Self-replicating RNA and uses thereof

Abstract

The technology described herein relates to compositions and methods for modifying and regulating cellular activity by expressing proteins from self-amplifying RNA (saRNA). Also described herein are compositions and methods for modifying and regulating cellular activity by expressing proteins from self-amplifying RNA substituted with chemically modified nucleotides. TIFF2025539792000052.tif82170

Description

[Technical Field]

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

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

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

[0004] Technical Field The technology described herein relates to methods and compositions for modifying and controlling cellular activity with self-replicating RNA.

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

[0006] background Adoptive cell therapy is a promising strategy for treating various cancers. Currently, there are five approved adoptive cell therapies for the treatment of leukemia, lymphoma, and multiple myeloma. Furthermore, applications of adoptive cell therapy beyond oncology are being explored. While clinical outcomes of approved therapies are promising, many challenges associated with ex vivo cell engineering remain. Cell isolation, genetic engineering, and expansion protocols are costly and time-consuming. Furthermore, genetic modification of cells poses the risk of off-target genome editing. An attractive alternative is to reprogram cells ex vivo or in vivo with messenger RNA (mRNA). In the oncology context, the use of immune cells programmed with mRNA has demonstrated efficacy in a limited number of cancer models. However, several challenges have been reported regarding expression levels and duration. As a result, repeated dosing is often required to achieve functional benefits. In contrast, self-replicating RNA can be used to express therapeutic programs at higher levels and for longer periods. Furthermore, self-replicating RNA systems can be used for the ex vivo or in vivo modification of cells.

[0007] Control is desirable to ensure safety and improve the activity of engineered cell therapies. Control is often achieved by including components that can be externally controlled to turn activity on or off. A common example of control in cell therapy is a kill switch that can be included in engineered cells. If the engineered cells pose a risk, they can be killed by injecting a small molecule that activates the kill switch present in the engineered cells. An additional strategy for controlling the activity of engineered cells is to utilize engineered proteins with externally controlled behavior. In the context of oncology, there are a series of chimeric antigen receptors that are regulated by drugs. The resulting activity of CAR-T cells engineered with drug-regulated receptors depends on the dose of the regulatory molecule administered. Another strategy for controlling cell activity is to utilize logic computation. By including components that interact with each other, it is possible to create circuits that sense and respond logically to various signals. This allows for the creation of therapies with enhanced specificity by performing therapeutic actions after satisfying a combination of input signals. The complexity of such logic computation circuits is often limited by the size capacity of genetic engineering methods. The development of a rapid, scar-free method to modify cells with self-replicating RNA and controllable activities is highly desirable.

[0008] Furthermore, the initial discovery by Kariko and Weissman of how modified nucleotides can enable mRNA to evade the immune system paved the way for the field of mRNA therapeutics (Kariko, K. Immunity, 2005. PMID: 16111635 (Non-Patent Document 1)) (Kariko, K. Molecular Therapy, 2008. PMID: 18797453 (Non-Patent Document 2)). The immunogenicity of mRNA containing chemically modified nucleotides is significantly reduced (Kariko, K. Molecular Therapy, 2008. PMID: 18797453) (Kormann, M. Nature Biotechnology, 2011. PMID: 21217696 (Non-Patent Document 3)) (Kariko, K. Immunity, 2005. PMID: 16111635). Currently, the clinical gold standard for mRNA therapeutics is the complete substitution of uridine with N1-methylpseudouridine (m1Ψ), which traditionally has the greatest effect on suppressing type I interferon (IFN) responses induced by dsRNA and ssRNA (Kariko, K. Immunity, 2005. PMID: 16111635). At the molecular level, modified nucleotides alter the stability or accessibility of specific base pairs, alter hydrogen bonding patterns, and shift RNA hydrophobicity. Some of these novel interactions alter RNA stability and alter the primary or secondary structure of RNA in a way that stabilizes or inhibits RNA-protein interactions (Kierzek, E. Nature Communications, 2022. PMID: 35277476 (Non-Patent Document 4)) (Harcourt, E. Nature, 2017. PMID: 28102265 (Non-Patent Document 5)) (Davis, D. Nucleic Acids Research, 1995. PMID: 8559660 (Non-Patent Document 6)). It is through these mechanisms, in part, that modified nucleotides imbue RNA with immune evasion properties. However, these same RNA-RNA and RNA-protein interactions are essential for self-amplifying RNA (saRNA) function.

[0009] saRNA is a type of RNA that has the ability to replicate and amplify itself in situ. This is achieved by encoding both an RNA-dependent RNA polymerase (RdRp) and a protein of interest (Bloom, K. Gene Therapy, 2021. PMID: 33093657 (Non-Patent Document 7)). Some of the advantages of saRNA include higher potency per μg of RNA and a longer duration of action compared to non-replicating mRNA (nrRNA) (Minnaert, K. Advanced Drug Delivery Reviews, 2021. PMID: 34324884 (Non-Patent Document 8)) (Geall, A. Expert Opinion on Drug Discovery, 2022. PMID: 36384351 (Non-Patent Document 9)). The RdRp encoded by the saRNA recognizes conserved secondary structures and sequences, called conserved sequence elements, and subgenomic promoters (SGPs), thereby enabling transcription of the minus- and plus-strand saRNAs and mRNA encoding the cargo of interest. The current understanding in the saRNA field is that incorporating modified nucleotides into saRNA inactivates replicase activity and abolishes 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 demonstrate that incorporating modified nucleotides at >25% substitution results in saRNA that does not produce sufficient antigen to be therapeutically effective.

[0010] Since 2015, there have been over 10 clinical trials using saRNA, and although preclinical evidence is promising, human data have demonstrated lower seroconversion rates, lower neutralizing antibody levels, and less antibody production after booster immunization compared to non-replicating mRNA (Geall, A. Expert Opinion on Drug Discovery, 2022. PMID: 36384351). One hypothesized reason for this is the early and potent activation of the innate immune response by saRNA containing unmodified nucleotides, which prevents both saRNA replication and the launch of cargo from the saRNA SGP. In knockout mice lacking type I IFN-α and β receptor subunit 1, saRNA resulted in higher IgG-specific antibody titers and seroconversion compared to wild-type mice (Pepini, T. The Journal of Immunology, 2017. PMID: 28416600 (Non-Patent Document 12)) (Zhong, Z. Nano Today, 2018. DOI: 10.1016 / J.NANTOD.2018.10.005 (Non-Patent Document 13)). Recently, corticosteroid immunosuppression was explored as a combination therapy to minimize innate immune responses, but only resulted in suppression of seroconversion. This same study demonstrated that removing dsRNA contaminants that activate TLR3 and stimulate type I IFN expression increased vaccine immunogenicity (Zhong, Z. Molecular Therapy, 2021. PMID: 33484964 (Non-Patent Document 14)). The suppression of type I IFN by saRNA is further supported by the fact that intramuscular injection (IM) is the optimal delivery route for saRNA, as intradermal injection has been shown to induce a higher type I IFN response than IM (Zhong, Z. Vaccines, 2019. PMID: 31450775). Collectively, previous studies have demonstrated that overcoming the initial interferon response could significantly increase saRNA efficacy. Part of what makes saRNA effective as a vaccine candidate is that saRNA acts as its own adjuvant.However, controlling the early IFN response is essential to achieve a balance between the adjuvant activity of saRNA launch and IFN-mediated suppression (Zhong, Z. Nano Today, Z23. (2018) DOI: 10.1016 / J.NANTOD.2018.10.005). Without mechanisms to avoid early recognition and pre-expression during endosomal escape, saRNAs may continue to stumble in the clinic.

[0011] Considerable efforts have been made to develop self-amplifying RNAs as therapeutic agents, but there is a continuing need to improve the stability and potency of self-amplifying RNAs to achieve this goal. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] 12 / 831,252 [Non-patent literature]

[0013] [Non-Patent Document 1] Kariko, K. Immunity, 2005. PMID: 16111635 [Non-patent document 2] Kariko, K. Molecular Therapy, 2008. PMID: 18797453 [Non-patent document 3] Kormann, M. Nature Biotechnology, 2011. PMID: 21217696 [Non-patent document 4] Kierzek, E. Nature Communications, 2022. PMID: 35277476 [Non-Patent Document 5] Harcourt, E. Nature, 2017. PMID: 28102265 [Non-patent document 6] Davis, D. Nucleic Acids Research, 1995. PMID: 8559660

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Summary of the Invention

[0014] overview The technology described herein relates to a self-replicating RNA system. In particular, methods and compositions are described herein for modifying cells using self-replicating RNA and introducing elements that can be externally controlled or establish logic-based computational control. Self-replicating RNA is composed of a sequence derived from an RNA virus and an additional cargo sequence. External control is established by utilizing a cargo protein domain that is responsive to external input (e.g., small molecules, light, proteins). Logic-based computational control is established by including components that sense external or internal input and interact with each other directly or indirectly to perform logic operations.

[0015] Described herein is a general framework for modifying cells with self-replicating RNA and providing them with controllable behavior: (1) expression of cargo proteins in the cell; (2) external control of cell behavior by administration of external factors that increase or decrease the activity of the cargo proteins; and (3) internal control of cell behavior by logic computation circuits that sense inputs and perform logic computations.

[0016] In some embodiments, self-replicating RNA is produced by utilizing the sequence derived from virus elements that can copy and produce additional RNA.Exemplary viruses include alphavirus, flavivirus, measles virus, and rhabdovirus.In certain embodiments, self-replicating RNA is produced by utilizing the sequence derived from 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 certain embodiments, replication can be the result of including conserved non-coding sequence elements at the 5'-end and 3'-end of RNA chain, in addition to including the coding sequence of proteins nsp1, nsp2, nsp3, and nsp4.The expression of any protein that can be encoded by gene can be achieved by placing the coding sequence behind a subgenomic promoter sequence or an internal ribosome entry site (IRES) sequence.

[0017] 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 polyA tail.

[0018] In one embodiment of any aspect herein, the self-replicating RNA is produced by in vitro transcription. In other embodiments, the self-replicating RNA is transcribed into RNA and delivered to cells as plasmid DNA containing the necessary sequences for replication.

[0019] In one embodiment of any aspect herein, the RNA contains a 5' cap structure.

[0020] In one embodiment of any aspect herein, the RNA comprises a 3' poly A tail.

[0021] In one embodiment of any aspect herein, the RNA contains a 5'UTR, a 3'UTR, or a combination of both.

[0022] In one embodiment of any aspect herein, the inclusion of sequence elements derived from the VEEV virus enhances expression of functional protein for a longer period of time than conventional mRNA.

[0023] In one embodiment of any aspect herein, the functional protein is expressed from a self-replicating RNA containing a protein-coding sequence downstream of a subgenomic promoter.

[0024] In one embodiment of any aspect herein, the functional protein is expressed from a self-replicating RNA containing a protein-coding sequence downstream of an internal ribosome entry site (IRES) sequence.

[0025] In one embodiment of any aspect herein, the functional protein is a chimeric antigen receptor.

[0026] 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.

[0027] In one embodiment of any aspect herein, the functional protein is a chimeric antigen receptor having a domain that interacts with a small molecule, and the stability of the receptor is regulated in the presence of the small molecule by known and unknown mechanisms.

[0028] Another aspect details how the activity of a functional protein depends on the concentration of the administered small molecule.

[0029] Another aspect details how the activity of a functional protein depends on the concentration of multiple administered small molecules.

[0030] In one embodiment of any aspect herein, the functional protein is a chimeric antigen receptor having a domain that interacts with a protein, the stability of which is regulated by known and unknown mechanisms in the presence of the protein.

[0031] Another aspect details how the activity of a functional protein depends on the concentration of the administered protein.

[0032] Another aspect details methods in which the activity of a functional protein depends on the concentration of multiple administered proteins.

[0033] In one embodiment of any aspect herein, the expression level of the functional protein is equal to or greater than the expression level resulting from other methods for establishing constitutive expression of the protein.

[0034] In one embodiment of any aspect herein, multiple proteins are expressed from the same self-replicating RNA strand by including an IRES sequence between the coding sequences for each protein.

[0035] In one embodiment of any aspect herein, multiple proteins are expressed from the same self-replicating RNA strand by including a 2A sequence between the coding sequences for each protein.

[0036] In one embodiment of any aspect herein, the expression level of multiple proteins expressed from the same self-replicating RNA is equal to or greater than the expression level resulting from other methods for establishing constitutive expression of multiple proteins.

[0037] In one embodiment of any aspect herein, multiple chimeric antigen receptors with opposite functions are expressed from the same self-replicating RNA strand.

[0038] Another aspect details a method in which the resulting cellular activity depends on external signals sensed by multiple proteins with different functions. The resulting activity performed by the cell depends 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, etc.

[0039] In one embodiment of any aspect herein, a functional protein with expression enhancing capabilities by known or unknown mechanism is co-expressed, which is used to enhance the level and duration of expression.

[0040] In one embodiment of any aspect herein, the self-replicating RNA is delivered to a cell via electroporation.

[0041] In one embodiment of any aspect herein, the self-replicating RNA is delivered to a cell via nucleofection.

[0042] In one embodiment of any aspect herein, the RNA is delivered to the cell via a lipid nanoparticle.

[0043] In one embodiment of any aspect herein, the RNA is delivered to the cell via a nanoparticle.

[0044] In one embodiment of any aspect herein, the RNA is delivered to a designated cell type by delivery using lipid nanoparticles that contain an antibody on the surface of the nanoparticle.

[0045] In one embodiment of any aspect herein, the function of the chimeric antigen receptor is to activate immune cells.

[0046] In one embodiment of any aspect herein, the function of the chimeric antigen receptor is a function of inhibiting an immune cell.

[0047] In one embodiment of any aspect herein, a reporter protein is co-expressed with the cargo protein to measure the level of expression resulting from the self-replicating RNA.

[0048] In one embodiment of any aspect herein, multiple cell types are modified with the self-replicating RNA.

[0049] In one embodiment of any aspect herein, one or more cell types are modified with self-replicating RNA.

[0050] In one embodiment of any aspect herein, one or more cell types are modified with a self-replicating RNA prior to administration to a patient.

[0051] In one embodiment of any aspect herein, one or more cell types are modified with the self-replicating RNA, where the cells are within a patient.

[0052] In one embodiment of any aspect herein, one or more cell types modified with self-replicating RNA are administered to a patient via intraosseous (IO), intraperitoneal (IP), subcutaneous (SC), intravenous (IV), intramuscular (IM), and intra-articular administration, or via inhalation or topical administration.

[0053] Previous studies have suggested that the level of modified nucleotide substitution in SaRNA should be less than 25%; otherwise, expression of the transgene encoded by the SaRNA will be reduced compared to the equivalent SaRNA lacking such modifications. (See, e.g., Voigt, E. NPJ vaccines, 7. (2022), (USSN 12 / 831,252), (WO2011005799), (US10532067B2), (US11291682B2), (US20220054525A1), (US10487332B2), (US20200048636A1), (US20220056449A1), (EP3964584A1), (WO2012006 376), (US20220192997A1), (US11058762B2), (US20210290755A1), (US20140242152), (US20220313815A1), (US20210347828A1), (WO2022137128A2), (US20140271829A1). For example, USSN 12 / 831,252 discloses that incorporation of 0.01% to 25% modified nucleotides is the optimal substitution ratio for maintaining expression of transgene cargo, either a reporter construct or a vaccine antigen. At higher substitution ratios, expression of the transgene cargo is reduced.

[0054] 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% to 100%, or even 100%, and the resulting saRNA can still express one or more cargoes of interest encoded on the saRNA. Furthermore, in certain circumstances, the expression level of a transgene encoded by the saRNA can be even higher than that of a comparable saRNA lacking such modifications. These specific modifications allow the saRNA to maintain its self-amplifying function and maintain expression of the cargo protein encoded by one or more cargoes.

[0055] Described herein is a general framework for expressing cargo from self-amplifying RNA that contains modified nucleotides at more than 25% substitutions and can maintain or increase expression capacity compared to unmodified saRNA. This allows for the expression of vaccine antigens, protein replacement therapies, antibodies, enzymes, or for modifying cells with modified self-replicating RNA to equip cells with controllable behaviors: (1) expression of cargo proteins in the cell; (2) external control of cellular behavior by administering external factors that increase or decrease the activity of the cargo proteins; and (3) internal control of cellular behavior by logic-based computational circuits that sense inputs and perform logic-based computations.

[0056] Disclosed herein are self-amplifying RNAs containing more than 25% substitution of certain nucleotides with modified nucleotides. These highly substituted saRNAs not only maintain saRNA function but also confer suppression of innate immune activation. Furthermore, these highly substituted saRNAs are more effective, increasing cargo expression and transfection efficiency. The saRNAs described herein have the potential to open the next frontier in RNA therapeutics, which has been hindered by the inherent immunogenicity of this construct. This research provides a blueprint for modifying and enhancing saRNA therapeutics as vaccines, long-lasting cell therapies, protein replacement therapies, and any other conceivable form of RNA therapeutics.

[0057] In some embodiments, self-replicating RNA is produced by utilizing sequences derived from viral elements that can copy and produce additional RNA.Exemplary viruses include alphavirus, flavivirus, measles virus, coronavirus, and rhabdovirus.In certain embodiments, self-replicating RNA is produced by utilizing sequences derived from 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 certain embodiments, replication can be the result of including conserved non-coding sequence elements at the 5'-end and 3'-end of the RNA chain in addition to including the coding sequences of proteins nsp1, nsp2, nsp3, and nsp4.The expression of any protein that can be encoded by a gene can be achieved by placing the coding sequence behind a subgenomic promoter sequence or an internal ribosome entry site (IRES) sequence. Additionally, non-coding RNAs can be transcribed by placing microRNA or siRNA sequences behind a subgenomic promoter.

[0058] 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 nucleotide comprises a pyrimidine nucleoside phosphate with a moiety on carbon 5 of the pyrimidine, the moiety being selected from the group consisting of methyl, ethyl, propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, and hydroxypropyl functional groups.

[0059] In some embodiments of any aspect, the modified nucleotide comprises 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, 5-hydroxymethylcytidine, or a combination thereof.

[0060] In some embodiments of any aspect, the saRNA expresses the cargo at a level greater than or equal to that of a corresponding saRNA having fewer than 25% modified nucleotides.

[0061] In one embodiment of any aspect herein, the self-amplifying RNA is composed of nucleotides, wherein one or more of the unmodified nucleotides, including cytidine, adenosine, uridine, and guanosine, are replaced with a percentage of modified analogs of the corresponding base, the percentage being at least 25%, for example, 25-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, or 90-100%, 25-95%. , 30-95%, 40-95%, 50-95%, 60-95%, 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 level of substitution of modified nucleotides is between 25% and 50%; 51% and 75%; 75% and 99%; 99.1% and 99.9%; or 100%.

[0062] In one embodiment of any aspect herein, a self-amplifying RNA (saRNA) encodes a cargo of interest, wherein the saRNA contains certain nucleotides at a level of greater than 25% substitution with modified nucleotides, the modified nucleotides being 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.

[0063] In one embodiment of any aspect herein, a self-amplifying RNA (saRNA) encodes a cargo of interest, wherein the saRNA contains certain nucleotides at a level of greater than 25% substitution with modified nucleotides, the modified nucleotides being 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.

[0064] In one embodiment of any aspect herein, a self-amplifying RNA (saRNA) encodes a cargo of interest, wherein the saRNA contains certain nucleotides at a level of greater than 25% substitution with modified nucleotides, wherein the modified nucleotides are composed of pyrimidine nucleoside triphosphates having a moiety on the 5th carbon, wherein the moiety is selected from the list including methyl, ethyl, propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, or hydroxypropyl functional groups, and optionally, the saRNA expresses the cargo at a level greater than or equal to that of the same saRNA without the modified nucleotides.

[0065] In one embodiment of any aspect herein, the substitution of 5-methyluridine with one or both of 5-methylcytidine and 5-hydroxymethylcytidine occurs on the same saRNA molecule.

[0066] In one embodiment of any aspect herein, substitution of 5-methyluridine or 5-hydroxymethyluridine with one or both of 5-methylcytidine and 5-hydroxymethylcytidine occurs on the same saRNA molecule.

[0067] In one embodiment of any aspect herein, the initial nucleotide, which is the nucleotide immediately adjacent to the 5' cap, is adenosine or an adenosine analog.

[0068] In one embodiment of any aspect herein, the initial nucleotide, which is the nucleotide immediately adjacent to the 5' cap, is guanosine or a guanosine analog.

[0069] In one embodiment of any aspect herein, the initial nucleotide of the saRNA and, optionally, subsequent nucleotides are methylated at the 2'O position of the ribose.

[0070] In some embodiments of any aspect, the start nucleotide comprises: (a) an adenosine or adenosine analog, wherein the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1); (b) an adenosine or adenosine analog, wherein both the start nucleotide and the following nucleotide of the saRNA are methylated at the 2'O position of the ribose (Cap 2); (c) a guanosine or guanosine analog, wherein the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1); or (d) a guanosine or guanosine analog, wherein both the start nucleotide and the following nucleotide of the saRNA are methylated at the 2'O position of the ribose (Cap 2).

[0071] In one embodiment of any aspect herein, the saRNA contains components 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).

[0072] In some embodiments of any aspect, 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 polyA tail.

[0073] In some embodiments of any aspect, 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 type of virus.

[0074] In some embodiments of any aspect, at least one virus is an alphavirus.

[0075] 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), Getah virus (GETV), Ross River virus (RRV), Una virus (UNAV), Middleburg virus (MIDV), O'nyong-nyong virus (ONNV), Barmah Forest virus (BFV), Mucambo virus (MUCV), Tonate virus (TONV), Everglades virus (EVEV), Rio Negro virus (RNV), Turnip Rosette virus (TROV), Highlands virus (HFV), and the like. J virus (HJV), Western equine encephalitis virus (WEEV), Fig Mosaic Emaravirus (FMV), Aura virus (AURAV), Kunjin virus (KUN), measles virus (MV), coronavirus (CoV), rabies virus (RABV), and vesicular stomatitis virus (VSV).

[0076] In one embodiment of any aspect herein, the saRNA expresses a cargo that is one or more proteins of viral, bacterial, protozoan, mammalian, or plant origin.

[0077] 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-related antigen comprising at least one protein encoded by the genome of a virus, and at least one transcription factor; at least one growth factor and / or cytokine; one or both of papalysin-A1 (PAPPA1) and papalysin-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 having antioxidant activity; or glucagon-like peptide-1 (GLP-1) or a fragment thereof.

[0078] In one embodiment of any aspect herein, the saRNA expresses a chimeric antigen receptor containing an extracellular domain that senses an input signal.

[0079] In one embodiment of any aspect herein, the saRNA expresses a ligand, cell surface receptor, transcription factor, cytokine, chemokine, enzyme, antibody, or other protein with biological activity.

[0080] In one embodiment of any aspect herein, the saRNA expresses one or more cargoes of interest that include one or more domains that are responsive to external inputs.

[0081] In one embodiment of any aspect herein, the saRNA transcribes one or more non-coding RNAs selected from the list including siRNA, shRNA, or microRNA.

[0082] In one embodiment of any aspect herein, the cargo of interest is one or more vaccine-associated antigens, wherein the vaccine-associated antigens are one or more proteins encoded in the genome of a virus selected from the list including respiratory syncytial virus, hemagglutinin virus, human immunodeficiency virus, influenza virus, Zika virus, sudden acute respiratory syndrome coronavirus 2, human papillomavirus, herpesvirus, rotavirus, chickenpox, dengue virus, hepatitis A virus, hepatitis B virus, rubella virus, poliovirus, or rabies virus.

