridine. In one embodiment, the saRNA comprises 100% 5 -trifluoromethyl substituted for uridine. In one embodiment, the saRNA comprises 100% 5- difluoromethyluridine substituted for uridine. In one embodiment, the saRNA comprises 100% 5-hydroxymethyluridine substituted for uridine. In some embodiment, the saRNA comprises 100% 5-ethyluridine substituted for uridine. In one embodiment, the saRNA comprises 100% 5-propyluridine substituted for uridine. In one embodiment, the saRNA comprises 100% 5- nitrouridine substituted for uridine. In some embodiments, the modified saRNA comprises 100% 5-difluorohydroxymethyluridine substituted for uridine. In one embodiment, the modified saRNA comprises 100% 5-monofluorohydroxymethyluridine substituted for uridine. In one embodiment, the saRNA comprises 100% 5 -hydroxy ethyluridine substituted for uridine. In one embodiment, the saRNA comprises 100% 5-hydroxypropyluridine substituted for uridine.

[0532]

[0546] In embodiments wherein the saRNA comprises multiple types of modified nucleotides (e.g., at least two of 5 -methyluridine, 5-fluorouridine, 5-bromouridine, 5-iodouridine, 5- azidouridine, 5-trifluoromethyluridine, 5-difluoromethyluridine, 5-hydroxymethyluridine, 5- ethyluridine, 5-propyluridine, 5-nitrouridine, 5-difluorohydroxymethyluridine, 5- monofluorohydroxymethyluridine, 5-hydroxyethyluridine, 5-hydroxypropyluridine), the percentage of each modified nucleotide can be the same as each other or can be different from each other.

[0533]

[0547] As a non-limiting example, in an saRNA comprising 25% modified cytidines, the saRNA can comprise 0% 5mC and 25% 50HmC; 1% 5mC and 24% 50HmC; 2% 5mC and 23% 50HmC; 3% 5mC and 22% 50HmC; 4% 5mC and 21% 50HmC; 5% 5mC and 20% 50HmC; 6% 5mC and 19% 50HmC; 7% 5mC and 18% 50HmC; 8% 5mC and 17% 50HmC; 9% 5mC and 16% 50HmC; 10% 5mC and 15% 50HmC; 11% 5mC and 14% 50HmC; 12% 5mC and 13% 50HmC; 12.5% 5mC and 12.5% 50HmC; 13% 5mC and 12% 50HmC; 14% 5mC and 11% 50HmC; 15% 5mC and 10% 50HmC; 16% 5mC and 9% 50HmC; 17% 5mC and 8% 50HmC; 18% 5mC and 7% 50HmC; 19% 5mC and 6% 50HmC; 20% 5mC and 5% 5OHmC; 21% 5mC and 4% 5OHmC; 22% 5mC and 3% 5OHmC; 23% 5mC and 2% 5OHmC; 24% 5mC and 1% 5OHmC; or 25% 5mC and 0% 50HmC.

[0534]

[0548] As a non-limiting example, the modified saRNA comprising 25% modified uridines, the saRNA can comprise 0% 5mU and 25% 50HmU; 1% 5mU and 24% 50HmU; 2% 5mU and 23% 50HmU; 3% 5mU and 22% 50HmU; 4% 5mU and 21% 50HmU; 5% 5mU and 20% 50HmU; 6% 5mU and 19% 50HmU; 7% 5mU and 18% 50HmU; 8% 5mU and 17% 50HmU; 9% 5mU and 16% 50HmU; 10% 5mU and 15% 50HmU; 11% 5mU and 14% 50HmU; 12% 5mU and 13% 50HmU; 12.5% 5mU and 12.5% 50HmU; 13% 5mU and 12% 50HmU; 14% 5mU and 11% 50HmU; 15% 5mU and 10% 50HmU; 16% 5mU and 9% 50HmU; 17% 5mU and 8% 50HmU; 18% 5mU and 7% 50HmU; 19% 5mU and 6% 50HmU; 20% 5mU and 5% 50HmU; 21% 5mU and 4% 50HmU; 22% 5mU and 3% 50HmU; 23% 5mU and 2% 50HmU; 24% 5mU and 1% 50HmU; or 25% 5mU and 0% 50HmU.

