Alphaviral encrypted RNAS and their methods of use

Abstract

The present disclosure relates to alphaviral encrypted RNAs, and DNAs that encode the alphaviral encrypted RNAs, that enable increased translation of a polypeptide, including a therapeutic polypeptide, after being contacted by alphaviral translation activators, and methods of their use. Methods of manufacturing the encrypted RNAs, and DNAs that encode the alphaviral encrypted RNAs are also provided.

Description

ALPHAVIRAL ENCRYPTED RNAS AND THEIR METHODS OF USEU.S. GOVERNMENT STATEMENT

[0001] This invention was made with U.S. Government support under Agreement No. W15QKN-16-9-1002 awarded by the United States Army Contracting Command — New Jersey Contracting Activity (ACC-NJ) to the Medical CBRN Defense Consortium (MCDC). The Government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The application claims benefit to U.S. Provisional Application No. 63 / 619,726, filed January 10, 2024, which is incorporated by reference herein in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing, which has been submitted electronically in .xml format. The contents of the electronic sequence listing (009796_00016_WO_SL.xml; Size 767,838 bytes, and Date of Creation: January 9, 2025) is incorporated by reference herein in its entirety.FIELD OF THE INVENTION

[0004] The disclosure relates to alphaviral encrypted RNAs and DNAs that encode alphaviral encrypted RNAs, that enable increased translation of polypeptides, including therapeutic polypeptides, after being contacted by translation activators, and methods of their use.BACKGROUND

[0005] Small molecules and proteins represent the two major classes of FDA-approved drugs. Each class has its limitations. The small-molecule drugs predominantly target proteins via competitive binding. However, the targeted proteins have been estimated to account for only 2-5% of the proteins encoded by the human genome because of the structural requirements (e.g., binding pockets for the small-molecule drags) (Hopkins AL. et al., Nat. Rev. Drug Discov. 2002; 1: 727-30). On the other hand, the size, specificity, and stability of protein drags (e.g., antibodies) significantly limit their utility towards therapeutic targets

[0006] In contrast, RNA drugs may circumvent these limitations because RNA molecules can specifically bind to the nucleic acid targets via base pairing and / or aim to permanently change the host’s DNA (e.g., gene editing). Over the past few decades, RNA drugs have emerged as promising candidates to address diseases at the gene and / or RNA levels. However, therapeutic RNA delivery has faced a few obstacles. For example, naked, single-stranded RNA is prone to nuclease degradation, can activate the immune system, and is challenging to passively cross the cell membrane because of its size and negative charge. The nucleic acid delivery field thus has centered on the design of delivery methods and materials that will transport RNA drugs to the site of interest.

[0007] Despite the recent successes of RNA therapeutics (e.g., mRNA COVID-19 vaccines), there is still a need in the art to identify RNA medicines that are safe and effective in treating disease, including RNA medicines that have an increased specificity to diseases or therapeutic targets.SUMMARY

[0008] Provided herein are new RNA therapeutics that have improved efficacy in treating infectious diseases as well as cancers and genetic conditions. The compounds and compositions comprising the compounds also have improved specificity in treating a disease or condition and can be more effectively and / or precisely modulated.Methods of making and using the compounds, compositions containing the compounds, and uses of medicaments comprising these compounds and compositions are also disclosed.

[0009] The present disclosure is related to an “alphaviral encrypted RNA” that encodes a polypeptide of interest, which is translated at reduced levels until the encrypted RNA is contacted by a “target-specific translation activator.” The target-specific translation activator directs increased translation of the polypeptide of interest by transcribing the encrypted RNA into a distinct mRNA species that is more translatable by the cellular ribosomal machinery. In some embodiments, an encrypted RNA encodes a therapeutic polypeptide of interest. The present disclosure is also related to DNA that encodes encrypted RNA. In some embodiments, the target-specific translation activator comprises an RNA-dependent RNA polymerase or an RNA-dependent DNA polymerase.

[0010] In one aspect, provided(i) a coding region comprising a coding sequence encoding a therapeutic polypeptide,(ii) a left flanking region (L region) adjacent to and contiguous with a 5’ end of the coding region; and(iii) a right flanking region (R region) adjacent to and contiguous with a 3’ end of the coding region; wherein both the L region and the R region of the alphaviral encrypted RNA are derived from an alphavirus, and wherein, once the alphaviral encrypted RNA is inside a cell containing a translation activator, the L region and the R region interact with the translation activator, thereby resulting in translation of the therapeutic polypeptide.Each of the L region and the R region can be derived from the reverse complement of a corresponding region that is native to the alphavirus. The alphavirus can be Chikungunya virus, Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Sindbis Virus (SINV), Semliki Forest Virus (SFV), or Western Equine Encephalitis Virus (WEEV). The translation activator can be an RNA dependent polymerase, wherein the RNA dependent polymerase is a viral RNA dependent polymerase, optionally an RNA-dependent RNA polymerase or an RNA- dependent DNA polymerase.

[0011] In another aspect, the coding sequence within the alphaviral encrypted RNA is in an antisense orientation. A combination of the L region and the R region may satisfy one of the following:(i) the L region comprises SEQ ID NO: 249 or a variant of SEQ ID NO: 249, wherein the variant of SEQ ID NO: 249 comprises a variation at one or more positions of positions 21-354 of SEQ ID NO: 249, and the R region comprises SEQ ID NO: 441 or a variant of SEQ ID NO: 441, wherein the variant of SEQ ID NO: 441 comprises a variation at one or more positions of positions 21-323 of SEQ ID NO: 441;(ii) the L region comprises SEQ ID NO: 438 or a variant of SEQ ID NO: 438, wherein the variant of SEQ ID NO: 438 comprises a variation at one or more positions of positions 21-113 of SEQ ID NO: 438 and the R region comprises SEQ ID NO: 442 or a variant of SEQ ID NO: 442, wherein the variant of SEQ ID NO: 442 comprises a variation at one or more positions of positions 21-353 of SEQ ID NO: 442;(iii) the L region comprises SEQ ID NO: 438 or a variant of SEQ ID NO: 438, wherein the variant of SEQ ID NO: 438 comprises a variation at one or more positions of positions 21-113 of SEQ ID NO: 438 and the R region comprises SEQ ID NO: 443or a variant of SEQ ID NO: 443, wherein the variant of SEQ ID NO: 443 comprises a variation at one or more positions of positions 21-517 of SEQ ID NO: 443;(iv) the L region comprises SEQ ID NO: 438 or a variant of SEQ ID NO: 438, wherein the variant of SEQ ID NO: 438 comprises a variation at one or more positions of positions 21-113 of SEQ ID NO: 438 and the R region comprises SEQ ID NO: 444 or a variant of SEQ ID NO: 444, wherein the variant of SEQ ID NO: 444 comprises a variation at one or more positions of positions 21-168 of SEQ ID NO: 444;(v) the L region comprises SEQ ID NO: 439 or a variant of SEQ ID NO: 439, wherein the variant of SEQ ID NO: 439 comprises a variation at one or more positions of positions 21-256 of SEQ ID NO: 439, and the R region comprises SEQ ID NO: 445 or a variant of SEQ ID NO: 445, wherein the variant of SEQ ID NO: 445 comprises a variation at one or more positions of positions 21-355 of SEQ ID NO: 445;(vi) the L region comprises SEQ ID NO: 439 or a variant of SEQ ID NO: 439, wherein the variant of SEQ ID NO: 439 comprises a variation at one or more positions of positions 21-256 of SEQ ID NO: 439, and the R region comprises SEQ ID NO: 446 or a variant of SEQ ID NO: 446, wherein the variant of SEQ ID NO: 446 comprises a variation at one or more positions of positions 21-286 of SEQ ID NO: 446;(vii) the L region comprises SEQ ID NO: 439 or a variant of SEQ ID NO: 439, wherein the variant of SEQ ID NO: 439 comprises a variation at one or more positions of positions 21-256 of SEQ ID NO: 439, and the R region comprises SEQ ID NO: 447 or a variant of SEQ ID NO: 447, wherein the variant of SEQ ID NO: 447 comprises a variation at one or more positions of positions 21-293 of SEQ ID NO: 447;(viii) the L region comprises SEQ ID NO: 440 or a variant of SEQ ID NO: 440, wherein the variant of SEQ ID NO: 440 comprises a variation at one or more positions of positions 21-74 of SEQ ID NO: 440, and the R region comprises SEQ ID NO: 445 or a variant of SEQ ID NO: 445, wherein the variant of SEQ ID NO: 445 comprises a variation at one or more positions of positions 21-355 of SEQ ID NO: 445;(ix) the L region comprises SEQ ID NO: 440 or a variant of SEQ ID NO: 440, wherein the variant of SEQ ID NO: 440 comprises a variation at one or more positions of positions 21-74 of SEQ ID NO: 440, and the R region comprises SEQ ID NO: 446 or a variant of SEQ ID NO: 446, wherein the variant of SEQ ID NO: 446 comprises a variation at one or more positions of positions 21-286 of SEQ ID NO: 446 and(x) the L region comprises SEQ ID NO: 440 or a variant of SEQ ID NO: 440, wherein the variant of SEQ ID NO: 440 comprises a variation at one or more positionsof positions 21-74 of SEQ ID NO: 440, and the R region comprises SEQ ID NO: 447 or a variant of SEQ ID NO: 447, wherein the variant of SEQ ID NO: 447 comprises a variation at one or more positions of positions 21-293 of SEQ ID NO: 447.

[0012] In another aspect, the coding sequence within the alphaviral encrypted RNA is in a sense orientation. A combination of the L region and the R region may satisfy one of the following:(i) the L region comprises SEQ ID NO: 421 or a variant of SEQ ID NO: 421, wherein the variant of SEQ ID NO: 421 comprises a variation at one or more positions of positions 21-323 of SEQ ID NO: 421, and the R region comprises SEQ ID NO: 432 or a variant of SEQ ID NO: 432, wherein the variant of SEQ ID NO: 432 comprises a variation at one or more positions of positions 21-363 of SEQ ID NO: 432;(ii) the L region comprises SEQ ID NO: 422 or a variant of SEQ ID NO: 422, wherein the variant of SEQ ID NO: 422 comprises a variation at one or more positions of positions 21-515 of SEQ ID NO: 422, and the R region comprises SEQ ID NO: 432 or a variant of SEQ ID NO: 432, wherein the variant of SEQ ID NO: 432 comprises a variation at one or more positions of positions 21-363 of SEQ ID NO: 432;(iii) the L region comprises SEQ ID NO: 423 or a variant of SEQ ID NO: 423, wherein the variant of SEQ ID NO: 423 comprises a variation at one or more positions of positions 21-398 of SEQ ID NO: 423, and the R region comprises SEQ ID NO: 432 or a variant of SEQ ID NO: 432, wherein the variant of SEQ ID NO: 432 comprises a variation at one or more positions of positions 21-363 of SEQ ID NO: 432;(iv) the L region comprises SEQ ID NO: 421 or a variant of SEQ ID NO: 421, wherein the variant of SEQ ID NO: 421 comprises a variation at one or more positions of positions 21-323 of SEQ ID NO: 421, and the R region comprises SEQ ID NO: 433 or a variant of SEQ ID NO: 433, wherein the variant of SEQ ID NO: 433 comprises a variation at one or more positions of positions 21-399 of SEQ ID NO: 433;(v) the L region comprises SEQ ID NO: 422 or a variant of SEQ ID NO: 422, wherein the variant of SEQ ID NO: 422 comprises a variation at one or more positions of positions 21-515 of SEQ ID NO: 422, and the R region comprises SEQ ID NO: 433 or a variant of SEQ ID NO: 433, wherein the variant of SEQ ID NO: 433 comprises a variation at one or more positions of positions 21-399 of SEQ ID NO: 433;(vi) the L region comprises SEQ ID NO: 423 or a variant of SEQ ID NO: 423, wherein the variant of SEQ ID NO: 423 comprises a variation at one or more positions of positions 21-398 of SEQ ID NO: 423 and the R region comprises SEQ ID NO: 433or a variant of SEQ ID NO: 433, wherein the variant of SEQ ID NO: 433 comprises a variation at one or more positions of positions 21-399 of SEQ ID NO: 433;(vii) the L region comprises SEQ ID NO: 424 or a variant of SEQ ID NO: 424, wherein the variant of SEQ ID NO: 424 comprises a variation at one or more positions of positions 21-574 of SEQ ID NO: 424, and the R region comprises SEQ ID NO: 434 or a variant of SEQ ID NO: 434, wherein the variant of SEQ ID NO: 434 comprises a variation at one or more positions of positions 21-122 of SEQ ID NO: 434;(viii) the L region comprises SEQ ID NO: 425 or a variant of SEQ ID NO: 425, wherein the variant of SEQ ID NO: 425 comprises a variation at one or more positions of positions 21-589 of SEQ ID NO: 425, and the R region comprises SEQ ID NO: 434 or a variant of SEQ ID NO: 434, wherein the variant of SEQ ID NO: 434 comprises a variation at one or more positions of positions 21-122 of SEQ ID NO: 434;(ix) the L region comprises SEQ ID NO: 426 or a variant of SEQ ID NO: 426, wherein the variant of SEQ ID NO: 426 comprises a variation at one or more positions of positions 21-517 of SEQ ID NO: 426, and the R region comprises SEQ ID NO: 434 or a variant of SEQ ID NO: 434, wherein the variant of SEQ ID NO: 434 comprises a variation at one or more positions of positions 21-122 of SEQ ID NO: 434;(x) the L region comprises SEQ ID NO: 424 or a variant of SEQ ID NO: 424, wherein the variant of SEQ ID NO: 424 comprises a variation at one or more positions of positions 21-574 of SEQ ID NO: 424, and the R region comprises SEQ ID NO: 260 or a variant of SEQ ID NO: 260, wherein the variant of SEQ ID NO: 260 comprises a variation at one or more positions of positions 21-124 of SEQ ID NO: 260;(xi) the L region comprises SEQ ID NO: 425 or a variant of SEQ ID NO: 425, wherein the variant of SEQ ID NO: 425 comprises a variation at one or more positions of positions 21-589 of SEQ ID NO: 425, and the R region comprises SEQ ID NO: 260 or a variant of SEQ ID NO: 260, wherein the variant of SEQ ID NO: 260 comprises a variation at one or more positions of positions 21-124 of SEQ ID NO: 260;(xii) the L region comprises SEQ ID NO: 426 or a variant of SEQ ID NO: 426, wherein the variant of SEQ ID NO: 426 comprises a variation at one or more positions of positions 21-517 of SEQ ID NO: 426, and the R region comprises SEQ ID NO: 260 or a variant of SEQ ID NO: 260, wherein the variant of SEQ ID NO: 260 comprises a variation at one or more positions of positions 21-124 of SEQ ID NO: 260;(xiii) the L region comprises SEQ ID NO: 431 or a variant of SEQ ID NO: 431, wherein the variant of SEQ ID NO: 431 comprises a variation at one or more positionsof positions 21-306 of SEQ ID NO: 431, and the R region comprises SEQ ID NO: 437 or a variant of SEQ ID NO: 437, wherein the variant of SEQ ID NO: 437 comprises a variation at one or more positions of positions 21-304 of SEQ ID NO: 437;(xiv) the L region comprises SEQ ID NO: 427 or a variant of SEQ ID NO: 427, wherein the variant of SEQ ID NO: 427 comprises a variation at one or more positions of positions 21-355 of SEQ ID NO: 427, and the R region comprises SEQ ID NO: 435 or a variant of SEQ ID NO: 435, wherein the variant of SEQ ID NO: 435 comprises a variation at one or more positions of positions 21-265 of SEQ ID NO: 435;(xv) the L region comprises SEQ ID NO: 428 or a variant of SEQ ID NO: 428, wherein the variant of SEQ ID NO: 428 comprises a variation at one or more positions of positions 21-418 of SEQ ID NO: 428, and the R region comprises SEQ ID NO: 435 or a variant of SEQ ID NO: 435, wherein the variant of SEQ ID NO: 435 comprises a variation at one or more positions of positions 21-265 of SEQ ID NO: 435;(xvi) the L region comprises SEQ ID NO: 429 or a variant of SEQ ID NO: 429, wherein the variant of SEQ ID NO: 429 comprises a variation at one or more positions of positions 21-355 of SEQ ID NO: 429, and the R region comprises SEQ ID NO: 435 or a variant of SEQ ID NO: 435, wherein the variant of SEQ ID NO: 435 comprises a variation at one or more positions of positions 21-265 of SEQ ID NO: 435;(xvii) the L region comprises SEQ ID NO: 448 or a variant of SEQ ID NO: 448, wherein the variant of SEQ ID NO: 448 comprises a variation at one or more positions of positions 21-286 of SEQ ID NO: 448, and the R region comprises SEQ ID NO: 450 or a variant of SEQ ID NO: 450, wherein the variant of SEQ ID NO: 450 comprises a variation at one or more positions of positions 21-83 of SEQ ID NO: 450;(xviii) the L region comprises SEQ ID NO: 449 or a variant of SEQ ID NO: 449, wherein the variant of SEQ ID NO: 449 comprises a variation at one or more positions of positions 21-393 of SEQ ID NO: 449, and the R region comprises SEQ ID NO: 450 or a variant of SEQ ID NO: 450, wherein the variant of SEQ ID NO: 450 comprises a variation at one or more positions of positions 21-83 of SEQ ID NO: 450; and(xix) the L region comprises SEQ ID NO: 430 or a variant of SEQ ID NO: 430, wherein the variant of SEQ ID NO: 430 comprises a variation at one or more positions of positions 21-333 of SEQ ID NO: 430, and the R region comprises SEQ ID NO: 436 or a variant of SEQ ID NO: 436, wherein the variant of SEQ ID NO: 436 comprises a variation at one or more positions of positions 21 323 of SEQ ID NO: 436

[0013] Each of the L region and the R region may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a corresponding region that is native to the alphavirus, or wherein each of the L region and the R region may comprise fewer than 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleoside variations relative to the corresponding region that is native to the alpha virus. Alternatively, each of the L region and the R region can vary from a corresponding region that is native to the alpha virus by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside substitution that is / are not involved in 5’ capping.

[0014] In another aspect, the alphaviral encrypted RNA may comprise at least one nucleoside modification. For example, each of the L region and the R region comprises no more than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% modified nucleosides, or each of the L region and the R region can comprise at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 100% modified nucleosides. The nucleoside modification can be a nonimmunogenic uridine modification; the percentage of modified uridine modifications can be (i) no more than 40%, 35%, 30%, 25%, 20% 15% or 10%, or (ii) more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%, or is 100% of all uridines. The nucleoside modification can be a nonimmunogenic cytidine modification; the percentage of modified cytidine modifications can be (i) no more than 40%, 35%, 30%, 25%, 20% 15% or 10%, or (ii) more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%, or is 100% of all cytidines. The nucleoside modification can be a nonimmunogenic adenosine modification, and the percentage of modified adenosine modifications can be between 1% and 30%, optionally, about 1%, 5%, 10%, 15%, 20%, 25%, or 30% of all adenosine.

[0015] In yet another aspect, the alphaviral encrypted RNA can comprise a 5* cap structure. Typically the 5’ cap structure can be selected from Cap 0, Cap 0 (3’-O-Me), Cap 1, Cap 1 (3*-O-Me), Cap 2, Cap 2 (3’-O-Me), Anti-Reverse Cap Analog (ARCA), inosine, Nl-methyl-guanosine, 2’ -fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, locked nucleic acid guanosine (LNA-guanosine), and 2- azido-guanosine structure, or selected from any combination or subcombination thereof. The 5’ end of the L region can comprise a 5’ cap structure. The 5’ end of the L region may comprise one or more variations associated with a 5’ cap structure. The alphaviral encrypted RNA may not comprise a 5’ cap structure (uncapped). Alternatively, the 5’ end of the L region may not comprise a 5’ cap structure(uncapped). The 5’ end of the alphaviral encrypted RNA comprises a 5’- monophosphate, 5 ’-diphosphate, or 5-triphosphate. Alternatively, the 5’ end of the alpha encrypted RNA does not comprise a 5’-phosphate (dephosphorylated).

[0016] In a further aspect, the therapeutic polypeptide encoded by the alphaviral encrypted RNA can be a secreted polypeptide, optionally an antibody. The therapeutic polypeptide may be one selected from the group consisting of an interferon, an interferon-stimulated gene product, a cytokine, a chemokine, an antibody, a signaling molecule, a cytotoxic protein, a protein that causes cell death, an antineoplastic protein, an immunomodulatory protein, a protein toll-like receptor agonist, and a dominant negative protein, optionally wherein the cytokine is (i) an inflammatory cytokine, optionally TNF-o, or (ii) an anti-inflammatory cytokine, optionally an interleukin- 1 receptor antagonist (IL-1RN), or wherein the therapeutic polypeptide is an interleukin, optionally IL-12A, IL-12B, or IL-2, or wherein the therapeutic polypeptide is a caspase, or wherein the therapeutic polypeptide is an interferon, optionally an IFN-α, IFN-p, IFN-e, IFN-K, IFN-ω, IFN-y, or IFN-X, further optionally IFN-al, IFN-a2, IFN- a4, IFN-a5, IFN-a6, IFN-a7, IFN-a8, IFN-alO, IFN-al3, IFN-al4, IFN-al6, IFN-al7, IFN-a21, IFN-β1, IFN-e, IFN-K, IFN-col, IFN-y, IFN-X1 (IL28A), IFN- X2 (IL28B), IFN- X3 (IL29), or IFN- X4. The coding sequence of the alphaviral encrypted RNA may encode two or more therapeutic polypeptides, which are separated by one or more ribosomal skipping sequences, or the coding region may further comprise one or more regulatory elements selected from the group consisting of a ribosomal binding site, a Kozak sequence, a Shine-Dalgamo sequence, a ribozyme, a riboswitch, a promoter, a microRNA binding site, and an internal ribosomal entry site (IRES). The one or more regulatory elements optionally may be operably linked to the coding sequence. The alphaviral encrypted RNA may comprise a polyadenylation signal and / or a 3’ poly(A) tail. The alphaviral encrypted RNA may be in a linear form or a covalently-closed circular form.

[0017] Also provided is an isolated DNA polynucleotide encoding the alphaviral encrypted RNA. Also contemplated is a cell or a cell line comprising the isolated DNA polynucleotide. Further provided is a vector comprising the alphaviral encrypted RNA or the isolated DNA polynucleotide, optionally wherein the vector can be a viral vector an expression vector. Further provided is a lipid nanoparticle encapsulating the alphaviral encrypted RNA.

[0018] In a further aspect, provided is a method that may comprise administering to a subject in need thereof a therapeutically effective amount of the alphaviral encrypted RNA, the isolated DNA polynucleotide, the cell or the cell line, the vector, or the lipid nanoparticle. The method may further comprise administering a polynucleotide encoding the translation activator to initiate the translation of the therapeutic polypeptide. The subject can be a human, a cow, a pig, a sheep, a horse, a deer, a ruminant, a rodent, a fish, or a fowl. The subject may have a disease or a disorder resulting from a viral infection. Alternatively, the subject may have an infection with a virus. For the method, administering is by intratracheal or inhalation, intranasal, oral, rectal, vaginal, transmucosal, or intestinal administration; or by parenteral delivery, optionally intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections; or by intrathecal, direct intraventricular, intravenous, or intraperitoneal administration.

[0019] Each of the limitations of the compositions and methods described in this disclosure may encompass various described embodiments. It is, therefore, anticipated that each of the limitations of the disclosed compositions and methods involving any one element or combinations of elements can be included in each aspect of the invention. This present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are not intended to be drawn to scale. The drawings are illustrative only and should not be interpreted to limit any one claim. Not every component may be labeled in every drawing. In the drawings:

[0021] FIGs. 1A and IB show schematics of how some embodiments of encrypted RNAs function. FIG. 1A shows an encrypted RNA in the absence of a target-specific translation activator. FIG. IB shows an encrypted RNA in the presence of a targetspecific translation activator.

[0022] FIGs. 2A and 2B show schematics comparing an encrypted RNA with an mRNA in the presence or absence of a translation activator of the encrypted RNA. FIG. 2A shows that the level of protein translation from the mRNA is not dependent on the presence of the translation activator in a cell. In contrast, FIG. 2B shows that theactivation of the encrypted RNA is dependent on the presence of the target-specific translation activator in a cell.