[0083] In one embodiment of any aspect herein, the one or more cargoes encoded by the saRNA are one or more chimeric antigen receptors selected from the list including CD19, CD22, CD30, b-cell maturation antigen (BCMA), disialoganglioside GD2, human estrogen receptor 2 (HER2), GPR87, fibroblast activator protein (FAP), CD20, receptor tyrosine kinase-like orphan receptor 1 (ROR1), carcinoembryonic antigen (CEA), mesothelin (MSLN), prostate-specific membrane antigen (PSMA), EGFRvIII, IL13Rα2, and NKG2D.

[0084] In one embodiment of any aspect herein, the one or more cargoes encoded by the saRNA are one or more transcription factors, including transcription factors selected from the list of Oct3 / 4, Sox2, Klf4, and c-Myc.

[0085] In one embodiment of any aspect herein, the one or more cargoes encoded by the saRNA are one or more growth factors or cytokines selected from the list including 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), neurotrophin, migration stimulating factor (MSF), sarcoma growth factor (SGF).

[0086] In one embodiment of any aspect herein, the one or more cargoes encoded by the saRNA are one or both of papalysin-A1 (PAPPA1) and papalysin-A2 (PAPPA2).

[0087] In one embodiment of any aspect herein, the one or more cargoes encoded by the saRNA are one or more interleukins selected from the list including IL-2, IL-4, IL-7, IL-10, IL-13, and IL-15, or their cognate receptors and receptor subunits.

[0088] In one embodiment of any aspect herein, the one or more cargoes encoded by the saRNA are one or more enzymes having antioxidant activity selected from the list including phospholipid hydroperoxide glutathione peroxidase, superoxide dismutase-1, and superoxide dismutase-2; Bruton's tyrosine kinase; adenosine deaminase; and ecto-nucleoside triphosphate diphosphydrolase.

[0089] In one embodiment of any aspect herein, the invention is a pharmaceutical composition comprising saRNA and a pharmaceutically acceptable carrier.

[0090] In one aspect, the present invention describes a method for expressing at least one cargo of interest in cells, comprising contacting cells with at least one saRNA described herein.In one embodiment of any aspect herein, cells are transfected with the saRNA described herein.

[0091] In one embodiment of any aspect herein, the cargo of the described saRNA is expressed in a cell (e.g., a eukaryotic cell).

[0092] In one aspect, the present invention describes a method for expressing at least one cargo in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition that comprises or expresses at least one saRNA described herein.In one embodiment of any aspect herein, one or more preselected cargoes are expressed in a subject in need thereof, and this embodiment comprises administering to the subject (e.g., human, livestock) an effective amount of saRNA.

[0093] In one embodiment of any aspect herein, the saRNA maintains or increases initial saRNA replication and cargo expression compared to the same dose of unmodified saRNA.

[0094] In one embodiment of any aspect herein, expression of one or more cargoes of interest encoded by the saRNA is equivalent to or increased compared to expression of unmodified saRNA.

[0095] In one embodiment of any aspect herein, the transfection efficiency of the self-amplifying RNA molecule is increased compared to the transfection efficiency of unmodified saRNA.

[0096] In one embodiment of any aspect herein, the early interferon response resulting from introduction of the saRNA is reduced compared to the early interferon response of unmodified saRNA.

[0097] In one embodiment of any aspect herein, the length of time that the cargo is expressed at a detectable level from the saRNA is increased compared to the length of time from unmodified saRNA.

[0098] In one embodiment of any aspect herein, the saRNA is delivered to a human cell.

[0099] In one embodiment of any aspect herein, the saRNA is administered to a subject with cancer.

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

[0101] In one embodiment of any aspect herein, the saRNA is administered to a subject in need of protein replacement therapy.

[0102] In one embodiment of any aspect herein, the saRNA is administered to a subject in need of antibody therapy.

[0103] In one embodiment of any aspect herein, the saRNA enables tissue-, organ-, or cell-type-specific expression of the cargo.

[0104] In one embodiment of any aspect herein, the saRNA contains greater than 50% uridine substitutions with 5-methyluridine to facilitate kidney-specific expression of the cargo.

[0105] In one embodiment of any aspect herein, the saRNA modulates cell differentiation of cells containing the saRNA at a level equal to or greater than the level of modulation achieved with an equivalent dose of unmodified saRNA.

[0106] In a preferred embodiment, the self-amplifying RNA highly substituted with modified nucleotides expresses more cargo when compared to unmodified saRNA.

[0107] In a preferred embodiment of any aspect herein, the self-replicating RNA is produced by in vitro transcription.

[0108] In one embodiment of any aspect herein, the RNA contains a 5' cap structure.

[0109] In a preferred embodiment of any aspect herein, the 5' cap structure is immediately upstream of the adenosine nucleotide or analogue.

[0110] In a preferred embodiment of any aspect herein, the 5' cap structure is immediately upstream of the guanosine nucleotide or analogue.

[0111] In one embodiment of any aspect herein, the RNA comprises a 3' poly A tail.

[0112] In one embodiment of any aspect herein, the RNA contains a 5'UTR, a 3'UTR, or a combination of both.

[0113] In one embodiment of any aspect herein, the inclusion of the selected modified nucleotides increases or enhances expression of the cargo protein compared to the unsubstituted saRNA.

[0114] In one embodiment of any aspect herein, the inclusion of selected unmodified nucleotides increases or enhances the transfection efficiency of the saRNA into a cell of interest.

[0115] In one embodiment of any aspect herein, the cargo protein is expressed from a self-replicating RNA containing a protein-coding sequence downstream of a subgenomic promoter.

[0116] In one embodiment of any aspect herein, the functional protein is expressed from a self-replicating RNA containing a protein-coding sequence downstream of an internal ribosome entry site (IRES) sequence.

[0117] In one embodiment of any aspect herein, the cargo protein is a chimeric antigen receptor.

[0118] In preferred embodiments, the vaccine-associated antigen is a protein encoded by the viral genome of any strain of respiratory syncytial virus, hemagglutinin virus, human immunodeficiency virus, influenza virus, Zika virus, sudden acute respiratory syndrome coronavirus 2, human papillomavirus, herpesvirus, rotavirus, chickenpox, dengue virus, hepatitis A virus, hepatitis B virus, rubella virus, poliovirus, or rabies virus.

[0119] In preferred embodiments, the vaccine-associated antigen is a protein encoded by the genome of any of Mycobacterium tuberculosis, diptheriae, Neisseria meningitidis, Streptococcus pneumoniae, or Clostridium tetani.

[0120] In one embodiment of any aspect herein, the functional protein is a chimeric antigen receptor.

[0121] 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.

[0122] In one embodiment of any aspect herein, the functional protein is a chimeric antigen receptor having a domain that interacts with a small molecule, and the stability of the receptor is regulated in the presence of the small molecule by known and unknown mechanisms.

[0123] Another aspect details how the activity of a functional protein depends on the concentration of the administered small molecule.

[0124] Another aspect details how the activity of a functional protein depends on the concentration of multiple administered small molecules.

[0125] In one embodiment of any aspect herein, the expression level of functional protein from the highly substituted saRNA is equal to or greater than the expression level resulting from the unsubstituted saRNA.

[0126] In one embodiment of any aspect herein, the initial immune response elicited by the highly substituted saRNA is smaller than the initial immune response of the unsubstituted saRNA.

[0127] In one embodiment of any aspect herein, multiple chimeric antigen receptors with opposite functions are expressed from the same highly substituted self-replicating RNA strand.

[0128] Another aspect details a method in which the resulting cellular activity depends on external signals sensed by multiple proteins with different functions. The resulting activity performed by the cell depends 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, etc.

[0129] In one embodiment of any aspect herein, a functional protein with expression enhancing capabilities by known or unknown mechanism is co-expressed, which is used to enhance the level and duration of expression.

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

[0131] In one embodiment of any aspect herein, the highly substituted saRNA is delivered to cells via nucleofection.

[0132] In one embodiment of any aspect herein, the highly substituted saRNA is delivered to cells via lipid nanoparticles.

[0133] In one embodiment of any aspect herein, the highly substituted saRNA is delivered to a designated cell type by delivery using lipid nanoparticles bearing a targeting moiety.

[0134] In a preferred embodiment of any aspect herein, the function of the chimeric antigen receptor is to activate immune cells.

[0135] In a preferred embodiment of any aspect herein, the function of the chimeric antigen receptor is to inhibit immune cells.

[0136] In one embodiment of any aspect herein, a reporter protein is co-expressed with the cargo protein to measure the level of expression resulting from the highly permuted self-replicating RNA.

[0137] In one embodiment of any aspect herein, multiple cell types are modified with highly substituted self-replicating RNA.

[0138] In one embodiment of any aspect herein, cell type specific expression is achieved by selecting appropriate modified nucleotides for hypersubstitution. [Brief explanation of the drawings]

[0139] [Figure 1A] Figures 1A-1B show a series of schematic diagrams. Figure 1A illustrates conventional versus self-replicating RNA modifications in cells. In this illustration, immune cells are used as a non-limiting example. Self-replicating RNAs self-replicate and amplify, resulting in larger and longer-lasting expression of cargo proteins. Conventional mRNAs are degraded and diluted by proliferation. [Figure 1B]Figures 1A-1B show a series of schematic diagrams. Figure 1B shows a schematic diagram detailing the use of self-replicating RNA to modify cells through protein expression. The delivered proteins (e.g., reporters, enzymes, receptors, ligands, transcription factors) can respond to external inputs (e.g., small molecules, light) or perform logic-based computations to control the modified cells. [Figure 2] A schematic diagram detailing an exemplary format of a self-replicating RNA is shown. The developed self-replicating RNA format contains a conserved sequence element (CSE) derived from an RNA virus (e.g., Venezuelan equine encephalitis virus) and a coding sequence for a nonstructural protein (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. Multiple cargo proteins can be simultaneously expressed by separating the coding sequences with an IRES or 2A sequence or an additional SGP sequence. [Figure 3] Figure 1 shows the expression of a conventional chimeric antigen receptor via self-replicating RNA. Self-replicating RNA can be used to express chimeric antigen receptors that target various antigens. [Figure 4] Figure 1 shows the longer duration of expression of the HER2 chimeric antigen receptor from self-replicating RNA (black) compared to conventional mRNA (gray). Cells expressing mCherry-tagged HER2 CAR were analyzed by flow cytometry 48 and 96 hours after transfection. The percentage of cells expressing the receptor was determined by measuring mCherry fluorescence. The bars above the bar graph indicate the fold change between each time point for each condition. [Figure 5]This shows that expression of the CD19 chimeric antigen receptor from self-replicating RNA is longer. Cells expressing the CD19 CAR were analyzed by flow cytometry up to 12 days after transfection. The percentage of cells expressing the receptor was determined by staining the cells with an anti-myc antibody, which binds to the myc tag on the receptor. Lentivirally transduced cells were used as a control to account for any discrepancies in staining. [Figure 6] Long-term detection of CD19 chimeric antigen receptors in CD3+ primary T cells transfected with self-replicating RNA. Primary CD3+ T cells were transfected with self-replicating (left) or conventional (right) RNA encoding an intracellular mCherry-tagged CD19 chimeric antigen receptor. The plot shows measurements of mCherry fluorescence (x-axis) and anti-myc staining (y-axis) (anti-myc antibody binding to the extracellular myc tag present on the receptor) at multiple time points post-transfection. [Figure 7] Long-term HER2 chimeric antigen receptor function after transfection with self-replicating RNA is shown. Activation of Jurkat T cells expressing HER2 CAR from conventional or self-replicating RNA 72 hours after transfection (top). Cells were cultured on plates with or without HER2 antigen. Activation was measured by GFP fluorescence from the NFAT GFP reporter system. Fold activation was read from the mean GFP fluorescence intensity (bottom). ****=p<0.0001. ns=not significant. N=3 independent replicates. [Figure 8] Long-term CD19 chimeric antigen receptor function after transfection with self-replicating RNA. The percentage of activated CAR-T cells was measured by flow cytometry during overnight co-culture with CD19-expressing Nalm6 cells. 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. [Figure 9]Figure 1 shows CD19 chimeric antigen receptor function in primary CD3+ T cells transfected with self-replicating RNA. Bar graph shows the percent of CAR-T cells positive for CD69, a marker of early T cell activation. Primary T cells transfected with conventional or self-replicating RNA encoding the CD19 CAR were co-cultured with CD19-expressing Nalm6 cells. N=3 independent replicates. ****=p<0.0001. ns=not significant. [Figure 10] Enhancement of CD19 chimeric antigen receptor function in primary CD3+ T cells transfected with self-replicating RNA. Bar graph shows fold change in mean fluorescence intensity of CAR-T stained with an antibody targeting CD69 after overnight co-culture with Nalm6 cells. N=3 independent replicates. *=p<0.05. [Figure 11] Enhanced cytotoxic function of CAR-T cells generated from self-replicating RNA. Target cell killing was observed after co-culture of CAR-T cells with Nalm6-Luc cells for 24 hours. CAR-T cells were primary CD3+ T cells transfected with mRNA or self-replicating RNA 48 hours prior to assay. N=3 independent replicates. [Figure 12] Figure 1 shows that transfection with self-replicating RNA results in higher levels of chimeric antigen receptor expression per cell. Chimeric antigen receptor expression levels were measured by flow cytometry. Bars report the normalized mean fluorescence intensity of a fluorescently labeled anti-myc antibody that binds to the myc tag on the receptor. Intensity values ​​were normalized to the intensity of cells transduced with a lentivirus encoding a CD19 CAR. The fold difference is indicated above each bar. [Figure 13]Figure 1 shows increasing levels of chimeric antigen receptor (mCherry-tagged) expression per cell over extended periods of time following transfection with self-replicating RNA. CAR expression levels over time via measurement of mCherry fluorescence by flow cytometry. Resulting mean fluorescence intensity values ​​were normalized to the values ​​obtained for lentivirally transduced cells at each time point. [Figure 14] Figure 1 shows the abundance of chimeric antigen receptor RNA transcripts at multiple time points post-transfection. RNA levels of CD19 CAR transcripts were measured via RT-qPCR. Resulting transcript levels were normalized to the values ​​obtained for lentivirally transduced cells. N=3 independent replicates. [Figure 15] A schematic diagram illustrating the expression of an exemplary chimeric antigen receptor with external control and logic function by self-replicating RNA is shown. The schematic diagram shows the structure of a chimeric antigen receptor with external control and logic function. Some receptors have domains that turn their function on or off in response to external molecules (e.g., on-CAR, off-CAR, on / off-CAR). Furthermore, some receptors function as activating or inhibitory receptors, allowing conditional activity depending on the presence or absence of target antigen (e.g., inhibitory CAR, supra-CAR). [Figure 16] This demonstrates the function of a drug-controlled "on" chimeric antigen receptor ("on-CAR"). After the CAR is expressed, the NS3 protease domain cleaves the adjacent cleavage site, resulting in the release of the signaling domain from the extracellular scFv. Addition of an NS3 protease inhibitor (e.g., grazoprevir) prevents autocleavage, allowing the CAR to be turned on in the presence of target antigen. [Figure 17]This demonstrates the function of a drug-controlled "off" chimeric antigen receptor ("off-CAR"). After the two components are expressed, the NS3-binding peptide in component 1 binds to the catalytically dead NS3 domain in component 2, allowing the CAR to be activated upon target antigen binding. When a protease inhibitor (e.g., grazoprevir) is added, the binding peptide is displaced and the CAR cannot be activated. [Figure 18] This demonstrates the function of a chimeric antigen receptor ("on / off-CAR") with drug control for "off" and "on." After CAR expression, the NS3 protease domain cleaves the adjacent cleavage site, resulting in the release of the signaling domain from the extracellular scFv. Addition of an NS3 protease inhibitor (e.g., grazoprevir) prevents autocleavage and allows the CAR to function. Addition of lenalidomide also recruits an E3 ligase to the IKZF3 domain, resulting in degradation of the CAR. [Figure 19] This shows that self-replicating RNA efficiently expresses chimeric antigen receptors with external control functions. The expression levels of self-replicating RNA-controlled CD19 CARs and conventional CD19 CARs (e.g., on-CARs, on / off CARs), conventional mRNA, and lentiviral transduced cells were measured by staining with an anti-myc antibody and measuring the fluorescence intensity of the mCherry tag on the receptor. [Figure 20] This figure shows that the activity of chimeric antigen receptors expressed by self-replicating RNA is increased by small molecule administration. The percentage of activated Jurkat CAR-T cells expressing CD19 CARs with the NS3 domain (on-CAR) was measured by flow cytometry. Cells were transfected with self-replicating RNA encoding the receptor 24 hours prior to the assay. CAR-T cells were co-cultured overnight in the absence or presence of Nalm6 and 1 μM grazoprevir. A CD19 CAR expressed by self-replicating RNA lacking the NS3 domain was included as a control (right). N=3 independent replicates. Ns=not significant. **=p<0.01. ****=p<0.0001. [Figure 21] This study demonstrates that the activity of a chimeric antigen receptor expressed by self-replicating RNA increases or decreases with the administration of two different small molecules. The degree of activation of Jurkat CAR-T cells expressing a CD19 CAR (on / off-CAR) with NS3 and IKZF3 domains was measured by flow cytometry. Cells were transfected with self-replicating RNA encoding the receptor 24 hours before the assay. CAR-T cells were co-cultured overnight with Nalm6 without grazoprevir or lenalidomide, with 1 μM grazoprevir, and with 1 μM grazoprevir and 1 μM lenalidomide. N=3 independent replicates. [Figure 22] We demonstrate that the activity of chimeric antigen receptors expressed by self-replicating RNA (on-CAR) can be controlled by the administration of small molecules in primary T cells. The cytotoxicity of CAR-T cells expressing the CD19 CAR and NS3 domain was measured by co-culturing them with the Nalm6 luciferase cell line at a 2:1 effector:target ratio. CAR-T cells and Nalm6 cells were cultured overnight in the absence or presence of 1 μM GZV. As a control, CD19 CAR-T cells generated using self-replicating RNA were included alongside CD19 CAR-T cells generated by lentiviral transduction. [Figure 23] A schematic diagram detailing the functions of chimeric antigen receptors with activating or inhibitory functions that can be expressed by self-replicating RNA is shown. Activating receptors bind to antigens present on target cells, leading to their killing by CAR-T cells. Inhibitory receptors bind to antigens expressed on off-target cells, leading to the inhibition 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. [Figure 24]This demonstrates that self-replicating RNA can efficiently express multiple chimeric antigen receptors with different functions in the same cells. The flow chart shows the expression of CD19-activating CAR and HER2-inhibitory CAR in Jurkat cells transfected with self-replicating RNA. Expression of CD19-activating CAR was measured by staining with anti-myc antibody. 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. [Figure 25] We demonstrate that expressing multiple chimeric antigen receptors with self-replicating RNA enables logic-based computational control of cell function. Activation of Jurkat CAR-T cells expressing CD19-activating and HER2-inhibitory CARs with self-replicating RNA was measured by flow cytometry. CAR-T cells were cultured on plates coated with CD19 antigen alone or CD19 and HER2 antigens together. The extent of NFAT activation was measured by mean fluorescence intensity after gating on CAR+ cells. As controls, cells were lentivirally transduced or transfected with self-replicating RNA encoding only the CD19-activating CAR. N=3 independent replicates. Ns=not significant. ****=p<0.0001. [Figure 26] A schematic diagram detailing the split, universal, and programmable (SUPRA) CAR system, which can be expressed by self-replicating RNA, is shown. The signaling domain and antigen recognition domain are separated into two polypeptide sequences that can interact via a leucine zipper pair. The divisibility of the SUPRA CAR system allows for tunable signaling by swapping the signaling domain recognized by a specific recognition domain. Furthermore, the SUPRA CAR system can be used to perform logic-based computations by recognizing multiple antigens and performing different signaling operations for each. [Figure 27]Figure 1 shows that self-replicating RNA reduces basal activation of T cells expressing CARs with drug-controllable domains (e.g., On-CAR, On / Off-CAR). Bar graphs show the percentage of activated Jurkat CAR-T cells under resting conditions due to tonic signaling. Activation was measured by flow cytometry in Jurkat cells with an NFAT-GFP reporter. N=3 independent replicates. Ns=not significant. ****=p<0.0001. [Figure 28] These results demonstrate that self-replicating RNA-mediated activation of T cells expressing CARs with drug-controllable domains (e.g., On-CARs) is comparable to that of lentivirally generated T cells. The extent of NFAT activation was measured in Jurkat CAR-T cells after co-culture with CD19-expressing Nalm6 cells. Fold NFAT activation was determined by measuring mean fluorescence intensity after gating on CAR+ cells. N=3 independent replicates. ****=p<0.0001. [Figure 29] We demonstrate that self-replicating RNA-mediated chimeric antigen receptor expression is enhanced by coexpression of an expression-enhancing protein. Jurkat T cells were transfected with self-replicating RNA encoding a CD19 CAR or a CD19 CAR plus B18R or E3L. The percentage of cells expressing the CAR was tracked over time by flow cytometry, measuring the fluorescence of the mCherry tag at the C-terminus of the CAR. [Figure 30] We demonstrate that the temporal expression level of chimeric antigen receptors expressed by self-replicating RNA can be controlled by coexpression of an expression-enhancing protein. Jurkat T cells were transfected with self-replicating RNA encoding CD19 CAR or CD19 CAR and either B18R or E3L. CAR expression levels were tracked over time by measuring the fluorescence of the mCherry tag at the C-terminus of the CAR using flow cytometry. MFI values ​​were normalized to the MFI of the self-renewing CD19 CAR alone on day 1. [Figure 31]Jurkat T cells carrying the NFAT-GFP reporter were transfected with self-replicating RNA encoding either the CD19 CAR or the CD19 CAR and either B18R or E3L. Activation of the NFAT pathway was measured by analyzing GFP expression in cells expressing the constructs. [Figure 32] This shows that the activity of chimeric antigen receptors expressed by self-replicating RNA is not affected by co-expression of additional proteins. The extent of NFAT activation was measured in Jurkat CAR-T cells after co-culture with CD19-expressing Nalm6 cells. Fold NFAT activation was determined by measuring mean fluorescence intensity after gating on CAR+ cells. N=3 independent replicates. [Figure 33] This figure shows that T cells can be transfected with lipid nanoparticles containing mRNA with or without antibody conjugation to the nanoparticle surface. The bar graph shows luminescence after transfection of luciferase-encoding mRNA in Jurkat cells with bare Dlin-MC3-PEG-DBCO ("LNP"), isotype antibody-conjugated ("α-isoLNP"), or anti-CD3 antibody-conjugated ("α-CD3LNP") lipid nanoparticles. Electroporation was performed as a positive transfection control. Transfection was verified by lysing cells with a commercially available luciferase assay reagent and measuring emitted light using a plate reader. n=3 independent replicates. [Figure 34] We demonstrate that self-replicating RNA can be used to express proteins and track their activity over extended periods of time. Flow plots show mCherry expression from self-replicating RNA as a model cargo over a 10-day period in Jurkat T cells. [Figure 35] Figure 1 shows that primary cells can be engineered ex vivo to express proteins via self-replicating RNA. Flow plots show mCherry expression from self-replicating RNA as a model cargo over a 7-day period in primary CD3+ T cells. [Figure 36]Figure 1 shows a schematic diagram detailing an exemplary self-amplifying RNA (saRNA) containing modified nucleotides. A linearized plasmid encoding an RNA-dependent RNA polymerase (RdRp) and the cargo of interest was utilized as a template for in vitro transcription (IVT). The IVT reaction contains a nucleoside triphosphate (NTP) mixture of 25%-100% modified nucleotides and may include a cap analog for co-transcriptional capping. The resulting composition is a saRNA with 25%-100% substitution of modified nucleotides. [Figure 37]