[0535]

[0549] In one embodiment, the modified nucleotide is not a modified purine. In one embodiment, the modified nucleotide is not a modified guanosine or adenosine. In one embodiment, the modified nucleotide is not 7-Deazaad enosine, N1 -Methyladenosine, N6- Methyladenosine, 6-Chloropurineriboside, 2-Amino-6-chloropurineriboside, 2- Aminoadenosine, 5-Methoxycytidine, 5-Formylcytidine, 5-Aminoallylcytidine, 5- Hydroxycytidine, Isoguanosine, Thienoguanosine, 2-Aminopurine-riboside, 8-Oxoguanosine, 5-Carboxymethylesteruridine, Thienouridine, 5-Methoxyuridine, 5-Carboxyuridine, 2- Thiouridine, N1 -Propylpseudouridine, N1 -Methoxymethylpseudouridine, Nl- Ethylpseudouridine, Pseudouridine, or N1 -Methylpseudouridine.

[0536]

[0550] In some embodiments of any of the aspects, an saRNA as described herein comprises less than 25% modified nucleotides. In some embodiments of any of the aspects, an saRNA as described herein comprises 0%, at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 11%, at most 12%, at most 13%, at most 14%, at most 15%, at most 16%, at most 17%, at most 18%, at most 19%, at most 20%, at most 21%, at most 22%, at most 23%, at most 24%, at most 25% modified nucleotides. In some embodiments of any of the aspects, an saRNA as described herein does not comprise any modified nucleotides.

[0537]

[0551] In some embodiments of any of the aspects, the modified saRNA as described herein is substituted using the template of SEQ ID NO: 1 or a nucleic acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least at least about 98%, at least about 99%, or 100%, identical to SEQ ID NO: 1 that maintains the same function (e.g., self-replication), or a codon-optimized version thereof. In some embodiments of any of the aspects, at least one cargo is inserted between the Aflll and Ndel cut sites of SEQ ID NO: 1. In some embodiments of any of the aspects, at least one cargo is inserted between nucleotides 7627 and 7628 of SEQ ID NO: 1.

[0538]

[0552] In one embodiment, the modified saRNA as described herein saRNA is substituted using the template of SEQ ID NO: 5 or a nucleic acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least at least about 98%, at least about 99%, or 100%, identical to SEQ ID NO: 5 that maintains the same function (e.g., self-replication), or a codon-optimized version thereof.

[0539]

[0553] In some embodiments of any of the aspects, the pyrimidine comprises cytidine, and the modified nucleotide comprises 5-methylcytidine, 5-fluorocytidine, 5-bromocytidine, 5- iodocytidine, 5-azidocytidine, 5-trifluoromethylcytidine, 5-difluoromethylcytidine, 5- hydroxymethylcytidine, 5-ethylcytidine, 5-propylcytidine, 5-nitrocytidine, 5- difluorohydroxymethylcytidine, 5-monofluorohydroxymethylcytidine, 5- hydroxyethylcytidine, and / or 5-hydroxypropylcytidine, and the substituted saRNA comprises SEQ ID NO: 6 or a nucleic acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least at least about 98%, at least about 99%, or 100%, identical to SEQ ID NO: 6 that maintains the same function (e.g., self-replication), or a codon-optimized version thereof.

[0540]

[0554] In some embodiments of any of the aspects, the pyrimidine comprises uridine, and the modified nucleotide comprises 5-methyluridine, 5-fluorouridine, 5 -bromouridine, 5- iodouridine, 5-azidouridine, 5-trifluoromethyluridine, 5-difluoromethyluridine, 5- hydroxymethyluridine, 5-ethyluridine, 5-propyluridine, 5-nitrouridine, 5- difluorohydroxymethyluridine, 5-monofluorohydroxymethyluridine, 5-hydroxy ethyluridine, and / or 5-hydroxypropyluridine, and the substituted saRNA comprises SEQ ID NO: 7 or a nucleic acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least at least about 98%, at least about 99%, or 100%, identical to SEQ ID NO: 7 that maintains the same function (e.g., selfreplication), or a codon-optimized version thereof.

