Self-replicating RNA and uses thereof

Abstract

The technology described herein relates to compositions and methods for modifying and controlling cellular activity by expression of proteins from self-amplifying RNA (saRNA). Also described herein are compositions and methods for modifying and controlling cellular activity by expressing proteins from self-amplifying RNAs substituted with chemically modified nucleotides.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 538,540, filed on September 15, 2023, U.S. Provisional Application No. 63 / 460,506, filed on April 19, 2023, and U.S. Provisional Application No. 63 / 426,597, filed on November 18, 2022, the contents of each of which are incorporated herein by reference in their entirety. Government support

[0002] This invention was made with government support under Grant No. R01AR079489 and Contract No. CA265713-02 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Sequence Listing

[0003] This application contains a sequence listing, which has been submitted in XML format through the Patent Center and is incorporated herein by reference in its entirety. The XML copy was created on November 15, 2023 and is named 701586-000105WOPT_USPT_SL.xml, which is 74,643 bytes in size. Technical Field

[0004] The technology described herein relates to methods and compositions for modifying and controlling cellular activities through self-replicating RNA.

[0005] The technology described herein relates to methods and compositions for the use of self-amplifying RNA that is highly or fully substituted with chemically modified nucleotides. Background Art

[0006] Adoptive cell therapy represents a promising strategy for treating a variety of cancers. Currently, there are five approved adoptive cell therapies for the treatment of leukemia, lymphoma, and multiple myeloma. In addition, the application of adoptive cell therapy is expanding beyond oncology. Although the clinical results of approved therapies are encouraging, there are still many challenges associated with in vitro cell engineering. Cell isolation, genetic engineering, and expansion protocols are costly and time-consuming. In addition, there is a risk of off-target genome editing in cell gene modification. An attractive alternative is to reprogram cells in vivo or ex vivo with messenger RNA (mRNA). In the field of oncology, the use of mRNA-programmed immune cells has shown efficacy in a limited number of cancer models. However, some challenges have been reported in terms of expression levels and duration of expression. Therefore, repeated administration is usually required to achieve functional benefits. In contrast, self-replicating RNA can be used to express therapeutic programs at higher levels and for longer periods of time. In addition, self-replicating RNA systems can be used for ex vivo or in vivo modification of cells.

[0007] In order to ensure the safety of engineered cell therapy and improve its activity, it is desirable to control. Control is usually achieved by including components that can be externally controlled to turn on or off activity. A common example of control in cell therapy is a kill switch that can be included in engineered cells. If engineered cells pose a risk, they can be killed by injecting small molecules that activate the kill switch present in the engineered cells. There are other strategies to control the activity of engineered cells by utilizing engineered proteins with external control behavior. In the context of oncology, there are a series of chimeric antigen receptors regulated by drugs. The resulting activity of CAR-T cells manufactured with drug-controlled receptors depends on the dose of the control molecule administered. Another strategy for controlling cell activity is to utilize logical calculations. By including components that interact with each other, a circuit that can sense multiple signals and perform logical responses can be created. This enables the creation of a therapy with enhanced specificity by requiring a combination of input signals to be satisfied before therapeutic activity is performed. The complexity of such logical calculation circuits is generally limited by the size capacity of genetic engineering methods. It is highly desirable to develop a method for modifying cells with controllable activity in a fast, scar-free manner using self-replicating RNA.

[0008] In addition, Karikó and Weissman initially discovered how modified nucleotides enable mRNA to evade attack by the immune system, paving the way for the field of mRNA therapy (Karikó, K. Immunity, 2005. PMID: 16111635) (Karikó, K. Molecular Therapy, 2008. PMID: 18797453). mRNA containing chemically modified nucleotides has significantly less immunogenicity (Karikó, K. Molecular Therapy, 2008. PMID: 18797453) (Kormann, M. Nature Biotechnology, 2011. PMID: 21217696) (Karikó, K. Immunity, 2005. PMID: 16111635). Currently, the clinical gold standard for mRNA therapy is to completely replace uridine with N1-methylpseudouridine (m1ψ), which has the greatest impact on suppressing the type I interferon (IFN) response traditionally induced by dsRNA and ssRNA (Karikó, K. Immunity, 2005. PMID: 16111635). At the molecular level, modified nucleotides change the stability or accessibility of specific base pairs, alter hydrogen bonding patterns, and transform RNA hydrophobicity. Along with altering RNA stability, some of these new interactions also change the primary or secondary structure of RNA, stabilizing or inhibiting RNA-protein interactions (Kierzek, E. Nature Communications, 2022. PMID: 35277476) (Harcourt, E. Nature, 2017. PMID: 28102265) (Davis, D. Nucleic Acids Research, 1995. PMID: 8559660). In part through these mechanisms, modified nucleotides confer immune evasion properties to RNA. However, these same RNA-RNA and RNA-protein interactions are crucial for self-amplifying RNA (saRNA) function.

[0009] saRNA is a kind of RNA with the ability of in situ replication and amplification itself.This is achieved by encoding RNA-dependent RNA polymerase (RdRp) and protein of interest (Bloom, K.Gene Therapy, 2021.PMID: 33093657). Compared with non-replicative mRNA (nrRNA), some advantages of saRNA include increasing the effectiveness of each μg RNA and prolonging the action time (Minnaert, K.Advanced Drug Delivery Reviews, 2021.PMID: 34324884) (Geall, A.Expert Opinion on Drug Discovery, 2022.PMID: 36384351). The RdRp encoding saRNA recognizes the conserved secondary structure and sequence and subgenomic promoter (SGP) referred to as conserved sequence elements, which enable transcription of both negative and positive strand saRNA and the mRNA encoding goods of interest. The current understanding in the saRNA field is that the incorporation of modified nucleotides into saRNA leads to inactivation of replicase activity and elimination of downstream efficacy (Geall, A. Expert Opinion on Drug Discovery, 2022. PMID: 36384351) (Voigt, E. NPJ vaccines, 2022. PMID: 36323666) (Novartis AG, 12 / 831,252) (Kairuz, D. Frontiers in Immunology, 2022. PMID: 36353641) (Minnaert, K. Advanced Drug Delivery Reviews, 2021. PMID: 34324884). These studies teach that the incorporation of modified nucleotides at > 25% substitutions does not produce enough antigen to achieve therapeutically effective saRNA.

[0010] Since 2015, there have been 10+ clinical trials using saRNA, and although preclinical evidence has prospects, human data show that after strengthening, lower serum conversion, lower neutralizing antibody levels and reduced antibody production compared with non-replicating mRNA (Geall, A.Expert Opinion on Drug Discovery, 2022.PMID: 36384351). The reason for this hypothesis is that saRNA containing unmodified nucleotides strongly activates the innate immune response in the early stage, which hinders saRNA replication and launching (launch) goods from saRNA SGP. In knockout mice lacking type I IFN α and β receptor subunit 1, saRNA leads to higher IgG specific antibody titers and serum conversion compared with wild-type mice (Pepini, T.TheJournal of Immunology, 2017.PMID: 28416600) (Zhong, Z.Nano Today, 2018.DOI: 10.1016 / J.NANTOD.2018.10.005). Recently, corticosteroid immunosuppression has been explored as a combination therapy to minimize the innate immune response, but ultimately suppressed seroconversion. The same study did show that removing dsRNA contaminants that activate TLR3 and stimulate type I IFN expression can increase the immunogenicity of the vaccine (Zhong, Z. Molecular Therapy, 2021. PMID: 33484964). The fact that IM is the best delivery route for saRNA further supports the inhibition of saRNA by type I IFN, as intradermal injection has been shown to induce a higher type I IFN response than IM (Zhong, Z. Vaccines, 2019. PMID: 31450775). In summary, previous studies have shown that if the early interferon response can be overcome, the efficacy of saRNA is likely to be substantially improved. Part of the reason why saRNA is so effective as a vaccine candidate is that it itself acts as an adjuvant. However, in order to strike a balance between the adjuvant effect and the interferon-mediated suppression initiated by saRNA, it is crucial to control the early interferon response (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 initial expression, saRNA may still be difficult to play a role in clinical practice.

[0011] Although considerable effort has been devoted to developing self-amplifying RNAs as therapeutic agents, there remains a need to improve the stability and efficacy of self-amplifying RNAs to achieve this goal. Summary of the Invention

[0012] The technology described herein relates to a self-replicating RNA system. In particular, methods and compositions for modifying cells using self-replicating RNA and introducing elements that can be externally controlled or set up for logical computational control are described herein. Self-replicating RNA comprises sequences derived from RNA viruses and additional cargo sequences. External control is established by utilizing cargo protein domains that respond to external inputs (e.g., small molecules, light, proteins). Logical computational control is established by comprising components that sense external or internal inputs and interact directly or indirectly with each other to perform logical operations.

[0013] This paper describes a general framework for modifying cells with self-replicating RNA to impart controllable behavior: (1) cargo proteins are expressed in cells; (2) cellular behavior is externally controlled by administering external factors that increase or decrease cargo protein activity; and (3) cellular behavior is internally controlled by logical computation circuits that sense inputs and perform logical computations.

[0014] In some embodiments, a sequence derived from a viral element capable of replicating and generating additional RNA is utilized to create self-replicating RNA. Exemplary viruses include alphaviruses, flaviviruses, measles viruses, and rhabdoviruses. In a specific embodiment, a sequence derived from the following is utilized to create self-replicating RNA: Venezuelan 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 a specific embodiment, in addition to comprising the coding sequence of proteins nsp1, nsp2, nsp3, and nsp4, replication can also be achieved by comprising conserved non-coding sequence elements at the 5' and 3' ends of the RNA chain. The expression of any genetically encodable protein can be achieved by placing the coding sequence after a subgenomic promoter sequence or an internal ribosome entry site (IRES) sequence.

[0015] In one aspect, described herein are self-amplifying RNAs (saRNAs) comprising, from 5' to 3': (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural 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.

[0016] In one embodiment of any aspect herein, the self-replicating RNA is generated by in vitro transcription. In other embodiments, the self-replicating RNA is delivered to the cell as a plasmid DNA that is transcribed into RNA and comprises the sequences required for replication.

[0017] In one embodiment of any aspect herein, the RNA comprises a 5' cap structure.

[0018] In one embodiment of any aspect herein, the RNA comprises a 3' poly-A tail.

[0019] In one embodiment of any aspect herein, the RNA comprises a 5'UTR, a 3'UTR, or a combination of both.

[0020] In one embodiment of any aspect herein, inclusion of sequence elements from the VEEV virus results in enhanced expression of a functional protein for a longer duration than conventional mRNA.

[0021] In one embodiment of any aspect herein, the functional protein is expressed by a self-replicating RNA comprising a protein coding sequence downstream of a subgenomic promoter.

[0022] In one embodiment of any aspect herein, the functional protein is expressed by a self-replicating RNA comprising a protein coding sequence downstream of an internal ribosome entry site (IRES) sequence.

[0023] In one embodiment of any aspect herein, the functional protein is a chimeric antigen receptor.

[0024] In one embodiment of any aspect herein, the functional protein is a chimeric antigen receptor having a protease domain that is responsive to a protease inhibitor.

[0025] In one embodiment of any aspect herein, the functional protein is a chimeric antigen receptor having a domain that interacts with a small molecule. In the presence of the small molecule, the stability of the receptor is regulated by known and unknown mechanisms.

[0026] Another aspect details a method wherein the activity of the functional protein is dependent on the concentration of the administered small molecule.

[0027] Another aspect details a method wherein the activity of the functional protein is dependent on the concentration of a plurality of administered small molecules.

[0028] In one embodiment of any aspect herein, the functional protein is a chimeric antigen receptor having a domain that interacts with a protein. In the presence of a protein, the stability of the receptor is regulated by known and unknown mechanisms.

[0029] Another aspect details a method wherein the activity of the functional protein is dependent on the concentration of the administered protein.

[0030] Another aspect details a method wherein the activity of the functional protein is dependent upon the concentration of multiple administered proteins.

[0031] In one embodiment of any aspect herein, the functional protein is expressed at a level equal to or greater than that obtained by other methods for establishing constitutive expression of proteins.

[0032] In one embodiment of any of the aspects herein, multiple proteins are expressed from the same self-replicating RNA strand by including an IRES sequence between the coding sequences for each protein.

[0033] In one embodiment of any of the aspects herein, multiple proteins are expressed from the same self-replicating RNA strand by including a 2A sequence between the coding sequences for each protein.

[0034] In one embodiment of any aspect herein, the expression levels of the multiple proteins expressed from the same self-replicating RNA are equal to or greater than expression levels obtained by other methods for establishing constitutive expression of the multiple proteins.

[0035] In one embodiment of any aspect herein, multiple chimeric antigen receptors with opposing functions are expressed from the same self-replicating RNA strand.

[0036] Another aspect details a method in which the activity of a resulting cell is dependent on an external signal sensed by a plurality of proteins having different functions. The resulting activity performed by the cell is dependent on the presence or absence of the sensed external signal. In some embodiments, a small molecule is administered as the external signal. Non-limiting examples of other external signals that can be used to control activity include light, ultrasound, proteins, ligands, antibodies, antibody fragments, and the like.

[0037] In one embodiment of any aspect herein, a functional protein having expression-enhancing ability by known or unknown mechanisms is co-expressed, the functional protein being used to enhance expression levels and duration.

[0038] In one embodiment of any aspect herein, the self-replicating RNA is delivered to the cell by electroporation.

[0039] In one embodiment of any aspect herein, the self-replicating RNA is delivered to the cell by nucleofection.

[0040] In one embodiment of any aspect herein, the RNA is delivered to the cell via lipid nanoparticles.

[0041] In one embodiment of any aspect herein, the RNA is delivered to the cell via nanoparticles.

[0042] In one embodiment of any of the aspects herein, RNA is delivered to a specific cell type via lipid nanoparticles containing antibodies on the surface of the nanoparticles.

[0043] In one embodiment of any aspect herein, the function of the chimeric antigen receptor is to activate an immune cell.

[0044] In one embodiment of any of the aspects herein, the chimeric antigen receptor functions to inhibit immune cells.

[0045] In one embodiment of any aspect herein, a protein reporter is co-expressed with the cargo protein to measure the expression level caused by the self-replicating RNA.

[0046] In one embodiment of any aspect herein, multiple cell types are modified with the self-replicating RNA.

[0047] In one embodiment of any aspect herein, one or more cell types are modified with a self-replicating RNA.

[0048] In one embodiment of any of the aspects herein, one or more cell types are modified with the self-replicating RNA prior to administration to a patient.

[0049] In one embodiment of any aspect herein, one or more cell types are modified with the self-replicating RNA while the cells are in a patient.

[0050] In one embodiment of any aspect herein, one or more cell types modified with self-replicating RNA are administered to a patient intraosseously (IO), intraperitoneally (IP), subcutaneously (SC), intravenously (IV), intramuscularly (IM), and intraarticularly, or by inhalation or topically.

[0051] Previous studies have shown that the level of substitution of modified nucleotides for SaRNA should be less than 25%, otherwise the expression of the transgene encoded by the SaRNA will be reduced relative to a comparable SaRNA without such modifications (see, for example, Voigt, E. NP Jvaccines, 7. (2022) (USSN 12 / 831,252) (WO2011005799) (US10532067B2), (US11291682B2), (US20220054525A1), (US10487332B2), (US20200048636A1), (US20220056449A1), (EP3964584A1), (WO20120063 76), (US20220192997A1), (US11058762B2), (US20210290755A1), (US20140242152), (US20220313815A1), (US20210347828A1), (WO2022137128A2), (US20140271829A1). For example, USSN 12 / 831,252 teaches that incorporation of 0.01%-25% modified nucleotides is the optimal substitution rate for maintaining expression of transgenic cargo (reporter construct or vaccine antigen). At higher substitution rates, expression of transgenic cargo decreases.

[0052] The present invention is based in part on the discovery that certain modified nucleotides can be incorporated into saRNA at levels greater than 25%, including greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%-100%, or even 100%, and the resulting saRNA can still express the cargo of interest encoded on the saRNA. Moreover, in some cases, the expression level of the transgene encoded by the saRNA can even be higher than that of a comparable saRNA that does not contain such modifications. These specific modifications allow for the maintenance of the self-amplification function of the saRNA and the maintenance of expression of the cargo protein encoded by one or more cargos.

[0053] This paper describes a general framework for expressing cargo from self-amplifying RNA that contains modified nucleotides with greater than 25% substitutions and maintains or improves expression capacity compared to unmodified saRNA. This enables the expression of vaccine antigens, protein replacement therapies, antibodies, enzymes, or the modification of cells with controllable behavior through modified self-replicating RNA: (1) expression of cargo proteins in cells; (2) external control of cell behavior by administering external factors that increase or decrease cargo protein activity; (3) internal control of cell behavior by logical computation circuits that sense inputs and perform logical computations.

[0054] Disclosed herein are self-amplifying RNAs containing greater than 25% of a given nucleotide replaced with modified nucleotides. These highly substituted saRNAs not only maintain saRNA functionality but also confer inhibition of innate immune activation. Furthermore, these highly substituted saRNAs are more potent, improving cargo expression and transfection efficiency. The saRNAs described herein have the potential to unlock the next frontier in RNA therapeutics, which has been hampered by the inherent immunogenicity of the constructs; this work provides a blueprint for modifying and enhancing saRNA therapeutics as vaccines, long-acting cell therapies, protein replacement therapies, and any other envisioned implementation of RNA therapeutics.

[0055] In some embodiments, a sequence derived from a viral element capable of replicating and generating additional RNA is utilized to create self-replicating RNA. Exemplary viruses include alphaviruses, flaviviruses, measles viruses, coronaviruses, and rhabdoviruses. In a specific embodiment, a sequence derived from the following is utilized to create self-replicating RNA: Venezuelan 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 a specific embodiment, in addition to the coding sequence comprising proteins nsp1, nsp2, nsp3, and nsp4, replication can also be achieved by including conserved non-coding sequence elements at the 5' and 3' ends of the RNA chain. The expression of any genetically encodable protein can be achieved by placing the coding sequence after a subgenomic promoter sequence or an internal ribosome entry site (IRES) sequence. In addition, non-coding RNA can be transcribed by placing a microRNA or siRNA sequence after a subgenomic promoter.

[0056] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising: at least 25% modified nucleotides and at least one cargo of interest, wherein the modified nucleotides comprise a pyrimidine nucleoside phosphate having a moiety at the 5-carbon of the pyrimidine, wherein the moiety is selected from the group consisting of methyl, ethyl, propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, and hydroxypropyl functional groups.

[0057] In some embodiments of any of the aspects, the modified nucleotides comprise 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, 5-hydroxymethylcytidine, or a combination thereof.

[0058] In some embodiments of any of the aspects, the saRNA expresses the cargo at a level greater than or equal to that of a corresponding saRNA having less than 25% modified nucleotides.

[0059] In one embodiment of any aspect herein, the self-amplifying RNA comprises nucleotides, wherein one or more unmodified nucleotides including cytidine, adenosine, uridine and guanosine are replaced by a percentage of modified analogs of the corresponding bases, wherein the percentage is at least 25%, e.g., 25%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100% or 90%-100%, 25%-95%, 30%-95%, 40%-95%, 50%-100%, 60%-100%, 70%-100%, 80%-100% or 90%-100%, 25%-95%, 30%-95%, 40%-95%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, or 90%-100%. 0%-95%, 70%-95%, 80%-95%, 25%-90%, 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-90% or 80%-90%, 25%-30%; 30%-35%; 40%-45%; 45%-50%; 50%-55%; 55%-60%; 60%-65%; 65%-70%; 70%-75%; 75%-80%; 80%-85%, 85%-90%, 90%-95%, 95%-99%, 99%%-100% or 100%. In some embodiments of any aspect, the substitution level of modified nucleotides is: 25%-50%, 51%-75%, 75%-99%, 99.1%-99.9%, or 100%.

[0060] In one embodiment of any of the aspects herein, the self-amplifying RNA (saRNA) encodes a cargo of interest, wherein the saRNA comprises a substitution level of modified nucleotides for a given nucleotide of greater than 25%, wherein the modified nucleotide is selected from the group consisting of 5-methylcytidine, 5-methyluridine, and 5-hydroxymethylcytidine, and optionally, the saRNA expresses the cargo at a level greater than or equal to that of the same saRNA without the modified nucleotides.

[0061] In one embodiment of any of the aspects herein, the self-amplifying RNA (saRNA) encodes a cargo of interest, wherein the saRNA comprises a substitution level of greater than 25% of modified nucleotides for a given nucleotide, wherein the modified nucleotide is selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine, and optionally, the saRNA expresses the cargo at a level greater than or equal to that of the same saRNA without the modified nucleotides.

[0062] In one embodiment of any of the aspects herein, the self-amplifying RNA (saRNA) encodes a cargo of interest, wherein the saRNA comprises a substitution level of modified nucleotides for a given nucleotide greater than 25%, wherein the modified nucleotide comprises a pyrimidine nucleoside triphosphate having a moiety at the 5-carbon, wherein the moiety is selected from the list comprising a methyl, ethyl, propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, or hydroxypropyl functional group, and optionally, the saRNA expresses the cargo at a level greater than or equal to that of the same saRNA without the modified nucleotide.

[0063] In one embodiment of any of the aspects herein, the substitutions with one or both of 5-methylcytidine and 5-hydroxymethylcytidine, and 5-methyluridine occur in the same saRNA molecule.

[0064] In one embodiment of any of the aspects herein, the substitutions with one or both of 5-methylcytidine and 5-hydroxymethylcytidine, and 5-methyluridine or 5-hydroxymethyluridine occur in the same saRNA molecule.

[0065] In one embodiment of any aspect herein, the initial nucleotide (the nucleotide immediately adjacent to the 5' cap) is adenosine or an adenosine analog.

[0066] In one embodiment of any aspect herein, the initial nucleotide (the nucleotide immediately adjacent to the 5' cap) is guanosine or a guanosine analog.

[0067] In one embodiment of any of the aspects herein, the initial nucleotide and optionally the subsequent nucleotides of the saRNA are methylated at the 2'0 position of the ribose sugar.

[0068] In some embodiments of any of the aspects, the starting nucleotide comprises: (a) adenosine or an adenosine analog, and the starting nucleotide of the saRNA is methylated at the 2'O position of ribose (Cap 1); (b) adenosine or an adenosine analog, and the starting nucleotide of the saRNA and subsequent nucleotides are both methylated at the 2'O position of ribose (Cap 2); (c) guanosine or a guanosine analog, and the starting nucleotide of the saRNA is methylated at the 2'O position of ribose (Cap 1); or (d) guanosine or a guanosine analog, and the starting nucleotide of the saRNA and subsequent nucleotides are both methylated at the 2'O position of ribose (Cap 2).

[0069] In one embodiment of any of the aspects herein, the saRNA contains a component derived from Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), Sindbis virus (SIN), Kunjin virus (KUN), measles virus (MV), coronavirus (CoV), rabies virus (RABV), or vesicular stomatitis virus (VSV).

[0070] In some embodiments of any of the aspects, the saRNA comprises, from 5' to 3': (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural 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.

[0071] In some embodiments of any of the aspects, the saRNA further comprises at least one 5' conserved sequence element (CSE) and / or at least one 3' conserved sequence element (CSE) derived from at least one virus.

[0072] In some embodiments of any aspect, the at least one virus is an alphavirus.

[0073] In some embodiments of any aspect, the at least one virus is selected from the group consisting of Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), Sindbis virus (SIN), Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Mayaro virus (MAYV), Geta virus (GETV), Ross River virus (RRV), Una virus (UNAV), Middleburg virus (MIDV), O'nyong nyong virus (ONNV), Bama 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 virus (FMV), Aura virus (AURAV), Kunjin virus (KUN), Measles virus (MV), Coronavirus (CoV), Rabies virus (RABV) and Vesicular stomatitis virus (VSV).

[0074] In one embodiment of any of the aspects herein, the cargo of saRNA expression is one or more proteins of viral, bacterial, protozoan, mammalian, or plant origin.

[0075] In some embodiments of any aspect, the cargo comprises: a chimeric antigen receptor (CAR) comprising an extracellular domain that specifically binds to an antigen of interest; a ligand, a cell surface receptor, a transcription factor, a cytokine, a chemokine, an enzyme, and / or an antibody or fragment thereof; a bispecific T cell engager (BiTE); at least one non-coding RNA; at least one vaccine-associated antigen comprising at least one protein encoded by a viral genome; at least one transcription factor; at least one growth factor and / or cytokine; one or both of Pappalysin-A1 (PAPPA1) and Pappalysin-A2 (PAPPA2); at least one interleukin and / or a cognate receptor for an interleukin and / or a receptor subunit for an interleukin; at least one enzyme with antioxidant activity; and glucagon-like peptide-1 (GLP-1) or a fragment thereof.

[0076] In one embodiment of any of the aspects herein, the saRNA expresses a chimeric antigen receptor containing an extracellular domain that senses an incoming signal.

[0077] In one embodiment of any of the aspects herein, the saRNA expresses a ligand, cell surface receptor, transcription factor, cytokine, chemokine, enzyme, antibody, or other protein with biological activity.

[0078] In one embodiment of any of the aspects herein, the saRNA expresses a cargo of interest, said cargo comprising a domain that responds to an external input.

[0079] In one embodiment of any of the aspects herein, the saRNA transcripts are selected from one or more non-coding RNAs of the list comprising siRNA, shRNA, or microRNA.

[0080] In one embodiment of any aspect herein, the cargo of interest is one or more vaccine-associated antigens, wherein the vaccine-associated antigen is one or more proteins encoded on the genome of a virus selected from the list consisting of respiratory syncytial virus, hemagglutinin virus, human immunodeficiency virus, influenza virus, Zika virus, acute respiratory syndrome coronavirus 2, human papillomavirus, herpes virus, rotavirus, varicella virus (chicken pox), dengue virus, hepatitis A virus, hepatitis B virus, rubella virus, polio virus, or rabies virus.

[0081] In one embodiment of any aspect herein, the cargo encoded by the saRNA is one or more chimeric antigen receptors selected from the list comprising: CD19, CD22, CD30, b-cell maturation antigen (BCMA), disialoganglioside GD2, human estrogen receptor 2 (HER2), GPR87, fibroblast activation protein (FAP), CD20, receptor tyrosine kinase-like orphan receptor 1 (ROR1), carcinoembryonic antigen (CEA), mesothelin (MSLN), prostate-specific membrane antigen (PSMA), EGFRvIII, IL13Rα2, NKG2D.

[0082] In one embodiment of any of the aspects herein, the cargo encoded by the saRNA is one or more transcription factors, including those selected from the list Oct3 / 4, Sox2, Klf4, and c-Myc.

[0083] In one embodiment of any aspect herein, the cargo encoded by the saRNA is one or more growth factors or cytokines selected from the list comprising platelet-derived growth factor (PDGF), erythropoietin (EPO), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), fibroblast growth factor (FGF), human relaxin-2 (RLX2), alpha-melanocyte stimulating hormone (α-MSH), insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), nerve growth factor (NGF), granulocyte-monocyte colony-stimulating factor (GMCSF), thrombopoietin (TPO), bone morphogenetic protein (BMP), hepatocyte growth factor (HGF), growth / differentiation factor (GDF), neurotrophic factor, migration stimulating factor (MSF), sarcoma growth factor (SGF).

[0084] In one embodiment of any of the aspects herein, the cargo encoded by the saRNA is one or both of Pappalysin-A1 (PAPPA1) and Pappalysin-A2 (PAPPA2).

[0085] In one embodiment of any of the aspects herein, the cargo encoded by the saRNA is one or more interleukins or their cognate receptors and receptor subunits selected from the list comprising IL-2, IL-4, IL-7, IL-10, IL-13 and IL-15.

[0086] In one embodiment of any of the aspects herein, the cargo encoded by the saRNA is one or more enzymes with antioxidant activity selected from the list comprising: phospholipid hydroperoxide glutathione peroxidase, superoxide dismutase-1, and superoxide dismutase-2; Bruton's tyrosine kinase; adenosine deaminase; ectonucleoside triphosphate diphosphohydrolase.

[0087] In one embodiment of any of the aspects herein, the present invention is a pharmaceutical composition comprising saRNA and a pharmaceutically acceptable carrier.

[0088] In one aspect, described herein is a method of expressing at least one cargo of interest in a cell, the method comprising contacting the cell with at least one saRNA described herein. In one embodiment of any aspect herein, the cell is transfected with the saRNA.

[0089] In one embodiment of any aspect herein, the saRNA cargo is expressed in a cell (e.g., a eukaryotic cell).

[0090] In one aspect, described herein are methods of expressing at least one cargo in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition comprising or expressing at least one saRNA described herein. In one embodiment of any aspect herein, one or more preselected cargos are expressed in a subject in need thereof, comprising administering to the subject (e.g., human, livestock) an effective amount of a saRNA.

[0091] In one embodiment of any of the aspects herein, the saRNA maintains or increases initial saRNA replication and cargo expression compared to the same dose of unmodified saRNA.

[0092] In one embodiment of any of the aspects herein, the expression of the cargo of interest encoded by the saRNA is comparable to or increased compared to the expression of unmodified saRNA.

[0093] In one embodiment of any of the aspects herein, the transfection efficiency of the self-amplifying RNA molecule is increased compared to the transfection efficiency of unmodified saRNA.

[0094] In one embodiment of any of the aspects herein, the early interferon response resulting from the introduction of the saRNA is reduced compared to the response of unmodified saRNA.

[0095] In one embodiment of any of the aspects herein, the length of time that the cargo is expressed at detectable levels by the saRNA is increased compared to the length of time that the unmodified saRNA is expressed.

[0096] In one embodiment of any of the aspects herein, the saRNA is delivered to human cells.

[0097] In one embodiment of any of the aspects herein, the saRNA is administered to a subject having cancer.

[0098] In one embodiment of any of the aspects herein, the saRNA is administered to a subject in need of vaccination.

[0099] In one embodiment of any of the aspects herein, the saRNA is administered to a subject in need of protein replacement therapy.

[0100] In one embodiment of any of the aspects herein, saRNA is administered to a subject in need of antibody therapy.

[0101] In one embodiment of any aspect herein, the saRNA allows for tissue, organ, or cell type specific expression of the cargo.

[0102] In one embodiment of any of the aspects herein, the saRNA comprises greater than 50% substitution of uridine with 5-methyluridine to promote kidney-specific expression of the cargo.

