PROGRAMMABLE, RNA EDITOR-CONTROLLED NUCLEIC ACID DOSE AMPLIFIERS AND THEIR METHODS OF USE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The application claims benefit to U.S. Provisional Application No. 63/619,723, filed January 10, 2024, which is incorporated by reference herein in its entirety .
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing, which has been submitted electronically in ,xml format. The contents of the electronic sequence listing (009796..00015_W0_.SL.xml; Size 1,137,828 bytes, and Date of Creation: January 9, 2025) is incorporated by reference herein in its entirety.
FIELD
[0003] The disclosure relates to RNA editor-controlled nucleic acid dose amplifiers (e.g., encrypted RNAs and target- specific translation activators controlled by an RNA editor), that enable programmable, increased translation of polypeptides, including therapeutic polypeptides, and methods of their use.
BACKGROUND
[0004] Over the past few decades, RNA drugs have emerged as promising candidates to address diseases at the gene and/or RNA levels. 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). RNA drugs may change the traditional landscape in which small molecules and proteins represent the two major classes of FDA-approved drugs. 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.
[0005] Despite the recent successes of RNA therapeutics (e.g., mRNA CO VID- 19 vaccines), there is still a need in the ait to identify RNA medicines that are safe and effective in treating disease, including (1) RNA medicines that have an increased specificity to diseases or therapeutic targets, and/or (2) a dosage of RNA drugs that can be more effectively and precisely modulated.
SUMMARY
[0006] 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 (e.g., resulting in a lOOOx or more increase of therapeutic polypeptide production). Methods of making and using the compounds, compositions containing the compounds, and uses of medicaments comprising these compounds and compositions are also disclosed.
[0007] The present disclosure is related to a programmable, RNA editor-controlled nucleic acid dose amplification system, comprising an “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,” wherein the production of the target- specific translation activator is modulated by an RNA editor. For example, the presence of an RNA editor may edit an RNA sequence encoding the target- specific translation activator, thereby resulting in the effective translation of the 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 rnRNA species that is more translatable by the cellular ribosomal machinery. In some embodiments, the target-specific translation activator comprises an RNA- dependent RNA polymerase or an RNA-dependent DNA polymerase.
[0008] In one aspect, provided is a composition comprising:
(a) an 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: and
(b) a target mRNA comprising an edit tract, wherein a stop codon within the edit tract prevents translation of a translation activator or a translation activator component encoded by the target mRNA. Delivering the composition into a cell can form an intracellular protein-nucleic acid complex comprising:
(i) the target mRN A
(ii) an RNA editor, and
(iii) a guide RNA, and wherein the guide RNA binds to the target mRNA to form a double- stranded RNA complex as a substrate for the RNA editor, wherein the substrate comprises a mispairing within the stop codon, wherein the RNA editor converts the mispairing into a pairing in the substrate, thereby allowing translation of the translation activator or the translation activator component from the target mRNA, and wherein the encrypted RNA contacts the translation activator, thereby resulting in translation of the therapeutic polypeptide. The composition may comprise a lipid nanoparticle encapsulating the encrypted RNA and the target mRNA.
[0009] In another aspect, provided is a composition comprising:
(a) an 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;
(b) an RNA editor;
(c) a guide RNA;
(d) two or more mRNA molecules, each of which encodes a portion of a translation activator or a portion of a translation activator component, wherein each portion is not functional. Delivering the composition into a cell can result in (i) activation of the RNA editor by the guide RNA; (ii) a ligation of the two or more mRNA molecules by the RNA editor to form a mRNA product encoding the translation activator or the translation activator component; and (iii) translation of the translation activator or the translation activator component, wherein the encrypted RNA contacts the translation activator, thereby resulting in translation of the therapeutic polypeptide. The RNA editor can be eDisCas7-l 1. [0010] In another aspect, both the L region and the R region of the encrypted RNA can be derived from a virus. Each of the L region and the R region can be the reverse complement of a corresponding region that is native to the virus. The translation activator can be an RNA dependent polymerase. The RNA dependent polymerase can be a viral RNA dependent polymerase, optionally an RNA-dependent RNA polymerase or an RNA-dependent DNA polymerase. The virus can be selected from the group consisting of: Alphacoronavirus 229E, Alphacoronavirus NL.63, Alphacoronavirus WA2028, Avian metapneumovirus (AMPV), Betacoronavirus HKU1, Betacoronavirus HKU15, Betacoronavirus HKU33, Betacoronavirus OC43, Chikungunya virus, Crimean-Congo Hemorrhagic Fever Virus, Dengue Virus, Eastern Equine Encephalitis Virus (EEEV), Enterovirus D68 (EV-D68), Foot and Mouth Disease Virus, Hanta Virus, Hendra Virus, Hepatitis B Virus, Hepatitis C Virus, HMPV, Human Parainfluenzavirus 1 (HPIV1), Human Parainfluenzavirus 3 (HPIV3), Infectious Salmon Anemia Vims, Influenza A Vims, Influenza B Virus, Lassa Vims, Marburg Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Newcastle Disease Virus (NDV), Nipah Virus, Norwalk Virus, Rabies Virus, Respiratory Syncytial Virus, Reston Ebola virus, Rhinovirus, Rift Valley Fever Virus, Rubella virus, SARS-CoV-1, SARS-CoV-2, Sudan Ebola vims, Venezuelan Equine Encephalitis Virus (VEEV), Vesicular Stomatitis Vims, Western Equine Encephalitis Virus (WEEV), Yellow Fever Vims, Zaire Ebola virus, and Zika Vims. Alternatively, the virus may not be an alphavirus. The L region and R region can be native to the virus. 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 virus, 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 vims. Alternatively, each of the L region and the R region can vary from a corresponding region that is native to the 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.
[0011] In another aspect, the encrypted RNA can comprise at least one nucleoside modification. For example, each of the L region and the R region can comprise no more than about 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, and the percentage of modified uridine modifications can be (i) no more than about 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, and the percentage of modified cytidine modifications can be (i) no more than about 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 about 1% and about 30%, optionally, about 1%, 5%, 10%, 15%, 20%, 25%, or 30% of all adenosine.
[0012] In yet another aspect, the encrypted RNA can comprise a 5’ cap structure.
Typically, the 5’ cap structure can be selected from the group consisting of: 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 (ARC A), inosine, Nl-methyl-guanosine, 2’ -fluoro-guanosine, 7 -deaza- guanosine, 8-oxo-guanosine, 2-ammo-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 I, 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 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 encrypted RNA comprises a 5 ’-monophosphate, 5’- di phosphate, or 5-triphosphate. Alternatively, the 5’ end of the encrypted RNA does not comprise a 5’-phosphate (dephosphorylated).
[0013] In a further aspect, the coding sequence within the encrypted RNA is in an antisense orientation. The virus may be an influenza virus, and a combination of the L region and the R region may satisfy one of the following:
(i) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 2; or a variant of SEQ ID NO: 2, wherein the variant of SEQ ID NO: 2 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14- 26 of SEQ ID NO: 2, and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 20, 21, 22, or 23: or a variant of any one of SEQ ID NOs: 20,
21, 22, or 23, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-15 of any one of SEQ ID NOs: 20, 21, 22, or 23;
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 3; or a variant of SEQ ID NO: 3, wherein the variant of SEQ ID NO: 3 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14- 35 of SEQ ID NO: 3; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 24, 25, 26, or 27; or a variant of any one of SEQ ID NOs: 24, 25, 26, or 27, wherein the variant of SEQ ID NO: 24 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-12 of SEQ ID NO: 24; the variant of SEQ ID NO: 25 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-12 of SEQ ID NO: 25; the variant of SEQ ID NO: 26 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-12 of SEQ ID NO: 26; or the variant of SEQ ID NO: 27 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-12 of SEQ ID NO: 27;
(iii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 4; or a variant of SEQ ID NO: 4, wherein the variant of SEQ ID NO: 4 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14- 50 of SEQ ID NO: 4; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 28, 29, 30, or 31; or a variant of any one of SEQ ID NOs: 28, 29, 30, or 31, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-12 of any one of SEQ ID NOs: 28, 29, 30, or 31;
(iv) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 1 or 5; or a variant of SEQ ID NO: 1 or 5, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-37 of SEQ ID NO: 1 or 5: and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 18 or 19; or a variant of SEQ ID NO: 18 or 19, wherein the variant compri ses a variation at one or more nucleotide positions selected from the group consisting of positions 8-20 of SEQ ID NO: 18 or 19;
(v) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 6; or a variant of SEQ ID NO: 6, wherein the variant of SEQ ID NO: 6 comprises a variation at one or more nucleotide positions selected from position 14 or 15 of SEQ ID NO: 6; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 32 or 33; or a variant of SEQ ID NO: 32 or 33, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-33 of SEQ ID NO: 32 or 33;
(vi) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 7; or a variant of SEQ ID NO: 7, wherein the variant of SEQ ID NO: 7 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14- 20 of SEQ ID NO: 7; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 34, 35, 36, or 37: or a variant of any one of SEQ ID NOs: 34, 35, 36, or 37, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 5-8 of SEQ ID NO: 34, 35, 36, or 37;
(vii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 8; or a variant of SEQ ID NO: 8, wherein the variant of SEQ ID NO: 8 comprises a variation at one or more nucleotide positions selected from position 14 or 15 of SEQ ID NO: 8; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 38, 39, 40, or 41; or a variant of any one of SEQ ID NOs: 38, 39, 40, or 41 , wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-13 of SEQ ID NO: 38, 39, 40, or 41;
(viii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 9; or a variant of SEQ ID NO: 9, wherein the variant of SEQ ID NO: 9 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-18 of SEQ ID NO: 9; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 42 or 43; or a variant of SEQ ID NO: 42 or 43, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-14 of SEQ ID NO: 42 or 43;
(ix) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 11; or a variant of SEQ ID NO: 11, wherein the variant of SEQ ID NO: 11 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-81 of SEQ ID NO: 11; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 46 or 47; or a variant of SEQ ID NO: 46 or 47, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 5-9 of SEQ ID NO: 46 or 47;
(x) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 12; or a variant of SEQ ID NO: 12, wherein the variant of SEQ ID NO: 12 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-52 of SEQ ID NO: 12; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NO: 48 or 49; or a variant of any one of SEQ ID NO: 48 or 49, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-11 of SEQ ID NO: 48 or 49;
(xi) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 13; or a variant of SEQ ID NO: 13, wherein the variant of SEQ ID NO: 13 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-87 of SEQ ID NO: 13; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 50 or 51; or a variant of SEQ ID NO: 50 or 51, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-17 of SEQ ID NO: 50 or 51;
(xii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NO: 10; or a variant of any one of SEQ ID NO: 10, wherein the variant of SEQ ID NO: 10 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-86 of SEQ ID NO: 10; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 44 or 45; or a variant of SEQ ID NO: 44 or 45, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-21 of SEQ ID NO: 44 or 45;
(xiii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 14; or a variant of SEQ ID NO: 14, wherein the variant of SEQ ID NO: 14 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-93 of SEQ ID NO: 14; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NO: 52 or 53; or a variant of any one of SEQ ID NO: 52 or 53, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-48 of SEQ ID NO: 52 or 53;
(xiv) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 15; or a variant of SEQ ID NO: 15, wherein the variant of SEQ ID NO: 15 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-95 of SEQ ID NO: 15; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 54 or 55; or a variant of any one of SEQ ID NO: 54 or 55, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-34 of SEQ ID NO: 54 or 55;
(xv) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 16; or a variant of SEQ ID NO: 16, wherein the variant of SEQ ID NO: 16 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-81 of SEQ ID NO: 16; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NO: 56 or 57; or a variant of any one of SEQ ID NO: 56 or 57, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-12 of SEQ ID NO: 56 or 57; and
(xvi) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 17; or a variant of SEQ ID NO: 17, wherein the variant of SEQ ID NO: 17 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-22 of SEQ ID NO: 17; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NO: 58 or 59; or a variant of any one of SEQ ID NO: 58 or 59, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-32 of SEQ ID NO: 58 or 59.
[0014] The virus may be a sarbecovirus virus, and a combination of the L region and the R region may satisfy one of the following:
(i) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 137; or a variant of SEQ ID NO: 137, wherein the variant of SEQ ID NO: 137 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-1557 of SEQ ID NO: 137; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 128; or a variant of any one of SEQ ID NO: 128, wherein the variant of SEQ ID NO: 128 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-30 of SEQ ID NO: 128; and
(ii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 138, 139, 140, 141, 142, 143, or 144; or a variant of any one of SEQ ID NOs: 138, 139, 140, 141, 142, 143, or 144, wherein the valiant of SEQ ID NO: 138 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-312 of SEQ ID NO: 138; the variant of SEQ ID NO: 139 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1567 of SEQ ID NO: 139; the variant of SEQ ID NO: 140 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1488 of SEQ ID NO: 140; the variant of SEQ ID NO: 141 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1593 of SEQ ID NO: 141; the variant of SEQ ID NO: 142 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1570 of SEQ ID NO: 142; the variant of SEQ ID NO: 143 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1488 of SEQ ID NO: 143; or the variant of SEQ ID NO: 144 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1593 of SEQ ID NO: 144; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 130, 136, 145, 146, or 147; or a variant of any one of SEQ ID NOs: 130, 136, 145, 146, or 147, wherein the variant of SEQ ID NO: 130 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130; the variant of SEQ ID NO: 136 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-33 of SEQ ID NO: 136: the variant of SEQ ID NO: 145 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1461 of SEQ ID NO: 145; the variant of SEQ ID NO: 146 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-1441 of SEQ ID NO: 146; or the variant of SEQ ID NO: 147 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-897 of SEQ ID NO: 147. [0015] The virus may be a Respiratory Syncytial Virus (RSV), and the L region may comprise a nucleotide sequence set forth as any one of SEQ ID NOs: 158, 163, 165, 166, or 419; or a variant of any one of SEQ ID NOs: 158, 163, 165, 166, or 419, wherein the valiant of SEQ ID NO: 158 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-207 of SEQ ID NO: 158; the variant of SEQ ID NO: 163 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 18-210 of SEQ ID NO: 163: the variant of SEQ ID NO: 165 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-147 of SEQ ID NO: 165; the variant of SEQ ID NO: 166 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15—32 of SEQ ID NO: 166: or the variant of SEQ ID NO: 419 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 18-35 of SEQ ID NO: 419; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 169, 170, 176, 177, or 420: or a variant of any one of SEQ ID NOs: 169, 170, 176, 177, or 420, wherein the variant of SEQ ID NO: 169 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-78 of SEQ ID NO: 169; the variant of SEQ ID NO: 170 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-80 of SEQ ID NO: 170; the variant of SEQ ID NO: 176 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-36 of SEQ ID NO: 176; the variant of SEQ ID NO: 177 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-33 of SEQ ID NO: 177; or the variant of SEQ ID NO: 420 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-35 of SEQ ID NO: 420.
[0016] The virus may be a parainfluenzavirus, and a combination of the L region and the R region may satisfy one of the following:
(i) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 181, 182, or 183; or a variant of any one of SEQ ID NOs: 181 , 182, or 183, wherein the variant of SEQ ID NO: 181 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-136 of SEQ ID NO: 181 : the variant of SEQ ID NO: 182 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-142 of SEQ ID NO: 182; or the valiant of SEQ ID NO: 183 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-136 of SEQ ID NO: 183; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 184; or a variant of SEQ ID NO: 184, wherein the variant of SEQ ID NO: 184 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-98 of SEQ ID NO: 184: and
(ii) the L, region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 187, 188, or 189; or a variant of any one of SEQ ID NOs: 187, 188, or 189, wherein the variant of SEQ ID NO: 187 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-95 of SEQ ID NO: 187; the variant of SEQ ID NO: 188 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-101 of SEQ ID NO: 188: or the variant of SEQ ID NO: 189 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-95 of SEQ ID NO: 189; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 190; or a variant of SEQ ID NO: 190, wherein the variant of SEQ ID NO: 190 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-93 of SEQ ID NO: 190.
[0017] The virus may be a metapneumovirus, and a combination of the L region and the R region may satisfy one of the following:
(i) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 196, 197, or 199; or a variant of any one of SEQ ID NOs: 196, 197, or 199, wherein the variant of SEQ ID NO: 196 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-224 of SEQ ID NO: 196; the variant of SEQ ID NO: 197 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21 -230 of SEQ ID NO: 197: the variant of SEQ ID NO: 199 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-140 of SEQ ID NO: 199; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 201 : or a valiant of SEQ ID NO: 201, wherein the variant of SEQ ID NO: 201 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-32 of SEQ ID NO: 201; and
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 195; or a variant of SEQ ID NO: 195, wherein the variant of SEQ ID NO: 195 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-224 of SEQ ID NO: 195; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 200; or a variant of SEQ ID NO: 200, wherein the variant of SEQ ID NO: 200 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-32 of SEQ ID NO: 200.
[0018] The virus may be a henipavirus, and a combination of the L region and the R region may satisfy one of the following:
(i) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 204; or a variant of SEQ ID NO: 204, wherein the variant of SEQ ID NO: 204 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-77 of SEQ ID NO: 204; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 206; or a variant of SEQ ID NO: 206, wherein the variant of SEQ ID NO: 206 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-91 of SEQ ID NO: 206; and
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 209 or 210; or a variant of SEQ ID NO: 209 or 210, wherein the variant of SEQ ID NO: 209 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-77 of SEQ ID NO: 209; or the variant of SEQ ID NO: 210 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-83 of SEQ ID NO: 210; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 211; or a variant of SEQ ID NO: 211, wherein the variant of SEQ ID NO: 211 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-91 of SEQ ID NO: 211.
[0019] The virus may be a hepadnavirus, the L region may comprise a nucleotide sequence set forth as SEQ ID NO: 222 or 223; or a variant of SEQ ID NO: 222 or 223, wherein the variant of SEQ ID NO: 222 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-639 of SEQ ID NO: 222; or the variant of SEQ ID NO: 223 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101—186 of SEQ ID NO: 223; and the R region may comprise the nucleotide sequence set forth as SEQ ID NO: 225; or a variant of SEQ ID NO: 225, wherein the variant of SEQ ID NO: 225 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-1023 of SEQ ID NO: 225.
[0020] The virus may be a filovirus, and a combination of the L region and the R region may satisfy one of the following:
(i) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 227, 228, 229, or 230; or a variant of any one of SEQ ID NOs: 227, 228, 229, or 230, wherein the variant of SEQ ID NO: 227 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-710 of SEQ ID NO: 227; the variant of SEQ ID NO: 228 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 23-713 of SEQ ID NO: 228; the variant of SEQ ID NO: 229 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-707 of SEQ ID NO: 229; or the variant of SEQ ID NO: 230 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-707 of SEQ ID NO: 230; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 231; or a variant of SEQ ID NO: 231, wherein the variant of SEQ ID NO: 231 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-449 of SEQ ID NO: 231 ;
(ii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 232, 233, 234, or 235; or a variant of any one of SEQ ID NOs: 232, 233, 234, or 235, wherein the variant of SEQ ID NO: 232 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-678 of SEQ ID NO: 232; the valiant of SEQ ID NO: 233 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 23-681 of SEQ ID NO: 233; the variant of SEQ ID NO: 234 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 23-678 of SEQ ID NO: 234; or the variant of SEQ ID NO: 235 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 23-678 of SEQ ID NO: 235; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 236; or a variant of SEQ ID NO: 236, wherein the variant of SEQ ID NO: 236 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-437 of SEQ ID NO: 236;
(iii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 237, 238, or 239; or a variant of any one of SEQ ID NOs: 237, 238, or 239, wherein the valiant of SEQ ID NO: 237 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-605 of SEQ ID NO: 237; the variant of SEQ ID NO: 238 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-606 of SEQ ID NO: 238; or the variant of SEQ ID NO: 239 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-605 of SEQ ID NO: 239; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 240; or a variant of SEQ ID NO: 240, wherein the variant of SEQ ID NO: 240 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-83 of SEQ ID NO; 240;
(iv) the L region comprises a nucleotide sequence set forth as SEQ ID NO; 241 : or a variant of SEQ ID NO: 241, wherein the variant of SEQ ID NO: 241 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-34 of SEQ ID NO: 241; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 242; or a variant of any one of SEQ ID NO: 242, wherein the variant of SEQ ID NO: 242 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 100-593 of SEQ ID NO: 242. (v) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 243: or a variant of SEQ ID NO: 243, wherein the variant of SEQ ID NO: 243 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 30-45 of SEQ ID NO: 243; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 244; or a variant of SEQ ID NO: 244, wherein the variant of SEQ ID NO: 244 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 100-677 of SEQ ID NO: 244; and
(vi) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 245, 246, or 247; or a variant of any one of SEQ ID NOs: 245, 246, or 247, wherein the variant of SEQ ID NO: 245 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 29—171 of SEQ ID NO: 245; the variant of SEQ ID NO: 246 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 30-171 of SEQ ID NO: 246; or the variant of SEQ ID NO: 247 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 29-171 of SEQ ID NO: 247: and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 248; or a variant of SEQ ID NO: 248, wherein the variant of SEQ ID NO: 248 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-91 of SEQ ID NO: 248.