[0541]

[0555] In one embodiment, the modified saRNA is provided wherein nucleotides 7634 to 8347 of SEQ ID NO: 5, nucleotides 7617 to 8330 of SEQ ID NO: 6, or nucleotides 7617 to 8330 of SEQ ID NO: 7, each corresponding to mCherry, is replaced with at least one alternative cargo of interest. In one aspect, described herein is a self-amplifying RNA (saRNA) comprising: (a) at least one non-structural protein derived from at least one virus; (b) a subgenomic promoter (SGP) derived from at least one virus; and (c) at least one cargo of interest; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-fluorocytidine, 5-bromocytidine, 5-iodocytidine, 5- azidocytidine, 5-trifluoromethylcytidine, 5 -difluoromethylcytidine, 5-fluorouridine, 5- bromouridine, 5-iodouridine, 5-azidouridine, 5-trifluoromethyluridine, 5- difluoromethyluridine, 5 -methyluridine, 5-hydroxymethyluridine, 5-hydroxymethylcytidine, 5-ethyluridine, 5-ethylcytidine, 5 -propyluridine, 5-propylcytidine, 5-nitrouridine, 5- nitrocytidine, 5-difluorohydroxymethyluridine, 5-difluorohydroxymethylcytidine, 5- monofluorohydroxymethyluridine, 5-monofluorohydroxymethylcytidine, 5- hydroxyethylcytidine, 5-hydroxyethyluridine, 5-hydroxypropylcytidine, and / or 5- hydroxypropyluridine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2'0 position of the ribose (Capl).

[0542]

[0556] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus; (c) a subgenomic promoter (SGP) derived from at least one virus; (d) a 5’ untranslated region (UTR) derived from at least one virus; (e) at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one virus; and (g) a poly -A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-fluorocytidine, 5-bromocytidine, 5-iodocytidine, 5-azidocytidine, 5- trifluoromethylcytidine, 5-difluoromethylcytidine, 5-fluorouridine, 5 -bromouridine, 5- iodouridine, 5-azidouridine, 5-trifluoromethyluridine, 5 -difluoromethyluridine, 5-methyluridine, 5-hydroxymethyluridine, 5-hydroxymethylcytidine, 5-ethyluridine, 5-ethylcytidine, 5- propyluridine, 5-propylcytidine, 5-nitrouridine, 5 -nitrocytidine, 5-difluorohydroxymethyluridine, 5-difluorohydroxymethylcytidine, 5-monofluorohydroxymethyluridine, 5- monofluorohydroxymethylcytidine, 5-hydroxyethylcytidine, 5-hydroxyethyluridine, 5- hydroxypropylcytidine, and / or 5-hydroxypropyluridine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2'0 position of the ribose (Capl).

[0557] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) at least one non-structural protein derived from at least one virus; (b) a subgenomic promoter (SGP) derived from at least one virus; and (c) at least one cargo of interest; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-fluorocytidine, 5 -bromocytidine, 5- iodocytidine, 5-azidocytidine, 5-trifluoromethylcytidine, 5-difhioromethylcytidine, 5- fluorouridine, 5-bromouridine, 5-iodouridine, 5-azidouridine, 5-trifluoromethyluridine, 5- difluorom ethyluridine, 5 -methyluridine, 5 -hydroxymethyluridine, 5 -hydroxymethyl cytidine, 5- ethyluridine, 5-ethylcytidine, 5-propyluridine, 5-propylcytidine, 5-nitrouridine, 5-nitrocytidine, 5-difluorohydroxymethyluridine, 5-difluorohydroxymethylcytidine, 5- monofluorohydroxymethyluridine, 5-monofluorohydroxymethylcytidine, 5- hydroxyethylcytidine, 5-hydroxyethyluridine, 5-hydroxypropylcytidine, and / or 5- hydroxypropyluridine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2'0 position of the ribose (Capl).