[0103] In one embodiment of any of the aspects herein, the saRNA modulates cellular differentiation of cells containing the saRNA at a level comparable to or greater than that achieved with an equivalent dose of unmodified saRNA.

[0104] In a preferred embodiment, the self-amplifying RNA highly substituted with modified nucleotides expresses more cargo compared to unmodified saRNA.

[0105] In a preferred embodiment of any aspect herein, the self-replicating RNA is produced by in vitro transcription.

[0106] In one embodiment of any aspect herein, the RNA comprises a 5' cap structure.

[0107] In a preferred embodiment of any aspect herein, the 5' cap structure is directly upstream of the adenosine nucleotide or analogue.

[0108] In a preferred embodiment of any aspect herein, the 5' cap structure is directly upstream of the guanosine nucleotide or analogue.

[0109] In one embodiment of any aspect herein, the RNA contains a 3' poly-A tail.

[0110] In one embodiment of any aspect herein, the RNA contains a 5'UTR, a 3'UTR, or a combination of both.

[0111] In one embodiment of any of the aspects herein, the inclusion of the selected modified nucleotides increases or enhances expression of the cargo protein compared to unsubstituted saRNA.

[0112] In one embodiment of any of the aspects herein, the inclusion of selected unmodified nucleotides increases or enhances the efficiency of saRNA transfection into the cells of interest.

[0113] In one embodiment of any aspect herein, the cargo protein is expressed by a self-replicating RNA comprising a protein coding sequence downstream of a subgenomic promoter.

[0114] In one embodiment of any aspect herein, the functional protein is expressed by a self-replicating RNA comprising a protein-coding sequence downstream of an internal ribosome entry site (IRES) sequence.

[0115] In one embodiment of any of the aspects herein, the cargo protein is a chimeric antigen receptor.

[0116] In a preferred embodiment, the vaccine-associated antigen is a protein encoded on the viral genome of any of the following strains: respiratory syncytial virus, hemagglutinin virus, human immunodeficiency virus, influenza virus, Zika virus, acute respiratory syndrome coronavirus 2, human papillomavirus, herpes virus, rotavirus, varicella virus, dengue virus, hepatitis A virus, hepatitis B virus, rubella virus, poliovirus or rabies virus.

[0117] In a preferred embodiment, the vaccine-associated antigen is a protein encoded on the genome of any tuberculosis, diphtheria, meningococcus, pneumococcus or tetanus bacteria.

[0118] In one embodiment of any aspect herein, the functional protein is a chimeric antigen receptor.

[0119] In one embodiment of any aspect herein, the functional protein is a chimeric antigen receptor having a protease domain that is responsive to a protease inhibitor.

[0120] In one embodiment of any aspect herein, the functional protein is a chimeric antigen receptor having a domain that interacts with a small molecule. In the presence of small molecules, the stability of the receptor is regulated by known and unknown mechanisms.

[0121] Another aspect details a method wherein the activity of a functional protein is dependent on the concentration of an administered small molecule.

[0122] Another aspect details a method wherein the activity of a functional protein is dependent upon the concentration of multiple administered small molecules.

[0123] In one embodiment of any of the aspects herein, the expression level of functional protein from the highly substituted saRNA is equal to or higher than the expression level from the unsubstituted saRNA.

[0124] In one embodiment of any of the aspects herein, the early immune response elicited by the highly substituted saRNA is less than the early immune response elicited by the unsubstituted saRNA.

[0125] In one embodiment of any aspect herein, multiple chimeric antigen receptors with opposing functions are expressed from the same highly substituted self-replicating RNA strand.

[0126] Another aspect details a method in which the activity of a resulting cell is dependent on an external signal sensed by a plurality of proteins having different functions. The resulting activity performed by the cell is dependent on the presence or absence of the sensed external signal. In some embodiments, a small molecule is administered as the external signal. Non-limiting examples of other external signals that can be used to control activity include light, ultrasound, proteins, nucleic acids, ligands, antibodies, antibody fragments, and the like.

[0127] In one embodiment of any aspect herein, a functional protein having the ability to enhance expression by known or unknown mechanisms is co-expressed, and the functional protein is used to enhance the level and duration of expression.

[0128] In one embodiment of any of the aspects herein, the highly substituted saRNA is delivered to the cells by electroporation.

[0129] In one embodiment of any of the aspects herein, the highly substituted saRNA is delivered to the cells by nucleofection.

[0130] In one embodiment of any of the aspects herein, the highly substituted saRNA is delivered to cells via lipid nanoparticles.

[0131] In one embodiment of any of the aspects herein, the highly substituted saRNA is delivered to specific cell types via lipid nanoparticles bearing targeting moieties.

[0132] In a preferred embodiment of any aspect herein, the function of the chimeric antigen receptor is to activate immune cells.

[0133] In a preferred embodiment of any aspect herein, the chimeric antigen receptor functions to inhibit immune cells.

[0134] In one embodiment of any aspect herein, a protein reporter is co-expressed with the cargo protein to measure the expression level caused by the highly substituted self-replicating RNA.

[0135] In one embodiment of any aspect herein, multiple cell types are modified with the highly substituted self-replicating RNA.

[0136] In one embodiment of any aspect herein, cell type specific expression is achieved by selecting appropriate modified nucleotides for hypersubstitution. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figure 1A-1B A series of schematic diagrams are shown. Figure 1A Figure 2 shows conventional RNA modifications of cells relative to self-replicating RNA modifications. In this figure, immune cells are used as non-limiting examples. Due to self-renewal and amplification, self-replicating RNA can cause higher and longer-lasting expression of cargo proteins. Conventional mRNA undergoes degradation and dilution through proliferation. Figure 1B A schematic diagram detailing the use of self-replicating RNA to modify cells by expressing proteins is shown. The delivered proteins (e.g., reporters, enzymes, receptors, ligands, transcription factors) can respond to external inputs (e.g., small molecules, light) or perform logical computations to allow control of the modified cells.

[0138] Figure 2 Shown is a schematic diagram of an exemplary format for describing self-replicating RNA in detail. Developed self-replicating RNA formats include conserved sequence elements (CSEs) derived from RNA viruses (e.g., Venezuelan equine encephalitis virus), and coding sequences for nonstructural proteins (e.g., nsp1, nsp2, nsp3, nsp4). The cargo coding sequence is placed downstream of a subgenomic promoter (SGP) or internal ribosome entry site (IRES) sequence. By separating the coding sequence with IRES or 2A sequences or extra SGP sequences, multiple cargo proteins can be co-expressed.

[0139] Figure 3 Shown is the expression of a conventional chimeric antigen receptor by self-replicating RNA. Self-replicating RNA can be used to express chimeric antigen receptors targeting multiple antigens.

[0140] Figure 4 Shown, compared with conventional mRNA (grey), the duration of HER2 chimeric antigen receptor expression from self-replicating RNA (black) is longer. 48 and 96 hours after transfection, cells expressing mCherry-labeled HER2CAR were analyzed by flow cytometry. The percentage of cells expressing the receptor was determined by measuring mCherry fluorescence. The bars above the histogram show the fold change between each time point under the corresponding conditions.

[0141] Figure 5It is shown that the duration of CD19 chimeric antigen receptor expression by self-replicating RNA is longer. After transfection, cells expressing CD19 CAR were analyzed by flow cytometry for up to 12 days. The percentage of cells expressing the receptor was determined by staining the cells with an anti-myc antibody that binds to the myc tag on the receptor. Cells transduced with lentivirus were used as a control to explain any differences in staining.

[0142] Figure 6 Shown is the detection of CD19 chimeric antigen in CD3+ primary T cells transfected with self-replicating RNA over a longer duration. Primary CD3+ T cells were transfected with self-replicating RNA (left) or conventional RNA (right) encoding the CD19 chimeric antigen receptor, which was labeled with intracellular mCherry. The graph shows the measured values ​​of mCherry fluorescence (x-axis) and anti-myc staining (y-axis) (anti-myc antibody that binds to the extracellular myc tag on the receptor) at multiple time points after transfection.

[0143] Figure 7 Shown is the function of HER2 chimeric antigen receptor in a longer duration after transfection with self-replicating RNA. 72 hours after transfection, activation of Jurkat T cells expressing HER2 CAR by conventional RNA or self-replicating RNA (upper figure). Cells were cultured in culture dishes containing or without HER2 antigen. Activation was measured by GFP fluorescence of the NFAT GFP reporter system. Fold activation was read out from the mean fluorescence intensity of GFP (lower figure). ****=p<0.0001. ns=not significant. N=3 independent repeats.

[0144] Figure 8 The function of the CD19 chimeric antigen receptor is shown to be longer-lasting after transfection with self-replicating RNA. The percentage of CAR-T cells activated during overnight co-culture with CD19-expressing Nalm6 cells was measured by flow cytometry. The degree of activation was measured by GFP fluorescence in Jurkat T cells containing the NFAT-GFP reporter system. N = 3 independent replicates. ** = p < 0.001, **** = p < 0.0001

[0145] Figure 9 The function of CD19 chimeric antigen receptor in primary CD3+T cells transfected with self-replicating RNA is shown. The bar graph shows the percentage of CAR-T cells positive for CD69 (a marker of early T cell activation). Primary T cells transfected with conventional RNA or self-replicating RNA encoding CD19CAR were co-cultured with Nalm6 cells expressing CD19. N=3 independent replicates. ****=p<0.0001. ns=not significant.

[0146] Figure 10 This figure shows enhanced CD19 chimeric antigen receptor function in primary CD3+ T cells transfected with self-replicating RNA. The bar graph shows the fold change in mean fluorescence intensity of CAR-T cells stained with an antibody targeting CD69 after overnight co-culture with Nalm6 cells. N = 3 independent replicates. * = p < 0.05.

[0147] Figure 11 The cytotoxic function of CAR-T cells generated by self-replicating RNA is enhanced. Target cell killing was observed after 24 hours of co-culture of CAR-T cells with Nalm6-Luc cells. CAR-T cells were primary CD3+ T cells transfected with mRNA or self-replicating RNA 48 hours before the assay. N = 3 independent replicates.

[0148] Figure 12 Shown is the expression level of higher chimeric antigen receptor in each cell after transfection with self-replicating RNA. The expression level of chimeric antigen receptor was measured by flow cytometry. The column reports the normalized mean fluorescence intensity of the fluorescently labeled anti-myc antibody bound to the myc tag on the receptor. The intensity value has been normalized to the intensity of the cell transduced with the lentivirus encoding CD19 CAR. The double difference is shown above each column.

[0149] Figure 13 Shown after being transfected with self-replicating RNA, the higher expression level of chimeric antigen receptor (marked with mCherry) in each cell in the extended time.The expression level of CAR of mCherry fluorescence is measured by flow cytometry over time.At each time point, the mean fluorescence intensity value obtained is normalized to the fluorescence intensity value obtained by the cell transduced with lentivirus.

[0150] Figure 14 Shown are greater amounts of chimeric antigen receptor RNA transcripts at multiple time points after transfection. RNA levels of CD19 CAR transcripts were measured by RT-qPCR. The resulting transcript levels were normalized to the values ​​obtained for cells transduced with lentivirus. N = 3 independent replicates.

[0151] Figure 15 A schematic diagram of the expression of an exemplary chimeric antigen receptor with external control and logic functions by self-replicating RNA is shown. The schematic diagram shows the structure of a chimeric antigen receptor with external control and logic functions. Some receptors have domains (e.g., ON-CAR, OFF-CAR, ON / OFF CAR) that respond to external molecules to turn on or off functions. In addition, some receptors play the function of activating or inhibitory receptors, allowing conditional activity (e.g., inhibitory CAR, SUPRA CAR) based on the presence or absence of target antigens.

[0152] Figure 16 The function of the drug-controlled "ON" chimeric antigen receptor ("ON-CAR") is shown. After CAR expression, the NS3 protease domain will cleave the flanking cleavage site, resulting in the separation of the signaling domain from the extracellular scFv. When an NS3 protease inhibitor (e.g., grazoprevir) is added, self-cleavage is prevented and the CAR can be turned on in the presence of the target antigen.

[0153] Figure 17 The function of an "OFF" chimeric antigen receptor ("OFF-CAR") under drug control is shown. After the two components are expressed, the NS3 binding peptide on component 1 will bind to the catalytically dead NS3 domain on component 2. When the target antigen is bound, this will lead to CAR activation. If a protease inhibitor (such as grazoprevir) is added, the binding peptide will be displaced and the CAR cannot be activated.

[0154] Figure 18 The function of a chimeric antigen receptor ("ON / OFF-CAR") with "OFF" and "ON" drug control is shown. After CAR expression, the NS3 protease domain will cut the flanking cleavage site, resulting in the separation of the signaling domain from the extracellular scFv. When an NS3 protease inhibitor (such as grazoprevir) is added, self-cleavage is prevented and the CAR is functional. If lenalidomide is also added, the E3 ligase will be recruited to the IKZF3 domain, and the CAR will be degraded.

[0155] Figure 19 The results show that self-replicating RNA can effectively express chimeric antigen receptors with external control functions. The expression levels of controllable and conventional CD19 CARs (e.g., ON-CAR, ON / OFF CAR) were measured by staining with anti-myc antibodies and measuring the fluorescence intensity of the mCherry tag on the receptor.

[0156] Figure 20It is shown that the activity of the chimeric antigen receptor expressed by self-replicating RNA can be increased by administering small molecules. The percentage of activated Jurkat CAR-T cells expressing CD19 CAR (ON-CAR) with NS3 domain was measured by flow cytometry. 24 hours before the assay, cells were transfected with self-replicating RNA encoding the receptor. In the absence or presence of 1 μM grazoprevir, CAR-T cells were co-cultured with Nalm6 overnight. CD19 CAR without NS3 domain expressed by self-replicating RNA was included as a control (right). N=3 independent repeats. Ns=not significant. **=p<0.01. ****=p<0.0001.

[0157] Figure 21 It is shown that the activity of chimeric antigen receptors expressed by self-replicating RNA can be increased or decreased by administering two different small molecules. The degree of activation of Jurkat CAR-T cells expressing CD19 CAR (ON / OFF-CAR) with NS3 and IKZF3 domains was measured by flow cytometry. 24 hours before the assay, cells were transfected with self-replicating RNA encoding the receptor. CAR-T cells were co-cultured with Nalm6 overnight in the absence of grazoprevir or lenalidomide, with 1 μM grazoprevir, and with 1 μM grazoprevir and 1 μM lenalidomide. N = 3 independent replicates.

[0158] Figure 22 It is shown that the activity of chimeric antigen receptors expressed by self-replicating RNA can be controlled in primary T cells by administering small molecules (ON-CAR). The cytotoxicity of CAR-T cells expressing CD19 CAR with NS3 domain was measured by co-culturing with the Nalm6 luciferase cell line at a ratio of 2: 1 effector cells to target cells. CAR-T cells and Nalm6 cells were cultured overnight in the absence or presence of 1 μM GZV. CD19 CAR-T cells generated with self-replicating RNA and CD19 CAR-T cells generated by lentiviral transduction were included as controls.

[0159] Figure 23 A schematic diagram details the function of a chimeric antigen receptor with either activating or inhibitory functions, which can be expressed via self-replicating RNA. Activating receptors bind to antigens on target cells, triggering CAR-T cells to kill the target cells. Inhibitory receptors bind to antigens expressed on off-target cells, leading to suppression of the killing response. This helps enhance the specificity of CAR-T therapy by improving the ability of CAR-T cells to distinguish between target and non-target cells.

[0160] Figure 24It is shown that, through self-replicating RNA, multiple chimeric antigen receptors with different functions can be efficiently expressed in the same cell. The flow chart shows the expression of CD19 activating CAR and HER2 inhibitory CAR in Jurkat cells transfected with self-replicating RNA. The expression of CD19 activating CAR was measured by staining with anti-myc antibody. The expression of HER2 inhibitory CAR was measured by staining with anti-V5 antibody. As a control, cells were transduced with a lentivirus encoding an activating CAR targeting CD19.

[0161] Figure 25 It is shown that the expression of multiple chimeric antigen receptors by self-replicating RNA enables the control of cell functions by logical calculations. The activation of Jurkat CAR-T cells expressing CD19 activating CAR and HER2 inhibitory CAR by self-replicating RNA was measured by flow cytometry. CAR-T cells were cultured on plates coated with only CD19 antigen or co-coated with CD19 and HER2 antigens. The degree of activation of NFAT was measured by the mean fluorescence intensity after gating CAR+ cells. As a control, cells were transduced with lentivirus or transfected with self-replicating RNA encoding only CD19 activating CAR. N=3 independent repeats. Ns=not significant. ****=p<0.0001.

[0162] Figure 26 Shown is a schematic diagram of a split, universal, programmable and reconfigurable (SUPRA) CAR system that can be expressed by self-replicating RNA. The signal transduction and antigen recognition domains are split into two polypeptide sequences that can interact through leucine zipper pairs. The split characteristics of the SUPRACAR system allow adjustable signal transduction to be achieved by swapping the signal transduction domains recognized by specific recognition domains. In addition, the SUPRA CAR system can perform logical calculations by recognizing multiple antigens and performing different signal transduction operations on each antigen.

[0163] Figure 27 The basic activation of T cells expressing CAR (e.g., ON-CAR, ON / OFF-CAR) with drug-controllable domains by self-replicating RNA is shown to be reduced. The histogram shows the percentage of Jurkat CAR-T cells activated by tonic signaling (tonicsignaling) in a resting state. Activation was measured in Jurkat cells using NFAT-GFP reporters by flow cytometry. N=3 independent repeats. Ns=not significant. ****=p<0.0001.

[0164] Figure 28It is shown that the activation of T cells expressing CAR (e.g., ON-CAR) with a drug-controllable domain by self-replicating RNA is comparable to the activation of T cells generated by lentivirus. After co-culturing with Nalm6 cells expressing CD19, the degree of NFAT activation in Jurkat CAR-T cells was measured. The NFAT multiple was determined by measuring the mean fluorescence intensity after gating on CAR+ cells. N=3 independent repeats. ****=p<0.0001.

[0165] Figure 29 The results show that the expression of chimeric antigen receptors by self-replicating RNA is enhanced by co-expression of an expression enhancing protein. Jurkat T cells were transfected with self-replicating RNA encoding CD19 CAR or CD19 CAR and B18R or E3L. The percentage of cells expressing CAR was longitudinally tracked by flow cytometry by measuring the fluorescence of the mCherry tag at the C-terminus of the CAR.

[0166] Figure 30 The results show that the expression level of chimeric antigen receptors expressed by self-replicating RNA can be controlled by the co-expression of expression-enhancing proteins over time. Jurkat T cells were transfected with self-replicating RNA encoding CD19 CAR or CD19 CAR and B18R or E3L. CAR expression levels were tracked longitudinally by flow cytometry by measuring the fluorescence of the mCherry tag on the C-terminus of the CAR. The MFI values ​​were normalized to the MFI of the self-replicating CD19 CAR alone on day 1.

[0167] Figure 31 Shown are Jurkat T cells with NFAT-GFP reporters transfected with self-replicating RNA encoding CD19 CAR or CD19 CAR and B18R or E3L. Activation of the NFAT pathway was measured by analyzing GFP expression in cells expressing the construct.

[0168] Figure 32 It is shown that the co-expression of additional proteins does not affect the activity of chimeric antigen receptors expressed by self-replicating RNA. The degree of NFAT activation in Jurkat CAR-T cells was measured after co-culture with Nalm6 cells expressing CD19. The fold NFAT activation was determined by measuring the mean fluorescence intensity after gating on CAR+ cells. N = 3 independent replicates.

[0169] Figure 33Shown, T cells can be transfected with lipid nanoparticles containing mRNA, with or without antibody conjugation on the nanoparticle surface. The histogram shows the luminescence after the mRNA encoding luciferase is transfected into Jurkat cells by lipid nanoparticles exposed by Dlin-MC3-PEG-DBCO (" LNP "), conjugated with isotype antibodies (" α-Iso LNP ") or conjugated with anti-CD3 antibodies (" α-CD3 LNP "). As a positive transfection control, electroporation was performed. Transfection was verified by lysing cells using commercially available luciferase assay reagent and measuring emitted light with a microplate reader. N=3 independent repeats.

[0170] Figure 34 Shown that self-replicating RNA can be used to express proteins and track their activity over extended durations. Flowchart shows the expression of mCherry from self-replicating RNA as a model cargo in Jurkat T cells over a 10-day period.

[0171] Figure 35 Shows that primary cells can be modified in vitro to express proteins via self-replicating RNA. Flowchart shows expression of mCherry from self-replicating RNA as a model cargo in primary CD3+ T cells over a 7 day period.

[0172] Figure 36 Schematic diagrams detailing an exemplary self-amplifying RNA (saRNA) comprising modified nucleotides. A linearized plasmid encoding an RNA-dependent RNA polymerase (RdRp) and a cargo of interest is shown as a template for in vitro transcription (IVT). The IVT reaction includes a mixture of 25%-100% nucleoside triphosphates (NTPs) of modified nucleotides and may include a cap analog for co-transcriptional capping. The resulting composition is a saRNA with 25%-100% modified nucleotide substitutions.

[0173] Figure 37 is a schematic diagram detailing the expression profile of saRNAs with >25% substitution of the specified modified nucleotides. The top portion of the schematic diagram details the results for unmodified saRNA, where a small population of cells takes up the saRNA and expresses the cargo. Aspects of the present disclosure detail the unexpected result that incorporation of modified nucleotides into self-amplifying RNA increases the percentage of transfected cells and the amount of protein expressed in each transfected cell.

[0174] Figure 38Depicted are the transfection efficiencies of a self-amplified RNA library encoding mCherry fully substituted with 28 individually modified nucleotides in HEK293T cells. This IVT was performed using a template harboring a T7 promoter and m7G(5')ppp(5')(2'OMeA)pU (CLEANCAP AU) as the 5' cap.

[0175] Figure 39 Depicted are the median expression intensities of a self-amplified RNA library encoding mCherry that was fully substituted with 28 modified nucleotides individually in HEK293T cells. This IVT was performed using a template with a T7 promoter and m7G(5')ppp(5')(2'OMeA)pU (CLEANCAP AU) as the 5' cap.

[0176] Figure 40 Depicted are the transfection efficiencies of a self-amplified RNA library encoding mCherry fully substituted with 28 individually modified nucleotides in HEK293T cells. This IVT was performed using a template harboring a T7 promoter and 3′-O-Me-m7G(5′)ppp(5′)G (ARCA) as the 5′ cap.

[0177] Figure 41 Depicted are the median expression intensities of a self-amplified RNA library encoding mCherry fully substituted with 28 modified nucleotides individually in HEK293T cells. This IVT was performed using a template with a T7 promoter and 3′-O-Me-m7G(5′)ppp(5′)G (ARCA) as the 5′ cap.

[0178] Figure 42 Depicts the relative expression of cargo proteins from self-amplified RNAs with different starting nucleotides, measured by the MFI of mCherry. The figure shows the Cap 0 structure in HEK293T cells. The left-right order of the columns in each group corresponds to the top-bottom order in the legend.

[0179] Figure 43 Depicts the relative expression of cargo proteins from self-amplified RNAs with different starting nucleotides, measured by the MFI of mCherry. The figure shows the Cap 1 structure in HEK293T cells. The left-right order of the columns in each group corresponds to the top-bottom order in the legend.

[0180] Figure 44 Depicts the relative expression of cargo protein from self-amplified RNA with different starting nucleotides, measured by the percentage of positive mCherry expression. The figure shows the Cap 0 structure in HEK293T cells. The left-right order of the columns in each group corresponds to the top-bottom order in the legend.

[0181] Figure 45 Depicted are the relative expression of cargo proteins from self-amplified RNAs with different starting nucleotides, measured by the percentage of mCherry-positive expression, showing the Cap 1 structure in HEK293T cells. The left-right order of the columns in each group corresponds to the top-bottom order in the legend.

[0182] Figures 46A-46B The protein level early interferon α and β responses in human peripheral blood mononuclear cells (PBMCs) using constructs initiated with Cap-1 and ATP are depicted. PBMCs were exposed to RNA (saRNA or traditional highly substituted mRNA) for 6 hours and analyzed by ELISA for IFNα (all subtypes; see e.g., Figure 46A ) and IFNβ (see e.g. Figure 46B ) concentration. Cell culture supernatant was diluted 1:1 in reagent buffer. The left-right order of the columns corresponds to the top-bottom order in the figure legend.

[0183] Figures 47A-47B Transcriptional early interferon alpha and beta responses in human peripheral blood mononuclear cells using constructs initiated with Cap-1 and ATP are depicted. PBMCs were exposed to saRNA with wild-type or modified nucleotides for 6 hours, and IFNA1 was measured by qPCR (see, e.g., Figure 47A ) and IFNB1 (see e.g. Figure 47B ) mRNA expression levels. Data represent transcript levels from three unique human donors, who were expressed at 100 ng / 1×10 5 The left-right order of the columns corresponds to the top-bottom order in the legend.

[0184] Figure 48 Depicts the expression of spike protein (Wuhan-1 variant) from self-amplified RNA in C2C12 mouse myoblasts measured by MFI from AF647-conjugated anti-spike antibody. The right side of the figure shows the different highly substituted modified nucleotides. The left and right order of the columns corresponds to the top and bottom order in the legend. Cap-1 and ATP-initiated saRNA constructs were used.

[0185] Figure 49 Depicts the expression of spike protein (Wuhan-1 variant) from self-amplified RNA in C2C12 mouse myoblasts measured by the percentage of positive expressing cells. The right side of the figure shows the different highly substituted modified nucleotides. The left and right order of the columns corresponds to the top and bottom order in the legend. Cap-1 and ATP-initiated saRNA constructs were used.

[0186] Figure 50Depicts cell-type-specific expression of vaccine antigens from highly substituted, self-amplifying RNA. This IVT was performed using a template harboring a T7 promoter and m7G(5')ppp(5')(2'OMeA)pU (CLEANCAP AU) as a 5' cap. The left-right order of the bars in each group corresponds to the top-bottom order in the figure legend.

[0187] Figures 51A-51C Depicted is the enhanced expression of saRNA constructs in human CD3+ T cells using Cap-1 and ATP initiation resulting from 100% substitution of 5-methylcytidine. Untreated cells were compared to saRNA encoding the fluorescent reporter protein mCherry with or without nucleotide modifications. Figure 51A Representative flow cytometry plots for each treatment condition are shown. Figure 51B Representative live-cell fluorescence microscopy images are shown. Figure 51C Quantification of flow cytometry data from biological replicates is shown; the left-right order of the columns corresponds to the top-bottom order in the legend.

[0188] Figure 52 Depicts prolonged and robust expression of saRNA constructs initiated with Cap-1 and ATP in human CD3+ T cells, resulting from 100% substitution of 5-methylcytidine. Untreated cells were compared with saRNA encoding the fluorescent reporter protein mCherry with or without nucleotide modifications and analyzed over time by flow cytometry.

[0189] Figures 53A-53C Depicted is the enhanced expression of saRNA constructs in Jurkat cells using Cap-1 and ATP initiation resulting from 100% substitution of 5-methylcytidine. Untreated cells were compared to saRNA encoding the fluorescent reporter protein mCherry with or without nucleotide modifications. Figure 53A Representative flow cytometry plots for each treatment condition are shown. Figure 53B Representative live-cell fluorescence microscopy images are shown. Figure 53C Quantification of flow cytometry data from biological replicates is shown; the left-right order of the columns corresponds to the top-bottom order in the legend.

[0190] Figure 54A-Figure 54B Depicts the prolonged and robust expression of saRNA constructs initiated with Cap-1 and ATP in Jurkat cells, resulting from 100% substitution of 5-methylcytidine. Untreated cells were compared with saRNA encoding the fluorescent reporter protein mCherry with or without nucleotide modifications and analyzed over time by flow cytometry. Figure 54A Histograms from these experiments are shown, and Figure 54B A line graph of the time course of these experiments is shown.

[0191] Figure 55 Figure 3: Effects of increasing substitution rates of N1-methylpseudouridine and 5-methylcytidine on the efficacy of Cap-1 and ATP-initiated saRNAs in Jurkat cells. Transfection efficiency of saRNA encoding a fluorescent reporter protein was synthesized with increasing % substitution of modified nucleotides. 0% substitution refers to unmodified saRNA.

[0192] Figure 56 Depicted is the effect of increasing 5-methylcytidine substitution on the immunogenicity of Cap-1 and ATP-initiated saRNAs. Human PBMCs were treated with saRNA-loaded LNPs for 6 hours and then harvested for gene expression analysis. 0% substitution refers to unmodified saRNA. The left-right order of the columns corresponds to the top-bottom order in the figure legend.

[0193] Figure 57 Depicts increased chimeric antigen receptor expression conferred by 100% substitution of 5-methylcytidine in saRNA constructs initiated by Cap-1 and ATP in human CD3+ T cells. Chimeric antigen receptor-encoding saRNA was delivered to CD3+ T cells via lipid nanoparticles synthesized with two different helper lipids with or without a CD3 targeting domain.

[0194] Figures 58A-58I is a series of schematics and graphs illustrating the identification of modified nucleotides compatible with self-amplifying RNA and their in vitro biological activities. Figure 58A Shown is a schematic diagram illustrating the limitations of unmodified saRNA, N1mΨ-modified saRNA, and the advantages of saRNA with compatible modNTPs. Figure 58B Shown is the workflow for synthesizing a library of fully substituted saRNAs with modified nucleotides and transfecting the library encoding an mCherry reporter into HEK293 cells using lipofection. Figure 58C Shown are flow cytometry results measuring the percentage of expressing cells in HEK293 cells transfected with a library of modified saRNAs. Error bars represent the standard deviation of n=3 biological replicates. Figure 58D Live cell fluorescence microscopy images and representative histograms in HEK293 cells of selected modified nucleotides are shown. Error bars represent standard deviation of n=4 biological replicates. Figure 58E Shown are expression levels 24 hours after transfection of 10 ng of RNA encoding a luciferase reporter in HEK293 cells with SM102 LNPs. Luciferase signal is expressed as fold change relative to untransfected mock cells. Error bars represent standard deviation of n=4 biological replicates. Figure 58F Shown are expression levels 24 hours after transfection of 10 ng of RNA encoding a luciferase reporter in C2C12 cells with SM102 LNPs. Luciferase signal is expressed as fold change relative to untransfected mock cells. Error bars represent standard deviation of n=4 biological replicates. Figure 58G Transfection efficiency is shown 24 hours after transfection of 25 ng and 250 ng of RNA encoding the mCherry reporter in Jurkat cells with SM102 LNPs. Error bars represent standard deviation of n=3 biological replicates. Figure 58H Shown are transfection efficiencies 24 hours after transfection of 500 ng of RNA encoding the mCherry reporter in CD3+ T cells from two different donors. n=3 biological replicates per group. Figure 58I The expression time course of 100 ng of unmodified or 5mC-modified saRNA in Jurkat cells is shown. Error bars represent the standard deviation of n=3 biological replicates. Statistical significance was determined by two-way ANOVA with Tukey's multiple comparison correction, ****p<0.0001, **p<0.01, *p<0.05. Figure 58E-Figure 58H The left-right order of the columns in each group corresponds to the top-bottom order in the legend.