[0021] The virus may be an alphavirus, and a combination of the L region and the R region sati sfies one of the following:
(i) the L region comprises a nucleotide sequence set forth as 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 nucleotide positions selected from the group consisting of positions 60-274 of SEQ ID NO: 249; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 250 or 251: or a variant of SEQ ID NO: 250 or 251, wherein the variant of SEQ ID NO: 250 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-183 of SEQ ID NO: 250; or the valiant of SEQ ID NO: 251 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-375 of SEQ ID NO: 251;
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 255; or a variant of SEQ ID NO: 255, wherein the variant of SEQ ID NO: 255 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-35 of SEQ ID NO: 255; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 256 or 257; or a variant of SEQ ID NO: 256 or 257, wherein the variant of SEQ ID NO: 256 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 600-273 of SEQ ID NO: 256; or the variant of SEQ ID NO: 257 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-377 of SEQ ID NO: 257; and
(iii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 261; or a variant of SEQ ID NO: 261 , wherein the variant of SEQ ID NO: 261 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-215 of SEQ ID NO: 261 ; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 262 or 263; or a variant of SEQ ID NO: 262 or 263, wherein the variant of SEQ ID NO: 262 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60- 166 of SEQ ID NO: 262; or the variant of SEQ ID NO: 263 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-379 of SEQ ID NO: 263. [0022] In a further aspect, the coding sequence within the encrypted RNA can be in a sense orientation. The virus may be a sarbecovirus, and a combination of the L region and the R region may satisfy one of the following:
(i) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, or 67; or a variant of any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, or 67, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1426- 1493 of any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, or 67; and the R region comprises the nucleotide sequence set forth SEQ ID NO: 129; or a variant of SEQ ID NO: 129, wherein the variant of SEQ ID NO: 129 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 129;
(ii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 68, 69, 70, 71, 72, 73, 74, 75, 76, or 77; or a variant of any one of SEQ ID NO: 68, 69, 70, 71, 72, 73, 74, 75, 76, or 77, wherein the variant of SEQ ID NO: 68 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 68; the variant of SEQ ID NO: 69 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 69; the variant of SEQ ID NO: 70 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1446-1513 of SEQ ID NO: 70; the variant of SEQ ID NO: 71 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1455-1522 of SEQ ID NO: 71; the variant of SEQ ID NO: 72 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1462-1529 of SEQ ID NO: 72; the variant of SEQ ID NO: 73 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1469-1536 of SEQ ID NO: 73; the variant of SEQ ID NO: 74 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1485-1552 of SEQ ID NO: 74; the variant of SEQ ID NO: 75 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1686-1753 of SEQ ID NO: 75; the valiant of SEQ ID NO: 76 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1704-1771 of SEQ ID NO: 76; or the variant of SEQ ID NO: 77 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1720-1787 of SEQ ID NO: 77; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 130; or a variant of SEQ ID NO: 130, wherein the variant of SEQ ID NO: 130 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130;
(iii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 78, 79, 80, 81, 82, 83, 85, 86, 87, or 88: or a variant of any one of SEQ ID NOs: 78, 79, 80, 81, 82, 83, 85, 86, 87, or 88, wherein the variant of SEQ ID NO: 78 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1734-1801 of SEQ ID NO: 78; the variant of SEQ ID NO: 79 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1687-1754 of SEQ ID NO: 79; the variant of SEQ ID NO: 80 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1695-1762 of SEQ ID NO: 80; the variant of SEQ ID NO: 81 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 81; the variant of SEQ ID NO: 82 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1443-1510 of SEQ ID NO: 82; the variant of SEQ ID NO: 83 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1459-1526 of SEQ ID NO: 83; the variant of SEQ ID NO: 85 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 85; the valiant of SEQ ID NO: 86 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 86; the variant of SEQ ID NO: 87 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1435-1502 of SEQ ID NO: 87; or the variant of SEQ ID NO: 88 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1463-1530 of SEQ ID NO: 88; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 130; or a variant of SEQ ID NO: 130, wherein the variant of SEQ ID NO: 130 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130;
(iv) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 89, 90, 91, 92, 96, 104, 105, 106, 107, or 108; or a variant of any one of SEQ ID NOs: 89, 90, 91, 92, 96, 104, 105, 106, 107, or 108, wherein the variant of SEQ ID NO: 89 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1466-1533 of SEQ ID NO: 89; the variant of SEQ ID NO: 90 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1425-1492 of SEQ ID NO: 90; the variant of SEQ ID NO: 91 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1425-1492 of SEQ ID NO: 91; the variant of SEQ ID NO: 92 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1425-1492 of SEQ ID NO: 92; the variant of SEQ ID NO: 96 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-769 or 1471-1471 of SEQ ID NO: 96; the variant of SEQ ID NO: 104 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1446-1513 of SEQ ID NO: 104; the valiant of SEQ ID NO: 105 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1455-1522 of SEQ ID NO: 105; the variant of SEQ ID NO: 106 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1462-1529 of SEQ ID NO: 106; the variant of SEQ ID NO: 107 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1469-1536 of SEQ ID NO: 107; or the variant of SEQ 119 NO: 108 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 89-839 or 1485-1552 of SEQ ID NO: 108; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 130; or a variant of SEQ ID NO: 130, wherein the variant of SEQ ID NO: 130 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130;
(v) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 109, 110, 111, 112, 113, 114, 115, 116, 117, or 118; or a variant of any one of SEQ ID NOs: 109, 110, 111, 112, 113, 114, 115, 116, 117, or 118, wherein the variant of SEQ ID NO: 109 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1686-1753 of SEQ ID NO: 109; the variant of SEQ ID NO: 110 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1704-1771 of SEQ ID NO: 110; the variant of SEQ ID NO: 111 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1720-1787 of SEQ ID NO: 111; the variant of SEQ ID NO: 112 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1734-1801 of SEQ ID NO: 112; the variant of SEQ ID NO: 113 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1687-1754 of SEQ ID NO: 113; the valiant of SEQ ID NO: 114 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1695-1762 of SEQ ID NO: 114; the variant of SEQ ID NO: 115 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 115; the variant of SEQ ID NO: 116 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 116; the variant of SEQ ID NO: 1 17 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 117; or the variant of SEQ ID NO: 118 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 118; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 130; or a variant of SEQ ID NO: 130, wherein the variant of SEQ ID NO: 130 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130; and
(vi) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 126, or 127; or a variant of any one of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 126, or 127, wherein the variant of SEQ ID NO: 119 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1443-1510 of SEQ ID NO: 119; the variant of SEQ ID NO: 120 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1459-1526 of SEQ ID NO: 120: the variant of SEQ ID NO: 122 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 122; the variant of SEQ ID NO: 123 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 123; the valiant of SEQ ID NO: 124 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434-1501 of SEQ ID NO: 124; the variant of SEQ ID NO: 125 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1463-1530 of SEQ ID NO: 125; the variant of SEQ ID NO: 126 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1466-1533 of SEQ ID NO: 126; the variant of SEQ ID NO: 127 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1425-1492 of SEQ ID NO: 127; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 130; or a variant of SEQ ID NO: 130, wherein the variant of SEQ ID NO: 130 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130.
[00231 The virus may be a Respiratory Syncytial Virus (RSV), wherein the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 148, 149, 150, 151, or 152; or a variant of any one of SEQ ID NOs: 148, 149, 150, 151, or 152, wherein the variant of SEQ ID NO: 148 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15—78 of SEQ ID NO: 148; the variant of SEQ ID NO: 149 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-33 of SEQ ID NO: 149; the variant of SEQ ID NO: 150 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-35 of SEQ ID NO: 150; the variant of SEQ ID NO: 151 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 18-36 of SEQ ID NO: 151; or the variant of SEQ ID NO: 152 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-38 of SEQ ID NO: 152; and wherein the R region comprises the nucleotide sequence set forth as SEQ ID NO: 154 or 155: or a variant of SEQ ID NO: 154 or 155, wherein the valiant of SEQ ID NO: 154 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-207 of SEQ ID NO: 154; or the variant of SEQ ID NO: 155 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-32 of SEQ ID NO: 155. [0024] The virus may be a parainfluenzavirus, and a combination of the L region and the R region may satisfy one of the following:
(1) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 180: or a variant of SEQ ID NO: 180, wherein the variant of SEQ ID NO: 180 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-136 of SEQ ID NO: 180: and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 179; or a variant of SEQ ID NO: 179, wherein the variant of SEQ ID NO: 179 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-98 of SEQ ID NO: 179;
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 186; or a variant of SEQ ID NO: 186, wherein the variant of SEQ ID NO: 186 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-95 of SEQ ID NO: 186; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 185; or a variant of SEQ ID NO: 185, wherein the variant of SEQ ID NO: 185 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-93 of SEQ ID NO: 185.
[0025] The virus may be a metapneumovirus, and a combination of the L region and the R region sati sfies one of the following:
(i) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 194; or a variant of SEQ ID NO: 194, wherein the variant of SEQ ID NO: 194 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-220 of SEQ ID NO: 194; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 192; or a variant of any one of SEQ ID NO: 192, wherein the variant of SEQ ID NO: 192 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-32 of SEQ ID NO: 192; and
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 193; or a variant of SEQ ID NO: 193, wherein the variant of SEQ ID NO: 193 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-220 of SEQ ID NO: 193: and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 191; or a variant of SEQ ID NO: 191 , wherein the variant of SEQ ID NO: 191 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-32 of SEQ ID NO: 191 .
[0026] The virus may be a henipaviras, and a combination of the L region and the R region satisfies one of the following:
(i) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 203: or a variant of SEQ ID NO: 203, wherein the variant of SEQ ID NO: 203 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-77 of SEQ ID NO: 203: and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 202; or a variant of SEQ ID NO: 202, wherein the variant of SEQ ID NO: 202 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-91 of SEQ ID NO: 202; and
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 207; or a variant of SEQ ID NO: 207, wherein the variant of SEQ ID NO: 207 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-77 of SEQ ID NO: 207; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 208; or a variant of SEQ ID NO: 208, wherein the variant of SEQ ID NO: 208 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-91 of SEQ ID NO: 208.
[0027] The virus may be a hepadnavirus, the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 212, 213, 214, 215, or 216: or a variant of any one of SEQ ID NOs: 212, 213, 214, 215, or 216, wherein the variant of SEQ ID NO: 212 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-1326 of SEQ ID NO: 212; the variant of SEQ ID NO: 213 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-1291 of SEQ ID NO: 213; the variant of SEQ ID NO: 214 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-1325 of SEQ ID NO: 214: the valiant of SEQ ID NO: 215 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-15 of SEQ ID NO: 215; or the variant of SEQ ID NO: 216 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-21 1 of SEQ ID NO: 216; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 217, 218, 219, or 220; or a variant of any one of SEQ ID NOs: 217, 218, 219, or 220, wherein the variant of SEQ ID NO: 217 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-754 of SEQ ID NO: 217; the variant of SEQ ID NO: 218 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101 -790 of SEQ ID NO: 218; the variant of SEQ ID NO: 219 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-892 of SEQ ID NO: 219; or the variant of SEQ ID NO: 220 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-2309 of SEQ ID NO: 220. [0028] For the composition, the therapeutic polypeptide 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-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, IL-12B, or IL-2, or wherein the therapeutic polypeptide is a caspase, or wherein the therapeutic polypeptide is an interferon, optionally an IFN-a, IFN-P, IFN-e, IFN-K, IFN-CD, IFN-y, or IFN-k, further optionally IFN-al, IFN-a2, IFN-a4, IFN-a5, IFN-a6, IFN-a7, IFN-a8, IFN-alO, IFN- al3, !FN-al4, IFN-al6, IFN-al7, IFN-a21, IFN-pi, IFN-e, IFN-K, IFN-rol, IFN-y, IFN-AI (IL28A), IFN- X2 (IL28B), IFN- X3 (IL29), or IFN- M.
[0029] For the composition, the coding sequence of the 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- Dal garno 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 encrypted RNA or the target mRNA may comprise a polyadenylation signal and/or a 3’ poly(A) tail. The encrypted RNA or the target mRNA may be in a linear form or a covalently-closed circular form.
[0030] For the composition, the RNA editor can be (i) an endogenous ADAR, (ii) an exogenous ADAR, or (iii) an engineered ADAR. The substrate for the RNA editor may comprise an adenosine to cytidine mispairing within the stop codon. The RNA editor can convert the adenosine to cytidine mispairing into an inosine to cytidine pairing in the substrate. The RNA editor can be one selected from the group consisting of: ADAR2, AD ARI, AD ARI pl50, AD ARI pl 10, ADAR2 R455G, ADAR2 S486T, ADAR2 T375G E488Q T490A, ADAR2 T375G, ADAR2 T375S, ADAR2 N473D, ADAR2 deaminase domain, ADAR2 T490S, ADAR2 T490A, MCP-ADAR2 deaminase domain, ADAR2 R455E, ADAR2 T375G T490A, ADAR2 E488Q, MCP- ADAR2 deaminase domain E488Q T490A, ADAR? R510E, ADAR2 R455S, and ADAR2 V351L.
[0031] For the composition, the target mRNA may comprise, in 5’ to 3’ order, the following operably linked elements: a 5’ UTR, an upstream coding sequence (uCDS), the edit tract, and a downstream coding sequence (dCDS) encoding the translation activator or the translation activator component.
[0032] The edit tract can comprise two or more stop codons that prevent translation of the translation activator or the translation activator component encoded by the target mRNA. The edit tract and the dCDS can be operably linked via (i) a ribosomal skipping sequence; (ii) a coding sequence encoding a protease cleavage site (CDS-PS); or (iii) a coding sequence encoding a flexible linker (CDS-FL). The guide RNA may have a length of 200-10,000 nucleotides. The guide RNA may be (i) native to the cell; (ii) delivered to the cell; or (ii) exogenous to the cell and transcribed by the cell.
[0033] In yet another aspect, provided is a method of modulating expression of a therapeutic polypeptide in a cell of a subject, comprising introducing the composition into the cell. The subject can be a human, a cow, a pig, a sheep, a horse, a deer, a ruminant, a rodent, fish, or a fowl.
[0034] The method may further comprise introducing an amplification target mRNA into the cell, wherein the amplification target mRNA comprises an edit tract, and wherein a stop codon within the edit tract prevents translation of the RNA editor encoded by the amplification target mRNA; wherein an amplification guide RNA binds to the amplification target mRNA to form a double-stranded RNA complex as a substrate for the RNA editor, wherein the substrate comprises a mispairing within the stop codon, and wherein the RNA editor acts upon the substrate to convert the mispairing into a pairing, thereby allowing translation of the RNA editor from the amplification target mRNA. The RNA editor may be (i) an endogenous ADAR or (ii) an exogenous ADAR, or (iii) an engineered ADAR. The substrate may comprise an adenosine to cytidine mispairing within the stop codon. The RNA editor can convert the adenosine to cytidine mispairing into an inosine to cytidine pairing in the substrate. The RNA editor may be one selected from the group consisting of: ADAR2, AD ARI, ADAR1 pl50, ADAR1 pl 10, ADAR2 R455G, ADAR2 S486T, ADAR2 T375G E488Q T490A, ADAR2 T375G, ADAR2 T375S, ADAR2 N473D, ADAR2 deaminase domain, ADAR2 T490S, ADAR2 T490A, MCP-ADAR2 deaminase domain, ADAR2 R455E, ADAR2 T375G T490A, ADAR2 E488Q, MCP- AD AR2 deaminase domain E488Q T490A, ADAR2 R510E, ADAR2 R455S, and ADAR2 V351L. The amplification guide RNA may be (1) native to the cell; (ii ) delivered to the cell; or (iii) exogenous to the cell and transcribed by the cell. The amplification guide RNA may have a length of 200-10,000 nucleotides.
[0035] 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 invention 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
[0036] The accompanying drawings are not intended to be drawn to scale. For purposes of clarity, the drawings are illustrative only and are not required for enablement of the disclosure. Not every component may be labeled in every drawing. In the drawings:
[0037] FIG. 1 shows a schematic of how targeted RNA-editing of an mRNA encoding a translation activator component can lead to increased translation of a translation activator component.
[0038] FIG. 2A shows a schematic of an editable target mRNA comprising a 5' UTR (UTR); an upstream CDS encoding a secreted embryonic alkaline phosphatase (SEAP); an Edit Tract that is the target of an RNA editor, and a downstream CDS sequence encoding a luciferase to report on the simulated translation of a Translation Activator. FIG. 2B shows that translation of downstream luciferase is increased when both the target editable mRNA and a guide RNA are simultaneously introduced into the same cells (RNA editor: an endogenous ADAR). Increased translation occurs when an exogenous RNA editor (“Hyp. ADAR” means hyperactive ADAR) is introduced in addition to the guide and target RNAs,
[0039] FIGs. 3A and 3B show that the regulation of levels of a component of a translation activator can dramatically modulate the translated levels of a polypeptide of interest. FIG. 3A shows that increasing levels of a translation activator agonist by only lOx (above a threshold of -1000 AU) can increase translation of the polypeptide of interest by ~5 logs. FIG. 3B shows that increasing levels of a translation activator antagonist just lOx (above the threshold of ~109 AU) can decrease translation of the polypeptide of interest by ~3 logs. Dashed lines indicate a 4 PL model fit to the experimental data.
[0040] FIG. 4 shows a schematic of how RNA editing of an mRNA encoding a translation activator component can lead to increased translation of a translation activator component and subsequent amplification and production of a payload encoded in an encrypted RNA.
[0041] FIG. 5A shows a schematic of an editable target mRNA comprising a 5' UTR (UTR); an upstream CDS; an Edit Tract that is the target of an RNA editor, and a downstream CDS sequence encoding a component of a Translation Activator. FIG. 5B shows a schematic of how an Edit Tract and Translation Activator can be operably linked via a Ribosomal Skipping Sequence. FIG. 5C shows a schematic of how an Edit Tract and Translation Activator can be operably linked via a Protease Cleavage Site. FIG. 5D shows a schematic of how an Edit Tract and Translation Activator can be operably linked via a Flexible Linker. FIG. 5D discloses SEQ ID NO: 583 (GSSG).
[0042] FIGs. 6A, 6B, 6C, 6D, and 6E show a mathematical comparison of idealized models of RNA-editing systems, comparing RADAR, DART VADAR, and EncRNA, and demonstrate that mRNA amplification via EncRNA can lead to higher expression levels of payload protein. FIG. 6A shows an ordinary differential equation (ODE) model where untranslatable mRNA (x) is converted to translatable mRNA (y) via RNA editing and the encoded protein is translated (z). FIG. 6B describes the three key parameters of the model: d, the decay rate of mRNA and edited mRNA; b, the editing rate of mRN A; and r, the translation rate of edited mRNA. FIG. 6C shows the closed form solutions of the family of ODE. FIG. 6D plots the solution with parameters d - 0.0866/h, b = d/10, and r = lOOd. As shown in FIG. 6E, only EncRNA can amplify mRNA post-activation, allowing for high theoretical production of an encoded payload.
[0043] FIG. 7A shows that encRNA amplification after ADAR-editing can lead to increased expression of the payload sequence (here, about a lOx increase in signal) when one of four components of a translation activator is ADAR-controlled. FIG. 7B shows that encRNA amplification after ADAR-editing can lead to increased expression of the payload sequence (in this case, about a lOOOx increase in signal) when two of four components of a translation activator are ADAR-controlled.
[0044] FIG. 8 depicts one example of how 1 -component, 2-component, 3-component, or 4-component Translation Activators can be controlled by encoding each component of a Translation Activator as an individual Editable mRNA. In essence, all elements of a translation activator must be provided to activate the Encrypted RN A. Therefore, the system functions as an “AND-gate ladder.”
[0045] FIG. 9 shows that a preliminary system utilizing well-known ribosomal skipping sequences can be activated after editing. The system comprises one to four editable mRNA, with each editable mRNA encoding a distinct Translation Activator component of an influenza Translation Activator (PB2, PB 1 , PA, and NP) in addition to an influenza encrypted RNA. Joining PB1, PA, and NP to an upstream Edit Tract has a severe effect on the activation of the encrypted RN A.
[0046] FIG. 10A shows a schematic of an editable target mRNA comprising a 5' UTR (UTR); an upstream CDS; an Edit Tract that is the target of an RNA editor, and a downstream CDS sequence encoding a component of a Translation Activator where the Edit Tract and Translation Activator Component are operably linked via a Ribosomal Skipping Sequence. FIG. 10B provides additional detail on the linkage at nucleotide (RNA) and amino acid (translated polypeptide) levels. FIG. 10B discloses SEQ ID NOs: 584-588, respectively, in order of appearance.
[0047] FIG. 11 shows strategies for improving the Translation Activator Component function when the component is operably linked to and positioned downstream of the Edit Tract. FIG. 11 discloses SEQ ID NOs: 589-605, respectively, in order of appearance.
[0048] FIG. 12 lists some alternatives to RNA editor-controlled activation of encrypted RNA translation activators at four different levels of gene expression: transcriptional control, post-transcriptional control, translational control, and post-translational control.
[0049] FIG. 13A shows the cross-activation of alphavirus encRNA by alphavirus translation activators (nsPl, nsP2, nsP3, nsP4). FIG. 13B displays a phylogenetic tree computed 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,
[0050] FIG. 14 shows that some nucleoside-modified VEEV sense-encrypted RNAs retain activation function and have lower levels of background translation than corresponding nonmodified encrypted RNAs.
[0051] FIG. 15 shows that RNA Editor Controlled Decryption of Encrypted RNA (RECDER) systems lead to elevated levels of a polypeptide of interest when RNA editing controls production of 2 translation activator components. FIG. 15 shows a RECDER system in which RN A editing controls translation of influenza PB2 and PA translation activator components to control activation of an influenza encRNA.
[0052] FIGs. 16A, 16B, and 16C show an ordinary differential equation (ODE) mathematical model of an encrypted RNA amplified RNA-editing system.