[0543]

[0558] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to

[0544] 3’: (a) a 5’ cap; (b) non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus; (c) a subgenomic promoter (SGP) derived from at least one virus; (d) a 5’ untranslated region (UTR) derived from at least one virus; (e) at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one virus; and (g) a poly -A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-fluorocytidine, 5-bromocytidine, 5-iodocytidine, 5-azidocytidine, 5- trifluoromethylcytidine, 5-difluoromethylcytidine, 5-fluorouridine, 5-bromouridine, 5- iodouridine, 5-azidouridine, 5-trifluoromethyluridine, 5 -difluoromethyluridine, 5-methyluridine, 5-hydroxymethyluridine, 5-hydroxymethylcytidine, 5-ethyluridine, 5-ethylcytidine, 5- propyluridine, 5-propylcytidine, 5-nitrouridine, 5-nitrocytidine, 5-difluorohydroxymethyluridine, 5-difluorohydroxymethylcytidine, 5-monofluorohydroxymethyluridine, 5- monofluorohydroxymethylcytidine, 5-hydroxyethylcytidine, 5-hydroxyethyluridine, 5- hydroxypropylcytidine, and / or 5-hydroxypropyluridine; wherein the initiating nucleotide comprises an adenosine or adenosine analog; and wherein the initiating nucleotide of the saRNA is methylated at the 2'0 position of the ribose (Capl).

[0545]

[0559] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising from 5’ to 3’: (a) a 5’ cap; (b) non- structural protein 1 (nspl), non-structural protein 2 (nsp2), non- structural protein 3 (nsp3), and / or non-structural protein 4 (nsp4), each derived from at least one virus, wherein the nsPl comprises at least one conserved 5' sequence element (5' CSE); (c) a subgenomic promoter (SGP) derived from at least one virus; (d) a 5’ untranslated region (UTR) derived from at least one virus; (e) at least one cargo of interest; (f) a 3’ untranslated region (UTR) derived from at least one virus, wherein the 3’ UTR comprises at least one 3'- conserved sequence element (3' CSE); and (g) a poly -A tail; wherein the saRNA comprises at least 25% modified nucleotides, wherein the modified nucleotides are selected from the group consisting of 5- methylcytidine, 5 -fluorocytidine, 5-bromocytidine, 5-iodocytidine, 5-azidocytidine, 5- trifluoromethylcytidine, 5-difluoromethylcytidine, 5-fluorouridine, 5 -bromouridine, 5- iodouridine, 5-azidouridine, 5-trifluoromethyluridine, 5 -difluoromethyluridine, 5-methyluridine, 5-hydroxymethyluridine, 5-hydroxymethylcytidine, 5-ethyluridine, 5-ethylcytidine, 5- propyluridine, 5-propylcytidine, 5 -nitro...

Claims

CLAIMSWhat is claimed is:

1. A self-amplifying RNA (saRNA) comprising a nucleotide sequence encoding (i) a mutated non- structural protein 2 (nsp2), wherein the mutation is D584N and encoded by a nucleotide substitution from GAC to AAC and (ii) at least one protein or fragment thereof, and wherein the saRNA is derived from VEEV.

2. The saRNA of claim 1, wherein the saRNA exhibits an increase in protein expression when introduced into a cell compared to a comparable VEEV saRNA lacking the D584N mutation.

3. The saRNA of claim 2, wherein the increase in expression is at least about 2-fold, about 3-fold, about 4-fold, or about 5-fold greater or more compared to a comparable saRNA lacking the mutation.

4. The saRNA of claims 1-3, wherein the saRNA exhibits protein expression that is specifically enhanced in immune cells compared to a comparable saRNA lacking the mutation when introduced in a subject.

5. The saRNA of claim 4, wherein the immune cell is selected from T cells, dendritic cells, macrophages, monocytes, B cells or a combination thereof.

6. The saRNA of claim 4-5, wherein the protein expression is specifically enhanced by greater than about 2-fold.

7. The saRNA of claims 4-6, wherein the saRNA enhances antigen-specific immune responses when introduced into a subject.

8. The saRNA of claims 1-7, wherein activation of the innate immune response is decreased compared to a comparable saRNA lacking the D584N mutation.

9. The saRNA claim 8, wherein the decrease is at least about 20%, at least about 60%, or at least about 80% or more.

10. The saRNA of claims 1-9, further comprising a chimeric 3’UTR sequence.

11. The saRNA of claim 10, wherein saRNA exhibits protein expression when introduced into a cell that is synergistically enhanced by the presence of the chimeric 3’UTR and the D584N mutation.

12. The saRNA of claims 1-11, wherein the at least one protein or fragment thereof is a selected from an antigen or therapeutic protein.