[0023] FIGs. 3A, 3B, and 3C show schematics of some embodiments of therapeutic encrypted RNAs, which encode a therapeutic polypeptide of interest and for which the translation activator is provided by virus infection of a cell. FIG. 3A shows a schematic of some embodiments of therapeutic encrypted RNAs, wherein negligible levels of the therapeutic polypeptide of interest are translated in a cell in the absence of a translation activator, such as viral infection. FIG. 3B is a schematic demonstrating that, in some embodiments, viral infection of a cell in the absence of therapeutic encrypted RNA treatment can result in high levels of viral replication. FIG. 3C is a schematic showing that, in some embodiments, upon virus infection of a cell treated with a therapeutic encrypted RNA, increased translation of the therapeutic polypeptide of interest occurs. FIG. 3C also shows that in some embodiments, a therapeutic polypeptide of interest is a secreted protein, for example, a cytokine that induces an antiviral response after it binds to its receptor on the surface of a cell.

[0024] FIGs. 4A, 4B, and 4C describe and show experiments to test the level of activation of alphavirus encrypted RNA after treating cells with a target-specific translation activator comprising nsPl, nsP2, nsP3, and nsP4 of an alphavirus. FIG. 4A shows a schematic of the experiment. FIG. 4B shows that an EEEV encrypted RNA can be activated by EEEV nsPl-4. FIG. 4C shows that a VEEV encrypted RNA can be activated by VEEV nsPl-4.

[0025] FIGs. 5A & 5B describe and show experiments to test the level of activation of alphavirus encrypted RNA after treating different cell lines (FIG. 5A: BHK-21 cells; FIG. 5B: Vero cells) with a target-specific translation activator comprising nsPl, nsP2, nsP3, and nsP4 of an alphavirus.

[0026] FIGs. 6A and 6B show that activation of an EEEV encrypted RNA, encoding an antiviral cytokine (human interferon beta, hIFN-P), by EEEV nsPl-4 can lead to secreted levels of interferon that are sufficient to control viral infection, as measured by supernatant transfer to a (surrogate) RSV infection. FIG. 6A shows that only an EEEV encrypted RNA encoding an antiviral payload (i.e., hIFN-P) yields the antiviral effect when activated in BHK-21 cells. FIG. 6B shows the same, but when treatment was performed in Vero cells.

[0027] FIG. 7 A shows the cross-activation of alphavirus encRNA by alphavirus translation activators (nsPl, nsP2 nsP3 nsP4) FIG 7B displays a phylogenetic treecomputed from a whole genome alignment of viral species within the alphavirus genus. This computed phylogenetic tree indicates that VEEV, WEEV, EEEV, and SINV are more closely related to each other than to SFV.

[0028] FIGs. 8A and 8B show that alphaviral encrypted RNAs encoding an antiviral polypeptide of interest (the cytokine human interferon beta) have antiviral activity in Vero E6 cells against EEEV or VEEV, reducing virus levels by >3000 x with a single treatment at 100 pM concentration. In contrast, analogous alphaviral encrypted RNAs that encode a non-antiviral polypeptide of interest (Gaussia luciferase) (“sham encrypted RNA”) have negligible antiviral activity. FIG. 8A shows that an EEEV encrypted RNA encoding human interferon beta has antiviral activity against EEEV (strain V105). FIG. 8B shows that a VEEV encrypted RNA encoding human interferon beta has antiviral activity against VEEV (strain INH9813).DETAILED DESCRIPTIONDefinitions:

[0029] So that the present disclosure can be more readily understood, certain terms are first defined. As used in this specification, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the specification.

[0030] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. If “or” is used with “and” as in “and / or”, then the conjunctions are interpreted as either “and” or “or”.

[0031] As used herein, the indefinite articles “a”, “an”, or “some" should be understood to refer to “one or more" of any recited or enumerated component. As such, the terms “a”, “an”, “some”, “one or more”, and “at least one” can be used interchangeably.

[0032] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has," “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has" or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps.Similarly, any composition that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0033] The use of any examples, or exemplary language (e.g., “such as”) provided herein is intended to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. This disclosure is not limited to the particular methodology, protocols, reagents, etc., described herein, which can be modified as would be known in the art. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure. The terminology used herein is to describe particular embodiments and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the CONCISE DICTIONARY OF BIOMEDICINE AND MOLECULAR BIOLOGY, JUO, Pei-Show, 2nded., 2002, CRC Press; THE DICTIONARY OF CELL AND MOLECULAR BIOLOGY, 5thed., 2012, Elsevier; TABER’S CYCLOPEDIC MEDICAL DICTIONARY, 23rded., (2017), and the OXFORD DICTIONARY OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, 2nd ed., 2008, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0035] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges include the numbers that define the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0036] The terms “about”, “substantially”, “approximately”, or “comprising essentially of’ refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, e.g., on the limitations of the measurement system. For example, “about”, “substantially”, “approximately", or “comprising essentially of’ can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about,” “substantially”, “approximately”, or “comprising essentially of’ can mean a range of up to 20%. Furthermore, for biological systems or processes, the terms can mean up to 5-fold or 10-fold of a value. When particular values or compositions are provided in theapplication and claims unless otherwise stated, the meaning of “about”, “substantially”, “approximately”, or “comprising essentially of’ should be assumed to be within an acceptable scientific error range for that particular value or composition.

[0037] As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth or one-hundredth of an integer), unless otherwise indicated. In addition, all ranges are intended to expressly include the boundaries of the range individually. For clarity, the range 3-6 is intended to include individually 3, 4, 5, and 6 as well as any fraction (e.g., about 0.1 fraction) within that range.

[0038] As used herein, a “target-specific translation activator” is one or more polypeptides that direct synthesis of a coding region of an encrypted RNA, which coding region encodes a polypeptide of interest that is translated at increased levels when the target-specific translation activator contacts the encrypted RNA. As used herein, “translation activator” means “target-specific translation activator”. In some embodiments, the target-specific translation activator is a polymerase. In some embodiments, the polymerase is an RNA-dependent RNA polymerase. In some embodiments, the polymerase is an RNA-dependent DNA polymerase.

[0039] As used herein, an “encrypted RNA” is an isolated ribonucleic acid (RNA) polynucleotide, comprising: (a) a “coding region” that encodes a polypeptide of interest; and (b) “template regions” for binding a target-specific translation activator; wherein the target-specific translation activator directs transcription of mRNA that is distinct from the isolated RNA, and wherein translation of the polypeptide of interest is increased in a cell containing said RNA polynucleotide when the RNA polynucleotide is contacted in said cell with the target- specific translation activator. As used herein, a “polypeptide of interest” or “protein of interest” is a polypeptide encoded within a coding region of an encrypted RNA according to the invention.

[0040] The template regions are comprised of two distinct regions, a left flanking region (“L region”) of a virus and a right flanking region (“R region”) of the virus. The L region is 5' to and contiguous with the coding region and the R region is 3' to and contiguous with the coding region. Examples of the L and R regions of various viruses and variants thereof are provided in the sequence listing and the examples herein. In some embodiments, the L and the R regions of a virus each do not contain a polynucleotide sequence encoding a polypeptide In some embodiments the L or the Rregion can contain a polynucleotide sequence encoding a polypeptide, wherein the polypeptide is homologous to the virus. If the L and / or the R region contain(s) a polynucleotide sequence, then that polynucleotide sequence contributes to the interaction of the L or R region, as appropriate, with the translation activator.

[0041] A coding region comprises one or more coding sequences. In addition, a coding region may contain one or more non-coding sequences. Typically, a coding region contains a 5' untranslated region (5' UTR), a coding sequence, and a 3' untranslated region (3' UTR).

[0042] A “coding sequence” is a sequence of nucleotides that encodes the complete amino acid sequence of at least one polypeptide. As used herein, “a polypeptide of interest" is a polypeptide encoded by the coding sequence of a coding region. In some embodiments, the coding sequence of a coding region encodes a polypeptide that is heterologous to the virus from which the L and R regions of the encrypted RNA are derived. As used herein, “heterologous to the virus” means the coding sequence encodes a polypeptide that is not found in the species of the virus from which the L and R regions are obtained. As used herein, “homologous to the virus” means the coding sequence encodes a polypeptide or non-coding domain that is found in the same species of the virus as the L and R regions. Classification of species is according to internationally accepted standards established by the International Committee on Taxonomy of Viruses (“ICTV”). The coding sequence is comprised of a series of three-nucleotide units, known as codons. The first three nucleotides of a coding sequence, the “start codon”, initiate translation of the polypeptide(s) of interest and typically encode for methionine or N-formylmethionine. An exemplary start codon is “atg”. The final three nucleotides of a coding sequence, the “stop codon”, encode a stop codon or termination codon, which terminates translation elongation of the polypeptide(s) of interest. Some examples of stop codons are “tag” (amber stop codon), “taa” (ochre stop codon), and “tga” (opal stop codon).

[0043] A “non-coding sequence” is a contiguous sequence of nucleotides that does not contain a coding sequence. Non-coding sequences can be used to alter the expression of a polypeptide of interest. Some examples of non-coding sequences are a 5 -UTR, a 3'- UTR, promoters, introns, ribozymes, riboswitches, ribosome binding sites, Kozak sequences, Shine-Dalgamo sequences, Internal Ribosomal Entry Site(s) (IRES), polyadenylation signals, poly- A sequences, microRNA binding sites, and other regulatory elements. As mentioned above in some embodiments either or both of the L and the Rregions consist of non-coding sequences. In some embodiments, the L or the R region can contain a polynucleotide sequence encoding a polypeptide, which polypeptide is homologous to the virus.

[0044] A “5 -UTR of a coding sequence” or “5' untranslated region of a coding sequence” is a non-coding sequence located adjacent to and contiguous with the 5' start codon of a coding sequence. When a coding sequence is the first coding sequence 3' of an L region, the 5 -UTR of the coding sequence begins at the first nucleotide of the first 5' non-coding sequence in the coding region and ends one nucleotide before the start codon of the coding sequence. If there are two or more coding sequences in the coding region, then the coding sequences can be separated by untranslated regions. When a coding sequence is not the first coding sequence 3' of an L region, there can be a second 5'-UTR for the second coding sequence, which separates the coding sequences from one another. The S'-UTR of a coding sequence may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites, Kozak sequences, Shine-Dalgamo sequences, ribozymes, riboswitches, promoters, microRNA binding sites, or IRES elements.

[0045] A “3 -UTR of a coding sequence” or “3' untranslated region of a coding sequence” is a non-coding sequence located adjacent to and contiguous with the 3' stop codon of a coding sequence. When a coding sequence is the first coding sequence adjacent to the 5' end of an R region, the 3'-UTR of the coding sequence begins at the first nucleotide following the stop codon of the coding sequence and terminates at the last 3' nucleotide of the coding region before the 5' end of the R region. If there are two or more coding sequences in the coding region, then the first and the second coding sequences can be separated by untranslated regions. A first 3 -UTR of the first coding sequence can separate the first coding sequence from the following adjacent coding sequence nearer the R region. The 3'-UTR of a coding sequence may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribozymes, micro RNA binding sites, poly(A) sequences, and polyadenylation signals.

[0046] Translation of a “polypeptide of interest” or “protein of interest” in an encrypted RNA of the invention is increased when the encrypted RNA contacts a target-specific translation activator of the encrypted RNA that encodes said polypeptide or protein of interest.

[0047] The polypeptide of interest or protein of interest can be a “therapeutic polypeptide”. A therapeutic polypeptide, exemplified in greater detail below, is a polypeptide that treats or ameliorates one or more symptoms of a disease or condition in a subject. In some embodiments, the treatment is of an existing condition in said subject. In some embodiments, the treatment is a prophylactic treatment for a subject. In some embodiments, the therapeutic polypeptide encoded by an encrypted RNA is heterologous to the virus from which the L and R regions of the encrypted RNA are derived for use in a subject or for the manufacture of a medicament for use in a subject. In some embodiments, the coding sequence for a therapeutic polypeptide does not naturally occur in the same position in a viral genome. In some embodiments, the therapeutic polypeptide can be an immunomodulatory protein, such as a human immunomodulatory protein known to exert an activity on the human immune system or in the immune system of another subject (e.g., a domesticated animal or farmed species of animal). Examples of immunomodulatory proteins include proteins such as a chemokine, a cytokine, an interleukin, a factor (e.g., hormones, growth factors, blood factors), an antibody, or an immune checkpoint inhibitor. A therapeutic polypeptide in some embodiments is a native human protein or an analog of a human protein (e.g., a truncated version of the protein or variant of the protein having one or more amino acid substitutions). In some embodiments, the therapeutic polypeptide is an antigen, such as a cancer antigen or an antigen present in a virus.

[0048] A “therapeutic polypeptide of interest”, a “therapeutic polypeptide”, or a “therapeutic protein” (the terms can be used interchangeably) has an advantageous effect on the condition or disease state of a subject when administered to the subject in a therapeutically effective amount. In some aspects, a therapeutic polypeptide has one or more curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay the onset of, or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic polypeptide may have prophylactic properties and can be used to delay the onset of a disease or infection or to lessen the severity of such disease, infection, or other pathological condition. The term therapeutic polypeptide includes full-length / mature proteins or polypeptides as well as their precursors and can also refer to active fragments thereof; “therapeutic polypeptide” can also include active analogs of a peptide or protein. A pharmaceutically active peptide or protein can also be referred to as a therapeutic peptide or protein.

[0049] In some embodiments, the polypeptide of interest is a polypeptide that when administered to a particular subject, does not provoke or induce a medically significant antigen-specific response to the polypeptide of interest. In some embodiments, the polypeptide of interest is a polypeptide that, when administered to a particular subject, provokes or induces a medically significant antigen-specific response to the polypeptide of interest. In some embodiments, the polypeptide of interest can be a reporter polypeptide. Examples of reporter polypeptides are provided in the Examples and are well known to those of ordinary skill in the art.

[0050] As used herein, “activation” or “activate” describes the process or action or series of processes or series of actions by which translation of a polypeptide of interest is increased when an encrypted RNA encoding the polypeptide of interest is contacted by a translation activator of the encrypted RNA. As used herein, an encrypted RNA is said to be “activated” by contact with a translation activator.

[0051] As shown in FIGs. 1A and IB and FIGs. 2A and 2B, in some embodiments, the contact between an encrypted RNA and a translation activator increases the translation of the polypeptide of interest.

[0052] As used herein, an “encrypted protein” or an “encrypted polypeptide” is a polypeptide of interest encoded by an encrypted RNA.

[0053] As used herein, a “therapeutic encrypted RNA” is an encrypted RNA wherein the coding region encodes a therapeutic polypeptide. As used herein, “SHIELD”, “SHIELD RNA", or “SHIELD encrypted RNA” have the same meanings as “therapeutic encrypted RNA”, and can be used interchangeably.

[0054] As used herein, a “DNA-encoded encrypted RNA” is a DNA sequence that encodes an encrypted RNA cassette.

[0055] As used herein, an “encrypted nucleic acid” means an encrypted RNA or a DNA-encoded encrypted RNA.

[0056] As used herein, “antisense encrypted RNA” means that the coding region that encodes the polypeptide of interest is positioned in an antisense orientation to the encrypted RNA sequence. As used herein, the phrase “negative-sense encrypted RNA” and “(-)-sense encrypted RNA” are equivalent to the phrase “antisense encrypted RNA”.

[0057] As used herein, “sense encrypted RNA” means that the coding region that encodes the polypeptide of interest is positioned in a sense orientation to the encryptedRNA sequence. As used herein, the phrase “positive-sense encrypted RNA” and “(+)- sense encrypted RNA” are equivalent to the phrase “sense encrypted RNA”.

[0058] As used herein, an “alphaviral encrypted RNA” is an encrypted RNA with a target-specific translation activator comprising an alphavirus polypeptide. An encrypted RNA with a target-specific translation activator comprising an alphavirus polypeptide means the encrypted RNA is activated by an alphavirus polypeptide.

[0059] As used herein, a “therapeutic alphaviral encrypted RNA” or an “alphaviral SHIELD” is an alphaviral encrypted RNA that is a therapeutic encrypted RNA.

[0060] As used herein, an “alphaviral antisense encrypted RNA”, an “alphaviral negative-sense encrypted RNA”, or an “alphaviral (-)-sense encrypted RNA” is an alphaviral encrypted RNA that is an antisense encrypted RNA.

[0061] As used herein, an “alphaviral sense encrypted RNA”, an “alphaviral positivesense encrypted RNA”, or an “alphaviral (+)-sense encrypted RNA” is an alphaviral encrypted RNA that is a sense encrypted RNA.

[0062] As used herein, a carrier or polymeric carrier is typically a compound that facilitates the transport or complexation of another compound (e.g., cargo). A polymeric carrier is typically a carrier that is formed of a polymer. A carrier may be associated with its cargo by covalent or non-covalent interaction. A carrier may transport nucleic acids, e.g., RNA or DNA, to the target cells. The carrier may, for some embodiments, be a cationic component.

[0063] The term “cationic component” typically refers to a charged molecule, which is positively charged (cation) at a pH value typically from about 1 to about 9.Accordingly, a cationic component may be any positively charged compound or polymer, such as a cationic peptide, protein, or lipid, that is positively charged under physiological conditions, such as those that occur in vivo. A “cationic peptide or protein" may contain at least one positively charged amino acid, or more than one positively charged amino acid, e.g., selected from Arg, His, Lys, or Asn. Accordingly, “polycationic” components are also within the scope of exhibiting more than one positive charge under the given conditions.

[0064] The term “subject" refers to an animal, for example, a human, to whom treatment, including prophylactic treatment, with methods and compositions described herein, is provided. For treatment of those conditions or disease states specific to a specific animal, such as a human subject, the term “subject" refers to that specific animal. Cells, tissues, and cells derived from a biological entity’s (subject’s) tissueobtained in vivo or cultured in vitro are also included. In addition to humans, subjects include all domesticated, research, and / or agricultural animals such as dogs, cats, cows, pigs, sheep, horses, camels, llamas, sheep, goats, deer, other ruminants, primates or monkeys, rodents (e.g., mice, rats, guinea pigs), fish (e.g., salmon, tilapia), and fowl (e.g., chickens, turkeys, and ducks).

[0065] The term “tissue” refers to a group or layer of similarly specialized cells which together perform certain special functions.

[0066] “Gene therapy” may typically be understood to mean a treatment of a subject’s body or isolated elements of a subject’s body, for example, isolated tissues / cells, by nucleic acids encoding a peptide or protein. It typically may comprise at least one of the steps of a) administration of a nucleic acid directly to the patient — by whatever administration route — or in vitro to isolated cells / tissues of the patient, which results in transfection of the patient’s cells either in vivo, ex vivo, or in vitro; b) transcription or translation of the introduced nucleic acid molecule; and optionally c) re-administration of isolated, transfected cells to the patient, if the nucleic acid has not been administered directly to the patient. The term “gene therapy,” as used herein, is meant to include treatment as well as prevention or prophylaxis of a disease.

[0067] As used herein, it is understood that RNA polynucleotides are comprised of ribonucleotide monomers and that DNA polynucleotides are comprised of deoxyribonucleotide monomers. As ribonucleotides are nucleotides and deoxyribonucleotides are nucleotides, the leading “ribo” or “deoxyribo” can be omitted when the meaning is clear. As an example, “an RNA polynucleotide comprised of nucleotides” has the same meaning as “an RNA polynucleotide comprised of ribonucleotides”. Likewise, “a DNA polynucleotide comprised of nucleotides” has the same meaning as “a DNA polynucleotide comprised of deoxyribonucleotides”.

[0068] “RNA" is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule or polynucleotide, i.e., a polymer consisting of ribonucleotides (nucleotides). These nucleotides are usually adenosine monophosphate (AMP), cytidine monophosphate (CMP), guanosine-monophosphate (GMP), and uridine monophosphate (UMP) monomers, which are connected to each other along a so-called backbone or phosphodiester backbone. When the meaning is clear, RNA polynucleotides may be said to be comprised of their nucleotide triphosphates, e.g., adenine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), or uridine triphosphate (UTP) indicating that an RNA polynucleotide wassynthesized or transcribed using nucleotide triphosphate monomers to form a usual RNA polynucleotide.

[0069] The backbone is formed by phosphodiester bonds between the sugar, i.e., ribose, of a first monomer and a phosphate moiety of a second, adjacent monomer. The specific succession of the monomers is called the RNA sequence. Usually, RNA may be obtainable by transcription of a DNA sequence, e.g., inside a cell. In eukaryotic cells, transcription generally occurs inside the nucleus or the mitochondria. In vivo transcription of DNA usually results in the so-called premature RNA, which has to be processed into so-called messenger RNA, usually abbreviated as mRNA. Processing of the premature RNA, e.g., in eukaryotic organisms, can comprise a variety of different post-transcriptional modifications such as splicing, 5 '-capping, polyadenylation, export from the nucleus or the mitochondria, and the like. The sum of these processes is also called the maturation of RNA. The mature messenger RNA usually provides the nucleotide sequence that may be translated into an amino-acid sequence of a particular polypeptide or protein. Typically, a mature mRNA comprises a 5 -UTR, an open reading frame, and a 3'-UTR. Aside from messenger RNA (mRNA), several types of RNA exist, which may be involved in the regulation of transcription or translation.

[0070] As used herein, “nucleoside-modified" means that an RNA polynucleotide is comprised of at least one nucleotide that is not AMP, CMP, GMP, or UMP.

[0071] As used herein, the terms “nucleoside-modified RNA”, “nucleoside-modified encrypted RNA”, “nucleoside-modified therapeutic encrypted RNA" or “nucleoside- modified SHIELD”, or “nucleoside-modified mRNA” refer to RNA molecules containing one, two, or more than two nucleoside modifications compared to adenosine (A) ((2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol), guanosine (G) (2-amino-9-[3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-3H-purin-6- one), cytidine (C) (4-amino-l-[3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2- yl]pyrimidin-2-one), or uridine (U) (l-[(3R,4S,5R)-3,4-dihydroxy-5- (hydroxymethyl)oxolan-2-yl]pyrimidine-2, 4-dione), or compared to AMP, GMP, CMP, or UMP respectively, in RNA molecules, or a portion thereof. Non-limiting examples of nucleoside modifications are provided elsewhere in this specification. Where the nucleotide sequence of a particular claimed RNA is otherwise identical to the sequence of a naturally-existing RNA molecule, the nucleoside-modified RNA is understood to be an RNA molecule with at least one modification different from those existing in the natural counterpart. The difference can be either in the chemical change to thenucleoside / nucleotide. In some embodiments, a nucleoside-modified RNA includes at least one UMP that is modified to form Nl-methyl-pseudo-UMP (Nl- methylpseudouridine). In some embodiments, a nucleoside-modified RNA includes at least one UMP that is modified to form pseudo-UMP (pseudouridine). In a nucleoside- modified RNA, not all nucleosides need to be modified. In some embodiments, between about 10% and 100% of UMP nucleotides within a nucleoside-modified RNA are replaced with pseudo-UMP or with Nl-methyl-pseduo-UMP. In some embodiments, about 10%, 20%, 30%, 40%, 50%, 60%, and about 70% of UMP nucleotides within a nucleoside-modified RNA are replaced with pseudouridine or Nl- methyl-pseduo-UMP. In some embodiments, between about 10% and about 35% of UMP nucleotides within a nucleoside-modified RNA are replaced with pseudo-UMP or Nl-methyl-pseduo-UMP. In some embodiments, about 100% of UMP nucleotides within a nucleoside-modified RNA are replaced with pseudo-UMP or Nl-methyl- pseudo-UMP. In some embodiments, about 70% of UMP nucleotides within a nucleoside-modified RNA are replaced with pseudo-UMP nucleotides. In some embodiments, about 100% of UMP nucleotides within a nucleoside-modified RNA are replaced with pseudo-UMP nucleotides. In some embodiments, a nucleoside-modified RNA includes at least one AMP modified to form N6-methyl-AMP. In some embodiments, a nucleoside-modified RNA includes at least one CMP modified to form s-methyl-CMP. In some embodiments, a nucleoside-modified RNA includes at least one UMP modified to form 5-methoxy-UMP.

[0072] As used herein, “capped RNA” or “5 '-capped RNA” refers to RNA molecules incorporating a Cap structure at their 5' end. Cap structures are present on the 5 '-end of many mRNAs in eukaryotic organisms as well as on the viral RNA of some viruses.