[0023] Figure 1 is a schematic diagram detailing the expression profile of saRNA with >25% substitution of the indicated modified nucleotides. The top of the 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 when modified nucleotides are incorporated into self-amplifying RNA, both the percentage of transfected cells and the amount of protein expressed in each transfected cell increase. [Figure 38] Figure 1 shows the transfection efficiency of a library of self-amplifying RNAs encoding mCherry, each individually fully substituted with 28 modified nucleotides, in HEK293T cells. The IVT was performed using a T7 promoter-bearing template with m7G(5')ppp(5')(2'OMeA)pU(CLEANCAP AU) as the 5' cap. [Figure 39] Median expression intensity of a library of self-amplified mCherry RNAs individually and fully substituted with 28 modified nucleotides in HEK293T cells was measured using a T7-promoted template with m7G(5')ppp(5')(2'OMeA)pU(CLEANCAP AU) as the 5' cap. [Figure 40]Figure 1 shows the transfection efficiency of a library of self-amplifying RNAs encoding mCherry, each individually fully substituted with 28 modified nucleotides, in HEK293T cells. The IVT was performed using a T7-promoted template with a 5' cap of 3'-O-Me-m7G(5')ppp(5')G (ARCA). [Figure 41] Figure 1 shows the median expression intensity of a library of self-amplified mCherry-encoding RNAs individually and fully substituted with 28 different modified nucleotides in HEK293T cells. The IVT was performed using a T7-promoted template with a 5' cap of 3'-O-Me-m7G(5')ppp(5')G (ARCA). [Figure 42] Relative expression of cargo proteins from self-amplifying RNAs with different initiating nucleotides, as measured by mCherry MFI, is shown. The figure shows the cap-0 structure in HEK293T cells. The left-to-right order of the bars in each group corresponds to the top-to-bottom order in the legend. [Figure 43] Relative expression of cargo proteins from self-amplifying RNAs with different initiating nucleotides, as measured by mCherry MFI, is shown. The figure shows Cap1 structures in HEK293T cells. The left-to-right order of the bars in each group corresponds to the top-to-bottom order in the legend. [Figure 44] Figure 1 illustrates the relative expression of cargo proteins from self-amplifying RNAs with different initiating nucleotides as measured by percent positive mCherry expression. The figure shows the cap 0 constructs in HEK293T cells. The left-to-right order of the bars in each group corresponds to the top-to-bottom order in the legend. [Figure 45] Figure 1 illustrates the relative expression of cargo proteins from self-amplifying RNAs with different initiating nucleotides as measured by percent positive mCherry expression. The figure shows Cap1 structures in HEK293T cells. The left-to-right order of the bars in each group corresponds to the top-to-bottom order in the legend. [Figure 46] Figures 46A-46B illustrate protein-level early interferon α and β responses in human peripheral blood mononuclear cells (PBMCs) using the Cap-1·ATP initiator construct. PBMCs were exposed to either saRNA or conventional highly substituted mRNA for 6 hours, and the concentrations of IFNα (all subtypes; see, e.g., Figure 46A) and IFNβ (see, e.g., Figure 46B) were analyzed by ELISA. Cell culture supernatants were diluted 1:1 with reagent buffer. The left-to-right order of the bars corresponds to the top-to-bottom order in the legend. [Figure 47] Figures 47A-47B illustrate the transcriptional early interferon α and β response in human peripheral blood mononuclear cells (PBMCs) using the Cap-1·ATP initiation construct. PBMCs were exposed to saRNA with wild-type or modified nucleotides for 6 hours, and mRNA expression levels of IFNA1 (e.g., see Figure 47A) and IFNB1 (e.g., see Figure 47B) were measured by qPCR. Data represent transcript levels from three unique human donors dosed with 100 ng / 1 x 10 cells. The left-to-right order of the bars corresponds to the top-to-bottom order in the legend. [Figure 48] Figure 1 illustrates spike protein (Wuhan-1 variant) expression in C2C12 mouse myoblasts from self-amplified RNA as measured by MFI from AF647-conjugated anti-spike antibody. Different highly substituted modified nucleotides are indicated on the right side of the figure. The left-to-right order of the bars corresponds to the top-to-bottom order in the legend. A Cap-1·ATP-initiated saRNA construct was used. [Figure 49] Figure 1 illustrates spike protein (Wuhan-1 variant) expression in C2C12 mouse myoblasts as measured by the percentage of positive expressing cells from self-amplified RNA. Different highly substituted modified nucleotides are indicated on the right side of the figure. The left-to-right order of the bars corresponds to the top-to-bottom order in the legend. A Cap-1·ATP primed saRNA construct was used. [Figure 50]This diagram illustrates cell-type-specific expression of vaccine antigens from highly substituted self-amplifying RNA. This IVT was performed using a T7 promoter-driven template with m7G(5')ppp(5')(2'OMeA)pU(CLEANCAP AU) as the 5' cap. The left-to-right order of the bars in each group corresponds to the top-to-bottom order in the legend. [Figure 51A] Figures 51A-51C illustrate the enhanced expression conferred by 100% substitution with 5-methylcytidine using a Cap-1·ATP-initiated saRNA construct in human CD3+ T cells. Untreated cells were compared with saRNA encoding the fluorescent reporter protein, mCherry, with or without the nucleotide modification. Figure 51A shows representative flow cytometry plots for each treatment condition. [Figure 51B] Figures 51A-51C illustrate the enhanced expression conferred by 100% substitution with 5-methylcytidine using a Cap-1·ATP-initiated saRNA construct in human CD3+ T cells. Untreated cells were compared with saRNA encoding the fluorescent reporter protein, mCherry, with or without the nucleotide modification. Figure 51B shows representative live-cell fluorescence microscopy images. [Figure 51C] Figures 51A-51C illustrate the enhanced expression conferred by 100% substitution with 5-methylcytidine using a Cap-1·ATP-initiated saRNA construct in human CD3+ T cells. Untreated cells were compared with saRNA encoding the fluorescent reporter protein, mCherry, with or without the nucleotide modification. Figure 51C shows quantification of flow cytometry data from biological replicate experiments. The left-to-right order of the bars corresponds to the top-to-bottom order in the legend. [Figure 52]Figure 1 illustrates the long-term and robust expression conferred by 100% 5-methylcytidine substitution using a Cap-1·ATP-initiated saRNA construct in human CD3+ T cells. Untreated cells were compared with saRNA encoding the fluorescent reporter protein mCherry with or without the nucleotide modification and analyzed over time by flow cytometry. [Figure 53A] Figures 53A-53C illustrate the enhanced expression conferred by 100% substitution with 5-methylcytidine using a Cap-1·ATP-initiated saRNA construct in Jurkat cells. Untreated cells were compared with saRNA encoding the fluorescent reporter protein, mCherry, with or without the nucleotide modification. Figure 53A shows representative flow cytometry plots for each treatment condition. [Figure 53B] Figures 53A-53C illustrate the enhanced expression conferred by 100% substitution with 5-methylcytidine using a Cap-1·ATP-initiated saRNA construct in Jurkat cells. Untreated cells were compared with saRNA encoding the fluorescent reporter protein, mCherry, with or without the nucleotide modification. Figure 53B shows representative live-cell fluorescence microscopy images. [Figure 53C] Figures 53A-53C illustrate the enhanced expression conferred by 100% substitution with 5-methylcytidine using a Cap-1·ATP-initiated saRNA construct in Jurkat cells. Untreated cells were compared with saRNA encoding the fluorescent reporter protein, mCherry, with or without the nucleotide modification. Figure 53C shows quantification of flow cytometry data from biological replicate experiments. The left-to-right order of the bars corresponds to the top-to-bottom order in the legend. [Figure 54A]Figures 54A-54B illustrate the long-term, robust expression conferred by 100% substitution with 5-methylcytidine using a Cap-1·ATP-initiated saRNA construct in Jurkat cells. Untreated cells were compared with saRNA encoding the fluorescent reporter protein, mCherry, with or without the nucleotide modification and analyzed over time by flow cytometry. Figure 54A shows a histogram from these experiments, and Figure 54B shows a time course line graph of these experiments. [Figure 54B] See legend to Figure 54A. [Figure 55] Figure 1 illustrates the effect of increasing substitution ratios of N1-methylpseudouridine and 5-methylcytidine on the potency of Cap-1·ATP-initiated saRNA in Jurkat cells. Transfection efficiency of saRNA encoding a fluorescent reporter protein synthesized with increasing % substitution of modified nucleotides. 0% substitution refers to unmodified saRNA. [Figure 56] Figure 1 illustrates the effect of increasing substitution ratios of 5-methylcytidine on Cap-1·ATP-initiated saRNA immunogenicity. Human PBMCs were treated with saRNA-loaded LNPs for 6 hours and then collected for gene expression analysis. 0% substitution indicates unmodified saRNA. The left-to-right order of the bars corresponds to the top-to-bottom order in the legend. [Figure 57] Figure 1 illustrates the increased expression of chimeric antigen receptors in human CD3+ T cells conferred by 100% substitution of 5-methylcytidine in the Cap-1·ATP-initiated saRNA construct. saRNA encoding chimeric antigen receptors was delivered to CD3+ T cells by lipid nanoparticles synthesized with two different helper lipids, with or without a CD3 targeting domain. [Figure 58-1]Figures 58A-58I are a series of diagrams and graphs illustrating the identification of modified nucleotides compatible with self-amplifying RNA and their in vitro bioactivity. Figure 58A shows a diagram illustrating the limitations of unmodified saRNA and N1mΨ-modified saRNA and the advantages of saRNA with compatible modNTPs. Figure 58B shows the workflow for synthesizing a library of saRNAs fully substituted with modified nucleotides and transferring the library encoding an mCherry reporter into HEK293 cells using lipofection. Figure 58C shows flow cytometry results measuring the percentage of expressing cells in HEK293 cells transfected with the modified saRNA library. Error bars represent the standard deviation of n=3 biological replicates. Figure 58D shows live-cell fluorescence microscopy images and representative histograms of selected modified nucleotides in HEK293 cells. Error bars represent the standard deviation of n=4 biological replicates. Figure 58E shows the expression levels in HEK293 cells 24 hours after transfection with 10 ng of luciferase reporter-encoded RNA using SM102 LNPs. Luciferase signals are shown as fold changes compared to untransfected mock cells. Error bars represent standard deviations for n=4 biological replicates. Figure 58F shows the expression levels in C2C12 cells 24 hours after transfection with 10 ng of luciferase reporter-encoded RNA using SM102 LNPs. Luciferase signals are shown as fold changes compared to untransfected mock cells. Error bars represent standard deviations for n=4 biological replicates. Figure 58G shows the transfection efficiency in Jurkat cells 24 hours after transfection with 25 ng and 250 ng of mCherry reporter-encoded RNA using SM102 LNPs. Error bars represent standard deviations for n=3 biological replicates. Figure 58H shows transfection efficiency in CD3+ T cells from two different donors 24 hours after transfection with 500 ng of mCherry reporter-encoding RNA. n=3 biological replicates / group.Figure 58I shows the expression time course in Jurkat cells using 100 ng of unmodified saRNA or 5mC-modified saRNA. Error bars represent standard deviation of n=3 biological replicates. Statistical significance was determined by two-way ANOVA with Tukey's multiple comparisons correction. ****p<0.0001, **p<0.01, *p<0.05. The left-to-right order of the bars in each group in Figures 58E-58H corresponds to the top-to-bottom order in the legend. [Figure 58-2] See description of Figure 58-1. [Figure 58-3] See description of Figure 58-1. [Figure 58-4] See description of Figure 58-1. [Figure 58-5] See description of Figure 58-1. [Figure 58-6] See description of Figure 58-1. [Figure 58-7] See description of Figure 58-1. [Figure 59A]Figures 59A-59G are a series of diagrams and graphs showing the evaluation of the immunogenicity of modified saRNA in human PBMCs. Figure 59A shows a diagram illustrating modified saRNA that escaped TLR detection, thereby leading to reduced interferon production. Figure 59B shows an assay for detecting early interferon responses from human PBMC transfection with unmodified or modified saRNA. Figures 59C-59E show gene expression analysis of IFN-α1 (Figure 59C), IFN-α2 (Figure 59D), and IFN-β1 (Figure 59E) in RNA collected from unique human PBMCs (n=3) 6 hours after saRNA treatment. Figure 59F shows serum analysis of IFN-α (all subtypes) in a unique donor 6 hours after saRNA treatment. Figure 59G shows 6-hour and 24-hour serum analysis of IFN-β levels from saRNA-treated PBMCs. Error bars represent standard deviation of n=3 biological replicates. Statistical significance was determined by ANOVA, taking into account multiple comparisons using Dunnett's test. ***p<0.001, ****p<0.0001. nd Not determined / Below detection limit. The left-to-right order of the bars in each group in Figures 59C-59G corresponds to the top-to-bottom order in the legend. [Figure 59B] See legend to Figure 59A. [Figure 59C] See legend to Figure 59A. [Figure 59D] See legend to Figure 59A. [Figure 59E] See legend to Figure 59A. [Figure 59F] See legend to Figure 59A. [Figure 59G] See legend to Figure 59A. [Figure 60A]Figures 60A-60H are a series of diagrams and graphs showing the development and characterization of a fully modified saRNA vaccine against SARS-CoV-2. Figure 60A shows a diagram illustrating different RNA formats for expressing the SARS-CoV-2 spike protein compared in vitro and in vivo. These include non-replicating N1mΨ mRNA, wild-type self-amplifying RNA, and 5mC-modified self-amplifying RNA. Figure 60B shows spike protein expression in HEK293 cells 24 hours after transfection with 100 ng of RNA in SM102 LNPs. Figure 60C shows spike protein expression in C2C12 cells 24 hours after transfection with 100 ng of RNA in SM102 LNPs. Figure 60D shows the median fluorescence intensity (MFI) of anti-spike AF647 staining in C2C12 cells. MFI is relative to untreated cells. Error bars represent standard deviations for n=3 biological replicates. Figure 60E shows the study design for a SARS-CoV-2 challenge study in C57BL / 6 mice. Mice were vaccinated using a prime-boost scheme, serum was collected for interferon response and titer analysis, and on day 35, mice were challenged with SARS-CoV-2 MA30. Figure 60F shows IFN-α1 expression in serum collected 24 or 48 hours after primary vaccination with 1000 ng of RNA in LNPs. Error bars represent standard deviations for n=5 biological replicates. Figure 60G shows mouse survival following lethal challenge with MA30 virus. n=10 mice per group. Figure 60H shows mouse weight change following challenge with mouse-adapted SARS-CoV-2 MA30 virus. Error bars represent SEM. Statistical significance was determined by ANOVA, taking into account multiple comparisons using Dunnett's test. **p<0.005, ***p<0.001, ****p<0.0001. For survival study statistics, the log-rank (Mantel-Cox) test was used between groups. ****=p<0.0001, ***=p<0.001. The left-to-right order of the bars in each group in Figures 60D-60F corresponds to the top-to-bottom order in the legend. [Figure 60B] See legend to Figure 60A. [Figure 60C] See legend to Figure 60A. [Figure 60D] See legend to Figure 60A. [Figure 60E] See legend to Figure 60A. [Figure 60F] See legend to Figure 60A. [Figure 60G] See legend to Figure 60A. [Figure 60H] See legend to Figure 60A. [Figure 61A] Figures 61A-61D are a series of graphs, plots, and images showing testing of modified saRNAs. Figure 61A shows a comparison of flow cytometry results from a modified saRNA screen to a wild-type unmodified control construct. [Figure 61B] Figures 61A-61D are a series of graphs, plots, and images showing testing of modified saRNAs. Figure 61B shows the median fluorescence intensity of HEK293 cells transfected with a library of modified saRNAs. [Figure 61C] Figures 61A-61D are a series of graphs, plots, and images showing the testing of modified saRNA. Figure 61C shows a live cell microscope image of the control sample from Figure 58D. Wild type refers to unmodified saRNA. [Figure 61D] Figures 61A-61D are a series of graphs, plots, and images showing the testing of modified saRNA. Figure 61D shows representative histograms of HEK293 cells transfected with modified saRNA synthesized with 3'-O-Me-m7G(5')ppp(5')G(ARCA) (top row) or m7G(5')ppp(5')(2'OMeA)pU(CLEANCAP AU) (bottom row). [Figure 62A]Figures 62A-62E are a series of graphs and images showing the testing of modified saRNAs. Figure 62A shows the dose response of HEK293 transfected with SM102 LNPs containing N1mΨ mRNA, WT saRNA, or 5mC saRNA encoding luciferase. [Figure 62B] Figures 62A-62E are a series of graphs and images showing testing of modified saRNAs. Figure 62B shows representative flow plots of Jurkat cells transfected with WT or 5mC saRNA encoding mCherry. [Figure 62C] Figures 62A-62E are a series of graphs and images showing testing of modified saRNAs. Figure 62C shows live cell microscopy of Jurkat cells transfected with WT or 5mC saRNA encoding mCherry. [Figure 62D] Figures 62A-62E are a series of graphs and images showing the testing of modified saRNA. Figure 62D shows expression levels in Jurkat cells 24 hours after transfection with 250 ng of RNA encoding a luciferase reporter using SM102 LNP. Luciferase signal is shown as fold change compared to untransfected mock cells. The left-to-right order of the bars corresponds to the top-to-bottom order in the legend. [Figure 62E] Figures 62A-62E are a series of graphs and images showing testing of modified saRNAs. Figure 62E shows the MFI of Jurkat cells transfected with WT or 5mC saRNA encoding mCherry over a 7-day period. [Figure 63A] Figures 63A-63B are a series of plots and images showing testing of modified saRNAs. Figure 63A shows representative flow plots of primary T cells from two different donors transfected with WT or 5mC saRNA encoding mCherry. [Figure 63B]Figures 63A-63B are a series of plots and images showing testing of modified saRNA. Figure 63B shows live cell microscopy of primary T cells transfected with WT or 5mC encoding mCherry. [Figure 64A] Figures 64A-64D are a series of graphs showing the testing of modified saRNA as a SARS-CoV-2 vaccine. Figure 64A shows the transfection efficiency 24 hours after transfection of C2C12 cells with 100 ng of modified spike-encoding mRNA or saRNA. Figure 64B shows spike protein detection by ELISA in lysed C2C12 cells after transfection with 100 ng of modified spike-encoding mRNA or saRNA. Figure 64C shows the transfection efficiency 24 hours after transfection of C2C12 cells with 25 ng of modified HA-encoding mRNA or saRNA. Figure 64D shows the median fluorescence intensity (MFI) of anti-HA AF647 staining in C2C12 cells. The left-to-right order of the bars in each group in Figures 64A-64D corresponds to the top-to-bottom order in the legend. [Figure 64B] See legend to Figure 64A. [Figure 64C] See legend to Figure 64A. [Figure 64D] See legend to Figure 64A. [Figure 65A] Figures 65A-65B are a series of graphs and plots showing the testing of modified saRNA as a SARS-CoV-2 vaccine. Figure 65A shows IFN-β expression in serum collected 24 or 48 hours after primary vaccination with 1000 ng of RNA in LNP. Error bars represent standard deviation of n=5 biological replicates. The left-to-right order of the bars within each group corresponds to the top-to-bottom order in the legend. [Figure 65B]Figures 65A-65B are a series of graphs and plots showing the testing of modified saRNA as a SARS-CoV-2 vaccine. Figure 65B shows the survival rate of mice after lethal challenge with MA30 virus. n=10 mice / group. For survival study statistics, the log-rank (Mantel-Cox) test was used between groups. ****=p<0.0001. [Figure 66A] Figures 66A-66D are a series of graphs showing the testing of modified saRNA as a SARS-CoV-2 vaccine. Figure 66A shows the encapsulation efficiency of spike-coded LNPs used in in vivo studies after dialysis. Figure 66B shows the size of the LNPs used in in vivo studies. Figure 66C shows the polydispersity index (PDI) of the LNPs used in in vivo studies. Figure 66D shows the RNA concentration in ng / uL. The left-to-right order of the bars in each group in Figures 66A-66D corresponds to the top-to-bottom order in the legend. [Figure 66B] See legend to Figure 66A. [Figure 66C] See legend to Figure 66A. [Figure 66D] See legend to Figure 66A. [Figure 67A] Figures 67A-67D show the flow cytometry gating strategy used in Example 20. Figure 67A shows the gating strategy used in screening the modNTP saRNA library by HEK293 transfection. [Figure 67B] Figures 67A-67D show the flow cytometry gating strategy used in Example 20. Figure 67B shows the gating strategy used in analyzing the transfection efficiency of saRNA LNPs in Jurkat T cells. [Figure 67C] Figures 67A-67D show the flow cytometry gating strategy used in Example 20. Figure 67C shows the gating strategy to confirm expression of SARS-CoV-2 spike protein in HEK or C2C12 cells. [Figure 67D]Figures 67A-67D show the flow cytometry gating strategy used in Example 20. Figure 67D shows the gating strategy used in analyzing the transfection efficiency of saRNA LNPs in primary T cells. [Figure 68] Yields of self-amplifying RNA constructs after in vitro transcription are shown with no modified nucleotides (wild-type) or with 100% substitution of cytidine or uridine with 5-hydroxymethylcytidine, 5-methylcytidine, 5-methyluridine, or N1-methylpseudouridine, respectively. Reported yields are relative to the wild-type of each construct. The left-to-right order of the bars in each group corresponds to the top-to-bottom order in the legend. [Figure 69] 10 is a series of flow cytometry results showing the distribution of mCherry expression 24 hours after LNP-mediated transfection of Jurkat cells. [Figure 70] 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 is shown. [Figure 71] Analysis of IL12 p70 expression in HEK293-T cells transfected with LNPs containing unmodified (wild-type) saRNA or 5-methylcytidine-modified saRNA is shown. [Figure 72] Figure 1 shows target cell lysis of primary human T cells cocultured with supernatants derived from 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). [Figure 73] Figure 1 shows target cell lysis of primary human T cells cocultured with supernatants derived from 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). [Figure 74]Figure 1 shows target cell lysis of primary human T cells cocultured with supernatants derived from 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). [Figure 75] 10 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 a HER2 bispecific antibody. [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 measuring the mCherry reporter. [Figure 77] Relative expression of bispecific T cell engagers in C2C12 cells transfected with LNPs containing each RNA construct is shown, as measured by median fluorescence intensity of the mCherry reporter. [Figure 78] 10 is a series of flow cytometry data illustrating GFP expression in Jurkat cells carrying an NFAT GFP reporter co-cultured with supernatants derived from HER2-overexpressing Nalm6 cells and C2C12 cells transfected with saRNA or mRNA constructs encoding BiTEs. [Figure 79] Representative bioluminescence images of mice at different time points after intramuscular injection of LNPs containing 2.5 μg of luciferase-encoding N1mΨ mRNA (left) or 5mC saRNA (right) are shown. Scale bars indicate radians. [Figure 80] Figure 1 shows total flux from BLI imaging of mice intramuscularly injected with LNPs containing 2.5 μg of luciferase-encoding N1mΨ mRNA or 5mC saRNA (n=5 biological replicates). The dashed line indicates the average signal for the PBS group over the study period. Error bars indicate the standard error of the mean. [Figure 81] Schematic diagram of the HER2 CAR saRNA plasmid. [Figure 82] Schematic diagram of the CD19 CAR saRNA plasmid. [Figure 83] Schematic diagram of the CD19 NS3 CAR saRNA plasmid. [Figure 84] Schematic diagram of the CD19 NS3-IKZF3 CAR saRNA plasmid. [Figure 85] Schematic diagram of CD19 aCAR+HER2 iCAR saRNA plasmid. [Figure 86] Schematic diagram of the mCherry saRNA plasmid. [Figure 87] Schematic diagram of the SARS-CoV-2 spike saRNA plasmid. [Figure 88] Schematic diagram of influenza HA saRNA plasmid. [Figure 89] Schematic diagram of the firefly luciferase saRNA plasmid. [Figure 90] Schematic diagram of the HER2 CD3 scFv BITE saRNA plasmid. [Figure 91] Schematic diagram of the IL12 saRNA plasmid. [Figure 92] 1 is a bar graph showing that the bioactive GLP-1 peptide (amino acids 7-37) was detected in the supernatant of HEK293 cells transfected with 5-methylcytidine-substituted self-amplifying RNA. DETAILED DESCRIPTION OF THE INVENTION

[0140] Detailed Description The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description and examples illustrate exemplary embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0141] Disclosed herein are methods and compositions for modifying and controlling cellular activity by expressing proteins from self-replicating RNA. The technology disclosed herein allows for protein expression at levels comparable to other methods used to establish constitutive protein expression. The protein cargo can be any gene-encodable protein with known or unknown function. The system is modular, allowing for the expression of multiple proteins with various functions. Furthermore, the activity of cells transfected with self-replicating RNA can be controlled by expressing proteins that respond to external inputs or proteins that interact with each other to perform logic-based computations. This technology is useful for rapidly creating cell therapies with controllable activity without gene editing. Furthermore, this technology can be combined with RNA delivery strategies (e.g., lipid nanoparticles) to transfect cells in vivo.