13. The saRNA of claim 12, wherein the sequence encoding the therapeutic protein is operably linked to a sequence encoding an Fc domain or an albumin-binding domain by a peptide linker.

14. The saRNA of claim 12, wherein the antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a cancer antigen or a cancer-associated antigen.

15. The saRNA of claims 1-13, wherein the at least one protein is a therapeutic protein selected from enzymes, antibodies, bi-specific antibodies, cytokines, transcription factors, receptors, or peptides.

16. The saRNA of claims 1-11, wherein the at least one protein or fragment thereof is a chimeric antigen receptor (CAR).

17. The saRNA of claims 1-14, wherein the saRNA comprises sequences encoding at least two protein sequences or fragment thereof.

18. The saRNA of claims 17, wherein at least one of the at least two protein sequences is an adaptive immune response inhibitor protein.

19. The saRNA of claim 18, wherein the increase in protein expression is synergistic in the presence of the mutation and the adaptive immune inhibitor protein and / or the 3’ UTR.

20. The saRNA of claim 17-19, wherein a 2A sequence or an IRES sequence operably links the nucleotide sequences encoding the at least two proteins or fragments thereof.

21. The saRNA of claims 1-20, wherein the nucleotide sequence comprises modified nucleotides.

22. The saRNA of claims 1-21, wherein the nucleotide sequence comprises greater than about 25% modified pyrimidines.

23. The saRNA of claims 18-22, wherein the increase in protein expression is synergistic in the presence of the mutation and the adaptive immune response inhibitor and / or the 3’ UTR and / or the modified nucleotides.

24. An saRNA comprising a nucleotide sequence encoding (i) a chimeric 3 ’UTR sequence; and (ii) at least one protein or fragment thereof.

25. The saRNA of claim 22, wherein the chimeric 3 ’UTR comprises sequences from at least two RNA viruses.

26. The saRNA of claim 20, wherein the RNA viruses are alphaviruses are selected from Venezuela Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV), Sindbis Virus (SIN), Chikungunya Virus (CHIKV), Eastern Equine Encephalitis Virus (EEEV), Mayaro Virus (MAYV), Getah Virus (GETV), Ross River Virus (RRV), Una Virus (UNAV),Middleburg Virus (MIDV), O'nyong nyong virus (ONNV), Barmah Forest Virus (BFV), Mucambo Virus (MUCV), Tonate Virus (TONV), Everglades Virus (EVEV), Rio Negro Virus (RNV), Turnip Rosette Virus (TROV), Highlands J Virus (HJV), Western Equine Encephalitis Virus (WEEV), Fig Mosaic Emaravirus (FMV), Aura Virus (AURAV), Kunjin Virus (KUN)27. The saRNA of claims 24-26, wherein the at least one protein or fragment thereof is an antigen or therapeutic protein.

28. The saRNA of claim 27, wherein the sequence encoding the therapeutic protein is operably linked to a sequence encoding an Fc domain or an albumin-binding domain by a peptide linker.

29. The saRNA of claim 24-27, wherein the at least one protein or fragment thereof is an antigen selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a cancer antigen or a cancer-associated antigen.

30. The saRNA of claims 24-28, wherein the at least one protein is a therapeutic protein selected from enzymes, antibodies, bi-specific antibodies cytokines, transcription factors, receptors, or peptides.

31. The saRNA of claims 24-26, wherein the at least one protein is a chimeric antigen receptor (CAR).

32. The saRNA of claims 24-31, wherein the saRNA reduces off-target immune activation compared to a comparable saRNA lacking the chimeric 3’ UTR.

33. The saRNA of claim 32, wherein the reduction is at least about 2-fold.

34. The saRNA of claims 24-33, wherein the saRNA enhances protein expression levels compared to a comparable saRNA lacking the chimeric 3’ UTR.

35. The saRNA of claims 24-34, wherein the saRNA exhibits altered or restricted cellular tropism compared to a comparable saRNA lacking the chimeric 3’ UTR.

36. The saRNA of claim 24-35, wherein the comparable saRNA comprises greater than about 25% modified nucleotides.

37. The saRNA of claims 24-36, wherein the saRNA comprises at least two protein sequences.

38. The saRNA of claim 37, wherein at least one of the protein sequences is an adaptive immune inhibitor protein.

39. The saRNA of claim 30, wherein the nucleotide sequence encoding the at least two proteins are operably linked by a 2A sequence or an IRES sequence.