[0053] FIGs. 17A and 17B show that a programmable RNA-editing controlled system is dependent on the level of translation activator component produced (after editing) and that the system is payload independent, as both Gaussia luciferase (FIG. 17A) and Renilla luciferase (FIG. 17B) produce equivalent results.
[0054] FIGs. 18A, 18B, and 18C show schematics of how RECDER systems can be designed to enable preferential activation of an encRNA in cancer cells. FIG. 18A shows how a RECDER system can be designed for activation in cancer cells, with lower levels of activation in healthy (non -cancerous) cells. In one example, increased translation in cancer cells occurs, because cancer-associated RNA transcripts can serve as guide RNAs to program the removal of a stop codon from an RNA editor controlled mRNA (REC mRNA) that encodes a translation activator component of an encRNA, leading to activation of the encRNA and increased translation of the polypeptide of interest in cancer cells. FIG. 18B shows that RECDER systems can achieve dose amplification via two or more processes, including (i) exponential amplification of an encRNA after activation or (ii) paracrine signaling if the polypeptide of interest is a molecule with paracrine-signaling properties, FIG. 18C shows that the design of an encRNA within a RECDER system can be used to enable preferential translation of virtually any polypeptide of interest in cancer cells, including secreted immunotherapeutic proteins or reporter proteins.
[0055] FIG. 19A shows that cancer-associated transcripts, such as MYC, that can serve as guide RNAs in RECDER systems can differ dramatically between healthy cells and cancer cells over a range of up to 1,000 x differences in expression (units are nTPM, the number of transcripts per million transcripts) as measured by RNA-seq. Similarly, FIG. 19B shows a specific example of MYC expression differences between a colorectal cancer cell line (COLO320DM) and a normal colon cell line (CCDlSCo).
[0056] FIG. 19C shows that a programmable RNA-editing controlled system and an encrypted RNA using a 2-component translation activator (RECDER #3) leads to elevated production of the polypeptide of interest in a colorectal cancer cell line and to greater levels of the polypeptide of interest than an unamplified system. Increased payload production in the colorectal cancer cell line is driven by the increased presence of a cancer-associated transcript (MYC). The colorectal cancer cell line shown is COLO320DM and the normal colon cell line is CCDlSCo.
[0057] FIG. 20 shows the functionality of an influenza B virus-based RECDER system that can be programmed for activation in response to unique RNA transcripts, The system shown uses a 2-component translation activator (RECDER #5) controlled by the presence of a cancer-associated transcript (MYC), which leads to elevated levels of payload production in a colon cancer cell line (COLO320DM) compared to a normal colon cell line (CCDI8C0).
[0058] FIGs 21A, 21B, and 21C show experimental results that together demonstrate the functionality of two RSV-based RECDER systems (RECDER #6 and RECDER #7) that utilize either 1 or 2 RNA Editor Controlled mRNA (REC mRNA) that can be programmed for activation in response to unique RNA transcripts. FIG. 21A shows that both 1 REC mRN A (RECDER #6) and 2 REC mRNA (RECDER #7) systems are controlled by the presence of a synthetic guide RNA and are sensitive to RNA editing activity provided by an RNA editor (ADAR). FIG. 21 B shows that both systems are functional in multiple species (mouse and human) and achieve similar' levels of controlled protein translation when activated. FIG. 21C shows that both systems can be programmed for responsiveness to a cancer-associated transcript (MYC) and achieve a 50-75 x increase in translation in high MYC-expressing cells (COLO320DM) (“CR cancer cells” in legend) compared to low MYC-expressing cells (293T) (“Normal cells” in legend).
[0059] FIG. 22 shows the function of a RECDER system (RECDER #8), where the RNA editor is not a base editor, but is an RNA editor with ligase activity (eDC7„l 1). The two bi-partite gRNAs evaluated were: DR+cMycIntr2_tg2 (“MYC Target 2”) and DR+cMycIntr2_tg3 (“MYC Target 3”). Error bars correspond to 1 standard deviation from the mean (N=3 biological replicates),
DETAILED DESCRIPTION
Definitions:
[0060] 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.
[0061] 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”.
[0062] 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.
[0063] 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.
[0064] The use of any examples, or exemplary language (e.g., “such as”) provided for certain embodiments, 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 and as such can vary. 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.
[0065] 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 thi s disclosure is related. For example, the CONCISE DICTIONARY OF BIOMEDICINE AND MOLECULAR BIOLOGY, JUO, Pei-Show, 2Hd ed„ 2002, CRC Press; THE DICTIONARY OF CELL AND MOLECULAR BIOLOGY, 5th ed., 2012, Elsevier; TABER’S CYCLOPEDIC MEDICAL DICTIONARY, 23rd ed., (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,
[0066] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining 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.
[0067] 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 up to 10-fold of a value. When particular values or compositions are provided in the application 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.
[0068] 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.
[0069] 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.
[0070] 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. The disclosure of PCT/US2023/069976 (entitled “Encrypted RNA and Methods of Its Use”) filed on July 11, 2023, is incorporated by reference in its entirety for all purposes. 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.
[0071] 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 R region 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.
[0072] 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).
[0073] 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 example 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).
[0074] 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 R regions 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.
[0075] 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 5'-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.
[0076] 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 i s 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 next 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 .
[0077] 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.
[0078] 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.
[0079] 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.
[ 0080] 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.
[0081] 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.
[0082] As shown in FIGs. 1A and IB and FIGs. 2A and 2B of PCT7US2023/069976 filed on July 11 , 2023 (incorporated by reference in its entirety for all purposes), contact between an encrypted RNA and a translation activator increases the translation of the polypeptide of interest.
[0083] As used herein, an “encrypted protein” or an “encrypted polypeptide” is a polypeptide of interest encoded by an encrypted RNA.
[0084] 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.
[0085] As used herein, a “DNA-encoded encrypted RNA” is a DNA sequence that encodes an encrypted RNA cassette.
[0086] As used herein, an “encrypted nucleic acid” means an encrypted RNA or a DNA-encoded encrypted RNA. [0087] 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 phrases “negative-sense encrypted RNA” and “(-)-sense encrypted RNA” are equivalent to the phrase “antisense encrypted RNA”.
[0088] 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 encrypted RNA sequence. As used herein, the phrases “positive-sense encrypted RNA” and “(+)■ sense encrypted RNA” are equivalent to the phrase “sense encrypted RNA”.
[0089] As used herein, an “influenza encrypted RNA” is an encrypted RNA with a target- specific translation activator comprising an influenza virus polypeptide. An encrypted RNA with a target- specific translation activator comprising an influenza virus polypeptide means the encrypted RNA is activated by an influenza virus polypeptide.
[0090] As used herein, an “influenza A encrypted RNA” is an encrypted RNA with a target- specific translation activator comprising an influenza A virus polypeptide. An influenza A encrypted RNA is activated by an influenza A virus polypeptide.
[0091] As used herein, an “influenza B encrypted RNA” is an encrypted RNA activated by an influenza B virus polypeptide.
[0092] As used herein, a “therapeutic influenza encrypted RNA” or an “influenza SHIELD” is an influenza encrypted RNA that is a therapeutic encrypted RNA.
[0093] As used herein, the phrases an “influenza antisense encrypted RNA”, “influenza negative- sense encrypted RNA”, and “influenza (-)- sense encrypted RNA” are interchangeable in meaning and mean an influenza encrypted RNA that is an antisense encrypted RNA.
[0094] As used herein, an “influenza sense encrypted RNA”, an “influenza positivesense encrypted RNA”, or an “influenza (+)- sense encrypted RNA” are interchangeable phrases and mean an influenza encrypted RNA that is a sense encrypted RNA.
[0095] As used herein, a “sarbecovirus encrypted RNA” is an encrypted RNA activated by a sarbecovirus polypeptide. Examples of sarbecovirus peptides include but are not limited to peptides from SARS-CoV-1, MERS, SARS-CoV-2, or similar members of the genus betacoronavirus (group 2). [0096] As used herein, a “therapeutic sarbecovirus encrypted RNA” or a “sarbecovirus SHIELD” is a sarbecovirus encrypted RNA that is a therapeutic encrypted RNA.
[0097] As used herein, a “sarbecovirus antisense encrypted RNA” or a “sarbecovirus negative-sense encrypted RNA” or a “sarbecovirus (-)-sense encrypted RNA” is a sarbecovirus encrypted RNA that is an antisense encrypted RNA.
[0098] As used herein, a “sarbecovirus sense encrypted RNA” or a “sarbecovirus positive-sense encrypted RNA” or a “sarbecovirus (+)-sense encrypted RNA” is a sarbecovirus encrypted RNA that is a sense encrypted RNA.
[0099] As used herein, “SARS-2” is the SARS-CoV-2 virus (i.e., severe acute respiratory syndrome coronavirus 2, a strain of coronavirus).
[0100] As used herein, an “RSV encrypted RNA” is an encrypted RNA activated by a respiratory syncytial virus (RSV) polypeptide.
[0101] As used herein, a “therapeutic RSV encrypted RNA” or an “RSV SHIELD” is an RSV encrypted RNA that is a therapeutic encrypted RNA,
[0102] As used herein, an “RSV antisense encrypted RNA”, an “RSV negative-sense encrypted RNA”, or an “RSV (-)-sense encrypted RNA” is an RSV encrypted RNA that is an antisense encrypted RNA.
[0103] As used herein, an “RSV sense encrypted RNA”, an “RSV positive-sense encrypted RNA”, or an “RS V (+)-sense encrypted RNA” is an RSV encrypted RNA that is a sense encrypted RNA.
[0104] As described herein, the encrypted RNA can have the template regions (the L region and the R region) derived from various viruses, including but not limited to: Alphacoronavirus 229E, Alphacoronavirus NL63, Alphacoronavirus WA2028, Avian metapneumovirus (AMPV), Betacoronavirus HKU1, Betacoronavirus HKU15, Betacoronavirus HKU33, Betacoronavirus OC43, Chikungunya virus, Crimean-Congo Hemorrhagic Fever Virus, Dengue Virus, Eastern Equine Encephalitis Virus (EEEV), Enterovirus D68 (EV-D68), Foot and Mouth Disease Virus, Hanta Virus, Hendra Virus, Hepatitis B Virus, Hepatitis C Virus, HMPV, Human Parainfluenzavirus 1 (HPIV1), Human Parainfluenza virus 3 (HPIV3), Infectious Salmon Anemia Virus, Influenza A Virus, Influenza B Virus, Lassa Virus, Marburg Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Newcastle Disease Virus (NOV). Nipah Virus, Norwalk Virus, Rabies Virus, Respiratory Syncytial Virus, Reston Ebola virus, Rhinovirus, Rift Valley Fever Virus, Rubella virus, SARS-CoV-1, SARS-CoV-2, Sudan Ebola virus, Venezuelan Equine Encephalitis Virus (VEEV), Vesicular Stomatitis Virus, Western Equine Encephalitis Virus (WEEV), Yellow Fever Virus, Zaire Ebola virus, or Zika Virus.
[0105] As used herein, a carrier or polymeric carrier is typically a compound that facilitates the transport or complex ation 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 earner may, for some embodiments, be a cationic component.
[0106] 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 in a subject. 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,
[0107] 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 which are 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) tissue obtained 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). In the case of some animal and plant species, it may be possible to introduce DNA polynucleotides into the germline, such that the population of somatic cells is permanently rendered transgenic.
[0108] The term “tissue” refers to a group or layer of similarly specialized cells which together perform certain special functions. [0109] "‘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 typically comprises treatment as well as prevention or prophylaxis of a disease.
[0110] 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”,
[0111] “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 (UM P) 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 was synthesized or transcribed using nucleotide triphosphate monomers to form a usual RNA polynucleotide.
[0112] 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 is typically performed 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, 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, several types of RNA exist, which may be involved in the regulation of transcription or translation.
[0113] 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,
[0114] 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 RN A 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 the nucleoside/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 RN A 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 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 wdthin 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 that is modified to form N6-methyl-AMP. In some embodiments, a nucleoside- modified RNA includes at least one CMP that is modified to form 5 -methyl -CMP. In some embodiments, a nucleoside-modified RNA includes at least one UMP that is modified to form 5-methoxy-UMP.
[0115] 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.
[0116] Naturally occurring Cap structures typically comprise a riboguanosine residue that is methylated at position N7 of the guanine base. This N7-methylguanosine (m'G) 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 m'’G), the structure is referred to 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. [0117] Cap 0 (3'-O~Me) is Cap 0 in which the 3' -OH (i.e., 3' hydroxyl group) of the 5’ N7-methylguanosine (m''G) 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 (m'G) cap relative to the respective Cap 1 or Cap 2.
[0118] 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'-O-Me), a Cap 2, a Cap 2 (3'-O-Me), and an AntiReverse Cap Analog (ARCA), or selected from any combination or subcombination thereof. All of these represent nucleoside-modified RNA molecules.
[0119] As used herein, "‘uncapped RNA” or “noncapped RNA” refers to RNA molecules that lack a 5 '-Cap.
[0120] 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 “non phosphoryl a ted” or “5 '-nonphosphorylated” have no 5' terminal phosphate (zero phosphate or dephosphorylated).
[0121] 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 deoxyribonucleotide building 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. Typically, 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).
[0122] “RNA-dependent RNA polymerases” or “RdRPs” are multi-domain (a and p) proteins that catalyze RNA-temp late-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.
[0123] Some viral polymerases possess additional domains such as methyltransferase or 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. The host factors include translation factors, protein chaperones, RNA- modifying enzymes, or other cellular proteins. These 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.
[0124] As defined herein, a “ribozyme” is a catalytic macromolecular complex comprising an RNA with catalytic activity. Examples of ribozymes include: 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 nuclease activity may cleave within the molecule in which they are embedded or may cleave RNA outside of the molecule in which they are embedded.
[0125] 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 identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for 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: INI- ORM A TICS 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, New Jersey, 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. [0126] Polynucleotide or polypeptide sequences can be compared by performing a sequence alignment, which may be gapped or ungapped. In an un gapped 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 “gaps”) may be inserted to optimally align the sequences.
[0127] As used herein, “sequence similarity”, is used in a like manner to “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.
[0128] Methods to determine identity and similarity are codified in publicly available algorithms or software, and can include but are not limited to: BLAST, FASTA, T- COFFEE, or M-COFFEE. hi 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/bioinformatics/btt509).
[0129] 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.
[0130] 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 wall 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.
[0131] 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.
[0132] 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. [0133] 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, hi 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.
[0134] 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.
[0135] 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, which allows the convenient storage of a nucleic acid molecule, for example, of 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 needed for transcription of a sequence stretch of the vector, such as a promoter sequence, e.g., an RN A 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, e.g., a plasmid vector or a bacteriophage vector. A transfer vector may be a vector, which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors. In some embodiments, the viral vector can be a 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, such as 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.
[0136] A “lentivirus” as used herein 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 DN A of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV-1, SIV, and FIV are all examples of lentiviruses, Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.
[0137] 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,
[0138] 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, it may be a physiologically acceptable liquid, which is suitable for storing, transporting, or administering a pharmaceutically active compound. Encrypted RNAs
[0139] In some embodiments, an encrypted RNA is a ssRNA (single- stranded RNA).
[0140] In some embodiments, an encrypted RNA is a capped ssRNA.
[0141] In some embodiments, an encrypted RNA is an uncapped ssRNA.
[0142] In some embodiments, an encrypted RNA is a 5 '-triphosphorylated uncapped ssRNA.
[0143] In some embodiments, an encrypted RNA is a 5 '-diphosphorylated uncapped ssRNA.
[0144] In some embodiments, an encrypted RNA is a 5 '-monophosphorylated uncapped ssRNA. [0145] In some embodiments, an encrypted RNA is a 5 '■■nonphosphorylated uncapped ssRNA.
[0146] In some embodiments, an encrypted RNA is an uncapped ssRNA with four or more 5 '-terminal phosphates.
[0147] In some embodiments, an encrypted RNA is a stabilized nucleic acid molecule.
[0148] In some embodiments, an encrypted RNA is a circular RNA.
[0149] In some embodiments, the template region of an encrypted RNA or the translation activator is not derived from an alphavirus genome.
[01591 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.
[0151] In some embodiments, an encrypted RNA is delivered to cells together with a target- specific translation activator.
[0152] In some embodiments, DNA is used to encode an encrypted RNA.
[01531 In some embodiments, an encrypted nucleic acid is a stabilized nucleic acid molecule.
[0154] 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.
[0155] In some embodiments, a DNA-encoded encrypted RNA cassette can be repurposed to produce other RNA species by substitution of the encrypted RNA sequence with an alternative, non-encrypted RNA sequence encoding 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. [0156] In some embodiments, the DNA encoding an encrypted RNA is delivered to cells in a viral vector.
[0157] In some embodiments, the DNA encoding an encrypted RNA is delivered to cells in a plasmid.
[0158] In some embodiments, an encrypted RNA or the DNA encoding the encrypted RNA encodes a therapeutic protein, for example, an immunomodulatory protein.
[0159] 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 the use of 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 the 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).
[0160] 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.
[0161] RNA viruses are quite diverse in virus particle and genome structure and in virus entry and assembly mechanisms. However, they do 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 often 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 picornavirus 3Dpol, to the — 100-kDa flavi virus 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 the seven classic RdRP catalytic motifs, A-E are within the most conserved palm domain, and F and G are located in the fingers; they 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 for finding intervention strategies targeting these enzymes with possible broad-spectrum potential.
[0162] 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 RN A 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).
[0163] In some embodiments, an encrypted RNA resembles a viral RNA and possesses sufficient ds-acting sequences to be encapsidated into viral particles. In some further embodiments, encrypted RNA possesses sufficient cA-acting sequences to be encapsidated into viral particles that are infectious and can transmit and deliver encrypted RNA to additional cells via viral infection. In some embodiments, the RN A 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 cri-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.
[0164] 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, 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 negative- strand RNA viruses is the antisense strand.
[0165] 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.
[0166] 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 Picomaviridae), Coxsackie virus, and echovirus.
[0167] 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 pro vims or proviral DNA can be used to describe the DNA copy of a retroviral genome.
[01681 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.
[0169] 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. [0170] 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 RN A polymerases are typically thought to be primarily DNA-dependent RNA polymerases but may be able to synthesize RNA from a DNA template or an RNA template,
[0171] 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 pro tei n production ,
[0172] 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 mRN A in the absence of the translation activator.
[0173] 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, for example, FIG. 4B of PCT/US2023/069976 filed on July 11 , 2023, incorporated by reference in its entirety for all purposes).
[0174] 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.
[0175] 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 the precursor encrypted RNA by the ribozyme portion, the encrypted RNA is 5'- monopho sphorylated .
[0176] Exemplary sequence elements of encrypted RNAs or DNA-encoded encrypted RNAs 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 sequences for producing some encrypted RNAs or for DNA-encoding encrypted RNAs are listed in Table 4, Some exemplary amino acid sequences of polypeptides of interest are listed in Table 5.
[0177] 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).
TABLE 1: Sequence elements of L and R regions of encrypted RNAs
TABLE 2: Exemplary Encrypted RNA Scaffolds
TABLE 3: Coding Sequences and Reverse Complements of Coding Sequences for Exemplary Encrypted RNAs
TABLE 4: Additional Genetic Elements
TABLE 5: Amino Add Sequences for Exemplary Polypeptides of Interest / Other Peptides
Proteins of interest
[0178] 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.
[0179] 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 therefore reduces the expected lifetime of the cell. 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; receptorinteracting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL); caspases, including caspase-3, caspase-6, and caspase-7; and gasdermin D.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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 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) .
[0184] 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 CO VID- 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,
[0185] It is recognized that some viral RNAs are modified by cellular enzymes in select positions during their natural lifecycle. For examples, 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. & Horner S; PLoS Pathog. 2017 Mar; 13(3): el006188).
[0186] 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 is useful for treating a wide range of TH2- mediated diseases/conditions (e.g., atopic dermatitis, allergic rhinitis, and asthma).
[0187] 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” relates to any substance that induces in the subject being treated an anti gen- specific antibody or T- lymphocyte (T-cell) response. The term “antigen” comprises any molecule which comprises 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.
[0188] 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), fungi, as well as viruses.
[0189] 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 a non -self- antigen. A non-self- antigen may be a viral antigen, a bacterial antigen, a fungal antigen, an allergen, or a parasite antigen.
[0190] 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. In this 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.
[0191] 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.
[0192] In some embodiments, background translation of the polypeptide of interest is not substantially immunogenic.
[0193] 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.
[0194] 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 III, glucocerebrosidase, erythropoietin granulocyte colony stimulating factor (GCSF) or modified Factor VIII, anticoagulants, and the like),
[0195] In some embodiments, the therapeutic polypeptide of interest is comprised of 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-al3, IFN-al4, IFN-al6, IFN-al7, IFN-a21, IFN-pl , IFN-s, IFN-K, IFN-®1, IFN-y, IFN-M (IL28A), IFN-X2 (IL28B), IFN-X3 (IL29), or IFN-X4. Exemplary nucleotide sequences encoding proteins of interest are listed in Table 3. Exemplary amino acid sequences of proteins of interest are listed in Table 5. Additional nucleotide and amino acid sequences would be understood to be capable of substitution with those listed in the Tables,
[0196] 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.
[0197] In some embodiments, the therapeutic polypeptide of interest is comprised of an “antineoplastic protein”, An antineoplastic protein is a polypeptide effective in the treatment of cancer. 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.).