[0142] As a non-limiting example of clinical relevance, the present disclosure outlines the development of a self-replicating RNA system used to engineer immune cells to express a reporter protein and a chimeric antigen receptor that targets CD19 and HER2. In some embodiments, the activity of the engineered cells is externally controlled or controlled by a logic-based computational circuit established by the delivered self-replicating RNA.

[0143] Disclosed herein are methods and compositions for modifying and controlling cellular activity by expressing proteins from self-replicating RNAs highly substituted with chemically modified nucleotides. Using the technology disclosed herein, highly substituted saRNAs can express proteins at levels comparable to or greater than those of non-substituted self-amplifying RNAs. The protein cargo can be any gene-encodable protein with known or unknown function. The system is modular, allowing for the expression of multiple proteins with various functions. Furthermore, the activity of cells transfected with highly substituted self-amplifying RNAs can be controlled by expressing proteins that respond to external inputs or proteins that interact with each other to perform logic-based computations. This technology is useful for generating low-dose saRNA-based vaccines that express equivalent or greater cargo compared to unmodified saRNAs. A further aspect of the present invention is the reduced immunogenicity that enhances highly substituted saRNAs. This technology is useful for creating vaccines, cell therapies either in situ or ex vivo, and protein replacement therapies. Furthermore, this technology can be combined with RNA delivery strategies (e.g., lipid nanoparticles) to transfect cells in vivo.

[0144] Self-amplifying RNA In some aspects, self-amplifying RNA is described herein. As used herein, the term " self-amplifying RNA " or " saRNA " or " self-replicating RNA " or " srRNA " is used interchangeably and refers to an RNA strand that can undergo replication activity, generating a replicative strand from the original strand.

[0145] In some aspects, described herein is an saRNA that includes (a) at least one nonstructural protein from at least one virus, (b) a subgenomic promoter (SGP) from at least one virus, and (c) at least one cargo of interest. In a further aspect, described herein is an saRNA that includes, from 5' to 3', (a) at least one nonstructural protein from at least one virus, (b) a subgenomic promoter (SGP) from at least one virus, and (c) at least one cargo of interest.

[0146] In another aspect, described herein is an saRNA that includes (a) at least one nonstructural protein from at least one alphavirus, (b) a subgenomic promoter (SGP) from at least one alphavirus, and (c) at least one cargo of interest. In a further aspect, described herein is an saRNA that includes, from 5' to 3', (a) at least one nonstructural protein from at least one alphavirus; (b) a subgenomic promoter (SGP) from at least one alphavirus; and (c) at least one cargo of interest.

[0147] 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 polyA tail.

[0148] In one aspect, described herein is a saRNA that includes, 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 polyA tail.

[0149] 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 polyA tail.

[0150] In one aspect, described herein is a saRNA that includes, 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 polyA tail.

[0151] In one aspect, described herein is a saRNA comprising: (a) a 5' cap; (b) nonstructural protein 1 (nsp1) (comprising at least one conserved 5' sequence element (5' CSE)), 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 comprising at least one 3' conserved sequence element (3' CSE); and (g) a polyA tail.

[0152] In one aspect, described herein is a saRNA that includes, from 5' to 3', (a) a 5' cap; (b) nonstructural protein 1 (nsp1) (containing at least one conserved 5' sequence element (5' CSE)), 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 containing at least one 3' conserved sequence element (3' CSE); and (g) a polyA tail.

[0153] In one aspect, described herein is a saRNA comprising: (a) a 5' cap; (b) nonstructural protein 1 (nsp1) (comprising at least one conserved 5' sequence element (5' CSE)), 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 comprising at least one 3' conserved sequence element (3' CSE); and (g) a polyA tail.

[0154] In one aspect, described herein is a saRNA that includes, from 5' to 3', (a) a 5' cap; (b) nonstructural protein 1 (nsp1) (containing at least one conserved 5' sequence element (5' CSE)), 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, the 3' untranslated region (UTR) containing at least one 3' conserved sequence element (3' CSE); and (g) a polyA tail.

[0155] In some aspects, the saRNAs described herein comprise at least one nucleotide modification as further described herein. In some aspects, the saRNAs described herein do not comprise a nucleotide modification.

[0156] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising: (a) at least one nonstructural protein from at least one virus; (b) a subgenomic promoter (SGP) from at least one virus; and (c) at least one cargo of interest, wherein the saRNA comprises at least 25% modified nucleotides, the modified nucleotides being selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; the start nucleotide of the saRNA comprises adenosine or an adenosine analog; and the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1).

[0157] In one aspect, the nucleic acid sequence comprises (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural protein 4 (nsp4), each from at least one virus; (c) a subgenomic promoter (SGP) from at least one virus; (d) a 5' untranslated region (UTR) from at least one virus; (e) at least one cargo of interest; and (f) a 3' untranslated region (UTR) from at least one virus. Described herein is a self-amplifying RNA (saRNA) comprising (R), and (g) a poly-A tail, wherein the saRNA comprises at least 25% modified nucleotides, the modified nucleotides being selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine, the start nucleotide of the saRNA comprises adenosine or an adenosine analog, and the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1).

[0158] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising, from 5' to 3', (a) at least one nonstructural protein from at least one virus, (b) a subgenomic promoter (SGP) from at least one virus, and (c) at least one cargo of interest, wherein the saRNA comprises at least 25% modified nucleotides, the modified nucleotides being selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine, the start nucleotide of the saRNA comprises adenosine or an adenosine analog, and the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1).

[0159] In one aspect, the nucleic acid sequence 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 from at least one virus; (c) a subgenomic promoter (SGP) from at least one virus; (d) a 5' untranslated region (UTR) from at least one virus; (e) at least one cargo of interest; and (f) a 3' untranslated region (UTR) from at least one virus. Described herein is a self-amplifying RNA (saRNA) comprising: (a) a universally translated region (UTR); and (b) a poly-A tail, wherein the saRNA comprises at least 25% modified nucleotides, the modified nucleotides being selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; the start nucleotide of the saRNA comprises adenosine or an adenosine analog; and the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1).

[0160] In one aspect, the nucleic acid sequence comprises, from 5' to 3', (a) a 5' cap; (b) nonstructural protein 1 (nsp1) (containing at least one conserved 5' sequence element (5' CSE)), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural protein 4 (nsp4), each from at least one virus; (c) a subgenomic promoter (SGP) from at least one virus; (d) a 5' untranslated region (UTR) from at least one virus; (e) at least one cargo of interest; and (f) at least one 3' conserved sequence element (3' CSE). Described herein is a self-amplifying RNA (saRNA) comprising: (a) a 3' untranslated region (UTR) derived from at least one virus comprising a 3' CSE; and (b) a poly-A tail, wherein the saRNA comprises at least 25% modified nucleotides, the modified nucleotides being selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; the start nucleotide of the saRNA comprises adenosine or an adenosine analog; and the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1).

[0161] In one aspect, described herein is a 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, the modified nucleotides being selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; the start nucleotide comprises adenosine or an adenosine analog; and the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1).

[0162] In one aspect, the vector comprises (a) a 5' cap; (b) nonstructural protein 1 (nsp1), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural protein 4 (nsp4), each from at least one alphavirus; (c) a subgenomic promoter (SGP) from at least one alphavirus; (d) a 5' untranslated region (UTR) from at least one alphavirus; (e) at least one cargo of interest; and (f) a 3' untranslated region (UTR) from at least one alphavirus. Described herein is a self-amplifying RNA (saRNA) comprising an untranslated region (UTR); and (g) a poly-A tail, wherein the saRNA comprises at least 25% modified nucleotides, the modified nucleotides being selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine, the start nucleotide of the saRNA comprises adenosine or an adenosine analog, and the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1).

[0163] In one aspect, described herein is a self-amplifying RNA (saRNA) comprising, from 5' to 3', (a) at least one nonstructural protein from at least one alphavirus, (b) a subgenomic promoter (SGP) from at least one alphavirus, and (c) at least one cargo of interest, wherein the saRNA comprises at least 25% modified nucleotides, the modified nucleotides being selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine, the start nucleotide of the saRNA comprises adenosine or an adenosine analog, and the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1).

[0164] In one aspect, the nucleic acid sequence 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 from at least one alphavirus; (c) a subgenomic promoter (SGP) from at least one alphavirus; (d) a 5' untranslated region (UTR) from at least one alphavirus; (e) at least one cargo of interest; and (f) a nucleic acid sequence derived from at least one alphavirus. Described herein is a self-amplifying RNA (saRNA) comprising: (a) a 3' untranslated region (UTR) derived from a nucleotide sequence of ...

[0165] In one aspect, the nucleic acid sequence comprises, from 5' to 3', (a) a 5' cap; (b) nonstructural protein 1 (nsp1) (containing at least one conserved 5' sequence element (5' CSE)), nonstructural protein 2 (nsp2), nonstructural protein 3 (nsp3), and / or nonstructural protein 4 (nsp4), each from at least one alphavirus; (c) a subgenomic promoter (SGP) from at least one alphavirus; (d) a 5' untranslated region (UTR) from at least one alphavirus; (e) at least one cargo of interest; and (f) at least one 3' conserved sequence element (5' CSE).

[0003] Described herein is a self-amplifying RNA (saRNA) comprising: (a) a 3' untranslated region (UTR) from at least one alphavirus, the 3' untranslated region (UTR) comprising a 3' CSE; and (b) a polyA tail, wherein the saRNA comprises at least 25% modified nucleotides, the modified nucleotides being selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; the start nucleotide of the saRNA comprises adenosine or an adenosine analog; and the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1).

[0166] In some embodiments of any aspect, the nucleic acid (e.g., DNA) encoding the saRNA described herein comprises one of SEQ ID NOs: 2 or 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 NOs: 2 or 5, or a codon-optimized version thereof, that maintains the same function (e.g., self-replication).

[0167] 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 7634-8347 of SEQ ID NO: 5, nucleotides 7617-8330 of SEQ ID NO: 6, or nucleotides 7617-8330 of SEQ ID NO: 7, each corresponding to mCherry, are replaced with at least one cargo of interest as further described herein.

[0168] In some embodiments of any aspect, the saRNA described herein comprises one of SEQ ID NOs:6-7, 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 NOs:6-7, or a codon-optimized version thereof that maintains the same function (e.g., self-replication).

[0169] Table 6 shows non-limiting examples of domains contained in exemplary saRNAs and exemplary template DNA for the saRNAs, as further described herein.

[0170] Table 6: Exemplary saRNAs TIFF2025539792000002.tif92160

[0171] Nucleic acid modification In some embodiments of any aspect, the saRNA described herein comprises at least one type of nucleic acid modification that can enhance the efficacy of the saRNA. In some embodiments of any aspect, the saRNA described herein comprises modified nucleotides, for example, 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 a terminal modification, a backbone modification, and / or a sugar modification.

[0172] Modified Nucleotides In several aspects, saRNA containing modified nucleotides is 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, and may include or exclude natural and synthetically introduced chemical groups on any side of the nitrogenous base.It is expressly stated herein that the definition of the term "modified nucleotide" does not include modifications to the sugar-phosphate backbone.This exception is not intended to exclude methylation at the 2'O position of the first and second starting nucleotides, also known as cap-1 and cap-2 structures.

[0173] 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 with a moiety on carbon 5 of the pyrimidine. In some embodiments of any aspect, the moiety on carbon 5 of the pyrimidine is selected from the group consisting of methyl, ethyl, propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, and hydroxypropyl functional groups. In some embodiments of any aspect, the pyrimidine comprises cytidine and / or uridine.

[0174] 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, for example, Formula I). ​​In some embodiments of any aspect, the pyrimidine comprises cytidine, and the modified nucleotide comprises 5-hydroxymethylcytidine (see, for example, II). In some embodiments of any aspect, the pyrimidine comprises cytidine, and the modified nucleotide comprises 5-methylcytidine and 5-hydroxymethylcytidine.

[0175] 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.

[0176] 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 can include any combination of modified nucleotides listed 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.

[0177] Table 4: Exemplary modified nucleotide combinations in the saRNAs described herein TIFF2025539792000003.tif128155

[0178] In some embodiments of any aspect, the modified nucleotides include 5-methyluridine or 5-hydroxymethyluridine and one or both of 5-methylcytidine and 5-hydroxymethylcytidine in the same saRNA molecule. In some embodiments of any aspect, the modified nucleotides include 5-methyluridine and one or both of 5-methylcytidine and 5-hydroxymethylcytidine in the same saRNA molecule.

[0179] In some embodiments of any aspect, the saRNA described herein comprises 5-methylcytidine as a nucleotide modification in combination 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.

[0180] In some embodiments of any aspect, the saRNA described herein contains 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 a particular nucleotide in the corresponding unmodified saRNA. For example, if the unmodified saRNA contains 1,000 uridine nucleosides and the modified saRNA contains 250 5-methyluridine (5mU) instead of uridine, the saRNA contains 25% modified nucleotides. As another non-limiting example, if the unmodified saRNA contains 1,000 uridine nucleosides and the modified saRNA contains 125 5-methyluridine (5mU) and 125 5-hydroxymethyluridine (5OHmU) instead of uridine, the saRNA contains 25% modified nucleotides.

[0181] 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 to generate 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 molar % 5-methyluridine (5 mU) and 75 molar % uridine.

[0182] In some embodiments of any aspect, the saRNA described herein contains at least 25% modified nucleotides, and up to 100% modified nucleotides, corresponding to at least one particular nucleotide (e.g., pyrimidine; e.g., cytidine and / or uridine). In some embodiments of any aspect, the saRNA described herein has 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 Also 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% modified nucleotides.

[0183] In some embodiments of any aspect, the saRNA described herein has up to 25%, up to 26%, up to 27%, up to 28%, up to 29%, up to 30%, up to 31%, up to 32%, up to 33%, up to 34%, up to 35%, up to 36%, up to 37%, up to 38%, up to 39%, 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 100%, up to 101%, up to 102%, up to 103%, up to 104%, up to 105%, up to 10 8%, up to 39%, 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 Contains 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.

[0184] In some embodiments of any aspect, the saRNA described herein contains 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, 90% to 100%, 95% to 100%, or 99% to 100% modified nucleotides corresponding to at least one particular type of nucleotide (e.g., pyrimidine; e.g., cytidine and / or uridine).

[0185] In some embodiments of any aspect, the saRNA described herein comprises 25% to 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% to 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% to 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).

[0186] In some embodiments of any aspect, the saRNA described herein contains 100% 5-methylcytidine (5mC) substituted for cytidine. In some embodiments of any aspect, the saRNA described herein contains 100% 5-hydroxymethyl-cytidine (5OHmC) substituted for cytidine. In some embodiments of any aspect, the saRNA described herein contains 100% 5-methyluridine (5mU) substituted for uridine. In some embodiments of any aspect, the saRNA described herein contains 100% 5-hydroxymethyl-uridine (5OHmU) substituted for uridine.

[0187] 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 contains 100% 5-hydroxymethyl-cytidine (5OHmC) substituted for cytidine and 100% 5-hydroxymethyl-uridine (5OHmU) substituted for uridine.

[0188] In some embodiments of any aspect, the saRNA containing at least 25% modified nucleotides expresses at least one cargo at a level greater than or equal to that of the corresponding saRNA containing less than 25% modified nucleotides. As used herein, the phrase "corresponding saRNA containing less than 25% modified nucleotides" refers to a saRNA having the same base sequence as the RNA containing at least 25% modified nucleotides when adenosine, guanosine, uridine (and analogs thereof), and cytidine (and analogs thereof) are used in the base sequence. 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).

[0189] In some embodiments of any aspect, the saRNA containing at least 25% modified nucleotides expresses at least one cargo at a level equal to that of a corresponding saRNA having less than 25% modified nucleotides. In some embodiments of any aspect, the saRNA containing at least 25% modified nucleotides expresses at least one cargo at a level 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 that of a corresponding saRNA having less than 25% modified nucleotides.

[0190] In embodiments in which the saRNA contains more than one type of modified nucleotide (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 different from each other. As a non-limiting example, in a saRNA containing 25% modified cytidines, the saRNA may have 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.

[0191] As a non-limiting example, in a saRNA containing 25% modified uridines, the saRNA may have 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.

[0192] 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.

[0193] 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 riboside, 2-amino-6-chloropurine riboside, 2-aminoadenosine, 5-methoxycytidine, 5-formylcytidine, 5-aminoallylcytidine, 5-hydroxycytidine, isoguanosine, or thienoguanosine. Not 2-aminopurine-riboside, not 8-oxoguanosine, not 5-carboxymethylester uridine, not thienouridine, not 5-methoxyuridine, not 5-carboxyuridine, not 2-thiouridine, not N1-propylpseudouridine, not N1-methoxymethylpseudouridine, not N1-ethylpseudouridine, not pseudouridine, not N1-methylpseudouridine.

[0194] In some embodiments of any aspect, the saRNA described herein is replaced with a template of SEQ ID NO:2, or a nucleic acid sequence 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, or a codon-optimized version thereof, that maintains the same function (e.g., self-replication). 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.

[0195] In some embodiments of any aspect, the saRNA saRNA described herein is replaced 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, or a codon-optimized version thereof, that maintains the same function (e.g., self-replication).

[0196] In some embodiments of any aspect, the pyrimidine comprises cytidine, the modified nucleotide comprises 5-methylcytidine and / or 5-hydroxymethylcytidine, and the substituted saRNA comprises SEQ ID NO:6, or a nucleic acid sequence 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, or a codon-optimized version thereof, that maintains the same function (e.g., self-replication).

[0197] In some embodiments of any aspect, the pyrimidine comprises a uridine, the modified nucleotide comprises 5-methyluridine and / or 5-hydroxymethyluridine, and the substituted saRNA comprises SEQ ID NO:7, or a nucleic acid sequence 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, or a codon-optimized version thereof, that maintains the same function (e.g., self-replication).

[0198] Cap and start nucleotides In several aspects, the present specification describes saRNAs that include 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 saRNA stabilization to resist 5'-exonuclease degradation.

[0199] In eukaryotes, a 5' cap, called Cap-0, is found at the 5' end of RNA molecules. Cap-0 consists of a guanine nucleotide attached to the mRNA via a 5'-5' triphosphate linkage. This guanosine is methylated at the 7-position by a methyltransferase immediately after capping in vivo. Cap-0 is also sometimes 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 saRNA containing 5' Cap0.

[0200] Multicellular eukaryotes and some viruses have additional 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 a methylated 2'-hydroxyl group on the first two ribose sugars. In some embodiments of any aspect, the initiating nucleoside of the saRNA described herein is methylated at the 2'O position of the ribose (Cap 1). In some embodiments of any aspect, Cap 1 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 generating saRNA containing 5' Cap 1. In some embodiments of any aspect, both the initiating nucleotide and the following nucleotide of the saRNA described herein are methylated at the 2'O position of the ribose (Cap 2). In some embodiments of any aspect, cap 2 has the following chemical structure: m7GpppNmNm.

[0201] In some embodiments of any aspect, the start nucleotide immediately adjacent to the 5' cap in the saRNA described herein comprises an adenosine or an adenosine analog. In some embodiments of any aspect, the start nucleotide immediately adjacent to the 5' cap in the saRNA described herein comprises a guanosine or a guanosine analog. In some embodiments of any aspect, the first two start nucleotides immediately adjacent to the 5' cap in the saRNA described herein comprise an adenosine or an adenosine analog at position 1 and a uridine or a uridine analog at position 2. In some embodiments of any aspect, the first three start nucleotides immediately adjacent to the 5' cap in the saRNA described herein comprise a guanosine or a guanosine analog at position 1, an adenosine or an adenosine analog at position 2, and a uridine or a uridine analog at position 3.

[0202] In some embodiments of any aspect, the start nucleotide of the saRNA described herein comprises adenosine or an adenosine analog, and the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1).

[0203] In some embodiments of any aspect, the start nucleotide of the saRNA described herein comprises adenosine or an adenosine analog, and both the start nucleotide and the following nucleotide of the saRNA are methylated at the 2'O position of the ribose (Cap 2).

[0204] In some embodiments of any aspect, the start nucleotide of the saRNA described herein comprises guanosine or a guanosine analog, and the start nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1).

[0205] In some embodiments of any aspect, the start nucleotide of the saRNA described herein comprises guanosine or a guanosine analog, and both the start nucleotide and the following nucleotide of the saRNA are methylated at the 2'O position of the ribose (Cap 2).

[0206] In some embodiments of any aspect, the start nucleotide of the saRNA described herein comprises an adenosine or an adenosine analog, and the 5' cap is m7G 5'Cap-0. In some embodiments of any aspect, the start nucleotide of the saRNA described herein comprises a guanosine or a guanosine analog, and the 5' cap is m7G 5'Cap-0.

[0207] Non-limiting examples of nucleic acid modifications It is further contemplated herein that the nucleic acids (e.g., DNA, saRNA) described herein can be chemically modified to enhance stability or other beneficial characteristics. The nucleic acids described herein can be synthesized and / or modified by methods well established in the art, such as those described in "Current Protocols in Nucleic Acid Chemistry," Beaucage, SL et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Modifications include, for example, (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, inverted linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, inverted linkage, etc.), (b) sugar modifications (e.g., sugar modifications at the 2' or 4' position) or sugar replacement, and (c) backbone modifications, including phosphodiester bond modification or replacement. Specific examples of nucleic acid compounds useful in the embodiments described herein include, but are not limited to, nucleic acids containing modified backbones or nucleic acids that do not contain natural internucleoside linkages. Nucleic acids having modified backbones particularly include nucleic acids that do not have a phosphorus atom in the backbone.For the purposes of this specification, and as sometimes referred to in the art, modified nucleic acids that do not have a phosphorus atom in the internucleoside backbone can also be considered oligonucleosides.In some embodiments of any aspect, the modified nucleic acid may have a phosphorus atom in its internucleoside backbone.