[0195] Figures 59A-59G is a series of schematic diagrams and graphs showing the evaluation of the immunogenicity of modified saRNA in human PBMCs. Figure 59A Shown is a graph depicting that modified saRNA evades TLR detection, resulting in reduced interferon production. Figure 59B Shown is an assay for detecting early interferon responses from human PBMCs transfected with unmodified or modified saRNA. Figure 59C-Figure 59E Shown are the expression of IFN-α1 ( Figure 59C ), IFN-α2( Figure 59D ) and IFN-β1( Figure 59E ) gene expression analysis. Figure 59F Shown is IFN-α (all subtypes) serum analysis performed 6 hours after saRNA treatment in a unique donor. Figure 59G 6-hour and 24-hour serum analysis of IFN-β levels from saRNA-treated PBMCs is shown. Error bars represent standard deviations of n=3 biological replicates. Statistical significance was determined by ANOVA using Dunnett's method to control for multiple comparisons. ***p<0.001, ****p<0.0001. nd Not determined / below the limit of detection. Figure 59C-59G The left-right order of the columns in each group corresponds to the top-bottom order of the legend.

[0196] Figures 60A-60H is a series of schematics and diagrams illustrating the development and characterization of a fully modified saRNA vaccine against SARS-CoV-2. Figure 60A A schematic diagram illustrating different RNA formats used to express the SARS-CoV-2 spike protein, compared in vitro and in vivo. This includes non-replicating N1mΨ mRNA, wild-type self-amplifying RNA, and 5mC-modified self-amplifying RNA. Figure 60B Shown is the expression of spike protein 24 hours after transfection with 100 ng RNA in SM102 LNPs in HEK293 cells. Figure 60C Shown is the expression of spike protein 24 hours after transfection with 100 ng RNA in SM102 LNPs in C2C12 cells. Figure 60D The median fluorescence intensity (MFI) of anti-Spike AF647 staining in C2C12 cells is shown. MFI is relative to untreated cells. Error bars represent the standard deviation of n=3 biological replicates. Figure 60E Figure 2 shows the study design for a SARS-CoV-2 challenge study in C57BL / 6 mice. Mice were vaccinated using a prime-boost schedule, sera were collected for analysis of interferon responses and titers, and challenged with SARS-CoV-2 MA30 on day 35. Figure 60F Shown is IFN-α1 expression in sera collected 24 or 48 hours after initial vaccination with 1000 ng RNA in LNPs. Error bars represent standard deviation of n=5 biological replicates. Figure 60G Shown are the survival of mice after lethal challenge with MA30 virus. n=10 mice per group. Figure 60H Figure 2 shows the weight change of mice after challenge with mouse-adapted SARS-CoV-2 MA30 virus. Error bars represent standard error (SEM). Statistical significance was determined by ANOVA, using Dunnett's method to control for multiple comparisons. **p<0.005, ***p<0.001, ****p<0.0001. For survival study statistics, the log-rank (Matel-Cox) test was used between groups. ****=p<0.0001, ***=p<0.001. Figure 60D and 60F The left-right order of the columns in each group corresponds to the top-bottom order of the legend.

[0197] Figures 61A-61D is a series of charts, graphs and images illustrating the testing of modified saRNA. Figure 61A Shown are flow cytometry results comparing modified saRNA screening with wild-type unmodified control constructs. Figure 61BShown are the median fluorescence intensities of HEK293 cells transfected with the modified saRNA library. Figure 61C Shown from Figure 58D

[00145] Live cell microscopy images of a control sample of WT saRNA. Wild type refers to unmodified saRNA. Figure 61D Shown are representative histograms of HEK293 cells transfected with modified saRNA synthesized with 3′-O-Me-m7G(5′)ppp(5′)G(ARCA) (upper row) or m7G(5′)ppp(5′)(2′OMeA)pU(CLEANCAP AU) (lower row).

[0198] Figures 62A-62E is a series of charts and images showing the testing of modified saRNA. Figure 62A Shown are the dose responses of HEK293 cells transfected with SM102 LNPs containing N1mΨ mRNA encoding luciferase, WT saRNA, or 5mC saRNA. Figure 62B Shown are representative flow cytometry plots of Jurkat cells transfected with WT or 5mC saRNA encoding mCherry. Figure 62C Shown are live cell microscopy images of Jurkat cells transfected with WT or 5mC saRNA encoding mCherry. Figure 62D Shown is the expression level 24 hours after 250ng of RNA encoding luciferase reporter was transfected with SM102 LNP in Jurkat cells. Luciferase signal is expressed as a fold change compared to untransfected mock cells. The left and right order of the columns corresponds to the top and bottom order in the legend. Figure 62E Shown are the MFI of Jurkat cells transfected with WT or 5mC saRNA encoding mCherry over 7 days.

[0199] Figure 63A-Figure 63B is a series of graphs and images showing the testing of modified saRNA. Figure 63A Shown are representative flow diagrams of primary T cells from two different donors transfected with WT or 5mC saRNA encoding mCherry. Figure 63B Shown are live cell microscopy images of primary T cells transfected with WT or 5mC encoding mCherry.

[0200] Figures 64A-64D is a series of graphs showing the testing of modified saRNA as a SARS-CoV-2 vaccine. Figure 64A Shown are the transfection efficiencies 24 hours after C2C12 cells were transfected with 100 ng of modified mRNA encoding Spike or saRNA. Figure 64BShown is the detection of Spike protein by ELISA in lysed C2C12 cells after transfection with 100 ng of modified Spike-encoding mRNA or saRNA. Figure 64C Shown are the transfection efficiencies 24 hours after C2C12 were transfected with 25 ng of modified HA-encoding mRNA or saRNA. Figure 64D Shown are the median fluorescence intensity (MFI) of anti-HA AF647 staining in C2C12 cells. Figures 64A-64D The left-right order of the columns in each group corresponds to the top-bottom order in the legend.

[0201] Figure 65A-Figure 65B is a series of graphs showing the testing of modified saRNA as a SARS-CoV-2 vaccine. Figure 65A The expression of IFN-β in serum collected 24 or 48 hours after initial vaccination with 1000 ng RNA in LNP is shown. Error bars represent the standard deviation of n=5 biological replicates. The left-right order of the columns in each group corresponds to the top-bottom order in the legend. Figure 65B Shown is the survival of mice after a lethal challenge with MA30 virus. n = 10 mice per group. For survival study statistics, the log-rank (Matel-Cox) test was used between groups. **** = p < 0.0001.

[0202] Figures 66A-66D is a series of graphs showing the testing of modified saRNA as a SARS-CoV-2 vaccine. Figure 66A Shown are the encapsulation efficiencies of post-dialysis spike-encoding LNPs used in in vivo studies. Figure 66B The sizes of the LNPs used in the in vivo studies are shown. Figure 66C Shown are the polydispersity index (PDI) of the LNPs used in the in vivo studies. Figure 66D RNA concentrations in ng / uL are shown. Figures 66A-66D The left-right order of the columns in each group corresponds to the top-bottom order of the legend.

[0203] Figures 67A-67D The flow cytometry gating strategy used in Example 20 is shown. Figure 67A Shown is the gating strategy used when screening the modNTP saRNA library by HEK293 transfection. Figure 67B Shown is the gating strategy used to analyze saRNA LNP transfection efficiency in Jurkat T cells. Figure 67C Shown is the gating strategy used to determine the expression of SARS-CoV-2 spike protein in HEK or C2C12 cells. Figure 67DShown is the gating strategy used to analyze the transfection efficiency of saRNA LNPs in primary T cells.

[0204] Figure 68 The yield of self-amplified RNA constructs after in vitro transcription is shown, without modified nucleotides (wild type) or with 100% substitution of cytidine or uridine with 5-hydroxymethylcytidine, 5-methylcytidine, or 5-methyluridine, N1-methylpseudouridine, respectively. The reported yield is relative to the wild type of each construct. The left-right order of the columns in each group corresponds to the top-bottom order in the legend.

[0205] Figure 69 is a series of flow cytometry results showing the distribution of mCherry expression 24 hours after LNP-mediated transfection of Jurkat cells.

[0206] Figure 70 Shown is analysis of firefly luciferase reporter expression in HEK293-T cells transfected with LNPs containing N1-methylpseudouridine mRNA, unmodified (wild-type) saRNA, or 5-methylcytidine-modified saRNA.

[0207] Figure 71 Shown is the analysis of IL12 p70 expression in HEK293-T cells transfected with LNPs containing unmodified (wild-type) saRNA or 5-methylcytidine-modified saRNA.

[0208] Figure 72 Shown are target cell lysis of primary human T cells co-cultured with supernatants of HER2-expressing Nalm6 cells and C2C12 cells transfected with unmodified (WT) or 5-methylcytidine (5mC)-modified saRNA encoding a HER2 bispecific T cell engager (BiTE).

[0209] Figure 73 Shown are target cell lysis of primary human T cells co-cultured with supernatants of HER2-expressing Nalm6 cells and C2C12 cells transfected with unmodified (WT) or 5-methylcytidine (5mC)-modified saRNA encoding a HER2 bispecific T cell engager (BiTE).

[0210] Figure 74 Shown are target cell lysis of primary human T cells co-cultured with supernatants of HER2-negative wild-type Nalm6 cells or HER2-expressing Nalm6 cells, and C2C12 cells transfected with unmodified (WT) or 5-methylcytidine (5mC)-modified saRNA encoding a HER2 bispecific T cell engager (BiTE).

[0211] Figure 75 is a series of flow cytometry results showing the distribution of mCherry reporter expression 24 hours after LNP-mediated transfection of C2C12 cells with saRNA or mRNA encoding HER2 bispecific antibodies.

[0212] Figure 76 The percentage of C2C12 cells transfected with LNPs containing the indicated RNA constructs is shown. The percentage of cells expressing the encoded protein was determined by measurement of the mCherry reporter.

[0213] Figure 77 Shown are the relative expression of bispecific T cell engagers in C2C12 cells transfected with LNPs containing each RNA construct, as measured by the median fluorescence intensity of the mCherry reporter.

[0214] Figure 78 Figure 2 is a series of flow cytometry data illustrating the expression of GFP in Jurkat cells carrying a NFAT GFP reporter co-cultured with supernatant from Nalm6 cells overexpressing HER2 and C2C12 cells transfected with saRNA or mRNA constructs encoding BiTEs.

[0215] Figure 79 Representative bioluminescence images of mice at different time points after intramuscular injection of LNPs containing 2.5 μg of N1mΨ mRNA encoding luciferase (left) or 5mC saRNA (right) are shown. The scale bar represents the radiance.

[0216] Figure 80 The total flux of BLI imaging of mice (n=5 biological replicates) injected intramuscularly with 2.5 μg of LNPs encoding luciferase N1mΨ mRNA or 5mC saRNA is shown. The dotted line represents the average signal of the PBS group during the study. The error bars represent the standard error mean.

[0217] Figure 81 is a schematic diagram of the HER2 CAR saRNA plasmid.

[0218] Figure 82 is a schematic diagram of the CD19 CAR saRNA plasmid.

[0219] Figure 83 is a schematic diagram of the CD19 NS3 CAR saRNA plasmid.

[0220] Figure 84 is a schematic diagram of the CD19 NS3-IKZF3 CAR saRNA plasmid.

[0221] Figure 85Schematic diagram of the CD19 aCAR+HER2 iCAR saRNA plasmid.

[0222] Figure 86 is a schematic diagram of the mCherry saRNA plasmid.

[0223] Figure 87 Schematic diagram of the SARS-CoV-2 Spike saRNA plasmid.

[0224] Figure 88 is a schematic diagram of the influenza HA saRNA plasmid.

[0225] Figure 89 Schematic diagram of firefly luciferase saRNA plasmid.

[0226] Figure 90 Schematic diagram of HER2 CD3 scFv BITE saRNA plasmid.

[0227] Figure 91 is a schematic diagram of the IL12 saRNA plasmid.

[0228] Figure 92 is a bar graph showing the detection of bioactive GLP-1 peptide (amino acids 7-37) in supernatants of HEK293 cells transfected with self-amplifying RNA substituted with 5-methylcytidine. DETAILED DESCRIPTION

[0229] The above summary of the present invention is not intended to describe every disclosed embodiment or every implementation of the present invention. The following description and examples illustrate illustrative embodiments. In several places throughout the application, guidance is provided by lists of examples, which can be used in various combinations. In each case, the enumerated list serves only as a representative group and should not be construed as an exclusive list.

[0230] Disclosed herein are methods and compositions for modifying and controlling cell activity by expressing proteins from self-replicating RNA. The technology disclosed herein enables expression of proteins at levels comparable to other methods for establishing constitutive protein expression. Protein cargo can be any genetically codable protein with known or unknown functions. The system is modular and allows the expression of a variety of proteins with various functions. In addition, the activity of cells transfected with self-replicating RNA can be controlled by expressing proteins that respond to external inputs or by expressing proteins that interact with each other for logical calculations. This technology can be used to rapidly generate cell therapies with controllable activity without the need for gene editing. In addition, this technology can be combined with RNA delivery strategies (such as lipid nanoparticles) for in vivo transfection of cells.

[0231] As a clinically relevant, non-limiting example, this disclosure outlines the development of a self-replicating RNA system for modifying immune cells to express reporter proteins and chimeric antigen receptors targeting CD19 and HER2. In some embodiments, the activity of the modified cells is controlled by external control or by a logical computational circuit established by the delivered self-replicating RNA.

[0232] Disclosed herein are methods and compositions for modifying and controlling cell activity by expressing proteins from self-amplifying RNAs that are highly substituted with chemically modified nucleotides. The technology disclosed herein allows highly substituted saRNAs to express proteins at levels comparable to or higher than unsubstituted self-amplifying RNAs. Protein cargo can be proteins encoded by any gene with known or unknown functions. The system is modular and allows the expression of a variety of proteins with various functions. In addition, the activity of cells transfected with highly substituted self-amplifying RNAs can be controlled by expressing proteins that respond to external inputs or by expressing proteins that interact with each other to perform logical calculations. This technology can be used to generate low-dose saRNA-based vaccines that express equal or greater cargo than unmodified saRNA. Another aspect of the present invention is to reduce immunogenicity, and highly substituted saRNAs are thereby enhanced. This technology can be used to generate vaccines, in situ or ex vivo cell therapies, and protein replacement therapies. In addition, this technology can be combined with RNA delivery strategies (e.g., lipid nanoparticles) to transfect cells in vivo. Self-amplifying RNA

[0233] Self-amplifying RNA is described herein in various aspects. As used herein, the terms "self-amplifying RNA" or "saRNA" or "self-replicating RNA" or "srRNA" are used interchangeably and refer to an RNA chain that is capable of undergoing replication activity such that a replicated chain is generated from an original chain.

[0234] In various aspects, described herein are saRNAs comprising: (a) at least one nonstructural 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 are saRNAs comprising, from 5' to 3': (a) at least one nonstructural 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.

[0235] In other aspects, described herein are saRNAs comprising: (a) at least one nonstructural 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. In further aspects, described herein are saRNAs comprising, from 5' to 3': (a) at least one nonstructural 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.

[0236] In one aspect, described herein is a saRNA comprising: (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural 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.

[0237] In one aspect, described herein is a saRNA comprising, from 5' to 3': (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3) and / or nonstructural 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.

[0238] In one aspect, described herein is a saRNA comprising: (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural 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.

[0239] In one aspect, described herein is a saRNA comprising, from 5' to 3': (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural 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.

[0240] In one aspect, described herein is a saRNA comprising: (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural protein 4 (nsp4), each derived from at least one virus, wherein nsP1 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.

[0241] In one aspect, described herein is a saRNA comprising, from 5' to 3': (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3) and / or nonstructural protein 4 (nsp4), each derived from at least one virus, wherein nsP1 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.

[0242] In one aspect, described herein is a saRNA comprising: (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural protein 4 (nsp4), each derived from at least one alphavirus, wherein nsP1 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.

[0243] In one aspect, described herein is a saRNA comprising, from 5' to 3': (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural protein 4 (nsp4), each derived from at least one alphavirus, wherein nsP1 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.

[0244] In some embodiments of this aspect, the saRNA described herein comprises at least one nucleotide modification, as further described herein. In some embodiments of this aspect, the saRNA described herein does not comprise a nucleotide modification.

[0245] In one aspect, described herein are self-amplifying RNA (saRNA) comprising: (a) at least one nonstructural 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 start nucleotide comprises adenosine or an adenosine analog; and wherein the start nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap1).

[0246] In one aspect, described herein are self-amplifying RNA (saRNA) comprising: (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural 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 start nucleotide comprises adenosine or an adenosine analog; and wherein the start nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap1).

[0247] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising, from 5' to 3': (a) at least one nonstructural 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 start nucleotide comprises adenosine or an adenosine analog; and wherein the start nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap1).

[0248] In one aspect, described herein is a self-amplifying RNA (saRNA), comprising, from 5' to 3': (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural 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 start nucleotide comprises adenosine or an adenosine analog; and wherein the start nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap1).

[0249] In one aspect, described herein are self-amplifying RNAs (saRNAs) comprising, from 5' to 3': (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural protein 4 (nsp4), each derived from at least one virus, wherein nsP1 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 start nucleotide comprises adenosine or an adenosine analog; and wherein the start nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap1).

[0250] In one aspect, described herein are self-amplifying RNA (saRNA) comprising: (a) at least one nonstructural 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 start nucleotide comprises adenosine or an adenosine analog; and wherein the start nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap1).

[0251] In one aspect, described herein are self-amplifying RNA (saRNA) comprising: (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural 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 start nucleotide comprises adenosine or an adenosine analog; and wherein the start nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap1).

[0252] In one aspect, described herein are self-amplifying RNA (saRNA) comprising, from 5' to 3': (a) at least one nonstructural 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 start nucleotide comprises adenosine or an adenosine analog; and wherein the start nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap1).

[0253] In one aspect, described herein are self-amplifying RNAs (saRNAs) comprising, from 5' to 3': (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural 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 start nucleotide comprises adenosine or an adenosine analog; and wherein the start nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap1).

[0254] In one aspect, described herein are self-amplifying RNAs (saRNAs) comprising, from 5' to 3': (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural protein 4 (nsp4), each derived from at least one alphavirus, wherein nsP1 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 start nucleotide comprises adenosine or an adenosine analog; and wherein the start nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap1).

[0255] In some embodiments of any aspect, the nucleic acid (e.g., DNA) encoding the saRNA described herein comprises one of SEQ ID NO: 2 or 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 about 98%, at least about 99%, or 100% identical to one of SEQ ID NO: 2 or SEQ ID NO: 5 and maintains the same function (e.g., self-replication), or a codon-optimized version thereof.

[0256] In some embodiments of any aspect, at least one cargo is inserted between the AflII and NdeI cleavage sites of SEQ ID NO: 2. In some embodiments of any aspect, at least one cargo is inserted between nucleotides 7627 and 7628 of SEQ ID NO: 2. In some embodiments of any aspect, 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) are replaced with at least one cargo of interest, as further described herein.

[0257] In some embodiments of any aspect, the saRNA described herein comprises one of SEQ ID NO: 6-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 about 98%, at least about 99%, or 100% identical to one of SEQ ID NO: 6-SEQ ID NO: 7 and maintains the same function (e.g., self-replication), or a codon-optimized version thereof.

[0258] Table 6 provides non-limiting examples of domains comprised by exemplary saRNAs, as well as exemplary template DNA for the saRNAs, as further described herein.

[0259] Table 6: Exemplary saRNAs Nucleic acid modification

[0260] In some embodiments of any aspect, the saRNA described herein comprises at least one nucleic acid modification, which can improve the efficacy of the saRNA. In some embodiments of any aspect, the saRNA described herein comprises modified nucleotides, such as at least 25% modified nucleotides. In some embodiments of any aspect, the saRNA described herein comprises a specific combination of 5' and start nucleotides. In some embodiments of any aspect, the saRNA described herein comprises terminal modifications, backbone modifications, and / or sugar modifications. Modified nucleotides

[0261] In various aspects, saRNAs comprising modified nucleotides are described herein. The term "modified nucleotide" refers to any analog of cytidine, adenosine, guanosine, uridine, or pseudouridine. These analogs may include isomers of nitrogenous bases, as well as the inclusion or exclusion of chemical groups (naturally occurring and synthetically introduced) in any aspect of the nitrogenous bases. 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 methylation of the 2'O position of the first and second starting nucleotides (also known as Cap-1 and Cap-2 structures).

[0262] In some embodiments of any aspect, the modified nucleotide comprises a modified pyrimidine nucleoside phosphate. In some embodiments of any aspect, the modified nucleotide comprises a pyrimidine nucleoside phosphate moiety at the 5-carbon of the pyrimidine. In some embodiments of any aspect, the moiety at the 5-carbon of the pyrimidine is selected from the group consisting of a methyl, ethyl, propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, and hydroxypropyl functional group. In some embodiments of any aspect, the pyrimidine comprises cytidine and / or uridine.

[0263] In some embodiments of any aspect, the pyrimidine comprises cytidine. In some embodiments of any aspect, the pyrimidine comprises cytidine, and the modified nucleotide comprises 5-methylcytidine (see, e.g., Formula I). ​​In some embodiments of any aspect, the pyrimidine comprises cytidine, and the modified nucleotide comprises 5-hydroxymethylcytidine (see, e.g., Formula II). In some embodiments of any aspect, the pyrimidine comprises cytidine, and the modified nucleotide comprises 5-methylcytidine and 5-hydroxymethylcytidine.

[0264] In some embodiments of any aspect, the pyrimidine comprises uridine. In some embodiments of any aspect, the pyrimidine comprises uridine, and the modified nucleotide comprises 5-methyluridine (see, e.g., Formula III). In some embodiments of any aspect, the pyrimidine comprises uridine, and the modified nucleotide comprises 5-hydroxymethyluridine (see, e.g., Formula IV). In some embodiments of any aspect, the pyrimidine comprises uridine, and the modified nucleotide comprises 5-methyluridine and 5-hydroxymethyluridine.

[0265] In some embodiments of any aspect, the modified nucleotide is selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine, or any combination thereof. For example, the saRNA described herein may comprise any combination of the modified nucleotides shown in Table 4. In some embodiments of any aspect, the modified nucleotide is selected from the group consisting of 5-methylcytidine, 5-methyluridine, and 5-hydroxymethylcytidine, or any combination thereof. In some embodiments of any aspect, the modified nucleotide is selected from the group consisting of 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine, or any combination thereof.

[0266] Table 4: Exemplary modified nucleotide combinations in saRNA described herein.

[0267] In some embodiments of any of the aspects, the modified nucleotides comprise one or both of 5-methylcytidine and 5-hydroxymethylcytidine, and 5-methyluridine or 5-hydroxymethyluridine in the same saRNA molecule. In some embodiments of any of the aspects, the modified nucleotides comprise one or both of 5-methylcytidine and 5-hydroxymethylcytidine, and 5-methyluridine in the same saRNA molecule.

[0268] In some embodiments of any aspect, the saRNA described herein comprises 5-methylcytidine as a nucleotide modification and is combined with at least one of 5-methyluridine, 5-hydroxymethyluridine, and / or 5-hydroxymethylcytidine, or any combination thereof. In some embodiments of any aspect, the saRNA described herein does not comprise 5-methylcytidine as a nucleotide modification. In some embodiments of any aspect, the saRNA described herein does not comprise 5-methylcytidine as the only nucleotide modification.

[0269] In some embodiments of any aspect, the saRNA described herein comprises at least 25% modified nucleotides. The percentage of modified nucleotides can be determined by dividing the total number of modified nucleotides in the modified saRNA by the total number of specific nucleotides in the corresponding unmodified saRNA. For example, if the unmodified saRNA comprises 1000 uridine nucleosides and the modified saRNA comprises 250 5-methyluridine (5mU) instead of uridine, the saRNA comprises 25% modified nucleotides. As another non-limiting example, if the unmodified saRNA comprises 1000 uridine nucleosides and the modified saRNA comprises 125 5-methyluridine (5mU) and 125 5-hydroxymethyluridine (5OHmU) instead of uridine, the saRNA comprises 25% modified nucleotides.

[0270] In some embodiments of any aspect, the percentage of modified nucleotides in the saRNA can be controlled by the molar percentage of modified nucleotides included in the in vitro transcription (IVT) reaction that generates the saRNA from the DNA template. For example, to generate a saRNA containing 25% 5-methyluridine (5 mU), the IVT reaction mixture can contain 25 mole% 5-methyluridine (5 mU) and 75 mole% uridine.

[0271] In some embodiments of any aspect, the saRNA described herein comprises at least 25% modified nucleotides and at most 100% modified nucleotides corresponding to at least one specific nucleotide (e.g., pyrimidine; e.g., cytidine and / or uridine). In some embodiments of any aspect, the saRNA 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 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at 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.

[0272] In some embodiments of any aspect, the saRNA 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 5 ... Up to 40%, up to 41%, up to 42%, up to 43%, up to 44%, up to 45%, up to 46%, up to 47%, up to 48%, up to 49%, up to 50%, up to 51%, up to 52%, up to 53%, up to 54%, up to 55%, up to 56%, up to 57%, up to 58%, up to 59%, up to 60%, up to 61%, up to 62%, up to 63%, up to 64%, Up to 65%, up to 66%, up to 67%, up to 68%, up to 69%, up to 70%, up to 71%, up to 72%, up to 73%, up to 74%, up to 75%, up to 76%, up to 77%, up to 78%, up to 79%, up to 80%, up to 81%, up to 82%, up to 83%, up to 84%, up to 85%, up to 86%, up to 87%, up to 88%, up to 89% , up to 90%, up to 91%, up to 92%, up to 93%, up to 94%, up to 95%, up to 96%, up to 97%, up to 98%, up to 99%, up to 99.1%, up to 99.2%, up to 99.3%, up to 99.4%, up to 99.5%, up to 99.6%, up to 99.7%, up to 99.8%, up to 99.9%, or up to 100% modified nucleotides.

[0273] In some embodiments of any aspect, the saRNA described herein comprises 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-100%, or 99-100% modified nucleotides corresponding to at least one specific nucleotide (e.g., pyrimidine; e.g., cytidine and / or uridine).

[0274] In some embodiments of any aspect, the saRNA described herein comprises 25-50% modified nucleotides corresponding to at least one specific nucleotide (e.g., pyrimidine; e.g., cytidine and / or uridine). In some embodiments of any aspect, the saRNA described herein comprises 51-75% modified nucleotides corresponding to at least one specific nucleotide (e.g., pyrimidine; e.g., cytidine and / or uridine). In some embodiments of any aspect, the saRNA described herein comprises 75-99% modified nucleotides corresponding to at least one specific nucleotide (e.g., pyrimidine; e.g., cytidine and / or uridine). In some embodiments of any aspect, the saRNA described herein comprises 100% modified nucleotides corresponding to at least one specific nucleotide (e.g., pyrimidine; e.g., cytidine and / or uridine).

[0275] In some embodiments of any aspect, the saRNA described herein comprises 100% 5-methylcytidine (5mC) substituted for cytidine. In some embodiments of any aspect, the saRNA described herein comprises 100% 5-hydroxymethyl-cytidine (5OHmC) substituted for cytidine. In some embodiments of any aspect, the saRNA described herein comprises 100% 5-methyluridine (5mU) substituted for uridine. In some embodiments of any aspect, the saRNA described herein comprises 100% 5-hydroxymethyluridine (5OHmU) substituted for uridine.

[0276] In some embodiments of any aspect, the saRNA described herein comprises 100% 5-methylcytidine (5mC) substituted for cytidine, and 100% 5-methyluridine (5mU) substituted for uridine. In some embodiments of any aspect, the saRNA described herein comprises 100% 5-methylcytidine (5mC) substituted for cytidine, and 100% 5-hydroxymethyl-uridine (5OHmU) substituted for uridine. In some embodiments of any aspect, the saRNA described herein comprises 100% 5-hydroxymethyl-cytidine (5OHmC) substituted for cytidine, and 100% 5-methyluridine (5mU) substituted for uridine. In some embodiments of any aspect, the saRNA described herein comprises 100% 5-hydroxymethyl-cytidine (5OHmC) substituted for cytidine, and 100% 5-hydroxymethyl-uridine (5OHmU) substituted for uridine.

[0277] In some embodiments of any aspect, the saRNA comprising at least 25% modified nucleotides expresses at least one cargo at a level greater than or equal to that of a corresponding saRNA having less than 25% modified nucleotides. As used herein, the phrase "a corresponding saRNA having less than 25% modified nucleotides" refers to a saRNA having the same base sequence as an RNA comprising at least 25% modified nucleotides when the base sequence uses adenosine, guanosine, uridine (and its analogs), and cytidine (and its analogs); modified nucleotides can be classified as analogs of unmodified nucleotides (e.g., 5-methylcytidine and 5-hydroxymethylcytidine are classified as cytidine analogs; e.g., 5-methyluridine and 5-hydroxymethyluridine are classified as uridine analogs).

[0278] In some embodiments of any aspect, the saRNA comprising at least 25% modified nucleotides expresses a level of at least one cargo that is equivalent to the level of a corresponding saRNA having less than 25% modified nucleotides. In some embodiments of any aspect, the saRNA comprising at least 25% modified nucleotides expresses a level of at least one cargo that is at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold, or more higher than the level of a corresponding saRNA having less than 25% modified nucleotides.