40. The saRNA of claims 24-39, wherein expression of the at least one protein is increased by about 3-fold or greater compared to a comparable saRNA lacking a chimeric 3’ UTR sequence.

41. An saRNA comprising a nucleotide sequence encoding at least two proteins or fragments thereof, wherein one of the at least two proteins or fragments thereof is an adaptive immune response inhibitor protein.

42. The sRNA of claim 41, wherein the adaptive immune response inhibitor is a viral immune evasion protein.

43. The saRNA of claim 42, wherein the viral immune evasion protein is selected from BNLF2A or UL49.5.

44. The saRNA of claims 41-43, wherein activation of the innate immune response is decreased compared to a comparable saRNA lacking the adaptive immune response inhibitor protein when introduced into a subject.

45. The saRNA of claim 44, wherein the decrease is at least about 20%, at least about 60%, or at least about 80% or more.

46. The saRNA of claims 41-46, wherein the expression of the adaptive immune response inhibitor protein is transient and sufficient to inhibit the adaptive immune response during an initial expression period of the at least one protein or fragment thereof when introduced into a subject.

47. The saRNA of claims 41-46, wherein at least one of the at least two proteins or fragments thereof is an antigen or therapeutic protein.

48. The saRNA of claim 47, wherein the sequence encoding the antigen or therapeutic protein is operably linked to a sequence Fc domain or an albumin-binding domain by a peptide linker.

49. The saRNA of claims 47-48, wherein the adaptive immune response inhibitor protein transiently modulates the immune response to prolong antigen availability and / or reduce immunogenicity of the saRNA when introduced into a subject.

50. The saRNA of claims 1-49, wherein the nucleotide sequence comprises greater than about 25% modified pyrimidines selected from cytidine, uridine or a combination thereof.

51. The saRNA of claims 1-50, wherein the nucleotide sequence comprises between about 26% and about 100% modified pyrimidines.

52. The saRNA of claims 1-51, wherein the nucleotide sequence comprises between about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% modified pyrimidines.

53. The saRNA of claims 1-52, wherein the nucleotide sequence comprises greater than about 25% modified pyrimidines, wherein modified pyrimidines are selected from 5- fhiorocytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-methyluridine, 5-fluorouridine or combinations thereof.

54. The saRNA of claims 1-43, wherein the nucleotide sequence comprises between about 50% and 100% 5-fluorocytidine.

55. The saRNA of claim 54, wherein the nucleotide sequence comprises about 100% 5- flourocytidine.

56. A saRNA comprising a nucleotide sequence comprising greater than about 25% of cytidines substituted with 5-flourocytidine and encoding at least one protein or a fragment thereof.

57. The saRNA of claim 56, wherein the nucleotide sequence comprises between about 50% and about 100% of cytidines substituted with 5-flourocytidine.

58. The saRNA of claims 56-57, wherein the saRNA expresses the at least one protein or fragment thereof at a level equal to or greater than a comparable saRNA lacking 5- flourocytidine substitution or having a reduced percentage of substitution.

59. The saRNA of claims 56-58, wherein the saRNA reduces innate immune response activation compared to a comparable saRNA lacking 5-flourocytidine substitution or having a reduced percentage of substitution.

60. An saRNA comprising a nucleotide sequence encoding (i) at first nucleotide sequence encoding a first protein or fragment thereof; (ii) a second nucleotide sequence encoding a second protein of fragment thereof; and (ii) an IRES which operably links the first and second nucleotide sequence, and wherein the nucleotide sequence comprises greater than 25% modified pyrimidines.

61. The saRNA of claim 60, wherein the saRNA further comprises (iii) a third nucleotide sequence encoding a third protein or fragment thereof and (iv) a second IRES sequence operably linking the second nucleotide sequence to the third nucleotide sequence.

62. The saRNA of claim 60, wherein the saRNA further comprises (v) a fourth nucleotide sequence encoding a fourth protein or fragment thereof and (vi) a third IRES sequence operably linking the third nucleotide sequence to the fourth nucleotide sequence.