[0208] Modified nucleic acid backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Modified nucleic acid backbones that do not contain phosphorus atoms have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages.These include morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and mixed backbones. Included are others having combined N, O, S, and CH component moieties, as well as oligonucleosides having heteroatom backbones, particularly --CH--NH--CH--, --CH--N(CH)--O--CH-- [known as a methylene (methylimino) or MMI backbone], --CH--O--N(CH)--CH--, --CH--N(CH)--N(CH)--CH--, and --N(CH)--CH--CH-- [a naturally occurring phosphodiester backbone is represented by --O--P--O--CH--].

[0209] In other nucleic acid mimetics, the sugar and internucleoside linkages, i.e., backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, specifically an aminoethylglycine backbone. The nucleobases are retained and are attached directly or indirectly to aza nitrogen atoms in the amide portion of the backbone.

[0210] The nucleic acid can also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribose moieties, which contain an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce 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).

[0211] Modified nucleic acids may also contain one or more substituted sugar moieties. The nucleic acids described herein may contain 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, where alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2) n O]mCH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2)nONH2, and O(CH2) n ON[(CH2) nCH3)]2, where n is 1 to about 10. In some embodiments of any aspect, the nucleic acid includes one of the following at the 2' position: C1-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, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a nucleic acid, or a group for improving the pharmacodynamic properties of a nucleic acid, and other substituents with similar properties. In some embodiments of any aspect, the modification includes 2'-methoxyethoxy (2'-O-CH2CHOCH3, 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 the O(CH2)2ON(CH3)2 group, also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, as described in the Examples herein 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), as described in the Examples herein below.

[0212] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Other positions on the nucleic acid can also be similarly modified, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in 2'-5'-linked dsRNA, and the 5' position of the 5'-terminal nucleotide. Nucleic acids can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar.

[0213] Preparations of the above modified nucleic acids, backbones, and sugars are well known in the art.

[0214] Another nucleic acid modification featured in the invention involves chemically linking the nucleic acid to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the nucleic acid. Such moieties include lipid moieties, e.g., cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, e.g., 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), thiocholesterols (Oberhauser et al., Nucl. Acids Res., 1992, 4:1053-1060), and the like. 20:533-538), aliphatic chains such as 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 such as dihexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0215] Exemplary viral components 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, subgenomic promoter (SGP), 5' conserved sequence element (5'CSE), 5' untranslated region (5'UTR), 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 because it contains 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 a subgenomic RNA encoding a cargo of interest from a subgenomic promoter (SGP) derived from a virus, which is recognized by the RdRp.

[0216] As used herein, the term "derived from" refers to origin or source and may include natural, recombinant, unpurified, or purified molecules, e.g., nucleic acids or polypeptides. In some embodiments of any aspect, "derived from" includes mutation and / or maturation of any of the nucleic acids or polypeptides 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, e.g., to increase the amount and / or duration of cargo expression. Non-limiting examples of nonstructural 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 rate of RNA replication. For example, at least one of non-coding conserved sequence elements (for example, 5'UTR) can be mutated and / or matured to comprise an A-rich region that can accelerate RNA replication.See, for example, Li et al., Scientific Reports volume 9, Article number: 6932 (2019); Perkovic et al., Molecular Therapy Volume 31, Issue 6, P1636-1646 (2023).The contents of each of these are incorporated herein by reference in their entirety.

[0217] In some embodiments of any aspect, 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 nsp1, nsp2, nsp3, nsp4, SGP, 5'UTR, 3'UTR, 5'CSE, and / or 3'CSE in the saRNA described herein are derived from a different virus.

[0218] In some embodiments of any aspect, the component, e.g., RNA or polypeptide, derived from at least one virus comprises a naturally occurring component, e.g., RNA or polypeptide, found in the 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 naturally occurring component, e.g., RNA or polypeptide, found in the virus.

[0219] In some embodiments of any aspect, the component, e.g., RNA or polypeptide, derived from at least one virus maintains the function of the native component, e.g., RNA or polypeptide, found 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 native component, e.g., RNA or polypeptide, found in the virus.

[0220] The phrase "derived from at least one virus" may include any virus, including, but not limited to, Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), Sindbis virus (SIN), Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Mayaro virus (MAYV), Getah virus (GETV), Ross River virus (RRV), Una virus (UNAV), Middleburg virus (MIDV), O'Nyong-Nyong virus (ONNV), Barmah Forest virus (BFV), and 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 emara virus (FMV), Aura virus (AURAV), Kunjin virus (KUN), Measles virus (MV), Coronavirus (CoV), Rabies virus (RABV), and Vesicular stomatitis virus (VSV).

[0221] 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), Barmah 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, Getah virus (GETV), Highlands J virus (HJV), Madariaga virus, Mayaro virus (MAYV), Middleburg virus (MIDV), Mosso das Pedras virus, Mucambo virus (MUCV), Nudum virus, O'nyong-nyong virus (ONNV), Pixuna virus, Rio Negro virus (RNV), Ross River virus (RRV), Salmon pancreas disease virus (SPVV), Salmon flu ... 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).

[0222] Alphaviruses are a genus of RNA viruses and the only genus in the Togaviridae family. They belong to group IV of the Baltimore classification of viruses, with a positive-sense, single-stranded RNA genome. Alphaviruses are small, spherical, enveloped viruses with a positive-sense, single-stranded RNA genome. Their genomes range from 11,000 to 12,000 nucleotides in length and contain a 5' cap and a 3' poly(A) tail. Four nonstructural protein genes are encoded in the 5' two-thirds of the genome, while three structural proteins are translated from subgenomic mRNAs colinear with the 3' one-third of the genome. The genome contains two open reading frames (ORFs): a nonstructural and a structural one. The first ORF is nonstructural and encodes proteins (nsP1 to nsP4) required for viral RNA transcription and replication. The second open reading frame (ORF) encodes three structural proteins: the core nucleocapsid protein C and the envelope proteins P62 and E1.

[0223] In some embodiments of any aspect, the saRNA described herein does not contain structural proteins derived from at least one virus. In some embodiments of any aspect, the saRNA described herein does not contain structural proteins derived from at least one alphavirus. In some embodiments of any aspect, the saRNA described herein does not contain structural proteins derived from VEEV (e.g., does not contain C, P62, or E1).

[0224] In some embodiments of any aspect, the saRNA described herein does not comprise capsid or envelope proteins derived from at least one virus.In some embodiments of any aspect, the saRNA described herein does not comprise capsid or envelope proteins derived from at least one alphavirus.In some embodiments of any aspect, the saRNA described herein does not comprise capsid or envelope proteins derived from VEEV.

[0225] Nonstructural proteins 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. Because one polyprotein constitutes the viral replication machinery, the alphavirus nonstructural protein can be selected from the four nonstructural proteins (nsP1-4) produced. For example, in VEEV and other alphaviruses, nsp1 is a methyl / guanylyl transferase involved in RNA capping. nsp2 is a cysteine ​​protease, helicase, and NTPase. nsp3 is a poly (ADP-ribose) hydroxylase. 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), enabling replication of the saRNA (e.g., +RNA to -RNA to +RNA) and / or production of subgenomic RNA (e.g., -RNA to sgRNA using SGP).

[0226] 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 nonstructural proteins listed in Table 5, each derived from an alphavirus, where the alphaviruses may be from the same or different species of alphavirus. 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 derived from an alphavirus species, where the alphavirus species may be from the same or different species of alphavirus. 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 derived from an alphavirus species, which may each be the same or different alphavirus species.

[0227] Table 5. Exemplary nonstructural protein combinations in the saRNAs described herein TIFF2025539792000004.tif75128

[0228] In some embodiments of any aspect, the saRNA described herein comprises nsP1 from a strain of VEEV, nsP2 from a strain of VEEV, nsP3 from a strain of VEEV, and / or nsP4 from a strain of VEEV, or any combination thereof. For example, the saRNA described herein may comprise any combination of nonstructural proteins listed in Table 5, each from a strain of VEEV, which may be the same or different strains of VEEV. As a non-limiting example, the saRNA described herein may comprise nsP1 from a strain of VEEV, nsP2 from a strain of VEEV, nsP3 from a strain of VEEV, and nsP4 from a strain of VEEV. In some embodiments of any aspect, the saRNA described herein comprises, from 5' to 3', nsP1, nsP2, nsP3, and nsP4, each from a strain of VEEV, which may be the same or different strains of VEEV. 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 derived from a strain of VEEV, which may be the same or different strains of VEEV.

[0229] In some embodiments of any aspect, the saRNA described herein encodes 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, or that maintains a function thereof (e.g., at least one nsP1-nsP4 function; e.g., saRNA replication; e.g., RNA-dependent RNA polymerase).

[0230] In some embodiments of any aspect, the saRNA described herein is one of nucleotides 62 to 7543 of SEQ ID NO:2 (DNA or corresponding RNA sequence), nucleotides 62 to 7543 of SEQ ID NO:5, nucleotides 45 to 7526 of SEQ ID NO:6, or nucleotides 45 to 7526 of SEQ ID NO:7, or nucleotides 62 to 7543 of SEQ ID NO:2 (DNA or corresponding RNA sequence), nucleotides 62 to 7543 of SEQ ID NO:5, nucleotides 45 to 7526 of SEQ ID NO:6, or nucleotides 45 to 7526 of SEQ ID NO:7. and nonstructural proteins comprising a nucleic acid sequence 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 to 7526 of NO:7, or a corresponding RNA sequence from a codon-optimized version thereof.

[0231] Conserved sequence elements 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 across multiple species of alphavirus. 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 an RdRp (e.g., nsP1-4) encoded by the saRNA and enable replication of the saRNA. In some embodiments of any aspect, the conserved sequence element comprises a secondary structure (e.g., a hairpin) for interacting with an RdRp (e.g., nsP1-4) encoded by the saRNA. In some embodiments of any aspect, the conserved sequence element is non-coding, i.e., it does not encode a polypeptide and is not translated.

[0232] As a non-limiting example, analysis of alphavirus genome sequences has identified four types of sequence elements (CSEs) that are conserved throughout the genus: the 5' untranslated region (UTR), a 51-nt 5' CSE within nsP1, the 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: a 5' UTR, a 5' CSE, an SGP, and / or a 3' CSE within the 3' UTR, each 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 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 derived from VEEV. See, e.g., 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 entireties.

[0233] 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.

[0234] 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) in length, or about 40-60 nt in length. The 5'CSE may depend on the viral machinery (e.g., nsP1-4) used to generate saRNA. In the case of VEEV, the 5'CSE may be: TIFF2025539792000005.tif4128 (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).

[0235] 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.

[0236] The conserved 3' conserved sequence element (3'CSE) may be encoded in the 3'UTR. The conserved 3' sequence element (3'CSE) may be about 70 nucleotides (nt) in length, or about 60-80 nt in length. The 3'CSE may rely on the viral machinery encoded by the saRNA (e.g., nsP1-4) to replicate itself. In the case of VEEV, the 3'CSE is located in the 3'UTR sequence. TIFF2025539792000006.tif4128 (see, e.g., nucleotides 7673-7742 of SEQ ID NO:2, nucleotides 8393-8462 of SEQ ID NO:5, and nucleotides 8376-8445 of SEQ ID NOs:6-7). The 3' CSE is highly conserved in at least 27 alphavirus genomes and may contain 85%-90% AU-rich sequences.

[0237] In some embodiments of any aspect, the conserved sequence element comprises SEQ ID NO:9, SEQ ID NO:10, or a nucleic acid sequence 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 that maintains its function (e.g., at least one nsP1-nsP4 function; e.g., saRNA replication; e.g., RNA-dependent RNA polymerase).

[0238] 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.

[0239] 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 located 3' (downstream) of 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.

[0240] The subgenomic promoter (SGP) may be located at the end of nsp4 and contain sequences within and following the coding sequence of nsp4. The SGP may rely on viral machinery (e.g., nsP1-4; e.g., RdRP) to generate the subgenomic RNA (sgRNA) of the saRNA. The subgenomic promoter controls 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) leads to a higher number of sgRNA copies compared to the full-length +RNA. In the context of alphaviruses, a higher concentration of sgRNA compared to the full-length +RNA can increase the concentration of translated structural proteins compared to translated nonstructural proteins. In the context of the saRNA described herein, a higher concentration of sgRNA compared to the full-length +RNA can increase the concentration of at least one cargo (which may be a translated protein or a noncoding RNA, as further described herein) compared to the translated nonstructural proteins.

[0241] The SGP sequence of VEEV is TIFF2025539792000007.tif24159 (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:6-7).

[0242] In some embodiments of any aspect, the subgenomic promoter (SGP) comprises SEQ ID NO:11, or a nucleic acid sequence 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 that maintains its function (e.g., replication of a subgenomic RNA (sgRNA); e.g., replication of at least one type of cargo encoded by a saRNA).

[0243] 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.

[0244] 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 located 3' (downstream) of the SGP and 5' (upstream) of 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.

[0245] 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 located 3' (downstream) of at least one cargo and 5' (upstream) of the polyA 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.

[0246] 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.

[0247] Exemplary Cargo 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, at least one cargo is located 3' (downstream) of the subgenomic promoter and 5' untranslated region (UTR), and 5' (upstream) of the 3' untranslated region (UTR) and poly A tail.

[0248] 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 ways to separate each cargo protein 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 may be, for example, a 2A peptide selected from the group consisting of P2A, E2A, F2A, and T2A. An IRES is an RNA element that enables translation initiation in a cap-independent manner.

[0249] In some embodiments of any aspect, at least one cargo protein is of viral, bacterial, protozoan, mammalian, or plant origin. In some embodiments of any aspect, at least one cargo peptide is of viral, bacterial, protozoan, mammalian, or plant origin.

[0250] In some embodiments of any aspect, the cargo comprises a chimeric antigen receptor (CAR). In some embodiments of any aspect, the saRNA described herein encodes and expresses a CAR selected from the group consisting of: (a) a conventional CAR; (b) an on-CAR; (c) an off-CAR system; (d) an on / off-CAR; (e) an inhibitory CAR; or (f) a split, universal, programmable, and reconfigurable (SUPRA) CAR system. For further details regarding such exemplary CARs, see, for example, U.S. Patent Nos. 11,059,864 and 11,530,252; Li et al. Cancer Cell 40, 1-12 (2022); Li et al. Nat Med. 28(10): 2133-2144 (2022). The contents of each of these are incorporated herein by reference in their entirety.

[0251] In some embodiments of any aspect, the saRNA described herein encodes and expresses at least one conventional CAR. In some embodiments of any aspect, the 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.

[0252] 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 cleaves and degrades the on-CAR in the absence of a protease inhibitor.

[0253] 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) an inhibitory protease domain capable of specifically binding 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 is transferred 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 the inhibitory protease, and the second polypeptide may comprise the peptide domain. In some embodiments of any aspect, the second polypeptide further comprises a transmembrane domain.

[0254] 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 apoNS3a leader (ANR) peptide.

[0255] 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 suppressible protease domain that cleaves and degrades the on / off-CAR in the absence of a protease inhibitor; and (e) a drug-inducible degron domain.

[0256] 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, NS3 is catalytically active (e.g., on-CAR, on / off-CAR). In some embodiments of any aspect, NS3 is catalytically inactive (i.e., dead; e.g., off-CAR). For NS3, the catalytic triad may comprise His-57, Asp-81, and Ser-139. Thus, a catalytically inactive NS3 protease may comprise a nonsynonymous mutation at any one of His-57, Asp-81, and Ser-139, or may comprise another inactivating mutation described herein. In some embodiments of any aspect, a catalytically inactive N33 protease comprises a S139A mutation.

[0257] In some embodiments of any aspect, a CAR described herein comprises 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 cleaved by an inhibitory protease. A protease cleavage site comprises a specific amino acid sequence or motif that is recognized by a protease during proteolytic cleavage and typically comprises 1 to 6 amino acids surrounding a scissile bond that binds to the active site of the protease and is used for recognition as a substrate. In some embodiments of any aspect, a protease cleavage site can be any site that is specifically bound and cleaved by an inhibitory protease. In some embodiments of any aspect, a CAR polypeptide described herein (or collectively, a CAR polypeptide system) comprises one, two, three, four, five, or more protease cleavage sites. In some embodiments of any aspect, a CAR polypeptide comprises two protease cleavage sites. In embodiments comprising multiple protease cleavage sites, the multiple protease cleavage sites may be different individual protease cleavage sites, multiple copies of the same protease cleavage site, or combinations of the above.

[0258] As a non-limiting example, during HCV replication, the NS3-4A serine protease is responsible for proteolytic cleavage at four junctions of the HCV polyprotein precursor: NS3 / NS4A (autocleavage), NS4A / NS4B, NS4B / NS5A, and NS5A / NS5B. Thus, the protease cleavage site of a CAR polypeptide described herein can be an NS3 / NS4A cleavage site, an NS4A / NS4B cleavage site, an NS4B / NS5A cleavage site, or an NS5A / NS5B cleavage site.

[0259] In some embodiments of any aspect, the CAR polypeptide described herein is combined with a protease inhibitor. As used herein, "in combination with" refers to two or more substances being present in the same formulation, e.g., in an admixture, solution, mixture, suspension, colloid, or emulsion, in any molecular or physical arrangement. The formulation may be a homogeneous mixture or a heterogeneous mixture. In some embodiments of any aspect, the active compound may be included in a superstructure, e.g., a nanoparticle, liposome, vector, cell, scaffold, etc., which is in a solution, mixture, admixture, suspension, etc., together with the CAR polypeptide or CAR polypeptide system. In some embodiments of any aspect, the CAR polypeptide binds to a protease inhibitor bound to an inhibitory protease. In some embodiments of any aspect, the CAR polypeptide specifically binds to a protease inhibitor bound to an inhibitory protease.

[0260] In some embodiments of any aspect, the CAR polypeptide is combined with one, two, three, four, five, or more protease inhibitors. In some embodiments of any aspect, the CAR polypeptide is combined with one protease inhibitor. In embodiments including multiple protease inhibitors, the multiple protease inhibitors can be different individual protease inhibitors, multiple copies of the same protease cleavage site, or combinations of the foregoing.

[0261] 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, including, but not limited to, grazoprevir, danoprevir, simeprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir. Further 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 incorporated herein by reference in their entirety.

[0262] In some embodiments of any aspect, the drug-inducible degron domain comprises an IKAROS family zinc finger 3 (IKZF3) domain that can bind to and activate CAR for degradation by the drugs lenalidomide or pomalidomide.

[0263] In some embodiments of any aspect, the saRNA described herein encodes and expresses at least one inhibitory CAR. In some embodiments of any aspect, the inhibitory 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) an inhibitory domain.

[0264] 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, the ITIM comprises S / I / V / LxYxxI / V / L, SEQ ID NO:20, where 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., an ITIM-containing domain) from FcγRIIB, CTLA-4, PD-1, BTLA, CD72, NKG2A, CD31, SIGLEC, CD66, ILT, or LIR.

[0265] 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 the first member of the extracellular protein interaction domain of the first polypeptide; (ii) a transmembrane domain; and (iii) at least one type of intracellular signaling domain.

[0266] In some embodiments of any aspect, at least one domain is moved from a first polypeptide to a second polypeptide of the SUPRA CAR system, or from a second polypeptide to a first polypeptide of the SUPRA CAR system. 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.

[0267] In some embodiments of any aspect, at least the intracellular signaling domain present in a CAR described herein 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. In some embodiments of any aspect, at least the intracellular signaling domain in a CAR described herein is derived from CD28 or CD3ζ.

[0268] 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.

[0269] In some embodiments of any aspect, the extracellular binding domain of a CAR described herein comprises an antigen-binding domain derived from an antibody. In some embodiments of any aspect, the extracellular binding domain of a CAR described herein comprises a single-chain variable fragment (scFv).

[0270] In some embodiments of any aspect, the cargo comprises at least one domain that is responsive to an external input. In some embodiments of any aspect, the cargo comprises an antibody or 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 engager (BiTE). In some embodiments of any aspect, the BiTE is a fusion protein comprising the extracellular binding domains of two antibodies. For example, one arm of the BiTE comprises an extracellular binding domain specific for a protein found on the surface of cytotoxic T cells (e.g., CD3), and the other arm of the BiTE comprises an extracellular binding domain specific for a particular protein found primarily on tumor cells (e.g., HER2, or other antigens described herein). Upon binding to both targets, the BiTE molecule forms a bridge between the cytotoxic T cell and the tumor cell, allowing the T cell to recognize the tumor cell and combat it by injecting toxic molecules.

[0271] In some embodiments of any aspect, the cargo comprises an extracellular domain that specifically binds to an antigen of interest. In some embodiments of any aspect, the antigen of interest is specific to and / or upregulated on certain cancer cells. Non-limiting examples of antigens of interest for cargo proteins (e.g., CARs, antibodies, BiTEs, etc.) include CD19, CD22, CD30, b-cell maturation antigen (BCMA), disialoganglioside GD2, human estrogen receptor 2 (HER2), G-protein coupled receptor 87 (GPR87), fibroblast activator 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 group 2 member D (NKG2D).

[0272] Further 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 a, GD2, GD3, HLA-AI MAGE A1, HLA-A2, IL-1 These include 1Ra, IL13Ra2, KDR, Lambda, Lewis-Y, MCSP, mesothelin, Mucl, Mucl6, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, survivin, TAG72, TEM1, TEM8, VEGRR2, carcinoembryonic antigen, HMW-MAA, and VEGF receptors. Other exemplary antigens that can be targeted are antigens present in the extracellular matrix of tumors, such as oncofetal variants of fibronectin, tenascin, or necrotic regions of tumors.

[0273] Additional tumor-selective molecules that can be targeted include 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 (VEGFR- 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 a, transmembrane glycoprotein NMB (GPNMB), CC chemokine receptor (e.g., CCR4), prostate-specific membrane antigen (PSMA), récepteur d'origine nantais (RON) receptor, cytotoxic T-lymphocyte antigen 4 (CTLA4), as well as other tumor-specific receptors or antigens.

[0274] Non-limiting examples of tumor antigens include the following: differentiation antigens, such as MART-1 / Melan-A (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed fetal antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from 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, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA These include 242, 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.

[0275] 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 US20170209492 or US20170335281, the contents of each of which are incorporated by reference in their entirety. In some embodiments, the tumor antigen is CD19; CD123; CD22; CD30; CD171; CS-1 (CD2 subset 1, also known as 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 α-2 (IL-13Ra2 or CD213A2); mesothelial Phosphoryltransferase (PTS) receptor; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (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 α (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta type 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of the breakpoint cluster region (BCR) and Abelson murine leukemia viral 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 (HMW-MSA) antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (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); hexasaccharide portion of globoH glycoceramide (GloboH) glycoceramide); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 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 (Tie2); 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 mutant; prostein; survivin; telomerase; prostate cancer tumor antigen 1 (PCTA-1 or galectin-8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (type II transmembrane serine protease (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis 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) Sites), squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAXS); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma X breaking point 2 (SSX2); receptor for advanced glycation end products (RAGE-1); kidney ubiquitous 1 (RU1);Renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (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 Medullary Thyroid Cancer," Mar. 13, 2015).