[0279] In embodiments where the saRNA comprises multiple types of modified nucleotides (e.g., at least two of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine), the percentage of each modified nucleotide may be the same as or may be different from one another. As a non-limiting example, in a saRNA comprising 25% modified cytidine, the saRNA may comprise 0% 5mC and 25% 5OHmC; 1% 5mC and 24% 5OHmC; 2% 5mC and 23% 5OHmC; 3% 5mC and 22% 5OHmC; 4% 5mC and 21% 5OHmC; 5% 5mC and 20% 5OHmC; 6% 5mC and 19% 5OHmC; 7% 5mC and 18% 5OHmC; 8% 5mC and 17% 5OHmC; 9% 5mC and 16% 5OHmC; 10% 5mC and 15% 5OHmC; 11% 5mC and 14% 5OHmC; 12% 5mC and 13% 5OHmC; 12.5% ​​5mC and 12.5% ​​5OHmC; 13% 5mC and 12% 5OHmC; 14% 5mC and 11% 5OHmC; 15% 5mC and 10% 5OHmC; 16% 5mC and 9% 5OHmC; 17% 5mC and 8% 5OHmC; 18% 5mC and 7% 5OHmC; 19% 5mC and 6% 5OHmC; 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% 5OHmC.

[0280] As a non-limiting example, in a saRNA comprising 25% modified uridine, the saRNA may comprise 0% 5mU and 25% 5OHmU; 1% 5mU and 24% 5OHmU; 2% 5mU and 23% 5OHmU; 3% 5mU and 22% 5OHmU; 4% 5mU and 21% 5OHmU; 5% 5mU and 20% 5OHmU; 6% 5mU and 19% 5OHmU; 7% 5mU and 18% 5OHmU; 8% 5mU and 17% 5OHmU; 9% 5mU and 16% 5OHmU; 10% 5mU and 15% 5OHmU; 11% 5mU and 14% 5OHmU; 12% 5mU and 13% 5OHmU; 12.5% ​​5mU and 12.5% ​​5OHmU; 13% 5mU and 12% 5OHmU; 14% 5mU and 11% 5OHmU; 15% 5mU and 10% 5OHmU; 16% 5mU and 9% 5OHmU; 17% 5mU and 8% 5OHmU; 18% 5mU and 7% 5OHmU; 19% 5mU and 6% 5OHmU; 20% 5mU and 5% 5OHmU; 21% 5mU and 4% 5OHmU; 22% 5mU and 3% 5OHmU; 23% 5mU and 2% 5OHmU; 24% 5mU and 1% 5OHmU; or 25% 5mU and 0% 5OHmU.

[0281] In some embodiments of any aspect, the saRNA described herein comprises less than 25% modified nucleotides. In some embodiments of any aspect, the saRNA 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 aspect, the saRNA described herein does not comprise any modified nucleotides.

[0282] In some embodiments of any aspect, the modified nucleotide is not a modified purine. In some embodiments of any aspect, the modified nucleotide is not a modified guanosine or adenosine. In some embodiments of any aspect, the modified nucleotide is not 7-deazaadenosine, N1-methyladenosine, N6-methyladenosine, 6-chloropurine nucleoside, 2-amino-6-chloropurine nucleoside, 2-aminoadenosine, 5-methoxycytidine, 5-formylcytidine, 5-aminoallylcytidine, 5-hydroxycytidine, isoguanosine, thienoguanosine, 2-aminopurine-nucleoside, 8-oxoguanosine, 5-carboxymethyluridine, thienouridine, 5-methoxyuridine, 5-carboxyuridine, 2-thiouridine, N1-propylpseudouridine, N1-methoxymethylpseudouridine, N1-ethylpseudouridine, pseudouridine or N1-methylpseudouridine.

[0283] In some embodiments of any aspect, the saRNA described herein is substituted with a template of SEQ ID NO: 2, 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 about 98%, at least about 99%, or 100% identical to SEQ ID NO: 2 and maintains the same function (e.g., self-replication), or a codon-optimized version thereof. In some embodiments of any aspect, at least one cargo is inserted between the AflII and NdeI cleavage sites of SEQ ID NO: 2. In some embodiments of any aspect, at least one cargo is inserted between nucleotides 7627 and 7628 of SEQ ID NO: 2.

[0284] In some embodiments of any aspect, the saRNA described herein is substituted with a 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 about 98%, at least about 99%, or 100% identical to SEQ ID NO: 5 and maintains the same function (e.g., self-replication), or a codon-optimized version thereof.

[0285] In some embodiments of any aspect, the pyrimidine comprises cytidine, and the modified nucleotide comprises 5-methylcytidine and / or 5-hydroxymethylcytidine, 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 about 98%, at least about 99%, or 100% identical to SEQ ID NO: 6 and maintains the same function (e.g., self-replication), or a codon-optimized version thereof.

[0286] In some embodiments of any aspect, the pyrimidine comprises uridine, and the modified nucleotides comprise 5-methyluridine and / or 5-hydroxymethyluridine, 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 about 98%, at least about 99%, or 100% identical to SEQ ID NO: 7 and maintains the same function (e.g., self-replication), or a codon-optimized version thereof. Cap and start nucleotides

[0287] In various aspects, described herein are saRNAs comprising a 5' cap found at the 5' end of the saRNA molecule. In some embodiments of any aspect, the 5' cap is derived from at least one virus. In some embodiments of any aspect, the 5' cap is derived from at least one alphavirus. In some embodiments of any aspect, the 5' cap is selected from the group consisting of cap-0, cap-1, and cap-2. The 5' cap may be involved in translation, nucleocytoplasmic transport, splicing, and / or stabilization against 5' exonucleolytic degradation of the saRNA.

[0288] In eukaryotes, a 5' cap (called cap-0) is found at the 5' end of the RNA molecule. Cap-0 consists of a guanine nucleotide linked to the mRNA by a 5' to 5' triphosphate linkage. This guanine nucleotide is methylated at position 7 by a methyltransferase immediately after capping in vivo. Cap-0 may also be referred to as a 7-methylguanylate cap, abbreviated as m7G. In some embodiments of any aspect, the saRNA described herein comprises cap-0. In some embodiments of any aspect, the saRNA described herein comprises an m7G 5' cap. ARCA (anti-reverse cap analog) consisting of 3'-O-Me-m7G(5')ppp(5')G is a non-limiting example of a reagent for generating a saRNA comprising 5'Cap0.

[0289] In multicellular eukaryotes and some viruses, there are further 5' cap modifications, including methylation of the 2' hydroxyl groups of the first two ribose sugars at the 5' end of the RNA. Cap-1 has a methylated 2' hydroxyl group on the first ribose sugar, while cap-2 has methylated 2' hydroxyl groups on the first two ribose sugars. In some embodiments of any aspect, the starting nucleoside of the saRNA described herein is methylated at the 2'O position of the ribose sugar (Cap1). In some embodiments of any aspect, Cap1 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 a saRNA comprising 5' Cap1. In some embodiments of any aspect, both the starting nucleotide and the subsequent nucleotides of the saRNA described herein are methylated at the 2'O position of the ribose sugar (Cap2). In some embodiments of any aspect, Cap2 has the following chemical structure: m7GpppNmNm.

[0290] In some embodiments of any aspect, the first nucleotide immediately adjacent to the 5' cap of the saRNA described herein comprises adenosine or an adenosine analog. In some embodiments of any aspect, the first nucleotide immediately adjacent to the 5' cap of the saRNA described herein comprises guanosine or a guanosine analog. In some embodiments of any aspect, the first two nucleotides immediately adjacent to the 5' cap of the saRNA described herein comprise adenosine or an adenosine analog at position 1, and uridine or a uridine analog at position 2. In some embodiments of any aspect, the first three nucleotides immediately adjacent to the 5' cap of the saRNA described herein comprise guanosine or a guanosine analog at position 1, adenosine or an adenosine analog at position 2, and uridine or a uridine analog at position 3.

[0291] In some embodiments of any of the aspects, the starting nucleotide of the saRNA described herein comprises an adenosine or an adenosine analog, and the starting nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap 1).

[0292] In some embodiments of any of the aspects, the initial nucleotide of the saRNA described herein comprises an adenosine or an adenosine analog, and both the initial and subsequent nucleotides of the saRNA are methylated at the 2'O position of the ribose sugar (Cap2).

[0293] In some embodiments of any of the aspects, the starting nucleotide of the saRNA described herein comprises guanosine or a guanosine analog, and wherein the starting nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap 1).

[0294] In some embodiments of any of the aspects, the initial nucleotide of the saRNA described herein comprises guanosine or a guanosine analog, and wherein both the initial nucleotide and the subsequent nucleotides of the saRNA are methylated at the 2'O position of the ribose sugar (Cap2).

[0295] In some embodiments of any of the aspects, the starting nucleotide of the saRNA described herein comprises adenosine or an adenosine analog, and the 5' cap is m7G 5'cap-0. In some embodiments of any of the aspects, the starting nucleotide of the saRNA described herein comprises guanosine or a guanosine analog, and the 5' cap is m7G 5'cap-0. Non-limiting examples of nucleic acid modifications

[0296] It is further contemplated herein that the nucleic acids described herein (e.g., DNA, saRNA) can be chemically modified to enhance stability or other beneficial features. The nucleic acids described herein can be synthesized and / or modified by methods well known in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, SL et al. (ed.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Modifications include, for example, (a) end modifications, such as 5' end modifications (phosphorylation, conjugation, reverse linking, etc.), 3' end modifications (conjugation, DNA nucleotides, reverse linking, etc.); (b) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and (c) backbone modifications, including modification or substitution of phosphodiester linkages. Specific examples of nucleic acid compounds useful in the embodiments described herein include, but are not limited to, nucleic acids containing modified backbones or non-natural internucleoside linkages. Nucleic acids with modified backbones particularly include those that do not contain phosphorus atoms in the backbone. For the purposes of this application document, and as sometimes referred to in the art, modified nucleic acids that do not contain a phosphorus atom in the internucleoside backbone can also be considered oligonucleosides. In some embodiments of any aspect, the modified nucleic acid can have a phosphorus atom in its internucleoside backbone.

[0297] Modified nucleic acid backbone can comprise for example phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonate (comprising 3 '-alkylene phosphonate and chiral phosphonate), phosphinate, phosphoramidate (comprising 3 '-amino phosphoramidate and aminoalkyl phosphoramidate), thiocarbonyl phosphoramidate, thiocarbonyl alkyl phosphonate, thiocarbonyl alkyl phosphotriester and the borane phosphate with normal 3 '-5 ' link, these 2 '-5 ' link analogs and those with reversed polarity (wherein adjacent nucleoside unit is to with 3 '-5 ' to 5 '-3 ' or 2 '-5 ' to 5 '-2 ' link).Also comprise various salts, mixed salts and free acid form.The modified nucleic acid backbone that does not contain phosphorus atom has the skeleton that is formed by linking between short chain alkyl or cycloalkyl nucleoside, mixing heteroatoms and alkyl or cycloalkyl nucleoside, or linking between one or more short chain heteroatoms or heterocycle nucleoside. These include backbones with morpholino linkages (formed in part by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; olefin-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; other oligonucleosides with mixed N, O, S and CH2 components, as well as heteroatom backbones, particularly - -CH2--NH--CH2--, --CH2--N(CH3)--O--CH2-- [called methylene (methylimino) or MMI skeleton], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- [where the natural phosphodiester skeleton is represented as --O--P--O--CH2--].

[0298] In other nucleic acid mimics, the sugar and internucleoside linkages (i.e., backbones) of the nucleotide units are replaced by new groups. The base unit is retained to hybridize with a suitable nucleic acid target compound. A type of such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is referred to as peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone (particularly an aminoethylglycine backbone). The core base is retained and is bound directly or indirectly to the nitrogen atoms of the amide portion of the backbone.

[0299] Nucleic acids can also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribose moieties, wherein the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo structural conformation. It has been shown that adding locked nucleic acids to siRNA improves the stability of siRNA in serum and reduces off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, Or. et al., (2007) Mol. Canc. Ther. 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0300] The modified nucleic acids may also contain one or more substituted sugar moieties. The nucleic acids described herein may comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl groups. Exemplary suitable modifications include O[(CH2) n O]mCH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) nCH3)]2, wherein n is 1 to about 10. In some embodiments of any aspect, the nucleic acid comprises at the 2' position one of the following: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of nucleic acid, or group for improving the pharmacodynamic properties of nucleic acid, as well as other substituents with similar properties. In some embodiments of any aspect, the modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., an O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the Examples below; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, also described in the Examples below.

[0301] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropyloxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the nucleic acid, particularly at the 3' position of the sugar of the 3' terminal nucleotide, or in 2'-5' linked dsRNAs, and at the 5' position of the sugar of the 5' terminal nucleotide. Nucleic acids can also have sugar mimetics (e.g., cyclobutyl moieties) in place of the pentofuranosyl sugar.

[0302] The preparation of the above-mentioned modified nucleic acids, backbones and sugars is well known in the art.

[0303] Another modification of the nucleic acids described herein involves chemically linking the nucleic acids to one or more ligands, moieties, or conjugates to enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the nucleic acids. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acids. Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20: 533-538), aliphatic chains, for example dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10: 1111-1118; Kabanov et al., FEBS Lett., 1990, 259: 327-330; Svinarchuk et al., Biochimie, 1993, 75: 49-54), phospholipids, for example di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937). Exemplary viral components

[0304] In some embodiments of any aspect, at least one component of the saRNA described herein is derived from at least one virus. As a non-limiting example, at least one nonstructural protein, a subgenomic promoter (SGP), a 5' conserved sequence element (5'CSE), a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), and / or at least one 3' conserved sequence element (3'CSE) may be derived from at least one virus. For example, in some embodiments, the saRNA described herein can self-replicate due to the inclusion of conserved sequence elements (CSEs) derived from at least one virus, located at the 5' and 3' ends of the RNA, in combination with a protein machinery (RNA-dependent RNA polymerase or RdRp derived from at least one virus). In some embodiments, the saRNA may also include amplification of subgenomic RNA from a subgenomic promoter (SGP) derived from a virus that is recognized by the RdRp, which may encode a cargo of interest.

[0305] As used herein, the term "derived from" refers to origin or source and can include naturally occurring, recombinant, unpurified or purified molecules, such as nucleic acids or polypeptides. In some embodiments of any aspect, "derived from" includes mutation and / or maturation of any nucleic acid or polypeptide described herein. As a non-limiting example, at least one of the nonstructural proteins (nsp1-4) each derived from at least one virus can be mutated and / or matured compared to the wild-type sequence, for example, to increase the amount and / or duration of expression of the cargo. Non-limiting examples of non-structural protein mutations include: nsP2 A1979G (nucleic acid), G656G (amino acid); nsP2 G3936C (nucleic acid), G1309R (amino acid); nsP3 A4311G (nucleic acid), K1434E (amino acid); nsP3 A4758G (nucleic acid), S1583G (amino acid); nsP3 G4796T (nucleic acid), E1595D (amino acid); and / or nsP3 G4944A (nucleic acid), V1645M (amino acid). As another non-limiting example, at least one of the non-coding conserved sequence elements (e.g., 5'UTR, 5'CSE, SGP, 3'CSE, 3'UTR), each derived from at least one virus, can be mutated and / or matured compared to the wild-type sequence, for example, to increase the replication rate of the RNA. For example, at least one of the non-coding conserved sequence elements (e.g., 5'UTR) can be mutated or matured to include an A-rich region, which can accelerate RNA replication. See, for example, Li et al., Scientific Reports Vol. 9, Article No. 6932 (2019); Perkovic et al., Molecular Therapy Vol. 31, No. 6, P1636-1646 (2023); the contents of which are incorporated herein by reference in their entirety.

[0306] In some embodiments of any aspect, the nsp1, nsp2, nsp3, nsp4, SGP, 5'UTR, 3'UTR, 5'CSE and / or 3'CSE in the saRNA described herein are derived from the same virus. In some embodiments of any aspect, at least one of the nsp1, nsp2, nsp3, nsp4, SGP, 5'UTR, 3'UTR, 5'CSE and / or 3'CSE in the saRNA described herein are derived from different viruses.

[0307] In some embodiments of any aspect, the component (e.g., RNA or polypeptide) derived from at least one virus comprises a component (e.g., RNA or polypeptide) naturally occurring in a virus, or 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 about 98%, at least about 99%, or 100% identical to a component (e.g., RNA or polypeptide) naturally occurring in a virus.

[0308] In some embodiments of any aspect, the component (e.g., RNA or polypeptide) derived from at least one virus maintains the function of the component (e.g., RNA or polypeptide) naturally occurring in the virus, or maintains 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 about 98%, at least about 99%, or 100% of the function of the component (e.g., RNA or polypeptide) naturally occurring in the virus.

[0309] The phrase "derived from at least one virus" can include any virus. Non-limiting examples of such viruses include: Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), Sindbis virus (SIN), Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Mayaro virus (MAYV), Geta virus (GETV), Ross River virus (RRV), Una virus (UNAV), Middleburg virus (MIDV), Arara virus (ONNV), Bama Forest virus (BFV), Mucambo virus (MUCV), Tonate virus (TONV), Everglades virus (EVEV), Rio Negro virus (RNV), Turnip bush virus (TROV), Highlands J virus (HJV), Western equine encephalitis virus (WEEV), Fig mosaic virus (FMV), Aura virus (AURAV), Kunjin virus (KUN), measles virus (MV), coronavirus (CoV), rabies virus (RABV), and vesicular stomatitis virus (VSV).

[0310] In some embodiments of any aspect, at least one component of the saRNA described herein is derived from at least one alphavirus. Non-limiting examples of such alphaviruses include: Aura virus (AURAV), Bama Forest virus (BFV), Bebaru virus, Caaingua virus, Cabassou virus, Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Eliat virus, Everglades virus (EVEV), Fort Morgan virus, Geta virus (GETV), Highlands J virus (HJV), Madariaga virus, Mayaro virus (MAYV), Middleburg virus (MIDV), Mosso das Pedras virus, Mucambo virus (MUCV), Ndumu virus, Arliang virus (ONNV), Pixuna virus, Rio de Janeiro virus (RIV), Negro virus (RNV), Ross River virus (RRV), salmon pancreatic disease virus, Semliki Forest virus (SFV), Sindbis virus (SIN), Southern elephant seal virus, Tonate virus (TONV), Trocara virus, Una virus (UNAV), Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus (WEEV), and Whataroa virus. In some embodiments of any aspect, at least one component of the saRNA described herein is derived from Venezuelan equine encephalitis virus (VEEV).

[0311] Alphavirus is a genus of RNA viruses, the only genus in the Togaviridae family. Alphaviruses belong to group IV of the Baltimore Virus Classification and have positive-sense, single-stranded RNA genomes. Alphaviruses are small, spherical, enveloped viruses with single-stranded, positive-sense RNA genomes. The total genome length ranges from 11,000 to 12,000 nucleotides and possesses a 5' cap and a 3' poly-A tail. The four nonstructural protein genes are encoded in the 5'-terminal two-thirds of the genome, while the three structural proteins are translated from subgenomic mRNAs colinear with the 3'-terminal third of the genome. The genome contains two open reading frames (ORFs), one nonstructural and one structural. The first is nonstructural and encodes proteins (nsP1-nsP4) essential for viral RNA transcription and replication. The second encodes three structural proteins: the core nucleocapsid protein C, and the envelope proteins P62 and E1.

[0312] In some embodiments of any aspect, the saRNA described herein does not comprise structural proteins derived from at least one virus. In some embodiments of any aspect, the saRNA described herein does not comprise structural proteins derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein does not comprise structural proteins derived from VEEV (e.g., does not comprise C, P62, or E1).

[0313] In some embodiments of any aspect, the saRNA described herein does not comprise a capsid protein or envelope protein derived from at least one virus. In some embodiments of any aspect, the saRNA described herein does not comprise a capsid protein or envelope protein derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein does not comprise a capsid protein or envelope protein derived from VEEV. Nonstructural proteins

[0314] In some embodiments of any aspect, the saRNA described herein comprises at least one nonstructural protein derived from at least one virus. In some embodiments of any aspect, the saRNA described herein comprises at least one nonstructural protein derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein comprises at least one nonstructural protein derived from VEEV. Alphavirus nonstructural proteins can be selected from four nonstructural proteins (nsP1-4), which are produced in the form of a single polyprotein that constitutes the replication machinery of the virus. For example, in VEEV and other alphaviruses, nsp1 is a methyl / guanylyltransferase involved in RNA capping; nsp2 is a cysteine ​​protease, helicase, and NTPase; nsp3 is a poly ADP-ribose hydroxylase; and nsp4 is an RNA-dependent RNA polymerase (RdRp). In some embodiments of any aspect, the saRNA described herein comprises at least one nonstructural protein that functions as an RNA-dependent RNA polymerase (RdRp) and allows saRNA replication (e.g., +RNA to -RNA to +RNA) and / or the production of subgenomic RNA (e.g., from -RNA to sgRNA using SGP).

[0315] In some embodiments of any aspect, the saRNA described herein comprises nsP1 derived from an alphavirus, nsP2 derived from an alphavirus, nsP3 derived from an alphavirus, and / or nsP4 derived from an alphavirus, or any combination thereof. For example, the saRNA described herein may comprise any combination of the nonstructural proteins shown in Table 5, each of which is derived from an alphavirus, which may be of the same or different species. As a non-limiting example, the saRNA described herein may comprise nsP1 derived from an alphavirus, nsP2 derived from an alphavirus, nsP3 derived from an alphavirus, and nsP4 derived from an alphavirus. In some embodiments of any aspect, the saRNA described herein comprises, from 5' to 3', nsP1, nsP2, nsP3, and nsP4, each of which is derived from an alphavirus species, each of which may be of the same or different species. In some embodiments of any aspect, the 5' region of the saRNA described herein comprises, from 5' to 3', nsP1, nsP2, nsP3, and nsP4, each of which is derived from an alphavirus species, each of which may be of the same or different species.

[0316] Table 5: Exemplary non-structural protein combinations in saRNAs described herein

[0317] In some embodiments of any aspect, the saRNA described herein comprises nsP1 derived from a VEEV strain, nsP2 derived from a VEEV strain, nsP3 derived from a VEEV strain, and / or nsP4 derived from a VEEV strain, or any combination thereof. For example, the saRNA described herein may comprise any combination of the non-structural proteins shown in Table 5, each of which is derived from a VEEV strain, each of which may be the same or a different VEEV strain. As a non-limiting example, the saRNA described herein may comprise nsP1 derived from a VEEV strain, nsP2 derived from a VEEV strain, nsP3 derived from a VEEV strain, and nsP4 derived from a VEEV strain. In some embodiments of any aspect, the saRNA described herein comprises, from 5' to 3', nsP1, nsP2, nsP3, and nsP4, each of which is derived from a VEEV strain, each of which may be the same or a different VEEV strain. In some embodiments of any of the aspects, the 5' region of the saRNA described herein comprises, from 5' to 3', nsP1, nsP2, nsP3, and nsP4, each of which is derived from a VEEV strain, which can each be the same or a different VEEV strain.

[0318] In some embodiments of any aspect, the saRNA described herein encodes the amino acid sequence of SEQ ID NO: 4 and / or SEQ ID NO: 23, or an amino 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 about 98%, at least about 99%, or 100% identical to SEQ ID NO: 4 and / or SEQ ID NO: 23 and retains the function thereof (e.g., at least one nsP1-nsP4 function; e.g., saRNA replication; e.g., RNA-dependent RNA polymerase).

[0319] In some embodiments of any aspect, the saRNA described herein comprises a non-structural protein comprising one of nucleotides 62-7543 of SEQ ID NO: 2 (DNA or corresponding RNA sequence), nucleotides 62-7543 of SEQ ID NO: 5, nucleotides 45-7526 of SEQ ID NO: 6, nucleotides 45-7526 of SEQ ID NO: 7, or a combination of nucleotides 62-7543 of SEQ ID NO: 2 (DNA or corresponding RNA sequence), nucleotides 62-7543 of SEQ ID NO: 5, nucleotides 45-7526 of SEQ ID NO: 6, nucleotides 45-7526 of SEQ ID NO: 7. 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 about 98%, at least about 99%, or 100% identical to one of nucleotides 45-7526 of NO:7, or the corresponding RNA sequence of a codon-optimized version thereof. Conserved sequence elements

[0320] In some embodiments of any aspect, the saRNA described herein comprises at least one conserved sequence element (CSE) derived from at least one virus. In some embodiments of any aspect, the saRNA described herein comprises at least one conserved sequence element (CSE) derived from at least one alphavirus. In some embodiments of any aspect, the conserved sequence element is conserved between multiple alphavirus species. In some embodiments of any aspect, the saRNA described herein comprises at least one conserved sequence element (CSE) derived from VEEV. In some embodiments of any aspect, the conserved sequence element can interact with the RdRp (e.g., nsP1-4) encoded by the saRNA and allow saRNA replication. In some embodiments of any aspect, the conserved sequence element comprises a secondary structure (e.g., a hairpin structure) for interacting with the RdRp (e.g., nsP1-4) encoded by the saRNA. In some embodiments of any aspect, the conserved sequence element is non-coding, i.e., does not encode a polypeptide and is not translated.

[0321] As a non-limiting example, analysis of alphavirus genomic sequences has identified four sequence elements (CSEs) that are conserved across genera: a 5' untranslated region (UTR), a 51nt 5'CSE within nsP1, a subgenomic promoter (or junction) region, and a 3'CSE within the 3'UTR. In some embodiments of any aspect, the saRNA described herein comprises at least one of the following conserved sequence elements, each of which is derived from at least one alphavirus: a 5'UTR, a 5'CSE, an SGP, and / or a 3'CSE within the 3'UTR. In some embodiments of any aspect, the saRNA described herein comprises a 5'UTR, a 5'CSE, an SGP, and / or a 3'CSE within the 3'UTR, each of which is derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein comprises a 5'UTR, a 5'CSE, an SGP, and / or a 3'CSE within the 3'UTR, each of which is derived from VEEV. See, eg, Hyde et al., “The 5′ and 3′ ends of alphavirus RNAs—Non-coding is not non-functional,” Virus Research 206 (2015): 99-107, the contents of which are incorporated herein by reference in their entirety.

[0322] In some embodiments of any aspect, the saRNA described herein comprises at least one 5' conserved sequence element (5'CSE) derived from at least one virus. In some embodiments of any aspect, the saRNA described herein comprises at least one 5' conserved sequence element (5'CSE) derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein comprises at least one 5' conserved sequence element (5'CSE) derived from VEEV.

[0323] A conserved 5' sequence element (5'CSE) may be included in the nsp1 coding sequence. The conserved 5' sequence element (5'CSE) may be about 51 nucleotides (nt) long, or about 40-60 nt long. The 5'CSE may depend on the viral machinery used to generate saRNA (e.g., nsP1-4). For VEEV, the 5'CSE may comprise: aagcaggtcactgataatgaccatgctaatgccagagcgtttcgcatctg, SEQ ID NO: 10 (see, e.g., nucleotides 149-199 of SEQ ID NO: 2, nucleotides 149-199 of SEQ ID NO: 5, nucleotides 132-182 of SEQ ID NO: 6-7).

[0324] In some embodiments of any aspect, the saRNA described herein comprises at least one 3' conserved sequence element (3'CSE) derived from at least one virus. In some embodiments of any aspect, the saRNA described herein comprises at least one 3' conserved sequence element (3'CSE) derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein comprises at least one 3' conserved sequence element (3'CSE) derived from VEEV.

[0325] A conserved 3'-conserved sequence element (3'CSE) can be encoded in the 3'UTR. The conserved 3' sequence element (3'CSE) can be about 70 nucleotides (nt) long, or about 60-80 nt long. The 3'CSE can rely on the viral machinery encoded in the saRNA (e.g., nsP1-4) for self-replication. For VEEV, the 3'CSE is located in the 3'UTR sequence and may comprise: catgccgccttaaaatttttattttattttttcttttcttttccgaatcggattttgtttttaatatttc, SEQ ID NO: 9 (see, e.g., nucleotides 7673-7742 of SEQ ID NO: 2, nucleotides 8393-8462 of SEQ ID NO: 5, nucleotides 8376-8445 of SEQ ID NO: 6-SEQ ID NO: 7). The 3'CSE is highly conserved in at least 27 alphavirus genomes and can contain 85% to 90% AU-rich sequences.

[0326] In some embodiments of any aspect, the conserved sequence element comprises SEQ ID NO: 9, SEQ ID NO: 10, 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 about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9 or SEQ ID NO: 10 and retains its function (e.g., at least one nsP1-nsP4 function; e.g., saRNA replication; e.g., an RNA-dependent RNA polymerase).

[0327] In some embodiments of any aspect, the saRNA described herein comprises at least one 5' conserved sequence element (5'CSE) and at least one 3' conserved sequence element (3'CSE) each derived from at least one virus. In some embodiments of any aspect, the saRNA described herein comprises at least one 5' conserved sequence element (5'CSE) and at least one 3' conserved sequence element (3'CSE) each derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein comprises at least one 5' conserved sequence element (5'CSE) and at least one 3' conserved sequence element (3'CSE) each derived from VEEV.

[0328] In some embodiments of any aspect, the saRNA described herein comprises at least one subgenomic promoter (SGP) derived from at least one virus. In some embodiments of any aspect, the SGP is 3' (downstream) of the at least one nonstructural protein and 5' (upstream) of the 5' UTR and at least one cargo. In some embodiments of any aspect, the saRNA described herein comprises at least one subgenomic promoter (SGP) derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein comprises at least one subgenomic promoter (SGP) derived from VEEV.

[0329] The subgenomic promoter (SGP) can be located at the end of nsp4 and contain sequences within and after the nsp4 coding sequence. The SGP can rely on viral machinery (e.g., nsP1-4; e.g., RdRP) to generate subgenomic RNA (sgRNA) of the saRNA. The subgenomic promoter controls the expression of the sgRNA from the antisense template RNA, independent of its genomic length counterpart. In some embodiments of any aspect, the strength of the subgenomic promoter (SGP) produces more coated sgRNA compared to the full-length +RNA. In the context of alphaviruses, a higher concentration of sgRNA compared to the full-length +RNA can allow for a higher concentration of translated structural proteins compared to translated non-structural proteins. In the context of the saRNA described herein, a higher concentration of sgRNA compared to the full-length +RNA can allow for a higher concentration of the at least one cargo (which can be a translated protein or non-coding RNA, as further described herein) compared to translated non-structural proteins.

[0330] The sequence of SGP in VEEV can comprise: SEQ ID NO: 11, agcttggcaaacctctggcagcagacgatgaacatgatgatgatgacaggagaagggcattgcatgaagagtcaacacgctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaaaccgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaatcattcagctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctagtccgccaag (see, e.g., nucleotides 7308-7578 of SEQ ID NO: 2; see, e.g., nucleotides 7308-7578 of SEQ ID NO: 5; see, e.g., nucleotides 7291-7561 of SEQ ID NO: 6-SEQ ID NO: 7).