[0073] Naturally occurring Cap structures typically comprise a riboguanosine residue that is methylated at position N7 of the guanine base. This N7-methylguanosine (m7G) is linked via a 5'- to 5 '-triphosphate chain at the 5'-end of the mRNA molecule. 5'- capping of RNA can facilitate resistance to degradation by exonucleases and facilitate the transport of mRNAs from the nucleus to the cytoplasm. Naturally occurring examples of Cap structures include Cap 0, Cap 1, and Cap 2. When the only capping modification is an N7-methylguanosine linked to the terminal nucleotide of the RNA via a 5 '-to-5 '-triphosphate linkage, the structure is referred to as Cap 0. When the RNA additionally incorporates a 2'-O-methylation of only the first nucleoside 5' of Cap 0 (i.e., the penultimate nucleoside of the RNA inclusive of m7G) the structure is referredto as Cap 1. When the RNA additionally incorporates 2'-O-methylation of the first two nucleosides 5' of Cap 0 (i.e., both the penultimate and the antepenultimate nucleoside, inclusive of m7G), the structure is referred to as Cap 2.

[0074] Cap 0 (3'-0-Me) is Cap 0 in which the 3' -OH (i.e., 3' hydroxyl group) of the 5'N7-methylguanosine (m7G) cap of Cap 0 is replaced by -OCH3 (i.e., 3' methoxy group). Similarly, Cap 1 (3'-0-Me) and Cap 2 (3'-0-Me) are Cap 1 and Cap 2 structures, which include a 3'-O-methylation of the 5' N7-methylguanosine (m7G) cap relative to the respective Cap 1 or Cap 2.

[0075] In some embodiments, a capped RNA contains a 5 '-Cap structure that is selected from the group consisting of a Cap 0, a Cap 0 (3'-0-Me), a Cap 1, a Cap 1 (3'- O-Me), a Cap 2, a Cap 2 (3'-0-Me), an Anti-Reverse Cap Analog (ARCA), an inosine, an Nl-methyl-guanosine, a 2'-fluoro-guanosine, a 7-deaza-guanosine, an 8-oxo- guanosine, a 2-amino-guanosine, a locked nucleic acid guanosine (LNA-guanosine), and a 2-azido-guanosine structure, or selected from any combination or subcombination thereof. In other embodiments, a 5 '-Cap structure can be selected from the group consisting of a Cap 1, a Cap 1 (3'-0-Me), a Cap 2, a Cap 2 (3'-0-Me), and an AntiReverse Cap Analog (ARCA), or selected from any combination or subcombination thereof. All of these represent nucleoside-modified RNA molecules.

[0076] As used herein, “uncapped RNA” or “noncapped RNA" refers to RNA molecules that lack a 5 '-Cap.

[0077] As used herein, “5 '-phosphorylation” refers to the number of consecutive phosphate molecules attached to the 5 '-end of uncapped RNA. RNA molecules that are “triphosphorylated” or “5 '-triphosphorylated” are uncapped and have a 5 '-terminal triphosphate (3 phosphates). RNA molecules that are “5 '-diphosphorylated” or “5 - biphosphorylated” are uncapped and have a 5 '-terminal diphosphate (2 phosphates). RNA molecules that are “monophosphorylated” or “5 '-monophosphorylated” are uncapped and have a 5 '-terminal monophosphate or 5 '-terminal phosphate (1 phosphate). RNA molecules that are “nonphosphorylated” or “5 '-nonphosphorylated" have no 5' terminal phosphate (zero phosphate or dephosphorylated).

[0078] A “polymerase” generally refers to a molecular entity capable of catalyzing the synthesis of a polymeric molecule from monomeric building blocks. An “RNA polymerase” is a molecular entity capable of catalyzing the synthesis of an RNA molecule from ribonucleotide building blocks. A “DNA polymerase” is a molecular entity capable of catalyzing the synthesis of a DNA molecule from deoxyribonucleotidebuilding blocks. In the case of DNA polymerases or RNA polymerases, the molecular entity is typically a protein or an assembly or complex of multiple proteins. A DNA polymerase synthesizes a DNA molecule based on a template nucleic acid, which is typically a DNA molecule. Some DNA polymerases are RNA-dependent DNA polymerases and synthesize DNA molecules based on template nucleic acids. Some RNA-dependent DNA polymerases are termed “reverse transcriptases”. Typically, an RNA polymerase synthesizes an RNA molecule based on a template nucleic acid, which is either a DNA molecule (in that case, the RNA polymerase is a DNA- dependent RNA polymerase, DdRP), or an RNA molecule (in that case the RNA polymerase is an RNA-dependent RNA polymerase, RdRP).

[0079] “RNA-dependent RNA polymerases” or “RdRPs” are multi-domain (a and P) proteins that catalyze RNA-template-dependent formation of phosphodiester bonds between ribonucleotides in the presence of divalent metal ions. The initiation of synthesis occurs at the 3 '-end of the template in a primer-dependent or independent manner and proceeds on the synthesized strand in the 5' — » 3' direction (i.e., sense). The average length of the core RdRP domain is less than 500 amino acids and is folded into three subdomains. The active sites of RdRPs from different RNA viruses are conserved and show resemblances to those of other enzymes, such as reverse transcriptases and DNA polymerases, indicating their similar role in nucleotidyl transfer reactions.

[0080] Some viral polymerases possess additional domains, such as a methyltransferase or an endonuclease domain, to carry out functions associated with RNA synthesis. The polymerase domain may also interact with other host factors for efficient polymerization and to discriminate activities such as genome replication and mRNA transcription. Host factors can include translation factors, protein chaperones, RNA-modifying enzymes, or other cellular proteins. These factors together with the RdRPs, constitute the viral replication complexes (VRCs). The VRCs differ in their composition, subcellular location, and interaction with the viral RNA templates.

[0081] As defined herein, a “ribozyme” is a catalytic macromolecular complex comprising an RNA with catalytic activity. Examples of ribozymes include but are not limited to: an RNA molecule with a self-splicing intron sequence, an RNA molecule comprised of the Hepatitis Delta Virus (HDV) antigenomic ribozyme, an RNA molecule comprised of a “Hammerhead” ribozyme, or a two-component ribonucleoprotein system comprising a guide RNA (gRNA) complexed with a Cas protein (“CRISPR-Cas”). RNA molecules comprising a ribozyme with nucleaseactivity may cleave within the molecule in which they are embedded or may cleave RNA outside of the molecule in which they are embedded.

[0082] As used herein, “sequence identity”, is used to mean a relationship between two or more protein (polypeptide) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Two or more sequences are identical if they exhibit the same length and order of nucleotides or amino acids. Calculation of the percent identity (or % identity) of two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, the length of a sequence aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the length of a reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the molecules are considered identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences while accounting for the number of gaps and the length of each gap, which is necessary to achieve optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using an algorithm. For example, the percent identity between two nucleotide sequences or two polypeptide sequences can be determined using methods such as those described in Y. Zang, COMPUTATIONAL MOLECULAR BIOLOGY, Oxford Academic Press, 2006; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W„ ed., Academic Press, New York, 1994; BIOINFORMATICS: SEQUENCES, STRUCTURES, PHYLOGENY, Asheesh Shanker ed.. Springer, 2018; BIOINFORMATICS: VOLUME II: STRUCTURE, FUNCTION AND APPLICATIONS, Jonathan M. Keith ed., Springer Science and Business, New York, 2017; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; ADVANCES IN SEQUENCE ANALYSIS: THEORY, METHOD, APPLICATIONS, Philippe Blanchard et al. eds., Springer Cham, New York 2014; COMPUTER ANALYSIS OF SEQUENCE DATA, Part I, Griffin, A. M„ and Griffin, H. G„ eds., Humana Press, NewJersey, 1994; and SEQUENCE ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; each of which is incorporated herein by reference.

[0083] Polynucleotide or polypeptide sequences can be compared by performing a sequence alignment, which may be gapped or ungapped. In an ungapped alignment, two or more sequences are compared as “contiguous” sequences, i.e., one sequence is aligned with the other sequence, and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. In an ungapped alignment, in an otherwise identical pair of sequences, one insertion or deletion may cause the other nucleotide or amino acid residues to be put out of alignment, thus resulting in a potentially non-optimal global alignment. In a gapped alignment, sequences are compared “non-contiguously”, and insertions and deletions (collectively referred to generally as “gaps") may be inserted to optimally align the sequences.

[0084] As used herein, “sequence similarity", is used like “sequence identity", but captures aspects of relatedness between two sequences, such as functional or phenotypic relatedness, that may not be fully explained by methods to determine sequence identity.

[0085] Methods to determine sequence identity and similarity are codified in publicly available algorithms or software, and can include but are not limited to: BLAST, PASTA, T-COFFEE, or M-COFFEE. In some methods, a scaled similarity score matrix or equivalent can be used to assign a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix — the default matrix for the BLAST suite of programs. There are also alternative computational methods used to determine identity or similarity (e.g., INFERNAL or R-COFFEE) which also consider aspects of sequence relatedness (e.g., covariance models, secondary structure, or tertiary structure) in addition to the primary sequence (Eddy & Durbin, Nucleic Acids Research (1994); DOI: 10.1093 / nar / 22.11.2079) (Rivas et al., Bioinformatics (2020); DOI: 10.1093 / bioinformatics / btaa080) (Nawrocki & Eddy; Bioinformatics (2013); DOI: 10.1093 / bioinfonnatics / btt509).

[0086] In some embodiments, encrypted RNAs with different template regions can be activated by the same translation activator. Therefore, template regions may share a common structure and function although their primary nucleotide sequences differ i.e.,template regions of the same translation activator may be non-identical but similar sequences.

[0087] In some embodiments, an encrypted RNA with a variant template region will have the same or similar activation in the presence or absence of a translation activator as an encrypted RNA with a reference template region. Alternatively, an encrypted RNA with the variant template region may have altered activation (e.g., increased or decreased) relative to the encrypted RNA with a reference template region. Generally, the variant template region will have similarity or identity to the reference template region of at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. As used herein, a “variant” of a nucleotide sequence is one that has less than 100% identity due to a substitution of at least one nucleotide for another, an addition of one or more nucleotides, or a deletion of one or more nucleotides relative to a reference sequence. As used herein, a “variant” of a polypeptide sequence is one that has less than 100% identity due to a substitution of at least one amino acid for another, an addition of one or more amino acids, or a deletion of one or more amino acids relative to a reference sequence.

[0088] In some embodiments, two different translation activators can activate the same encrypted RNA. Therefore, translation activators may share a common structure and function, although their primary polypeptide sequences differ.

[0089] In some embodiments, a translation activator comprising a variant polypeptide will similarly activate an encrypted RNA as a translation activator comprising a reference polypeptide. Alternatively, a translation activator comprising a variant polypeptide may have altered activation of an encrypted RNA (e.g., increased or decreased) relative to a translation activator comprising a reference polypeptide. Generally, the variant polypeptide will have similarity or identity to the reference template region of at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.

[0090] A “stabilized nucleic acid molecule” is a nucleic acid molecule, typically a DNA or RNA molecule, that is modified such that it is more stable to disintegration or degradation, e.g., by environmental factors or enzymatic digestion such as by exo- or endonuclease degradation / digestion than the nucleic acid molecule without the modification. In some embodiments, a stabilized nucleic acid molecule is stabilized against degradation in a cell, such as a prokaryotic or eukaryotic cell. In some further embodiments, a stabilized nucleic acid molecule is stabilized against degradation in a mammalian cell, such as a human cell. The stabilization effect may also be exerted outside of cells, e.g., in a buffer solution, etc., for example, in a manufacturing process for a pharmaceutical composition comprising the stabilized nucleic acid molecule.

[0091] The term “transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, such as eukaryotic cells. In the context of the present invention, the term “transfection” encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, such as mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g. based on cationic lipids or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine, etc.

[0092] The term “vector” refers to a nucleic acid molecule that is suitable for incorporating or harboring a desired nucleic acid sequence, such as a nucleic acid sequence comprising an open reading frame. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. A “storage vector” is a vector that allows the convenient storage of a nucleic acid molecule, such as an mRNA molecule. Thus, the vector may comprise a sequence corresponding, e.g., to a desired mRNA sequence or a part thereof, such as a sequence corresponding to the coding sequence and the 3 -UTR of an mRNA. An “expression vector” may be used for the production of expression products, such as RNA, encrypted RNA, mRNA, peptides, polypeptides, or proteins. An expression vector may comprise sequences required to transcribe a sequence stretch of the vector, such as a promoter sequence, e.g., an RNA polymerase promoter sequence. A “cloning vector" is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector. A cloning vector may be a plasmid vector or a bacteriophage vector. A “transfer vector” can include a vector suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors In some embodiments the viral vector can bea lenti viral vector (HIV-1, HTLV, SIV, etc., especially recombinant forms of such vectors). A vector in the context of the present invention may be, e.g., an RNA vector or a DNA vector. In some embodiments, the vector is a DNA molecule. In some embodiments, the vector comprises a cloning site, a selection marker (e.g., an antibiotic resistance factor), and a sequence suitable for the multiplication of the vector, such as an origin of replication. In some embodiments, the vector is a plasmid vector.

[0093] A “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in being able to infect non-dividing cells, whereas non- lentivirus retroviruses can only transduce cells during mitosis; lentiviruses can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0094] As used herein, a “lentiviral vector” is a viral vector derived from a lentivirus. See Munis, “Gene Therapy Applications of Non-human Lentiviral Vectors," Viruses 12(10) doi.org / 10.3390 / vl2101106.

[0095] A “vehicle" is typically understood to be a material that is suitable for storing, transporting, or administering a compound, such as a pharmaceutically active compound. For example, the vehicle may be a physiologically acceptable liquid, which is suitable for storing, transporting, or administering a pharmaceutically active compound.Encrypted RNAs

[0096] In some embodiments, an encrypted RNA is a ssRNA (single-stranded RNA).

[0097] In some embodiments, an encrypted RNA is a capped ssRNA.

[0098] In some embodiments, an encrypted RNA is an uncapped ssRNA.

[0099] In some embodiments, an encrypted RNA is a 5 '-triphosphorylated uncapped ssRNA.

[0100] In some embodiments, an encrypted RNA is a 5 '-diphosphorylated uncapped ssRNA.

[0101] In some embodiments, an encrypted RNA is a 5 '-monophosphorylated uncapped ssRNA.

[0102] In some embodiments, an encrypted RNA is a 5 '-nonphosphorylated uncapped ssRNA.

[0103] In some embodiments, an encrypted RNA is an uncapped ssRNA with four or more 5 '-terminal phosphates.

[0104] In some embodiments, an encrypted RNA is a stabilized nucleic acid molecule.

[0105] In some embodiments, an encrypted RNA is a circular RNA.

[0106] In some embodiments, the template region of an encrypted RNA or the translation activator is not derived from an alphavirus genome.

[0107] In some embodiments, an encrypted RNA is delivered to cells and is translated at low levels until it contacts a polymerase encoded by an infectious virus.

[0108] In some embodiments, an encrypted RNA is delivered to cells with a targetspecific translation activator.

[0109] In some embodiments, DNA is used to encode an encrypted RNA.

[0110] In some embodiments, an encrypted nucleic acid is a stabilized nucleic acid molecule.

[0111] In some embodiments, a DNA sequence that flanks an encrypted RNA within a DNA-encoded encrypted RNA cassette can have a desirable effect on the production of the encrypted RNA by inducing one or more outcomes, including altering the level (abundance) of encrypted RNA produced, altering the average molecular structure of the encrypted RNA, or altering the rate at which encrypted RNA is produced from the DNA template.

[0112] In some embodiments, a DNA-encoded encrypted RNA cassette can be repurposed to produce other RNA species, substituting the encrypted RNA sequence with an alternative, non-encrypted RNA sequence that encodes an RNA. In some embodiments, a DNA-encoded encrypted RNA cassette can be repurposed to encode non-encrypted RNA sequences that encode viral genetic elements. In some embodiments, a DNA-encoded encrypted RNA cassette can be repurposed to encode non-encrypted RNA sequences that are antisense to a targeted sequence. In some embodiments, a DNA-encoded encrypted RNA cassette can be repurposed to encode non-encrypted RNA sequences, which encode guide RNAs for a CRISPR-Cas system. In some embodiments, a DNA-encoded encrypted RNA cassette can be repurposed to encode non-encrypted RNA sequences, which encode mRNA sequences.

[0113] In some embodiments, the DNA encoding an encrypted RNA is delivered to cells in a viral vector.

[0114] In some embodiments, the DNA encoding an encrypted RNA is delivered to cells in a plasmid.

[0115] In some embodiments, an encrypted RNA or the DNA encoding the encrypted RNA encodes a therapeutic protein, such as an immunomodulatory protein.

[0116] In some embodiments, an encrypted RNA or a DNA encoding an encrypted RNA encodes more than one polypeptide of interest. Strategies for encoding multiple polypeptides are well-known to practitioners skilled in the art (see, e.g., Liu et al., Scientific Reports (2017) DOI: 10.1038 / s41598-017-02460-2). Such strategies include using multiple promoters, fusion proteins, proteolytic cleavage sites within polypeptides, internal ribosome entry sites, and “ribosomal skipping" 2A peptides. In some embodiments, more than one polypeptide of interest is encoded in a coding sequence that is translated as two or more polypeptides through (he action of one or more “2A” like sequences present in a coding sequence (e.g., the 2A sequence from a porcine teschovirus-1, the 2A sequence from a foot-and-mouth disease virus, the 2A sequence from an equine rhinitis A virus, or the 2A sequence from Thosea asigna virus).

[0117] In some embodiments, a target-specific translation activator comprises a polymerase. In some embodiments, a translation activator comprises an RNA- Dependent RNA Polymerase (“RdRP”) or an RNA-Dependent DNA Polymerase (“RdDP"). In some further embodiments, a translation activator comprises additional polypeptides that facilitate mRNA synthesis. In some further embodiments, the additional polypeptides can include matrix proteins, nucleoproteins, or non-structural proteins.

[0118] RNA viruses are quite diverse in virus particle and genome structure and virus entry and assembly mechanisms. However, they share fundamental features in their genome replication and transcription, often using a virally encoded RdRP to carry out the biosynthesis of an RNA product directed by an RNA template. Although the genome replication machinery frequently requires the participation of other factors, typically at the initiation phase of synthesis, the RdRP governs the elongation phase of synthesis that includes thousands of efficient nucleotide addition cycles (NACs). Viral RdRPs vary greatly in size and structural organization, from the ~50-kDa picomavirus 3Dpol to the ~100-kDa flavivirus NS5 that contains a naturally fused methyltransferase domain to the ~250-kDa RSV L protein harboring at least three enzymatic domains, to the ~260-kDa three-subunit PA-PB1-PB2 influenza virus replicase complex. On the other hand, all RdRPs share a 50- to 70-kDa polymerase core that forms a unique encircled right-hand structure with palm, fingers, and thumb domains. Among theseven classic RdRP catalytic motifs, A-E are within the most conserved palm domain, and F and G are located in the fingers; the motifs are all arranged similarly around the active site. The structural conservation of the RdRP polymerase core and the seven motifs form the basis for understanding the common features in viral RdRP catalytic mechanism and finding intervention strategies targeting these enzymes with possible broad-spectrum potential.

[0119] In some embodiments, an encrypted RNA resembles an RNA that is viral in origin, but instead of encoding a polypeptide of interest that is native to the virus, the encrypted RNA encodes a therapeutic polypeptide of interest that is not native to the virus. In this instance, the construct of the encrypted RNA encoding the therapeutic polypeptide of interest is arranged with a flanking L region and a flanking R region and the arrangement is not “native” to the virus. In some embodiments, the flanking L region and the flanking R region can be derived from two different, but closely related viruses (e.g., if recombination between the viruses is possible).

[0120] In some embodiments, an encrypted RNA resembles a viral RNA and possesses sufficient cis-acting sequences to be encapsidated into viral particles. In some further embodiments, encrypted RNA possesses sufficient cis-acting sequences to be encapsidated into viral particles that are infectious and can transmit and deliver the encrypted RNA to additional cells via viral infection. In some embodiments, the RNA species produced after an encrypted RNA is contacted by a target-specific translation activator is competent for encapsidation into viral particles. In some further embodiments, the RNA species produced after an encrypted RNA is contacted by a target-specific translation activator possesses sufficient czs-acting sequences to be encapsidated into viral particles that are infectious and can transmit and deliver the produced RNA species to additional cells via viral infection.

[0121] The negative-strand RNA viruses of animals are divided into several families and include the agents of well-known diseases such as rabies, mumps, measles, and influenza, as well as more emerging pathogens such as Ebola virus or Hendra virus. In all of these negative-strand RNA viruses, the single-stranded RNA in the virus particle is complementary to the mRNA and is, therefore, the minus strand. These viruses vary in shape and structure but are similar in having an outer envelope derived from the membrane of the host cell where they were assembled. Thus, the RNA in negativestrand RNA viruses is the antisense strand.

[0122] After infiltrating the cell, a key mission of a negative-strand RNA virus is to make its RNA double-stranded by synthesizing the corresponding positive RNA strand. Once it becomes double-stranded, it uses both RNA strands as templates. The plus strand (alternatively written as “+ strand”) is used as a template to manufacture more negative strands for the next generation of virus particles. The minus strand (alternatively written as ‘ - strand”) is used as a template to manufacture multiple positive strands that act as mRNA molecules. This strategy is not only an effective division of labor but also avoids the problem of translating multiple reading frames on a single incoming virus RNA molecule.

[0123] Positive-strand RNA viruses, also known as sense-strand RNA viruses, are viruses whose genetic information consists of a single strand of RNA that is the positive (or sense) strand, which encodes mRNA and protein. Replication in positivestrand RNA viruses proceeds through a negative-strand intermediate. Examples of positive-strand RNA viruses include coronaviruses, poliovirus (a Picornaviridae), Coxsackie virus, and echovirus.

[0124] Some RNA viruses, including all retroviruses and lentiviruses, produce a DNA copy of their RNA genome during an aspect of their natural viral lifecycle. A virally- encoded RdDP or reverse transcriptase is the polymerase that reverse transcribes the viral genomic RNA into a DNA copy, which can be subsequently integrated into a host cell chromosome or be retained extrachromosomally as an episome. The term provirus or proviral DNA can be used to describe the DNA copy of a retroviral genome.

[0125] Some DNA viruses, such as those of the family Hepadnaviridae (a member of which is Hepatitis B Virus, “HBV”), replicate their DNA viral genome through an RNA intermediate and possess an RdDP or reverse transcriptase to convert the RNA intermediate into DNA template molecule for further genome amplification.

[0126] In some embodiments, a sequence within the encrypted RNA is converted to DNA by a translation activator comprised of an RdDP or a reverse transcriptase. The DNA sequence can then be further transcribed into mRNA by a translation activator.

[0127] Some RNA viruses, such as Hepatitis Delta Virus (HDV), are thought to use the minor RdRP activity of certain host RNA polymerases, including human RNA Polymerase I (human Pol I), human RNA Polymerase II (human Pol II), or human RNA Polymerase III (human Pol III) to transcribe their viral RNA to produce mRNA. These host RNA polymerases are typically thought to be primarily DNA-dependentRNA polymerases but may be able to synthesize RNA from a DNA template or an RNA template.

[0128] In some embodiments, the translation activator of an encrypted RNA is comprised of viral RdRPs. In some embodiments, activation of an encrypted RNA into mRNA occurs because the encrypted RNA contains virus-derived sequences that can bind to viral RdRP complexes. In some embodiments, activation of an encrypted RNA occurs because the encrypted RNA contains virus-derived sequences that can bind to viral RdDP complexes. In some embodiments, the polypeptide (or protein) of interest of a therapeutic encrypted RNA is translated at reduced levels by host cell ribosomal machinery in the absence of viral infection, enabling virus-dependent therapeutic protein production.

[0129] In some embodiments, an encrypted RNA can resemble viral replication intermediates that a virus synthesizes into mRNA to replicate its genome. In other words, both the encrypted RNA and the reverse complement of the encrypted RNA can be activated by a translation activator. In some embodiments, when virus infection ends, translation of the polypeptide of interest of an encrypted RNA substantially ends due to the short half-life of produced mRNA and of the polypeptide of interest and an inability to substantially produce new mRNA in the absence of the translation activator.

[0130] In some embodiments, encrypted RNAs do not contain internal ribosome entry site (IRES) sequences. In some embodiments, encrypted RNAs are engineered to lack features that are important for efficient protein translation by host cell ribosomes, such as a 5 '-Cap or a 3' poly(A) tail. In some embodiments, the translation of a polypeptide of interest encoded by an encrypted RNA can be increased by more than 4-log fold in the presence of virus infection (see, e.g., FIG. 4B).