63. The saRNA of claims 60-62, wherein the nucleotide sequence comprises between 26% and 100% modified pyrimidines.

64. The saRNA of claims 60-63, wherein the nucleotide sequence comprises between about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% modified pyrimidines.

65. The saRNA of claims 60-64, wherein the nucleotide sequence comprises greater than about 25% modified pyrimidines selected from 5-fluorocytidine, 5-methylcytidine, 5- hydroxymethylcytidine, 5-methyluridine, 5-fluorouridine or combinations thereof.

66. The saRNA of claims 60-65, wherein the nucleotide sequence comprises between about 50% and about 100% 5-fluorocytidine.

67. The saRNA of claim 66, wherein the nucleotide sequence comprises about 100% 5- flourocytidine.

68. The saRNA of claims 60-67, wherein expression of the protein downstream of the IRES sequence is enhanced relative to a comparable saRNA having about 25% or less modified pyrimidines.

69. The saRNA of claim 68, wherein expression of the downstream protein is enhanced by least about 2-fold, about 3-fold, about 4-fold, or about 5-fold greater or more compared to a comparable saRNA having about 25% or less modified pyrimidines.

70. The saRNA of claims 60-69, wherein at least one of the at least two proteins is an adaptive immune response inhibitor protein.

71. The saRNA of claim 55 or 70, wherein the adaptive immune response inhibitor protein is a viral immune evasion protein.

72. The saRNA of claim 71, wherein the viral immune evasion protein is selected from BNLF2A or UL49.5.

73. The saRNA of claims 60-72, wherein at least one of the at least two proteins or fragments thereof is an antigen.

74. The saRNA of claim 61, wherein at least two of the at least three proteins or fragments thereof is an antigen.

75. The saRNA of claim 62, wherein at least three of the at least three proteins or fragments thereof is an antigen.

76. The saRNA of claim 73-75, wherein the antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a cancer antigen or a cancer-associated antigen.

77. The saRNA of claims 60-72, wherein at least one of the at least two proteins is a therapeutic protein.

78. The saRNA of claim 77, wherein the therapeutic protein is selected from the group consisting of therapeutic proteins include enzymes, antibodies, cytokines, transcription factors, receptors, or peptides.

79. The sRNA of claims 60-72, at least one of the at least two proteins is a chimeric antigen receptor (CAR).

80. The saRNA of claim 20, 39 or 60-72, wherein the IRES sequence(s) is derived from one or more of the following: encephalomyocarditis virus (EMCV), hepatitis C virus (HCV), cricket paralysis virus (CrPV), poliovirus (PV), foot-and-mouth disease virus (FMDV), Theiler's murine encephalomyelitis virus (TMEV), human rhinovirus (HRV), Coxsackievirus B, enterovirus 71 (EV71), hepatitis A virus (HAV), classical swine fever virus (CSFV), Drosophila C virus (DCV), or murine leukemia virus (MLV), or combinations thereof.

81. The saRNA of claims 1-80, comprising a unique barcode sequence inserted into the 5’ or 3’ untranslated region (UTR) of the saRNA.

82. A lipid nanoparticle encapsulating the saRNA of claims 1-81.

83. The lipid nanoparticle of claim 82, wherein the lipid nanoparticle comprises an ionizable lipid and a helper lipid.

84. The lipid nanoparticle of claim 83, wherein the helper lipid is cholesterol, a polyethylene glycol (PEG)-modified lipid or a combination thereof.

85. The lipid nanoparticle of claim 82-84, further comprising a targeting moiety.

86. A cell comprising the lipid nanoparticle of claims 82-84.

87. The cell of claim 86, wherein the cell is in vitro.

88. The cell of claim 86, wherein the cell is in vivo.

89. The cell of claims 86-88, wherein the cell is a human cell.

90. A pharmaceutical composition comprising (i) a pharmaceutically acceptable excipient and (ii) the lipid nanoparticle of claims 82-85.

91. A method of increasing expressing at least one protein, comprising administering an saRNA to a cell, wherein the saRNA comprises a nucleotide sequence encoding one or more features selected from (i) a D584N mutation in non- structural protein 2 (NSP2), (ii) a chimeric 3’ UTR, (ii) at least two proteins or fragments thereof, wherein at least one protein is an adaptive immune response protein, (iv) at least two or more proteins operably linked by an IRES sequence, wherein greater than about 25% of the nucleotide sequence comprises modified pyrimidines, wherein administration results in increased expression of at least one protein relative to an saRNA lacking one or more of the features.