[0331] In some embodiments of any aspect, the subgenomic promoter (SGP) comprises SEQ ID NO: 11, 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 about 98%, at least about 99%, or 100% identical to SEQ ID NO: 11 and retains its function (e.g., replication of the subgenomic RNA (sgRNA); e.g., replication of at least one cargo encoded by the saRNA).

[0332] In some embodiments of any aspect, the saRNA described herein comprises at least one 5' conserved sequence element (5'CSE), at least one 3' conserved sequence element (3'CSE), and at least one subgenomic promoter (SGP), each derived from at least one virus. In some embodiments of any aspect, the saRNA described herein comprises at least one 5' conserved sequence element (5'CSE), at least one 3' conserved sequence element (3'CSE), and at least one subgenomic promoter (SGP), each derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein comprises at least one 5' conserved sequence element (5'CSE), at least one 3' conserved sequence element (3'CSE), and at least one subgenomic promoter (SGP), each derived from VEEV.

[0333] In some embodiments of any aspect, the saRNA described herein comprises at least one 5' untranslated region (5'UTR) derived from at least one virus. In some embodiments of any aspect, the 5'UTR is 3' (downstream) of the SGP and 5' (upstream) of the at least one SGP. In some embodiments of any aspect, the saRNA described herein comprises at least one 5' untranslated region (5'UTR) derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein comprises at least one 5' untranslated region (5'UTR) derived from VEEV.

[0334] In some embodiments of any aspect, the saRNA described herein comprises at least one 3' untranslated region (3'UTR) derived from at least one virus. In some embodiments of any aspect, the 3'UTR is 3' (downstream) of the at least one cargo and 5' (upstream) of the poly-A tail. In some embodiments of any aspect, the saRNA described herein comprises at least one 3' untranslated region (3'UTR) derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein comprises at least one 3' untranslated region (3'UTR) derived from VEEV.

[0335] In some embodiments of any aspect, the saRNA described herein comprises at least one 5' untranslated region (5'UTR) and at least one 3' untranslated region (3'UTR) derived from at least one virus. In some embodiments of any aspect, the saRNA described herein comprises at least one 5' untranslated region (5'UTR) and at least one 3' untranslated region (3'UTR) derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein comprises at least one 5' untranslated region (5'UTR) and at least one 3' untranslated region (3'UTR) derived from VEEV. Exemplary goods

[0336] The saRNA described herein encodes and expresses at least one cargo of interest. In some embodiments of any aspect, the saRNA described herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cargoes. In some embodiments of any aspect, the at least one cargo is 3' (downstream) of the subgenomic promoter and 5' untranslated region (UTR), and 5' (upstream) of the 3' untranslated region (UTR) and polyA tail.

[0337] In some embodiments of any aspect, the cargo comprises at least one cargo protein. In some embodiments of any aspect, the cargo comprises at least one cargo peptide. In some embodiments of any aspect, the cargo comprises at least one cargo protein and at least one cargo peptide. In some embodiments of any aspect, the cargo comprises at least two cargo proteins. Non-limiting examples of methods for separating individual cargo proteins include: a self-cleaving peptide domain, an internal ribosome entry site (IRES), or a separate promoter (e.g., a subgenomic promoter) between each cargo protein. The self-cleaving peptide domain can be a 2A peptide, for example, selected from the group consisting of P2A, E2A, F2A, and T2A. IRES is an RNA element that allows translation to be initiated in a cap-independent manner.

[0338] In some embodiments of any aspect, the at least one cargo protein is derived from a virus, bacteria, protozoa, mammal, or plant. In some embodiments of any aspect, the at least one cargo peptide is derived from a virus, bacteria, protozoa, mammal, or plant.

[0339] In some embodiments of any aspect, the cargo comprises a chimeric antigen receptor (CAR). In some embodiments of any aspect, saRNA as described herein encodes and expresses a CAR selected from the group consisting of: (a) conventional CAR; (b) ON-CAR; (c) OFF-CAR system; (d) ON / OFF-CAR; (e) inhibitory CAR; or (f) split, universal, programmable and reconfigurable (SUPRA) CAR system. For further details on such exemplary CARs, see, for example, U.S. Patents 11059864 and 11530252; Li et al., Cancer Cell 40, 1–12 (2022); Li et al., Nat Med. 28 (10): 2133–2144 (2022); their respective contents are incorporated herein by reference in their entirety.

[0340] In some embodiments of any aspect, saRNA described herein encodes and expresses at least one conventional CAR. In some embodiments of any aspect, conventional CAR comprises (e.g., from 5' to 3', or from N-terminus to C-terminus): (a) an extracellular binding domain; (b) a transmembrane domain; and (c) at least one intracellular signaling domain.

[0341] In some embodiments of any aspect, the saRNA described herein encodes and expresses at least one ON-CAR. In some embodiments of any aspect, the ON-CAR comprises (e.g., from 5' to 3', or from N-terminus to C-terminus): (a) an extracellular binding domain; (b) a transmembrane domain; (c) at least one intracellular signaling domain; and (d) a repressible protease domain that cuts and degrades the ON-CAR in the absence of a protease inhibitor.

[0342] In some embodiments of any aspect, the saRNA described herein encodes and expresses at least one OFF-CAR system, or at least one component thereof. In some embodiments of any aspect, the OFF-CAR system comprises: (a) a first polypeptide comprising (e.g., from 5' to 3', or from N-terminus to C-terminus): (i) an extracellular binding domain; (ii) a transmembrane domain; and (iii) a peptide domain; and (b) a second polypeptide comprising (e.g., from 5' to 3', or from N-terminus to C-terminus): (i) a repressible protease domain that can specifically bind to the peptide domain in the absence of a protease inhibitor; and (ii) at least one intracellular signaling domain. In some embodiments of any aspect, at least one domain moves from the first polypeptide of the OFF-CAR system to the second polypeptide, or from the second polypeptide of the OFF-CAR system to the first polypeptide. As a non-limiting example, the first polypeptide may comprise a repressible protease, and the second polypeptide may comprise a peptide domain. In some embodiments of any aspect, the second polypeptide further comprises a transmembrane domain.

[0343] In some embodiments of any aspect, the peptide domain is selected from the group consisting of K5-66, K5-66-A, K5-66-B, K6-10, K6-10A, K6-10B, K5-66-R, CP5-46, CP5-46-4D5E, CP5-46-A, CP5-46A-4D5E, Ant-CP5-46A-4D5E and apo NS3a reader (ANR) peptide.

[0344] In some embodiments of any aspect, the saRNA described herein encodes and expresses at least one ON / OFF-CAR. In some embodiments of any aspect, the ON / OFF-CAR comprises (e.g., from 5' to 3', or from N-terminus to C-terminus): (a) an extracellular binding domain; (b) a transmembrane domain; (c) at least one intracellular signaling domain; (d) a repressible protease domain that cuts and degrades the ON / OFF-CAR in the absence of a protease inhibitor; and (e) a drug-inducible degron domain.

[0345] In some embodiments of any aspect, the repressible protease domain comprises hepatitis C virus (HCV) nonstructural protein 3 (NS3). In some embodiments of any aspect, the NS3 has catalytic activity (e.g., ON-CAR, ON / OFF CAR). In some embodiments of any aspect, the NS3 is catalytically inactive (i.e., ineffective; e.g., OFF-CAR). For NS3, the catalytic triad can comprise His-57, Asp-81, and Ser-139. Therefore, the catalytically inactive NS3 protease can comprise a non-synonymous mutation of any one of His-57, Asp-81, and Ser-139, or other inactivating mutations as described herein. In some embodiments of any aspect, the catalytically inactive N33 protease comprises an S139A mutation.

[0346] In some embodiments of any aspect, CAR described herein includes at least one protease cleavage site. As used herein, the term "protease cleavage site" refers to a specific sequence or sequence motif that is recognized and cut by a repressible protease. The cleavage site of the protease is included in a specific amino acid sequence or motif that is recognized by the protease during the proteolytic cleavage process, and is generally included in 1 to 6 amino acids around either side of the scissile bond, which is bound to the active site of the protease and is used to be recognized as a substrate. In some embodiments of any aspect, the protease cleavage site can be any site that can be specifically bound and cut by a repressible protease. In some embodiments of any aspect, the CAR polypeptide described herein (or CAR polypeptide system as a whole) includes 1, 2, 3, 4, 5 or more protease cleavage sites. In some embodiments of any aspect, the CAR polypeptide includes two protease cleavage sites. In an embodiment comprising multiple protease cleavage sites, the multiple protease cleavage sites can be different separate protease cleavage sites or multiple copies of the same protease cleavage site, or a combination of the foregoing.

[0347] As a non-limiting example, during HCV replication, the NS3-4A serine protease is responsible for the proteolytic cleavage of the four junctions of the HCV polyprotein precursor: NS3 / NS4A (self-cleavage), NS4A / NS4B, NS4B / NS5A, and NS5A / NS5B. Thus, the protease cleavage site of the CAR polypeptide described herein can be a NS3 / NS4A cleavage site, a NS4A / NS4B cleavage site, a NS4B / NS5A cleavage site, or a NS5A / NS5B cleavage site.

[0348] In some embodiments of any aspect, the CAR polypeptide as described herein is combined with a protease inhibitor. As used herein, "with ... combination" refers to two or more substances being present in the same preparation in any molecular arrangement or physical arrangement (e.g., as an admixture, as a solution, as a mixture, as a suspension, as a colloid, as an emulsion). The preparation may be a homogeneous or heterogeneous mixture. In some embodiments of any aspect, the active compound may be included by a superstructure (e.g., nanoparticles, liposomes, carriers, cells, scaffolds, etc.), which is in the form of a solution, a mixture, an admixture, a suspension, etc. together with a CAR polypeptide or a CAR polypeptide system. In some embodiments of any aspect, the CAR polypeptide is bound to a protease inhibitor, which is bound to a repressible protease. In some embodiments of any aspect, the CAR polypeptide specifically binds to a protease inhibitor, which is bound to a repressible protease.

[0349] In some embodiments of any aspect, the CAR polypeptide is combined with 1, 2, 3, 4, 5 or more protease inhibitors. In some embodiments of any aspect, the CAR polypeptide is combined with one protease inhibitor. In embodiments comprising multiple protease inhibitors, the multiple protease inhibitors can be different individual protease inhibitors or multiple copies of the same protease inhibitor, or a combination of the foregoing.

[0350] In some embodiments of any aspect, the protease inhibitor is grazoprevir (abbreviated as GZV or GZP; see, e.g., PubChem CID: 44603531). In some embodiments of any aspect, the protease inhibitor is danoprevir (DNV; see, e.g., PubChem CID: 11285588). In some embodiments of any aspect, the protease inhibitor is an approved NS3 protease inhibitor, such as, but not limited to, grazoprevir, danoprevir, simeprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir. Additional non-limiting examples of NS3 protease inhibitors are listed in McCauley and Rudd, Hepatitis C virus NS3 / 4a protease inhibitors, Current Opinion in Pharmacology 2016, 30:84–92; the contents of which are herein incorporated by reference in their entirety.

[0351] In some embodiments of any aspect, the drug-inducible degron domain comprises an IKAROS family zinc finger 3 (IKZF3) domain, which can be bound and activated by the drug lenalidomide or pomalidomide to degrade CAR.

[0352] In some embodiments of any aspect, saRNA as described herein encodes and expresses at least one inhibitory CAR. In some embodiments of any aspect, inhibitory CAR includes (for example, from 5' to 3', or from N-terminus to C-terminus): (a) extracellular binding domain; (b) transmembrane domain; and (c) inhibitory domain.

[0353] In some embodiments of any aspect, the inhibitory domain comprises a killer cell inhibitory receptor (KIR) domain. In some embodiments of any aspect, the inhibitory domain comprises at least one immunoreceptor tyrosine-based inhibitory motif (ITIM). In some embodiments, ITIM comprises S / I / V / LxYxxI / V / L, SEQ ID NO: 20, wherein x is any amino acid, Y is a tyrosine residue that can be phosphorylated, S is the amino acid serine, I is the amino acid isoleucine, and V is the amino acid valine. In some embodiments of any aspect, the inhibitory domain comprises an inhibitory domain (e.g., a domain comprising an ITIM) from FcγRIIB, CTLA-4, PD-1, BTLA, CD72, NKG2A, CD31, SIGLEC, CD66, ILT, or LIR.

[0354] In some embodiments of any aspect, the saRNA described herein encodes and expresses at least one SUPRA CAR system or at least one component thereof. In some embodiments of any aspect, the SUPRA CAR system comprises: (a) a first polypeptide comprising (e.g., from 5' to 3', or from N-terminus to C-terminus): (i) an extracellular binding domain; and (ii) a first member of an extracellular protein interaction domain; and (b) a second polypeptide comprising (e.g., from 5' to 3', or from N-terminus to C-terminus): (i) a second member of an extracellular protein interaction domain that can specifically bind to a first member of the extracellular protein interaction domain of the first polypeptide; (ii) a transmembrane domain; and (iii) at least one intracellular signaling domain.

[0355] In some embodiments of any aspect, at least one domain is moved from the first polypeptide of the SUPRA-CAR system to the second polypeptide, or from the second polypeptide of the SUPRA-CAR system to the first polypeptide. As a non-limiting example, the first polypeptide may include a second member of an extracellular protein interaction domain, and the second polypeptide may include a first member of an extracellular protein interaction domain. In some embodiments of any aspect, the first member and the second member of the extracellular protein interaction domain include a leucine zipper pair.

[0356] In some embodiments of any aspect, at least one intracellular signaling domain in the CAR described herein is selected from the group consisting of: TCRC; FcRy; FcRp; CD3zeta; CD3y; CD35; CD3s; CD3C; CD22; CD79a; CD79b; CD66d; CARD11; CD2; CD7; CD27; CD28; CD30; CD40; CD54 (ICAM); CD83; CD134 (OX40); CD137 (4-1BB); CD150 (SLAMF1); CD152 (CTLA4); CD223 (LAG3); CD270 (HVEM); CD273 (PD-L2); CD274 (PD-L1); CD278 (ICOS); DAP10; LAT; KD2C SLP76; TRIM; ZAP70; and 41BB. In some embodiments of any of the aspects, at least one intracellular signaling domain in the CAR described herein is derived from CD28 or CD3ζ.

[0357] In some embodiments of any aspect, the cargo comprises a CAR and at least one expression enhancing protein. In some embodiments of any aspect, the expression enhancing protein is B18R. In some embodiments of any aspect, the expression enhancing protein is E3L.

[0358] In some embodiments of any aspect, the extracellular binding domain of CAR as described herein includes the antigen binding domains from antibody.In some embodiments of any aspect, the extracellular binding domain of CAR as described herein includes single-chain variable fragment (scFv).

[0359] In some embodiments of any aspect, the cargo comprises at least one domain responsive to external input. In some embodiments of any aspect, the cargo comprises an antibody or a fragment thereof. In some embodiments of any aspect, the antibody is a bispecific antibody. In some embodiments of any aspect, the cargo comprises a bispecific T cell adapter (BiTE). In some embodiments of any aspect, the BiTE is a fusion protein comprising two antibody extracellular binding domains. For example, one arm of the BiTE comprises an extracellular binding domain specific for a protein (such as CD3) found on the surface of cytotoxic T cells, and the other arm of the BiTE comprises an extracellular binding domain specific for a specific protein (such as HER2 or other antigens described herein) present primarily on tumor cells. When both targets are engaged, the BiTE molecule forms a bridge between the cytotoxic T cells and the tumor cells, thereby enabling the T cells to recognize tumor cells and to combat tumor cells by infusing toxic molecules.

[0360] In some embodiments of any aspect, goods include the extracellular domain of the antigen of specific binding interest.In some embodiments of any aspect, the antigen of interest is specific to some cancer cells and / or is expressed up-regulated on some cancer cells. Non-limiting examples of the antigen of interest of cargo proteins (such as CAR, antibodies, BiTE, etc.) include: CD19, CD22, CD30, b cell maturation antigen (BCMA), disialoganglioside GD2, human estrogen receptor 2 (HER2), G protein coupled receptor 87 (GPR87), fibroblast activation protein (FAP), CD20, receptor tyrosine kinase-like orphan receptor 1 (ROR1), carcinoembryonic antigen (CEA), mesothelin (MSLN), prostate-specific membrane antigen (PSMA), epidermal growth factor receptor variant III (EGFRvIII), interleukin 13 receptor alpha 2 (IL13Rα2) and natural killer cell group 2 member D (NKG2D).

[0361] Additional non-limiting examples of tumor antigens that can be targeted include EphA2, HER2, AXL, GD2, Glypican-3, 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, fetal AchR, folate receptor alpha, GD2, GD3, HLA-AIMAGE Al, HLA-A2, IL1 1Ra, IL13Ra2, KDR, Lambda, Lewis-Y, MCSP, mesothelin, Mucl, Mucl6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, carcinoembryonic antigen, HMW-MAA and VEGF receptor. Other exemplary antigens that can be targeted are those present in the extracellular matrix of tumor cells, such as fibronectin, oncofetal variants of tenascin, or necrotic areas of tumors.

[0362] Additional tumor-selective molecules that can be targeted include any membrane protein or biomarker expressed or overexpressed in tumor cells, including but not limited to integrins (e.g., integrin αvβ3, α5β1), EGF receptor family (e.g., EGFR2, Erbb2 / HER2 / neu, Erbb3, Erbb4), proteoglycans (e.g., heparan sulfate proteoglycans), disialogangliosides (e.g., GD2, GD3), B7-H3 (also known as CD276), cancer antigen 125 (CA-125), epithelial cell adhesion molecule (EpCAM), vascular endothelial growth factor receptors 1 and 2 (VEG FR-1, VEGFR-2), CD52, carcinoembryonic antigen (CEA), tumor-associated glycoproteins (e.g., TAG-72), cluster of differentiation 19 (CD19), CD20, CD22, CD30, CD33, CD40, CD44, CD74, CD152, mucin 1 (MUC1), tumor necrosis factor receptor (e.g., TRAIL-R2), insulin-like growth factor receptor, folate receptor alpha, transmembrane glycoprotein NMB (GPNMB), CC chemokine receptors (e.g., CCR4), prostate-specific membrane antigen (PSMA), recepteurd'origine nantais (RON) receptor, cytotoxic T lymphocyte antigen 4 (CTLA4), and other tumor-specific receptors or antigens.

[0363] Non-limiting examples of tumor antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed proto-oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens generated by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA15-3\CA 27.29\BCAA, CA 195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP and TPS.

[0364] In some embodiments of any aspect, the tumor antigen is a tumor antigen described in International Application PCT / US2015 / 020606 or U.S. Patent Application No. US20170209492 or US20170335281, the contents of each of which are herein incorporated by reference in their entirety. In some embodiments, the tumor antigen is selected from one or more of the following: CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDG1cp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine Testisin or PRSS21; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface-associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100); proto-oncogene fusion protein composed of the breakpoint cluster region (BCR) and Abelson murine leukemia virus proto-oncogene homolog 1 (Ab1) (bcr-ab1); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1;Sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDG1cp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related protein (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5 member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); the hexasaccharide portion of GloboH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pan-connexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutants; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); rat sarcoma (Ras) mutants; human telomerase reverse transcriptase ( hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytic neoplasia viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC);Tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); pre-acrosomal protein-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma breakpoint X 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPVE7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); IgA receptor Fc fragment (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A, member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12, member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1). In some embodiments, the tumor antigen is GFRa4 (see, e.g., Spinasanta, “The Endocrine Society's 97th Annual Meeting & Expo: Targeted Therapies in Medullar Thyroid Cancer” Mar. 13, 2015).

[0365] In some embodiments of any aspect, the extracellular binding domain comprises an anti-Her2 antibody. HER2 (human epidermal growth factor receptor 2) is a gene that plays a role in the development of breast cancer. Cancers that may be HER2 positive include breast cancer, bladder cancer, pancreatic cancer, ovarian cancer, and gastric cancer. Non-limiting examples of anti-Her2 antibodies include G98A, C6.5, ML39, H3B1 (e.g., SEQ ID NO: 8, 41), scFv800E6, and trastuzumab. See, e.g., Rudnick et al. Cancer Res. 2011 Mar 15, 71(6): 2250–2259; Sommaruga et al. Appl Microbiol Biotechnol. 2011 Aug, 91(3): 613-21; U.S. Patents US 5977322, US 8580263, US 8703427, US 8927694, US 10188742, US 10239951, the contents of each of which are incorporated herein by reference in their entirety.

[0366] In some embodiments of any of the aspects, the extracellular binding domain comprises an anti-Axl antibody. AXL overexpression has been demonstrated in a variety of cancer types, such as breast cancer (Meric et al., Clin. Cancer Res. 8:361-367, 2002; Berclaz et al., Ann. Oncol. 12:819-824, 2001), colon cancer (Chen et al., Int. J. Cancer 83:579-584, 1999; Craven et al., Int. J. Cancer 60:791-797, 1995), prostate cancer (Jacob et al., Cancer Detect. Prey. 23:325-332, 1999), lung cancer (Wimmel et al., Eur J Cancer 37:2264-2274, 2001), gastric cancer (Wu et al., Anticancer Res 22:1071-1078, 2002), ovarian cancer (Sun et al., Oncology 54:1061-1068, 2003), and pancreatic cancer (Wu et al., Anticancer Res 54:1061-1068, 2003). 66:450-457, 2004), endometrial cancer (Sun et al., Ann. Oncol. 14:898-906, 2003), renal cancer (Chung et al., DNA Cell Biol. 22:533-540, 2003), hepatocellular carcinoma (Tsou et al., Genomics 50:331-340, 1998), thyroid cancer (Ito et al., Thyroid 12:971-975, 2002; Ito et al., Thyroid 9:563-567, 1999) and esophageal cancer (Nemoto et al., 1997), and in CML (Janssen et al., A novel putative tyrosine kinase receptor with oncogenic potential. Oncogene, 6:2113-2120, 1991; Braunger et al., Oncogene 14:2619-2631, 1997). 1997; O'Bryan et al., Mol Cell Biol 11:5016-5031, 1991), AML (Rochlitz et al., Leukemia 13:1352-1358, 1999), osteosarcoma (Nakano et al., J. Biol. Chem. 270:5702-5705, 2003), melanoma (van Ginkel et al., Cancer Res 64:128-134, 2004), and head and neck squamous cell carcinoma (Green et al., Br J. Cancer. 2006 94:1446-5, 2006). In addition, AXL has been identified as a metastasis-associated gene that is upregulated in invasive breast cancer cell lines compared to non-invasive cells.Non-limiting examples of anti-Axl antibodies include 11B7, 11D5, 10D12, and h#11B7-T18. See, for example, International Patent Application WO2010130751 or US Patent No. 8,841,424, the contents of each of which are incorporated herein by reference in their entirety.

[0367] In some embodiments of any aspect, the extracellular binding domain comprises an anti-CD19 antibody. Because CD19 is a marker for B cells, the protein has been used to diagnose cancers originating from such cells, particularly B cell lymphomas, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). Most B cell malignancies express normal to high levels of CD19. Non-limiting examples of anti-CD19 antibodies include A3B1, FMC63, FMC63-28Z, SEQ ID NO: 94; see, e.g., U.S. Patents 10,221,245, 8,906,682, 10,421,810, the contents of each of which are incorporated herein by reference in their entirety.

[0368] In some embodiments of any aspect, the cargo comprises a ligand, a cell surface receptor, a transcription factor, a cytokine, a chemokine, an enzyme, and / or an antibody. In some embodiments of any aspect, the cargo comprises at least one ligand. In some embodiments of any aspect, the cargo comprises at least one cell surface receptor. In some embodiments of any aspect, the cargo comprises at least one transcription factor. In some embodiments of any aspect, the cargo comprises at least one cytokine. In some embodiments of any aspect, the cargo comprises at least one chemokine. In some embodiments of any aspect, the cargo comprises at least one enzyme. In some embodiments of any aspect, the cargo comprises at least one antibody.

[0369] In some embodiments of any aspect, the cargo comprises at least one non-coding RNA. For example, the non-coding RNA can be selected from the group consisting of short interfering RNA (siRNA), small hairpin RNA (shRNA), and microRNA. In some embodiments of any aspect, the cargo comprises at least one siRNA. In some embodiments of any aspect, the cargo comprises at least one shRNA. In some embodiments of any aspect, the cargo comprises at least one microRNA.

[0370] In some embodiments of any aspect, the cargo comprises at least one vaccine-related antigen. As used herein, the term "vaccine-related antigen" refers to a foreign (e.g., microbial) substance that can elicit an immune response in a subject to which the antigen is administered (e.g., as a vaccine against a foreign substance (e.g., microbial)).

[0371] In some embodiments of any aspect, the vaccine-associated antigen comprises at least one protein encoded by a viral genome. Non-limiting examples of viruses expressing such antigens include: Rift Valley fever, Crimean-Congo hemorrhagic fever, Lassa fever, Chikungunya virus (CHIKV), Nipah virus (NiV), respiratory syncytial virus (RSV), Ebola virus, Marburg virus, West Nile virus, Venezuelan equine encephalitis virus, yellow fever virus, Japanese encephalitis virus, western equine encephalitis virus, eastern equine encephalitis virus, cytomegalovirus (CMV), human immunodeficiency virus (HIV), influenza virus, Zika virus, Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human papillomavirus (HPV), herpes virus, rotavirus, varicella-zoster virus (VZV), dengue virus, hepatitis A virus, hepatitis B virus, rubella virus, poliovirus, and rabies virus.

[0372] In some embodiments of any aspect, the vaccine-associated antigen comprises an antigen selected from the group consisting of influenza virus hemagglutinin, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, and human respiratory syncytial virus (RSV) fusion glycoprotein.

[0373] In some embodiments of any aspect, the vaccine-associated antigen comprises one of SEQ ID NO: 13-SEQ ID NO: 16, SEQ ID NO: 22, or an amino 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 about 98%, at least about 99%, or 100% identical to one of SEQ ID NO: 13-SEQ ID NO: 16, SEQ ID NO: 22. In some embodiments of any aspect, the vaccine-associated antigen is encoded by nucleotides 7599-11384 of SEQ ID NO: 21, or is encoded by 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 about 98%, at least about 99%, or 100% identical to nucleotides 7599-11384 of SEQ ID NO: 21. In some embodiments, the vaccine-associated antigen encoded and expressed by the saRNA retains the same function as the wild-type viral protein (e.g., viral entry into cells). In some embodiments, the vaccine-associated antigen encoded and expressed by the saRNA has a weakened or inactivated function compared to the function of the wild-type viral protein.

[0374] In some embodiments of any aspect, the cargo comprises at least one transcription factor. In some embodiments of any aspect, the transcription factor is a stem cell transcription factor. In some embodiments of any aspect, the transcription factor is selected from the group consisting of octamer-binding transcription factor 3 (Oct3, Oct4), sex-determining region Y (SRY)-box protein transcription factor 2 (Sox2), Kruppel-like factor 4 (Klf4), and c-Myelocytic proto-oncogene (c-Myc).

[0375] In some embodiments of any aspect, the cargo comprises at least one growth factor and / or cytokine. In some embodiments of any aspect, the growth factor or cytokine is selected from the list consisting of platelet-derived growth factor (PDGF), erythropoietin (EPO), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), fibroblast growth factor (FGF), human relaxin-2 (RLX2), α-melanocyte stimulating hormone (α-MSH), insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), nerve growth factor (NGF), granulocyte-monocyte colony-stimulating factor (GMCSF), thrombopoietin (TPO), bone morphogenetic protein (BMP), hepatocyte growth factor (HGF), growth / differentiation factor (GDF), neurotrophic factor, migration stimulating factor (MSF), and sarcoma growth factor (SGF).

[0376] In some embodiments of any of the aspects, the cargo comprises one or both of Pappalysin-A1 (PAPPA1) and Pappalysin-A2 (PAPPA2).

[0377] In some embodiments of any aspect, the cargo comprises at least one interleukin. In some embodiments of any aspect, the cargo comprises at least one cognate receptor for an interleukin. In some embodiments of any aspect, the cargo comprises at least one receptor subunit for an interleukin. In some embodiments of any aspect, the interleukin is selected from the group consisting of IL-2, IL-4, IL-7, IL-10, IL-12, IL-13, and IL-15.

[0378] In some embodiments of any aspect, the cargo comprises at least one enzyme having antioxidant activity. In some embodiments of any aspect, the enzyme is selected from the group consisting of phospholipid hydroperoxidase glutathione peroxidase, superoxide dismutase-1, superoxide dismutase-2, Bruton's tyrosine kinase, adenosine deaminase, and ectonucleoside triphosphate diphosphohydrolase.

[0379] In some embodiments of any aspect, the cargo comprises glucagon-like peptide-1 (GLP-1) or a fragment thereof. As a non-limiting example, a biologically active form of glucagon-like peptide-1 (GLP-1) comprises amino acids 7-37 of GLP-1: HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 17). In some embodiments of any aspect, the GLP-1 comprises SEQ ID NO: 17, or an amino 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 about 98%, at least about 99%, or 100% identical to SEQ ID NO: 17 and maintains its function (e.g., stimulating insulin secretion and / or inhibiting glucagon secretion).

[0380] In some embodiments of any aspect, the cargo further comprises an endogenous insulin secretion signal (MALWMRLLPLLALLALWGPDPAAA, SEQ ID NO: 18) and / or a furin cleavage site (RGRR, SEQ ID NO: 19). In some embodiments of any aspect, the saRNA comprising glucagon-like peptide-1 (GLP-1) or a fragment thereof can be used to treat diabetes and / or obesity.

[0381] In some embodiments of any aspect, at least one cargo is inserted between the AflII and NdeI cleavage sites of SEQ ID NO: 2. In some embodiments of any aspect, at least one cargo is inserted between nucleotides 7627 and 7628 of SEQ ID NO: 2. In some embodiments of any aspect, 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) are replaced with at least one cargo of interest, as further described herein.