[0198] In some aspects, the polypeptide of interest is an antagonist of Programmed Cell Death Ligand 1 (PD-Ll) and/or Programmed Cell Death 1 (PD-1) or another immune checkpoint inhibitor. [0199] A PD-1 antagonist, as used herein, is an agent that inhibits or prevents PD-1 activity, e.g., by binding to PD-1.
[0200] 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 sixfold higher than that with PD-L1, suggesting that PD-L.2 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)).
[0201] Exemplary PD-1 antagonists include those described in U.S. Publications 20130280265, 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 ah, 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).
[0202] Exemplary PD-1 antagonists include nivolumab (e.g., OPD1VO® 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.
[0203] A PD-Ll antagonist, as used herein, is an agent that inhibits or prevents PD- Ll activity, e.g., by binding to PD-L1.
[0204] 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-Ll can also be inhibited or blocked by molecules other than antibodies that bind PD-LL Such molecules include proteins (such as fusion proteins and peptides.
[0205] Exemplary PD-Ll 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-Ll antagonists are described in Sunshine et al., Curr. Opin. in Pharm aco 1. 23 (2015).
[0206] PD-Ll antagonists include, for example: atezolizumab (also called MPDL3280A or TECENTRIQ™, Genentech/Roche), a human monoclonal antibody that binds to PD-Ll; durvalumab (also called MEDI4736 or IMFINZI™, AstraZeneca/Medlmmune), a human immunoglobulin IgGl kappa monoclonal antibody that binds to PD-Ll; BMS-936559 also known as MDX 1105 (Bristol-Myers Squibb), a fully human IgG4 monoclonal antibody that binds to PD-Ll ; avelumab (also called MSB 0010718C or BAVENCIO®, Merck KGaA/Pfizer), a fully human IgGl monoclonal antibody that binds to PD-Ll; 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-Ll.
[0207] 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-P'as ligand antibody, an anti-CXCR4 antibody, an anti- mesothelin antibody, an anti-CD27 antibody, an anti-GITR, or any combination thereof.
[0208] 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 gluclose 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.
[0209] In some aspects, the polypeptide of interest is a sodium glucose cotransporter 2 (SGLT-2) inhibitors. SGLT-2 inhibitors represent another class of drugs (other than GLP-1 agonist) 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).
RAA editor-controlled amplification
[0210] As described herein, translation of the target-specific translation activator(s) can be regulated by an RNA editor (e.g., FIGs. 1 and 4). The production, by translation, of the target- specific translation activator(s) can then activate the encrypted RNA, resulting in the translation of the polypeptide of interest encoded by the encrypted RNA. The RNA editor-controlled regulation mechanism described herein is similar to that described in WO 2022/266117.
[0211] As used herein, an "‘RNA editor” refers to an enzyme that has RNA as a substrate and has an activity (i) that can covalently modify the RNA nucleobase, such as adenosine deamination activity (i.e., capable of converting adenosine to inosine) (Cox et al., Science 358(6366): 1019-1027 (2017)); or (ii) capable of ligating two separate RNA fragments to form a functional mRNA encoding a desired product. When using the type (ii) RNA editor having a programmable RNA ligase activity, a translation activator gene or a gene encoding a translation activator component can be split into two parts, each encoded on a separate mRNA, and these two mRNA molecules can be ligated by the RNA editor into a single translatable product mRNA, ultimately resulting in the translation of the functional translation activator product or functional translation activator component product. [0212] A typical RNA editor capable of modifying the nucleobase is an adenosine deaminase acting on RNA (ADAR), which has been known to convert codons within a transcript such that the translation product can be functionally altered. Additional RNA editors include cytidine deaminases such as apolipoprotein B mRNA-editing enzymes, catalytic peptide (APOBEC), or fusions of RNA editing domains with Cas proteins.
[0213] ADAR enzymes are evolutionarily conserved among animals. For example, three known ADAR enzymes are present in mammalian cells: AD ARI, ADAR2, and ADAR3. ADAR 1 and AD AR 2 are known to be catalytically active. However, ADAR3, despite the substantial similarity to ADAR2, is generally considered catalytically inactive (see e.g., Savva et al., Genome Biol. 13(12): 252. (2012)). ADAR enzymes contemplated in the present disclosure include an endogenous ADAR, an exogenous ADAR, or an engineered ADAR. For example, ADAR enzymes may include ADAR2, AD ARI, AD ARI pl 50, AD ARI pl 10, ADAR2 R455G, ADAR2 S486T, ADAR2 T375G E488Q T490A, ADAR2 T375G, ADAR2 T375S, ADAR2 N473D, ADAR.?, deaminase domain, ADAR2 T490S, ADAR2 T490A, MCP-ADAR2 deaminase domain, ADAR2 R455E, ADAR2 T375G T490A, ADAR2 E488Q, MCP-ADAR2 deaminase domain E488Q T490A, ADAR? R510E, ADAR2 R455S, and ADAR2 V351L.
[0214] As described herein, a target mRNA is provided, which comprises an edit tract and a coding sequence for the translation activator or the translation activator component, wherein a stop codon within the edit tract prevents translation of the translation activator or the translation activator component. Once the RNA editor acts upon the edit tract of the target mRNA, in the presence of a guide, the conversion of adenosine in the stop codon to inosine occurs (i.e,, converting an adenosine to cytidine mispairing into an inosine to cytidine pairing; removing the stop codon), thereby allowing the translation of the translation activator or the translation activator component from the target mRNA (e.g., FIG. 1)
[0215] In some embodiments, the RNA editor may be an endogenous ADAR or an exogenous ADRA. For example, an engineered ADRA can be used.
[0216] In some embodiments, the RNA editor-controlled amplification may be achieved via an autocatalytic mechanism such as described in Gayet RV. et al., Nat. Commun. (2023) 14(1): 1339, doi: 10.1038/s41467 -023-36851-z. To achieve the amplification, an amplification target mRNA is provided, wherein the amplification target mRNA comprises an edit tract and a coding sequence for the RNA editor. The edit tract comprises a stop codon that prevents translation of the RNA editor. Once an RNA editor (e.g., an endogenous ADAR) acts upon the edit tract of the amplification target mRNA, in the presence of a guide, the conversion of adenosine in the stop codon to inosine occurs (i.e., removing the stop codon), thereby allowing the translation of the encoded RNA editor. The newly translated RNA editor (e.g., an ARD A) can act upon the amplification target mRNA, resulting in an autocatalytic amplification (see, e.g., FIG. 4).
RNA compositions
[0217] In some embodiments, an encrypted RNA or DNA encoding an encrypted RNA and a target mRNA or DNA encoding a target mRN A are 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.
[0218] 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).
[0219] In some embodiments, an encrypted RNA or a DNA encoding an encrypted RNA and a target mRNA or DNA encoding a target mRNA 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.
[0220] 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.
[0221] 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 (bit. J. Nanomedicine, 2014; 9: 1833-1843).
[0222] Cationic (including ionizable cationic) or neutral lipids have been widely studied as synthetic materials for the delivery of RNA. In some LNP embodiments, 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 genetic material 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.
[0223] 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 -00.358-0). Furthermore, an LNP can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, or carbohydrates) to its surface or to the terminal end of the attached PEG chains (see e.g.. Front Pharmacol. 2015 Dec. 1; 5:285).
[0224] IJpid 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-trimethylammomum- propane); DOTMA (N- [ 1 -(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 {see e.g., Adv. Drug Deliv. Rev. 2014 February; 55: 110-115) and newer lipid nanoparticles that utilize squaramide amino lipids {see e.g., Comebise et al., zWv. Functional Materials (2022), DOI: 10.1002/adfm.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.
[0225] In some embodiments, a composition can comprise an encrypted RNA or a DNA encoding an encrypted RNA and a target mRNA or DNA encoding a target mRNA that are formulated together with a cationic or polycationic compound or with a polymeric carrier. Accordingly, in a further embodiment of the invention, 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 as described herein can also be associated with a vehicle, transfection, or complexation agent for increasing the transfection efficiency or the expression of the RNA according to the invention or of optionally comprised further included nucleic acids.
[ 0226] 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.
[0227] In some embodiments, the composition comprises at least an encrypted RNA and a target mRNA as defined herein, which are 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 RNAs according to the invention that are 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”). [0228] In some embodiments, the complexed RNA in the compositions described herein is prepared according to a first step by complexing the RNA 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.
[0229] In other embodiments, the compositions comprising the disclosed compounds comprising the RNA may be administered naked without being associated with any further vehicle, transfection, or complexation agent.
[0230] 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 composition 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 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 such that more than one administration is required to administer the (therapeutic) encrypted nucleic acid species. If the 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 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.
[0231] The (pharmaceutical) composition according to the present invention may be provided in liquid or dry (e.g., lyophilized) form.
[0232] In some embodiments, the (pharmaceutical) composition comprises a safe and effective amount of (i) an encrypted nucleic acid, encoding a (therapeutic) polypeptide of interest or a combination of (therapeutic) polypeptides of interest; and (ii) a target nucleic acid (a target mRNA or the DNA encoding thereof), encoding the translation activator. As used herein, “safe and effective amount” means an amount of the encrypted nucleic acid and the target 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) and the target nucleic acid that is suitable for obtaining appropriate expression levels of the (therapeutic) polypeptide of interest. Such a “safe and effective amount” of the encrypted nucleic acid and the target nucleic acid of the (pharmaceutical) composition may furthermore be selected in dependence on the encrypted nucleic acid types and target nucleic acid types (e.g., nucleoside-modified vs. non-nucleoside-modified, or 5 '-triphosphorylated vs. 5 '-nonphosphorylated) or in the method of encrypted nucleic acid production, purification, or formulation. For example, 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 and the target nucleic acid of the (pharmaceutical) composition as defined above will furthermore vary in connection with: the particular disease/infection/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.
[0233] In some embodiments, the encrypted nucleic acid and the target 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 some embodiments, 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 to allow individual administration of each of the encrypted nucleic acids.
[0234] A composition according to the invention 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- free 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, NaaCOi, NaHCOi, NaaSCri; some examples of the optional potassium salts include KCI, KI, KBr, K2CO2, KHCO2, K2SO4; and some examples of calcium salts include CaCh, Cah, CaBi’2, CaCCh, CaSCh, 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 (KCI), wherein further anions may be present in addition to the chlorides. CaCh can also be replaced by another salt like KCI. 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
[0235] Suitable routes of administration include, for example, pulmonary including intratracheal or inhaled, intranasal, oral, rectal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including 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 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.
[0236] 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 (HSV) 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.
[0237] 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, integrationdeficient 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 extrachromosornal episomes. Some chromosomal integration can occur with IDLV technology, with 0.1 %— 1% of proviruses integrating into the genome.
[0238] 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 double- stranded DNA (dsDNA) product, which then enters the nucleus.
[0239] 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.
[0240] 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. Ad Vs are able to deliver larger transgenes than A A Vs. As with AAV, AdV -delivered DNA does not generally integrate into the host genome, but rather, 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.
[0241] 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 a 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.
[0242] 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.
[0243] In some embodiments, an encrypted RNA or DNA encoding an encrypted RNA and a target mRNA or DNA encoding thereof according to the invention are used to produce a medicament, wherein the medicament is for treatment or prophylaxis of a disease, disorder, or condition. In some embodiments, treatment of 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 (as shown in FIG. 3A of PCT7US2023/069976, filed on July 11, 2023, incorporated by reference in its entirety for all purposes). Conversely, 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 (as shown in FIG. 3B of PCT/US2023/069976, filed on July 11, 2023, incorporated by reference in its entirety for all purposes), If cells are both treated with a therapeutic encrypted RNA (or a DNA encoding a therapeutic encrypted RNA) and also infected with a vims 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 production of a distinct mRNA species and subsequent translation of the therapeutic polypeptide of interest (e.g., an interferon) (as shown in FIG. 3C of PCT/US2023/069976, filed on July 11, 2023, incorporated by reference in its entirety for all purposes). 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.
[0244] In some embodiments, encrypted nucleic acids and target nucleic acids or formulations thereof (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 target nucleic acids, or 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 the present 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.
[0245] 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.
[0246] In some embodiments, a subject is treated with a therapeutically effective amount of more than one encrypted RNA, wherein treatment with each encrypted RN A 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.
[0247] 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.
[0248] 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. [0249] 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.
[0250] 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.
[0251] 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
[0252] 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.
[0253] 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, or configurations will depend upon the specific application or applications for which the inventive teachings are used.
[0254] 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.
[0255] 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.
[0256] 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.
[0257] 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.
[02581 The present invention is further illustrated by the following examples, which in no way should be construed as further limiting.
Conventions
[0259] Unless otherwise noted, the following buffers have the below essential compositions:
WFI is nuclease-free, endotoxin- free water for injection. * PBS is phosph ate- 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 Serum (FBS), 100 U/mL penicillin, and 100 pg/mL of streptomycin (all concentrations final).
* D10 is Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) Fetal Bovine Serum, 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 RN Ase- free).
* PF A is paraformaldehyde.
[0260] Unless otherwise noted, water-soluble solutions were usually prepared using dII2O.
[0261] 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 chain reaction; 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.
[0262] Room temperature is any temperature in the interval from about 19 °C to about 26 °C.
[0263] Unless otherwise specified, kits and specified materials were used according to their manufacturer’s instructions.
Nanoparticle formulation
* FM00 is DPBS.
* FM01 is 5% (m/v) sucrose in DPBS-CMF.
Immuno staining
* PBSM is DPBS-CMF with 5% (m/v) dry nonfat milk.
* PBSMT is PBSM wdth 0.05% (v/v) Tween-20.
* PBST is DPBS-CMF with 0.05% (v/v) Tween-20,
Other 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 wdth 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 bysubtracting 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
[0264] To aid in the teaching of these Examples, example viral strains or example 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.
[0265] Influenza A virus abbreviations are: A/PR8 means Influenza A/PuertoRico/8/1934 (H1N1); A/SwOH means Influenza A Virus, A/swine/Ohio/09SW79M/2009 (H3N2); A/NewCaledonia means Influenza A Virus, A/New Caledonia/20/1999 (H1N1).
[0266] Influenza B virus abbreviations are: B/Texas means Influenza B Virus, B/Texas/06/2011 (Yamagata Lineage): B/Sydney means Influenza B virus B/Sydney/507/2006 (Yamagata Lineage); B/Brisbane means Influenza B Virus, B/Brisbane/60/2008 (Victoria Lineage); B/Ohio means Influenza B virus B/Ohio/01/2005 (Victoria Lineage).
[0267] Coronavirus abbreviations are: OC43 or OC43-CoV means Human
Coronavirus OC43 (unless otherwise noted, strain ATCC VR-759 is used for the OC43 experiments herein); SARS-CoV-2 means Severe acute respiratory syndrome coronavirus 2; Washington or WA-1 means SARS-CoV-2 Isolate USA-WA1/2020 (BEI Resources, cat. No. NR-52281); MA30 means the SARS2-N5O1YMA3O mouse- adapted SARS-CoV-2 virus described in (Wong et al. Nature (2022), DOI: 10.1038/s41586-022-04630-3); Oniicron BA.l means the SARS-CoV-2 isolate from Omicron variant lineage B.1.1.529; .Alpha means the SARS-CoV-2 isolate from Alpha variant lineage B.1.1.7; Beta means the SARS-CoV-2 isolate from Beta variant lineage B.1.351; Gamma means the SARS-CoV-2 isolate from Gamma variant lineage P. l; Delta means the SARS-CoV-2 isolate from Delta variant lineage B.1.617.2; MERS-CoV means Middle East Respiratory Syndrome Coronavirus (MERS) (unless otherwise noted, isolate EMC/2012 is used for the MERS experiments performed or prophesized herein).
[0268] EMCV abbreviations are: “EMCV” means Encephalomyocarditis virus, strain MM (BEI Resources; cat. No. NR-19846).
[0269] 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.
[0270] Human parainfluenza virus abbreviations are: “HPIV1” refers to human parainfluenza virus 1, and “HPIV3” refers to human parainfluenza virus 3.
[0271] Human metapneumovirus abbreviations are: “HMPV” means human metapneumovirus.
[0272] Henipavirus abbreviations are: “NiV” means Nipah virus; “NiV-Bangladesh” or “NIVB” means Nipah virus, Bangladesh strain; “NiV -Malaysia” or “NiVm” means Nipah virus, Malaysia stain; and “HeV” means Hendra virus.
Measurement ofLNP size via Dynamic Light Scattering and Calculation of Polydispersity Index (PDI ) from Particle Size Distribution
[0273] Particle size and particle size distribution of L.NP formulations were quantified using a standard technique: Dynamic Light Scattering (DLS). DLS was performed using a Zetasizer Pro (Malvern Panalytical, Malvern UK).
[0274] 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)). [0275] 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 of LNP mixture
[0276] The zeta-potential (C-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 a 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 of RNA Encapsulation efficiency via exclusion of membrane- impermeable RNA-specific dye
[0277] 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 CORNAIS 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 a <URNA > 0.85.
Measurement of pKa via TNS
[0278] 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: 10.1021/10.1021/lal04590k). Briefly, LNPs are diluted to 1 and 10 ng/uL (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 the amino lipids are in the unionized state, TNS has little fluorescence. The pH values at which half of the maximum fluorescence is reached are reported as the apparent p.% values of the LNP.
Measurement of protein translation via flow cytometry
[0279] 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 cm2 of cell 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).
[0280] 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.
[0281] 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 ]).
[0282] 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
[0283] Method A: The culture media samples or tissue homogenate were analyzed with luminescence assay by adding 50 pL QU ANTI- Luc luciferase substrate (InvivoGen, cat. No. rep-qlc2) to 10 uL of the sample in triplicates and measuring luminescence signal immediately on a multimode plate reader (PerkinElmer).
[0284] 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 samples were 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-
[0285] Culture media samples were analyzed with luminescence assay by adding 50 pL alkaline phosphatase substrate (Cayman Chemical, cat. No. 600183) to 10 uL 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)
[0286] 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.
[0287] Human interferon beta secreted into cell culture supernatant was detected using a LumiKine Xpress hIFN-P 2.0 ELISA (enzyme linked immunosorbent assay) kit (InvivoGen, cat. No. uex-hifnbv2) according to the manufacturer’s instructions. A standard 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.
[0288] Human interferon lambda (A.) 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.
[0289] 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.
[0290] Mouse interferon beta (INF-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.
[0291 ] Mouse interferon lambda 2 and 3 ( IFN-A2 and IFN-X3) 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.
[0292] 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
[0293] 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” specices for normalization of downstream realtime RT- PCR between samples and analysis runs.
Quantification of cellular/tissue gene expression changes via RT-qPCR
[0294] 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 DN Ase treatment’ was omitted.
[0295] 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.
[0296] 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. AMI 907), and the DNAse enzyme removed using the supplied inactivation reagent according to the manufacturer’s instructions.
[0297] Gene expression changes were quantified by performing reverse transcription quantitative real-time PCR (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.
[0298] Complementary DNA (cDNA) was prepared from DNAse-treated RNA in 20 pL reverse transcription reactions using reagents obtained from ProtoScript® 11 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 dTiaVN (SEQ ID NO: 582), 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.
[0299] 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 I pM reverse primer in NFW (125 nM final).
[0300] 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: Table 6. Standard qPCR thermocy cling conditions
Hydrolysis Probe -based qPCR
[0301] 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 was created 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 w'as 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.
[0302] 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.
Influenza infectious titer by focus-forming unit assay
[0303] Virus supernatants were serially diluted in DMEM, containing 0.035% BSA, 50 mM sodium bicarbonate, and antibiotics (typically 100 U/mL penicillin and 100 pg/mL streptomycin). Twelve- well dishes of MDCK cells were infected with various serial dilutions of the virus in 400 pL of the above media and incubated at 37°C in 5% CO2 for 90 min and gently rocked every 15 min. After removing the inoculum, the wells were overlaid with 2 mL of MEM with 0.001% (m/v) Dextran, 0.1% (m/v) sodium bicarbonate, 1 pg/mL of TPCK-trypsin, and 2% (m/v) low melt Oxoid agar. The purpose of adding TPCK-Trypsin was to allow' the influenza virus to spread cell- to-cell by maturation of the viral hemagglutinin protein. The plates were then incubated for 48 h in a humidified 5% CO2 cell incubator at one of two temperatures: 37°C (for most influenza A strains) or 33°C (for most influenza B strains).
[0304] After the 48 h incubation period, the plaquing monolayers were fixed using 1 mL of 4% PFA per well for a minimum of 4 hours to overnight (i.e., 16-20 hours). Following this, the agar overlay plugs were removed by gently tapping on the side of the microplate and rinsing the wells with dlfoO,
[0305] For influenza. A strains, plates were incubated with 1:4000 dilution of antiinfluenza A antibody (Chicken Influenza A, Puerto Rico 8/34 polyclonal antibody; MyBioSource Cat. No. MBS623909) in PBSM for 4 h at room temperature on a shaker. The plates were washed 3x with PBS and incubated with a 1:4000 dilution of Peroxide-Conjugated Affinipure Donkey anti-Chicken Antibody (secondary antibody) (Jackson Immunoresearch, cat no. 703-035-155) in PBSM for 1 h at room temperature on a shaker, Following incubation with the secondary antibody, the wells were washed 3x with PBS, and the plaques were developed using the TMB Solution (Ready-to-Use) for IMMUNOBLOT (Thermo Fisher Scientific, cat no. 002019).