[0131] In some embodiments, encrypted RNA is produced outside a cell via in vitro transcription (IVT) using an RNA polymerase and a DNA template molecule that encodes the encrypted RNA. In some embodiments, the encrypted RNA is prepared via IVT as a precursor molecule that is subsequently processed to yield the encrypted RNA.

[0132] In some embodiments, the precursor encrypted RNA is comprised of an encrypted RNA portion and a ribozyme portion, in which the ribozyme portion cleaves the precursor encrypted RNA to generate two shorter RNA products, including the encrypted RNA and the ribozyme. In some embodiments, after cleavage of theprecursor encrypted RNA by the ribozyme portion, the encrypted RNA is 5'- monophosphorylated.

[0133] Exemplary sequence elements of encrypted RNAs or DNA-encoded encryptedRNAs are listed in Table 1. Exemplary pairings of sequence elements that can be used together as elements of an encrypted RNA are listed in Table 2. Exemplary coding sequences and reverse complements of coding sequences (i.e., antisense coding sequences) are listed in Table 3. Exemplary useful sequences for producing some encrypted RNAs or DNA-encoding encrypted RNAs are listed in Table 4. Some exemplary amino acid sequences of polypeptides of interest are listed in Table 5.

[0134] A table below should be read to continue, potentially for multiple pages, until the next table is listed or the tables end; e.g., Table 1 continues over multiple pages until Table 2 begins. Similarly, Table 5 continues until the next section, entitled“Proteins of interest” begins. To the extent DNA sequences are listed, it is understood that the sequences also disclose and embody their RNA counterparts (T U).Similarly, when RNA sequences are listed, it is understood that the sequences also disclose and embody their DNA counterparts (U — * T).w 00oProteins of interest

[0135] The terms “polypeptide”, “peptide”, “amino acid sequence” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched.

[0136] In some embodiments, a therapeutic polypeptide of interest comprises a “protein that causes cell death”. A protein that causes cell death, when produced at a sufficient concentration within a cell, increases the death rate of the cell and reduces the cell’s expected lifetime. Proteins that induce cell death include, but are not limited to: granzymes, including Granzyme A and Granzyme K; perforins; pro-apoptotic members of the BCL-2 family such as BCL-2 homology domain 3-only proteins, the B-cell lymphoma-2 (Bcl-2) family proteins BIM, PUMA, BID, BMP, NOXA, BIK, BAD; herpesvirus thymidine kinase; vaccinia virus E3L; receptor-interacting protein kinase 3 (RIPK3) / mixed lineage kinase domain-like protein (MLKL); caspases, including caspase- 3, caspase-6, and caspase-7; and gasdermin D.

[0137] In some embodiments, a protein that causes cell death (apoptosis) can further increase the cell death rate when the surrounding milieu contains a sufficient concentration of a molecule that is contacted by the protein that causes cell death to produce a new cytotoxic molecule. Such a potentiating protein could include, by way of example, the use of herpesvirus thymidine kinase in conjunction with ganciclovir.

[0138] As used herein, an “immune response” may be a specific reaction of the adaptive immune system to a particular antigen (i.e., a specific or adaptive immune response), a nonspecific reaction of the innate immune system (i.e., a nonspecific or innate immune response), or a combination thereof.

[0139] As used herein, “immunogenicity” refers to the capacity of a polynucleotide that can induce an immune response. Some embodiments of the invention involve using RNA constructs that have altered nucleotides to reduce the immunogenicity of the polynucleotide in the absence of activation by a translation activator. An aspect of the compounds, compositions comprising the compounds, and methods described herein include the discovery that polynucleotides having L and R regions containing modified nucleotides can still be efficiently replicated by a viral polymerase.

[0140] This discovery that efficient replication could occur using a viral polymerase is surprising, in part because nucleotide modifications can alter the secondary or tertiary structures of a polynucleotide. Yet it was known that these secondary and tertiary structures are important for the polynucleotide to react with and enable polymerase activity. In addition, there are prior art reports that a viral RdRp, specifically an alphavirus RdRp, cannot replicate templates without unmodified uridine nucleotides for efficient protein translation (see, e.g., Beissert T et al., “A Trans-amplifying RNA Vaccine Strategy for Induction of Potent Protective Immunity,” Mol. Ther. 2020, DOI: 10.1016 / j .ymthe.2019.09.009).

[0141] It was noted that an attempt to use a nucleoside-modified alphavirus replicon (“a self-amplifying RNA”) encoding a SARS-CoV-2 vaccine antigen resulted in loss of antigen (protein) production in vivo (Voigt, E.A., et al. A self-amplifying RNA vaccine against COVID- 19 with long-term room-temperature stability. Npj Vaccines (2022). DOI: 10.1038 / s41541-022-00549-y). In contrast, the encrypted RNAs contemplated herein can be developed with 100% of uridine nucleotides modified and surprisingly retain at least equal or higher protein production in the presence of a viral polymerase — in addition to substantially reduced immunogenicity in the absence of the viral polymerase.

[0142] It is recognized that some viral RNAs are modified by cellular enzymes in select positions during their natural lifecycle. For example, the adenosines of a hepatitis C virus, a Zika virus, and a feline leukemia virus can be post-transcriptionally modified to N6- methyladenosine by cellular methyltransferases (Gokhale N. & Homer S; PLoS Pathog. 2017 Mar; 13(3): el(X)6188).

[0143] As used herein, an “immunomodulatory polypeptide” refers to a polypeptide that is able to alter an immune response, including by: inducing or suppressing maturation of immune cells, inducing or suppressing cytokine biosynthesis, or altering humoral immunity by stimulating antibody production by B cells. Immunomodulatory polypeptides may have antiviral and antitumor activity and may also down-regulate other aspects of the immune response, for example, shifting the immune response away from a TH2 immune response, which can be used to treat or moderate a wide range of TH2- mediated diseases / conditions (e.g., atopic dermatitis, allergic rhinitis, and asthma).

[0144] In some embodiments, the polypeptide of interest is an immunomodulatory polypeptide. In some further embodiments, the polypeptide of interest is an immunomodulatory polypeptide that is immunogenic in a subject, i.e., acts as an antigen in the subject to yield an immune response. As used herein, the term “antigen” means an immunogenic compound that elicits an adaptive immune response in a subject being treated with the antigen. In particular, an “antigen” is any substance that induces in the subject an antigen-specific antibody or T-lymphocyte (T-cell) response, when the subject is exposed to the antigen. The term “antigen” comprises any molecule that has at least one epitope. In one aspect, an antigen is a molecule that, optionally after processing, induces an immune reaction, which is specific for the antigen in the subject being treated. Antigens may include polypeptides derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens or from cancers, including tumor antigens. In one aspect, an antigen corresponds to a naturally occurring product, for example, a polypeptide naturally displayed on the surface of a cell, a pathogen, a bacterium, a virus, a fungus, a parasite, an allergen, or a tumor. The antigen may elicit an immune response against a cell, a pathogen, a bacterium, a virus, a fungus, a parasite, an allergen, or a tumor.

[0145] The term “pathogen” refers to pathogenic biological material capable of causing disease in an organism. Pathogens include microorganisms such as bacteria, unicellular eukaryotic organisms (protozoa), fimgi, and viruses.

[0146] In some embodiments, the polypeptide of interest comprises an antigen suitable for vaccination of a target organism. In some embodiments, an antigen is selected from the group comprising a self-antigen and non-self-antigen. A non-self-antigen may be a viral antigen, a bacterial antigen, a fungal antigen, an allergen, or a parasite antigen.

[0147] In some aspects, the antigen is a self-antigen, particularly a tumor antigen. The term “tumor antigen” or “tumor-associated antigen” refers to proteins that, under normal conditions, are specifically expressed in a limited number of tissues or organs or in specific developmental stages; for example, the tumor antigen may be under normal conditions specifically expressed in stomach tissue, for example in the gastric mucosa, in reproductive organs, e.g., in testis, in trophoblastic tissue, e.g., in placenta or germ line cells, and are expressed or aberrantly expressed in one or more tumor or cancer tissues. Inthis context, “a limited number” can be not more than 3, or not more than 2. Tumor antigens in the context of the present invention can include, for example, differentiation antigens, cell type-specific differentiation antigens, i.e., proteins that are under normal conditions specifically expressed in a certain cell type at a certain differentiation stage, cancer / testis antigens, i.e., proteins that are under normal conditions specifically expressed in testis and sometimes in the placenta, and germ line specific antigens. In one aspect, the tumor antigen is associated with the cell surface of a cancer cell and is not or only rarely expressed in normal tissues. In one aspect, the tumor antigen or the aberrant expression of the tumor antigen identifies cancer cells. In one aspect, the tumor antigen that is expressed by a cancer cell in a subject, e.g., a patient suffering from a cancer disease, is a self-protein in said subject. The tumor antigen in the context of the present invention can be expressed under normal conditions specifically in a tissue or organ that is non-essential, i.e., tissues or organs which, when damaged by the immune system, do not lead to the death of the subject, or in organs or structures of the body which are not or only hardly accessible by the immune system.

[0148] As used herein, "background translation" of a polypeptide of interest means the translation of the polypeptide of interest in the absence of a translation activator.

[0149] In some embodiments, background translation of the polypeptide of interest is not substantially immunogenic.

[0150] In some embodiments, background translation of the polypeptide of interest is not substantially immunogenic; however, translation of the polypeptide of interest in the presence of a translation activator (e.g., in a viral infection) is substantially immunogenic.

[0151] Therapeutic polypeptides of interest can be selected from the group comprised of: cytokines and immune system proteins such as immunogenic or immunomodulatory proteins (e.g., interferons, interleukins, colony stimulating factors (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factors (TNF), integrins, addressins, selectins, homing receptors, T cell receptors, immunoglobulins); hormones (e.g., insulin, thyroid hormone, catecholamines, gonadotropins, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptins and the like); growth hormones (e.g., human grown hormone); growth factors (e.g., epidermal growth factor, nerve growth factor,insulin-like growth factor and the like); growth factor receptors; enzymes (e.g., tissue plasminogen activator, streptokinase, cholesterol biosynthetic or degradative, steroidogenic enzymes, kinases, phosphodiesterases, methylases, de-methylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatases, cytochromes, adenylate or guanylate cyclases, neuraminidases and the like); receptors (e.g., steroid hormone receptors, peptide receptors); binding proteins (e.g., growth hormone or growth factor binding proteins and the like); transcription and translation factors; tumor growth suppressing proteins (e.g., proteins which inhibit angiogenesis); structural proteins (e.g., collagen, fibrin, fibrinogen, elastin, tubulin, actin, and myosin); or blood proteins / blood factors (e.g., thrombin, serum albumin, Factor VII, Factor VIII, insulin, Factor IX, Factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin HI, glucocerebrosidase, erythropoietin granulocyte colony stimulating factor (GCSF) or modified Factor VIII, anticoagulants and the like).

[0152] In some embodiments, the therapeutic polypeptide of interest can comprise an interferon. In some further embodiments, the therapeutic polypeptide of interest is comprised of an interferon (IFN) selected from: IFN-al, IFN-a2, IFN-a4, IFN-a5, IFN- a6, IFN-a7, IFN-a8, IFN-alO, IFN-al 3, IFN-al4, IFN-al6, IFN-al7, IFN-a21, IFN-pi, IFN-e, IFN-K, IFN-col, IFN-y, IFN-11 (IL28A), IFN- A2 (IL28B), IFN- A3 (IL29), or IFN- A4. Exemplary nucleotide sequences encoding proteins of interest are listed in Table 3. Exemplary sequences of proteins of interest are listed in Table 5. Additional nucleotide and amino acid sequences would be understood to be capable of being substituted with those listed in the tables.

[0153] In some embodiments, the therapeutic polypeptide of interest is comprised of a cytokine that is involved in regulating lymphoid homeostasis, such as a cytokine that is involved in and induces or enhances the development, priming, expansion, differentiation, or survival of T cells. In some embodiments, the cytokine is an interleukin (IL), such as IL-1 to IL-40 (e.g., IL-2, IL-6, IL-7, IL-12, IL-15, IL-21, or IL-23). In some embodiments, the interleukin is an anti-inflammatory cytokine, such as IL-1 receptor antagonist (IL-1RN or IL-IRA), IL-23RA, IL-36RA, or IL-37.

[0154] In some embodiments, the therapeutic polypeptide of interest is comprised of an “antineoplastic protein”. An antineoplastic protein is a polypeptide that is effective intreating cancer in a subject. Particular classes of antineoplastic proteins include, but are not limited to: monoclonal antibodies, nanobodies, hormones, proteins that cause cell death, immune checkpoint inhibitors, interleukins, and immunogens. Immune checkpoint inhibitors bind to and inhibit the activity of an immune checkpoint protein (e.g., PD-1, PD-L1, PD-L2, CTLA4, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3, VISTA, etc.).

[0155] In some aspects, the polypeptide of interest is an antagonist of Programmed Cell Death Ligand 1 (PD-L1) and / or Programmed Cell Death 1 (PD-1) or another immune checkpoint inhibitor.

[0156] A PD-1 antagonist, as used herein, is an agent that inhibits or prevents PD-1 activity, e.g., by binding to PD-1.

[0157] PD-1 activity may be interfered with by antibodies that bind selectively to and block the activity of PD- 1. The activity of PD- 1 can also be inhibited or blocked by molecules other than antibodies that bind PD-1. Such molecules include proteins (such as fusion proteins) and peptides, e.g., peptide mimetics of PD-L1 and PD-L2 that bind PD-1 but do not activate PD-1. Although the structure of PD-L2 is similar to PD-L1, the binding affinity between PD-L2 and PD-1 is two- to six-fold higher than that with PD- Ll, suggesting that PD-L2 is an important molecule in immune escape as the strong interaction inhibits cytokines secretion and proliferation of T cells (Wang, Y., Du, J., Gao, Z. et al. Evolving landscape of PD-L2: bring new light to checkpoint immunotherapy. Br. J. Cancer. 128, 1196-1207 (2023)).

[0158] Exemplary PD-1 antagonists include those described in U.S. Publications20130280265, 20130237580, 20130230514, 20130109843, 20130108651, 20130017199, 20120251537, and 20110271358, and in European Patent EP2170959B1, the entire disclosures of which are incorporated herein by reference. Other exemplary PD-1 antagonists are described in Curran et al., PNAS, 107: 4275 (2010); Topalian et al., New Engl. J. Med. 366: 2443 (2012); Brahmer et al., New Engl. J. Med. 366: 2455 (2012); Dolan et al., Cancer Control 21: 3 (2014); and Sunshine et al., Curr. Opin. in Pharmacol. 23 (2015).

[0159] Exemplary PD-1 antagonists include: nivolumab (e.g., OPDIVO® from Bristol- Myers Squibb), a fully human IgG4 monoclonal antibody that binds PD-1; pidilizumab(e.g., CT-011 from CureTech), a humanized IgGl monoclonal antibody that binds PD-1; pembrolizumab (e.g., KEYTRUDA® from Merck), a humanized IgG4-kappa monoclonal antibody that binds PD-1; MEDI-0680 (AstraZeneca / Medlmmune) a monoclonal antibody that binds PD-1; dostarlimab is an anti-PD-1 monoclonal antibody (also known as TSR-042, JEMPERLI®, WBP-285, and dostarlimab-gxly) manufactured by Tesaro; retifanlimab (also known as Zynyz is a PD-1 blocking monoclonal antibody produced by Incyte Corp, and is also known as AEX-1188, INCMGA-00012, MGA-012, and retifanlimab-dlwr); and REGN2810 (Regeneron / Sanofi; also known as cemiplimab and LIBTAYO®) a monoclonal antibody that binds PD-1. Another exemplary PD-1 antagonist is AMP-224 (Glaxo Smith Kline and Amplimmune), a recombinant fusion protein composed of the extracellular domain of the Programmed Cell Death Ligand 2 (PD-L2) and the Fc region of human IgGl, that binds to PD-1.

[0160] A PD-L1 antagonist, as used herein, is an agent that inhibits or prevents PD-L1 activity, e.g., by binding to PD-L1.

[0161] PD-L1 activity may be blocked by molecules that selectively bind to and block the activity of PD-L1, e.g., by blocking the interaction with and activation of PD-1 and / or B7-1. The activity of PD-L1 can also be inhibited or blocked by molecules other than antibodies that bind PD-L1. Such molecules include proteins (such as fusion proteins and peptides.

[0162] Exemplary PD-L1 antagonists include those described in U.S. Publications 20090055944, 20100203056, 20120039906, 20130045202, 20130309250, and 20160108123, the entire disclosures of which are incorporated herein by reference. Other exemplary PD-L1 antagonists are described in Sunshine et ah, Curr. Opin. in Pharmacol. 23 (2015).

[0163] PD-L1 antagonists include, for example: atezolizumab (also called MPDL3280A or TECENTRIQ ™, Genentech / Roche), a human monoclonal antibody that binds to PD- Ll; durvalumab (also called MED 14736 or IMFINZI™, AstraZeneca / Medlmmune), a human immunoglobulin IgGl kappa monoclonal antibody that binds to PD-L1; BMS- 936559 also known as MDX 1105 (Bristol-Myers Squibb), a fully human IgG4 monoclonal antibody that binds to PD-L1 ; avelumab (also called MSB 0010718C or BAVENCIO®, Merck KGaA / Pfizer), a fully human IgGl monoclonal antibody thatbinds to PD-L1; and CA-170 (Aurigene / Curis) is an oral molecule VISTA / PD-L1 antagonist (wherein VISTA is a V-domain Ig suppressor of T-cell activation) and shares structural similarity to PD-L1.

[0164] Other exemplary immune checkpoint inhibitors can include an anti-CTLA-4 antibody (e.g., ipilimumab or tremelimumab), an anti-LAG-3 antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, an anti-NKG2a antibody, an anti-OX40 antibody, an anti-ICOS antibody, an anti-MICA antibody, an anti-CD137 antibody, an anti-KIR antibody, an anti-TGFP antibody, an anti-IL-10 antibody, an anti-IL-8 antibody, an anti- B7-H4 antibody, an anti-Fas ligand antibody, an anti-CXCR4 antibody, an anti- mesothelin antibody, an anti-CD27 antibody, an anti-GITR, or any combination thereof.

[0165] In some aspects, the polypeptide of interest is a glucagon-like peptide 1 (GLP-1) agonist. GLP-1 agonists are analogs of GLP-1, which is a gut-derived peptide hormone that exhibits a glucose-lowering effect via stimulation of insulin secretion from pancreatic islets in response to an oral glucose load, known as the incretin effect. It has been shown that GLP-1 agonists can help manage Type 2 diabetes and obesity. GLP-1 agonists approved in U.S. include Dulaglutide (Trulicity®), Exenatide (Byetta®), Exenatide extended-release (Bydureon®), Liraglutide (Victoza®), Lixisenatide (Adlyxin®), Semaglutide injection (Ozempic®), and Semaglutide tablets (Rybelsus®). Tirzepatide (Mounjaro®) binds to the glucose-dependent insulinotropic polypeptide and glucagon like peptide 1 (GLP-1 ) receptor. It is a first-in-class medicine that activates both the GLP-1 and GIP receptors (as an agonist for both), which leads to improved blood sugar control.

[0166] In some aspects, the polypeptide of interest is a sodium glucose cotransporter 2 (SGLT-2) inhibitor. SGLT-2 inhibitors represent another class of drugs (other than GLP-1 agonists) that may lead to weight loss and improved blood sugar control. These SGLT-2 inhibitors include canagliflozin (Invokana), ertugliflozin (Steglatro), dapagliflozin (Farxiga), and empagliflozin (Jardiance).RNA compositions

[0167] In some embodiments, an encrypted RNA or DNA encoding an encrypted RNA can be complexed with one or more cationic or polycationic compounds, for example,with cationic or polycationic polymers, cationic or polycationic peptides or proteins (e.g., protamine), cationic or polycationic polysaccharides, or cationic or polycationic lipids.

[0168] As used herein, “lipid nanoparticles” or “LNPs” are nanoscale structures comprised of one or more lipid-like compounds. LNPs include liposomes, lipoplexes, RNA-carrying lipid nanoparticles, DNA-carrying lipid nanoparticles, solid lipid nanoparticles, lipidoid nanoparticles, or cubosomes (see, e.g., Tenchov et al., ACS Nano (2021); DOI: 10.1021 / acsnano.lc04996).

[0169] In some embodiments, an encrypted RNA or a DNA encoding an encrypted RNA can be complexed with lipids to form lipid nanoparticles. Therefore, in some embodiments, the inventive composition comprises lipid nanoparticles comprising one or more encrypted RNAs or one or more DNAs encoding encrypted RNAs.

[0170] Lipid-based formulations have been increasingly recognized as promising delivery systems for RNA due, in part, to their biocompatibility and their ease of large- scale production.

[0171] Liposomes are colloidal lipid-based and surfactant-based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment. They may present as spherical vesicles and can range in size from about 20 nm to a few microns (e.g., 2-10 microns). Cationic lipid-based liposomes can complex with negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the possibility of the large-scale production required for in vivo clinical applications. Liposomes can fuse with the plasma membrane for uptake; once inside the cell, the liposomes are processed via the endocytic pathway, and the genetic material is then released from the endosome / carrier into the cytoplasm. Liposomes have long been perceived as drug delivery vehicles because of their superior biocompatibility, given that liposomes are analogs of biological membranes and can be prepared from both natural and synthetic phospholipids ( / nt. J. Nanomedicine, 2014; 9: 1833-1843).

[0172] Cationic (including ionizable cationic) or neutral lipids have been widely studied as synthetic materials for the delivery of RNA. In some LNPs, after mixing together, nucleic acids are condensed by lipids to form lipid / nucleic acid complexes. LNP complexes can protect genetic material from the action of nucleases and deliver geneticmaterial into cells by interacting with the negatively charged cell membrane. Some LNPs can be prepared by directly mixing positively charged lipids at physiological pH with negatively charged nucleic acids.

[0173] LNPs are typically comprised of four lipid or lipid-like components: (i) a cholesterol or cholesterol derivative; (ii) a cationic lipid, sometimes called an ionizable lipid; (iii) a structural lipid, sometimes called a phospholipid; and (iv) a PEG lipid, sometimes called a PEGylated lipid, which is a polyethylene glycol (PEG) functionalized lipid used to stabilize the particle and improve product stability and pharmacokinetic properties due to surfactant properties (see, e.g., Hou et al., Nat. Rev. Mater. (2021); DOI: 10.1038 / s41578-021-00358-0). Furthermore, an LNP can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, or carbohydrates) to its surface or the terminal end of the attached PEG chains (see, e.g., Front. Pharmacol. 2015 Dec. 1; 5:285).

[0174] Lipid nanoparticles comprised of cationic lipids have been commonly used in non-viral delivery systems for mRNA sequences, as well as oligonucleotides, including plasmid DNA, antisense oligos, or siRNA / small hairpin RNA-shRNA. Cationic lipids, such as DOTAP (l,2-dioleoyl-3-trimethylammonium-propane); DOTMA (N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methyl sulfate); or D-Lin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), can form complexes with negatively charged nucleic acids to form nanoparticles by electrostatic interaction, providing high in vitro transfection efficiency. Furthermore, lipid nanoparticles comprised of neutral lipids for RNA delivery have been developed, such as l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based liposomes (Adv. Drug Deliv. Rev. 2014 February; 55: 110-115) and newer lipid nanoparticles that utilize squaramide amino lipids (Comebise et al., Adv. Functional Materials (2022), DOI: 10.1002 / adfin.202106727). Therefore, in some embodiments, an encrypted RNA is complexed with a cationic lipid (e.g., an ionizable cationic lipid) or a neutral lipid and is thereby formulated into a lipid nanoparticle.

[0175] In some embodiments, a composition can comprise an encrypted RNA or a DNA encoding an encrypted RNA that is formulated together with a cationic or polycationic compound or with a polymeric carrier. Accordingly, in a further embodiment of theinvention, the RNA as defined herein or any other nucleic acid comprised in the composition is associated with or complexed with a cationic or polycationic compound or a polymeric carrier. Therein, the RNA as defined herein or any other nucleic acid comprised in the composition described herein can also be associated with a vehicle, transfection, or complexation agent for increasing the transfection efficiency or the expression of the RNAs described herein or optionally comprised to further include additional nucleic acids.

[0176] As defined above, a polymeric carrier, which may be used to complex the RNA compounds or any further nucleic acid comprised in the compositions described herein may be formed by disulfide-crosslinked cationic (or polycationic) components.