[0382] In some embodiments of any aspect, at least one cargo comprises a detectable marker or reporter molecule, including but not limited to a fluorescent protein or a detectable tag (e.g., c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin). In some embodiments of any aspect, the saRNA described herein, in particular the saRNA administered to a subject or as part of a pharmaceutical composition, does not encode or comprise an immunogenic detectable marker. In some embodiments of any aspect, the saRNA described herein does not comprise or encode GFP, mCherry, HA1, or any other immunogenic marker. In some embodiments of any aspect, the saRNA described herein comprising a detectable marker may have a detectable marker that is removed later, e.g., a removable (e.g., cleavable) detectable marker. In some embodiments of any aspect, the saRNA described herein comprising a detectable marker may have a detectable marker that is replaced with a different detectable marker, as known in the art or described herein, e.g., a replaceable (e.g., interchangeable) detectable marker. PolyA tail

[0383] In various aspects, described herein are saRNAs comprising a polyA tail found at the 3' end of the saRNA molecule. In some embodiments of any aspect, the polyA tail is derived from at least one virus. In some embodiments of any aspect, the polyA tail is derived from at least one alphavirus. In some embodiments of any aspect, the polyA tail is derived from VEEV.

[0384] The poly-A (polyadenylation) tail is a long chain of adenine nucleotides added to RNA molecules during RNA processing to increase molecular stability. In some embodiments, the length of the poly-A tail can be between 20 and 30 residues, between 30 and 50 residues, between 50 and 100 residues, or between 100 and 250 residues. The poly-A tail makes the RNA molecule more stable and prevents its degradation. In addition, the poly-A tail allows the mature RNA molecule to be transported out of the nucleus and / or translated into protein by ribosomes in the cytoplasm.

[0385] In some embodiments of any aspect, the saRNA comprises a polyA signal (e.g., AAUAAA) that results in cleavage of the 3' end of the RNA to release the 3' hydroxyl group and recruit poly-A polymerase to add adenine nucleotide chains to the RNA. In some embodiments of any aspect, the polyA tail is encoded in the saRNA, for example, by using a polyT sequence at the 5' end of the minus strand template of the saRNA. Nucleic acids and vectors

[0386] The saRNA described herein can be encoded and / or expressed by a nucleic acid and / or a vector. Thus, in one aspect herein, a nucleic acid encoding or comprising a saRNA described herein is described herein. In another aspect herein, a vector encoding or comprising a saRNA described herein is described.

[0387] In some embodiments of any aspect, the nucleic acid encoding or comprising the saRNA described herein comprises DNA. In some embodiments of any aspect, the nucleic acid encoding or comprising the saRNA described herein consists essentially of DNA. In some embodiments of any aspect, the nucleic acid encoding or comprising the saRNA described herein consists of DNA.

[0388] In some embodiments of any aspect, the DNA molecule encoding the saRNA described herein comprises one of SEQ ID NO: 2 or 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 about 98%, at least about 99%, or 100% identical to one of SEQ ID NO: 2 or SEQ ID NO: 5 and maintains the same function (e.g., self-replication), or a codon-optimized version thereof.

[0389] In some embodiments of any aspect, the DNA molecules encoding the saRNA described herein comprise at least one regulatory sequence upstream of the encoded saRNA. In some embodiments of any aspect, the DNA molecules encoding the saRNA described herein comprise a promoter for transcribing the saRNA using an RNA polymerase. In some embodiments of any aspect, the DNA molecules encoding the saRNA described herein comprise a T7 promoter (see, e.g., SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 12; see, e.g., nucleotides 1-17 of SEQ ID: 2, nucleotides 1-17 of SEQ ID: 5).

[0390] When the nucleic acid molecules encoding any of the saRNAs described herein are expressed in cells, a variety of transcriptional control sequences (e.g., promoter / enhancer sequences) can be used to direct their expression. The promoter can be a native promoter, for example, a promoter of the at least one cargo protein in its endogenous environment, which provides normal regulation of cargo protein expression. In some embodiments, the promoter can be constitutive, that is, the promoter is not regulated, allowing for continuous transcription of the saRNA and its associated cargo. A variety of conditional promoters can also be used, for example, promoters that are controlled by the presence or absence of a molecule.

[0391] The exact nature of the regulatory sequences required for expression may vary depending on the species or cell type, but generally may include 5' non-transcribed sequences and 5' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, etc. In particular, such 5' non-transcribed regulatory sequences may include a promoter region comprising a promoter sequence for transcriptional control of the encoded saRNA. The regulatory sequence may also include an enhancer sequence or an upstream activation sequence as desired.

[0392] As used herein, a saRNA coding sequence and a regulatory sequence are said to be "operably" linked when they are covalently linked in a manner that places the expression or transcription of the saRNA coding sequence under the influence or control of the regulatory sequence. If translation of the at least one cargo encoded in the saRNA into a functional protein is desired, then the two DNA sequences are said to be operably linked if induction of the promoter in the 5' regulatory sequence results in transcription of the saRNA and the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the saRNA, or (3) interfere with the ability of the at least one cargo encoded in the saRNA to be translated into a protein.

[0393] Nucleic acid molecules encoding saRNAs described herein can be introduced into cells using methods and techniques standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation (including chemical transformation and electroporation), transduction, particle bombardment, etc. Expression of nucleic acid molecules encoding saRNAs described herein can also be achieved by integrating the nucleic acid molecules into the genome.

[0394] In some embodiments, one or more saRNA described herein are expressed in a recombinant expression vector or plasmid. Figures 81-91Schematic diagram of an exemplary vector for in vitro transcription of exemplary saRNA. As used herein, the term "vector" refers to a polynucleotide sequence suitable for transferring a nucleic acid (e.g., DNA encoding a saRNA described herein) into a host cell. A vector can encompass any genetic element that can replicate when associated with appropriate control elements and can transfer a nucleic acid sequence into a cell. The term "vector" includes plasmids, cloning vectors, expression vectors, naked DNA, mini-chromosomes, chromosomes, transposons, cosmids, viruses, virions, bacteriophages, and the like. See, for example, U.S. Patent Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and 5,919,670 and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press (1989). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA fragments are attached. Another type of vector is a viral vector, in which additional DNA fragments are connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). In addition, some vectors can instruct the expression of its operably connected gene. This type of vector is referred to as "expression vector" in this article. Usually, the expression vector used for recombinant DNA technology is usually in plasmid form. In this application document, "plasmid" and "vector" are used interchangeably because plasmid is the most commonly used vector form. However, the present invention is intended to include the expression vectors of these other forms, such as viral vectors (for example, replication-defective retrovirus, adenovirus and adeno-associated virus), which play equivalent functions.

[0395] In some embodiments of any aspect, the vector is recombinant, for example, it comprises a sequence derived from at least two different sources. In some embodiments of any aspect, the vector comprises a sequence derived from at least two different species. In some embodiments of any aspect, the vector comprises a sequence derived from at least two different genes, for example, it comprises a nucleic acid or a fusion protein encoding an expression product operably linked to at least one non-natural (for example, heterologous) genetic control element (for example, a promoter, a repressor, an activator, an enhancer, a response element, etc.).

[0396] In some embodiments of any aspect, the vectors or nucleic acids described herein are codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that the altered or engineered nucleic acid encodes a polypeptide expression product identical to the native / wild-type sequence, but will be transcribed and / or translated with increased efficiency in the desired expression system. In some embodiments of any aspect, the expression system is an organism (or a cell obtained from such an organism) that is different from the source of the native / wild-type sequence. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in mammals or mammalian cells (e.g., mouse, murine, or human cells). In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in human cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in yeast or yeast cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in bacterial cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in E. coli cells.

[0397] A cloning vector is a vector capable of autonomous replication or integration into the host cell genome, and is further characterized by one or more endonuclease restriction sites at which the vector can be cut in a deterministic manner and to which a desired DNA sequence (e.g., a DNA template for a saRNA as described herein) can be attached so that the new recombinant vector retains its ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence can occur multiple times as the number of copies of the plasmid increases within the host cell (e.g., a host bacterium), or only once per host before the host reproduces by mitosis. In the case of a phage, replication can occur actively during the lytic phase or passively during the lysogenic phase.

[0398] An expression vector is a vector into which a desired DNA sequence (e.g., a DNA template for a saRNA as described herein) can be inserted by restriction and ligation so that it can be operably linked to a regulatory sequence comprising a DNA binding domain as described herein and can be expressed as a saRNA transcript. The vector may further comprise one or more marker sequences suitable for identifying cells that have been transformed or not yet transformed or transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease tolerance or sensitivity to antibiotics or other compounds, genes encoding enzymes whose activity can be detected by standard assays known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase), and genes that significantly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). In certain embodiments, the vectors used herein are capable of autonomous replication and expression of saRNAs present in the DNA fragments to which they are operably linked. The expressed sequences are typically heterologous to the cells but not necessarily so. The expression vector may contain additional elements, for example the expression vector may have two replication systems, thereby allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[0399] Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by introducing heterologous DNA (or RNA) into the cells. The heterologous DNA (or RNA) is placed under the operable control of transcriptional elements to allow expression of the heterologous DNA in the host cell.

[0400] The term "viral vector" as used herein refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into viral vector particles. The viral vector may comprise a nucleic acid encoding a polypeptide as described herein in place of a non-essential viral gene. Vectors and / or particles may be used for the purpose of transferring any nucleic acid into cells in vitro or in vivo. Many forms of viral vectors are known in the art. Non-limiting examples of viral vectors of the present invention include AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, herpes virus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors.

[0401] It should be understood that in some embodiments, the vectors described herein can be combined with other suitable compositions and therapeutic agents. In some embodiments, the vector is an additional type. The use of a suitable additional vector provides a means for maintaining the nucleic acid of interest (e.g., a nucleic acid encoding a saRNA described herein) in a subject at a high copy number of extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration. Composition and administration

[0402] The saRNA described herein may be included in a composition (e.g., a pharmaceutical composition) as further described herein. In some embodiments, the saRNA described herein may be included in a cell (e.g., a eukaryotic cell). In some embodiments of any aspect, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments of any aspect, the cell is a T cell, an NK cell, a macrophage, or a B cell. In some embodiments, the cell is a T lymphocyte. In some embodiments, the cell is a CD4+ T lymphocyte.

[0403] In one aspect, described herein are compositions comprising saRNAs described herein. In one aspect, described herein are compositions comprising nucleic acids or vectors comprising or expressing saRNAs described herein. In one aspect, described herein are compositions comprising cells comprising or expressing saRNAs described herein.

[0404] In one aspect, described herein are pharmaceutical compositions comprising a saRNA as described herein and a pharmaceutically acceptable carrier. In one aspect, described herein are pharmaceutical compositions comprising a nucleic acid or vector comprising or expressing a saRNA as described herein and a pharmaceutically acceptable carrier. In one aspect, described herein are pharmaceutical compositions comprising a cell comprising or expressing a saRNA as described herein and a pharmaceutically acceptable carrier.

[0405] In some embodiments of any aspect, saRNA is formulated as lipid nanoparticles (LNPs). In some embodiments of any aspect, the lipid nanoparticles include a targeting moiety that is specific for the cell or tissue of interest. As a non-limiting example, LNPs can be conjugated to antibodies (e.g., anti-CD3 antibodies) that are specific for T cells. In some embodiments, LNPs are formulated with a lipid:oligonucleotide weight ratio of about 10:1. In some embodiments, LNPs are formulated with an N:P ratio of about 10. In some embodiments, LNPs are formulated with an N:P ratio of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.

[0406] In another embodiment, the lipid nanoparticles have an average diameter of less than 1000 nm, such as less than 500 nm, less than 300 nm, less than 200 nm, less than 150 nm, less than 100 nm, or less than about 50 nm. In another embodiment, the diameter ranges from 1000 nm to 50 nm, or about 500 nm to 50 nm, or about 300 nm to 50, or 200 nm to 50, or 100 nm to 50 nm. In another embodiment, the average diameter of the nanoparticles is less than about 100 nm.

[0407] In another embodiment, the polydispersity index (PDI) of the population of nanoparticles is about 2 or less, or about 1 or less, or about 0.5 or less, or about 0.25 or less, or about 0.14 or less.

[0408] In another embodiment, the lipid nanoparticles have a positive charge.

[0409] In another embodiment, the lipid nanoparticles have a negative charge.

[0410] In another embodiment, the lipid nanoparticles have no overall charge.

[0411] In some embodiments, the lipid in the LNP comprises an ionizable lipid, cholesterol, a phospholipid and / or a polyethylene glycol lipid. Further non-limiting examples of lipids for LNP formulations include DSPE, SM-102, DMG-PEG2K, DOPE and / or cholesterol.

[0412] In some embodiments, the lipid nanoparticles can be formulated with one or more of cationic lipids, anionic lipids, neutral lipids, ionizable lipids, and / or zwitterionic lipids, non-limiting examples of which include: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, dilinoleoylphosphatidylcholine, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), diacylphosphatidylcholine, diacylphosphatidyl Acylethanolamine, ceramide, sphingomyelin, cerebroside, cholesterol, cerebroside and diacylglycerol, triglyceride, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphatidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), trimyristin (Trimyristin, DYNASAN 114), Tripalmitin (DYNASAN 116), Tristearin (DYNASAN 118), a mixture of monoglycerides, diglycerides and triglycerides, Stearin (IMWITOR 900), Behenin (COMPRITOL 888 ATO), Palmitostearin (PRECIROL ATO 5), Stearic acid, Palmitic acid, DPPC, MSPC, DSPE-PEG 2000, DSPE-PEG 2500, Phosphatidylcholine, Soybean phosphatidylcholine, (4-hydroxybutyl) azanediylbis(hexane-6,1-diyl)bis(2-hexyldecanoate), (2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecyl acetamide, 1,2-distearoyl-sn-glyceryl Oil-3-phosphocholine, cholesterol, heptadecan-9-yl 8-[(2-hydroxyethyl)(6-oxo-6-(undecanyloxy)hexyl)amino]octanoate, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, ALC-0315, ALC-0159, SM-102, DTOP, DDAB, DOGS, N-(1,2-dimyristyloxypropyl-3-yl)-N,N-Dimethyl-N-hydroxyethylammonium bromide ("DMRIE"), DODAP, DLinDMA, cKK-E12, OF-02, C12-200, MC3, DLinkC2DMA, ICE (imidazole-based), HGT5000, HGT5001, HGT4003, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"), N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA"), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride ("DODAP"), N,N-distearoyl-N,N-dimethylammonium bromide ("DDAB"), 3-(N-(N,N-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"), DOSPA, DODMA and DMDMA, DODAC, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione, 4-(2,2-diocta-9,12-dienyl-[1,3]dioxolan-4-ylmethyl)-dimethylamine, DLinKDMA (see, e.g., WO 2009 / 132131 A1, incorporated herein by reference in its entirety), DLin-K-C2-DMA (see, for example, WO 2010 / 042877, incorporated herein by reference in its entirety), DLin-M-C3-DMA (see, for example, WO 2010 / 146740 and / or WO 2010 / 105209, incorporated herein by reference in its entirety), 2-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dienyloxy]propan-1-amine) (CLinDMA), 3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione, N1GL, N2GL, V1GL, (Cationic lipid nanoparticles containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine ("DOPE")) and combinations thereof.

[0413] In some embodiments, the LNP comprises one or more PEG-modified lipids. In some embodiments, the one or more PEG-modified lipids comprise a polyethylene glycol chain up to 5 kDa in length covalently attached to a C6-C 20 Length of the alkyl chain of the lipid.

[0414] Other phosphorus-deficient compounds, such as sphingolipids, the glycosphingolipid family, diacylglycerols, and S-acyloxy acids, also belong to the group known as amphipathic lipids and can be included in the LNPs described herein.

[0415] In some embodiments, the one or more cationic lipids included in LNP as herein described are amino lipids. Suitable amino lipids for LNP as herein described include those described in WO 2017180917, which are hereby incorporated by reference in their entirety. Exemplary amino lipids in WO 2017180917 include those described in

[0744] paragraph, such as DLin-MC3-DMA (MC3), (13Z, 16Z)-N, N-dimethyl-3-nonyl docosyl-13,16-diene-1-amine (L608) and the compound 18 described in WO 2017180917. Other exemplary amino lipids include compound 2, compound 23, compound 27, compound 10 or compound 20 described in WO 2017180917. Other amino lipids suitable for LNP as herein described include those described in WO 2017112865, which are hereby incorporated by reference in their entirety. Exemplary amino lipids in WO 2017112865 include compounds according to one of Formula (I), (Ia1)-(Ia6), (Ib), (II), (IIa), (III), (IIIa), (IV), (I7-1), (19-1), (19-11), and (20-1), and compounds of paragraphs

[00185] ,

[00201] , and

[0276] of WO 2017112865. In some embodiments, cationic lipids suitable for LNPs described herein include those described in WO2016118725, which are incorporated herein by reference. Exemplary cationic lipids in WO2016118725 include those such as KL22 and KL25. In some embodiments, cationic lipids suitable for LNPs described herein include cationic lipids described in WO2016118724, which are incorporated herein by reference. Exemplary cationic lipids in WO2016118725 include those such as KL10, 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLin-DMA), and KL25.

[0416] In some embodiments, the saRNA is formulated in a polymer matrix. In some embodiments, the polymer matrix can comprise one or more polymers, with or without one or more lipids, to form nanoparticles.

[0417] In one embodiment, the polymer matrix containing saRNA is a reverse micelle nanoparticle comprising two or more polymers, such as polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropylfumarate (polypropylfumerates), polyglycerol, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly (orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, poly (β-amino ester), polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene or polyamine, or a combination thereof. In one embodiment, the polymer matrix comprises one or more polyesters, polyanhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates or polycyanoacrylates. In one embodiment, the polymer matrix comprises a polyalkylene glycol, such as polyethylene glycol. In another embodiment, the polymer matrix comprises PLGA, PLA, PGA or polycaprolactone. In another embodiment, the polymer matrix comprises a copolymer of two or more polymers, such as a copolymer of PLGA or PLA and PEG. The polymer matrix may comprise PLGA or PLA and copolymers of PLGA or PLA and PEG.

[0418] In another embodiment, the polymer matrix comprises lipid-terminated polyalkylene glycols and polyesters, such as lipid-terminated PEG and PLGA. The lipid can be a lipid such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and its salts.

[0419] In one embodiment, the reverse micelles comprise an amphiphilic lipid, such as phosphatidylcholine, phosphatidylcholine, lipid A, cholesterol, dolichol, sphingosine, sphingomyelin, ceramide, cerebroside, sulfatide, glycosylceramide, phytosphingosine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, cardiolipin, phosphatidic acid or lysophospholipid, or a combination thereof.

[0420] In some embodiments, the technology described herein relates to pharmaceutical compositions comprising a saRNA as described herein and an optional pharmaceutically acceptable carrier. In some embodiments, the active ingredient of the pharmaceutical composition comprises a saRNA as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists essentially of a saRNA as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of a saRNA as described herein. Pharmaceutically acceptable carriers and diluents include physiological saline, aqueous buffer solutions, solvents, and / or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository waxes. waxes); (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (24) C2-C 12 Alcohols, such as ethanol; and (25) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient," "vehicle," "pharmaceutically acceptable carrier," etc. are used interchangeably herein. In some embodiments, the carrier inhibits degradation of the active agent (e.g., saRNA described herein).

[0421] In some embodiments, the pharmaceutical composition comprising the saRNA as described herein may be in a parenteral dosage form (i.e., administered or administered at a site of the body other than the mouth and digestive tract). Because administration of parenteral dosage forms generally bypasses a patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a subject. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready for injection by dissolution or suspension in a pharmaceutically acceptable solvent, suspensions ready for injection, and emulsions.

[0422] Those skilled in the art are familiar with suitable vehicles that can be used to provide parenteral dosage forms of the saRNA disclosed herein. Non-limiting examples include, but are not limited to, sterile water; water for injection USP; saline solution; phosphate-based saline; dextrose solution; aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethanol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0423] Pharmaceutical compositions comprising the saRNA described herein can also be formulated for oral administration, for example, as discrete dosage forms such as, but not limited to, tablets (including but not limited to scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, lozenges, wafers, sprays, or liquids such as, but not limited to, syrups, elixirs, suspensions or solutions in aqueous liquids, non-aqueous liquids, water-in-oil emulsions, or oil-in-water emulsions. Such compositions contain a predetermined amount of saRNA and can be prepared by pharmaceutical methods known to those skilled in the art. See generally Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott, Williams, and Wilkins, Philadelphia PA (2005).

[0424] In some embodiments, the methods described herein comprise administering to a subject an effective amount of a composition described herein (e.g., a saRNA described herein) to alleviate a symptom of a disease or disorder. As used herein, "alleviating a symptom of a disease or disorder" is to alleviate any condition or symptom associated with a disease or disorder. The reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as compared to an equivalent untreated control, as measured by any standard technique. A variety of methods for administering the compositions described herein to a subject are known to those skilled in the art.

[0425] In some embodiments of any aspect, the saRNA is present at about 5×10 -4 In some embodiments of any aspect, the saRNA is formulated at a dose of about 5×10 -3 In some embodiments of any aspect, the saRNA is formulated at a dose of about 5×10 -2 In some embodiments of any aspect, the saRNA is formulated at a dose of about 5×10 -1 In some embodiments of any aspect, the saRNA is formulated at a dose of about 1 ng. In some embodiments of any aspect, the saRNA is formulated at a dose of about 10 ng. In some embodiments of any aspect, the saRNA is formulated at a dose of about 100 ng. In some embodiments of any aspect, the saRNA is formulated at a dose of about 1000 ng. In some embodiments of any aspect, the saRNA is formulated at a dose of about 10,000 ng. In some embodiments of any aspect, the saRNA is formulated at a dose of about 2.5 μg. In some embodiments of any aspect, the saRNA may be formulated at a dose of about 0.1 μg to about 100 μg. In embodiments of a subject (e.g., a human subject) of about 70 kg, the saRNA may be formulated at a dose of about 1×10 -6 mg / kg to 1×10 -3 mg / kg dose preparation.

[0426] For systemic administration, a therapeutic amount of a composition comprising a saRNA described herein, such as 1×10 -6 mg / kg, 1×10 -5 mg / kg, 1×10 -4 mg / kg, 1×10 -3 mg / kg, 1×10 -2 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg or more.

[0427] As used herein, the term "effective amount" refers to the amount of saRNA required to alleviate at least one or more symptoms of a disease or disorder, and relates to a sufficient amount of a pharmaceutical composition to provide the desired effect. Thus, the term "therapeutically effective amount" refers to the amount of saRNA sufficient to provide a specific alleviating effect when administered to a typical subject. In various cases, the effective amount described herein also includes an amount sufficient to delay the development of disease symptoms, alter the course of a symptomatic disease (such as, but not limited to, slowing the progression of disease symptoms), or reverse disease symptoms. Therefore, specifying an exact "effective amount" is generally not practical. However, for any given situation, one of ordinary skill in the art can determine an appropriate "effective amount" using only routine experimentation.

[0428] The effective amount, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as those used to determine the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending on the dosage form employed and the route of administration used. The therapeutically effective dose can be estimated initially from cell culture assays. In addition, the dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of saRNA that achieves half-maximum inhibition of symptoms) as determined in cell culture or in an appropriate animal model. Levels in plasma can be measured, for example, by PCR. The effect of any particular dose can be monitored by an appropriate bioassay. The dosage can be determined and adjusted by a physician, if necessary, to suit the observed therapeutic effect.

[0429] The dosage range for administering saRNA according to the methods described herein depends, for example, on the form of the saRNA, its potency, and the degree to which the symptoms, markers, or indicators of the conditions described herein are desired to be reduced. The dosage should not be so large as to cause adverse side effects, such as an autoimmune reaction. Generally, the dosage will vary with the patient's age, condition, and sex and can be determined by one skilled in the art. The dosage can also be adjusted by the individual physician in the event of any complications.

[0430] The efficacy of saRNA in, for example, treating a condition described herein, or inducing a response described herein, can be determined by a skilled clinician. However, if one or more signs or symptoms of a condition described herein are altered in a favorable manner, other clinically recognized symptoms are improved or even alleviated, or treatment according to the methods described herein induces, for example, at least 10% of the desired response, then the treatment is considered to be "effective treatment" as the term is used herein. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or incidence of a condition treated according to the methods described herein, or by measuring any other suitable measurable parameter. Efficacy can also be measured by whether an individual evaluated in hospital no longer worsens or requires medical intervention (i.e., disease progression has ceased). Methods for measuring these indicators are known to those skilled in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals) and includes: (1) inhibiting the disease, such as preventing worsening of symptoms (e.g., pain or inflammation); or (2) alleviating the severity of the disease, such as causing symptoms to subside. An effective amount for treating a disease is an amount that, when administered to a subject in need thereof, is sufficient to produce an effective treatment for the disease as that term is defined herein. The efficacy of an agent can be determined by evaluating the physical indicators of the condition or the desired response. Those skilled in the art are fully capable of monitoring the efficacy of administration and / or treatment by measuring any one of these parameters or any combination of these parameters.

[0431] Efficacy can be assessed in animal models of the conditions described herein or using in vitro assays. When using experimental animal models or in vitro assays, efficacy of the treatment is demonstrated when a statistically significant change in a marker is observed.

[0432] The subject is typically monitored by a skilled clinician regarding the duration and frequency of treatment to determine when the treatment provides a therapeutic benefit and to determine whether to increase or decrease the dose, increase or decrease the frequency of dosing, discontinue treatment, resume treatment, or make other changes to the treatment regimen.

[0433] In certain embodiments, an effective dose of a composition comprising a saRNA described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising a saRNA described herein can be administered to a patient repeatedly.

[0434] The dosing schedule can vary from once a week to once a day, depending on several clinical factors, such as the subject's sensitivity to saRNA. The desired dose or amount can be given once, or divided into sub-doses (e.g., 2-4 sub-doses) and given over a period of time, such as at appropriate intervals within a day or according to other appropriate schedules. In some embodiments, administration can be over a period of weeks or months, with one or more doses and / or treatments given daily. Examples of dosage and / or treatment schedules are once a day, twice a day, three times a day, or four times a day or more over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or longer. The compositions comprising the saRNA described herein can be administered over a period of time (e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes).

[0435] In some embodiments, after an initial treatment regimen, treatment may be administered on a less frequent basis. For example, after three months of biweekly treatment, treatment may be repeated monthly for six months or a year or longer.

[0436] A variety of methods for administering the compositions described herein to a subject are known to those skilled in the art. Such methods may include, but are not limited to, intraocular, intraosseous (IO), intraperitoneal (IP), subcutaneous (SC), intravenous (IV), intramuscular (IM), rectal, intravaginal, intraarticular (IA), inhalation, or topical administration. Other non-limiting methods of administration include oral, parenteral, intravenous, intramuscular, transdermal, airway (aerosol), pulmonary, cutaneous, injection, or intratumoral administration. Administration may be local or systemic.

[0437] In some embodiments of any of the aspects, the saRNA described herein is administered as monotherapy, e.g., the subject is not administered another treatment for the disease or disorder.

[0438] In some embodiments of any aspect, the methods described herein can further comprise administering a second agent and / or treatment to the subject, for example, as part of a combination therapy. Non-limiting examples of second agents and / or treatments can include cancer therapies selected from the group consisting of: radiation therapy, surgery, gemcitabine, cisplatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; polyacetogenins (particularly bullatacin and bullatacinone); inone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adolesin, carzelesin, and biszelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); e leutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozostatin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma ll and calicheamicin omega ll (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarcinogen chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycin, authramycin, azaserine, bleomycin, actinomycin C, carabicin, caminomycin, carzinophilin, chromomycins, actinomycin D, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, Doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, desmethoxydaunorubicin, masiromicin, mitomycins (e.g., mitomycin C), mycophenolic acid, nogamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rhodorubicin, streptozocin, streptozotocin, tuberculin, ubenimex, zoloft, daunorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); leaf Acid analogs, such as dimethylfolate, methotrexate, pteropterin, trimesate; purine analogs, such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as captestosterone, drostanolone propionate, cyclothiocarbamate, melastane, testolactone; antiadrenal drugs, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid (folinic acid); acid); acetylglucuronolactone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diaziquone; elformithine; elliptonium acetate; epothilones; etoglucose; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; ethylhydrazine podophyllotoxin; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triazinon; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, baculocin A, and anguidine); carbamates; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.) and doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; mitoxantrone hydrochloride; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including regimens of irinotecan with 5-fluorouracil and leucovorin); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin regimens (FOLFOX); lapatinib PKC-α, Raf, H-Ras, EGFR (such as erlotinib) that reduce cell proliferation ) and VEGF-A inhibitors, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0439] Those skilled in the art can readily identify the chemotherapeutic agents used (e.g., see Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th Edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff's Clinical Oncology, 2013 Elsevier; and Fischer DS (ed.): The Cancer Chemotherapy Handbook, 4th Edition, St. Louis, Mosby-Year Book, 2003).

[0440] In addition, the treatment method may further include the use of radiation or radiotherapy. In addition, the treatment method may further include the use of surgical treatment.

[0441] The methods described herein may further include administering a second agent and / or treatment to the subject, for example, as part of a combination therapy. As a non-limiting example, if the subject's pain or inflammation is to be treated according to the methods described herein, a second agent and / or treatment known to be beneficial to the subject with pain or inflammation may also be administered to the subject. Examples of such agents and / or treatments include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs-such as aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (such as cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclomethasone); methotrexate; sulfasalazine; leflunomide; anti-TNF drugs; cyclophosphamide; pro-resolving drugs; mycophenolate mofetil; or opioids (such as endorphins, enkephalins, and dynorphins), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, etc.

[0442] In some embodiments of any aspect, the saRNA described herein can be co-administered with a synergistic therapeutic agent. Non-limiting examples of such synergistic therapies include RNA (e.g., siRNA, shRNA, miRNA), small molecules, and / or checkpoint inhibitors. Such co-administration can enhance the activation, antigen presentation, and / or function of cells (including but not limited to T cells and dendritic cells).