[0276] 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 5,977,322, US 8,580,263, US 8,703,427, US 8,927,694, US 10,188,742, US 10,239,951, the contents of each of which are incorporated herein by reference in their entirety.

[0277] In some embodiments of any aspect, the extracellular binding domain comprises an anti-Axl antibody. AXL overexpression has been shown to be a risk factor for various cancer types, including 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), and gastric cancer (Wu et al., Anticancer Res 22: 1071-1078, 2001). 2002), ovarian cancer (Sun et al., Oncology 66: 450-457, 2004), endometrial cancer (Sun et al., Ann. Oncol. 14: 898-906, 2003), kidney cancer (Chung et al., DNA Cell Biol. 22: 533-540, 2003), hepatocellular carcinoma (Tsou et al. 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); Tyrosine kinase receptor with oncogenic potential. Oncogene, 6: 2113-2120, 1991; Braunger et al., Oncogene 14:2619-2631 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). Furthermore, AXL has been identified as a metastasis-associated gene that is upregulated in aggressive 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 U.S. Patent US8841424, the contents of each of which are incorporated herein by reference in their entirety.

[0278] In some embodiments of any aspect, the extracellular binding domain comprises an anti-CD19 antibody. Because CD19 is a B-cell marker, this protein has been used to diagnose cancers arising from this type of cell, particularly B-cell lymphoma, 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, and SEQ ID NO:94. See, e.g., U.S. Patent Nos. 10,221,245, 8,906,682, and 10,421,810, the contents of each of which are incorporated herein by reference in their entirety.

[0279] 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.

[0280] In some embodiments of any aspect, cargo comprises at least one kind of non-coding RNA.For example, non-coding RNA may be selected from the group consisting of small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA.In some embodiments of any aspect, cargo comprises at least one kind of siRNA.In some embodiments of any aspect, cargo comprises at least one kind of shRNA.In some embodiments of any aspect, cargo comprises at least one kind of microRNA.

[0281] In some embodiments of any aspect, the cargo comprises at least one vaccine-associated antigen. As used herein, the term "vaccine-associated 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 vaccination against the foreign substance (e.g., microorganism).

[0282] In some embodiments of any aspect, the vaccine-associated antigen comprises at least one protein encoded by the genome of the virus. Non-limiting examples of viruses that express 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, 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), herpesvirus, rotavirus, varicella-zoster virus (VZV), dengue virus, hepatitis A virus, hepatitis B virus, rubella virus, poliovirus, and rabies virus.

[0283] 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.

[0284] In some embodiments of any aspect, the vaccine-associated antigen comprises one of SEQ ID NOs: 13-16, 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 NOs: 13-16, 22. In some embodiments of any aspect, the vaccine-associated antigen is encoded by nucleotides 7599-11384 of SEQ ID NO:21, or a nucleic acid sequence 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 maintains the same function (e.g., viral entry into cells) of the wild-type viral protein. In some embodiments, the vaccine-associated antigen encoded and expressed by the saRNA has an attenuated or inactive function compared to the function of the wild-type viral protein.

[0285] 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 transcription factor 2 (Sox2), Krüppel-like factor 4 (Klf4), and cellular myelocytomatosis oncogene (c-Myc).

[0286] 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), neurotrophin, migration stimulating factor (MSF), and sarcoma growth factor (SGF).

[0287] In some embodiments of any aspect, the cargo comprises one or both of papalysin-A1 (PAPPA1) and papalysin-A2 (PAPPA2).

[0288] 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 the interleukin. In some embodiments of any aspect, the cargo comprises at least one receptor subunit for the 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.

[0289] In some embodiments of any aspect, the cargo comprises at least one enzyme having antioxidant activity, 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.

[0290] In some embodiments of any aspect, the cargo comprises glucagon-like peptide-1 (GLP-1) or a fragment thereof. As a non-limiting example, the bioactive form of glucagon-like peptide-1 (GLP-1) is amino acids 7-37 of GLP-1: TIFF2025539792000008.tif4128. In some embodiments of any aspect, the GLP-1 comprises SEQ ID NO:17 or an amino acid sequence 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 that maintains its function (e.g., stimulating insulin secretion and / or inhibiting glucagon secretion).

[0291] In some embodiments of any aspect, the cargo is a polypeptide that encodes an endogenous insulin secretory signal. TIFF2025539792000009.tif4128 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.

[0292] 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 7634-8347 of SEQ ID NO: 5, nucleotides 7617-8330 of SEQ ID NO: 6, or nucleotides 7617-8330 of SEQ ID NO: 7, each corresponding to mCherry, are replaced with at least one cargo of interest, as further described herein.

[0293] 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, particularly the saRNA described herein administered to a subject or that is part of a pharmaceutical composition, does not encode or contain a detectable immunogenic marker. In some embodiments of any aspect, the saRNA described herein does not contain or encode GFP, mCherry, HA1, or any other immunogenic marker. In some embodiments of any aspect, the saRNA described herein that comprises a detectable marker may have the detectable marker subsequently removed, for example, may have a removable (e.g., cleavable) detectable marker. In some embodiments of any aspect, the saRNA described herein that includes a detectable marker may have the detectable marker replaced with a different detectable marker known in the art or described herein, e.g., have interchangeable (e.g., interchangeable) detectable markers.

[0294] PolyA tail In some aspects, the present invention describes saRNA that comprises poly A tail found at the 3' end of saRNA molecule.In some embodiments of any aspect, poly A tail is derived from at least one virus.In some embodiments of any aspect, poly A tail is derived from at least one alphavirus.In some embodiments of any aspect, poly A tail is derived from VEEV.

[0295] A polyA (polyadenylation) tail is a long chain of adenine nucleotides added to an RNA molecule during RNA processing to increase the molecule's stability. In some embodiments, the polyA tail can be 20-30 residues long, 30-50 residues long, 50-100 residues long, or 100-250 residues long. The polyA tail increases the stability of the RNA molecule and prevents its degradation. Furthermore, the polyA tail allows the mature RNA molecule to be exported from the nucleus and / or translated into protein by ribosomes in the cytoplasm.

[0296] In some embodiments of any aspect, the saRNA contains a polyA signal (e.g., AAUAAA) that cleaves the 3' end of the RNA, liberating a 3' hydroxyl and recruiting polyA polymerase to add a chain of adenine nucleotides 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 a minus-strand template of the saRNA.

[0297] Nucleic acids and vectors The saRNA described herein can be encoded and / or expressed by nucleic acid and / or vector.Therefore, in one aspect, the nucleic acid that encodes or comprises the saRNA described herein is described herein.In another aspect, the vector that encodes or comprises the saRNA described herein is described herein.

[0298] 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 essentially consists of DNA.In some embodiments of any aspect, the nucleic acid encoding or comprising the saRNA described herein consists of DNA.

[0299] In some embodiments of any aspect, the DNA molecule encoding the saRNA described herein comprises one of SEQ ID NOs:2 or 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 NOs:2 or 5, or a codon-optimized version thereof, that maintains the same function (e.g., self-replication).

[0300] In some embodiments of any aspect, the DNA molecules encoding the saRNA described herein contain at least one regulatory sequence upstream of the encoded saRNA. In some embodiments of any aspect, the DNA molecules encoding the saRNA described herein contain a promoter for saRNA transcription using an RNA polymerase. In some embodiments of any aspect, the DNA molecules encoding the saRNA described herein contain 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).

[0301] When a nucleic acid molecule encoding any of the saRNAs described herein is expressed in a cell, its expression can be induced using various transcription control sequences (e.g., promoter / enhancer sequences). The promoter can be a native promoter, such as a promoter of at least one cargo protein in its endogenous context, that normally regulates the expression of the cargo protein. In some embodiments, the promoter can be a constitutive promoter, i.e., the promoter is not regulated and can continuously transcribe the saRNA and its associated cargo. Various conditional promoters, such as promoters controlled by the presence or absence of a molecule, can also be used.

[0302] The exact content of the regulatory sequences required for expression may vary between species or cell types, but generally, they may include, as necessary, 5' non-transcribed sequences involved in the initiation of transcription and 5' non-translated sequences involved in the initiation of translation, such as a TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed regulatory sequences may include a promoter region containing a promoter sequence for transcriptional control of the encoded saRNA. Regulatory sequences may also include, as necessary, enhancer sequences or upstream activator sequences.

[0303] As used herein, a saRNA-encoding sequence and a regulatory sequence are said to be "operably" linked when they are covalently linked in such a way that expression or transcription of the saRNA-encoding sequence is under the influence or control of the regulatory sequence. When it is desired that at least one cargo encoded by the saRNA be translated into a functional protein, the promoter in the 5' regulatory sequence is induced to transcribe the saRNA, and the nature of the linkage between the two DNA sequences is such that (1) it does not introduce frameshift mutations, (2) it does not interfere with the ability of the promoter region to induce saRNA transcription, and (3) it does not interfere with the ability of at least one cargo encoded by the saRNA to be translated into a protein.

[0304] The nucleic acid molecules encoding the saRNAs described herein can be introduced into one or more cells using standard methods and techniques in the art. For example, the nucleic acid molecules can be introduced by standard protocols such as chemical transformation and transformation, including electroporation, transduction, particle bombardment, etc. Expression of the nucleic acid molecules encoding the saRNAs described herein can also be achieved by integrating the nucleic acid molecules into the genome.

[0305] In some embodiments, one or more of the saRNAs described herein are expressed in a recombinant expression vector or plasmid. Figures 81-91 include schematic diagrams of exemplary vectors used for in vitro transcription of exemplary saRNAs. 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, when associated with appropriate control elements, is capable of replicating and transferring a nucleic acid sequence into a cell. The term "vector" includes plasmids, cloning vectors, expression vectors, naked DNA, minichromosomes, chromosomes, transposons, cosmids, viruses, virions, phages, 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, as well as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). One type of vector is a "plasmid." A "plasmid" refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Furthermore, certain vectors are capable of directing the expression of genes operatively linked to the vector. Such vectors are referred to herein as "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0306] In some embodiments of any aspect, the vector is a recombinant vector, e.g., the vector comprises sequences originating from at least two different sources. In some embodiments of any aspect, the vector comprises sequences originating from at least two different species. In some embodiments of any aspect, the vector comprises sequences originating from at least two different genes, e.g., the vector comprises a fusion protein or an expression product-encoding nucleic acid operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., promoter, suppressor, activator, enhancer, response element, etc.).

[0307] 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 the same polypeptide expression product as the native / wild-type sequence, but is transcribed and / or translated with improved efficiency in a desired expression system. In some embodiments of any aspect, the expression system is an organism other than the source of the native / wild-type sequence (or cells derived from such an organism). In some embodiments of any aspect, the vectors or nucleic acids described herein are codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a mouse cell, or a human cell. In some embodiments of any aspect, the vectors or nucleic acids described herein are codon-optimized for expression in a human cell. In some embodiments of any aspect, the vectors or nucleic acids described herein are codon-optimized for expression in a yeast or yeast cell. In some embodiments of any aspect, the vectors or nucleic acids described herein are codon-optimized for expression in a bacterial cell. In some embodiments of any aspect, the vectors or nucleic acids described herein are codon-optimized for expression in E. coli cells.

[0308] A cloning vector is a vector that can replicate autonomously or integrate into the genome of a host cell and is further characterized by one or more endonuclease restriction sites that allow the vector to be cut in a determinable manner and to which a desired DNA sequence (e.g., a DNA template for the saRNA described herein) can be ligated so that the new recombinant vector retains the ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence may occur multiple times as the copy number of the plasmid increases within a host cell, such as a host bacterium, or replication of the desired sequence may occur only once per host before the host multiplies by mitosis. In the case of a phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.

[0309] An expression vector is a vector into which a desired DNA sequence (e.g., a DNA template for a saRNA described herein) can be inserted by restriction and ligation so that it is operably linked to regulatory sequences and expressed as a saRNA transcript. A vector may further contain one or more marker sequences suitable for use in identifying cells transformed or transfected with the vector, or cells that have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds (e.g., ampicillin resistance), genes encoding enzymes with activities detectable by standard assays known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase), and genes that visibly 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 the saRNA present in the operably linked DNA segment. The expressed sequence is often, but not necessarily, heterologous to the cell. Expression vectors may contain additional elements, for example, they may have two replication systems and thus be able to be maintained in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification.

[0310] 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, Second Edition, Cold Spring Harbor Laboratory Press, 1989.Cells are genetically engineered by introducing heterologous DNA (or RNA) into cells.This heterologous DNA (or RNA) is placed under the operable control of transcriptional elements so as to allow the expression of heterologous DNA in host cells.

[0311] The term "viral vector" as used herein refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. The viral vector can contain a nucleic acid encoding a polypeptide described herein in place of a non-essential viral gene. The vector and / or particle may be used to transfer any nucleic acid into cells in vitro or in vivo. Numerous types of viral vectors are known in the art. Non-limiting examples of viral vectors of the present invention include AAV vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors.

[0312] It should be understood that in some embodiments, the vector described herein can be combined with other suitable compositions and therapies.In some embodiments, the vector is an episomal vector.The use of suitable episomal vector allows the nucleic acid of interest (for example, encoding the saRNA described herein) to be maintained in the form of high copy number extrachromosomal DNA in the subject, thereby eliminating the potential impact of chromosomal integration.

[0313] Composition and Administration The saRNA described herein may be contained in a composition, such as a pharmaceutical composition, as further described herein. In some embodiments, the saRNA described herein may be contained in a cell, such as 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, a NK cell, a macrophage, or a B cell. In some embodiments, the cell is a T lymphocyte cell. In some embodiments, the cell is a CD4+ T lymphocyte cell.

[0314] In one aspect, the composition described herein comprises the saRNA described herein.In one aspect, the composition described herein comprises the nucleic acid or vector that comprises or expresses the saRNA described herein.In one aspect, the composition described herein comprises the cell that comprises or expresses the saRNA described herein.

[0315] In one aspect, the pharmaceutical composition described herein comprises the saRNA described herein and pharmaceutically acceptable carrier.In one aspect, the pharmaceutical composition described herein comprises the nucleic acid or vector that comprises or expresses the saRNA described herein and pharmaceutically acceptable carrier.In one aspect, the pharmaceutical composition described herein comprises the cell that comprises or expresses the saRNA described herein and pharmaceutically acceptable carrier.

[0316] In some embodiments of any aspect, the saRNA is formulated as a lipid nanoparticle (LNP). In some embodiments of any aspect, the lipid nanoparticle comprises a targeting moiety specific to a cell or tissue of interest. As a non-limiting example, the LNP can be conjugated to an antibody, such as an anti-CD3 antibody, specific to T cells. In some embodiments, the LNP is formulated at a lipid:oligonucleotide weight ratio of about 10:1. In some embodiments, the LNP is formulated at an N:P ratio of about 10. In some embodiments, the LNP is formulated at 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.

[0317] In another embodiment, the average diameter of the lipid nanoparticles is less than 1000 nm, e.g., 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 is between 1000 nm and 50 nm, or about 500 nm and 50 nm, or about 300 nm and 50 nm, or 200 nm and 50 nm, or 100 nm and 50 nm. In another embodiment, the average diameter of the nanoparticles is less than about 100 nm.

[0318] In another embodiment, the polydispersion 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.

[0319] In another embodiment, the lipid nanoparticles have a positive charge.

[0320] In another embodiment, the lipid nanoparticles have a negative charge.

[0321] In another embodiment, the lipid nanoparticles have no overall charge.

[0322] In some embodiments, the lipids in the LNPs comprise an ionizable lipid, cholesterol, a phospholipid, and / or a polyethylene glycol lipid. Further non-limiting examples of lipids for use in LNP formulations include DSPE, SM-102, DMG-PEG2K, DOPE, and / or cholesterol.

[0323] In some embodiments, the lipid nanoparticles can be formulated from one or more cationic lipids, anionic lipids, neutral lipids, ionizable lipids, and / or zwitterionic lipids, non-limiting examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleryl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, ... phosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, dilinoleoylphosphatidylcholine, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglyeerol (POPG), diacylphosphatidylcholine, diacylphosphatidyletbanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols, triglycerides, 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-phosphotidylcholine), 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 (DYNASAN 114), Tripalmitin (DYNASAN 116), Tristearin (DYNASAN 118), Mono-, Di-, and Triglyceride Mixture, Glyceryl Stearate (IMWITOR 900), Glyceryl Behenate (COMPRITOL 888 ATO), Glyceryl Palmitostearate (PRECIROL ATO) 5), stearic acid, palmitic acid, DPPC, MSPC, DSPE-PEG2000, DSPE-PEG2500, phosphatidylcholine, soy phosphatidylcholine, (4-hydroxybutyl)azanediylbis(hexane-6,1-diyl)bis(2-hexyldecanoate), (2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N- Ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, ALC-0315, ALC-0159, SM-102, DTO P, DDAB, DOGS, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydmxyethyl ammonium 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-trimethylanthnium chloride ("DODAP, N,N-distearyl-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-DMDMA A, 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, which is incorporated by reference in its entirety), DLin-K-C2- ... See WO2010 / 042877, which is incorporated herein by reference), DLin-M-C3-DMA (see, e.g., WO2010 / 146740 and / or WO2010 / 10520, which are incorporated herein by reference in their entireties), 2-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dienlyloxyl]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, LIPOFECTIN® (a commercially available cationic lipid nanoparticle comprising DOTMA and 1,2 dioleoyl-sn-3-phosphoethanolamine (“DOPE”)), TRANSFECTAM®, and combinations thereof.

[0324] In some embodiments, the LNP comprises one or more PEG-modified lipids. In some embodiments, the one or more PEG-modified lipids are C6-C 20 They contain poly(ethylene) glycol chains up to 5 kDa in length covalently attached to lipids with long alkyl chains.

[0325] Other phosphorus-free compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and S-acyloxyacids, are also within the group called amphipathic lipids that can be included in the LNPs described herein.

[0326] In some embodiments, one or more cationic lipids included in the LNPs described herein are amino lipids. Amino lipids suitable for use in the LNPs described herein include those described in WO2017180917, the entire contents of which are incorporated herein by reference. Exemplary amino lipids in WO2017180917 include the amino lipids described in paragraph

[0744] , e.g., DLin-MC3-DMA (MC3), (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (L608), and compound 18 described in WO2017180917. Other exemplary amino lipids include compound 2, compound 23, compound 27, compound 10, or compound 20 described in WO2017180917. Additional amino lipids suitable for use in the LNPs described herein include those described in WO2017112865, the entire contents of which are incorporated herein by reference. Exemplary amino lipids in WO2017112865 include compounds according to one of formulas (I), (Ia1)-(Ia6), (1b), (II), (Ila), (III), (Ilia), (IV), (17-1), (19-1), (19-11), and (20-1) in WO2017112865, as well as the compounds in paragraphs

[0185] ,

[0201] , and

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

[0327] In some embodiments, the saRNA is formulated in a polymer matrix. In some embodiments, the polymer matrix may include one or more polymers with or without one or more lipids to form nanoparticles.

[0328] In one embodiment, the saRNA-containing polymer matrix is ​​a reverse micelle nanoparticle comprising two or more polymers, such as polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropylfumerate, polyglycerol, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, poly(β-aminoester), 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, for example, a copolymer of PLGA or PLA and PEG. The polymer matrix may comprise PLGA or PLA and a copolymer of PLGA or PLA and PEG.

[0329] In another embodiment, the polymer matrix comprises lipid-terminated polyalkylene glycols and polyesters, such as lipid-terminated PEG and PLGA. The lipid may be a lipid such as 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof.

[0330] In one embodiment, the reverse micelles comprise amphiphilic lipids, such as lecithin, phosphatidylcholine, lipid A, cholesterol, dolichol, shingosine, sphingomyelin, ceramide, cerebroside, sulfatide, glycosylceramide, phytosphingosine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, cardiolipin, phophatidic acid, or lysophophatide, or a combination thereof.

[0331] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising the saRNA described herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, the active ingredient of the pharmaceutical composition comprises the saRNA described herein. In some embodiments, the active ingredient of the pharmaceutical composition essentially consists of the saRNA described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of the saRNA described herein. Pharmaceutically acceptable carriers and diluents include 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) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; oils; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and dairy. (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, 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) ethyl alcohol; (20) pH buffer 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 12Alcohol; and (25) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient," "carrier," and "pharmaceutically acceptable carrier" are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, for example, the saRNA described herein.

[0332] In some embodiments, pharmaceutical compositions containing the saRNA described herein may be in parenteral dosage forms (i.e., administered or generated in locations within the body other than the mouth and digestive tract). Because parenteral dosage forms typically bypass the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or can be sterilized before being administered to a patient. Examples of parenteral dosage forms include, but are not limited to, ready-to-inject solutions, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, ready-to-inject suspensions, and emulsions.

[0333] Suitable vehicles that can be used to provide the disclosed saRNA parenteral dosage form are well known to those skilled in the art.Non-limiting examples include, but are not limited to, sterile water; water for injection USP; saline; phosphate-based saline; glucose 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 ethyl alcohol, 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.

[0334] Pharmaceutical compositions containing the saRNA described herein can also be formulated for oral administration, for example, as individual dosage forms, including, but not limited to, tablets (including, but not limited to, scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, lozenges, cachets, aerosol sprays, or liquids, including, but not limited to, syrups, elixirs, solutions, or suspensions in the form of aqueous liquids, non-aqueous liquids, oil-in-water emulsions, or water-in-oil emulsions. Such compositions contain a predetermined amount of saRNA and can be prepared by methods of pharmacy well 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).

[0335] In some embodiments, the methods described herein include administering to a subject an effective amount of a composition described herein, such as a saRNA described herein, to alleviate symptoms of a disease or disorder. As used herein, "alleviating symptoms of a disease or disorder" refers to the alleviation of any condition or symptom associated with a disease or disorder. When compared to an equivalent untreated control, such a reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or more, as measured by any standard technique. Various means for administering the compositions described herein to a subject are known to those skilled in the art.

[0336] In some embodiments of any aspect, the saRNA is about 5x10 -4 In some embodiments of any aspect, the saRNA is formulated at a dose of about 5x10 -3 In some embodiments of any aspect, the saRNA is formulated at a dose of about 5x10 -2In some embodiments of any aspect, the saRNA is formulated at a dose of about 5x10 -1 In some embodiments of any aspect, the saRNA is formulated at a dose of about 1 mg / kg. 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 can be formulated at a dose of about 0.1 μg to about 100 μg. In an embodiment using a subject (e.g., a human subject) weighing about 70 kg, the saRNA is formulated at a dose of about 1×10 -6 mg / kg~1x10 -3 It can be formulated in mg / kg doses.

[0337] For systemic administration, subjects receive, for example, 1x10 -6 mg / kg, 1x10 -5 mg / kg, 1x10 -4 mg / kg, 1x10 -3 mg / kg, 1x10 -2 A therapeutic amount of 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, or more of a composition comprising the saRNA described herein can be administered.

[0338] 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 pharmacological composition to produce the desired effect. Thus, the term "therapeutically effective amount" refers to an amount of saRNA sufficient to produce a specific alleviating effect when administered to a typical subject. In various contexts, the term "effective amount" as used herein also includes an amount of drug sufficient to delay the onset of disease symptoms, alter the course of disease symptoms (including, but not limited to, slowing the progression of disease symptoms), or reverse disease symptoms. Therefore, it is generally not feasible to specify an exact "effective amount." However, in any given case, one skilled in the art can determine the appropriate "effective amount" using only routine experimentation.