[0177] In some embodiments, the composition comprises at least one RNA as defined herein, which is complexed with one or more polycations and at least one free RNA, wherein the at least one complexed RNA is identical to the at least one free RNA. In this context, the composition of the present invention can comprise the RNA according to the invention that is complexed at least partially with a cationic or polycationic compound or a polymeric carrier, e.g., cationic lipids or peptides. In this context, the disclosures of WO 2010 / 037539 and WO 2012 / 113513 are incorporated herewith by reference. Partially means that only a part of the RNA as defined herein is complexed in the composition according to the invention with a cationic compound and that the rest of the RNA as defined herein is (comprised in the inventive (pharmaceutical) composition) in uncomplexed form (“free”).

[0178] In some embodiments, the complexed RNA in the compositions described herein is prepared according to a first step by complexing the RNA according to the invention with a cationic or polycationic compound or with a polymeric carrier in a specific ratio to form a stable complex. In this context, no free cationic or polycationic compound or polymeric carrier or only a negligibly small amount thereof remains in the component of the complexed RNA after complexing the RNA. Accordingly, the ratio of the RNA and the cationic or polycationic compound or the polymeric carrier in the component of the complexed RNA can be selected in a range so that the RNA is entirely complexed and no free cationic or polycationic compound or polymeric carrier or only a negligibly small amount thereof remains in the composition.

[0179] In other embodiments, the compositions comprising the RNAs described herein may be administered naked without being associated with any further vehicle, transfection, or complexation agent.

[0180] In embodiments wherein the composition comprises more than one encrypted nucleic acid species, these encrypted nucleic acid species may be provided as, for example, two, three, four, five, six, or more separate compositions, which may contain at least one encrypted nucleic acid species each, each encoding a polypeptide of interest. Also, the compositions described may be a combination of at least two distinct compositions, each composition comprising at least one encrypted nucleic acid. In some embodiments, the two distinct compositions can comprise a composition carrying the encrypted RNA or DNA encoding the encrypted RNA and a composition carrying an RNA or DNA encoding a translation activator that activates the encrypted nucleic acid. The composition may be combined to form a single composition before its use, or it may be used in such a way that more than one administration is required to administer the (therapeutic) encrypted nucleic acid species. If the (pharmaceutical) composition contains at least one (therapeutic) encrypted nucleic acid species, for example, at least two encrypted nucleic acid species, encoding a combination of (therapeutic) proteins, it may e.g. be administered by one single administration (combining all encrypted nucleic acid species), or by at least two separate administrations (either sequentially or contemporaneously administered). Accordingly, any combination of (therapeutic) encrypted nucleic acid species encoding at least one (therapeutic) polypeptide of interest or any combination of (therapeutic) polypeptides of interest, provided as separate entities (each containing one encrypted nucleic acid species) or as a combined entity (containing more than one encrypted nucleic acid species), is understood as a (pharmaceutical) composition according to the present invention. Another exemplary composition contemplated encodes at least one encrypted nucleic acid encoding at least two (therapeutic) polypeptides of interest.

[0181] The (pharmaceutical) composition described herein may be provided in a liquid or a dry (e.g., lyophilized) form.

[0182] In some embodiments, the (pharmaceutical) composition comprises a safe and effective amount of an encrypted nucleic acid, encoding a (therapeutic) polypeptide ofinterest or a combination of (therapeutic) polypeptides of interest. As used herein, “safe and effective amount” means an amount of the encrypted nucleic acid that is sufficient to significantly favorably affect a disease or disorder (or a symptom thereof). At the same time, however, a “safe and effective amount” is preferably small enough to avoid serious side-effects, that is to say, to permit a sensible relationship between advantage and risk. In relation to the (pharmaceutical) composition of the present invention, the expression “safe and effective amount” can mean an amount of the encrypted nucleic acid (and thus of the (therapeutic) polypeptide of interest) that is suitable for obtaining appropriate expression levels of the (therapeutic) polypeptide of interest when the translation activator is present or absent. Such a “safe and effective amount” of the encrypted nucleic acid of the (pharmaceutical) composition may furthermore be selected in dependence on the encrypted nucleic acid species (e.g., nucleoside-modified vs. non-nucleoside- modified, or 5 '-triphosphorylated vs. 5 '-nonphosphorylated) or on the method of encrypted nucleic acid production, purification, or formulation; for example, since some (therapeutic) encrypted nucleic acid species may lead to substantially higher translation of the (therapeutic) polypeptide of interest than would occur from treatment with an equal amount of an alternative (therapeutic) encrypted nucleic acid species encoding the same (therapeutic) polypeptide of interest. A “safe and effective amount” of the encrypted nucleic acid of the (pharmaceutical) composition as defined above will furthermore vary in connection with: the particular condition to be treated, the severity of the condition, the age and physical condition of the patient to be treated, the duration of the treatment, the nature of any accompanying therapy, the particular pharmaceutically acceptable carrier used, or similar factors within the knowledge and experience of the accompanying doctor. The (pharmaceutical) composition according to the invention can be used according to the invention for a subject, such as for human or veterinary medical purposes.

[0183] In some embodiments, the encrypted nucleic acid of the (pharmaceutical) composition or kit of parts according to the invention is provided in lyophilized form. The lyophilized RNA can be reconstituted in a suitable buffer advantageously based on an aqueous carrier before administration; suitable aqueous carriers can include, e.g., Ringer-Lactate solution, Ringer solution, or a phosphate buffered solution (PBS). In someembodiments, the (pharmaceutical) composition or the kit of parts according to the invention contains at least two, three, four, five, six, or more encrypted nucleic acid species, which are provided separately in lyophilized form (optionally together with at least one further additive) and which may be reconstituted separately in a suitable buffer (such as Ringer-Lactate solution) before their use so as to allow individual administration of each of the encrypted nucleic acids.

[0184] A composition described herein may contain a pharmaceutically acceptable carrier. The expression “pharmaceutically acceptable carrier” as used herein, may include the liquid or non-liquid basis of the composition. If the composition is provided in liquid form, the carrier can be water, typically pyrogen-fiee water; isotonic saline or buffered (aqueous) solutions, e.g., phosphate, citrate, etc. buffered solutions. Particularly for injection of the (pharmaceutical) composition, water or a buffer, such as an aqueous buffer, may be used, containing a sodium salt, a calcium salt, or a potassium salt. In some embodiments, the sodium, calcium, or potassium salts may occur in the form of their halogenides, e.g., chlorides, iodides, or bromides, or in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Some examples of sodium salts include NaCl, Nal, NaBr, Na2CCh, NaHCCh, Na2SO4; some examples of the optional potassium salts include KC1, KI, KBr, K2CO2, KHCO2, K2SO4; and some examples of calcium salts include CaCh, Cah, CaBt2, CaCCh, CaSO4, Ca(OH)2. Furthermore, organic anions of the aforementioned cations may be contained in the buffer. In some embodiments, the buffer suitable for injection purposes, as defined above, may contain salts selected from sodium chloride (NaCl), calcium chloride (CaCh), and potassium chloride (KC1), wherein further anions may be present in addition to the chlorides. CaCh can also be replaced by another salt like KC1. In some injection buffers, salts are present in a concentration of at least 50 mM sodium chloride (NaCl), and at least 3 mM potassium chloride (KC1) and at least 0.01 mM calcium chloride (CaCh) are present. The injection buffer may be hypertonic, isotonic, or hypotonic with reference to the specific reference medium, i.e., the buffer may have a higher, identical, or lower salt content with reference to the specific reference medium, and concentrations of the aforementioned salts may be used that do not lead to damage of cells due to osmosis or other concentration effects. Common buffers or liquids are known to a skilled person. Ringer-Lactate solution is one example of a liquid basis.Administration and Delivery

[0185] Suitable routes of administration include, for example, include pulmonary (e.g., intratracheal, inhaled, or intranasal), oral, rectal, vaginal, transmucosal, or intestinal administration, parenteral delivery (e.g., intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, or intraperitoneal). Inhaled administration includes delivery via a nebulizer device or a nasal spray. In some embodiments, inhaled delivery is to airway cells, including upper or lower airway cells. In some embodiments, intramuscular administration is made to a muscle selected from the group consisting of skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, intravenous administration results in the delivery of RNA to liver cells.

[0186] Delivery of an encrypted nucleic acid, such as an encrypted RNA of the invention or a DNA encoding an encrypted RNA, can be achieved using viral vectors. Viral vectors useful for delivery include but are not limited to: lentiviral vectors, adenovirus (Ad) vectors, herpes simplex virus (HS V) vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, or baculovirus vectors. Within the non-viral subclass of delivery methods, techniques utilizing naked DNA injection, electroporation, biolistic systems for DNA delivery (e.g., gene guns), sonoporation, magnetofection, or LNPs have also been developed for gene delivery.

[0187] Lentiviral (LV) vectors based on human immunodeficiency virus type I (HIV-1 ) have been developed to deliver genetic material to a broad range of cell types. Integration-proficient LV (IPLV) vectors are the conventional form of LV technology, in which vector proviruses permanently integrate into the transduced cell genome. However, these integration events sometimes occur within genes, which can dysregulate endogenous gene expression. To minimize integration events, integration-deficient LV (IDLV) vectors have also been developed by mutating the HIV-1 integrase component of LV vectors to ensure that the majority of proviral DNA remains as extrachromosomal episomes. Some chromosomal integration can occur with IDLV technology, with 0.1%- 1% of proviruses integrating into the genome.

[0188] HIV- 1 -based LV vectors offer a potential means to deliver RNA to a wide range of cell types in vivo and in vitro as they package their genomes in the form of ssRNA. In conventional LV vectors, the ssRNA genome is reverse-transcribed to give a doublestranded DNA (dsDNA) product, which then enters the nucleus.

[0189] Adeno-associated virus (AAV) is a small, helper-dependent, single-stranded DNA virus capable of transducing dividing or non-dividing cells by delivering a predominantly episomal transgene product. In comparison to adenoviral vectors, AAV vectors may provide a safer option for transduction, given their potentially diminished pathogenicity and immunogenicity in humans. One disadvantage of AAV vectors is their small transgene capacity of approximately 4.8 kb, which can restrict the breadth of the therapeutic genes (also referred to as transgenes) that may be delivered via the AAV vector.

[0190] Adenoviral vectors are able to transduce replicating or quiescent cell populations, making them a valuable tool in delivering transgenes in vivo and within mature tissues. AdVs are able to deliver larger transgenes than AAVs. As with AAV, AdV-delivered DNA does not generally integrate into the host genome but instead resides episomally in the host nucleus. Such episomal transduction minimizes the risks of insertional mutagenesis by minimizing direct integration into the host genome. Yet, transgene expression is transient, is vulnerable to cell silencing mechanisms, and is destined for dilution among progeny cells should cell division ensue.

[0191] The herpes simplex virus (HSV) is a double-stranded DNA virus capable of delivering up to 50 kbp of transgenic DNA when used as a vector. Similar to the adenovirus, pre-existing immunity to HSV infection is prevalent within the general population; however, HSV vectors can frequently evade inactivation by host’s immune response. HSV vector genomes also remain episomal like those of AdVs. As a result, they are expectedly burdened by the same limitations of transient expression faced by AdVs.

[0192] RNA delivery offers a means to transiently express exogenous genes in a target cell, as the delivered RNA often remains extranuclear. Non-viral vectors have been developed for in vivo RNA delivery, but tissue-specific targeting requires further optimization.

[0193] In some embodiments, an encrypted RNA or DNA encoding an encrypted RNA according to the invention is used to produce a medicament in the context of a viral infection, wherein the medicament is for treatment or prophylaxis of a disease, disorder, or condition caused by the viral infection. As shown in FIG. 3A, in some embodiments, treating a cell with a therapeutic encrypted RNA (or a DNA encoding a therapeutic encrypted RNA) in the absence of viral infection (e.g., prophylactic administration) does not result in translation of the therapeutic polypeptide of interest, because no translation activator of the encrypted RNA is present. Conversely, FIG. 3B shows that viral infection of a cell in the absence of treatment with a therapeutic encrypted RNA (or a DNA encoding a therapeutic encrypted RNA) can result in high levels of viral replication. FIG. 3C shows that if cells are both treated with a therapeutic encrypted RNA (or a DNA encoding a therapeutic encrypted RNA) and also infected with a virus encoding a translation activator of the therapeutic encrypted RNA, the therapeutic encrypted RNA can be activated by the virus-encoded translation activator, which results in the production of a distinct mRNA species and subsequent translation of the therapeutic polypeptide of interest (e.g., an interferon). In some embodiments, upon infection of encrypted RNA-treated cells by a virus encoding a translation activator, the therapeutic polypeptide(s) of interest are both translated and secreted to protect neighboring cells from virus infection (including cells that did not initially receive the encrypted nucleic acid treatment), to thereby inhibit virus spread across a subject, e.g., via paracrine signaling.

[0194] In some embodiments, encrypted nucleic acids or formulations of encrypted nucleic acids (e.g., LNP or viral vector formulations) can be administered systemically or locally. Examples of systemic administration are described above (e.g., intravenous or subcutaneous administration) and can be useful for delivering to regions of the body such as the liver. Alternatively or additionally, encrypted nucleic acids and compositions of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a targeted tissue (e.g., in a sustained release formulation). Local delivery can be affected in various ways, depending on the tissue to be targeted. For example, aerosols containing compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of thepresent invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines; can be supplied in suppository form for rectal or vaginal application; or can be delivered to the eye by use of creams, drops, or injection.Formulations containing provided compositions complexed with therapeutic molecules or ligands can be surgically administered, for example, in association with a polymer or other structure or substance that can allow the compositions to diffuse from the site of implantation to surrounding cells. Alternatively, they can be applied surgically without the use of polymers or supports.

[0195] In some embodiments, a subject is treated with a therapeutically effective amount of one or more encrypted RNAs. As described herein, treatment could occur via a variety of means, including by providing therapeutic encrypted RNAs directly in RNA form via formulation into suitable lipid nanoparticles or by providing suitable DNAs encoding for the encrypted RNA as plasmids or viral vectors.

[0196] In some embodiments, a subject is treated with a therapeutically effective amount of more than one encrypted RNA, wherein treatment with each encrypted RNA can be accomplished by the same delivery method, by different delivery methods, or by combinations thereof. If more than one encrypted RNA is delivered in DNA-encoded form, each encrypted RNA could be encoded in a unique DNA molecule, or more than one encrypted RNA could be encoded in a single DNA molecule.

[0197] In some embodiments, a subject is treated with more than one therapeutic encrypted RNA, wherein at least two therapeutic encrypted RNAs encode different polypeptides of interest.

[0198] In some embodiments, a subject is treated with more than one therapeutic encrypted RNA, wherein at least two therapeutic encrypted RNAs have template regions for binding the same translation activator.

[0199] In some embodiments, a subject is treated with more than one therapeutic encrypted RNA, wherein at least two therapeutic encrypted RNAs have template regions for binding different translation activators.

[0200] In some embodiments, a cell harboring at least one encrypted RNA is created by delivering one or more encrypted nucleic acids encoding one or more therapeutic polypeptides of interest into a cell. In some further embodiments, at least two of these encrypted RNAs encode different therapeutic polypeptides of interest. In some further embodiments, at least two therapeutic encrypted RNAs have unique template regions for binding the same translation activator. In some further embodiments, at least two therapeutic encrypted RNAs have template regions for binding different translation activators.

[0201] In some embodiments, the present invention comprises a plant or animal cell comprising an encrypted nucleic acid. For example, the encrypted nucleic acid of the present invention may be exogenous to the plant or animal cell, e.g., an encrypted nucleic acid in which the polypeptide of interest is an antiviral protein.EXAMPLES

[0202] The compositions, compounds, and methods of making and using the same are not limited in their application to the details of construction or to the arrangement of components that are described herein. The compositions, compounds, and methods of making and using the various embodiments and of being practiced or of being carried out in various ways. It should also be understood that, unless indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0203] While several inventive embodiments are described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means or structures for performing the function or obtaining the results or one or more of the advantages described herein, and each of such variations or modifications is deemed to be within the scope of the inventive embodiments described herein. Those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and any actual parameters, dimensions, materials, orconfigurations will depend upon the specific application or applications for which the inventive teachings are used.

[0204] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.

[0205] Inventive embodiments of the present disclosure are directed to each feature, system, article, material, kit, or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, or methods, if such features, systems, articles, materials, kits, or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0206] Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only. All of the claims in the claim listing are herein incorporated by reference into the specification in their entirety as additional embodiments.

[0207] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, or ordinary meanings of the defined terms. All references, patents, and patent applications disclosed herein are incorporated by reference to the subject matter for which each is cited, which in some cases may encompass the entirety of a cited document.

[0208] The present invention is further illustrated by the following examples, which in no way should be construed as further limiting.Conventions

[0209] Unless otherwise noted, the following buffers have the below essential compositions:• WFI is nuclease-free, endotoxin-free water for injection.• PBS is phosphate buffered saline (0.144 g / L KC1; 9.00 g / L NaCl; 0.795 g / L Na2HPO4-7H2O).• DPBS is Dulbecco’s Phosphate-Buffered Saline (0.10 g / L anhydrous CaCl2; 0.20 g / L KC1; 0.20 g / L KH2PO4; 0.10 g / L MgCl2-6H2O; 8.00 g / L NaCl; anhydrous Na2HPO4; 2.1716 g / L Na2HPO4-7H2O; pH 7.4).• DPBS-CMF is calcium and magnesium-free DPBS (0.10 g / L anhydrous CaCl2; 0.20 g / L KC1; 0.20 g / L KH2PO4; 8.00 g / L NaCl; anhydrous Na2HPO4; 2.1716 g / L Na2HPO4-7H2O; pH 7.4).• DMEM is Dulbecco’s Modified Eagle Medium using the Coming formulation.• D02 is Dulbecco’s Modified Eagle Medium supplemented with 2% (v / v) Fetal Bovine Seram, 100 U / mL penicillin, and 100 pg / mL of streptomycin (all concentrations final).• DIO is Dulbecco’s Modified Eagle Medium supplemented with 10% (v / v) Fetal Bovine Seram, 100 U / mL penicillin, and 100 pg / mL of streptomycin (all concentrations final).• MEM is a minimal essential media.• Oxoid agar is Oxoid™ Purified Agar (Thermo Fisher Scientific, cat. No. LP0028B).• sterile saline is 0.9 g / L NaCl in WFI.• 100% ethanol or absolute ethanol is >99.5% ethanol (e.g., MilliporeSigma, cat. No 459836).• 70% ethanol is 70% (v / v) ethanol in NFW (see below).• dH2O is nuclease-free water with resistivity >18.0 megaohm-cm.• NFW is dH2O that is certified nuclease-free (DNAse-free and RNAse-free)• PFA is paraformaldehyde.

[0210] Unless otherwise noted, water-soluble solutions were usually prepared using dH2O.

[0211] PCR means “polymerase chain reaction”; “RT” means reverse transcription unless used in reference to temperature, and then is interpreted to mean room temperature; RT-qPCR means reverse-transcription quantitative polymerase chainreaction; Cq is the cycle of quantification, in qPCR or RT-qPCR the cycle at which the reaction signal exceeds a threshold. Unless otherwise indicated, all reactions usually occurred at a pressure of 89-101 kPa.

[0212] Room temperature is any temperature in the interval from about 19 °C to about 26 °C.

[0213] Unless otherwise specified, kits and specified materials were used according to their manufacturers’ instructions.Nanoparticle formulation• FM00 is DPBS• FM01 is 5% (m / v) sucrose in DPBS-CMFImmunostaining• PBSM is DPBS-CMF with 5% (m / v) dry nonfat milk• PBSMT is PBSM with 0.05% (v / v) Tween-20• PBST is DPBS-CMF with 0.05% (v / v) Tween-20Other conventions• Opti-MEM means Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific, cat. No. 31985070).• TPCK-trypsin means modified trypsin (obtained from bovine pancreas) that has been treated with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) to inactivate extraneous chymotryptic activity (Millipore Sigma, cat. No. T8802-100MG).• BSA means globulin-free bovine serum albumin, typically sourced as a 35% (m / v) sterile solution from (MP Biomedicals; cat. No. 08810061).Additional Definitions• dpi means days post-infection.• hpi means hours post-infection.• PFU means Plaque Forming Unit.• FFU means Focus Forming Unit.• PFU and FFU, as measures of the level of infectious virus, are used interchangeably, whether a plaque assay or focus-forming unit assay was performed.• Unless otherwise specified, when “n” is any non-negative number, “n log” means 10“ (e.g., 2 log means 100, and 3 log means 1000).• “AU” means arbitrary units. This can correspond to “raw data” as obtained from a raw measurement (e.g., one reported by an instrument) or to consistently transformed “raw data” (e.g., data consistently normalized by subtracting a value or by dividing by a value) to aid comparison within an experiment or between experiments.• means approximately equal to.• Unless otherwise noted, “GFP” or “green fluorescent protein” means any protein that exhibits green fluorescence in cells that are exposed to light in the blue to the ultraviolet (UV) range and can thereby be used to report on the level of protein translation in cells.• Unless otherwise noted, “RFP” or “red fluorescent protein” means any protein that exhibits red to orange fluorescence in cells and can thereby be used to report on the level of protein translation in cells. Other fluorescent proteins are also available and can be substituted depending on their strength in detection.Virus strains

[0214] To aid in illustrating the various embodiments described herein, exemplary viral strains or virus isolates are provided that in no way limit the scope of the invention disclosed. Although particular strains or isolates are described here, other strains, species, or genera of virus may be used as appropriate.

[0215] Alphavirus abbreviations are: “EEEV” means Eastern Equine Encephalitis Virus; “VEEV” means Venezuelan Equine Encephalitis Virus.

[0216] RSV abbreviations are: “RSV A2” or “A2” means Human Respiratory Syncytial Virus, strain A2; and “RSV Bl” or “RBI” means Human Respiratory Syncytial Virus, strain B 1 / 18537.Measurement ofLNP size via Dynamic Light Scattering & Calculation of Polydispersity Index (PDI) from Particle Size Distribution

[0217] Particle size and particle size distribution of LNP formulations were quantified using a standard technique: Dynamic Light Scattering (DLS). DLS was performed using a Zetasizer Pro (Malvern Panalytical, Malvern UK).

[0218] Each LNP sample was diluted 100-fold with DPBS-CMF before analysis. Peak size was reported as the average diameter in nanometers (d.nm) for each separate peak of the distribution, as calculated by the first cumulant or moment of the distribution, using the method specified in ISO method 18022412:2017 (ISO standard on particle size analysis via Dynamic Light Scattering (DLS)).