[0443] Non-limiting examples of immune checkpoint inhibitors (ICIs) include: pembrolizumab Nivolumab Cemiplimab Spartalizumab, Camrelizumab TM ), Sintilimab Tislelizumab, toripalimab (Tuoyi TM ), dostarlimab (JEMPERLI), INCMGA00012, AMP-224, AMP-514 (MEDI0608), atezolizumab ( ), avelumab (avelumab, ), envafolimab (KN035), cosibelimab (CK-301), AUNP12, CA-170, BMS-986189, BMS-936559 (MDX-1105), durvalumab ( ), tremelimumab, and ipilimumab See, for example, U.S. Patents Nos. 5,811,097, 5,855,887, 6,051,227, 6,682,736, 6,984,720, 7,595,048, 7,605,238, 7,943,743, 8,008,449, 8,217,149, 8,354,509, 8,383,796, 8,728,474, 8,735,553, 8,779,105, 8,779,108, 8,907,05 ... 8900587, US8952136, US9067999, US9073994, US9683048, US9987500, US10160736, US10316089, US10441655, US10590199, US1122552, US Patent Publication No. US2014341917; Storz et al., MAbs. 2016 Jan;8(1):10–26; the contents of each of which are incorporated herein by reference in their entirety. Exemplary Uses of saRNA

[0444] Described herein are exemplary uses of the saRNA described herein. In one aspect, described herein are methods of expressing at least one cargo of interest in a cell, the method comprising contacting the cell with at least one saRNA described herein. In some embodiments of any aspect, the cell is a human cell. In some embodiments, the saRNA is delivered to the cell in situ. In some embodiments, the saRNA is delivered to the cell ex vivo. In some embodiments of any aspect, the saRNA is delivered to the cell by electroporation. In some embodiments of any aspect, the saRNA is delivered to the cell via lipid nanoparticles. In some embodiments of any aspect, a plurality of different saRNAs are delivered to a mixture of cells.

[0445] In another aspect, described herein is a method of expressing at least one cargo in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition described herein, e.g., encoding, expressing, or comprising at least one saRNA described herein.

[0446] As used herein, "subject" means a person or an animal. Typically, the animal is a vertebrate, such as a primate, a rodent, a domesticated animal, or a game animal. Primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, such as rhesus macaques. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domesticated animals and game animals include cows, horses, pigs, deer, bison, buffaloes, felines (e.g., house cats), canines (e.g., dogs, foxes, wolves), birds (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0447] The subject is preferably 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 advantageously be used as subjects representing animal models of diseases or disorders. In some embodiments of any aspect, the subject is a human. In some embodiments of any aspect, the subject is a domestic animal. In some embodiments of any aspect, the subject is a domesticated or domesticated 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 aspect, the subject is a fish or a bird or a lizard or a snake. The subject can be male or female.

[0448] In some embodiments of any aspect, the subject has cancer. In some embodiments of any aspect, the subject is in need of a vaccine against an infectious disease. In some embodiments of any aspect, the subject is in need of protein replacement therapy. In some embodiments of any aspect, the subject is in need of antibody therapy. In some embodiments of any aspect, the subject is in need of treatment for diabetes and / or obesity. In some embodiments of any aspect, the subject is in need of BITE therapy.

[0449] The subject can be a subject who has previously been diagnosed with or identified as suffering from or suffering from such a condition in need of treatment, or one or more complications relevant to such a condition, and optionally has experienced the treatment of such a disease or disorder or one or more complications relevant to the disease or disorder. Alternatively, the subject can also be a subject who has not been previously diagnosed as suffering from the disease or disorder or one or more complications relevant to the disease or disorder. For example, the subject can be a subject who exhibits one or more risk factors for the disease or disorder or one or more complications relevant to the disease or disorder, or a subject who does not exhibit a risk factor. For treating a particular condition, a "subject in need" can refer to a subject who suffers from the condition, is diagnosed as suffering from the condition, or is at risk of developing the condition.

[0450] In some embodiments of any aspect, the saRNA described herein increases the expression level of a cargo protein in a cell, e.g., by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold or more, compared to a cell that does not comprise the saRNA.

[0451] In some embodiments of any aspect, use of a modified saRNA (e.g., comprising at least 25% modified nucleotides) has beneficial, unexpected results compared to a corresponding saRNA having less than 25% modified nucleotides.

[0452] In some embodiments of any aspect, the level of replication of the modified saRNA is greater than or equal to the level of an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides. In some embodiments of any aspect, the level of replication of the modified saRNA is equal to the level of an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides. In some embodiments of any aspect, the level of replication of the modified saRNA is at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold greater than the level of an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides.

[0453] In some embodiments of any aspect, the level of expression of the modified saRNA cargo is greater than or equal to the level of an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides. In some embodiments of any aspect, the level of expression of the modified saRNA cargo is equal to the level of an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides. In some embodiments of any aspect, the level of expression of the modified saRNA cargo is at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold higher than the level of expression of the corresponding saRNA cargo having less than 25% modified nucleotides.

[0454] In some embodiments of any aspect, the transfection efficiency of the modified saRNA is greater than or equal to the transfection efficiency of an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides. In some embodiments of any aspect, the transfection efficiency of the modified saRNA is equal to the transfection efficiency of an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides. In some embodiments of any aspect, the transfection efficiency of the modified saRNA is at a level that is at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold greater than the level of an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides.

[0455] In some embodiments of any aspect, the modified saRNA generates an early interferon response in the subject. In some embodiments of any aspect, the early interferon response is reduced compared to an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides. In some embodiments of any aspect, the early interferon response is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold or more compared to an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides.

[0456] In some embodiments of any aspect, the time for which the cargo is expressed at detectable levels from the modified saRNA is increased compared to an equivalent dose of a corresponding saRNA with less than 25% modified nucleotides. In some embodiments of any aspect, the cargo is expressed at detectable levels from the modified saRNA for a time period that is increased by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold, or more, compared to an equivalent dose of the corresponding saRNA with less than 25% modified nucleotides.

[0457] In some embodiments of any aspect, the saRNA described herein allows for tissue, organ, or cell type-specific expression of a cargo. In some embodiments of any aspect, the modified saRNA described herein allows for tissue, organ, or cell type-specific expression of a cargo.

[0458] In some embodiments of any aspect, the saRNA comprises greater than 50% uridine substituted with 5-methyluridine. In some embodiments of any aspect, the saRNA comprising greater than 50% uridine substituted with 5-methyluridine has increased kidney-specific expression of the cargo compared to an equivalent dose of a corresponding saRNA comprising less than 50% uridine substituted with 5-methyluridine. In some embodiments of any aspect, the kidney-specific expression is increased by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold or more.

[0459] In some embodiments of any aspect, the modified saRNA modulates differentiation of cells containing the saRNA at a level comparable to or greater than the level of modulation achieved with an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides. In some embodiments of any aspect, the modified saRNA modulates differentiation of cells containing the saRNA at a level comparable to the level of modulation achieved with an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides. In some embodiments of any aspect, the modified saRNA modulates differentiation of cells containing the saRNA at a level comparable to the level of modulation achieved with an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides. In some embodiments of any aspect, the modified saRNA modulates differentiation of cells containing the saRNA at a level that is at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold greater than the level of modulation achieved with an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides.

[0460] In some embodiments of any aspect, the cell is regulated by expression of a stem cell transcription factor encoded by a saRNA. In some embodiments of any aspect, the cell is regulated to become a stem cell by a saRNA described herein.

[0461] In some embodiments of any aspect, at least one cargo is constitutively expressed in the cell (ie, in a sustained manner). In some embodiments of any aspect, at least one cargo is constitutively expressed in the subject (ie, in a sustained manner).

[0462] In some embodiments of any aspect, the cargo comprises a chimeric antigen receptor. In some embodiments of any aspect, the cargo comprises multiple chimeric antigen receptors. In some embodiments of any aspect, the saRNA encodes and expresses multiple cargo proteins capable of interacting with each other. In some embodiments of any aspect, the interaction of the multiple cargo proteins results in conditional activity of cells transfected with the saRNA. In some embodiments of any aspect, the cargo proteins capable of interacting with each other are chimeric antigen receptors with activating and / or inhibitory function. In some embodiments of any aspect, the cargo comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chimeric antigen receptors. In some embodiments of any aspect, the cargo comprises a protein comprising a chimeric antigen receptor comprising an extracellular domain that senses at least one input signal. In some embodiments of any aspect, the cargo is a protein comprising at least one chimeric antigen receptor, and the input is a small molecule and / or protein. In some embodiments of any aspect, the cargo is a protein comprising at least one domain that responds to an external input. In some embodiments of any aspect, the cargo is a protein comprising at least one repressible protease domain that is responsive to at least one protease inhibitor to regulate the activity of the cargo.

[0463] In some embodiments of any aspect, the activity of the cargo protein expressed by saRNA is controlled by administering small molecules and / or proteins and / or RNA molecules. In some embodiments of any aspect, the RNA molecule is an aptamer. In some embodiments of any aspect, the protein is a chimeric antigen receptor (CAR), and the activity of the CAR is regulated by at least one small molecule response domain. In some embodiments of any aspect, the activity of the cargo protein expressed by saRNA is increased and / or decreased by administering at least one small molecule. In some embodiments of any aspect, the at least one small molecule is a protease inhibitor or a molecule capable of interacting with at least one protein domain in a cargo protein.

[0464] In some embodiments of any aspect, the methods described herein comprise contacting a cell comprising at least one saRNA with at least one input to control the fate or function of the cell. In some embodiments of any aspect, the methods described herein comprise administering at least one input to control the fate or function of at least one cell in a subject, wherein the subject has previously been administered at least one saRNA described herein.

[0465] In some embodiments of any aspect, the input is endogenous to the environment of the cell comprising the saRNA. In some embodiments of any aspect, the input is exogenous to the environment of the cell comprising the saRNA. In some embodiments of any aspect, the input comprises a cell, a protein, a small molecule, a specific wavelength of light, a specific temperature, and / or a magnetic field.

[0466] In some embodiments of any aspect, the input results in inhibition of a repressible protease domain in a cargo encoded by a saRNA described herein. In some embodiments of any aspect, the input results in activation of a repressible protease domain in a cargo encoded by a saRNA described herein. In some embodiments of any aspect, the input results in oligomerization and thereby activation of the cargo. In some embodiments of any aspect, the input results in oligomerization and thereby activation of the cargo.

[0467] In some embodiments of any aspect, control of the cell requires the simultaneous presence of all inputs. In some embodiments of any aspect, control of the cell requires the presence of any input. In some embodiments of any aspect, control of the cell requires both the presence and absence of different combinations of inputs.

[0468] In some embodiments of any aspect, the addition or removal of at least one saRNA and / or at least one input results in an altered fate and / or function of at least one cell. In some embodiments of any aspect, the fate of the cell (e.g., after adding or removing at least one input) is an altered cell type and / or altered cell localization. In some embodiments of any aspect, the altered cell function is lytic. In some embodiments of any aspect, the altered cell function is stimulatory. In some embodiments of any aspect, the altered cell function is immunomodulatory. In some embodiments of any aspect, the input results in cell death comprising at least one saRNA described herein. In some embodiments of any aspect, the input results in increased clearance of cells comprising at least one saRNA described herein. In some embodiments of any aspect, the input results in cell cycle arrest of cells comprising at least one saRNA described herein.

[0469] In some embodiments of any aspect, the saRNA cargo encodes and expresses at least one cargo protein, at least one protein reporter, and / or at least one expression-enhancing protein. In some embodiments of any aspect, the saRNA cargo encodes and expresses at least one cargo protein and at least one protein reporter. In some embodiments of any aspect, the saRNA cargo encodes and expresses at least one cargo protein and at least one expression-enhancing protein. In some embodiments of any aspect, the saRNA cargo encodes and expresses at least one protein reporter and at least one expression-enhancing protein. In some embodiments of any aspect, the saRNA cargo encodes and expresses at least one cargo protein, at least one protein reporter, and at least one expression-enhancing protein.

[0470] In some embodiments of any aspect, the cargo protein is a chimeric antigen receptor. In some embodiments of any aspect, the expression enhancing protein is B18R. In some embodiments of any aspect, the expression enhancing protein is E3L. In some embodiments of any aspect, the expression enhancing protein is B18R and E3L.

[0471] In some embodiments of any aspect, the saRNA described herein comprises a targeting domain to increase the transfection efficiency of the saRNA into a specific cell type. In some embodiments of any aspect, the composition comprising the saRNA described herein further comprises a targeting domain to increase the transfection efficiency of the saRNA into a specific cell type. definition

[0472] The invention exemplarily described herein can be implemented in the absence of any elements or limitations not specifically disclosed herein. Thus, for example, the terms "comprise", "include", "contain" etc. should be understood broadly and not restrictively. The terms and expressions used are used only as descriptive and not restrictive terms, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or parts thereof, but it should be recognized that, within the scope of the claimed invention, various modifications may be made. Therefore, it should be understood that although the present invention has been specifically disclosed by preferred methods, embodiments and optional features, those skilled in the art may modify and change the concepts disclosed herein, and such modifications and changes should be considered to be within the scope of the invention defined in the embodiments and other parts of the present invention. In the event of a conflict, the present application document (including definitions) shall prevail.

[0473] The contents of the articles, patents, and patent applications, and all other literature and electronically available information mentioned or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated as being incorporated by reference. Applicants reserve the right to actually incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.

[0474] Certain aspects and embodiments of the invention have been described broadly and generally herein. Each of the narrower species and subgeneric groupings falling within the scope of this general invention also form part of certain aspects and embodiments of the invention contemplated herein. This includes the general description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether the deleted material is specifically recited herein.

[0475] For convenience, the meanings of some terms and phrases used in the present invention, examples, and appended claims are provided below. Unless otherwise specified or implied by the context, the following terms and phrases include the meanings provided below. These definitions are provided to help describe specific embodiments and are not intended to limit the claimed technology, as the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the technology belongs. If there is a significant difference between the use of a term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0476] The terms "protein," "cargo," or "protein of interest" refer to any gene-encodable polypeptide or nucleic acid sequence with a known or unknown function. Examples of nucleic acid cargoes of interest include non-coding RNA, long non-coding RNA, microRNA, and siRNA. Examples of polypeptide cargoes of interest include receptors, ligands, enzymes, transmembrane receptors, transcription factors, viral components, and the like. These are examples of proteins with known functions and should not be construed as an exclusive list. Proteins with unknown functions may also be encoded by genes.

[0477] The term "protein" refers to any gene-encoded polypeptide sequence with a known or unknown function. Examples of proteins include receptors, ligands, enzymes, transcription factors, etc. These are examples of proteins with known functions and should not be construed as an exclusive list. Proteins with unknown functions may also be encoded by genes.

[0478] The term "cargo" refers to a polypeptide sequence encoded by conventional or self-replicating RNA. The polypeptide sequence may represent a polypeptide with known or unknown function. A non-exhaustive list of polypeptides that can be expressed by RNA includes fluorescent proteins, enzymes, transcription factors, receptors, ligands, and antibodies.

[0479] Chimeric antigen receptor (CAR) combines the components or functions of T cell receptor (TCR) and related molecules into a single polypeptide. In some embodiments of any aspect, the polypeptide described herein is a second or third generation CAR, which includes an extracellular binding domain, a hinge region, a transmembrane domain and one or more intracellular signal transduction domains. The extracellular binding domain generally contains a single chain variable fragment (scFv) derived from an antigen-reactive antibody, which is generally highly specific to a specific antigen. Most CARs contain CD3 ζ chain domains as intracellular signal transduction domains, which are the main transmitters of T cell activation signals. In addition to scFv, non-antibody-based methods have also been used to guide CAR specificity, generally utilizing ligand / receptor pairs that are generally bound to each other. In some embodiments of any aspect, the polypeptide described herein may include cytokines, innate immune receptors, TNF receptors, growth factors and structural proteins, which have all been successfully used as CAR antigen recognition domains.

[0480] The term "self-replicating RNA" refers to an RNA chain that is known to undergo replication, which generates a replica chain from the original chain. Self-replicating RNA is known to exist in the form of RNA virus genomes, and components from RNA viruses and sequences of interest can be used to create artificial self-replicating RNA. Components from RNA viruses can be used in combination or only from specific RNA viruses.

[0481] The term "self-amplifying RNA" refers to a nucleic acid polymer that is capable of replicating the entire nucleic acid polymer in both negative and positive strand conformations. It is capable of synthesizing additional self-amplifying RNA, in part by producing various non-structural proteins of viral origin that, among other activities, can act as RNA-dependent RNA polymerases. Self-amplifying RNA can be created by utilizing components from RNA viruses and sequences encoding cargoes of interest. An important aspect of saRNA is that the non-structural proteins are capable of generating both new full-length saRNA chains and RNA produced by subgenomic promoters. In therapeutic saRNA, the RNA produced by transcription from a subgenomic promoter encodes the cargo of interest. Throughout the disclosure of the present invention, self-amplifying RNA, saRNA, self-replicating RNA, and srRNA can be used interchangeably.

[0482] The term "expression" refers to the production of a protein from an RNA strand. For example, "high expression" can refer to the production of sufficient or large amounts of a protein. The desired expression level depends on the application of interest. The terms "high expression" and "low expression" can be used in embodiments to describe observations made between cells of interest.

[0483] The term "expression" refers to the production of a cargo from an RNA strand. In some embodiments, "expression" can refer to the production of a protein to elicit a desired therapeutic effect. In some embodiments, "expression" can refer to the percentage of a cell population that expresses the cargo. In preferred embodiments, "high expression" refers to overproduction of the cargo of interest relative to the total amount. In another preferred embodiment, "high expression" refers to the percentage of a cell population that expresses the cargo of interest that is equal to or greater than a control. The terms "high expression" and "low expression" can be used in embodiments to compare cargo expression between cells of interest relative to the total amount or percentage of cellular expression.

[0484] The term "conventional mRNA" refers to messenger RNA that does not have the ability to self-replicate. In some embodiments, conventional mRNA is generated by in vitro transcription. 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.

[0485] The term "transfection" or "delivery" refers to the introduction of exogenous RNA into the intracellular space of a cell. In some embodiments, transfection or delivery is performed by electroporation. In some embodiments, transfection or delivery is performed by lipid nanoparticles. In some embodiments, transfection or delivery is performed directly without modifying the RNA or a vehicle for carrying the RNA. In some embodiments, transfection or delivery occurs by conjugating a cell-reactive or targeting moiety to the RNA.

[0486] The term "input" refers to a stimulus that interacts with the cargo of the self-replicating RNA. Inputs include, but are not limited to, cells, proteins, enzymes, small molecules, light of a specific wavelength, thermal stimulation, or an applied magnetic field. Inputs include, but are not limited to, cells, mediator proteins, enzymes, small molecules, DNA, RNA, light of a specific wavelength, thermal stimulation, or an applied magnetic field.

[0487] The term "modified nucleotide" refers to any analog of cytidine, adenosine, guanosine, uridine, or pseudouridine. These analogs can include isomers of the nitrogenous bases, as well as the addition or removal of chemical groups (both naturally occurring and synthetically introduced) from any aspect of the nitrogenous bases. It is explicitly stated herein that the definition of the term "modified nucleotide" does not include modifications to the sugar-phosphate backbone. This exception does not exclude methylation of the 2'O position of the first and second initiating nucleotides, also known as Cap-1 and Cap-2 structures.

[0488] The term "highly substituted" or "high substitution" refers to the replacement of a high percentage of natural nucleotides with corresponding analogs. Wherein the high percentage is greater than 25%; wherein the percentage is greater than 30%; wherein the percentage is greater than 35%; wherein the percentage is greater than 40%; wherein the percentage is greater than 45%; wherein the percentage is greater than 50%; wherein the percentage is greater than 55%; wherein the percentage is greater than 60%; wherein the percentage is greater than 65%; wherein the percentage is greater than 70%; wherein the percentage is greater than 75%; wherein the percentage is greater than 80%; wherein the percentage is greater than 85%; wherein the percentage is greater than 90%; wherein the percentage is greater than 95%; wherein the percentage is 100%. In addition, the terms "100% substitution," "100% replaced," "completely substituted," "completely substituted," and "completely replaced" are used interchangeably throughout the disclosure of the present invention. Completely substituted means that a given unmodified nucleotide is absent from the synthesis or final product of the self-amplifying RNA. A given substitution percentage means that the fractional mixture of a given nucleotide comprises both an analog and naturally occurring cytidine, adenosine, guanosine, or uridine. In a preferred embodiment, 50%-100% of one of the combination of 5-methyluridine, 5-methylcytidine, and 5-hydroxymethylcytidine is substituted into the saRNA. In a preferred embodiment, 50%-100% of one of the combination of 5-methyluridine, 5-methylcytidine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine is substituted into the saRNA. In a preferred embodiment, 100% of the cytidines are replaced with 5-methylcytidine, and the resulting composition of the fully replaced self-amplifying RNA is entirely composed of adenosine, guanosine, uridine, and 5-methylcytidine, and the cargo level of its expression is equivalent to or greater than that of the unmodified saRNA. In another preferred embodiment, 100% of the cytidines are replaced with 5-hydroxymethylcytidine, and the resulting composition of the fully replaced self-amplifying RNA is adenosine, guanosine, uridine, and 5-hydroxymethylcytidine, and the cargo level of its expression is equivalent to or greater than that of the unmodified saRNA. In another preferred embodiment, 100% of the uridine is replaced with 5-methyluridine, and the resulting composition of the fully replaced self-amplifying RNA is adenosine, guanosine, cytidine, and 5-methyluridine, which expresses cargo levels comparable to or greater than unmodified saRNA. In another preferred embodiment, 100% of the uridine is replaced with 5-hydroxymethyluridine, and the resulting composition of the fully replaced self-amplifying RNA is adenosine, guanosine, cytidine, and 5-hydroxymethyluridine, which expresses cargo levels comparable to or greater than unmodified saRNA.

[0489] As used herein, the terms "reduce", "lower", "lower" or "inhibit" refer to a statistically significant reduction. In some embodiments, "reduce", "reduce" or "lower" or "inhibit" generally means reducing by at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and can include, for example, reducing 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, "reduce" or "inhibit" does not include complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. For example, for an individual without a given disorder, reduction can preferably be reduced to a level within an acceptable normal range.

[0490] As used herein, the terms "increased," "increase," "enhance," or "activate" refer to an increase in a statistically significant amount. In some embodiments, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, such as 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 an increase of 100% or any increase between 10%-100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level. In the context of a marker or symptom, "increase" refers to a statistically significant increase in the level.

[0491] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues that are interconnected by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" refer to polymers of amino acids, including modified amino acids (e.g., phosphorylation, glycosylation, glycation, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are typically used to refer to relatively large polypeptides, while the term "peptide" is typically used to refer to smaller polypeptides, but the use of these terms in the art is overlapping. When referring to gene products and fragments thereof, the terms "protein" and "polypeptide" are used interchangeably herein. Therefore, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the aforementioned.

[0492] Further contemplated within the various embodiments described herein are variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any of the specific polypeptides described. With respect to amino acid sequences, one of skill will recognize that a single substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, or protein sequence that changes a single amino acid or a small number of amino acids in the encoded sequence is a "conservatively modified variant," where such changes result in the replacement of an amino acid with a chemically similar amino acid and retain the desired activity of the polypeptide. Such conservatively modified variants are in addition to, but do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0493] A given amino acid may be substituted with a residue having similar physicochemical characteristics, for example, by substituting one aliphatic residue for another (e.g., Ile, Val, Leu, or Ala), or by substituting one polar residue for another (e.g., between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conservative substitutions (e.g., substitutions of entire regions having similar hydrophobicity characteristics) are well known. Polypeptides containing conservative amino acid substitutions can be tested to confirm that the desired activity of the native or reference polypeptide, such as activity and specificity, is retained.

[0494] Amino acids can be grouped according to the similarity of their side chain properties (see ALLehninger, Biochemistry, 2nd ed., pp. 73-75, Worth Publishers, New York (1975)): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic: Asp (D), Glu (E); (4) Basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into the following groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one class for another. Specific conservative substitutions include, for example: Ala for Gly or Ser; Arg for Lys; Asn for Gln or His; Asp for Glu; Cys for Ser; Gln for Asn; Glu for Asp; Gly for Ala or Pro; His for Asn or Gln; Ile for Leu or Val; Leu for Ile or Val; Lys for Arg, Gln or Glu; Met for Leu, Tyr or Ile; Phe for Met, Leu or Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp; and / or Phe for Val, Ile or Leu.

[0495] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be functional fragments of one of the amino acid sequences described herein. As used herein, "functional fragment" refers to a polypeptide fragment or segment that retains at least 50% of the activity of the wild-type reference polypeptide. Functional fragments may comprise conservative substitutions of the sequences disclosed herein.

[0496] In some embodiments, the polypeptides described herein may be variants of the polypeptide sequences described herein. In some embodiments, the variants are conservatively modified variants. Conservative substitution variants can be obtained by, for example, mutations of native nucleotide sequences. "Variants" as described herein are substantially homologous to native or reference polypeptides, but have a polypeptide having an amino acid sequence different from that of a native or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding the variant polypeptide encompasses additions, deletions, or substitutions comprising one or more nucleotides compared to a native or reference DNA sequence, but encodes a protein or a fragment thereof that retains the activity of a native or reference polypeptide. Various site-specific mutagenesis methods (e.g., based on PCR) are known in the art, and conventionally, those of ordinary skill in the art can apply these methods to generate and test artificial variants.

[0497] The 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 the native or reference sequence. For example, the degree of homology (percent identity) between the native sequence and the mutant sequence can be determined by comparing the two sequences using freely available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0498] Variant amino acid sequences may 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 identical amino acids or conservatively substituted amino acids, as described herein. Thus, the percentage of "sequence similarity" is the percentage of identical or conservatively changed amino acids; for example, "sequence similarity" = (sequence identity %) + (conservative change %). It should be understood that a sequence having a specified percentage of similarity to a reference sequence necessarily includes a sequence having the same specified percentage of identity as the reference sequence. Those skilled in the art will appreciate the various computer programs that use different mathematical algorithms that can be used to determine the identity or similarity between two sequences. For example, a computer program using the algorithm of Needleman and Wunsch (Needleman et al. (1970)); the GAP program in the Accelrys GCG software package (Accelerys Inc., San Diego USA); 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 BLAST (Basic Local Alignment Tool using default parameters); see, for example, U.S. Patent 10,023,890, the contents of which are incorporated herein by reference in their entirety.

[0499] As used herein, the phrase "maintains the same function" when used in reference to an enzyme, catalyzes the same reaction as a reference enzyme.

[0500] The change of the native amino acid sequence can be achieved by any of many techniques known to those skilled in the art.For example, mutations can be introduced at specific sites by synthesizing oligonucleotides containing mutant sequences, which are flanked by restriction sites that enable connection to the fragment of the native sequence. After connection, the reconstructed sequence encoding obtained has an analogue with the desired amino acid insertion, substitution or deletion. Alternatively, an oligonucleotide-mediated site-specific mutagenesis program can be used to provide the nucleotide sequence of the change, which has the specific codons changed according to the desired substitution, deletion or insertion. A variety of site-specific mutagenesis methods are known in the art and can be used by ordinary technicians for introducing mutations into specific nucleic acid sites, such as the Kunkel method, cassette mutagenesis, PCR site-directed mutagenesis (such as traditional PCR, primer extension or inverse PCR), full plasmid mutagenesis, in vivo site-directed mutagenesis, CRISPR / Cas guided mutagenesis. The techniques for making such changes are well established and include, for example, 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 Edition); 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. Patent Nos. 4,518,584 and 4,737,462, the contents of which are incorporated herein by reference in their entirety. Any cysteine ​​residue that is not involved in maintaining the correct conformation of the polypeptide may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds may be added to polypeptides to increase their stability or promote oligomerization.

[0501] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or their analogs. A nucleic acid can be 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 that is not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, for example, template DNA, plasmid DNA, or vector DNA. Suitable RNA can include, for example, saRNA or mRNA.

[0502] The term "expression" refers to the cellular processes involved in the production of RNA and protein, and secretion of protein (as appropriate), including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, as applicable. 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.

[0503] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequence and 3'UTR or "tail" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0504] The terms "treat / treating / treatment" or "relieve" as used herein refer to therapeutic treatment, wherein the purpose is to reverse, alleviate, alleviate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term "treatment" includes reducing or alleviating at least one adverse reaction or symptom of a condition, disease or disorder. A treatment is generally "effective" if one or more symptoms or clinical indicators are reduced. Alternatively, a treatment is "effective" if the progression of the disease is reduced or stopped. That is, "treatment" includes not only the improvement of symptoms or indicators, but also the cessation or at least slowing down of the progression or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, alleviating one or more symptoms, narrowing the scope of the disease, stabilizing the disease (i.e., no longer worsening), delaying or slowing the progression of the disease, alleviating or relieving the disease, alleviating (whether partially or completely) and / or reducing mortality (whether detectable or not). The term "treatment" of a disease also includes providing relief (including palliative care) of the symptoms or side effects of the disease.

[0505] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and a pharmaceutically acceptable carrier (e.g., a carrier commonly used in the pharmaceutical industry). As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be a carrier other than water. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticles, and / or ointment. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be an artificial or engineered carrier, for example, a carrier in which the active ingredient is not found in nature or in practice.

[0506] As used herein, the term "administering" refers to placing a saRNA, or a nucleic acid, or a cell, or a composition comprising a saRNA, as disclosed herein, into a subject by a method or route that results in at least partial delivery of the saRNA, or a nucleic acid, or a cell, or a composition comprising a saRNA, at a desired site. Pharmaceutical compositions comprising the saRNA, or a nucleic acid, or a cell, or a composition comprising a saRNA, as disclosed herein, can be administered by any appropriate route that results in effective treatment of the subject. In some embodiments, administration comprises physical action by the human body, such as injection, an act of ingestion, an act of administration, and / or manipulation of a delivery device or machine. For example, such actions can be performed by a medical professional and / or the subject being treated.

[0507] As used herein, "contacting" 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 a cell culture medium, transfection, transduction, perfusion, injection, or other delivery methods known to those skilled in the art. In some embodiments, contacting includes physical human activity, such as injection, dispensing, mixing, and / or decanting, and / or manipulation of a delivery device or machine.

[0508] In some embodiments of any aspect, the cells can be maintained in culture. As used herein, "maintain" refers to maintaining the viability of a cell or cell population. A maintained cell population will have at least a subpopulation of metabolically active cells.

[0509] As used herein, the term "specific binding" refers to a chemical or physical interaction between two molecules, compounds, cells, and / or particles wherein a first entity binds to a second, target entity with greater specificity and affinity than to a third, non-target entity. In some embodiments, specific binding can refer to an affinity of a first entity for a second, target entity that is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times, or more greater than its affinity for a third, non-target entity. An agent that is specific for a particular target is one that exhibits specific binding for that target under the assay conditions used.

[0510] As used herein, the term "analog" refers to a substance that shares one or more specific structural features, elements, components, or moieties with a reference substance. Typically, an "analog" exhibits significant structural similarity to a reference substance, such as sharing a core or common structure, but also differs in certain discrete aspects. In some embodiments, an analog is a substance that can be generated from a reference substance, for example, by chemical manipulation of the reference substance.