[0306] Viral titers were calculated by enumerating a countable number of discrete infection foci (e.g., >50) and multiplying by the nominal dilution factor. Viral titers were reported as plaque-forming units per mL (PFU/mL), regardless of whether the counting of discrete infection foci was enabled by immuno staining (focus -forming unit; FFU) or by clearing of a cell monolayer (plaque forming unit; PFU),
[0307] For Influenza B viruses, plates were incubated with 1 :4000 dilution of antiinfluenza B Rabbit polyclonal antibody HD09DE2801-B against B/Florida HA (Sino Biological, cat no. 11053 -T62) in PBSM for 4 h at room temperature. The plates were washed 3x with PBS and incubated with 1:4000 dilution of Peroxide-Conjugated Affinipure Donkey anti-Rabbit Antibody (Jackson Immunoresearch, cat no. 711-035- 152) in PBSM for 1 h at room temperature. Following incubation with the secondary' antibody, the plaques were developed, and the virus was quantified as described above.
SARS-CoV~2 & MERS-CoV infectious titer via plaque assay
[0308] The virus was titrated via plaque assay as described in (Zheng et al., Nature (2021); DOI: 10.1038/s41586-020-2943-z). Briefly, virus or tissue homogenate supernatants were serially diluted in DMEM and then used to inoculate 12 well plates of Vero E6 cells. Cells wzere incubated with inocula for 1 h at 37°C in 5% CO2 humidified incubators; cell plates were gently rocked every 15 min. After removing the inocula, plates were overlaid with D02 supplemented with 0.6% (m/v) low-melt agarose. After 3 days, overlays were removed, and the plaques were visualized by staining with 0.1% crystal violet. Viral titers were recorded as PFU/mL for cell culture samples or PFU/mL/mg tissue for tissue homogenate samples.
Infectious titration of generation-limited SARS-CoV-2
[0309] A BSL-2 infection model of SARS-CoV-2 (“generation-limited SARS-CoV- 2” or “SARS2-GL”) was used in some in vitro assays. Titration of infectious generation-limited SARS-CoV-2 virus was based upon a viral titration protocol outlined by (Mendoza et al., Curr. Protoc. Microbiol., (2020); DOI: 10.1002/cpmc,105). Supernatant samples were serially diluted in D02, then the 6- well plates of Vero-E6-hACE2+ORF3a/E cells were incubated with the diluted inocula for 1 h at 37°C in 5% CO2 humidified cell incubators, with gentle rocking by hand given every 15 min during the incubation.
[0310] After removing the inocula, plates were overlaid with 0.8% (m/v) Oxoid agarose in 1 x MEM (ThermoFisher Scientific, cat. No. 11935046) containing 3.5% FBS supplemented with 0.2 jig/mL doxycycline, 10 pg/mL puromycin, 1.8 mM L- glutamine (ThermoFisher Scientific, cat, No. 25030149), 0.9x nonessential amino acids (ThermoFisher Scientific, cat. No. 11140050), 0.7 x sodium bicarbonate (ThennoFisher Scientific, cat, No. 25080094). After 48 h, plaques were stained by adding an overlay additionally supplemented by 0.01% (m/v) neutral red. Plaques were enumerated at 24 h after adding the second overlay (at approximately 72 hpi).
SARS-CoV-2 infectious titer via RT-qPCR
[0311] SARS-CoV-2 viral RNA was extracted from infected cell culture supernatants as described above (“Standard Methods”). Further steps of DNAse treatment and RT- qPCR (dye-based or hydrolysis probe-based), were carried out as described above (“Standard Methods”). Virus genome titers were obtained by normalizing the viral RNA detected in the supernatant to the MS2 bacteriophage RNA spike-in. Table 7
OC43 infectious titer via RT-qPCR
[0312] OC43 viral RNA was extracted from infected cell culture supernatants as described above (“Standard Methods”). The further steps of DN Ase treatment and RT-qPCR were carried out as described above (“Standard Methods”). Virus titers were obtained by normalizing the viral RNA detected in the supernatant to the spikedin sample of MS2 phage RNA added during nucleic acid isolation.
Table 8
RV V' infectious titer via focus-forming unit assay
[0313] 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 uL 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 wdthout disturbing.
[0314] 6 days post-infection, the plaques in the monolayers were fixed using 1 mL of
4% PFA 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) CA-630 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),
[0315] 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 RSV bearing a fluorescent reporter gene-encoded into the viral genome, plaques were directly counted from the wells and imaged.
HPIV infectious titer via focus -forming unit assay or plaque -forming unit assay [0316] Virus supernatants were serially diluted in Opti-MEM. 24-well dishes of LLC-
MK2 cells or Hep2 cells were infected with various serial dilutions of human parainfluenza virus 1 (HPIV1) or human parainfluenza virus 3 (I IP1V3) in 200 pL of Opti-MEM and incubated at 37 °C in 5% CO? 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 carboxymethylcellulose, or HPIV3 overlay media (MEM containing 1 % Oxoid agar) for HPIV3 FFU assays. For the HPIV1 FFU assay, the cells were overlaid with HPIV1 overlay media (MEM containing 1% low melting point agarose and 1 mg/mL TPCK- Trypsin). The plates were incubated at 37 °C in a humidified 5% CO2 incubator for 4 days (HPIV3) or 6 days (HPIV 1) without disturbing.
[0317] After the 4-day (HPIV3) or 6-day incubation (HPIV1), the plates 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 rinsing with dHsO. Plates were then washed with PBS and treated with 0.5% (v/v) IGEPAL CA630 in DPBS for 10 min and w ashed 2x with PBS. Subsequently, the plates were incubated with PBSM to block for 1 h at room temperature on a shaker. After blocking, 1:1000 dilution of anti- HPIV1 or anti-HPIV3 guinea-pig antiserum (NIA1D, V321 -511-558 or V323-501- 558) in PBSMT, was added and the plates were incubated overnight at 4°C on a rocker, The plates were washed 3x with PBST and then incubated with a 1:1000 dilution of Peroxide-Conjugated AffiniPure Goat Anti-Guinea Pig antibody (Jackson Immunoresearch, cat no. 106-035-003) in PBSMT and the plates incubated for 1 h at room temperature on a rocker.
[0318] 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) according to manufacturer instructions. Viral titers were quantified as PFU/mL of tissue culture supernatant. Alternatively, for the FFU assay with an agarose overlay, an additional overlay with 1% agarose and 0.25 mg/ml neutral red was added to the wells for the 4- or 6-days post-infection cells. Forty-eight (48) hours after adding the second overlay, the plaques in the monolayers were fixed using 1 mL of 4% PFA per well for a minimum of 1 h. Following cell fixation, the overlay plugs were removed by gentle tapping, and water flow and plaques were visualized against the negative staining of the neutral red solution.
HMPV infectious titer via focus -forming unit assay
[0319] Virus supernatants were serially diluted in HMPV growth media (Opti-MEM containing 2% FBS, 100 U/mL penicillin, 100 pg/mL streptomycin, 2 mM glutamine, 50 pg/mL gentamycin, 2.5 ug/mL Amphotericin B, 100 ug/mL CaCh). 24- well dishes of LLC-MK2 cells were infected with various serial dilutions of HMPV stocks in 200 pL of HMPV growth media and incubated at 37 °C in 5% CO2 for 2 h, gently rocking every 15 min. After removing the inoculum, the wells wzere overlaid with HMPV overlay media containing up to 5 ug/ml TPCK-trypsin and 1% dissolved carboxymethylcellulose. The plates were incubated at 37°C in a humidified 5% CO2 incubator for 10 days without disturbing them.
[0320] Ten (10) days post-infection, the plaques in the monolayers were fixed using 1 mL of 4% PFA per well for a minimum of 1 h. Following fixation, 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 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-HMPV mouse monoclonal antibody (GeneTex, Cat. No. GTX36792) in PBSMT, was added and the plates were incubated overnight at 4 °C on a rocker. The plates were washed 3x with PBST and then incubated with a 1:1000 dilution of Peroxide-Conjugated Affinipure Goat antiMouse Antibody (Jackson Immunoresearch, cat no. 115-035-003) in PBSMT and the plates incubated for 1 h at room temperature on a rocker. 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) according to the manufacturer’s instructions. Viral titers were quantified as PFU/mL of tissue culture supernatant.
Encephalomyocarditis Virus (EMCV) titer via Median Tissue Culture Infectious Dose Assay
[0321] Encephalomyocarditis Virus (EMCV) stocks or tissue culture supernatants from infected cells were diluted from 10"jx to 10'12x via ten-fold dilutions in D02. Human HeLa or mouse L929 cells plated in a confluent monolayer in 96-well dishes were infected with 100 pL of the above virus dilutions per well and incubated for 48 h at 37 °C in 5% CO2. After 48 h or whenever clearance of wells was observed for lower dilutions of the virus, the plates were fixed with 100 uL of 4% PFA per well for at least 1 hour.
[0322] The fixatives were removed by tipping the plate and washed with PBS, The plates were then stained with 0.1% crystal violet and the completely cleared wells were recorded, TCID50 concentration is calculated by the Spearman & Karber algorithm as described in Hierholzer & Killington (1996), VIROLOGY METHODS MANUAL, p. 374. The viral titer in TCID50 is represented per mb of tissue culture supernatant.
Example 1: DNA constructs related to RNA~editing controlled mRNAs (REC mRNA)
Plasmids that encode guide RN As
[0323] Plasmids encoding guide mRNAs with regions complementary to REC mRNA targets were developed: (i) pCMV6-EGFP-stop-«mTyr», which encodes an mRNA comprising an EGFP CDS followed by a 70 nucleotide (nt) subsequence derived from the 3' end of the house mouse tyrosinase mRNA (NCBI Gene: 22173: mRNA: NM_011661.6) and (ii) pCMV-EGFP-stop-«Sl», which encodes an mRNA comprising an EGFP CDS followed by a synthetic 90 nt synthetic guide RNA sequence (Kaseniit KE et al., Nat. Biotech. (2023) 41 (4): 482-487, doi: 10.1038/s41587-022-01493-x). To enhance the readability of plasmid nomenclature, the sequence region of a guide RNA that is complementary to a target RNA is flanked by doubled-angled brackets (e.g., “«mTyr»”).
[0324] In each plasmid, an RNA Polymerase II promoter (CMV IE2) drives transcription of the encoded guide mRNA upon introduction of the plasmid to mammalian cells.
Base plasmid.s that encode Design 1 (DI) REC-mRNA
[0325] Plasmids that encode mRNAs with RNA editor ■■controlled expression were developed: pSFFV-SEAP-P2A-[[Sl]]-P2A-{ {GDura}} and pSFFV-SEAP-P2A- [[mTyrS]]-P2A-{ {GDura}}. The relevant mRNA transcripts, with poly(A) tail stripped, are listed as SEAP-P2A-[[Sl]]-P2A-{ {GDura} } (SEQ ID NO: 457) and SEAP-P2A-[[mTyrS]]-P2A-{ {GDura} } (SEQ ID NO: 458). Within each plasmid, a spleen focus-forming virus (SFFV) promoter drives the PolII transcription of an mRNA containing an editable stop codon. Numerous promoters exist that are acceptable alternatives to SFFV, the key criterion being sufficient transcriptional activity in the cell or tissue receiving the DNA. The stop codon is positioned within an Edit Tract that is targeted by a guide RNA containing a region imperfectly complementary to the Edit Tract and mismatched at one or more positions comprising the target sequence. Two example guide sequences are SI or mTyr (see, e.g., above). To enhance the readability of plasmid nomenclature, the sequence region of a target RNA that is complementary to a guide RNA is flanked by doubled square brackets (e.g., “[[SI]]”) while the sequence region flanked by doubled curly brackets (e.g., “{ {GDura} }”) refers to the protein payload controlled by RNA editing.
[0326] To quantify the degree of RNA editing, a simulated “100% editing” control was developed for each transcript in which the “tag” stop codon within the Edit Tract was genetically corrected to “tgg” (the codon for tryptophan) to determine the maximum GDura expression level (see, for example, SEQ ID NOs: 459 & 460), The control plasmids were termed pSFFV-SEAP-P2A-[[Sl„nostop]]-P2A-{ {GDura} } and pSFFV-SEAP-P2A-[[mTyr_ nostop] ]-P2 A- { {GDura} }.
Plasmids that encode proteins with programmable RNA editing activity
[0327] Some methods of increasing intracellular RN A editing activity utilize the treatment of cells with exogenous RNA editors, such as ADAR1 or ADAR2. A family of expression plasmids was developed to produce exogenous ADARs, including: (i) pCMV6-MCP-ADAR2, which expresses human ADAR2; (ii) pCMV6-ADARl, which expresses human AD ARI, and (iii) pCMV6-MCP-ADAR2dd(E488Q, T490A ), which produces functional ADAR editors and is detailed in (Kuttan A & Bass BL. Proc Natl Acad Sci USA (2012): 109(48): E3295-304; doi:
10.1073/pnas.1212548109). For brevity, ADAR2dd(E488Q, T490A) is also termed “ADAR2 hyp” for hyperactivity.
Plasmids that encode REC-mRNA with payloads of influenza encrypted RNA translation activators
[0328] A series of plasmids was developed to control the expression of components of translation activators of influenza encrypted RNAs, triggered by the presence of guide RNA sequence, SI. Four plasmids were initially engineered, each encoding a separate component of an influenza A translation activator derived from influenza strain A/PR8: pSFFV-SEAP-P2 A-[ [S 1]]-P2A- { { PR8_PB2} } , pSFFV-SEAP-P2A- [[ S 1JJ-P2A- { { PR8...PB 1 } } , pSFFV-SEAP-P2A- [[S 1]] -P2A-{ {PR8 P.A } } , and pSFFV-SEAP-P2A-[[S 1 ]] -P2A- { {PR8..NP} } .
[0329] An analogous series of four DI plasmids was also engineered, where SEAP was replaced by ADAR2_hyp: pSFFV-MCP-ADAR2„hyp-P2A-[[Sl]]-P2A- { { PR8. _PB2| }, pSFFV-MCP-ADAR2_hyp-P2A-[[Sl]]-P2A-{{PR8_PBl } }, pSFFV- MCP- AD AR2_hyp-P2A- [[SI]] -P2A- { { PR8_ PA } } , and pSFFV-MCP- AD AR2_ hyp- P2 A-[[S 1 ]]-P2 A-{ {PR8. _NP] } .
[0330] In addition, a simulated ” 100% editing” control was developed for each transcript in which the "‘tag” stop codon within the Edit Tract was genetically corrected to “tgg” to determine the maximum GDura expression level. The 100% editing control plasmids were termed: pSFFV-SEAP-P2A-[[Sl_nostop]]-P2A- { {PR8_PB2}}, pSFFV-SEAP-P2A-[[Sl_nostop]]-P2A-{{PR8_PB1 } }, pSFFV- SEAP-P2A-[[Sl„nostop]]-P2A-{ {PR8..PA} }, pSFFV-SEAP-P2A-[[Sl„nostop]]- P2A-( (PR8_NP}}, pSFFV-MCP-ADAR2_hyp-P2A-[[Sl_nostop]]-P2A- { {PR8..PB2} }, pSFFV-MCP- AD AR2..hyp-P2A-[[Sl..nostop]]-P2A-{ {PR8„ PB1 } }, pSFFV-MCP-ADAR2_hyp-P2A-[[Sl_nostop]]-P2A-{ {PR8_PA}}, and pSFFV- MCP- AD AR2 hyp-P2 A- [ [S 1 „ nostop] ] -P2 A- { { PR8 NP } } . Example 2: Post-transcriptional control of mRNA expression by RNA -editing [0331] Human 293T cells were treated, by transfection, with the plasmid pools described in ’Table 9. At 24 hours post-infection, the levels of translated SEAP and GDura (luciferase) were measured by quantifying SEAP or GDura activity as described above section: “Measurement of secreted luciferases that convert coelenterazine” and “Measurement of secreted embryonic alkaline phosphatase which converts 1,2-dioxetane CSPD (disodium 3-(4-methoxyspiro { l,2-dioxetane-3,2'-(5'~ chloro)tricyclo [3.3.1.13,7]decan}-4-yl)phenyl phosphate).”
[0332] FIG. 2B shows the results of the experiment. The results demonstrated that RNA-editing of target mRNA can increase the expression of an encoded payload. Treatment 1 reflects the background levels of SEAP and GDura activity in untreated cells. In Treatments 2 and 3, where either the guide RNA or the target RNA was provided individually, only background levels of GDura activity were observed, similar to Treatment 1. Treatments 2 and 3 demonstrate that the stop codon was sufficient to reduce translation to background levels in the absence of a guide RNA.
[0333] In contrast, Treatment 4 shows that application of both a guide RNA and a target REC- mRNA, leads to a statistically significant ~ lOOx increase of GDura translation owing to the activity of endogenous cellular ADAR. Providing additional exogenous ADAR (via transfection of pCMV-MCP-ADAR2..hyp) further increased expression to ~300x, indicating that higher ADAR activity can increase mRNA translation of the encoded polypeptide. Note that all four treatments in which the target REC-mRNA was delivered showed the equivalent level of SEAP activity, demonstrating that transfection levels and non-controlled expression were equivalent among the treatments. Example 3: Control of the encRNA expression by modulating levels of translation activator components
Activation Sensitivity of Translation Activators with Cooperative Response
[0334] Human A549 cells were treated with a pool of mRNAs encoding Translation Activators of influenza encrypted RNAs. The Translation Activators were provided as a pool of mRNAs, comprising an equimolar amount of PR8..PB2, PR8..PB1, PR8__PA, and PR8_NP mRNAs. During transfection treatment, the total pool of influenza encrypted RNA translation activators w'as provided across a dosage range from 0 to 50 ng/cm2. Each cell population was contemporaneously treated with the same dosage level of encrypted RNA (150 ng).
[0335] The activation response of the influenza encrypted RNA is shown in FIG. 3A; the response was fit to a 4-parameter logistic regression and the Hill coefficient was found to be -2.5, indicating that the activation response is cooperative (FIG. 3C), as the Hill coefficient was >1.
Repression Sensitivity of Translation Activators with Cooperative Response
[0336] Human A549 cells were treated with a pool of influenza translation activation mRN As, with upstream miR-145 binding sites. Cells were treated with an increasing dose of synthetic siRNA targeting miR-145 binding sites (hsa-miR- 145-5p, assay ID MCI 1480, cat. No. 4464066, ThermoFisher Scientific).
[0337] The repression response is shown in FIG. 3B. The repression response was fit to a 4-parameter logistic regression and the Hill coefficient was found to be ~9, indicating the statistically significant high degree of cooperativity in the response (FIG. 3C). Importantly, the half-repression threshold (IC50) is positioned in the low nM range, allowing for the sensitive detection of RN A species that may be present at concentrations of few' er than 1,000 molecules per cell.
Example 4: Modelling of improvement in amplification due to encrypted RNA amplification
Mathematical Model of RNA editing controlled expression when editable RNA is provided in a single bolus dose
[0338] FIG. 6A shows a first-order differential equation (ODE) model where untranslatable mRNA (x) is converted to translatable mRNA (y) via RNA editing (e.g., editing of a stop codon), which subsequently permits translation of a protein (<) from the edited mRNA. The system dynamics are governed by 3 parameters, shown in FIG. 6B: d, the decay rate of mRNA (and edited mRN A); b, the editing rate of mRNA; and r, the translation rate of edited mRNA. Given that the intracellular halflife of a typical mRNA in mammalian cells is about 8—10 h (Yang et al.: Genome Research (2003)), it was calculated that the decay rate was ln(2)/(8 h) or 0.0866/h. For a typical mRNA, -1% editing is typically observed in vitro (Kaseniit KE et al. Nat. Biotech. (2023), 41(4): 482-487, doi: 10.1038/s41587-022-01493-x), although there have been reports of editing rates of 10% (Jiang K et ah, Nat. Biotechnol. 2023; 41(5): 698-707. doi: 10.1038/s41587 -022-01534-5); therefore, b was optimistically set as 10% of d in this model, or 0.00866/h. Given these assumptions, each mRNA should produce ~ 100 proteins during the edited mRNA lifetime, implying r is d xlOO or 8.66/h.
[0339] FIG. 6C shows the closed-form solution of the system of ODEs. Each function (x(i), y(t), z(t)) evolves as a sum of exponentials. As t — » oo, both x(t) and y(t) vanish (x(t) 0, y(i) —> 0) while z(t) br/[cl(d.+b')]. Thus, the limit of maximal protein expression increases with r and b, but maximal protein expression is reduced as d increases.
[0340] FIG. 6D plots the closed-form solution using the parameters, d - 0.0866/h, b = <#10, and r - 100J. The plot show's the conversion of unedited mRNA to edited mRNA and finally to the protein via a standard exponential decay chain.
[0341] Importantly, others have proposed methods of increasing protein expression by enhancing the editing rate, for example, by adding exogenous ADAR (Jiang K et al., Nat. Biotechnol. 2023; 41(5): 698-707. doi: 10.1038/s41587-022-01534-5) (Kaseniit KE et al, Nat. Biotech. (2023), 41(4): 482-487, doi: 10.1038/s41587-022- 01493-x), triggering ADAR production (Gayet RV et ah, Nat. Commun. (2023) 14(1): 1339, doi: 10.1038/s41467-023-36851-z), stabilizing guide RNAs (Vogel P et al., Angew Chem. Int. Ed. Engl. (2014), 53(24): 6267-71, doi: 10.1002/anie.201402634) or improving the association between guide and target RNA (Kuttan A & Bass BL; Proc. Nall. Acad. Sci. USA (2012), 109(48): E3295-304, doi:
10.1073/pnas.1212548109). What is the maximum enhancement in protein expression achievable by these proposed approaches? If all of the RNA is immediately translatable (i.e., b approaches oo), then the limit of protein expression is rid. The ratio between suboptimal editing and optimal editing is thus b!(d+b). Editing enhancement and editing acceleration can increase expression only up to the limit achievable by introducing a fully-translatable mRNA.