[0339] Effective doses, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the ED50 (the dose therapeutically effective in 50% of the population). Dosages may vary depending on the dosage form used and the route of administration utilized. A therapeutically effective dose can be initially estimated from cell culture assays. Furthermore, doses 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-maximal inhibition of symptoms) determined in cell culture or an appropriate animal model. Plasma levels can be measured, for example, by PCR. The effects of any particular dosage can be monitored by an appropriate bioassay. Dosages can be determined by a physician and adjusted, if necessary, to suit the observed effects of treatment.

[0340] The dosage range for administering saRNA according to the methods described herein depends, for example, on the form of saRNA, its efficacy, and the desired degree of reduction of the symptoms, markers, or indicators of the conditions described herein.The dosage should not be so high as to cause adverse side effects, such as autoimmune reactions.Generally, dosage varies depending on the age, condition, and gender of the patient, and can be determined by those skilled in the art.Dosage can also be adjusted by individual physicians if any complications arise.

[0341] The efficacy of saRNA, for example, the efficacy of saRNA in treating a condition described herein or the efficacy of saRNA in inducing a response as described herein, can be confirmed by a skilled clinician. However, as the term "effective treatment" is used herein, if, after treatment according to the methods described herein, one or more of the signs or symptoms of a condition described herein are beneficially altered, other clinically recognized symptoms are improved or ameliorated, or a desired response is induced, for example, by at least 10%. 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 any other suitable measurable parameter. Efficacy can also be measured by the lack of deterioration of an individual, as assessed by hospitalization or the need for medical intervention (i.e., the progression of the disease is halted). 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), including (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation), or (2) reducing the severity of the disease, e.g., reversing symptoms. An effective amount for treating a disease means an amount sufficient to provide effective treatment, as that term is defined herein, for the disease when administered to a subject in need thereof. The efficacy of an agent can be ascertained by assessing physical indicators of the condition or desired response. It is well within the capabilities of one skilled in the art to monitor the efficacy of administration and / or treatment by measuring any one or any combination of such parameters.

[0342] Efficacy may 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 a treatment is demonstrated when a statistically significant change in a marker is observed.

[0343] With regard to duration and frequency of treatment, a skilled clinician will often monitor the subject to ascertain when therapeutic benefit is being obtained from the treatment and to determine whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment regimen.

[0344] In certain embodiments, an effective dose of a composition comprising the saRNA described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising the saRNA described herein can be administered to a patient repeatedly.

[0345] Dosing regimens can vary from once a week to daily, depending on numerous clinical factors, such as the subject's sensitivity to the saRNA. The desired dose or amount may be administered once or divided into subdoses, e.g., two to four subdoses, administered over a period of time, e.g., at appropriate intervals throughout the day, or any other suitable regimen. In some embodiments, administration may be one or more doses and / or treatments per day for a period of several weeks or months. Examples of dosing and / or treatment regimens include daily, twice-daily, three-daily, or four-daily administrations, or more frequently, for one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, or six months, or longer. The compositions containing the saRNA described herein can be administered over a period of time, e.g., 5, 10, 15, 20, or 25 minutes.

[0346] In some embodiments, after an initial treatment regimen, treatment can be administered less frequently, for example, after three months of biweekly treatment, treatment can be repeated monthly for six months or a year, or longer.

[0347] Various means 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 administration, intraosseous (IO), intraperitoneal (IP), subcutaneous (SC), intravenous (IV), intramuscular (IM), rectal, vaginal, intraarticular (IA), inhalation, or topical administration. Further non-limiting administration methods include oral, parenteral, intravenous, intramuscular, transdermal, airway (aerosol), pulmonary, cutaneous, injection, or intratumoral administration. Administration may be local or systemic.

[0348] In some embodiments of any aspect, the saRNA described herein is administered as a monotherapy, e.g., the subject is not receiving another treatment for the disease or disorder.

[0349] In some embodiments of any aspect, the methods described herein can further include administering to the subject a second agent and / or treatment, e.g., as part of a combination therapy. Non-limiting examples of second agents and / or treatments include radiation therapy, surgery, gemcitabine, cisplatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents, e.g., thiotepa and CYTOXAN® cyclophosphamide; alkylsulfonates, e.g., busulfan, improsulfan, and piposulfan; aziridines, e.g., benzodopa (ben zodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin ( callystatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatin; nitro Gen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega 11 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophore and related enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin , chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, e.g., mitomycin C, mycophenolic acid, nogalamycin, olivomycin, penicillin, puromycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as , fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane;Folic acid supplements, such as furolic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® Cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine;Mercaptopurine; methotrexate; platinum analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE®; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including a treatment regimen of irinotecan with 5-FU and leucovorin); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and inhibitors of VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing;

[0350] Those skilled in the art can readily identify useful chemotherapeutic agents (see, e.g., 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 ed. St. Louis, Mosby-Year Book, 2003).

[0351] Additionally, the treatment method may further include the use of radiation or radiotherapy. Additionally, the treatment method may further include the use of surgical procedures.

[0352] The methods described herein may further include administering a second agent and / or treatment to the subject, e.g., as part of a combination therapy. As a non-limiting example, if a subject is treated for pain or inflammation according to the methods described herein, the subject may also be administered a second agent and / or treatment known to be beneficial to subjects suffering from pain or inflammation. Examples of such agents and / or treatments include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs—e.g., aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (e.g., cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclomethasone); methotrexate; sulfasalazine; leflunomide; anti-TNF drugs; cyclophosphamide; pro-resolving drugs; mycophenolate; or opiates (e.g., endorphins, enkephalins, and dynorphins), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and the like.

[0353] 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 therapeutic agents 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.

[0354] Non-limiting examples of immune checkpoint inhibitors (ICIs) include pembrolizumab (Keytruda®), nivolumab (Opdivo®), cemiplimab (Libtayo®), spartalizumab, camrelizumab (AiRuiKa™), sintilimab (TYVYT®), tislelizumab, toripalimab (Tuoyi™), dostallimab (JEMPERLI), INCMGA00012, AMP-224, AMP-514 (M EDI0608), atezolizumab (Tecentriq®), avelumab (Bavencio®), envafolimab (KN035), cosibelimab (CK-301), AUNP12, CA-170, BMS-986189, BMS-936559 (MDX-1105), durvalumab (IMFINZI®), tremelimumab, and ipilimumab (Yervoy®). For example, U.S. Patents US5811097, US5855887, US6051227, US6682736, US6984720, US7595048, US7605238, US7943743, US8008449, US8217149, US8354509, US8383796, US8728474, US8735553, US8779105, See US8779108, US8907053, US8900587, US8952136, US9067999, US9073994, US9683048, US9987500, US10160736, US10316089, US10441655, US10590199, US11225522, US Patent Application 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.

[0355] Exemplary Uses of saRNA Exemplary uses of saRNA described herein are described herein.In one aspect, described herein is a method for expressing at least one cargo in a cell, 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 by lipid nanoparticles.In some embodiments of any aspect, multiple separate saRNAs are delivered to a mixture of cells.

[0356] In another aspect, described herein is a method for expressing at least one cargo in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition described herein, e.g., a pharmaceutical composition encoding, expressing, or comprising at least one saRNA described herein.

[0357] As used herein, "subject" refers to a human or animal. Typically, an animal is a vertebrate, such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, for example, a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0358] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. A non-human mammal can be advantageously used as a subject corresponding to an animal model of a disease or disorder. In some embodiments of any aspect, the subject is a human. In some embodiments of any aspect, the subject is a livestock animal. In some embodiments of any aspect, the subject is a livestock or domesticated animal, such as a pet, including, but not limited to, a dog, a cat, a guinea pig, a rabbit, a rat, a mouse, or a hamster. In some embodiments of any aspect, the subject is a fish, a bird, a lizard, or a snake. The subject may be male or female.

[0359] In some embodiments of any aspect, the subject has cancer. In some embodiments of any aspect, the subject needs vaccination against an infectious disease. In some embodiments of any aspect, the subject needs protein replacement therapy. In some embodiments of any aspect, the subject needs antibody therapy. In some embodiments of any aspect, the subject needs treatment for diabetes and / or obesity. In some embodiments of any aspect, the subject needs BITE therapy.

[0360] The subject is a subject who has been previously diagnosed or identified as suffering from or having a condition that requires treatment, or one or more complications associated with such a condition, and optionally has already received treatment for such a disease or disorder, or one or more complications associated with such a disease or disorder. Alternatively, the subject may also be a subject who has not previously been diagnosed with this disease or disorder, or one or more complications associated with this disease or disorder. For example, the subject may be a subject who exhibits one or more risk factors for this disease or disorder, or one or more complications associated with this disease or disorder, or a subject who does not exhibit risk factors. A "subject in need" of treatment for a particular condition may be a subject who has the condition, a subject who has been diagnosed with the condition, or a subject who is at risk of developing the condition.

[0361] In some embodiments of any aspect, the saRNA described herein increases the expression level of a cargo protein in a cell by, for example, 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 not containing the saRNA.

[0362] In some embodiments of any aspect, the use of modified saRNA (e.g., containing at least 25% modified nucleotides) has beneficial and unexpected results compared to corresponding saRNA having less than 25% modified nucleotides.

[0363] In some embodiments of any aspect, the modified saRNA replicates at a level greater than or equal to that of a corresponding saRNA at an equivalent dose with less than 25% modified nucleotides. In some embodiments of any aspect, the modified saRNA replicates at a level equal to that of a corresponding saRNA at an equivalent dose with less than 25% modified nucleotides. In some embodiments of any aspect, the modified saRNA replicates at a level 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 that of a corresponding saRNA at an equivalent dose with less than 25% modified nucleotides.

[0364] In some embodiments of any aspect, the modified saRNA expresses cargo at a level greater than or equal to that of a corresponding saRNA at an equivalent dose with less than 25% modified nucleotides. In some embodiments of any aspect, the modified saRNA expresses cargo at a level equal to that of a corresponding saRNA at an equivalent dose with less than 25% modified nucleotides. In some embodiments of any aspect, the modified saRNA expresses cargo at a level 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 that of a corresponding saRNA at an equivalent dose with less than 25% modified nucleotides.

[0365] In some embodiments of any aspect, the transfection efficiency of the modified saRNA is greater than or equal to the transfection efficiency of the corresponding saRNA at an equivalent dose with 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 the corresponding saRNA at an equivalent dose with less than 25% modified nucleotides. In some embodiments of any aspect, the transfection efficiency 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 higher than the transfection efficiency of the corresponding saRNA at an equivalent dose with less than 25% modified nucleotides.

[0366] In some embodiments of any aspect, modified saRNA generates an early interferon response in subjects.In some embodiments of any aspect, compared with the corresponding saRNA of equivalent dose with less than 25% modified nucleotides, the early interferon response is reduced.In some embodiments of any aspect, compared with the corresponding saRNA of equivalent dose with less than 25% modified nucleotides, 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 1 / 2, at least 1 / 3, at least 1 / 4, at least 1 / 5, or at least 1 / 10, or more.

[0367] In some embodiments of any aspect, the cargo is detectably expressed from the modified saRNA for an increased period of time compared to a corresponding saRNA at an equivalent dose with less than 25% modified nucleotides. In some embodiments of any aspect, the cargo is detectably expressed from the modified saRNA for an increased period of time 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 longer, or longer, compared to a corresponding saRNA at an equivalent dose with less than 25% modified nucleotides.

[0368] In some embodiments of any aspect, the saRNAs described herein allow for tissue-, organ-, or cell-type-specific expression of cargo. In some embodiments of any aspect, the modified saRNAs described herein allow for tissue-, organ-, or cell-type-specific expression of cargo.

[0369] In some embodiments of any aspect, the saRNA contains more than 50% of uridines substituted with 5-methyluridine. In some embodiments of any aspect, the saRNA containing more than 50% of uridines substituted with 5-methyluridine has increased kidney-specific expression of cargo compared to a corresponding saRNA at an equivalent dose containing less than 50% of uridines substituted with 5-methyluridine. In some embodiments of any aspect, 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.

[0370] In some embodiments of any aspect, the modified saRNA modulates cell differentiation of cells containing the saRNA at a level equivalent to or greater than the level of modulation achieved by an equivalent dose of a corresponding saRNA with less than 25% modified nucleotides. In some embodiments of any aspect, the modified saRNA modulates cell differentiation of cells containing the saRNA at a level equivalent to the level of modulation achieved by an equivalent dose of a corresponding saRNA with less than 25% modified nucleotides. In some embodiments of any aspect, the modified saRNA modulates cell differentiation of cells containing the saRNA at a level 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 greater, than the level of modulation achieved by an equivalent dose of a corresponding saRNA with less than 25% modified nucleotides.

[0371] In some embodiments of any aspect, the cells are conditioned by expression of stem cell transcription factors encoded by the saRNA. In some embodiments of any aspect, the cells are conditioned to become stem cells by the saRNA described herein.

[0372] In some embodiments of any aspect, at least one cargo is constitutively (i.e., continuously) expressed in the cell. In some embodiments of any aspect, at least one cargo is constitutively (i.e., continuously) expressed in the subject.

[0373] 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 that can interact with each other. In some embodiments of any aspect, interaction of multiple cargo proteins results in conditional activity of cells transfected with the saRNA. In some embodiments of any aspect, the cargo proteins that can interact with each other are chimeric antigen receptors with activating and / or inhibitory functions. In some embodiments of any aspect, the cargo comprises one, two, three, four, five, six, seven, eight, nine, ten, or more chimeric antigen receptors. In some embodiments of any aspect, the cargo comprises a protein comprising a chimeric antigen receptor that includes 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 a protein. In some embodiments of any aspect, the cargo is a protein comprising at least one domain that is responsive to an external input. In some embodiments of any aspect, the cargo is a protein comprising at least one repressible protease domain that regulates the activity of the cargo in response to at least one protease inhibitor.

[0374] In some embodiments of any aspect, the activity of the cargo protein expressed by the saRNA is controlled by administering a small molecule and / or a protein and / or an RNA molecule. 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-responsive domain. In some embodiments of any aspect, the activity of the cargo protein expressed by the 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 that can interact with at least one protein domain in the cargo protein.

[0375] In some embodiments of any aspect, the methods described herein include contacting a cell containing 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 include 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.

[0376] In some embodiments of any aspect, the input is endogenous to the environment of the cell containing the saRNA. In some embodiments of any aspect, the input is exogenous to the environment of the cell containing the saRNA. In some embodiments of any aspect, the input includes cells, proteins, small molecules, light of one or more specific wavelengths, specific temperatures, and / or magnetic fields.

[0377] In some embodiments of any aspect, the input results in the 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 the 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 the oligomerization of the cargo, resulting in activation. In some embodiments of any aspect, the input results in the oligomerization of the cargo, resulting in inhibition.

[0378] 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 distinct combinations of inputs.

[0379] In some embodiments of any aspect, adding or removing at least one saRNA and / or at least one input alters the 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 a changed cell type and / or a changed cellular location. In some embodiments of any aspect, the changed cellular function is lytic. In some embodiments of any aspect, the changed cellular function is stimulatory. In some embodiments of any aspect, the changed cellular function is immunomodulatory. In some embodiments of any aspect, the input causes death of cells containing at least one saRNA described herein. In some embodiments of any aspect, the input increases the clearance rate of cells containing at least one saRNA described herein. In some embodiments of any aspect, the input causes cell cycle arrest of cells containing at least one saRNA described herein.

[0380] In some embodiments of any aspect, 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. In some embodiments of any aspect, the cargo of the saRNA encodes and expresses at least one cargo protein and at least one protein reporter. In some embodiments of any aspect, the cargo of the saRNA encodes and expresses at least one cargo protein and at least one expression-enhancing protein. In some embodiments of any aspect, the cargo of the saRNA encodes and expresses at least one protein reporter and at least one expression-enhancing protein. In some embodiments of any aspect, the cargo of the saRNA encodes and expresses at least one cargo protein, at least one protein reporter, and at least one expression-enhancing protein.

[0381] 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 proteins are B18R and E3L.

[0382] 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.

[0383] definition The invention illustratively described herein can be practiced without elements, or limitations, not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" are to be interpreted broadly and without limitation. The terms and expressions used are used as terms of description and not of limitation, and the use of such terms and expressions is not intended to exclude all equivalents of the shown and described features or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the invention has been specifically disclosed by preferred methods, embodiments, and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be practiced by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the invention as defined by the embodiments and elsewhere herein. In case of conflict, the present specification, including definitions, will control.

[0384] The contents of all articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein are incorporated by reference in their entirety to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. Applicant reserves the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.

[0385] Certain aspects and embodiments of the present invention have been described broadly and generically herein. Each narrower species and subgeneric grouping falling within the scope of the generic invention also forms part of some aspects and embodiments of the invention contemplated herein. This includes the generic description of the invention with any condition or negative limitation excluding any subject matter from the genus, regardless of whether the excluded material is specifically recited herein.

[0386] For convenience, the meanings of some terms and phrases used in the present invention, examples, and the appended claims are provided. Unless otherwise specified or implied from the context, the following terms and phrases have the meanings provided below. The definitions are provided to help explain certain embodiments and are not intended to limit the claimed technology, since the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and the definition of a term provided herein, the definition provided herein shall prevail.

[0387] The terms "protein," "cargo," or "protein of interest" refer to any polypeptide or nucleic acid sequence of known or unknown function that can be encoded by a gene. 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 function and should not be construed as an exclusive list. Proteins with unknown function can also be encoded by a gene.

[0388] The term "protein" refers to any polypeptide sequence of known or unknown function that can be encoded by a gene. Examples of proteins include receptors, ligands, enzymes, transcription factors, etc. These are examples of proteins with known function and should not be construed as an exclusive list. Proteins of unknown function can also be encoded by a gene.

[0389] The term "cargo" refers to a polypeptide sequence encoded by conventional or self-replicating RNA. This polypeptide sequence can be a polypeptide of 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.

[0390] Chimeric antigen receptors (CARs) combine the components or functions of a T cell receptor (TCR) and related molecules into a single polypeptide. In some embodiments of any aspect, the polypeptides described herein are second- or third-generation CARs, which include an extracellular binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains. Typically, the extracellular binding domain contains a single-chain variable fragment (scFv), usually derived from an antigen-reactive antibody with high specificity for a particular antigen. Most CARs contain the CD3 zeta chain domain, the primary transmitter of T cell activation signals, as the intracellular signaling domain. In addition to scFvs, non-antibody-based approaches have also been used to direct CAR specificity, typically utilizing ligand / receptor pairs that normally bind to each other. In some embodiments of any aspect, the polypeptides described herein may include cytokines, innate immune receptors, TNF receptors, growth factors, and structural proteins, all of which have been successfully used as CAR antigen recognition domains.

[0391] The term "self-replicating RNA" refers to an RNA strand that is known to undergo replication activity, generating a replicative strand from the original strand. Self-replicating RNA is known to exist in the form of RNA virus genomes, and artificial self-replicating RNA can be created by utilizing components derived from RNA viruses along with a sequence of interest. Components derived from RNA viruses can be used in combination, or only from a specific RNA virus.

[0392] The term "self-amplifying RNA" refers to a nucleic acid polymer capable of replicating the entire nucleic acid polymer in both negative and positive strand conformations. Self-amplifying RNA can synthesize additional self-amplifying RNAs, in part, by producing various nonstructural proteins of viral origin that can act, among other activities, as RNA-dependent RNA polymerases. Self-amplifying RNAs can be generated by utilizing components derived from RNA viruses along with sequences encoding the cargo of interest. An essential aspect of saRNA is that the nonstructural proteins can generate both new full-length saRNA strands and RNA produced from subgenomic promoters. In therapeutic saRNAs, the RNA produced by transcription from the subgenomic protomer encodes the cargo of interest. Self-amplifying RNA, saRNA, self-replicating RNA, and srRNA may be used interchangeably throughout this disclosure.

[0393] The term "expression" refers to the production of protein from an RNA strand. For example, "high expression" can refer to the production of sufficient or large amounts of protein. The desired expression level depends on the intended use. The terms "high expression" and "low expression" can be used in embodiments to describe observations made between cells of interest.

[0394] The term "expression" refers to the production of cargo from an RNA strand. In some embodiments, "expression" may refer to the production of a protein to induce an intended therapeutic effect. In some embodiments, "expression" may refer to the percentage of a cell population that expresses a cargo. In a preferred embodiment, "high expression" refers to the overproduction of a 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 the control. The terms "high expression" and "low expression" may be used in embodiments comparing cargo expression relative to the total amount or cell expression percentage between cells of interest.

[0395] The term "conventional mRNA" refers to a messenger RNA that does not have the ability to self-replicate. In some embodiments, conventional mRNA is produced by in vitro transcription. In some embodiments, conventional mRNA contains a 5' cap structure and a polyA tail. In some embodiments, conventional mRNA contains a 5' untranslated region and a 3' untranslated region.

[0396] The term "transfection" or "delivery" refers to the introduction of exogenous RNA into the intracellular space of cells.In some embodiments, transfection or delivery is carried out by electroporation.In some embodiments, transfection or delivery is carried out by lipid nanoparticles.In some embodiments, transfection or delivery is carried out directly without modifying RNA or vehicle to carry RNA.In some embodiments, transfection or delivery is carried out by conjugating cell-reactive moiety or targeting moiety to RNA.

[0397] The term "input" or "inputs" refers to a stimulus that interacts with the cargo of a self-replicating RNA. The input or inputs include, but are not limited to, cells, proteins, enzymes, small molecules, light of one or more specific wavelengths, a thermal stimulus, or the application of a magnetic field. The input or inputs include, but are not limited to, cells, proteins, enzymes, small molecules, DNA, RNA, light of one or more specific wavelengths, a thermal stimulus, or the application of a magnetic field.

[0398] The term "modified nucleotide" refers to any analog of cytidine, adenosine, guanosine, uridine, or pseudouridine. These analogs may include isomers of the nitrogenous base, as well as the inclusion or exclusion of natural and synthetically introduced chemical groups on either side of the nitrogenous base. It is expressly stated herein that the definition of the term "modified nucleotide" does not include modifications to the sugar-phosphate backbone. This exception is not intended to exclude methylation at the 2'O position of the first and second starting nucleotides, also known as cap-1 and cap-2 structures.

[0399] The term "highly substituted" or "highly substituted" refers to the replacement of a high percentage of natural nucleotides with their corresponding analogs. A high percentage can be greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 100%. Furthermore, the terms "100% substitution," "100% replaced," "full substitution," "fully substituted," and "fully replaced" can be used interchangeably throughout this disclosure. Full substitution means that a given unmodified nucleotide is absent in the synthesis or final product of the self-amplifying RNA. A certain percentage of substitution refers to a fractional mixture of a certain nucleotide, consisting of analogs and natural cytidine, adenosine, guanosine, or uridine. In a preferred embodiment, 50-100% of one of a combination of 5-methyluridine, 5-methylcytidine, and 5-hydroxymethylcytidine is substituted into the saRNA. In a preferred embodiment, 50-100% of one of a combination of 5-methyluridine, 5-methylcytidine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine is substituted into the saRNA. In a preferred embodiment, 100% of cytidines are replaced with 5-methylcytidine, and the resulting composition of fully substituted self-amplifying RNA is composed entirely of adenosine, guanosine, uridine, and 5-methylcytidine and expresses cargo at levels comparable to or greater than unmodified saRNA.In another preferred embodiment, 100% of the cytidines are replaced with 5-hydroxymethylcytidine, and the resulting composition of the fully substituted self-amplifying RNA is adenosine, guanosine, uridine, and 5-hydroxymethylcytidine, and expresses cargo at levels comparable to or greater than unmodified saRNA. In another preferred embodiment, 100% of the uridines are replaced with 5-methyluridine, and the resulting composition of the fully substituted self-amplifying RNA is adenosine, guanosine, cytidine, and 5-methyluridine, and expresses cargo at levels comparable to or greater than unmodified saRNA. In another preferred embodiment, 100% of the uridines are replaced with 5-hydroxymethyluridine, and the resulting composition of the fully substituted self-amplifying RNA is adenosine, guanosine, cytidine, and 5-hydroxymethyluridine, and expresses cargo at levels comparable to or greater than unmodified saRNA.