[0511] The term "statistically significant" or "significantly" refers to statistical significance, and typically refers to a difference of two standard deviations (2SD) or greater.

[0512] Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about.” As noted above, when used with percentages, the term “about” can mean ±1%.

[0513] As used herein, the term "comprising" means that there may be additional elements in addition to the specified elements shown. The use of "comprising" means including rather than limiting.

[0514] The term "consisting of" refers to the compositions, methods, and respective components described herein, excluding any elements not listed in the description of the embodiment.

[0515] As used herein, the term "consisting essentially of refers to the required elements of a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiment of the invention.

[0516] Unless the context clearly indicates otherwise, the singular terms "a / an" and "the" include plural referents. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Therefore, the abbreviation "eg" is synonymous with the term "for example."

[0517] The grouping of the alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each group member may be cited and claimed individually or in any combination with other members of the group or other elements found herein. For convenience and / or patentability, one or more members of a group may be included in or deleted from a group. When any such inclusion or deletion occurs, this specification is deemed to comprise the group modified, thereby satisfying the written description of all Markush groups used in the appended claims.

[0518] Unless otherwise defined herein, scientific and technical terms related to this application should have the meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that the present invention is not limited to the particular methods, protocols, and reagents described herein, and therefore may be varied. The terms used herein are only used to describe the purpose of specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the claims. Definitions of commonly used terms in cell biology, immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al., eds., The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers, ed., Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Werner Luttmann, Immunology, published by Elsevier, 2006; Kenneth Murphy, Allan Mowat, Casey Weaver, ed. Janeway's Immunobiology, WWNorton&Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI,...

Claims

1. A self-amplifying RNA (saRNA) comprising at least 25% modified nucleotides and at least one cargo of interest, wherein The modified nucleotides include pyrimidine nucleoside phosphates having a moiety at the 5-carbon of the pyrimidine, wherein the moiety is selected from the group consisting of methyl, ethyl, propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, and hydroxypropyl functional groups.

2. The saRNA according to claim 1, wherein The modified nucleotides include 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, 5-hydroxymethylcytidine, or a combination thereof.

3. The saRNA according to claim 1 or 2, wherein The saRNA expresses the cargo at a level greater than or equal to that of a corresponding saRNA having less than 25% modified nucleotides.

4. The saRNA according to any one of claims 1-3, wherein The pyrimidine includes cytidine, and the modified nucleotide includes 5-methylcytidine.

5. The saRNA according to any one of claims 1-4, wherein The pyrimidine includes cytidine, and the modified nucleotide includes 5-hydroxymethylcytidine.

6. The saRNA according to any one of claims 1-5, wherein The pyrimidine includes uridine, and the modified nucleotide includes 5-methyluridine.

7. The saRNA according to any one of claims 1-6, wherein The pyrimidine includes uridine, and the modified nucleotide includes 5-hydroxymethyluridine.

8. The saRNA of any one of claims 1-7, wherein The substitution level of the modified nucleotides is 25%-50%.

9. The saRNA of any one of claims 1-8, wherein The substitution levels of the modified nucleotides ranged from 51% to 75%.

10. The saRNA of any one of claims 1-9, wherein The substitution level of the modified nucleotides is 75%-99%.

11. The saRNA of any one of claims 1-10, wherein The substitution level of the modified nucleotides is 99.1%-99.9%.

12. The saRNA of any one of claims 1-11, wherein The substitution level of the modified nucleotides was 100%.

13. The saRNA of any one of claims 1-12, wherein In the same saRNA molecule, the modified nucleotides include one or both of 5-methylcytidine and 5-hydroxymethylcytidine, and 5-methyluridine or 5-hydroxymethyluridine.

14. The saRNA of any one of claims 1-13, wherein The initial nucleotide immediately adjacent to the 5' cap in the saRNA comprises adenosine or an adenosine analog.

15. The saRNA of any one of claims 1-14, wherein The initial nucleoside immediately adjacent to the 5' cap in the saRNA comprises guanosine or a guanosine analog.

16. The saRNA of any one of claims 1-15, wherein The initial nucleoside of the saRNA is methylated at the 2'O position of the ribose (Cap 1).

17. The saRNA of any one of claims 1-16, wherein Both the initial nucleotide and the subsequent nucleotides of the saRNA are methylated at the 2'0 position of the ribose sugar (Cap2).

18. The saRNA of any one of claims 1-17, wherein The initiator nucleotide comprises adenosine or an adenosine analog, and wherein the initiator nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap1).

19. The saRNA of any one of claims 1-18, wherein The initiating nucleotide comprises adenosine or an adenosine analog, and wherein both the initiating nucleotide and the subsequent nucleotides of the saRNA are methylated at the 2'O position of the ribose sugar (Cap2).

20. The saRNA of any one of claims 1-19, wherein The starting nucleotide comprises guanosine or a guanosine analog, and wherein the starting nucleotide of the saRNA is methylated at the 2'O position of the ribose sugar (Cap1).

21. The saRNA of any one of claims 1-20, wherein The initiating nucleotide comprises guanosine or a guanosine analog, and wherein both the initiating nucleotide and subsequent nucleotides of the saRNA are methylated at the 2'O position of the ribose sugar (Cap2).

22. The saRNA of any one of claims 1-21, 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) the at least one good of interest.

23. The saRNA of any one of claims 1-22, comprising from 5' to 3': (a) 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3) and / or nonstructural 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) the at least one good of interest; (f) a 3' untranslated region (UTR) derived from at least one virus; and (g) poly-A tail.

24. The saRNA of any one of claims 1-23, further comprising at least one 5' conserved sequence element (CSE) and / or at least one 3' conserved sequence element (CSE) derived from at least one virus.

25. The saRNA of any one of claims 1-24, wherein The nsp1, nsp2, nsp3, nsp4, SGP, 5'UTR, 3'UTR, 5'CSE and / or 3'CSE are derived from the same virus.

26. The saRNA of any one of claims 1-25, wherein At least one of the nsp1, nsp2, nsp3, nsp4, SGP, 5'UTR, 3'UTR, 5'CSE and / or 3'CSE is derived from a different virus.

27. The saRNA of any one of claims 1-26, wherein The virus is an alphavirus.

28. The saRNA of any one of claims 1-27, wherein The at least one virus is selected from the group consisting of Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), Sindbis virus (SIN), Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Mayaro virus (MAYV), Geta virus (GETV), Ross River virus (RRV), Una virus (UNAV), Middleburg virus (MIDV), Arara virus (ONNV), Bama Forest virus (BFV), Mucambo virus (MUCV), Tonate virus (TONV), Everglades virus (EVEV), Rio Negro virus (RNV), Turnip bush virus (TROV), Highlands J virus (HJV), Western equine encephalitis virus (WEEV), Fig mosaic virus (FMV), Aura virus (AURAV), Kunjin virus (KUN), measles virus (MV), coronavirus (CoV), rabies virus (RABV) and vesicular stomatitis virus (VSV).

29. The saRNA of any one of claims 1-28, wherein The alphavirus is selected from the group consisting of Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), Sindbis virus (SIN), Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Mayaro virus (MAYV), Geta virus (GETV), Ross River virus (RRV), Una virus (UNAV), Middleburg virus (MIDV), Arara virus (ONNV), Bama 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).

30. The saRNA of any one of claims 1-29, wherein The saRNA expresses the cargo of interest.

31. The saRNA of any one of claims 1-30, wherein The cargo comprises at least one cargo protein or peptide.

32. The saRNA of any one of claims 1-31, wherein The cargo comprises at least two cargo proteins or peptides.

33. The saRNA of claims 1-32, wherein The at least one cargo protein or peptide is derived from a virus, bacteria, protozoa, mammal or plant.

34. The saRNA of any one of claims 1-33, wherein The cargo comprises a chimeric antigen receptor (CAR) comprising an extracellular domain that specifically binds to an antigen of interest.

35. The saRNA of any one of claims 1-34, wherein The antigen of interest of the CAR is selected from the group consisting of: CD19, CD22, CD30, b-cell maturation antigen (BCMA), disialoganglioside GD2, human estrogen receptor 2 (HER2), G protein-coupled receptor 87 (GPR87), fibroblast activation protein (FAP), CD20, receptor tyrosine kinase-like orphan receptor 1 (ROR1), carcinoembryonic antigen (CEA), mesothelin (MSLN), prostate-specific membrane antigen (PSMA), epidermal growth factor receptor variant III (EGFRvIII), interleukin 13 receptor alpha 2 (IL13Rα2) and natural killer cell group 2 member D (NKG2D).

36. The saRNA of any one of claims 1-35, wherein The CAR is selected from the group consisting of: (a) Conventional CAR; (b)ON-CAR; (c) OFF-CAR system; (d)ON / OFF-CAR; (e) inhibitory CAR; or (f) Split, universal, programmable and reconfigurable (SUPRA) CAR system.

37. The saRNA of claim 36, wherein The conventional CAR comprises: (a) Extracellular binding domain; (b) a transmembrane domain; and (c) at least one intracellular signaling domain.

38. The saRNA of claim 36, wherein The ON-CAR comprises: (a) Extracellular binding domain; (b) transmembrane domain; (c) at least one intracellular signaling domain; and (d) a repressible protease domain that cleaves and degrades the ON-CAR in the absence of a protease inhibitor.

39. The saRNA of claim 36, wherein The OFF-CAR system comprises: (a) a first polypeptide, comprising: (i) extracellular binding domain; (ii) a transmembrane domain; and (iii) a peptide domain; and (b) a second polypeptide, comprising: (i) a repressible protease domain that is capable of specifically binding to the peptide domain in the absence of a protease inhibitor; and (ii) at least one intracellular signaling domain.

40. The saRNA of claim 39, wherein The peptide domain is selected from the group consisting of: K5-66, K5-66-A, K5-66-B, K6-10, K6-10A, K6-10B, K5-66-R, CP5-46, CP5-46-4D5E, CP5-46-A, CP5-46A-4D5E, Ant-CP5-46A-4D5E and apo NS3a reader (ANR) peptide.

41. The saRNA of claim 36, wherein The ON / OFF-CAR comprises: (a) Extracellular binding domain; (b) transmembrane domain; (c) at least one intracellular signaling domain; (d) a repressible protease domain that cleaves and degrades the ON / OFF-CAR in the absence of a protease inhibitor; and (e) Drug-inducible degron domain.

42. The saRNA of any one of claims 38, 39, or 41, wherein The repressible protease domain comprises the hepatitis C virus (HCV) nonstructural protein 3 (NS3).

43. The saRNA of any one of claims 38, 39 or 41, wherein The protease inhibitor is selected from the group consisting of grazoprevir (GZV), danoprevir, simeprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir and telaprevir.

44. The saRNA of claim 41, wherein The drug-inducible degradation determinant domain comprises an IKAROS family zinc finger 3 (IKZF3) domain that can be bound and activated by the drugs lenalidomide or pomalidomide to degrade CAR.

45. The saRNA of claim 36, wherein The inhibitory CAR comprises: (a) Extracellular binding domain; (b) a transmembrane domain; and (c) Inhibitory domain.

46. ​​The saRNA of claim 45, wherein The inhibitory domain comprises a killer cell inhibitory receptor (KIR) domain.

47. The saRNA of claim 36, wherein The SUPRA CAR system includes: (a) a first polypeptide, comprising: (i) an extracellular binding domain; and (ii) a first member of an extracellular protein interaction domain; and (b) a second polypeptide, comprising: (i) a second member of the extracellular protein interaction domain, wherein the second member of the extracellular protein interaction domain is capable of specifically binding to the first member of the extracellular protein interaction domain of the first polypeptide; (ii) a transmembrane domain; and (iii) at least one intracellular signaling domain.

48. The saRNA of any one of claims 37-47, wherein The intracellular signaling domain is selected from the group consisting of: TCRC; FcRy; FcRp; CD3ζ; CD3y; CD35; CD3s; CD3C; CD22; CD79a; CD79b; CD66d; CARD11; CD2; CD7; CD27; CD28; CD30; CD40; CD54 (ICAM); CD83; CD134 (OX40); CD137 (4-1BB); CD150 (SLAMF1); CD152 (CTLA4); CD223 (LAG3); CD270 (HVEM); CD273 (PD-L2); CD274 (PD-L1); CD278 (ICOS); DAP10; LAT; KD2C SLP76; TRIM; ZAP70; and 41BB.

49. The saRNA of any one of claims 1-48, wherein The extracellular binding domain includes an antigen binding domain from an antibody.

50. The saRNA of any one of claims 1-49, wherein The cargo includes a ligand, a cell surface receptor, a transcription factor, a cytokine, a chemokine, an enzyme and / or an antibody.

51. The saRNA of any one of claims 1-50, wherein The cargo comprises an antibody or fragment thereof.

52. The saRNA of claim 51 , wherein The antibody is a bispecific antibody.

53. The saRNA of any one of claims 1-52, wherein The cargo comprises a bispecific T cell engager (BiTE).

54. The saRNA of any one of claims 1-53, wherein The cargo comprises at least one domain that responds to an external input.

55. The saRNA of any one of claims 1-54, wherein The cargo comprises at least one non-coding RNA.

56. The saRNA of claim 55, wherein The non-coding RNA is selected from the group consisting of siRNA, shRNA and microRNA.

57. The saRNA of any one of claims 1-56, wherein The cargo comprises at least one vaccine-relevant antigen.

58. The saRNA of claim 57, wherein The vaccine-associated antigen comprises at least one protein encoded by the viral genome.

59. The saRNA of claim 58, wherein The virus is selected from the group consisting of Rift Valley fever, Crimean-Congo hemorrhagic fever, Lassa fever, Chikungunya virus (CHIKV), Nipah virus (NiV), respiratory syncytial virus (RSV), Ebola virus, Marburg virus, West Nile virus, Venezuelan equine encephalitis virus, yellow fever virus, Japanese encephalitis virus, western equine encephalitis virus, eastern equine encephalitis virus, cytomegalovirus (CMV), human immunodeficiency virus (HIV), influenza virus, Zika virus, Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human papillomavirus (HPV), herpes virus, rotavirus, varicella-zoster virus (VZV), dengue virus, hepatitis A virus, hepatitis B virus, rubella virus, poliovirus and rabies virus.

60. The saRNA of any one of claims 57-59, wherein The vaccine-associated antigens include antigens selected from the group consisting of influenza virus hemagglutinin, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and human respiratory syncytial virus (RSV) fusion glycoprotein.

61. The saRNA of any one of claims 57-60, wherein The vaccine-associated antigen comprises one of SEQ ID NO: 13-SEQ ID NO: 16, SEQ ID NO: 22, or an amino acid sequence that is at least 80% identical to one of SEQ ID NO: 13-SEQ ID NO: 16, SEQ ID NO: 22 and maintains the same function.

62. The saRNA of any one of claims 1-61, comprising the sequence of SEQ ID NO: 21 or a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 21 and maintains the same function.

63. The saRNA of any one of claims 1-62, wherein The cargo comprises at least one transcription factor.

64. The saRNA of claim 63, wherein The transcription factor is a stem cell transcription factor.

65. The saRNA of claim 63 or 64, wherein The transcription factor is selected from the group consisting of octamer-binding transcription factor 3 (Oct3, Oct4), sex-determining region Y (SRY)-box protein transcription factor 2 (Sox2), Kruppel-like factor 4 (Klf4) and c-Myelocytic proto-oncogene (c-Myc).

66. The saRNA of any one of claims 1-65, wherein The cargo comprises at least one growth factor and / or cytokine.

67. The saRNA of claim 66, wherein The growth factor or cytokine is selected from the group consisting of platelet-derived growth factor (PDGF), erythropoietin (EPO), vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), fibroblast growth factor (FGF), human relaxin-2 (RLX2), α-melanocyte stimulating hormone (α-MSH), insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), nerve growth factor (NGF), granulocyte-monocyte colony-stimulating factor (GMCSF), thrombopoietin (TPO), bone morphogenetic protein (BMP), hepatocyte growth factor (HGF), growth / differentiation factor (GDF), neurotrophic factor, migration stimulating factor (MSF) and sarcoma growth factor (SGF).

68. The saRNA of any one of claims 1-67, wherein The cargo comprises one or both of Pappalysin-A1 (PAPPA1) and Pappalysin-A2 (PAPPA2).

69. The saRNA of any one of claims 1-68, wherein The cargo comprises at least one interleukin and / or a cognate receptor for the interleukin and / or a receptor subunit for the interleukin.

70. The saRNA of claim 69, wherein The interleukin is selected from the group consisting of IL-2, IL-4, IL-7, IL-10, IL-12, IL-13 and IL-15.

71. The saRNA of any one of claims 1-70, wherein The cargo comprises at least one enzyme having antioxidant activity.

72. The saRNA of claim 71 , wherein The enzyme is selected from the group consisting of phospholipid hydroperoxide glutathione peroxidase, superoxide dismutase-1, superoxide dismutase-2, Bruton's tyrosine kinase, adenosine deaminase, and ectonucleoside triphosphate diphosphohydrolase.

73. The saRNA of any one of claims 1-72, wherein The cargo comprises glucagon-like peptide-1 (GLP-1) or a fragment thereof.

74. The saRNA of any one of claims 1-73, wherein The saRNA is substituted with a template of SEQ ID NO: 2 or a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 2 and maintains the same function, and wherein the at least one cargo is inserted between the AflII and NdeI cleavage sites of SEQ ID NO: 2 or between nucleotides 7627 and 7628 of SEQ ID NO:

2.

75. The saRNA of any one of claims 1-74, wherein The nsp1, nsp2, nsp3 and nsp4 proteins encoded by the saRNA comprise SEQ ID NO: 4 and / or SEQ ID NO: 23, or at least one amino acid sequence that is at least 90% identical to SEQ ID NO: 4 and / or at least 90% identical to SEQ ID NO: 23 and maintains the same function.

76. The saRNA of any one of claims 1-75, wherein The saRNA is substituted with a template of SEQ ID NO: 5 or a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 5 and maintains the same function.

77. The saRNA of any one of claims 1-76, wherein The pyrimidine includes cytidine, and the modified nucleotide includes 5-methylcytidine and / or 5-hydroxymethylcytidine, and the substituted saRNA comprises SEQ ID NO: 6 or a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 6 and maintains the same function.

78. The saRNA of any one of claims 1-77, wherein The pyrimidine includes uridine, and the modified nucleotide includes 5-methyluridine and / or 5-hydroxymethyluridine, and the substituted saRNA comprises SEQ ID NO: 7 or a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 7 and maintains the same function.

79. The saRNA of any one of claims 1-78, 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, are replaced with at least one alternative cargo of interest.

80. 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 good of interest; in, 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 adenosine or an adenosine analog; and The starting nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap1).

81. A self-amplifying RNA (saRNA), comprising from 5' to 3': (a) 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3) and / or nonstructural 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 good of interest; (f) a 3' untranslated region (UTR) derived from at least one virus; and (g) poly-A tail; in, 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 adenosine or an adenosine analog; and The starting nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap1).

82. The saRNA of claim 80 or 81 , further comprising at least one 5' conserved sequence element (CSE) and / or at least one 3' conserved sequence element (CSE) derived from at least one virus.

83. 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 good of interest.

84. A self-amplifying RNA (saRNA), comprising from 5' to 3': (a) 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3) and / or nonstructural 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 good of interest; (f) a 3' untranslated region (UTR) derived from at least one virus; and (g) poly-A tail.

85. The saRNA of claim 83 or 84, further comprising at least one 5' conserved sequence element (CSE) and / or at least one 3' conserved sequence element (CSE) derived from at least one virus.

86. The saRNA of any one of claims 83-85, wherein The saRNA does not comprise modified nucleotides.

87. The saRNA of any one of claims 83-86, wherein The saRNA comprises less than 25% modified nucleotides.

88. The saRNA of any one of claims 83-87, wherein The cargo comprises a chimeric antigen receptor (CAR) comprising an extracellular domain that specifically binds to an antigen of interest.

89. The saRNA of any one of claims 83-88, wherein The CAR is selected from the group consisting of: (a) Conventional CAR; (b)ON-CAR; (c) OFF-CAR system; (d)ON / OFF-CAR; (e) inhibitory CAR; or (f) Split, universal, programmable and reconfigurable (SUPRA) CAR system.

90. A nucleic acid encoding or comprising the saRNA of any one of claims 1-89.

91. A vector encoding or comprising the saRNA of any one of claims 1-89.

92. A composition comprising the saRNA of any one of claims 1-89.

93. The composition of claim 92, wherein The saRNA was expressed at approximately 5×10 -4 mg / kg dose preparation.

94. The composition of claim 92, wherein The saRNA was expressed at approximately 5×10 -3 mg / kg dose preparation.

95. The composition of claim 92, wherein The saRNA was expressed at approximately 5×10 -2 mg / kg dose preparation.

96. The composition of claim 92, wherein The saRNA was expressed at approximately 5×10 -1 mg / kg dose preparation.

97. The composition of any one of claims 92-96, formulated as lipid nanoparticles.

98. The composition of any one of claims 92-96, formulated in a polymer matrix.

99. A cell contacted with the saRNA of any one of claims 1-89, the nucleic acid of claim 90, the vector of claim 91, or the composition of any one of claims 92-98.

100. The cell of claim 99, wherein The cell is a eukaryotic cell.

101. A pharmaceutical composition comprising the saRNA of any one of claims 1-89 and a pharmaceutically acceptable carrier.

102. A pharmaceutical composition comprising the nucleic acid of claim 90 and a pharmaceutically acceptable carrier.

103. A pharmaceutical composition comprising the vector of claim 91 and a pharmaceutically acceptable carrier.

104. A pharmaceutical composition comprising the cell of any one of claims 99-100 and a pharmaceutically acceptable carrier.

105. The pharmaceutical composition according to any one of claims 101-104, wherein The saRNA was expressed at approximately 5×10 - 4 mg / kg dose preparation.

106. The pharmaceutical composition according to any one of claims 101-104, wherein The saRNA was expressed at approximately 5×10 - 3 mg / kg dose preparation.

107. The pharmaceutical composition according to any one of claims 101-104, wherein The saRNA was expressed at approximately 5×10 - 2 mg / kg dose preparation.

108. The pharmaceutical composition according to any one of claims 101-104, wherein The saRNA was expressed at approximately 5×10 - 1 mg / kg dose preparation.

109. The pharmaceutical composition of any one of claims 101-108, formulated as lipid nanoparticles.

110. The pharmaceutical composition of any one of claims 101-108, formulated in a polymer matrix.

111. A method of expressing at least one cargo of interest in a cell, the method comprising contacting the cell with the saRNA of any one of claims 1-89.

112. A method of expressing at least one cargo in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 101-110.

113. The method according to claim 112, wherein The subject is a human.

114. The method according to claim 112, wherein The subject is a livestock or pet.

115. The method according to any one of claims 111 to 114, wherein The saRNA is a modified saRNA comprising at least 25% modified nucleotides.

116. The method of claim 115, wherein: The modified saRNA is replicated at a level greater than or equal to that of an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides.

117. The method of claim 115, wherein The modified saRNA expresses the cargo at a level greater than or equal to that of an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides.

118. The method of claim 115, wherein: The transfection efficiency of the modified saRNA is greater than the transfection efficiency of an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides.

119. The method of claim 115, wherein: The modified saRNA generates an early interferon response in the subject, and wherein the early interferon response is reduced compared to an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides.

120. The method of claim 115, wherein The cargo is expressed at detectable levels from the modified saRNA for an increased period of time compared to an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides.

121. The method according to any one of claims 111 to 120, wherein The cells are human cells.

122. The method according to any one of claims 111 to 121, wherein The subject has cancer.

123. The method according to any one of claims 111-122, wherein The subject is in need of vaccination against an infectious disease.

124. The method according to any one of claims 111 to 123, wherein The subject is in need of protein replacement therapy.

125. The method according to any one of claims 111 to 124, wherein The subject is in need of antibody therapy.

126. The method according to any one of claims 111 to 125, wherein The subject is in need of treatment for diabetes and / or obesity.

127. The method according to any one of claims 111 to 126, wherein The subject is in need of BITE therapy.

128. The method according to any one of claims 111 to 127, wherein The saRNA allows for tissue-, organ-, or cell-type-specific expression of the cargo.

129. The method of claim 128, wherein The saRNA comprises greater than 50% 5-methyluridine to uridine substitutions and has increased kidney-specific expression of the cargo compared to a corresponding saRNA comprising less than 50% 5-methyluridine to uridine substitutions.

130. The method according to any one of claims 111 to 129, wherein The modified saRNA modulates cellular differentiation of cells containing the saRNA at a level equal to or greater than that achieved with an equivalent dose of a corresponding saRNA having less than 25% modified nucleotides.

131. The method of claim 130, wherein: The cells are regulated by expression of a stem cell transcription factor encoded by the saRNA.

132. The method of claim 130 or 131, wherein: The cells are conditioned to become stem cells.

133. The method according to any one of claims 111-132, wherein The at least one cargo is constitutively expressed in the cell.

134. The method according to any one of claims 111 to 133, wherein The at least one cargo is constitutively expressed in the subject.

135. The method of any one of claims 111-134, further comprising contacting the cell with at least one input to control the fate or function of the cell.

136. The method of any one of claims 111-135, further comprising administering to the subject at least one input to control the fate or function of at least one cell in the subject.

137. The method according to any one of claims 135 or 136, wherein The input is endogenous to the environment of the cell comprising the saRNA.

138. The method according to any one of claims 135 or 136, wherein The input is exogenous to the environment of the cell comprising the saRNA.

139. The method according to any one of claims 135-138, wherein The input is a cell.

140. The method according to any one of claims 135-139, wherein The input is protein.

141. The method of any one of claims 135-140, wherein The input is a small molecule.

142. The method of any one of claims 135-141, wherein The input is light of a specific wavelength.

143. The method according to any one of claims 135-142, wherein The input is a specific temperature.

144. The method according to any one of claims 135 to 143, wherein The input is an applied magnetic field.

145. The method of any one of claims 135-144, wherein The infusion results in cell death comprising the saRNA, increased clearance of cells comprising the saRNA, and / or cell cycle arrest of cells comprising the saRNA.

146. The method of any one of claims 135-145, wherein The import results in inhibition of the repressible protease domain in the cargo.

147. The method of any one of claims 135-146, wherein The import results in activation of the repressible protease domain in the cargo.

148. The method of any one of claims 135-147, wherein The import leads to oligomerization of the cargo and thus activation.

149. The method of any one of claims 135-148, wherein The import leads to oligomerization of the cargo and thus inhibition.

150. The method according to any one of claims 111-149, wherein The cargo comprises a chimeric antigen receptor.

151. The method of any one of claims 111-150, wherein The cargo comprises a plurality of chimeric antigen receptors.

152. The method of any one of claims 111-151, wherein Control of the cell requires the simultaneous presence of all inputs.

153. The method according to any one of claims 111-152, wherein Control of the cells requires the presence of any input.

154. The method of any one of claims 111-153, wherein Control of the cells requires both the presence and absence of different combinations of inputs.

155. The method of any one of claims 111-154, wherein The fate of the cell is a changed cell type.

156. The method of any one of claims 111-155, wherein The fate of the cell is an altered cell positioning.

157. The method of any one of claims 111-156, wherein The function of the cells is lytic.

158. The method of any one of claims 111-157, wherein The function of the cells is stimulatory.

159. The method of any one of claims 111-158, wherein The function of these cells is immunoregulatory.

160. The method according to any one of claims 111-159, wherein The cargo is a protein comprising a chimeric antigen receptor comprising an extracellular domain that senses at least one input signal.

161. The method of any one of claims 111-160, wherein The cargo is a protein comprising at least one chimeric antigen receptor, and the input is a small molecule and / or a protein.

162. The method of any one of claims 111-161, wherein The cargo is a protein comprising at least one domain that responds to an external input.

163. The method according to any one of claims 111-162, wherein The cargo is a protein comprising at least one repressible protease domain that is responsive to at least one protease inhibitor to regulate the activity of the cargo.

164. The method of any one of claims 111-163, wherein The saRNA is delivered to the cells in situ.

165. The method of any one of claims 111-164, wherein The saRNA is delivered to the cells ex vivo.

166. The method of any one of claims 111-165, wherein The cargo of the saRNA encodes and expresses at least one cargo protein, at least one protein reporter and / or at least one expression-enhancing protein.

167. The method of claim 166, wherein The cargo protein is a chimeric antigen receptor, and the expression enhancing protein is B18R.

168. The method of claim 166, wherein The cargo protein is a chimeric antigen receptor, and the expression enhancing protein is E3L.

169. The method of any one of claims 111-168, wherein The saRNA is delivered to the cells by electroporation.

170. The method of any one of claims 111-169, wherein The saRNA is delivered to the cells via lipid nanoparticles.

171. The method of any one of claims 111-170, wherein The saRNA or the composition comprising the saRNA comprises a targeting domain to increase the transfection efficiency of the saRNA into a specific cell type.

172. The method of any one of claims 111-171, wherein The saRNA increases the expression level of the cargo protein in the cells.

173. The method of any one of claims 111-172, wherein The activity of the cargo protein expressed by the saRNA is controlled by the administration of small molecules and / or proteins and / or RNA molecules.

174. The method of claim 173, wherein The RNA molecule is an aptamer.

175. The method of any one of claims 111-174, wherein The protein is a chimeric antigen receptor (CAR), and the activity of the CAR is regulated by at least one small molecule response domain.

176. The method of any one of claims 111-175, wherein The activity of the cargo protein expressed by the saRNA is increased and / or decreased by the administration of at least one small molecule.

177. The method of claim 176, wherein The at least one small molecule is a protease inhibitor or a molecule capable of interacting with at least one protein domain in the cargo protein.

178. The method of any one of claims 111-177, wherein The saRNA encodes and expresses multiple cargo proteins that are capable of interacting with each other.

179. The method of claim 178, wherein The interaction of the multiple cargo proteins results in conditional activity of cells transfected by the saRNA.

180. The method of claim 178 or 179, wherein The cargo proteins capable of interacting with each other are chimeric antigen receptors with activation and / or inhibition functions.

181. The method of any one of claims 111-180, wherein Multiple different saRNAs were delivered to a mixture of cells.

182. The method of any one of claims 111-181, wherein The saRNA is administered to the subject by intraocular, intraosseous (IO), intraperitoneal (IP), subcutaneous (SC), intravenous (IV), intramuscular (IM), intrarectal, intravaginal, intraarticular (IA), inhalation, or topical administration.

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