Model of RNA editing of an editable RNA bolus followed by mRNA amplification [0342] The previous model is illustrated by the data in FIGS. 6A to 6D demonstrates the fundamental limits of protein translation from non-amplified REC mRNA templates, i.e., the final level of translated protein cannot exceed the level achievable by providing a completely translatable mRNA. However, downstream amplification may be possible if the protein payload encoded in REC mRNA is not the ultimately desired protein but rather an intermediate product that subsequently catalyzes the amplification and translation of the ultimately desired protein.
[0343] Encoding a component of the Translation Activator of an encrypted RN A can achieve this desired amplification. For example, a component of the viral polymerase Translation Activator can be encoded as a REC mRN A payload. After RN A-editing, the translated viral polymerase will act upon an encrypted RNA target to translate high levels of a polypeptide of interest.
[0344] FIG. 16A shows a mathematical model of the modified system. Here, an additional step was incorporated, wherein RNA editing of an mRNA (x) first triggers the formation of edited RNA (yj, which leads to the production of a translation activator component of an encrypted RNA (</), which subsequently generates an amplified pool of mRNA, translating the desired polypeptide of interest (z) at high abundance.
[0345] The system dynamics are governed by six (6) parameters, shown in FIG. 16A: d, the decay rate of mRNA (and edited mRNA); b, the editing rate of mRNA; and r, the translation rate of edited mRNA; c, the clearance rate of the translation activator component; k, the half-maximum translation activator response concentration; and h, the Hill coefficient. The same assumptions were used for h, d, and r as before, but set c to a higher degradation rate, at 3d.
[0346] The new, logistic-function-like activation function recapitulates the activation response seen in Example 3 (FIG. 3A), and is plotted in FIG. 16B, with k = 0.005 and h = 10.
[0347] FIG. 16B shows the numerically-solved solution of the system of ODEs. As t co, both x(f) and y(t) vanish (x(t) — » 0, y(t) 0). FIG. 16B shows that while q(t) tracks y(t) and z(t) rapidly increases at first (once c\(t) exceeds k), it rises to a limit, about lOOOx greater than the prior un-amplified model.
[0348] Thus, in comparison to earlier systems described in the art (per FIG. 6E) such as RADAR (Jiang K et al., Nat. Biotechnol. 2023: 41(5):698-707, doi:
10.1038/s41587-022-01534-5) and DART VADAR (Gayet RV et ah, Nat. Commun. (2023) 14(1): 1339, doi: 10.1038/s41467 -023-36851-z), the use of encRNA demonstrates that mRNA amplification can lead to significantly higher expression levels than previously obtained with RADAR or DART V ADAR.
Example 5: ADAR-controI of a single-component encrypted RNA translation activator leads to amplified translation of target payload
[0349] Current techniques in RNA editor-controlled expression are limited by the concentration of edited mRNAs, at maximum, the instantaneous concentration of editable mRNAs (assuming all editable mRNAs can be instantly edited). It was reasoned that incorporating an additional step, wherein RNA editing of an mRNA first triggers the production of a translation activator component of an encrypted RNA, which subsequently generates an amplified pool of mRNA, would increase the overall expression of a protein payload.
[0350] The new system, termed RNA Editor Controlled Decryption of Encrypted RNA, or RECDER, therefore utilizes encrypted RNAs to encode the payload sequence and sw'itches the context of the RNA-editing controlled mRNA to translate a component of the translation activator, rather than the ultimate pay load {see FIG. 4).
[0351] To test the RECDER system’s capability, an analogous study to Example 2 was performed, via a head-to-head comparison of conventional mRNA editing via ADAR versus using encRNA amplified editing as described above, Human 293T cells were transfected with pools of plasmids listed in Table 10, and the level of activated translation was quantified by measuring the level of secreted GDura and SEAP at 24 hours after infection.
[0352] Treatments 1201-1203 tested the specific activation of a classical system, i.e., cells that were treated with combinations of components of the unamplified classical system (guide RNA, target mRNA).
[0353] Treatments 1301-1303 tested the specific activation of an amplified system (RECDER #1), with cells being treated with plasmids to produce combinations of: guide RNA; encrypted RNA encoding a GDura payload: editable mRNA encoding a component of a translation activator of an influenza (PB2); and additional non- editable mRNA encoding remaining translation activator components,
[0354] The results of the study are shown in FIG. 7A demonstrate that using RECDER #1 leads to ~30x greater amplification than the classical REC system, as shown by a corresponding increase in payload expression, measured as an increase in luciferase activity level from of 3E5 in the classical REC system to 1E4 with RECDER #1 . Some translational leakiness was observed in the absence of a guide RNA (1E4 for prototype 2 vs 1E3 for conventional).
Table 10: Treatments to test the performance of
RECDER #1
— - indicates 0 ng plasmid
Example 6: ADAR-coutrol of a two-component encrypted RNA translation activator leads to amplified translation of target payload
[0355] Translation Activators of encrypted RNAs comprise one or more protein components and levels of each component can be modulated separately (FIG. 8). It was reasoned that RNA-editing control of more than one translation activator component could reduce the background translation in the absence of a guide RNA, as sufficient levels of two proteins would be required. Thus, in comparison to Example 3, RNA-editing control of two components of an influenza Translation Activator (PB2 and PB1) was added to engineer a new RECDER prototype, RECDER #2. [0356] Human 293T cells were transfected with pools of plasmids listed in Table 11, and the level of activated translation was quantified by measuring the level of secreted GDura at 24 hours after infection.
[0357] Treatments 1401-1404 tested the specific activation of an amplified system, with the cells being treated with plasmids to produce combinations of: guide RNA: encrypted RNA encoding a GDura payload: an editable mRNA encoding a component of a translation activator of an influenza virus (PB2); an editable mRNA encoding a component of a translation activator of an influenza virus (PB1); and additional non-editable mRNA encoding remaining translation activator components.
[0358] The results of the study are shown in FIG. 7B, demonstrating that using RECDER #2 leads to ~3000x signal above background translation, compared to ~1E4 in the classical system, when comparing a luciferase activity level of 3E5 to 1E4. Lower translational leakiness was observed in the absence of a guide RNA (1E4 for RECDER #2 vs 1E3 for conventional REC mRNA).
Table 11: Treatment Schedule to Test RNA-editing controlled mRNA vs. 2-component dose-amplified REC mRNA (RECDER #2)
— ifidicaLes 0 ng plasmid
[0359] A similar experiment was performed using human 293T cells. The human 293T cells were transfected with pools of plasmids listed in Table 12 (forming RECDER #3), and the level of activated translation was quantified by measuring the level of secreted GDura at 24 hours after infection.
[0360] Treatments 1501-1504 tested the specific activation of RECDER #3, with cells being treated with plasmids to produce combinations of: guide RNA; encrypted RNA encoding a GDura payload; an editable mRNA encoding a component of a translation activator of an influenza virus (PB2); an editable mRNA encoding a component of a translation activator of an influenza virus (PB1): and additional non- editable mRNA encoding remaining translation activator components,
[0361] The results of the study are shown in FIG. 15, demonstrating that using RECDER #3 leads to ~> 10,000x signal above the background translation signal, as compared to the results obtained when using the classical system. Lower translational leakiness was observed in the absence of a guide RNA (1E2 for RECDER #3 vs. 1E3 for conventional REC mRNA).
Table 12: Treatment Schedule to Test RNA-editing controlled mRNA vs. 2-component dose-amplified REC mRNA (RECDER #3)
— - indicates 0 ng phasmid
Example 7: RECDER can eonditionaHy control the expression of arbitrary payloads
[0362] The generality of the platform in the delivery and conditional activation of arbitrary genetic payloads, including a Renilla luciferase (RLuc8) and human interleukin 12 ( IL12) was next evaluated. The functional performance of the orthogonal payloads was determined using a procedure analogous to that described in Example 6 (Treatments 1501—1504, RECDER #3), with the encRNA payload encoding either RLuc8 or IL12.
[0363] When the encRNA encoded RLuc8, it was found that dynamic expression of RLuc8 (as measured by luciferase activity) was >l,000x above the background levels when activated in the presence of a guide RNA and <2x above the background levels in the absence of a guide RNA. [0364] When the encRNA encoded IL12, levels of IL- 12 exceeded 4,000 pg/mL when activated in the presence of a guide RNA and were less than 15 pg/mL when not activated by a guide RNA (as 15 pg/mL is the limit of detection of the IL-12 ELISA). This indicates the dynamic activation of the encrypted RNA via RNA-editor control of the Translation Activator (RECDER #3) was at least 266x (4000/15 is about 266) and may, in fact, be greater.
Example 8: Expression levels can be tuned by varying the concent ration of guide RNA or target RNA or both guide and target RNAs
[0365] A key result of the mathematical modeling in Example 4 was the identified monotonic relationship between editing rate, b, and the overall translation level achievable with the REC-mRNA/encRNA system, with translated levels directly increasing as the editing rate increases. The instantaneous editing rate, b, is dictated by the concentrations of guide RNA, where b = b'lguide RNA].
[0366] FIG. 17A and FIG. 17B show the increased level of expression of conditionally translated payloads (i.e., only in the presence of the guide RNA) with a direct dependence on the levels of delivered REC niRNA. Additionally, FIG. 17A and FIG. 17B show the independence of payload expression from levels of sham guide RNA (- RNA), based upon the weak (and not statistically significant) linear correlation between conditional expression and levels of sham guide RNA,
Example 9: Some REC-mRNA payloads are sensitive to N-terminal proline additions conferred by ribosomal skipping sequence in certain backgrounds [0367] The activation system was next extended by systematically testing whether a controllable 3-component translation activator using the same methods as in Example 4 can be generated. The treatment schedule and results are shown in FIG. 9.
[0368] In Treatments 1601—1607, cells were treated with plasmids to produce: guide RNA; encrypted RNA encoding a GDura payload; and different combinations of editable or non-editable mRNA encoding components of translation activators. By default, each translation component was constitutively translatable: PB2, PB1, PA, and NP. However, in some treatments, constitutively translatable mRNA was replaced with editable mRNA: (i) PB2 was editable in 1601, 1602, 1605, and 1606; (ii) PB1 w'as editable in 1601, 1603, and 1605; (iii) NP was editable in 1601, 1602, 1603, and 1604. [0369] In Treatments 1608-1614, all treatments included the same guide RNA and encrypted RNA encoding a GDura payload as in Treatments 1601-1607. However,
Treatments 1608-1607 used different combinations of editable or non-editable mRNA encoding components of translation activators. In this case, the “editable” versions used “nostop” constructs, in which the stop codon was corrected at the transcript level, simulating a 100% editing rate. The “nostop” plasmid simulates the maximal conversion possible, Thus, by default, each translation component was constitutively translatable: PB2, PB1, PA, and NP. However, in some treatments, constitutively translatable mRNA was replaced with “nostop” mRNA: (i) PB2 was editable in 1608, 1609, 1612, and 1613; (ii) PB1 was editable in 1608, 1610, and 1612; (iii) NP was editable in 1608, 1609, 1610, and 1611.
[0370] Results of the study are shown in FIG. 9; the results demonstrate that N- terminal tagging of some translation activator components with a P2A ribosomal skipping sequence can inhibit their activity. The control arm of the study (Treatments 1608-1614), tested the effect of expressing PB2, PB1, and NP components of an influenza translation activator as “edited” RNAs in pSFFV-SEAP-P2A-[[Sl]]-P2A background. By including an upstream P2A site, the downstream polypeptide will incorporate an additional N-terminal proline (see FIGs. 10A and 10B).
[0371] In particular, expression of PB1 and NP, in the “ADAR2...hyp-P2A” background led to suboptimal activation of the encrypted RNA. This suboptimal activation can be observed by comparing the suboptimal treatments 1608-1611 (wherein PB1 or NP was editable) to optimal treatments 1613 and 1614 (where neither PB1 nor NP was editable).
Example 10: Alphavirus-based RECDER systems
[0372] A variety of suitable translation activators that can be encoded as a REC mRNA are compatible with this invention. In this example, a functional alphavirus RdRP REC mRNA was demonstrated, and challenges in its development were highlighted.
[0373] Several candidate alphavirus RdRP REC mRNA were developed. The lead candidate, pCMV-EGFP-P2A-[[Sl]]-P2A-{{EEEV„RdRP}}, contained an editing tract inserted upstream of the nspl start, codon. Four additional candidates (A,B,C, and D), contained insertions of the editing tract amidst protein loop domains within nsp2, nsp3, and nsp4. [0374] Human 293T cells were transfected with pools of plasmids listed in Table 13, and the level of activated translation was quantified by measuring the level of secreted GDura at 24 hours after treatment.
[0375] Treatments 1701-1704 tested the specific activation of an amplified system (RECDER #4), with cells being treated with plasmids to produce combinations of: guide RNA; alphavirus encrypted RNA encoding a GDura payload; an editable mRNA encoding a component of a translation activator of alphavirus; and a hyperactive ADAR2.
[0376] It was found that Treatment 1704 produced the highest overall levels of conditional protein expression, approximately 1 log above background levels, while Treatment 1703 (without supplementation with an exogenous RNA editor) resulted in a 0.5 log increase over the level detected in the control treatments 1701 and 1702.
[0377] Although pCMV-EGFP-P2A-[[S 1 ]] -P2 A- { {EEEV . RdRP} } had demonstrated functionality, activation with candidates A, B, C, or D was unable to be detected.
Table 13: Treatment Schedule to Test RNA-edsting controlled nsRNA of Alphavirus encrypted RNA (RECDER #4)
— - indicates 0 ng plasmid
Example 11: Application to treating cancer in host (mouse example) [0378] Groups of C57BL/6 mice will be engrafted with a syngeneic tumor, such as a
B16-F10 melanoma, by injection of the tumor cells into the relevant anatomical location (such as by subcutaneous injection into both contralateral dorsal flanks). Once the tumor has engrafted, the initial tumor volume will be measured by determining the overall dimensions of the visible tumor to approximate the volume.
[0379] Distinct groups of engrafted mice (e.g., 5-10 mice) will be treated with the folio wi ng ex perimental treatmen ts :8 (Treatment 1) RECDER #3 comprising a target mRNA with an Edit Tract complementary to a transcript known to be upregulated in the tumor cells (e.g., mouse tyrosinase mRNA for B16-F10) and an influenza encrypted RNA harboring an antineoplastic polypeptide of interest (such as mouse I L 12 ):
8 (Treatment 2) RECDER #3 comprising a target mRNA with an Edit Tract complementary to a transcript known to be upregulated in the tumor cells (e.g., mouse tyrosinase mRNA for B16-F10) and an influenza encrypted RNA harboring a non-therapeutic payload (such as Gaussia luciferase);
8 (Treatment 3) RECDER #3 comprising a target mRNA with a scrambled Edit Tract sequence and non-complementary to a transcript known to be upregulated in the tumor cells (e.g., mouse tyrosinase mRNA for B16-F10) and an influenza, encrypted RNA harboring an antineoplastic polypeptide of interest (such as mouse IL 12 ); and (Treatment 4) comprising a formulation vehicle control.
The RECDER #3 systems described in Treatments 1-3 can be introduced by a suitable method, for example, an LNP capable of delivery to the tumor of interest. Similarly, the RECDER #3 systems can be DNA-encoded and delivered as plasmids or viral vectors (e.g., lenti viral vector).
[0380] The tumors of mice in each group will be treated using Treatments 1-4, and potentially, using a suitable range of dosages and dosing intervals (every day, every 2 days, every 3 days, or every 5 days). The tumor size of each animal will be monitored by measurement with calipers or medical imaging,
[0381] The particular RECDER #3 system, if effective and specific to activation within the tumor line, is expected to significantly reduce tumor size progression in Treatment 1 at a greater rate than Treatments 2-4.
[0382] Given the examples disclosed herein with encrypted RNA constructs derived from various exemplary viruses and exemplary therapeutic polypeptides, an ordinary practitioner in the field would have believed that similar results would have been obtained if using other viruses and/or other therapeutic polypeptides (i.e., achieving an equivalent log fold increase using the processes described herein). Example 12: RECDER #3 can trigger translation selectively in cancer cells that overexpress the proto-oncogene MYC
[0383] The proto-oncogene MYC is aberrantly expressed in >50% of cancers and has been contemplated as a potential cancer drug target (Hermeking, H. (2003). Current cancer drug targets, 3(3), 163-175)(Duffy, M. J., et al (2021 ). Cancer treatment reviews, 94, 102154). Yet, despite the appeal of MYC based on its high prevalence in many cancers, there are currently no licensed drugs that directly target MYC (Duffy, M. J., et al (2021). Cancer treatment reviews, 94, 102154). The absence of MYC drugs has been attributed to the difficulty of identifying small molecule inhibitors specific to the intrinsically disordered protein structure of MYC, which lacks classical druggable features such as a hydrophobic pocket (Duffy, M. J., et al (2021). Cancer treatment reviews, 94, 102154).
[0384] Although targeting of MYC is challenging, FIG. 19A shows that cancer- associated transcripts, such as MYC, that can serve as guide RNAs in RECDER systems, can differ dramatically in expression level between normal healthy cells and cancer cells over a range of up to 1 ,000 x (units are nTPM, the number of transcripts per million transcripts) as measured by RNA-seq. Similarly, FIG. 19B show's a specific example in MYC expression differences between a colorectal cancer cell line (COLO320DM) and a normal colon cell line (CCDI8C0) as measured by RT-qPCR and normalized to housekeeping transcript (beta-actin) expression levels,
[0385] FIGs. 18A, 18B, and 18C show schematics of how RECDER systems can be designed to enable preferential activation of an encRNA in cancer cells. FIG. 18A shows how a RECDER system can be designed for activation in cancer cells, with lower activation levels in normal (non-cancerous) cells. In one example, increased translation in cancer cells occurs, because cancer-associated RNA transcripts can serve as guide RNAs to program the removal of a stop codon from an RNA editor- controlled mRNA (REC mRNA) that encodes a translation activator component of an encRNA, leading to activation of the encRNA and increased translation of the polypeptide of interest in cancer cells. FIG. 18B show's that RECDER systems can achieve dose amplification via. two or more processes, including (i) exponential amplification of an encRNA after activation or (ii) paracrine signaling if the polypeptide of interest is a molecule with paracrine-signaling properties.
[0386] It was reasoned that RECDER #3 comprising a target mRNA with an Edit Tract complementary to MYC (known to be upregulated in the tumor cells) and an influenza encrypted RNA harboring an antineoplastic polypeptide of interest (such as IL12) could be used together to form an anti-neoplastic therapeutic that would allow for selective translation of the anti-neoplastic IL12 protein in MYC-high cancer cells while preventing translation of IL 12 in MYC-low healthy cells. In another way, endogenous MYC mRNA would serve as a guide RNA for a RECDER #3 system to produce IL12 selectively in (cancer) cells with high MYC expression. Use of additional or other polypeptides of interest can offer new or additional functionality to RECDER systems. FIG. 18C shows that the design of an encRNA within a RECDER system can be used to enable preferential translation of virtually any polypeptide of interest in cancer cells, including secreted immunotherapeutic proteins or reporter proteins.
[0387] For this, plasmids that encode MYC-responsive mRNAs with RNA editor- controlled expression were developed by substitution of [[SI]] in pSFFV-SEAP-P2A- [[Sl ]]-P2A-{ {GDura} with one of ten editing tract sequences complementary to human MYC: MYC ..T01 (SEQ ID NO: 488), MYC...T02 (SEQ ID NO: 489), MYC_T03 (SEQ ID NO: 490), MYC_T04 (SEQ ID NO: 491), MYC_T05 (SEQ ID NO: 492), MYC 106 (SEQ ID NO: 493), MYC T07 (SEQ ID NO: 494), MYC...T08 (SEQ ID NO: 495), MYC_T09 (SEQ ID NO: 496), or MYC_T10 (SEQ ID NO: 497). Similarly, an additional three plasmids were generated that encode murine MYC- responsive (mm_MYC-responsive) mRNAs with RNA editor-controlled expression by substitution of [[SI]] in pSFFV-SEAP-P2A-[[Sl]]-P2A-{ {GDura} with one of three editing tract sequences complementary to murine MYC (mm...MYC): mm_MYC_T01 (SEQ ID NO: 498), mmJM YC_T02 (SEQ ID NO: 499), or mm...MYC...T03 (SEQ ID NO: 500).