[0400] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a decrease by a statistically significant amount. In some embodiments, "reduce," "reduction," or "reducing," or "inhibiting" typically refers to a decrease of at least 10% compared to a reference level (e.g., the absence of a particular treatment and agent), and may include, for example, a decrease of 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 greater. As used herein, "reduction" or "inhibition" does not include complete inhibition or reduction compared to a reference level. "Complete inhibition" refers to 100% inhibition compared to a reference level. The decrease may preferably be to a level that is accepted as being within the normal range, for example, to a level in individuals without a particular disorder.

[0401] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, e.g., 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% compared to a reference level, or an increase up to and including 100%, or any increase between 10 and 100%, or 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 more increase compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

[0402] As used herein, the terms "protein" and "polypeptide" are used interchangeably to designate a series of amino acid residues joined together by peptide bonds between the α-amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated amino acids, glycated amino acids, glycosylated amino acids, etc.) and amino acid analogs, regardless of their size or function. While "protein" and "polypeptide" are often used in reference to relatively large polypeptides, the term "peptide" is often used in reference to small polypeptides, the usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0403] Furthermore, the various embodiments described herein are intended to encompass variants (natural or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any specific polypeptide described. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" in which the change substitutes an amino acid for a chemically similar amino acid, and the desired activity of the polypeptide is retained. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0404] Certain amino acids can be exchanged for residues with similar physicochemical characteristics, for example, one aliphatic residue can be substituted for another (e.g., Ile, Val, Leu, or Ala for another), or one polar residue can be substituted for another (e.g., Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, such as substitutions of entire regions with similar hydrophobic characteristics, are well known. Polypeptides containing conservative amino acid substitutions can be tested to confirm that the desired activity, e.g., the activity and specificity of the native or reference polypeptide, is retained.

[0405] Amino acids can be classified according to similarities in side chain properties (AL Lehninger, in Biochemistry, second 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 classified into groups based on shared 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 that influence chain direction: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another. Particular conservative substitutions include, for example, Ala → Gly or Ser; Arg → Lys; Asn → Gln or → His; Asp → Glu; Cys → Ser; Gln → Asn; Glu → Asp; Gly → Ala or → Pro; His → Asn or → Gln; Ile → Leu or → Val; Leu → Ile or → Val; Lys → Arg → Gln or → Glu; Met → Leu → Tyr or → Ile; Phe → Met → Leu or → Tyr; Ser → Thr; Thr → Ser; Trp → Tyr; Tyr → Trp; and / or Phe → Val → Ile or → Leu.

[0406] 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, a "functional fragment" is a fragment or segment of a polypeptide that retains at least 50% of the wild-type reference polypeptide activity. Functional fragments may also include conservative substitutions of the sequences disclosed herein.

[0407] In some embodiments, the polypeptides described herein may be variants of the polypeptide sequences described herein. In some embodiments, variants are conservatively modified variants. Conservative substitution variants may be obtained, for example, by mutation of a native nucleotide sequence. As referred to herein, a "variant" is a polypeptide that is substantially homologous to a native or reference polypeptide but has an amino acid sequence that differs from the amino acid sequence of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide includes a sequence encoding a protein or fragment thereof that contains one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but retains the activity of the native or reference polypeptide. A wide variety of approaches, for example, PCR-based site-directed mutagenesis, are known in the art and can be applied by those skilled in the art to create and test artificial variants.

[0408] A variant amino acid sequence or variant DNA sequence may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to a native or reference sequence. The degree of homology (percent identity) between a native and a variant sequence can be determined, for example, by comparing the two sequences using publicly available, free computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0409] A variant amino acid sequence 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 changes). It should be understood that a sequence with a specified percent similarity to a reference sequence necessarily encompasses sequences with the same specified percent identity to the reference sequence. Those skilled in the art are aware of a variety of computer programs employing different mathematical algorithms that can be used to determine identity or similarity between two sequences. For example, a computer program using the Needleman and Wunsch algorithm (Needleman et al. (1970)); the GAP program in the Accelrys GCG software package (Accelerys Inc., San Diego USA); the E. Meyers and W. Miller algorithm (Meyers et al. (1989)) incorporated into the ALIGN program (version 2.0); or more preferably, BLAST (Basic Local Alignment Tool using default parameters) can be utilized. See, e.g., U.S. Patent 10,023,890, the contents of which are incorporated herein by reference in their entirety.

[0410] As used herein, the phrase "maintain the same function" when used in reference to an enzyme catalyzes the same reaction as the reference enzyme.

[0411] Alterations in native amino acid sequences can be accomplished by any of a number of techniques known in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing the mutated sequence flanked by restriction sites that allow ligation with fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-directed mutagenesis procedures can be used to provide altered nucleotide sequences with specific codons altered according to the required substitution, deletion, or insertion. A wide variety of site-directed mutagenesis approaches, such as the Kunkel method, cassette mutagenesis, PCR site-directed mutagenesis (e.g., conventional PCR, primer extension, or inverse PCR), whole-plasmid mutagenesis, in vivo site-directed mutagenesis, and CRISPR / Cas-guided mutagenesis, are known in the art and can be applied by those skilled in the art to introduce mutations at specific nucleic acid loci.Techniques for making such alterations are well established and are described, for example, in Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Braman, Jeff, ed. (2002) In Vitro Mutagenesis Protocols, Methods in Molecular Biology, Vol. 182 (2nd ed.); Khudyakov and Fields (2002), Artificial DNA: Methods and Applications, CRC Press; Hsu et al. (2014), Cell 157 (6): 1262-78; Cerchione et al. (2020) PLOS ONE 15 (4): e0231716; and the techniques disclosed in U.S. Patent Nos. 4,518,584 and 4,737,462. Any cysteine ​​residue not involved in maintaining the correct conformation of the polypeptide can also be generally substituted with serine to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bonds can be added to polypeptides to improve their stability or facilitate oligomerization.

[0412] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acids 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, a single-stranded nucleic acid can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, a nucleic acid can be DNA. In another aspect, a nucleic acid can be RNA. Suitable DNA can include, for example, template DNA, plasmid DNA, and vector DNA. Suitable RNA can include, for example, saRNA and mRNA.

[0413] The term "expression" refers to the cellular processes involved in the production of RNA and proteins, including, but not limited to, transcription, transcription processing, translation, and protein folding, modification, and processing, as applicable, and, where appropriate, secretion of proteins. Expression can refer to the transcription and stable accumulation of sense RNA (e.g., saRNA, mRNA) or antisense RNA derived from a nucleic acid fragment, and / or the translation of saRNA or mRNA into a polypeptide.

[0414] "Expression product" includes RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate control sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0415] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatment whose purpose is to reverse, alleviate, ameliorate, inhibit, slow, or halt the progression or severity of a condition associated with a disease or disorder. The term "treat" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the disease is slowed or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least slowing of the progression or worsening of symptoms compared to that expected in the absence of treatment. Beneficial or desirable clinical results include, but are not limited to, alleviation of one or more symptoms, a decrease in the extent of disease, stabilization of the disease state (i.e., no worsening), a delay or slowing of disease progression, remission or palliation of the disease state, remission (whether partial or complete), and / or a decrease in mortality, whether detectable or undetectable. The term "treating" a disease also includes alleviating the symptoms or side effects of the disease (including symptomatic treatment).

[0416] As used herein, the term "pharmaceutical composition" refers to an active agent combined with a pharmaceutically acceptable carrier, e.g., a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problem or complication, and are commensurate with a reasonable benefit / risk ratio. 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, nanoparticle, and / or ointment. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be an artificial or engineered carrier, e.g., a carrier in which the active ingredient is not found to occur in or within nature.

[0417] As used herein, the term "administering" refers to placing the saRNA disclosed herein, or a nucleic acid, cell, or composition comprising the saRNA, into a subject by a method or route that results in at least partial delivery of the saRNA, or a nucleic acid, cell, or composition comprising the saRNA, at the desired site. Pharmaceutical compositions comprising the saRNA disclosed herein, or a nucleic acid, cell, or composition comprising the saRNA, can be administered by any suitable route that results in effective treatment in the subject. In some embodiments, administration includes human physical activity, such as injection, ingestion, application, and / or operation of a delivery device or machine. Such activities may be performed, for example, by a medical professional and / or the subject receiving treatment.

[0418] As used herein, " contacting " refers to any suitable means for delivering or exposing a drug to at least one cell.Exemplary delivery methods include, but are not limited to, direct delivery into cell culture medium, transfection, transduction, perfusion, injection or other delivery methods known to those skilled in the art.In some embodiments, contacting includes human physical activity, such as injection; dispensing, mixing, and / or decanting; and / or operating a delivery device or machine.

[0419] In some embodiments of any aspect, the cells can be maintained in culture. As used herein, "maintaining" refers to continuing the survival of a cell or population of cells. A maintained cell population includes at least a subpopulation of metabolically active cells.

[0420] As used herein, the term "specific binding" refers to a chemical or physical interaction between two molecules, compounds, cells, and / or particles in which the first entity binds to a second entity, a target entity, with greater specificity and affinity than the first entity binds to a third entity, a non-target entity. In some embodiments, specific binding can refer to an affinity of the first entity for the 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 than its affinity for a third, non-target entity. A reagent specific for a particular target is one that exhibits specific binding to that target under the conditions of the assay being utilized.

[0421] The term "analog" as used herein refers to a substance that shares one or more specific structural features, elements, components or parts with a reference substance.Typically, "analog" shows significant structural similarity with a reference substance, for example, shares a core or consensus structure, but differs in certain individual respects.In some embodiments, an analog is a substance that can be generated from a reference substance, for example, by chemically manipulating the reference substance.

[0422] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference of 2 standard deviations (2SD) or greater.

[0423] Except in the working examples or unless otherwise specified, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in conjunction with percentages, the term "about" can mean ±1%.

[0424] As used herein, the term "comprising" means that other elements may be present in addition to the defined elements set forth. The use of "comprising" indicates inclusion rather than limitation.

[0425] The term "consisting of" refers to the compositions, methods...

Claims

1. 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 with a moiety on carbon 5 of the pyrimidine, the moiety being selected from the group consisting of methyl, ethyl, propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, and hydroxypropyl functional groups.

2. The saRNA of claim 1, wherein the modified nucleotide comprises 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, 5-hydroxymethylcytidine, or a combination thereof.

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

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

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

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

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

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

9. The saRNA of any one of claims 1 to 8, wherein the level of substitution of the modified nucleotides is between 51% and 75%.

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

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

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

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

14. The saRNA of any one of claims 1 to 13, wherein the initiation 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 to 14, wherein the initiation 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 to 15, wherein the initiating 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 to 16, wherein the initiating nucleotide and the following nucleotide of the saRNA are both methylated at the 2'O position of the ribose (Cap 2).

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

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

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

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

22. From 5' to 3', (a) at least one nonstructural protein from at least one virus; (b) a subgenomic promoter (SGP) derived from at least one virus; (c) at least one cargo of interest; The saRNA of any one of claims 1 to 21, comprising:

23. 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 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; (g) polyA tail and The saRNA of any one of claims 1 to 22, comprising:

24. The saRNA of any one of claims 1 to 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 type of virus.

25. The saRNA of any one of claims 1 to 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 to 25, wherein at least one of 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 to 26, wherein the virus is an alphavirus.

28. At least one virus is present in the swine flu virus, including Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), Sindbis virus (SIN), Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Mayaro virus (MAYV), Getah virus (GETV), Ross River virus (RRV), Una virus (UNAV), Middleburg virus (MIDV), O'nyong-nyong virus (ONNV), Barmah Forest virus (BFV), Mucambo virus (MUCV), Tonate virus (TONV), Everglades virus (EVEV), Rio Negro virus (RNV), Turnip Rosette virus (TROV), Highlands J virus (HJV), Western equine encephalitis virus (WEEV), Fig Mosaic Emara virus (Fig Mosaic The saRNA of any one of claims 1 to 27, wherein the saRNA is selected from the group consisting of: FMV, Aura virus (AURAV), Kunjin virus (KUN), measles virus (MV), coronavirus (CoV), rabies virus (RABV), and vesicular stomatitis virus (VSV).

29. 29. The saRNA of any one of claims 1 to 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), Getah virus (GETV), Ross River virus (RRV), Una virus (UNAV), Middleburg virus (MIDV), O'nyong-nyong virus (ONNV), Barmah Forest virus (BFV), Mucambo virus (MUCV), Tonate virus (TONV), Everglades virus (EVEV), Rio Negro virus (RNV), Highlands J virus (HJV), Western equine encephalitis virus (WEEV), and Aura virus (AURAV).

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

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

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

33. The saRNA of any one of claims 1 to 32, wherein at least one cargo protein or peptide is of viral, bacterial, protozoan, mammalian, or plant origin.

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

35. 35. The saRNA of any one of claims 1 to 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 activator 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 group 2 member D (NKG2D).

36. The CAR is (a) Conventional CAR; (b) On-CAR; (c) Off-CAR system; (d) ON / OFF-CAR; (e) inhibitory CAR; or (f) Split Universal Programmable Reconfigurable (SUPRA) CAR System The saRNA of any one of claims 1 to 35, selected from the group consisting of:

37. Conventional CAR, (a) extracellular binding domain; (b) a transmembrane domain; and (c) at least one intracellular signaling domain; 37. The saRNA of claim 36, comprising:

38. On-CAR (a) extracellular binding domain; (b) transmembrane domain; (c) at least one intracellular signaling domain; and (d) an inhibitable protease domain that cleaves and degrades the on-CAR in the absence of a protease inhibitor.

37. The saRNA of claim 36, comprising:

39. Off-car system (a) (i) extracellular binding domain; (ii) a transmembrane domain; and (iii) peptide domain a first polypeptide comprising: (b) (i) an inhibitory protease domain capable of specifically binding to the peptide domain in the absence of a protease inhibitor; and (ii) at least one intracellular signaling domain; and a second polypeptide comprising 37. The saRNA of claim 36, comprising:

40. 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 apoNS3a leader (ANR) peptides.

41. On / Off-CAR (a) extracellular binding domain; (b) transmembrane domain; (c) at least one intracellular signaling domain; (d) a suppressible protease domain that cleaves and degrades the ON / OFF-CAR in the absence of a protease inhibitor; and (e) Drug-inducible degron domain 37. The saRNA of claim 36, comprising:

42. 42. The saRNA of any one of claims 38, 39, or 41, wherein the repressible protease domain comprises 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 grazoprevir (GZV), danoprevir, simeprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir.

44. The saRNA of claim 41, wherein the drug-inducible degron domain comprises an IKAROS family zinc finger 3 (IKZF3) domain that can bind to and activate the CAR for degradation by the drug lenalidomide or pomalidomide.

45. Inhibitory CAR (a) extracellular binding domain; (b) a transmembrane domain; and (c) Inhibitory domain 37. The saRNA of claim 36, comprising:

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

47. The SUPRA CAR system (a) (i) an extracellular binding domain; and (ii) the first member of the extracellular protein interaction domain a first polypeptide comprising: (b) (i) a second member of the extracellular protein interacting domain capable of specifically binding to the first member of the extracellular protein interacting domain of the first polypeptide; (ii) a transmembrane domain; and (iii) at least one intracellular signaling domain; and a second polypeptide comprising 37. The saRNA of claim 36, comprising:

48. The saRNA of any one of claims 37 to 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 to 48, wherein the extracellular binding domain comprises an antigen-binding domain derived from an antibody.

50. The saRNA of any one of claims 1 to 49, wherein the cargo comprises 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 to 50, wherein the cargo comprises an antibody or a fragment thereof.

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

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

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

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

56. 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 to 56, wherein the cargo comprises at least one vaccine-related antigen.

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

59. 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, yellow fever virus, Japanese encephalitis virus, Western equine encephalitis virus, Eastern equine encephalitis, 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), herpesvirus, 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 to 59, wherein 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.

61. The saRNA of any one of claims 57 to 60, wherein the vaccine-associated antigen comprises one of SEQ ID NOs: 13 to 16, 22, or an amino acid sequence at least 80% identical to one of SEQ ID NOs: 13 to 16, 22 that maintains the same function.

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

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

64. 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 transcription factor 2 (Sox2), Krüppel-like factor 4 (Klf4), and cellular myelocytomatosis oncogene (c-Myc).

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

67. 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), neurotrophin, migration stimulating factor (MSF), and sarcoma growth factor (SGF).

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

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

70. 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 to 70, wherein the cargo comprises at least one enzyme having antioxidant activity.

72. 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 ecto-nucleoside triphosphate diphosphydrolase.

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

74. The saRNA of any one of claims 1 to 73, wherein the saRNA is replaced with a template of SEQ ID NO: 2 or a nucleic acid sequence at least 90% identical to SEQ ID NO: 2 that maintains the same function, and 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 to 74, wherein the nsp1, nsp2, nsp3, and nsp4 proteins encoded by the saRNA comprise at least one amino acid sequence of SEQ ID NO:4 and / or SEQ ID NO:23, or at least one amino acid sequence that maintains the same function and is at least 90% identical to SEQ ID NO:4 and / or at least 90% identical to SEQ ID NO:

23.

76. The saRNA of any one of claims 1 to 75, wherein the template of SEQ ID NO: 5 or a nucleic acid sequence at least 90% identical to SEQ ID NO: 5 is used to replace the template, maintaining the same function.

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

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

79. The saRNA of any one of claims 1 to 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. From 5' to 3', (a) at least one nonstructural protein from at least one virus; (b) a subgenomic promoter (SGP) derived from at least one virus; (c) at least one cargo of interest; A self-amplifying RNA (saRNA) comprising: the saRNA comprises at least 25% modified nucleotides, the modified nucleotides being selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; the start nucleotide comprises adenosine or an adenosine analog; The initiating nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1), The self-amplifying RNA (saRNA).

81. 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 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; (g) polyA tail and A self-amplifying RNA (saRNA) comprising: the saRNA comprises at least 25% modified nucleotides, the modified nucleotides being selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine; the start nucleotide comprises adenosine or an adenosine analog; The initiating nucleotide of the saRNA is methylated at the 2'O position of the ribose (Cap 1), The self-amplifying RNA (saRNA).

82. 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 type of virus.

83. From 5' to 3', (a) at least one nonstructural protein from at least one virus; (b) a subgenomic promoter (SGP) derived from at least one virus; (c) at least one cargo of interest; Self-amplifying RNA (saRNA), including

84. 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 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; (g) polyA tail and Self-amplifying RNA (saRNA), including

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 type of virus.

86. 86. The saRNA of any one of claims 83 to 85, which does not contain modified nucleotides.

87. 87. The saRNA of any one of claims 83 to 86, comprising less than 25% modified nucleotides.

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

89. The CAR is (a) Conventional CAR; (b) On-CAR; (c) Off-CAR system; (d) ON / OFF-CAR; (e) inhibitory CAR; or (f) Split Universal Programmable Reconfigurable (SUPRA) CAR System The saRNA of any one of claims 83 to 88, selected from the group consisting of:

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

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

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

93. The saRNA is about 5x10 -4 93. The composition of claim 92, formulated in a dose of mg / kg.

94. The saRNA is about 5x10 -3 93. The composition of claim 92, formulated in a dose of mg / kg.

95. The saRNA is about 5x10 -2 93. The composition of claim 92, formulated in a dose of mg / kg.

96. The saRNA is about 5x10 -1 93. The composition of claim 92, formulated in a dose of mg / kg.

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

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 to 89, the nucleic acid of claim 90, the vector of claim 91, or the composition of any one of claims 92 to 98.

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

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

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

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

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

105. The saRNA is about 5x10 -4 105. The pharmaceutical composition of any one of claims 101-104, formulated in a mg / kg dose.

106. The saRNA is about 5x10 -3 105. The pharmaceutical composition of any one of claims 101-104, formulated in a mg / kg dose.

107. The saRNA is about 5x10 -2 105. The pharmaceutical composition of any one of claims 101-104, formulated in a mg / kg dose.

108. The saRNA is about 5x10 -1 105. The pharmaceutical composition of any one of claims 101-104, formulated in a mg / kg dose.

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

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

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

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

113. 113. The method of claim 112, wherein the subject is a human.

114. 113. The method of claim 112, wherein the subject is a livestock animal or pet.

115. The method of 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 replicates at a level greater than or equal to that of a corresponding saRNA at an equivalent dose having fewer 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 a corresponding saRNA at an equivalent dose 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 produces an early interferon response in the subject, and the early interferon response is reduced compared to a corresponding saRNA of an equivalent dose having less than 25% modified nucleotides.

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

121. The method of any one of claims 111 to 120, wherein the cell is a human cell.

122. The method of any one of claims 111-121, wherein the subject has cancer.

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

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

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

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

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

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

129. The method of claim 128, wherein the saRNA comprises greater than 50% substitution of uridines with 5-methyluridine, and the saRNA has increased kidney-specific expression of the cargo compared to a corresponding saRNA at an equivalent dose comprising less than 50% substitution of uridines with 5-methyluridine.

130. The method of any one of claims 111 to 129, wherein the modified saRNA modulates cell differentiation of cells containing the saRNA at a level equivalent to or greater than the level of modulation achieved by 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. 132. The method of claim 130 or 131, wherein the cells are conditioned to become stem cells.

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

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

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

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

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

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

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

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

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

142. 142. The method of any one of claims 135-141, wherein the input is light of a particular wavelength or wavelengths.

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

144. 144. The method of any one of claims 135-143, wherein the input is application of a magnetic field.

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

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

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

148. 148. The method of any one of claims 135-147, wherein said input results in oligomerization and resulting activation of said cargo.

149. 149. The method of any one of claims 135-148, wherein said input results in oligomerization of said cargo and resulting inhibition.

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

151. 151. The method of any one of claims 111-150, wherein said cargo comprises multiple species of chimeric antigen receptors.

152. 152. The method of any one of claims 111-151, wherein said control of the cell requires all inputs to be present simultaneously.

153. 153. The method of any one of claims 111-152, wherein said control of said cells requires the presence of any input.

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

155. The method of any one of claims 111-154, wherein said cell fate is a change in cell type.

156. The method of any one of claims 111 to 155, wherein said cell fate is a change in cell localization.

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

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

159. The method of any one of claims 111-158, wherein the function of said cells is immunomodulatory.

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

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

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

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

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

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

166. The method of any one of claims 111 to 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. 167. The method of claim 166, wherein said cargo protein is a chimeric antigen receptor and said expression-enhancing protein is B18R.

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

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

170. The method of any one of claims 111-169, wherein the saRNA is delivered to the cell by a lipid nanoparticle.

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

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

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

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

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

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

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

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

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

181. The method of any one of claims 111-180, wherein multiple distinct saRNAs are delivered to a mixture of cells.

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

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