[0219] Homogeneous monodisperse preparations were expected to have a low polydispersity index (PDI) (typically the PDI < 0.1 for monodisperse preparations), where the PDI was also determined using the Zetasizer Pro. The PDI was determined as the square of the standard deviation divided by the square of the mean particle diameter, and the PDI width is the square root of the PDI times the z-average.Measurement of zeta potential ofLNP mixture

[0220] The zeta-potential (^-potential) of formulated LNPs was also measured using the Zetasizer Pro. The parameters of the Zetasizer Pro were set as follows: the temperature at 25°C, viscosity at 0.8872 (cP), a dielectric constant of 78.6, and Henry function of 1.5. Disposable folded capillary cells (DTS1070, Malvern Instruments) were rinsed thoroughly before use with water, followed by ethanol, and finally, water again using a minimum of 1 mL for each rinse. After the final rinse, capillary cells were air-dried before use. Each LNP sample was prepared at three concentrations in 0.22 pm filtered 10 mM NaCl. Capillary cells were loaded with 1 mL of diluted LNP sample and the averaged phase (over 5 measurements), frequency, and zeta potential distribution were measured.Measurement ofRNA Encapsulation efficiency via exclusion of membrane-impermeableRNA-speciftc dye

[0221] The degree of encapsulation of an RNA within an LNP formulation was estimated by determining the exclusion of a membrane-impermeable, RNA-specific dye from RNA formulated into the LNPs when the LNPs are intact vs. when they are disrupted. As an example, the concentration of LNP-formulated RNA and the efficiency of encapsulation were measured using a membrane-impermeable, RNA-specific RiboGreen dye (Thermo Fisher Scientific, cat. No. R11490). Using this dye, the concentration and encapsulation efficiency of an LNP-formulated RNA with RiboGreen were measured in duplicate following the manufacturer’s directions. The RNA encapsulation efficiency was calculated using the following equation:where ωRNA is the RNA encapsulation efficiency, Ftotai is the total RNA fluorescence, and Fun is the fluorescence component attributable to the RNA outside of the nanoparticles (‘unencapsulated RNA’). LNP formulations suitable for in vitro and in vivo studies typically have CDRNA > 0.85.Measurement ofpKa via TNS

[0222] LNP pKa was determined using TNS (6-(p-Toluidino)-2-naphthalenesulfonic acid, Sigma (T9892)) and an assay according to (Zhang et al., Langmuir (2011); DOI: 10.1021 / 10.102 l / lal04590k). Briefly, LNPs are diluted to 1 and 10 ng / pL (as determined by the concentration of encapsulated mRNA) in a series of buffers with a pH ranging between 3 and 12. Buffered solutions are composed of 150 mM NaCl or 100 mM citric acid / citrate, sodium acetate, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), or 3-morpholinopropane-l -sulfonic acid (MOPS) and 150 mM NaCl. A stock solution of TNS is prepared as a 300 pM solution in DMSO (dimethyl sulfoxide) and then added to the buffered solution containing LNPs to a 6 pM final solution of TNS. The fluorescence of the resulting solution was read on a Spectra Max Ms5 fluorescence plate reader (Molecular Devices) with the excitation wavelength set at 325 nm and the emission wavelength set at 435 nm. The fluorescence of TNS was plotted against the pH and fitted using a three-parameter sigmoid function. In the presence of LNPs, TNS fluorescence reaches a maximum when 100% of the amino lipids are ionized; when theamino lipids are in the un-ionized state, TNS has little fluorescence. The pH values at which half of the maximum fluorescence is reached are reported as the apparent pXa values of the LNP.Measurement of protein translation via flow cytometry

[0223] LNPs containing RNA were applied to adherent cells (cells were at approximately 50-75% cell confluency) at a typical dosage of 5-50 ng of mRNA per cm2of plate surface area. At 24 h post mRNA application, adherent cell cultures were first washed with DPBS, then detached from their plastic substrate by enzymatic dissociation with TrypLE (Thermo Fisher Scientific, cat. No. 12604013) for 5 min at 37 °C. Dissociated cells were subsequently resuspended in a suitable isotonic buffer (often DPBS-CMF supplemented with 5% FBS and 2 mM EDTA, ethylenediaminetetraacetic acid) and loaded into a CytoFlex S flow cytometer (Beckman Coulter).

[0224] Typically, a total of about 10,000 live, single-cell events were recorded for analysis. Live cell events were gated on forward-scatter and side-scatter, and single live cells were further gated on forward scatter height vs. forward scatter area. Subgates corresponding to live fluorescent cell populations were further established based on the spectral characteristics of the fluorescent protein utilized. Single cells are gated by forward and side scatter, and live cells are further gated by fluorescence (e.g., GFP or RFP) intensity. Fluorescent protein gates were set so that no more than 1% of untreated cells fell within a fluorescent-protein positive gate.

[0225] Fluorescence values of cell populations under study were reported as: (i) the median fluorescence intensity of all single, live-cell events; (ii) the fraction of fluorescent-protein positive cells ((fluorescing live cells] / [total live cell population]).

[0226] When reported as normalized fluorescent intensity (fold-increase), the median intensity from samples incubated with empty LNP (without RNA) was used as a basis for normalization.Measurement of secreted, luciferases that convert coelenterazine

[0227] Method A: The culture media samples or tissue homogenate were analyzed with luminescence assay by adding 50 pL QUANTI-Luc luciferase substrate (InvivoGen, cat.No. rep-qlc2) to 10 pL of the sample in triplicates and measuring luminescence signal immediately on a multimode plate reader (PerkinElmer).

[0228] Method B; Luciferase-containing samples were serially diluted by at least 10-fold using DPBS-CMF, and 10 pL of the diluted sample were transferred to white opaque 96- well microplates. Immediately before the reading, coelenterazine substrate (GoldBio, cat. No. CZ2.5) was diluted in DPBS to a final concentration of 3.5 pM and 90 pL of the 3.5 pM coelenterazine solution was added to the sample shortly before the sample was read (i.e., ideally samples were read at < 5 min). Quantification of luciferase activity as measured by detected chemiluminescence activity was using a PerkinElmer VictorX3 2030 Multilabel Reader.Measurement of secreted embryonic alkaline phosphatase which converts 1,2-dioxetaneCSPD (disodium 3-(4-methoxyspiro (l,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3. l.l3,7]decanJ-4-yl)phenyl phosphate)

[0229] Culture media samples were analyzed with luminescence assay by adding 50 pL alkaline phosphatase substrate (Cayman Chemical, cat. No. 600183) to 10 pL of the sample in triplicates and measuring luminescence signal after 15 minutes of incubation at room temperature on a multi-modal plate reader (e.g., PerkinElmer VictorX3 2030 Multilabel Reader or Molecular Devices SpectraMax i3x Multi-Mode Microplate Reader).ELISA (human, mouse, hamster)

[0230] For all ELIS As, a standard curve was created using the manufacturer’s provided standards in the kit, and concentrations of analyte of interest were estimated by fitting a linear least squares regression model to the standard curve and interpolating the test sample analyte concentration from either the absorbance or luminescence of the recorded value.

[0231] Human interferon beta secreted into the cell culture supernatant was detected using a LumiKine Xpress hlFN-P 2.0 ELISA (enzyme linked immunosorbent assay) kit (InvivoGen, cat. No. uex-hifhbv2) according to the manufacturer’s instructions. Astandard curve was created using the manufacturer’s standards provided in the kit, and concentrations of human IFN-P in picomoles / mL (nM) were estimated as described above. Dilutions of the test samples were made at ratios 1:1, 1:25., 1:50, and 1:100 with the manufacturer’s supplied buffer.

[0232] Human interferon lambda (X) 1-3 secreted into cell culture supernatant were detected using a DIY Human IFN Lambda 1 / 2 / 3 (IL-29 / 28A / 28B) ELISA kit (TCM) (PBL Assay Science, cat. No. 61840-1) according to the manufacturer’s instructions. A standard curve was created using the manufacturer’s instructions and standards provided in the kit, and concentrations of human IFN-p in picomoles / mL (nM) were estimated. Dilutions of the test samples were made at ratios 1:1, 1:25, 1:50, and 1:100 with the manufacturer’s supplied buffer.

[0233] The human inflammatory cytokine TNF-a was detected from tissue culture supernatants using the Human TNF-alpha DuoSet ELISA kit (R&D Systems, cat. No. DY210) according to the manufacturer’s instructions. Concentrations of secreted TNF-a in the test samples were calculated as described above. Dilutions of the test samples were made at ratios 1 : 1 with the supplied buffer.

[0234] Mouse interferon beta (IFN-P) secreted into the cell culture supernatant was detected using a LumiKine Xpress mIFNb-2.0 ELISA kit (InvivoGen, cat. No. luex- mifnbv2) according to the manufacturer’s instructions. A standard curve was created using the manufacturer’s provided standards and instructions in the kit, and concentrations of human IFN-P in picomoles / mL (nM) were estimated. Dilutions of the test samples were made at ratios 1:1, 1 :25, and 1 :50 using the supplied buffer.

[0235] Mouse interferon lambda 2 and 3 (IFN-X2 and IFNX3) secreted into cell culture supernatant were detected using a DIY Mouse IFN Lambda 2 / 3 (IL-28A / B) ELISA kit (TCM) (PBL Assay Science, cat. No. 62830-1) according to the manufacturer’s instructions. A standard curve was created using the manufacturer’s provided standards in the kit, and concentrations of human IFN-P in picomoles / mL (nM) were estimated. Dilutions of the samples were made at ratios 1:1, 1:25., 1 :50, or 1 : 100 with the supplied buffer.

[0236] The murine inflammatory cytokine TNF-a was detected from tissue culture supernatants using the Mouse TNF-alpha DuoSet ELISA kit (R&D Systems, cat no.DY410) according to the manufacturer’s instructions. A standard curve was created using the manufacturer’s provided standards and instructions in the kit and concentrations of human TNF-a in picomoles / mL (nM) were estimated.Isolation of cell-free or viral RNA from cell-free materials

[0237] RNA was extracted from infected cell culture supernatants using the Qiagen Viral RNA Mini Kit (Qiagen, cat. No. 52906) according to the manufacturer’s protocol with one exception. During RNA extraction, 5 pL of isolated MS2 bacteriophage genomic RNA (typically < 100 pg) was added into each mL of the AVL buffer used for lysis as a spike-in standard for downstream quantitation to serve as a surrogate “housekeeping” species for normalization of downstream real-time RT-PCR between samples and analysis runs.Quantification ofcellular / tissue gene expression changes via RT-qPCR

[0238] RNA was purified from cultured cells using the Monarch Total RNA Miniprep Kit (New England Biolabs, cat. No. T2010S) according to the manufacturer’s protocol with one exception. As DNAse treatment was included as a part of subsequent downstream assays, the optional ‘on-column DNAse treatment’ was omitted.

[0239] RNA was purified from animal tissue using one of two methods: (i) Monarch Total RNA Miniprep Kit (New England Biolabs, cat. No. T2010S) according to the manufacturer’s protocol, but omitting the optional ‘on-column DNAse treatment’ as described above; (ii) TRIzol Reagent (Thermo Fisher Scientific, cat. No. 15596018) according to the manufacturer’s protocol.

[0240] Purified cellular RNA, whether obtained from in vitro cultured cells or animal tissues, was subsequently treated with DNAse from the TURBO DNAse-Free kit (Thermo Fisher Scientific, cat. No. AM1907), and the DNAse enzyme removed using the supplied inactivation reagent according to the manufacturer’s instructions.

[0241] Gene expression changes were quantified by performing reverse transcription quantitative real-time PGR (RT-qPCR) on purified RNA isolated from cells. RT-qPCR was performed using either dye-based (e.g., SYBR Green I) or hydrolysis-probe-based (e.g., TaqMan probes) chemistries.

[0242] Complementary DNA (cDNA) was prepared from DNAse-treated RNA in 20 pL reverse transcription reactions using reagents obtained from ProtoScript® II First Strand cDNA Synthesis Kit (New England Biolabs, cat no. E6560L). First, 6 pL of DNA-free RNA was combined with 2 pL Random Primer Mix, and the combined volume was heated to 65°C for 5 min before cooling on ice. Random Primer Mix (60 pM) contains 35 pM random hexamers, 25 pM dTasVN (SEQ ID NO: 451), and 1 mM dNTPs in 5 mM Tris-HCl (pH 8.0) and 0.5 mM EDTA.) Next, 10 pL of ProtoScript® II Reaction mix and 2 pL of ProtoScript® II Enzyme mix were added, and the reaction was gently mixed. The reverse transcription reaction was carried out for ~1 h according to the manufacturer’s protocol (25 °C for 5 min; 42 °C for 1 h; 80 °C for 5 min). The resultant cDNA was used for further qPCR analysis. All cDNA templates were diluted (typically within dilution ratios of 1 :2 to 1 : 10 in NFW) before use in qPCR reactions.

[0243] Dye-based qPCR was performed with 20 pL reaction volumes assembled by combining the following for each reaction: 10 pL of 2x Luna® Universal qPCR Master Mix (New England Biolabs. Cat no. M3003E), 5 pL of the diluted cDNA, 2.5 pL of 1 pM forward primer in NFW (125 nM final), 2.5 pL of 1 pM reverse primer in NFW (125 nM final).

[0244] Final reactions were set up in a MicroAmp Optical 96-well Reaction Plate(Thermo Fisher Scientific, cat. No. 4306737) and analyzed in an ABI 7300 Real-Time PCR machine (Applied Biosystems, Inc.). The thermocycling conditions are listed in Table 6 below:Hydrolysis Probe-based qPCR

[0245] For hydrolysis probe-based qPCR, hydrolysis probes were designed and obtained from MilliporeSigma or Integrated DNA Technologies and diluted, if necessary, to 100 pM concentration with 10 mM Tris-HCl, pH 8.0 or NFW. A lOx Probe / primer mix wascreated at the following concentrations for probe and primers: hydrolysis probe (1 pM), forward primer (4 pM), and reverse primer (4 pM) in NFW. The final reaction master mix was set up using 2x Luna® Universal Probe qPCR Master Mix (New England Biolabs, cat no. M3004E), lx the Probe / primer mix, and 5 pL of the diluted cDNA in a total reaction volume of 20 pL.

[0246] Final reactions were set up in a MicroAmp Optical 96-well Reaction Plate (Thermo Fisher Scientific, cat. No. 4306737) and analyzed in an ABI 7300 Real Time PCR machine (Applied Biosystems, Inc.). The thermocycling conditions used are listed in Table 6.RSV infectious titer via focus-forming unit assay

[0247] Virus supernatants were serially diluted in Opti-MEM. The 24-well dishes of Hep-2 cells were infected with various serial dilutions of Respiratory Syncytial Virus (RSV) stocks in 200 pL of Opti-MEM and incubated at 37 °C in 5% CO2 for 2 h, gently rocking every 15 min. After removing the inoculum, the wells were overlaid with 2 mL of DMEM containing 2% FBS and 1% dissolved medium-density carboxymethylcellulose (Millipore-Sigma, cat. No. C4888-500G). The plates were incubated at 37°C in a humidified 5% CO2 cell incubator for 5 days without disturbing.

[0248] Six (6) days post-infection, the plaques in the monolayers were fixed using 1 mL of 4% PF A per well for a minimum of 1 h. Following this, the overlay plugs were removed by gentle tapping and water flow. Plates were then washed with PBS and treated with 0.5% (v / v) IGEPAL (octylphenoxypolyethoxyethanol) CA630 for 10 min and washed 2x with PBS. Subsequently, plates were incubated with PBSM to block for 1 h at room temperature on a shaker. After blocking, 1: 1000 dilution of anti-RSV goat polyclonal antibody (Abeam, cat. No. ab20745) in PBSMT, was added, and the plates were incubated for 4 h at room temperature on a rocker. The plates were washed 3x with PBST and then incubated with a 1: 1000 dilution of Peroxide-Conjugated Affinipure Rabbit anti-Goat Antibody (Jackson Immunoresearch, cat no. 305-035-003) in PBSMT and the plates incubated for 1 h at room temperature on a rocker with gentle agitation (40 rpm, 10° tilt).

[0249] Following incubation with the secondary antibody, the wells were washed 3x with PBST, and the plaques were developed using the TMB solution for blotting (Life Technologies Inc. Cat No. 002019). Viral titers were quantified as PFU / mL of tissue culture supernatant. For RS V bearing a fluorescent reporter gene encoded into the viral genome, plaques were directly counted from the wells and imaged.Example 1: Construction of alphavirus encrypted RNA scaffolds and their DNA- encoded cassettes

[0250] DNA sequences were designed computationally and cloned by standard molecular biology methods. Source templates were typically obtained via amplification by PCR, restriction digest, synthetic oligonucleotides, or by custom synthesis of dsDNA via assembly of dsDNA from pools of overlapping and complementary oligonucleotides (e.g. gBlock or eBlock fragments available from Integrated DNA Technologies (IDT)). The identity of cloned DNA molecules was confirmed by a combination of restriction digests, Sanger sequencing (Genewiz), or next-generation sequencing (NGS) based on Illumina DNA sequencing technology.

[0251] The initial alphavirus antisense encrypted RNA payloads were obtained by combining three independent sequence blocks (L, C, and R regions) using the encrypted RNA scaffold assembly scheme designated in Table 2. For example, ERNA-EEEV-503- GDura was generated by concatenation of sense_5p_EEEV_24 (SEQ ID NO: 421 ), the GDura coding sequence (CDS_GDura, SEQ ID NO: 273), and sense_3p_EEEV_24 (SEQ ID NO: 432) in 5' to 3' order.

[0252] Introduction of additional protein payloads into alphaviral encrypted RNA scaffolds

[0253] The above alphaviral encrypted RNA scaffolds that can encode for GDura were used as a basis to similarly encode alternative polypeptides of interest listed in Table 3 or Table 5 or Table 7 or others, including: EGFP, RlucS, human interleukin 2 (IL-2), human interleukin 12 (IL- 12), human IFN-P, human IFN-lambdal, human IFN- lambda3, mouse IFN-P, mouse IFN-lambda2, mouse IFN-lambda3, mouse interleukin2 (IL-2), mouse interleukin 12 (IL-12), Syrian hamster IFN-P, or domestic ferret 1FN- P.

[0254] To avoid ambiguity, “hu_IFNB” means “human IFN-beta”; “m_IFNB” means“mouse IFN-beta”.

[0255] Similarly, alphaviral encrypted RNA scaffolds encoding alternative polypeptides of interest were created by replacing CDS_GDura or rcCDS_GDura with another sense or antisense CDS, respectively, selected from Table 3 or Table 5 or Table 10.DNA-encoding of alphaviral encrypted RNA for in vitro production of encrypted RNA

[0256] A DNA-encoded cassette for the production of encrypted RNA via in vitro transcription was generated by cloning an alphaviral encrypted RNA scaffold in between a T7 promoter on the 5' end (Promoter_T7_Core, SEQ ID NO: 331) and a convenient 3' 5apl restriction site (RE_SapI_3p, SEQ ID NO: 335) within the MCS of the pUC19 vector. For simplicity, this construct is referred to as pAT201-X, where X is the alphaviral encrypted RNA scaffold. After linearization of a pAT201 -series plasmid via SapI digestion, the linearized template can used to produce uncapped, 5'- triphosphorylated transcripts with standard in vitro transcription with four classic ribonucleotides (ATP, CTP, GTP, UTP) or 5'-capped transcripts (Cap 1) via co- transcriptional capping (e.g., by using CleanCap reagents, TriLink BioTechnologies).Example 2: Activation of alphaviral encrypted RNAs by providing isolated mRNA that can be translated to comprise a target-specific translation activator

[0257] In this example, alphavirus encrypted RNAs (alphaviral encrypted RNAs) were tested for functionality by quantifying the increase in activation in response to a suitable target-specific translation activator.

[0258] Alphaviral target-specific translation activators can be a set of one or more DNA- encoded or IVT-produced mRNAs, which together comprise an alphaviral target-specific translation activator comprising alphaviral non-structural proteins nsPl, nsP2, nsP3, and nsP4. When nsPl, nsP2, nsP3, and nsP4 were encoded as a single polyprotein, the nomenclature is “nsPl -4” or “single-chain”. When nsPl , nsP2, and nsP3 were encoded as single polyprotein (nsPl-3) but nsP4 was individually and separately encoded, the nomenclature is “nsP1-3+nsP4” or “split-chain”.

[0259] Single-chain and split-chain alphaviral target-specific translation activators for EEEV and VEEV were cloned into plasmid vectors suitable for in vitro transcription (via a T7 promoter) or intracellular production (via a polll promoter). The reference nucleotide sequence for EEEV was based on strain EEEV / Culex / USA / T-2061 / 2003 (NCBI GenBank with ID KJ469627.1). The reference nucleotide sequence for VEEV was based on strain VEEV / donkey / Trinidad / 1943 (NCBI GenBank ID: NC075022.1).

[0260] Both single-chain and split-chain alphaviral target-specific translation activators were provided directly as mRNA by performing in vitro transcription and incorporating aCap analog into the reaction (e.g., CleanCap AG). For single-chain translation activators, the resultant product was a single mRNA species encoding nsPl-4. For split-chain translation activators, the resulting product was a mixture of two mRNA species: one encoding nsPl-3 and the other encoding nsP4.

[0261] EEEV and VEEV encrypted RNAs were generally prepared by IVT as described above. In some instances, these encrypted RNAs were modified by capping or nucleoside modification. Some capping modifications included Clean Cap AU. For nucleoside modification, between 30% and 100% of the internal uridine nucleotides were modified to pseudouridine or N1 -methylpseudourine (e.g., 30%, 50%, 70%, or 100%).

[0262] A typical experiment, performed using a 48-well plate scale, utilized cells known to support alphavirus replication (e.g., Human 293T, simian Veto E6, or hamster BHK- 21 cells). When single-chain translation activators were employed (whether DNA or mRNA), cells were treated with “DNA or mRNA pool_ A” comprising 125 ng of nsPl-4 from either VEEV or EEEV. When split-chain translation activators were utilized (whether DNA or mRNA), cells were treated with a total mass of 125 ng of nsPl-3+nsp4 but with varied ratios of nsPl-3:nsP4 by mass; (i) “DNA or mRNA pool_ B” comprising a 1:1 (nsPl-3: nsP4) mass ratio, (ii) “DNA or mRNA pool_ C” comprising a 4:1 (nsPl- 3:nsP4) mass ratio; or (iii) “DNA or mRNA pool_ D” comprising a 1:4 (nsPl-3:nsP4) mass ratio.

[0263] When cells were treated with an encrypted RNA, the mass of encrypted RNA was identical to the amount of single-chain or split-component pool set, e.g., 125 ng of ERNA-EEEV-GDura matched to 125 ng of DNA or mRNA pool_ A.

[0264] In a typical experiment, GDura-encoding alphavirus encrypted RNAs were tested for functionality by quantifying the increase in activation in response to a suitable targetspecific translation activator. For instance, to test the function of ERNA-EEEV-503- GDura, Human 293T, simian Vero E6, or hamster BHK21 cells in 48-well plates (120K / well) were treated simultaneously by one of the following: (Treatment 1) no RNA; (Treatment 2) -125 ng of ERNA-EEEV-503-GDura; (Treatment 3) -125 ng of “DNA or mRNA Pool_A”; (Treatment 4) -125 ng of “DNA or mRNA Pool_B”; (Treatment 5) -125 ng of “DNA or mRNA Pool_C”; (Treatment 6) -125 ng of “DNA or mRNA Pool_D”; (Treatment 7) -125 ng of ERNA-EEEV-503-GDura and -125 ng of strain-specific “DNA or mRNA Pool_A”; (Treatment 8) -125 ng of ERNA-EEEV-503-GDura and -125 ng of strain-specific “DNA or mRNA PoolJB”; (Treatment 9) -125 ng of ERNA-EEEV-551 -GDura and -125 ng of strain-specific “DNA or mRNA Pool_C”, or (Treatment 10) -125 ng of ERNA-EEEV-503-GDura and -125 ng of strain-specific “DNA or mRNA PoolJD”. In some cases, additional treatment with the second dose of 125 ng of species-specific ERNA was applied 16-24 h after initial treatment.

[0265] An equivalent study was performed using ERNA-VEEV-503-GDura and VEEV target-specific translation activators.

[0266] When DNA-encoded activators were used, Lipofectamine 2000 transfection reagent was used for activator delivery or activator and ERNA co-delivery into the cells. For mRNA-encoded activators and ERNA delivery, Lipofectamine MessengerMax (LMAX) transfection reagent was used. The amount of the translated polypeptide of interest (secreted GDura luciferase) was quantified at 24 or 48 hours post-treatment as described previously.

[0267] In this example, if the level of GDura increased more than lOx relative to untransfected cells, then the polypeptide of interest (GDura) was said to have increased translation.

[0268] Only negligible background levels of translation of the polypeptide of interest were observed in untreated cells (Treatment 1) or in cells receiving only target-specific translation activators (Treatments 3-6). In cells that received only an encrypted RNA (Treatment 2), increased levels of GDura were observed, typically lOx above the background level. In contrast, the polypeptide of interest (GDura) had increased translation when cells were treated with both an encrypted RNA and a target-specific translation activator of the encrypted RNA (Treatments 7-10), reaching from ~100x to l,000x above background levels when measured 48 hours after treatments (or 10-100x above levels in the presence of an encrypted RNA only).

[0269] FIGs. 4A, 4B, and 4C describe and show experiments to test the level of activation of alphavirus encrypted RNA after treating cells with a target-specific translation activator comprising nsPl, nsP2, nsP3, and nsP4 of an alphavirus. FIG. 4A shows a schematic of the experiment. FIG. 4B shows that an EEEV encrypted RNA canbe activated by EEEV nsPl-4. FIG. 4C shows that a VEEV encrypted RNA can be activated by VEEV nsPl-4.Example 3: Alphaviral encrypted RNA support nucleoside-modification and S’ capping

[0270] To test the effect of nucleoside-modification or 5' capping on the activation of alphaviral encRNA (encrypted RNA), the experimental protocol of Example 2 was repeated, but using alphaviral encRNA (VEEV and EEEV scaffolds) prepared with nucleoside-modification and 5' CleanCap AU. It was found that alphaviral encRNA (both EEEV and VEEV) were tolerant to uridine modification to pseudouridine and Nl- methyl-pseudouridine at rates from 0-100% (except for the first uridine at the 5' end), and were able to be activated by the target-specific Translation Activator component.