[0388] The MYC-responsiveness of RNA editor-controlled mRNAs (SEQ ID NOs: 488-497) was quantified by measuring the level of GDura translated from these MYC-responsive mRNAs (e.g. activated by endogenous MYC mRNA targets) after treating cell lines having variable levels of MYC expression. The three cell lines used differed in the basal level of MYC mRNA expression as quantified by real-time RT- PCR: 293T (low' MYC), CCDI8-C0 healthy colon cells (low' MYC), and COLO320DM colorectal cancer cells (high MYC) (see FIG. 18). Each cell line was treated by ten or more independent transfections via the co-transfection w'ith 225 ng of pSFFV-SEAP-T2A-[[MYC„T]]-P2A-{ {GDura}} and 25 ng of pCMV-ADARl, where [[MYC_T]] was one of ten MYC targets selected from the group of [[MYC.T01]], [[MYC.T02J], [[MYC, „TO3J], [[MYC T04]], [[MYC. T05]], [[MYC.T06]], [[MYC_T07]], [[MYC_T08]], [[MYC_T09]], and [[MYC. _T10]] (SEQ ID NOs: 488-497). Transfections were performed using Lipofectainine 3000 transfection reagent according to the manufacturer’s protocol at a 1:2 ratio of (pg total nucleic acid):(pL of Lipofectainine 3000). As negative controls, cells were left untreated or co-transfected with 225 ng of the off-target (with respect to MYC) pSFFV-SEAP-P2A-[[Tl]]-P2A-{ {GDura}} and 25 ng of pCMV-ADARl. After transfection, the level of activated translation was quantified by measuring the level of secreted GDura at 24 hours after treatment,
[0389] As summarized in FIG. 19C, treatments including [[MYC.T02]], [[MYC..T03]], [[MYC..T04]], or [[MYC..T09]] targets were activated by high levels of endogenous MYC mRNA in COLO320DM colorectal cancer cells, as evidenced by a >3x increase in translation of the GDura polypeptide of interest in MYC-high colorectal cancer cells (COLO320DM) compared to MYC-low healthy colon cells (CCD18 -Co) or 293T cells. In all cases, the maximum increase in GDura translation was < ~10x, indicated by the horizontal dashed line marked “non-arnplified”,
[0390] MYC-responsive editable target mRNAs were further tested in the context of the amplified RECDER #3 system (Example 6) by including an influenza encrypted RNA and the RNA-editor controlled translation activators of the influenza encrypted RNA. To test the capacity of the amplified system to be triggered by endogenous MYC levels, cells being treated with plasmids to produce combinations of: encrypted RNA encoding a GDura payload; an editable mRNA encoding a component of a translation activator of an influenza virus (PA) and a MYC target; an editable mRNA encoding a component of a translation activator of an influenza virus (PB2) and a MYC target; and additional non-editable mRNA encoding remaining translation activator components. The exact levels of transfected plasmids are listed below in Table 13. Transfections were performed using Lipofectainine 3000 transfection reagent according to the manufacturer’s protocol at a 1:2 ratio of (pg total nucleic acid):(pL of Lipofectainine 3000).
[0391] The results of the study are shown in FIG. 19C, demonstrating that using RECDER #3 leads to -100-1000 x signal above background translation, compared to ~10x in the conventional non-amplified system that does not use encRNA, when comparing a luciferase activity level. In FIG. 19C, “MYC encRNA vl” corresponded to use of [[MYC.T04]], “MYC encRNA v2” to [[MYC..T02]], and “MYC encRNA v3” to [[MYC_T03]] targets within the REC mRNA.
Table 13: An example Treatment Schedule to Test 2-component dose-amplified REC mRNA
(RECDER #3) response to MYC proto-oncogene
— - indicates 0 ng plasmid
MYC_Txx indicates the same target sequence selected from SEQ ID NOs: 488-497
Example 13: RECDER #3 can trigger translation selectively in cancer cells which overexpress the cancer-associated transcripts GPX2, TSPAN8, ERBB2, or AFP [0392] The independence of the RECDER #3 system to transcriptome guide RNA w'as next demonstrated by applying the RECDER #3 system to uniquely respond to cancer-associated transcripts GPX2, TSPAN8, ERBB2, or AFP, Plasmids that encode GPX2-responsive mRNAs with RNA editor-controlled expression were developed by substitution of [[SI]] in pSFFV-SEAP-P2A-[[Sl]]-P2A-{{GDura} with one of eight editing tract sequences complementary to human GPX2 mRN A: GPX2_T01 to GPX2...T08 (SEQ ID NOs: 501-508). Similarly, plasmids that encode TSPAN8- responsive mRNAs with RNA editor-controlled expression were developed by substitution of [[SI]] in pSFFV-SEAP-P2A-[[Sl]]-P2A-{ {GDura} with one of three editing tract sequences complementary to human TPSAN8 mRNA: TSPAN_T01 to TSPAN...T03 (SEQ ID NOs: 509-511). Plasmids that encode ERBB2-responsive mRNAs with RNA editor-controlled expression were developed by substitution of [[SI]] in pSFFV-SEAP-P2A-[[Sl]]-P2A-{ {GDura] with one of ten editing tract sequences complementary to human ERBB2 mRNA: ERBB2...T01 to ERBB2..T10 (SEQ ID NOs: 512-521 ). Plasmids that encode AFP-responsive mRNAs with RNA editor-controlled expression were developed by substitution of [[SI]] in pSFFV- SEAP-P2A-[[Sl]]-P2A-{ {GDura} with one of nine editing tract sequences complementary to human AFP: AFP_T01 to AFP_ _T09 (SEQ ID NOs: 522-530).
[0393] The TSPAN8-responsiveness of the RNA editor-controlled mRNAs (SEQ ID NOs: 509-511) was quantified by measuring the level of GDura translated from these TSPAN8 responsive mRNAs (e.g., activated by endogenous TSPAN8 mRNA targets) after treating cell lines having variable levels of TSPAN8 expression. The three cell lines used differed in the basal level of TSPAN8 mRNA expression as quantified by real-time RT-PCR: 293T (low TSPAN8), CCDI8-C0 healthy colon cells (low TSPAN8), and LS513 colorectal cancer cells (high TSPAN8). Each cell line was treated by ten or more independent transfections via the co-transfection with 225 ng of pSFFV-SEAP-T2A-[[TSPAN8_T]]-P2A-{ {GDura}} and 25 ng of pCMV- ADAR1, where [[TSPAN8_T] | was one of ten TSP ANS target selected from the group of [[TSPAN8..T01]], [[TSPAN8.. T02]], or [[TSPAN8. T03]]. Nucleic acids were delivered to the cells via a commercial electroporation system (Lonza Nucleofector 4D) using EO-120 pulse parameters and SE kit reagents (Lonza). As negative controls, cells were left untreated or co-transfected with 225 ng of the off- target (with respect to TSPAN8) pSFFV-SEAP-P2A-[[Tl]]-P2A-{ {GDura} } and 25 ng of pCMV-ADARl. After transfection, the level of activated translation was quantified by measuring the level of secreted GDura at 24 hours after treatment.
[0394] TSPAN8-responsive editable target mRNAs were further tested in the context of the amplified RECDER #3 system via the methods detailed in Example 12, but with the following differences. To test the capacity of the amplified system to be triggered by endogenous TSPAN8 levels, cells were treated with plasmids to produce combinations of: encrypted RNA encoding a GDura payload: an editable mRNA encoding a component of a translation activator of an influenza virus (PA) and a TSPAN8 target; an editable mRNA encoding a component of a translation activator of an influenza virus (PB2) and a TSPAN8 target; and additional non-editable mRNA encoding remaining translation activator components. The exact levels of transfected plasmids are listed below in Table 13. Transfections were performed using Lipofectamine 3000 transfection reagent according to the manufacturer’s protocol at a 1:2 ratio of (pg total nucleic acid):(uL of Lipofectamine 3000, ThermoFisher).
[0395] In a typical and representative study, delivery of a functional RECDER #3 system to GPX2-high cells using functional GPX2 target mRNA (REC mRNA) led to approximately lOO-lOOOx increase in translation of the polypeptide of interest from the influenza encRNA than in GPX2-low cells. Analogously, delivery of a functional RECDER #3 system to TSPAN8-high cells using functional TSPAN8 target mRNA
(REC mRNA) led to approximately 50-200 x increase in translation of the polypeptide of interest from the influenza encRNA than in TSPAN8-low cells.
[0396] Similarly, delivery of a functional RECDER #3 system to ERBB2-high cells using functional ERBB2 target mRNA (REC mRNA) led to approximately lOO-lOOx increase in translation of the polypeptide of interest from the influenza encRNA than in ERBB2-low cells.
Tabk- 14: An example Treatment Schedule to Test 2-component dose-amplified REC mRNA (RECDER #3) response to GPX2, TSPANS, or ERBB2 cancer-associated transcripts
— indicates 0 ng plasmid
Txx indicate the same target sequence selected from SEQ ID NOs: 501-521
[0397] The AFP-responsiveness of RNA editor-controlled mRNAs containing target sequences selected from AFP_.T01_.119 to AFP_.T09_.1547 (SEQ ID NOs: 522-530) was quantified by measuring the level of GDura translated from these AFP-responsive mRNAs (e.g., activated by AFP mRNA targets) after treating cell lines having variable levels of AFP expression. The level of AFP mRNA was controlled above basal amounts via the transfection of an expression plasmid producing exogenous AFP (pCMV-EGFP-stop-«Tl»). The three cell lines used for testing had variable levels of endogenous AFP mRNA as quantified by real-time RT-PCR: 293T (low AFP), Huh7 hepatocytes (high AFP), and HepG2 hepatocytes (high AFP). Each cell line was treated by nine or more independent transfections via the co-transfection with 225 ng of pSFFV-SEAP-T2A-[[AFP_T]]-P2A-{ {GDura}} and 25 ng of pCMV- ADAR1, where [[AFP_T]] was one of nine AFP targets selected from the group of [[AFP..T01...H9]], [[AFP .T02...135]], [[AFP. T03..173]], [[AFP..T04..186]], [[AFP_T05_260]], [[AFP_T06_375]], [[AFP„T07„1050]], [[AFP_T08_1416]], and [[AFP_T09_1547]] (SEQ ID NOs: 522-530). Nucleic acids were delivered to the cells via a commercial transfection reagent (Lipofectamine MessengerMAX™, ThermoFisher) using the manufacturer’s instructions). As negative controls, cells were left untreated or co-transfected with 225 ng of the off-target (with respect to AFP) pSFFV-SEAP-P2A-[[Tl]]-P2A-{ {GDura}} and 25 ng of pCMV-ADARl. After transfection, the level of activated translation was quantified by measuring the level of secreted GDura at 24 hours after treatment.
Table 15: An example Treatment Schedule to Test 1 -component dose-amplified REC mRNA (RECDER #2) response to AFP cancer-associated transcripts
---- indicates 0 ng plasmid
TX indicates target sequence for AFP selected from SEQ ID NOs: 522-530
[0398] In a typical and representative study, delivery of a functional RECDER #2 system to AFP-high cells using functional AFP target rnRNAs (REC mRNA) led to approximately 100-1 OOOx increase in translation of the polypeptide of interest from the influenza encRNA when compared to delivery of the same system in AFP-low cells.
Example 14: RECDER #3 can trigger translation selectively in cells that overexpress HBV infection-associated transcripts
[0399] Plasmids that encode HBV-responsive rnRNAs with RNA editor-controlled expression were developed by substitution of [[SI]] in pSFFV-SEAP-P2A-[[Sl]]- P2A-{{GDura} with one of three edit tract sequences complementary to HBV- associated transcripts: HBV_ _T01 to HBV_T03 (SEQ ID NOs: 531-533).
[0400] In a typical and representative study, delivery of a functional RECDER #3 system (see Table 16) to cells that express HBV transcripts using functional HBV target mRNAs (REC mRNA) led to approximately 10-50 x increase in translation of the polypeptide of interest from the influenza encRNA when compared to delivery of the same system in cells that lacked HBV transcripts.
Table 16: An example Treatment Schedule to Test 1-component dose-amplified REC mRNA
(RECDER #2) in response to HBV-associated transcripts
Example 15: RECDER #3 powered selective translation in cells that overexpress human papillomavirus (HPV) infection-associated transcripts
[0401] Infection with human papillomaviruses (HPV) can lead to the formation of mucosal and oral cancers, including cervical cancer. In particular, overexpression of HPV proteins E6 & E7 is associated with the transformation of healthy tissue into neoplastic tissue (Roman, A., & Munger, K. (2013). Virology, 445(1-2), 138-168.). Thus, treatment with a RECDER 3 system that is activated by E6 or E7 transcripts may yield a cancer-selective or virus-selective therapeutic.
[0402] To engineer a RECDER #3 system that is designed to be activated by HPV E6 (E6) or HPV E7 (E7) transcripts, modify the base RECDER scaffolds from Example 6 be responsive to E6 or E7 transcripts, as taught in Example 6. As an example, develop plasmids that encode E6- or E7 -responsive mRNAs with RNA editor- controlled expression by substitution of [[SI]] in pSFFV-SEAP-P2A-[[Sl]]-P2A- {{GDura} with a suitable edit tract sequences complementary to E6- or E / -associated transcripts. As a specific example, a representative edit tract sequence may be chosen from the group HPV16_T01 to HPV16_T10 ( SEQ ID NOs: 534-543) that correspond to HPV 16 target sequences or from the group HPV18.. T01 to HPV18..T10 (SEQ ID NOs: 544-553) that correspond to HPV18 target sequences.
[0403] It can be expected that delivery of a RECDER #3 comprising HPV E6- or E7- responsive editable target mRNAs into cells producing HPV E6- or E7-tran scripts (such as HPV 16 or HPV 18 -infected cells) will lead to elevated levels of payload translation when compared to E6- or E7-expressing cells that are not treated with the HPV-specific RECDER #3 system, or when compared to treated cells that do not express HPV E6 or E7 transcripts.
Example 16: Influenza B-based RECDER systems (RECDER #5)
[0404] To further demonstrate the modularity of the RECDER systems, RECDER #3 w'as engineered to use the components of influenza B virus (IBV) translation activator rather than the components of an influenza A translation activator. This IBV-driven system was termed RECDER #5. IBV PB2 (SEQ ID NO: 554) and IB V PA (SEQ ID NO: 555) from the influenza strain B/Brisbane/60/2008.
[0405] The system was validated using a similar experiment to the one described in Example 12, which was performed using human 293T cells. The human 293T cells were transfected with pools of plasmids listed in Table 17 (forming RECDER #6), and the level of activated translation was quantified by measuring the level of secreted GDura at 24 hours after infection.
[0406] Treatments 2201-2204 tested the specific activation of RECDER #5, with cells being treated with plasmids to produce combinations of: guide RNA; encrypted RNA encoding a GDura payload; an editable mRNA encoding a component of a translation activator of an influenza virus (PB2); an editable mRNA encoding a component of a translation activator of an influenza virus (PB1): and additional non- editable mRNA encoding remaining translation activator components.
[0407] FIG. 20 show's the functionality of an influenza. B virus-based RECDER system that can be programmed for activation in response to unique RNA transcripts. The system shown uses a 2 -component translation activator (RECDER #5) controlled by the presence of a cancer-associated transcript (MYC), which leads to elevated levels of payload production in a colon cancer cell line (COLO320DM) compared to a normal colon cell line (CCDlSCo).
[0408] The results of the study are shown in FIG. 20, demonstrating that using RECDER #5 leads to an ~l,000x increase in production of the polypeptide of interest in the colon cancer cell line above the level in the normal colon cell line, similar to the increase in activation shown above for the influenza A based RECDER #3 system.
Table 17: An example Treatment Schedule to Test RNA-editisig controlled mRNA vs. 2-component dose- amplified REC mRNA (RECDER #5)
— indicates 0 ng plasmid
Example 17: RSV based RECDER systems (RECDER #6-7)
[0409] A variety of suitable translation activators that can be encoded as a REC mRNA are compatible with this invention. In this example, a RSV RdRP REC mRNA was demonstrated to be functional, and challenges in its development were highlighted,
[0410] Several candidate RSV RdRP REC rnRNA s were engineered to allow' conditional translation of the RSV L protein translation activator component. An initial candidate, pCMV-EGFP-P2A-[[Sl]]-P2A-{ {RSV L}} (SEQ ID NO: 556), contained an editing tract inserted upstream of the encoded portion of RSV L. In this example, the RSV_L sequence coding sequence without a start codon (SEQ ID NO: 557) is from a common RSV laboratory strain. [0411] An additional candidate system utilized protein splicing to enable the division of the RSV L across two editable mRNA. Splitting the long (-6.5 kb) RSV L coding sequence across two or more editable mRNAs both reduces the maximum length of each individual editable mRNA and allows for more complex control of the translation activator via “AND-gate” logic. In this split system, the coding sequence of RSV L (SEQ ID NO: 556) was divided across two editable mRNAs. The N- terminal portion of the RSV_L coding sequence (SEQ ID NO: 556), termed RSV_L_1 (SEQ ID NO: 557) was encoded on one mRNA: mRNA_RSV_L_l (SEQ ID NO: 558). Similarly, the C-terminal portion of RSV_L, termed RSV_L_2 (SEQ ID NO: 559) was encoded on the other editable mRNA: mRNA..RSV„L.2 (SEQ ID NO: 560). Each portion also encoded a flanking intein sequence, located at the C_terminus of RSVJLJ (SEQ ID NO: 561) and the N-terminus of RSV_L_2 (SEQ ID NO: 562). Translation of the RSV..L regions of both mRNAs produced two polypeptides, which are subsequently and autonomously ligated into a single functional RSV L protein via intein-driven ligation.
[0412] To test these RSV systems, human 293T cells were transfected with pools of plasmids listed in Tables 19 & 20, and the level of activated translation was quantified by measuring the level of secreted GDura at 24 hours after treatment,
[0413] Treatments 4101-4104 tested the specific activation of an amplified system (RECDER #6), with cells being treated with plasmids to produce combinations of: guide RNA; RSV encrypted RNA encoding a GDura payload; an editable mRNA encoding a component of a translation activator of RSV and a hyperactive ADAR2.
[0414] It was found that Treatment 4104 produced the highest overall levels of conditional protein expression, approximately 2 logs above background levels, while Treatment 4103 (without supplementation with an exogenous RN A editor) resulted in a 1.0 log increase over the level detected in the control treatments 4101 and 4102.
Table 19: An example Treatment Schedule to Test RNA-editing controlled mRNA of RSV encrypted RNA (RECDER #6)
— - indicates 0 ng plasmid
[0415] Treatments 4201-4206 tested the specific activation of the split and amplified system (RECDER #7), with cells being treated with plasmids to produce combinations of: guide RNA; RSV encrypted RNA encoding a GDura payload; one or more editable mRNA (REC mRNA) encoding a component of a translation activator of RSV; and a hyperactive ADAR2.
Table 20: An example Treatment Schedule to Test RNA-editing controlled mRNA of RSV encrypted RNA (RECDER #7)
— indicates 0 ng plasmid
[0416] It was found that Treatment 4206 produced the highest overall levels of conditional protein expression, approximately 2 logs above background levels, while Treatment 4205 (without supplementation with an exogenous RNA editor) resulted in a 1.0 log increase over the level detected in the control treatments 4201-4204.
Notably, the translation of the split translation activator was effective in activating the encRNA leading to at least a 1-2 log increase when both components were provided (Treatments 4205 and 4206), but no increase was readily observed when only one or no components were provided (4202-4204). As predicted, in the absence of a suitable guide mRNA (Treatment 4201), no significant expression was observed.
[0417] FIGs 21A, 21B, and 21C show experimental results that together demonstrate the functionality of two RSV-based RECDER sy stems (RECDER #6 and RECDER #7) that utilize either 1 or 2 RNA Editor Controlled mRNA (REC mRNA) that can be programmed for activation in response to unique RN A transcripts. FIG. 21 A shows that both 1 REC mRNA (RECDER #6) and 2 REC mRNA (RECDER #7) systems are controlled by the presence of a synthetic guide RNA and are sensitive to RNA editing activity provided by an RNA editor (ADAR). FIG. 21B shows that both systems are functional in multiple species (mouse and human) and achieve similar levels of controlled protein translation when activated. In this example, mouse cells are Bl 6F 10 mouse melanoma cells.
[0418] A further study was performed where the [[SI ]] target in the RSV REC mRNA was replaced with a MYC target sequence [[MYC_T03]] (SEQ ID NO: 490) and the RECDER #6 and RECDER #7 systems were tested in MYC-high or MYC- low cell lines as described above in Example 12.
[0419] FIG. 21C shows that both systems can be programmed for responsiveness to a cancer-associated transcript (MYC) and achieve a 50-75 x increase in translation in high MYC-ex pres sing cells (COLO320DM) (“CR cancer cells” in legend) compared to low7 MYC-expressing cells (293T) (“Normal cells” in legend).
Example 18: Use of RNA editors that are not base editors
[0420] In addition to RNA editors that are base editors (such as ADAR), RNA editors that are not base editors can be used to control the translation of a translation activator. Instead of directly modifying the nucleotide sequence of an mRNA (as in base editing), an alternative method involves ligating two separate RNA fragments to form a functional mRNA encoding a translation activator or a translation activator component.
[0421] As an example, an RN A editor with programmable RNA ligase activity can be used to ligate two or more precursor RNAs into a single product mRNA which is subsequently translatable. The translation activator gene is split into two parts, each encoded on a separate mRNA. These two mRNA fragments cannot be translated independently to form a functional translation activator component. [0422] A programmable RNA editor, eDC7 11, is used to catalyze ligation between the two mRNA using trans-splicing. When the eDC7_ll system is activated by a specific guide RNA, it ligates the two fragments together, creating a complete mRNA that can be translated into the functional translation activator protein.
[0423] Using this ligation scheme, specific control over the expression of a translation activator can be achieved if each precursor RNAs contain portions of the translation activator that, when trans- spliced, together reconstitute the complete open reading frame of the translation activator within a translatable mRNA.
[0424] Initial experiments were performed using a programmable eDisCas7-l 1 RNA editor (eDC7.._l 1) that can be recruited to an RNA target to achieve RNA ligation via trans-splicing through the action of a bi-partite structured gRNA sequence. The complete coding sequence for the eDC7_l 1 RNA editor assembly (SEQ ID NO: 564) is comprised of the following elements in sequence: an upstream nuclear localization signal sequence (SEQ ID NO: 565), the eDC7_ll RNA editor coding sequence (SEQ ID NO: 566), and an additional downstream nuclear localization signal sequence (SEQ ID NO: 567).