92. The method of claim 91, wherein the cell is in vitro.

93. The method of claim 91, wherein the cell is in vivo.

94. The method of claim 91-93, wherein the cell is a human cell.

95. A method of enhancing serum protein levels, comprising administering the saRNA of claims 13, 28 or 48 to a subject, wherein administration results in enhanced serum protein levels relative to a comparable saRNA encoding an antigen or protein lacking an Fc or albumin domain.

96. The method of claim 95, wherein the administration is local, and expression is increased by about at least fivefold compared to a comparable saRNA encoding an antigen protein lacking the Fc-domain or albumin domain.

97. The method of claim 80, wherein the administration is following intramuscular (IM) or subcutaneous (SC) injection.

98. The method of claim 82, wherein the serum protein levels following IM or SC are increased by at least about 2-fold, about 5-fold, about 10-fold, about 20-fold, or about 100- fold.

99. A method of reducing immunogenicity of a saRNA, comprising administering the saRNA of claims 1-81 to a subject, wherein administration results in reduced immunogenicity.

100. The method of claim 84, wherein immunogenicity is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold, or about 5-fold or about 10-fold or more.

101. A method of reducing an adaptive immune response to a saRNA, comprising administering the saRNA of claims 1-81 to a subject, wherein administration results in a reduced adaptive immune response.

102. The method of claim 101, wherein the adaptive immune response is reduced by about is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 2-fold, about 3-fold, about 4-fold, or about 5-fold or about 10-fold or more.

103. A method of treating a disease or disorder, comprising administering an saRNA to a cell, wherein the saRNA comprises a nucleotide sequence encoding one or more features selected from (i) a D584N mutation in non- structural protein 2 (NSP2), (ii) a chimeric 3’ UTR, (ii) at least two proteins or fragments thereof, wherein at least one protein is an adaptive immune response protein, (iv) at least two or more proteins operably linked by anIRES sequence, wherein greater than about 25% of the nucleotide sequence comprises modified pyrimidines, wherein administration results treatment of the disease or disorder.

104. The method of claim 103, wherein the disease or disorder is an infectious disease or disorder.

105. The method of claim 104, wherein the infectious disease or disorder is a viral disorder.

106. The method of claim 105, wherein the disease or disorder is cancer, a cardiovascular disorder, a neurological disorder, an autoimmune disorder, a metabolic disorder or a respiratory disorder.

107. A method of manufacturing a saRNA comprising: (i) preparing a linearized plasmid DNA template; (ii) performing in vitro transcription using a mutant T7 RNA polymerase that generates reduced double-stranded RNA (dsRNA) impurities; and (iii) purifying the saRNA, wherein reduced dsRNA impurities result in simplified downstream purification and decreased innate immune responses when administered to a subject and simplified downstream purification.

108. The method of claim 107, wherein the mutant T7 RNA polymerase reduces dsRNA impurities by at least 50% compared to wild-type T7 RNA polymerase.

109. The method of claims 107-108, wherein the saRNA comprises greater than about 25% modified nucleotides selected from 5-hydroxymethylcytidine, 5-methylcytidine, 5- methyluridine, 5-fluorocytidine, 5-fluorouridine or combinations thereof.

110. A method of high-throughput screening lipid nanoparticle (LNP) formulations comprising: (i) encapsulating multiple barcoded saRNAs into different LNP formulations, wherein each saRNA s associated with a unique barcode; (ii) administering the LNP formulations to a biological model; (iii) isolating saRNA from target tissues or cells; and (iv) quantifying barcode abundance by next-generation sequencing (NGS) to identify LNP formulations with optimized functional saRNA delivery.

111. The method of claim 110, further comprising (v) screening different modified nucleotides within the same LNP formulation or screening combinations of different LNP formulations and modified nucleotides to identify optimized formulations.

112. The method of claim 111, wherein the optimized formulations exhibit one or more properties selected from increased cellular uptake, endosomal escape, or translation efficiency relative to non-optimized formulations.