[0271] Further, background translation (in the absence of the cognate TranslationActivator was reduced by eliminating CleanCap AU or increasing the proportion of modified uridine nucleotides in the encrypted RNA (except for the first uridine at 5' end).Example 4: Cross-activation of encRNA by alternative alphaviral Translation Activators

[0272] The studies of Example 2 were repeated, but using alphaviral encrypted RNAs and translation activators derived from different alphavirus species (e.g., VEEV encRNA and EEEV nsPl-4). Not all pairings of encRNA and nsPl-4 were tested. The results shown in FIG. 7 A demonstrates that EEEV nsPl-4 was able to activate SINV, EEEV, VEEV, and WEEV encRNA, while VEEV nsPl-4 was able to activate SINV, EEEV, VEEV encRNA. VEEV encrypted RNA was strongly activated by EEEV (2 log), but EEEV encrypted was modestly activated by VEEEV (0.5 log). These results indicate that certain alphaviral encRNA may be activated by more than one species of alphaviral Translation Activator and may be a distinct species.Example 5: Alphavirus encrypted RNAs can express arbitrary genetic payloads

[0273] The generality of the platform in the delivery and conditional activation of arbitrary genetic payloads, including a Green Fluorescent Protein (mNeongreen) andhuman interferon beta (IL12), were evaluated next. The functional performance of the orthogonal payloads was determined using a procedure analogous to Example 2 with the EEEV encrypted RNA or VEEV encrypted RNA encoding either CDS_mNeongreen or CDSJFNB.

[0274] When the encRNA encoded IFNB, it was found that dynamic expression of IFN- beta (as measured by ELISA) was >l,00x above background levels when activated by the cognate translation activator and <2x above background levels in the absence of the cognate translation activator.

[0275] FIGs. 5 A and 5B show the activation of two preparations of EEEV encrypted RNA encoding human interferon-beta in BHK-21 cells (FIG. 5 A) and Vero cells (FIG. 5B) by various EEEV target-specific translation activators. In both cases, activation of the encrypted RNA by the target-specific translation activator leads to >100x increase in secreted interferon levels, exceeding an interferon-beta concentration of 10,000 pg / mL, which is significantly above the antiviral concentration threshold of approximately 100 pg / mL.Example 6: Antiviral effect mediated by an alphaviral encRNA that encodes antiviral polypeptides of interest

[0276] Supernatant from Example 5 was added to HEp-2 cells and then infected with RSV to test if secreted interferon levels were sufficient to initiate an innate antiviral response.

[0277] FIG. 6A & 6B show that activation of an EEEV encrypted RNA, encoding an antiviral cytokine (human interferon beta), by EEEV nsPl-4 can lead to secreted levels of interferon that are sufficient to control viral infection, as measured by supernatant transfer to a (surrogate) RSV infection. FIG. 6A shows that only an EEEV encrypted RNA encoding an antiviral payload (human interferon beta) yields the antiviral effect when activated in BHK-21 cells. FIG. 6B shows the same, but when treatment was performed in Vero cells.Example 7: Antiviral activity against replication-competent alphaviruses mediated by an alphaviral encRNA that encodes antiviral polypeptides of interest

[0278] To demonstrate the antiviral activity of alphaviral encRNA against pathogenic human alphaviruses (e.g., EEEV and VEEV), an in vitro antiviral activity assay of alphaviral encRNA was performed using Vero E6 cells. All studies with replication- competent EEEV and VEEV were performed in a BSL-3 laboratory.

[0279] At 24 h before infection, Vero E6 cells were treated with lipid nanoparticle formulated alphaviral encRNA or controls using one of five following treatments:■ (Treatment 1) DIO media only;■ (Treatment 2) 100 pM ERNA-EEEV-503-IFNB encRNA;■ (Treatment 3) 100 pM ERNA-EEEV-503-GDura encRNA;■ (Treatment 4) 100 pM ERNA-VEEV-503-IFNB encRNA; or■ (Treatment 5) 100 pM ERNA-VEEV-503-GDura encRNA.Importantly, no significant cytotoxicity was observed at the 100 pM dosage levels prior to viral infection. At 0 hpi, the culture media was exchanged with fresh D10 media, and cells were infected with EEEV (strain V105) or VEEV (strain INH9813) at an MOI of 0.01. At 48 hpi, the cell culture media was harvested, and the level of infectious virus in each sample was quantified by plaque assay titration.

[0280] FIGs. 8A and 8B show that alphaviral encrypted RNAs encoding an antiviral cytokine (human interferon beta) have antiviral activity in Vero E6 cells against EEEV or VEEV and reduced virus levels by >1000 x. FIG. 8A shows that an EEEV encrypted RNA encoding human interferon beta has antiviral activity against EEEV (strain V105) as Treatment 2 (100 pM ERNA-EEEV-503-IFNB) reduced EEEV viral load by approximately l,000,000x (6 loglO). In contrast, treatment with an analogous alphaviral encrypted RNA that encoded a non-antiviral Gaussia luciferase payload ERNA-EEEV- 503-GDura (Treatment 3) (“sham encrypted RNA” in FIG 8A) had negligible antiviral activity, reducing viral loads by <0.3 loglO. Similarly, FIG. 8B shows that a VEEV encrypted RNA encoding human interferon beta has antiviral activity against VEEV (strain INH9813) as Treatment 4 (100 pM ERNA-VEEV-503-IFNB) reduced VEEV viral loads by approximately 3,000x (3.5 loglO), while treatment with an analogous alphaviralencrypted RNAs that encoded the non-antiviral ERNA-VEEV-503-GDura (“sham encrypted RNA” in FIG. 8B) had only negligible antiviral activity.

[0281] Given the examples disclosed herein with encrypted alphaviral RNA constructs with exemplary therapeutic polypeptides, an ordinary practitioner in the field would have believed that similar results would have been obtained if using other therapeutic polypeptides (i.e., achieving an equivalent level of increased production using the processes described herein).

[0282] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls.EMBODIMENTSEmbodiment 1. An alphaviral encrypted RNA comprising:(i) a coding region comprising a coding sequence encoding a therapeutic polypeptide,(ii) a left flanking region (L region) adjacent to and contiguous with a 5’ end of the coding region; and(iii) a right flanking region (R region) adjacent to and contiguous with a 3* end of the coding region; wherein both the L region and the R region of the alphaviral encrypted RNA are derived from an alphavirus, and wherein, once the alphaviral encrypted RNA is inside a cell containing a translation activator, the L region and the R region interact with the translation activator, thereby resulting in translation of the therapeutic polypeptide.Embodiment 2. The alphaviral encrypted RNA of Embodiment 1, wherein each of the L region and the R region is derived from the reverse complement of a corresponding region that is native to the alphavirus.Embodiment 3. The alphaviral encrypted RNA of Embodiment 1 or Embodiment 2, wherein the alphavirus is Chikungunya virus, Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Sindbis Virus (SINV), Semliki Forest Virus (SFV), or Western Equine Encephalitis Virus (WEEV).Embodiment 4. The alphaviral encrypted RNA of any one of Embodiments 1 to 3, wherein the translation activator is an RNA dependent polymerase.Embodiment 5. The alphaviral encrypted RNA of Embodiment 4, wherein the RNA dependent polymerase is a viral RNA dependent polymerase, optionally an RNA- dependent RNA polymerase or an RNA-dependent DNA polymerase.Embodiment 6. The alphaviral encrypted RNA of any one of Embodiments 1 to 5, wherein the coding sequence within the alphaviral encrypted RNA is in an antisense orientation.Embodiment 7. The alphaviral encrypted RNA of Embodiment 6, wherein a combination of the L region and the R region satisfies one of the following:(i) the L region comprises SEQ ID NO: 249 or a variant of SEQ ID NO: 249, wherein the variant of SEQ ID NO: 249 comprises a variation at one or more positions of positions 21-354 of SEQ ID NO: 249, and the R region comprises SEQ ID NO: 441 or a variant of SEQ ID NO: 441, wherein the variant of SEQ ID NO: 441 comprises a variation at one or more positions of positions 21-323 of SEQ ID NO: 441;(ii) the L region comprises SEQ ID NO: 438 or a variant of SEQ ID NO: 438, wherein the variant of SEQ ID NO: 438 comprises a variation at one or more positions of positions 21- 113 of SEQ ID NO: 438 and the R region comprises SEQ ID NO: 442 or a variant of SEQ ID NO: 442, wherein the variant of SEQ ID NO: 442 comprises a variation at one or more positions of positions 21-353 of SEQ ID NO: 442;(iii) the L region comprises SEQ ID NO: 438 or a variant of SEQ ID NO: 438, wherein the variant of SEQ ID NO: 438 comprises a variation at one or more positions of positions 21- 113 of SEQ ID NO: 438 and the R region comprises SEQ ID NO: 443 or a variant of SEQ ID NO: 443, wherein the variant of SEQ ID NO: 443 comprises a variation at one or more positions of positions 21-517 of SEQ ID NO: 443;(iv) the L region comprises SEQ ID NO: 438 or a variant of SEQ ID NO: 438, wherein the variant of SEQ ID NO: 438 comprises a variation at one or more positions of positions 21 - 113 of SEQ ID NO: 438 and the R region comprises SEQ ID NO: 444 or a variant of SEQ ID NO: 444, wherein the variant of SEQ ID NO: 444 comprises a variation at one or more positions of positions 21-168 of SEQ ID NO: 444;(v) the L region comprises SEQ ID NO: 439 or a variant of SEQ ID NO: 439, wherein the variant of SEQ ID NO: 439 comprises a variation at one or more positions of positions 21- 256 of SEQ ID NO: 439, and the R region comprises SEQ ID NO: 445 or a variant of SEQ ID NO: 445, wherein the variant of SEQ ID NO: 445 comprises a variation at one or more positions of positions 21-355 of SEQ ID NO: 445;(vi) the L region comprises SEQ ID NO: 439 or a variant of SEQ ID NO: 439, wherein the variant of SEQ ID NO: 439 comprises a variation at one or more positions of positions 21- 256 of SEQ ID NO: 439, and the R region comprises SEQ ID NO: 446 or a variant of SEQ ID NO: 446, wherein the variant of SEQ ID NO: 446 comprises a variation at one or more positions of positions 21-286 of SEQ ID NO: 446;(vii) the L region comprises SEQ ID NO: 439 or a variant of SEQ ID NO: 439, wherein the variant of SEQ ID NO: 439 comprises a variation at one or more positions of positions 21- 256 of SEQ ID NO: 439, and the R region comprises SEQ ID NO: 447 or a variant of SEQ ID NO: 447, wherein the variant of SEQ ID NO: 447 comprises a variation at one or more positions of positions 21-293 of SEQ ID NO: 447;(viii) the L region comprises SEQ ID NO: 440 or a variant of SEQ ID NO: 440, wherein the variant of SEQ ID NO: 440 comprises a variation at one or more positions of positions 21-74 of SEQ ID NO: 440, and the R region comprises SEQ ID NO: 445 or a variant of SEQ ID NO:445, wherein the variant of SEQ ID NO: 445 comprises a variation at one or more positions of positions 21-355 of SEQ ID NO: 445;(ix) the L region comprises SEQ ID NO: 440 or a variant of SEQ ID NO: 440, wherein the variant of SEQ ID NO: 440 comprises a variation at one or more positions of positions 21-74 of SEQ ID NO: 440, and the R region comprises SEQ ID NO: 446 or a variant of SEQ ID NO:446, wherein the variant of SEQ ID NO: 446 comprises a variation at one or more positions of positions 21-286 of SEQ ID NO: 446 and(x) the L region comprises SEQ ID NO: 440 or a variant of SEQ ID NO: 440, wherein the variant of SEQ ID NO: 440 comprises a variation at one or more positions of positions 21 -74 of SEQ ID NO: 440, and the R region comprises SEQ ID NO: 447 or a variant of SEQ ID NO:447, wherein the variant of SEQ ID NO: 447 comprises a variation at one or more positions of positions 21-293 of SEQ ID NO: 447.Embodiment 8. The alphaviral encrypted RNA of any one of Embodiments 1 to 5, wherein the coding sequence within the alphaviral encrypted RNA is in a sense orientation.Embodiment 9. The alphaviral encrypted RNA of Embodiment 8, wherein a combination of the L region and the R region satisfies one of the following:(i) the L region comprises SEQ ID NO: 421 or a variant of SEQ ID NO: 421, wherein the variant of SEQ ID NO: 421 comprises a variation at one or more positions of positions 21-323 of SEQ ID NO: 421, and the R region comprises SEQ ID NO: 432 or a variant of SEQ ID NO: 432, wherein the variant of SEQ ID NO: 432 comprises a variation at one or more positions of positions 21-363 of SEQ ID NO: 432;(ii) the L region comprises SEQ ID NO: 422 or a variant of SEQ ID NO: 422, wherein the variant of SEQ ID NO: 422 comprises a variation at one or more positions of positions 21- 515 of SEQ ID NO: 422, and the R region comprises SEQ ID NO: 432 or a variant of SEQ ID NO: 432, wherein the variant of SEQ ID NO: 432 comprises a variation at one or more positions of positions 21-363 of SEQ ID NO: 432;(iii) the L region comprises SEQ ID NO: 423 or a variant of SEQ ID NO: 423, wherein the variant of SEQ ID NO: 423 comprises a variation at one or more positions of positions 21- 398 of SEQ ID NO: 423, and the R region comprises SEQ ID NO: 432 or a variant of SEQ ID NO: 432, wherein the variant of SEQ ID NO: 432 comprises a variation at one or more positions of positions 21-363 of SEQ ID NO: 432;(iv) the L region comprises SEQ ID NO: 421 or a variant of SEQ ID NO: 421, wherein the variant of SEQ ID NO: 421 comprises a variation at one or more positions of positions 21- 323 of SEQ ID NO: 421, and the R region comprises SEQ ID NO: 433 or a variant of SEQ ID NO: 433, wherein the variant of SEQ ID NO: 433 comprises a variation at one or more positions of positions 21-399 of SEQ ID NO: 433;(v) the L region comprises SEQ ID NO: 422 or a variant of SEQ ID NO: 422, wherein the variant of SEQ ID NO: 422 comprises a variation at one or more positions of positions 21 - 515 of SEQ ID NO: 422, and the R region comprises SEQ ID NO: 433 or a variant of SEQ ID NO: 433, wherein the variant of SEQ ID NO: 433 comprises a variation at one or more positions of positions 21-399 of SEQ ID NO: 433;(vi) the L region comprises SEQ ID NO: 423 or a variant of SEQ ID NO: 423, wherein the variant of SEQ ID NO: 423 comprises a variation at one or more positions of positions 21- 398 of SEQ ID NO: 423, and the R region comprises SEQ ID NO: 433 or a variant of SEQ ID NO: 433, wherein the variant of SEQ ID NO: 433 comprises a variation at one or more positions of positions 21-399 of SEQ ID NO: 433;(vii) the L region comprises SEQ ID NO: 424 or a variant of SEQ ID NO: 424, wherein the variant of SEQ ID NO: 424 comprises a variation at one or more positions of positions 21- 574 of SEQ ID NO: 424, and the R region comprises SEQ ID NO: 434 or a variant of SEQ ID NO: 434, wherein the variant of SEQ ID NO: 434 comprises a variation at one or more positions of positions 21-122 of SEQ ID NO: 434;(viii) the L region comprises SEQ ID NO: 425 or a variant of SEQ ID NO: 425, wherein the variant of SEQ ID NO: 425 comprises a variation at one or more positions of positions 21- 589 of SEQ ID NO: 425, and the R region comprises SEQ ID NO: 434 or a variant of SEQ ID NO: 434, wherein the variant of SEQ ID NO: 434 comprises a variation at one or more positions of positions 21-122 of SEQ ID NO: 434;(ix) the L region comprises SEQ ID NO: 426 or a variant of SEQ ID NO: 426, wherein the variant of SEQ ID NO: 426 comprises a variation at one or more positions of positions 21- 517 of SEQ ID NO: 426, and the R region comprises SEQ ID NO: 434 or a variant of SEQ ID NO: 434, wherein the variant of SEQ ID NO: 434 comprises a variation at one or more positions of positions 21-122 of SEQ ID NO: 434;(x) the L region comprises SEQ ID NO: 424 or a variant of SEQ ID NO: 424, wherein the variant of SEQ ID NO: 424 comprises a variation at one or more positions of positions 21- 574 of SEQ ID NO: 424, and the R region comprises SEQ ID NO: 260 or a variant of SEQ ID NO: 260, wherein the variant of SEQ ID NO: 260 comprises a variation at one or more positions of positions 21-124 of SEQ ID NO: 260;(xi) the L region comprises SEQ ID NO: 425 or a variant of SEQ ID NO: 425, wherein the variant of SEQ ID NO: 425 comprises a variation at one or more positions of positions 21 - 589 of SEQ ID NO: 425, and the R region comprises SEQ ID NO: 260 or a variant of SEQ ID NO: 260, wherein the variant of SEQ ID NO: 260 comprises a variation at one or more positions of positions 21-124 of SEQ ID NO: 260;(xii) the L region comprises SEQ ID NO: 426 or a variant of SEQ ID NO: 426, wherein the variant of SEQ ID NO: 426 comprises a variation at one or more positions of positions 21- 517 of SEQ ID NO: 426, and the R region comprises SEQ ID NO: 260 or a variant of SEQ ID NO: 260, wherein the variant of SEQ ID NO: 260 comprises a variation at one or more positions of positions 21-124 of SEQ ID NO: 260;(xiii) the L region comprises SEQ ID NO: 431 or a variant of SEQ ID NO: 431, wherein the variant of SEQ ID NO: 431 comprises a variation at one or more positions of positions 21- 306 of SEQ ID NO: 431, and the R region comprises SEQ ID NO: 437 or a variant of SEQ ID NO: 437, wherein the variant of SEQ ID NO: 437 comprises a variation at one or more positions of positions 21-304 of SEQ ID NO: 437;(xiv) the L region comprises SEQ ID NO: 427 or a variant of SEQ ID NO: 427, wherein the variant of SEQ ID NO: 427 comprises a variation at one or more positions of positions 21- 355 of SEQ ID NO: 427, and the R region comprises SEQ ID NO: 435 or a variant of SEQ ID NO: 435, wherein the variant of SEQ ID NO: 435 comprises a variation at one or more positions of positions 21-265 of SEQ ID NO: 435;(xv) the L region comprises SEQ ID NO: 428 or a variant of SEQ ID NO: 428, wherein the variant of SEQ ID NO: 428 comprises a variation at one or more positions of positions 21- 418 of SEQ ID NO: 428, and the R region comprises SEQ ID NO: 435 or a variant of SEQ ID NO: 435, wherein the variant of SEQ ID NO: 435 comprises a variation at one or more positions of positions 21-265 of SEQ ID NO: 435;(xvi) the L region comprises SEQ ID NO: 429 or a variant of SEQ ID NO: 429, wherein the variant of SEQ ID NO: 429 comprises a variation at one or more positions of positions 21- 355 of SEQ ID NO: 429, and the R region comprises SEQ ID NO: 435 or a variant of SEQ ID NO: 435, wherein the variant of SEQ ID NO: 435 comprises a variation at one or more positions of positions 21-265 of SEQ ID NO: 435;(xvii) the L region comprises SEQ ID NO: 448 or a variant of SEQ ID NO: 448, wherein the variant of SEQ ID NO: 448 comprises a variation at one or more positions of positions 21 - 286 of SEQ ID NO: 448, and the R region comprises SEQ ID NO: 450 or a variant of SEQ ID NO: 450, wherein the variant of SEQ ID NO: 450 comprises a variation at one or more positions of positions 21-83 of SEQ ID NO: 450;(xviii) the L region comprises SEQ ID NO: 449 or a variant of SEQ ID NO: 449, wherein the variant of SEQ ID NO: 449 comprises a variation at one or more positions of positions 21- 393 of SEQ ID NO: 449, and the R region comprises SEQ ID NO: 450 or a variant of SEQ ID NO: 450, wherein the variant of SEQ ID NO: 450 comprises a variation at one or more positions of positions 21-83 of SEQ ID NO: 450; and(xix) the L region comprises SEQ ID NO: 430 or a variant of SEQ ID NO: 430, wherein the variant of SEQ ID NO: 430 comprises a variation at oneor more positions of positions 21-333 of SEQ ID NO: 430, and the R region comprises SEQ ID NO: 436 or a variant of SEQ ID NO: 436, wherein the variant of SEQ ID NO: 436 comprises a variation at one or more positions of positions 21-323 of SEQ ID NO: 436.Embodiment 10. The alphaviral encrypted RNA of any one of Embodiments 1 to 9, wherein each of the L region and the R region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a corresponding region that is native to the alphavirus, or wherein each of the L region and the R region comprises fewer than 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleoside variations relative to the corresponding region that is native to the alphavirus.Embodiment 11. The alphaviral encrypted RNA of any one of Embodiments 1 to 9, wherein each of the L region and the R region varies from a corresponding region that is native to the alphavirus by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside substitution that is / are not involved in 5’ capping.Embodiment 12. The alphaviral encrypted RNA of any one of Embodiments 1 to 11, wherein the alphaviral encrypted RNA comprises at least one nucleoside modification.Embodiment 13. The alphaviral encrypted RNA of Embodiment 12, wherein each of the L region and the R region comprises no more than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% modified nucleosides, or wherein each of the L region and the R region comprises at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 100% modified nucleosides.Embodiment 14. The alphaviral encrypted RNA of Embodiment 12 or Embodiment 13, wherein the nucleoside modification is a nonimmunogenic uridine modification, and the percentage of modified uridine modifications is (i) no more than 40%, 35%, 30%, 25%, 20% 15% or 10%, or (ii) more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%, or is 100% of all uridines; orwherein the nucleoside modification is a nonimmunogenic cytidine modification, and the percentage of modified cytidine modifications is (i) no more than 40%, 35%, 30%, 25%, 20% 15% or 10%, or (ii) more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%, or is 100% of all cytidines; or wherein the nucleoside modification is a nonimmunogenic adenosine modification, and the percentage of modified adenosine modifications is between 1% and 30%, optionally, about 1%, 5%, 10%, 15%, 20%, 25%, or 30% of all adenosine.Embodiment 15. The alphaviral encrypted RNA of any one of Embodiments 12 to 14, wherein the alphaviral encrypted RNA comprises a 5’ cap structure, optionally the 5’ cap structure is selected from the group consisting of Cap 0, Cap 0 (3’-0-Me), Cap 1, Cap 1 (3’-O- Me), Cap 2, Cap 2 (3’-0-Me), Anti-Reverse Cap Analog (ARC A), inosine, N1 -methylguanosine, 2’-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, locked nucleic acid guanosine (LNA-guanosine), and 2-azido-guanosine structure, or selected from any combination or subcombination thereof.Embodiment 16. The alphaviral encrypted RNA of Embodiment 15, wherein the 5’ end of the L region comprises a 5’ cap structure, optionally the 5’ end of the L region comprising one or more variations associated with a 5’ cap structure.Embodiment 17. The alphaviral encrypted RNA of any one of Embodiments 12 to 14, wherein the alphaviral encrypted RNA does not comprise a 5’ cap structure (uncapped), or wherein the 5’ end of the L region does not comprise a 5’ cap structure (uncapped).Embodiment 18. The alphaviral encrypted RNA of Embodiment 17, wherein the 5’ end of the alphaviral encrypted RNA comprises a 5 ’-monophosphate, 5 ’-diphosphate, or 5- triphosphate, or wherein the 5’ end of the encrypted RNA does not comprise a 5 ’-phosphate (dephosphorylated).Embodiment 19. The alphaviral encrypted RNA of any one of Embodiments 1 to 18, wherein the therapeutic polypeptide is a secreted polypeptide, optionally an antibody, orwherein the therapeutic polypeptide is an interferon, an interferon-stimulated gene product, a cytokine, a chemokine, an antibody, a signaling molecule, a cytotoxic protein, a protein that causes cell death, an antineoplastic protein, an immunomodulatory protein, a protein toll-like receptor agonist, or a dominant negative protein, optionally wherein the cytokine is (i) an inflammatory cytokine, optionally TNF-a, or (ii) an anti-inflammatory cytokine, optionally an interleukin- 1 receptor antagonist (IL-1RN), or wherein the therapeutic polypeptide is an interleukin, optionally IL-12A, 1L-12B, or IL-2, or wherein the therapeutic polypeptide is a caspase, or wherein the therapeutic polypeptide is an interferon (IFN), optionally an IFN-a, IFN-0, IFN-e, IFN-K, IFN-CD, IFN-y, or IFN-A, further optionally IFN-al, IFN-a2, IFN-a4, IFN-a5, IFN- a6, IFN-a7, IFN-a8, IFN-alO, IFN-al3, IFN-al4, IFN-al6, IFN-al7, IFN-a21, IFN-pi, IFN-e, IFN-K, IFN-ω1, IFN-y, IFN-A1 (IL28A), IFN- A2 (IL28B), IFN- A3 (IL29), or IFN- A4.Embodiment 20. The alphaviral encrypted RNA of any one of Embodiments 1 to 19, wherein the coding sequence of the alphaviral encrypted RNA encodes two or more therapeutic polypeptides which are separated by one or more ribosomal skipping sequences, or wherein the coding region further comprises one or more regulatory elements selected from the group consisting of a ribosomal binding site, a Kozak sequence, a Shine-Dalgamo sequence, a ribozyme, a riboswitch, a promoter, a microRNA binding site, and an internal ribosomal entry site (IRES), optionally the one or more regulatory elements are operably linked to the coding sequence.Embodiment 21. The alphaviral encrypted RNA of any one of Embodiments 1 to 20, wherein the alphaviral encrypted RNA comprises a polyadenylation signal and / or a 3’ poly(A) tail, or wherein the encrypted RNA is in a linear form or a covalently-closed circular form.Embodiment 22. An isolated DNA polynucleotide encoding the alphaviral encrypted RNA of any one of Embodiments 1 to 21.Embodiment 23. A cell or a cell line comprising the isolated DNA polynucleotide ofEmbodiment 22.Embodiment 24. A vector comprising the alphaviral encrypted RNA of any one of Embodiments 1 to 21 or the isolated DNA polynucleotide of Embodiment 22, optionally wherein the vector is a viral vector an expression vector.Embodiment 25. A lipid nanoparticle encapsulating the alphaviral encrypted RNA of any one of Embodiments 1 to 21.Embodiment 26. A method comprising administering to a subject in need thereof a therapeutically effective amount of the alphaviral encrypted RNA of any one of Embodiments 1 to 21, the isolated DNA polynucleotide of Embodiment 22, the cell or the cell line of Embodiment 23, the vector of Embodiment 24, or the lipid nanoparticle of Embodiment 25.Embodiment27. The method of Embodiment 26, further comprising administering a polynucleotide encoding the translation activator to initiate translation of the therapeutic polypeptide.Embodiment 28. The method of Embodiment 26 or Embodiment 27, wherein the subject is a human, a cow, a pig, a sheep, a horse, a deer, a ruminant, a rodent, fish, or a fowl.Embodiment 29. The method of any one of Embodiments 26 to 28, wherein the subject has a disease or a disorder resulting from a viral infection.Embodiment 30. The method of any one of Embodiments 26 to 29, wherein the subject has an infection with a virus.Embodiment 31. The method of any one of Embodiments 26 to 30, wherein administering isby intratracheal or inhalation, intranasal, oral, rectal, vaginal, transmucosal, or intestinal administration; or by parenteral delivery, optionally intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections; or by intrathecal, direct intraventricular, intravenous, or intraperitoneal administration.

Claims

CLAIMSWhat is claimed is:

1. An alphaviral encrypted RNA comprising:(i) a coding region comprising a coding sequence encoding a therapeutic polypeptide,(ii) a left flanking region (L region) adjacent to and contiguous with a 5’ end of the coding region; and(iii) a right flanking region (R region) adjacent to and contiguous with a 3’ end of the coding region; wherein both the L region and the R region of the alphaviral encrypted RNA are derived from an alphavirus, and wherein, once the alphaviral encrypted RNA is inside a cell containing a translation activator, the L region and the R region interact with the translation activator, thereby resulting in translation of the therapeutic polypeptide.

2. The alphaviral encrypted RNA of claim 1, wherein each of the L region and the R region is derived from the reverse complement of a corresponding region that is native to the alphavirus.

3. The alphaviral encrypted RNA of claim 1 or claim 2, wherein the alphavirus is Chikungunya virus, Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Sindbis Virus (SINV), Semliki Forest Virus (SFV), or Western Equine Encephalitis Virus (WEEV).

4. The alphaviral encrypted RNA of any one of claims 1 to 3, wherein the translation activator is an RNA dependent polymerase.The alphaviral encrypted RNA of claim 4, wherein the RNA dependent polymerase is a viral RNA dependent polymerase, optionally an RNA-dependent RNA polymerase or an RNA- dependent DNA polymerase.

6. The alphaviral encrypted RNA of any one of claims 1 to 5, wherein the coding sequence within the alphaviral encrypted RNA is in an antisense orientation.

7. The alphaviral encrypted RNA of claim 6, wherein a combination of the L region and the R region satisfies one of the following:(i) the L region comprises SEQ ID NO: 249 or a variant of SEQ ID NO: 249, wherein the variant of SEQ ID NO: 249 comprises a variation at one or more positions of positions 21-354 of SEQ ID NO: 249, and the R region comprises SEQ ID NO: 441 or a variant of SEQ ID NO: 441, wherein the variant of SEQ ID NO: 441 comprises a variation at one or more positions of positions 21-323 of SEQ ID NO: 441;(ii) the L region comprises SEQ ID NO: 438 or a variant of SEQ ID NO: 438, wherein the variant of SEQ ID NO: 438 comprises a variation at one or more positions of positions 21- 113 of SEQ ID NO: 438 and the R region comprises SEQ ID NO: 442 or a variant of SEQ ID NO: 442, wherein the variant of SEQ ID NO: 442 comprises a variation at one or more positions of positions 21-353 of SEQ ID NO: 442;(iii) the L region comprises SEQ ID NO: 438 or a variant of SEQ ID NO: 438, wherein the variant of SEQ ID NO: 438 comprises a variation at one or more positions of positions 21- 113 of SEQ ID NO: 438 and the R region comprises SEQ ID NO: 443 or a variant of SEQ ID NO: 443, wherein the variant of SEQ ID NO: 443 comprises a variation at one or more positions of positions 21-517 of SEQ ID NO: 443;(iv) the L region comprises SEQ ID NO: 438 or a variant of SEQ ID NO: 438, wherein the variant of SEQ ID NO: 438 comprises a variation at one or more positions of positions 21- 113 of SEQ ID NO: 438 and the R region comprises SEQ ID NO: 444 or a variant of SEQ ID NO: 444, wherein the variant of SEQ ID NO: 444 comprises a variation at one or more positions of positions 21-168 of SEQ ID NO: 444;(v) the L region comprises SEQ ID NO: 439 or a variant of SEQ ID NO: 439, wherein the variant of SEQ ID NO: 439 comprises a variation at one or more positions of positions 21- 256 of SEQ ID NO: 439, and the R region comprises SEQ ID NO: 445 or a variant of SEQ ID NO: 445, wherein the variant of SEQ ID NO: 445 comprises a variation at one or more positions of positions 21-355 of SEQ ID NO: 445;(vi) the L region comprises SEQ ID NO: 439 or a variant of SEQ ID NO: 439, wherein the variant of SEQ ID NO: 439 comprises a variation at one or more positions of positions 21- 256 of SEQ ID NO: 439, and the R region comprises SEQ ID NO: 446 or a variant of SEQ ID NO: 446, wherein the variant of SEQ ID NO: 446 comprises a variation at one or more positions of positions 21-286 of SEQ ID NO: 446;(vii) the L region comprises SEQ ID NO: 439 or a variant of SEQ ID NO: 439, wherein the variant of SEQ ID NO: 439 comprises a variation at one or more positions of positions 21- 256 of SEQ ID NO: 439, and the R region comprises SEQ ID NO: 447 or a variant of SEQ ID NO: 447, wherein the variant of SEQ ID NO: 447 comprises a variation at one or more positions of positions 21-293 of SEQ ID NO: 447;(viii) the L region comprises SEQ ID NO: 440 or a variant of SEQ ID NO: 440, wherein the variant of SEQ ID NO: 440 comprises a variation at one or more positions of positions 21-74 of SEQ ID NO: 440, and the R region comprises SEQ ID NO: 445 or a variant of SEQ ID NO:445, wherein the variant of SEQ ID NO: 445 comprises a variation at one or more positions of positions 21-355 of SEQ ID NO: 445;(ix) the L region comprises SEQ ID NO: 440 or a variant of SEQ ID NO: 440, wherein the variant of SEQ ID NO: 440 comprises a variation at one or more positions of positions 21-74 of SEQ ID NO: 440, and the R region comprises SEQ ID NO: 446 or a variant of SEQ ID NO:446, wherein the variant of SEQ ID NO: 446 comprises a variation at one or more positions of positions 21-286 of SEQ ID NO: 446 and(x) the L region comprises SEQ ID NO: 440 or a variant of SEQ ID NO: 440, wherein the variant of SEQ ID NO: 440 comprises a variation at one or more positions of positions 21 -74 of SEQ ID NO: 440, and the R region comprises SEQ ID NO: 447 or a variant of SEQ ID NO:447, wherein the variant of SEQ ID NO: 447 comprises a variation at one or more positions of positions 21-293 of SEQ ID NO: 447.

8. The alphaviral encrypted RNA of any one of claims 1 to 5, wherein the coding sequence within the alphaviral encrypted RNA is in a sense orientation.

9. The alphaviral encrypted RNA of claim 8, wherein a combination of the L region and the R region satisfies one of the following:(i) the L region comprises SEQ ID NO: 421 or a variant of SEQ ID NO: 421, wherein the variant of SEQ ID NO: 421 comprises a variation at one or more positions of positions 21-323 of SEQ ID NO: 421, and the R region comprises SEQ ID NO: 432 or a variant of SEQ ID NO: 432, wherein the variant of SEQ ID NO: 432 comprises a variation at one or more positions of positions 21-363 of SEQ ID NO: 432;(ii) the L region comprises SEQ ID NO: 422 or a variant of SEQ ID NO: 422, wherein the variant of SEQ ID NO: 422 comprises a variation at one or more positions of positions 21- 515 of SEQ ID NO: 422, and the R region comprises SEQ ID NO: 432 or a variant of SEQ ID NO: 432, wherein the variant of SEQ ID NO: 432 comprises a variation at one or more positions of positions 21-363 of SEQ ID NO: 432;(iii) the L region comprises SEQ ID NO: 423 or a variant of SEQ ID NO: 423, wherein the variant of SEQ ID NO: 423 comprises a variation at one or more positions of positions 21- 398 of SEQ ID NO: 423, and the R region comprises SEQ ID NO: 432 or a variant of SEQ ID NO: 432, wherein the variant of SEQ ID NO: 432 comprises a variation at one or more positions of positions 21-363 of SEQ ID NO: 432;(iv) the L region comprises SEQ ID NO: 421 or a variant of SEQ ID NO: 421, wherein the variant of SEQ ID NO: 421 comprises a variation at one or more positions of positions 21- 323 of SEQ ID NO: 421, and the R region comprises SEQ ID NO: 433 or a variant of SEQ ID NO: 433, wherein the variant of SEQ ID NO: 433 comprises a variation at one or more positions of positions 21-399 of SEQ ID NO: 433;(v) the L region comprises SEQ ID NO: 422 or a variant of SEQ ID NO: 422, wherein the variant of SEQ ID NO: 422 comprises a variation at one or more positions of positions 21 - 515 of SEQ ID NO: 422, and the R region comprises SEQ ID NO: 433 or a variant of SEQ ID NO: 433, wherein the variant of SEQ ID NO: 433 comprises a variation at one or more positions of positions 21-399 of SEQ ID NO: 433;(vi) the L region comprises SEQ ID NO: 423 or a variant of SEQ ID NO: 423, wherein the variant of SEQ ID NO: 423 comprises a variation at one or more positions of positions 21- 398 of SEQ ID NO: 423, and the R region comprises SEQ ID NO: 433 or a variant of SEQ ID NO: 433, wherein the variant of SEQ ID NO: 433 comprises a variation at one or more positions of positions 21-399 of SEQ ID NO: 433;(vii) the L region comprises SEQ ID NO: 424 or a variant of SEQ ID NO: 424, wherein the variant of SEQ ID NO: 424 comprises a variation at one or more positions of positions 21- 574 of SEQ ID NO: 424, and the R region comprises SEQ ID NO: 434 or a variant of SEQ ID NO: 434, wherein the variant of SEQ ID NO: 434 comprises a variation at one or more positions of positions 21-122 of SEQ ID NO: 434;(viii) the L region comprises SEQ ID NO: 425 or a variant of SEQ ID NO: 425, wherein the variant of SEQ ID NO: 425 comprises a variation at one or more positions of positions 21- 589 of SEQ ID NO: 425, and the R region comprises SEQ ID NO: 434 or a variant of SEQ ID NO: 434, wherein the variant of SEQ ID NO: 434 comprises a variation at one or more positions of positions 21-122 of SEQ ID NO: 434;(ix) the L region comprises SEQ ID NO: 426 or a variant of SEQ ID NO: 426, wherein the variant of SEQ ID NO: 426 comprises a variation at one or more positions of positions 21- 517 of SEQ ID NO: 426, and the R region comprises SEQ ID NO: 434 or a variant of SEQ ID NO: 434, wherein the variant of SEQ ID NO: 434 comprises a variation at one or more positions of positions 21-122 of SEQ ID NO: 434;(x) the L region comprises SEQ ID NO: 424 or a variant of SEQ ID NO: 424, wherein the variant of SEQ ID NO: 424 comprises a variation at one or more positions of positions 21- 574 of SEQ ID NO: 424, and the R region comprises SEQ ID NO: 260 or a variant of SEQ ID NO: 260, wherein the variant of SEQ ID NO: 260 comprises a variation at one or more positions of positions 21-124 of SEQ ID NO: 260;(xi) the L region comprises SEQ ID NO: 425 or a variant of SEQ ID NO: 425, wherein the variant of SEQ ID NO: 425 comprises a variation at one or more positions of positions 21 - 589 of SEQ ID NO: 425, and the R region comprises SEQ ID NO: 260 or a variant of SEQ ID NO: 260, wherein the variant of SEQ ID NO: 260 comprises a variation at one or more positions of positions 21-124 of SEQ ID NO: 260;(xii) the L region comprises SEQ ID NO: 426 or a variant of SEQ ID NO: 426, wherein the variant of SEQ ID NO: 426 comprises a variation at one or more positions of positions 21- 517 of SEQ ID NO: 426, and the R region comprises SEQ ID NO: 260 or a variant of SEQ ID NO: 260, wherein the variant of SEQ ID NO: 260 comprises a variation at one or more positions of positions 21-124 of SEQ ID NO: 260;(xiii) the L region comprises SEQ ID NO: 431 or a variant of SEQ ID NO: 431, wherein the variant of SEQ ID NO: 431 comprises a variation at one or more positions of positions 21- 306 of SEQ ID NO: 431, and the R region comprises SEQ ID NO: 437 or a variant of SEQ ID NO: 437, wherein the variant of SEQ ID NO: 437 comprises a variation at one or more positions of positions 21-304 of SEQ ID NO: 437;(xiv) the L region comprises SEQ ID NO: 427 or a variant of SEQ ID NO: 427, wherein the variant of SEQ ID NO: 427 comprises a variation at one or more positions of positions 21- 355 of SEQ ID NO: 427, and the R region comprises SEQ ID NO: 435 or a variant of SEQ ID NO: 435, wherein the variant of SEQ ID NO: 435 comprises a variation at one or more positions of positions 21-265 of SEQ ID NO: 435;(xv) the L region comprises SEQ ID NO: 428 or a variant of SEQ ID NO: 428, wherein the variant of SEQ ID NO: 428 comprises a variation at one or more positions of positions 21- 418 of SEQ ID NO: 428, and the R region comprises SEQ ID NO: 435 or a variant of SEQ ID NO: 435, wherein the variant of SEQ ID NO: 435 comprises a variation at one or more positions of positions 21-265 of SEQ ID NO: 435;(xvi) the L region comprises SEQ ID NO: 429 or a variant of SEQ ID NO: 429, wherein the variant of SEQ ID NO: 429 comprises a variation at one or more positions of positions 21- 355 of SEQ ID NO: 429, and the R region comprises SEQ ID NO: 435 or a variant of SEQ ID NO: 435, wherein the variant of SEQ ID NO: 435 comprises a variation at one or more positions of positions 21-265 of SEQ ID NO: 435;(xvii) the L region comprises SEQ ID NO: 448 or a variant of SEQ ID NO: 448, wherein the variant of SEQ ID NO: 448 comprises a variation at one or more positions of positions 21 - 286 of SEQ ID NO: 448, and the R region comprises SEQ ID NO: 450 or a variant of SEQ ID NO: 450, wherein the variant of SEQ ID NO: 450 comprises a variation at one or more positions of positions 21-83 of SEQ ID NO: 450;(xviii) the L region comprises SEQ ID NO: 449 or a variant of SEQ ID NO: 449, wherein the variant of SEQ ID NO: 449 comprises a variation at one or more positions of positions 21- 393 of SEQ ID NO: 449, and the R region comprises SEQ ID NO: 450 or a variant of SEQ ID NO: 450, wherein the variant of SEQ ID NO: 450 comprises a variation at one or more positions of positions 21-83 of SEQ ID NO: 450; and(xix) the L region comprises SEQ ID NO: 430 or a variant of SEQ ID NO: 430, wherein the variant of SEQ ID NO: 430 comprises a variation at oneor more positions of positions 21-333 of SEQ ID NO: 430, and the R region comprises SEQ ID NO: 436 or a variant of SEQ ID NO: 436, wherein the variant of SEQ ID NO: 436 comprises a variation at one or more positions of positions 21-323 of SEQ ID NO: 436.

10. The alphaviral encrypted RNA of any one of claims 1 to 9, wherein each of the L region and the R region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a corresponding region that is native to the alphavirus, or wherein each of the L region and the R region comprises fewer than 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleoside variations relative to the corresponding region that is native to the alphavirus.

11. The alphaviral encrypted RNA of any one of claims 1 to 9, wherein each of the L region and the R region varies from a corresponding region that is native to the alphavirus by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleoside substitution that is / are not involved in 5’ capping.

12. The alphaviral encrypted RNA of any one of claims 1 to 11 , wherein the alphaviral encrypted RNA comprises at least one nucleoside modification.

13. The alphaviral encrypted RNA of claim 12, wherein each of the L region and the R region comprises no more than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% modified nucleosides, or wherein each of the L region and the R region comprises at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or 100% modified nucleosides.

14. The alphaviral encrypted RNA of claim 12 or claim 13, wherein the nucleoside modification is a nonimmunogenic uridine modification, and the percentage of modified uridine modifications is (i) no more than 40%, 35%, 30%, 25%, 20% 15% or 10%, or (ii) more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%, or is 100% of all uridines; or wherein the nucleoside modification is a nonimmunogenic cytidine modification, and the percentage of modified cytidine modifications is (i) no more than 40%, 35%, 30%, 25%, 20% 15% or 10%, or (ii) more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%, or is 100% of all cytidines; orwherein the nucleoside modification is a nonimmunogenic adenosine modification, and the percentage of modified adenosine modifications is between 1% and 30%, optionally, about 1%, 5%, 10%, 15%, 20%, 25%, or 30% of all adenosine.

15. The alphaviral encrypted RNA of any one of claims 12 to 14, wherein the alphaviral encrypted RNA comprises a 5’ cap structure, optionally the 5’ cap structure is selected from the group consisting of Cap 0, Cap 0 (3’-0-Me), Cap 1, Cap 1 (3’-0-Me), Cap 2, Cap 2 (3’-0-Me), Anti-Reverse Cap Analog (ARCA), inosine, Nl-methyl-guanosine, 2’-fluoro-guanosine, 7- deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, locked nucleic acid guanosine (LNA- guanosine), and 2-azido-guanosine structure, or selected from any combination or subcombination thereof.

16. The alphaviral encrypted RNA of claim 15, wherein the 5’ end of the L region comprises a 5’ cap structure, optionally the 5’ end of the L region comprising one or more variations associated with a 5’ cap structure.

17. The alphaviral encrypted RNA of any one of claims 12 to 14, wherein the alphaviral encrypted RNA does not comprise a 5’ cap structure (uncapped), or wherein the 5’ end of the L region does not comprise a 5’ cap structure (uncapped).

18. The alphaviral encrypted RNA of claim 17, wherein the 5’ end of the alphaviral encrypted RNA comprises a 5’-monophosphate, 5 ’-diphosphate, or 5-triphosphate, or wherein the 5’ end of the encrypted RNA does not comprise a 5 ’-phosphate (dephosphorylated).

19. The alphaviral encrypted RNA of any one of claims 1 to 18, wherein the therapeutic polypeptide is a secreted polypeptide, optionally an antibody, or wherein the therapeutic polypeptide is an interferon, an interferon-stimulated gene product, a cytokine, a chemokine, an antibody, a signaling molecule, a cytotoxic protein, a protein that causes cell death, an antineoplastic protein, an immunomodulatory protein, a protein toll-like receptor agonist, or a dominant negative protein,optionally wherein the cytokine is (i) an inflammatory cytokine, optionally TNF-a, or (ii)an anti-inflammatory cytokine, optionally an interleukin- 1 receptor antagonist (IL-1RN), orwherein the therapeutic polypeptide is an interleukin, optionally IL-12A, IL-12B, or IL-2,orwherein the therapeutic polypeptide is a caspase, orwherein the therapeutic polypeptide is an interferon (IFN), optionally an IFN-a, IFN-β,IFN-e, IFN-K, IFN-ω, IFN-y, or IFN-λ, further optionally IFN-al, IFN-α2, IFN-a4, IFN-a5, IFN-a6, IFN-a7, IFN-a8, IFN-alO, IFN-al3, IFN-al4, IFN-al6, IFN-al7, IFN-a21, IFN-pi, IFN-e,IFN-K, IFN-ω1, IFN-y, IFN-A.1 (IL28A), IFN- A2 (IL28B), IFN- A3 (IL29), or IFN- A.4.20.The alphaviral encrypted RNA of any one of claims 1 to 19,wherein the coding sequence of the alphaviral encrypted RNA encodes two or moretherapeutic polypeptides which are separated by one or more ribosomal skipping sequences, orwherein the coding region further comprises one or more regulatory elements selectedfrom the group consisting of a ribosomal binding site, a Kozak sequence, a Shine-Dalgamosequence, a ribozyme, a riboswitch, a promoter, a microRNA binding site, and an internalribosomal entry site (IRES), optionally the one or more regulatory elements are operably linkedto the coding sequence.21.The alphaviral encrypted RNA of any one of claims 1 to 20,wherein the alphaviral encrypted RNA comprises a polyadenylation signal and / or a 3’poly(A) tail, orwherein the encrypted RNA is in a linear form or a covalently-closed circular form.22.An isolated DNA polynucleotide encoding the alphaviral encrypted RNA of any one ofclaims 1 to 21.23.A cell or a cell line comprising the isolated DNA polynucleotide of claim 22.

24. A vector comprising the alphaviral encrypted RNA of any one of claims 1 to 21 or the isolated DNA polynucleotide of claim 22, optionally wherein the vector is a viral vector an expression vector.

25. A lipid nanoparticle encapsulating the alphaviral encrypted RNA of any one of claims 1 to 21.

26. A method comprising administering to a subject in need thereof a therapeutically effective amount of the alphaviral encrypted RNA of any one of claims 1 to 21, the isolated DNA polynucleotide of claim 22, the cell or the cell line of claim 23, the vector of claim 24, or the lipid nanoparticle of claim 25.

27. The method of claim 26, further comprising administering a polynucleotide encoding the translation activator to initiate translation of the therapeutic polypeptide.

28. The method of claim 26 or claim 27, wherein the subject is a human, a cow, a pig, a sheep, a horse, a deer, a ruminant, a rodent, fish, or a fowl.

29. The method of any one of claims 26 to 28, wherein the subject has a disease or a disorder resulting from a viral infection.

30. The method of any one of claims 26 to 29, wherein the subject has an infection with a virus.

31. The method of any one of claims 26 to 30, wherein administering is by intratracheal or inhalation, intranasal, oral, rectal, vaginal, transmucosal, or intestinal administration; or by parenteral delivery, optionally intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections; or by intrathecal, direct intraventricular, intravenous, or intraperitoneal administration.

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