[0425] Three bi-partite gRNA sequences were developed to recruit eDC7.._l 1 to MYC mRN A: DR+cMycIntr2_tgl (SEQ ID NO: 568), DR+cMycIntr2_tg2 (SEQ ID NO: 569), and DR+cMycIntr2..tg3 (SEQ ID NO: 570). In addition, a negative control bipartite gRNA sequence that was expected not to recruit eDC7_l 1 to MYC mRNA was developed: DR+cMycIntr2_NT (SEQ ID NO: 571). Each of the sequences (SEQ ID NOs: 568-572) is comprised of a fixed sequence DR (SEQ ID NO: 572) followed by a variable sequence that is complementary to the desired target. The variable portion (SEQ ID NOs: 573- 575) of each three MYC-directed bi-partite gRNAs (SEQ ID NOs: 568-570) was directed against a different region within the MYC RNA. The variable portion (SEQ ID NOs: 576) of the negative control bi-partite gRNAs (SEQ ID NO: 571) was not directed against the MYC RNA.
[0426] To accomplish programmable trans-splicing, a complementary guide RNA sequence corresponding to cMYC (SEQ ID NO: 577) was encoded on an mRNA to serve as a synthetic analog of the natural cMYC mRN A sequence. In particular, gRNAs were complementary to the second intron of MYC (SEQ ID NO: 578), corresponding to nucleotides 803-2178 of SEQ ID NO: 577. Separately, a component of the translation activator was encoded on a distinct mRNA termed EGFP-cMYCJntr2-P2A-PR8_PA (SEQ ID NO: 579). Importantly, nt 734-883 of SEQ ID NO: 579 correspond to cMyc...IntrTarget2 (SEQ ID NO: 580) that contains several stop codons to prevent translation (absent splicing) and nt 884-916 of SEQ ID NO: 579 correspond to branch_addn_3 sequence (SEQ ID NO: 581) that promotes trans-splicing via a prototypical splicing branch site and spice acceptor sequence. Within EGFP-cMYCJntr2-P2A-PR8_PA (SEQ ID NO: 579), the PR8_PA translation activator component is designed to be untranslatable (absent trans- splicing) through the presence of multiple upstream stop codons contained within SEQ ID NO: 580.
[0427] Conditional translation of the translation activator component was accomplished using programmable trans-splicing, with an upstream mRNA...A (mRNA.. A) corresponding to a guide RNA (SEQ ID NO: 577) and the downstream mRNA_B corresponding to the translation activator component (SEQ ID NO: 579) being trans- spliced to facilitate translation of the translation activator component. Importantly, neither mRN A_A nor mRNA_B can be translated to produce a complete translation activator component, even when both are cis- spliced. In contrast, trans- splicing between mRNA_A (“splice donor”) and mRNA_B (“splice acceptor”) through the recruitment of the RNA editor eDC7...11 to mRN A..A (via gRNA annealing) removes the upstream stop-codon containing introns from mRNA_B and forms a new mRNA...species (mRNA...C), which contains a now-translatable translation activator component, having removed the upstream stop codons via splicing.
[0428] To test these systems, human 293T cells were transfected with pools of plasmids listed in ’fable 21, and the level of activated translation was quantified by measuring the level of secreted GDura at 24 hours after treatment.
[0429] Treatments 5101-5106 tested the specific activation of an amplified system (RECDER #8), with cells being treated with plasmids to produce combinations of: guide RNA; influenza encrypted RNA encoding a GDura payload; the first editable mRNA (mRNA... A) encoding the first exon of a component of a translation activator of influenza A; the second editable (mRNA_B) encoding the second exon, and a programmable RNA editor eDC7_11.
Table 21: An example Treatment Schedule to Test RNA-editing controlled mRNA of influenza encrypted RNA using a programmable RNA ligase (RECDER #8) mass of plasmid transfected so each treatment
[0430] It was found that Treatment 5106 produced the highest overall levels of conditional protein expression, approximately >0.5 logs above background levels, while Treatments 5105-5105 resulted in <0.5 log increase. Notably, control treatments failed to substantially activate the encRNA, consistent with prediction: (i) individual expression of either mRNA_A or mRNA_B failed to activate the encRNA (Treatments 5103-5104); (ii) expression of both mRNA..A and mRNA„B without the eDC7_l 1 editor and bi-partite gRNA failed to activated the encRNA (Treatment 5105). Further, in the absence of a suitable guide niRNA (Treatment 5101), no significant expression was observed, FIG. 22 shows the difference in expression for Treatments 5101 (“sham guide RNA”) v.s 5106 (“MYC guide RNA”) for two bipartite gRNAs DR+cMycIntr2_tg2 (SEQ ID NO: 569 ) (labelled as “MYC Target 2” in FIG ) and DR+cMycIntr2„tg3 (SEQ ID NO: 570 ) (“MYC Target 3”). Error bars correspond to 1 standard deviation from mean (N=3 biological replicates).
[0431] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each publication, patent, or patent application w'as 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. EMBODIMENTS
Embodiment I, A composition comprising:
(a) an 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; and
(b) a target niRNA comprising an edit tract, wherein a stop codon within the edit tract prevents translation of a translation activator or a translation activator component encoded by the target mRNA, wherein delivering the composition into a cell forms an intracellular protein-nucleic acid complex comprising:
(i) the target mRNA
(ii) an RNA editor, and
(iii) a guide RNA, wherein the guide RNA binds to the target mRNA to form a double- stranded RNA complex as a substrate for the RNA editor, wherein the substrate comprises a mispairing within the stop codon, wherein the RNA editor converts the mispairing into a pairing in the substrate, thereby allowing translation of the translation activator or the translation activator component from the target mRNA, and wherein the encrypted RNA contacts the translation activator, thereby resulting in translation of the therapeutic polypeptide,
Embodiment 2. The composition of Embodiment 1, wherein both the L region and the R region of the encrypted RNA are derived from a vims, and optionally each of the L region and the R region is the reverse complement of a corresponding region that is native to the virus, and wherein the translation activator is an RNA dependent polymerase. Embodiment 3. The composition of Embodiment 2, wherein the RNA dependent polymerase is a viral RNA dependent polymerase, optionally an RNA-dependent RNA polymerase or an RNA-dependent DNA polymerase.
Embodiment 4. The composition of Embodiment 2 or Embodiment 3, wherein the virus is selected from the group consisting of: Alphacoronavirus 229E, Alphacoronavirus NL63, Alphacoronavirus WA2028, Avian metapneumovirus (AMPV), Betacoronavirus HKU1, Betacoronavirus HKU15, Betacoronavirus HKU33, Betacoronavirus OC43, Chikungunya virus, Crimean-Congo Hemorrhagic Fever Virus, Dengue Virus, Eastern Equine Encephalitis Virus (EEEV), Enterovirus D68 (EV-D68), Foot and Mouth Disease Virus, Hanta Virus, Hendra Virus, Hepatitis B Virus, Hepatitis C Virus, HMPV, Human Parainfluenzavirus 1 (HPIV1), Human Parainfluenzavirus 3 (HPIV3), Infectious Salmon Anemia Virus, Influenza A Virus, Influenza B Virus, Lassa Virus, Marburg Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Newcastle Disease Virus (NDV), Nipali Virus, Norwalk Virus, Rabies Virus, Respiratory Syncytial Virus, Reston Ebola virus, Rhinovirus, Rift Valley Fever Virus, Rubella vims, SARS-CoV-1, SARS-CoV-2, Sudan Ebola virus, Venezuelan Equine Encephalitis Virus (VEEV), Vesicular Stomatitis Virus, Western Equine Encephalitis Virus (WEEV), Yellow Fever Virus, Zaire Ebola virus, and Zika Virus.
Embodiment 5. The composition of any one of Embodiments 2 to 4, wherein the virus is not an alphavirus.
Embodiment 6. The composition of any one of Embodiments 2 to 5, wherein the L region and R region are native to the virus.
Embodiment 7. The composition of any one of Embodiments 2 to 5, 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 virus, 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 virus.
Embodiment 8. The composition of any one of Embodiments 2 to 5, wherein each of the L region and the R region varies from a corresponding region that is native to the 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.
Embodiment 9. The composition of any one of Embodiments 2 to 8, wherein the encrypted RNA comprises at least one nucleoside modification.
Embodiment 10. The composition of Embodiment 9, wherein each of the L region and the R region comprises no more than about 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 11. The composition of Embodiment 9 or Embodiment 10, wherein the nucleoside modification is a nonimmunogenic uridine modification, and the percentage of modified uridine modifications is (i) no more than about 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 about 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 about 1 % and about 30%, optionally, about 1%, 5%, 10%, 15%, 20%, 25%, or 30% of all adenosine.
Embodiment 12. The composition of any one of Embodiments 9 to 11, wherein the encrypted RNA comprises a 5’ cap structure, optionally the 5’ cap structure being selected from the group consisting of: 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. Embodiment 13. The composition of Embodiment 12, 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 14. The composition of any one of Embodiments 9 to 11, wherein the 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 15. The composition of Embodiment 14, wherein the 5’ end of the 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 (depho sphory lated) .
Embodiment 16. The composition of any one of Embodiments 2 to 15, wherein the coding sequence within the encrypted RNA is in an antisense orientation.
Embodiment 17. The composition of Embodiment 16, wherein the virus is an influenza virus, and wherein a combination of the L region and the R region sati sfies one of the following:
(i) the L. region comprises a nucleotide sequence set forth as SEQ ID NO: 2; or a variant of SEQ ID NO: 2, wherein the variant of SEQ ID NO: 2 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-26 of SEQ ID NO: 2, and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 20, 21, 22, or 23; or a variant of any one of SEQ ID NOs: 20, 21, 22, or 23, wherein the vaiaant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-15 of any one of SEQ ID NOs: 20, 21, 22, or 23;
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 3; or a variant of SEQ ID NO: 3, wherein the variant of SEQ ID NO: 3 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-35 of SEQ ID NO: 3; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 24, 25, 26, or 27; or a variant of any one of SEQ ID NOs: 24, 25, 26, or 27, wherein the variant of SEQ ID NO: 24 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-12 of SEQ ID NO: 24; the variant of SEQ ID NO: 25 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-12 of SEQ ID NO: 25; the variant of SEQ ID NO: 26 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-12 of SEQ ID NO: 26; or the variant of SEQ ID NO: 27 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-12 of SEQ ID NO: 27;
(iii) the L. region comprises a nucleotide sequence set forth as SEQ ID NO: 4; or a variant of SEQ ID NO: 4, wherein the variant of SEQ ID NO: 4 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-50 of SEQ ID NO: 4; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 28, 29, 30, or 31; or a variant of any one of SEQ ID NOs: 28, 29, 30, or 31 , wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-12 of any one of SEQ ID NOs: 28, 29, 30, or 31;
(iv) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 1 or 5: or a variant of SEQ ID NO: 1 or 5, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-37 of SEQ ID NO: 1 or 5; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 18 or 19; or a variant of SEQ ID NO: 18 or 19, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-20 of SEQ ID NO: 18 or 19;
(v) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 6; or a variant of SEQ ID NO: 6, wherein the variant of SEQ ID NO: 6 comprises a variation at one or more nucleotide positions selected from position 14 or 15 of SEQ ID NO: 6; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 32 or 33; or a variant of SEQ ID NO: 32 or 33, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-33 of SEQ ID NO: 32 or 33;
(vi) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 7: or a variant of SEQ ID NO: 7, wherein the variant of SEQ ID NO: 7 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-20 of SEQ ID NO: 7; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 34, 35, 36, or 37; or a variant of any one of SEQ ID NOs: 34, 35, 36, or 37, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 5-8 of SEQ ID NO: 34, 35, 36, or 37;
(vii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 8; or a variant of SEQ ID NO: 8, wherein the variant of SEQ ID NO: 8 comprises a variation at one or more nucleotide positions selected from position 14 or 15 of SEQ ID NO: 8; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 38, 39, 40, or 41; or a variant of any one of SEQ ID NOs: 38, 39, 40, or 41, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-13 of SEQ ID NO: 38, 39, 40, or 41;
(viii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 9; or a variant of SEQ ID NO: 9, wherein the variant of SEQ ID NO: 9 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 14-18 of SEQ ID NO: 9; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 42 or 43; or a variant of SEQ ID NO: 42 or 43, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-14 of SEQ ID NO: 42 or 43;
(ix) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 11; or a variant of SEQ ID NO: 11 , wherein the variant of SEQ ID NO: 11 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-81 of SEQ ID NO: 11; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 46 or 47; or a variant of SEQ ID NO: 46 or 47, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 5-9 of SEQ ID NO: 46 or 47;
(x) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 12; or a variant of SEQ ID NO: 12, wherein the variant of SEQ ID NO: 12 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 14-52 of SEQ ID NO: 12; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NO: 48 or 49; or a variant of any one of SEQ ID NO: 48 or 49, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-11 of SEQ ID NO: 48 or 49;
(xi) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 13; or a variant of SEQ ID NO: 13, wherein the variant of SEQ ID NO: 13 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-87 of SEQ ID NO: 13: and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 50 or 51; or a variant of SEQ ID NO: 50 or 51, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-17 of SEQ ID NO: 50 or 51 ;
(xii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NO: 10; or a variant of any one of SEQ ID NO: 10, wherein the variant of SEQ ID NO: 10 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-86 of SEQ ID NO: 10; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 44 or 45: or a variant of SEQ ID NO: 44 or 45, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-21 of SEQ ID NO: 44 or 45;
(xiii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 14; or a variant of SEQ ID NO: 14, wherein the variant of SEQ ID NO: 14 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 14-93 of SEQ ID NO: 14; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NO: 52 or 53; or a variant of any one of SEQ ID NO: 52 or 53, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-48 of SEQ ID NO: 52 or 53;
(xiv) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 15; or a variant of SEQ ID NO: 15, wherein the variant of SEQ ID NO: 15 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-95 of SEQ ID NO: 15; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 54 or 55; or a variant of any one of SEQ ID NO: 54 or 55, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-34 of SEQ ID NO: 54 or 55;
(xv) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 16; or a variant of SEQ ID NO: 16, wherein the variant of SEQ ID NO: 16 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 14-81 of SEQ ID NO: 16; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NO: 56 or 57; or a variant of any one of SEQ ID NO: 56 or 57, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-12 of SEQ ID NO: 56 or 57; and
(xvi) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 17; or a variant of SEQ ID NO: 17, wherein the variant of SEQ ID NO: 17 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 14-22 of SEQ ID NO: 17; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NO: 58 or 59; or a variant of any one of SEQ ID NO: 58 or 59, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 8-32 of SEQ ID NO: 58 or 59.
Embodiment 18. The composition of Embodiment 16, wherein the virus is a sarbecovirus virus, and wherein a combination of the L region and the R region satisfies one of the following:
(i) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 137; or a variant of SEQ ID NO: 137, wherein the variant of SEQ ID NO: 137 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-1557 of SEQ ID NO: 137: and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 128; or a variant of any one of SEQ ID NO: 128, wherein the variant of SEQ ID NO: 128 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-30 of SEQ ID NO: 128; and
(ii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 138, 139, 140, 141, 142, 143, or 144; or a variant of any one of SEQ ID NOs: 138, 139, 140, 141, 142, 143, or 144, wherein the variant of SEQ ID NO: 138 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-312 of SEQ ID NO: 138; the variant of SEQ ID NO: 139 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1567 of SEQ ID NO: 139: the variant of SEQ ID NO: 140 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1488 of SEQ ID NO: 140; the variant of SEQ ID NO: 141 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1593 of SEQ ID NO: 141; the variant of SEQ ID NO: 142 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1570 of SEQ ID NO: 142: the variant of SEQ ID NO: 143 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1488 of SEQ ID NO: 143; or the variant of SEQ ID NO: 144 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1593 of SEQ ID NO: 144; and the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs:, 136, 145, 146, or 147; or a variant of any one of SEQ ID NOs: 130, 136, 145, 146, or, wherein the variant of SEQ ID NO: 130 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130; the variant of SEQ ID NO: 136 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-33 of SEQ ID NO: 136; the variant of SEQ ID NO: 145 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 50-1461 of SEQ
ID NO: 145: the variant of SEQ ID NO: 146 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-1441 of SEQ ID NO: 146; or the variant of SEQ ID NO: 147 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-897 of SEQ ID NO: 147.
Embodiment 19. The composition of Embodiment 16, wherein the virus is a Respiratory Syncytial Virus (RSV), wherein the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 158, 163, 165, 166, or 419; or a variant of any one of SEQ ID NOs: 158, 163, 165, 166, or 419, wherein the variant of SEQ ID NO: 158 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15 -207 of SEQ ID NO: 158; the variant of SEQ ID NO: 163 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 18-210 of SEQ ID NO: 163; the variant of SEQ ID NO: 165 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-147 of SEQ ID NO: 165; the variant of SEQ ID NO: 166 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15— -32 of SEQ ID NO: 166; or the variant of SEQ ID NO: 419 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 18-35 of SEQ ID NO: 419; and wherein the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 169, 170, 176, 177, or 420; or a variant of any one of SEQ ID NOs: 169, 170, 176, 177, or 420, wherein the variant of SEQ ID NO: 169 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-78 of SEQ ID NO: 169; the variant of SEQ ID NO: 170 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-80 of SEQ ID NO: 170; the variant of SEQ ID NO: 176 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-36 of SEQ ID NO: 176; the variant of SEQ ID NO: 177 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-33 of SEQ ID NO: 177; or the variant of SEQ ID NO: 420 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-35 of SEQ ID NO: 420.
Embodiment 20. The composition of Embodiment 16, wherein the vims is a parainfluenzavirus, and wherein a combination of the L region and the R region satisfies one of the following:
(i) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 181, 182, or 183; or a variant of any one of SEQ ID NOs: 181, 182, or 183, wherein the variant of SEQ ID NO: 181 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-136 of SEQ ID NO: 181; the variant of SEQ ID NO: 182 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-142 of SEQ ID NO: 182; or the variant of SEQ ID NO: 183 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17- 136 of SEQ ID NO: 183; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 184; or a variant of SEQ ID NO: 184, wherein the variant of SEQ ID NO: 184 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-98 of SEQ ID NO: 184; and
(ii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 187, 188, or 189; or a variant of any one of SEQ ID NOs: 187, 188, or 189, wherein the variant of SEQ ID NO: 187 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-95 of SEQ ID NO: 187; the variant of SEQ ID NO: 188 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-101 of SEQ ID NO: 188; or the variant of SEQ ID NO: 189 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-95 of SEQ ID NO: 189; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 190; or a variant of SEQ ID NO: 190, wherein the variant of SEQ ID NO: 190 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-93 of SEQ ID NO: 190.
Embodiment 21. The composition of Embodiment 16, wherein the virus is a metapneumovirus, and wherein a combination of the L region and the R region satisfies one of the following:
(i) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 196, 197, or 199; or a variant of any one of SEQ ID NOs: 196, 197, or 199, wherein the variant of SEQ ID NO: 196 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21 -224 of SEQ ID NO: 196; the variant of SEQ ID NO: 197 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-230 of SEQ ID NO: 197; the variant of SEQ ID NO: 199 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 21-140 of SEQ ID NO: 199; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 201; or a variant of SEQ ID NO: 201, wherein the variant of SEQ ID NO: 201 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-32 of SEQ ID NO: 201; and
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 195; or a variant of SEQ ID NO: 195, wherein the variant of SEQ ID NO: 195 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-224 of SEQ ID NO: 195; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 200; or a variant of SEQ ID NO: 200, wherein the variant of SEQ ID NO: 200 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 17-32 of SEQ ID NO: 200.
Embodiment 22. The composition of Embodiment 16, wherein the virus is a henipavims, and wherein a combination of the L region and the R region satisfies one of the following:
(i) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 204; or a variant of SEQ ID NO: 204, wherein the variant of SEQ ID NO: 204 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-77 of SEQ ID NO: 204; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 206; or a variant of SEQ ID NO: 206, wherein the variant of SEQ ID NO: 206 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-91 of SEQ ID NO: 206; and
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 209 or 210; or a variant of SEQ ID NO: 209 or 210, wherein the variant of SEQ ID NO: 209 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17 -77 of SEQ ID NO: 209; or the variant of SEQ ID NO: 210 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-83 of SEQ ID NO: 210; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 211 ; or a variant of SEQ ID NO: 211, wherein the variant of SEQ ID NO: 211 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-91 of SEQ ID NO: 211 .
Embodiment 23. The composition of Embodiment 16, wherein the virus is a hepadnavirus, wherein the L region comprises a nucleotide sequence set forth as SEQ ID NO: 222 or 223; or a variant of SEQ ID NO: 222 or 223, wherein the variant of SEQ ID NO: 222 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-639 of SEQ ID NO: 222; or the variant of SEQ ID NO: 223 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101—186 of SEQ ID NO: 223: and wherein the R region comprises the nucleotide sequence set forth as SEQ ID NO: 225; or a variant of SEQ ID NO: 225, wherein the variant of SEQ ID NO: 225 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 101-1023 of SEQ ID NO: 225.
Embodiment 24. The composition of Embodiment 16, wherein the virus is a filovirus, and wherein a combination of the L region and the R region satisfies one of the following:
(i) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 227, 228, 229, or 230; or a variant of any one of SEQ ID NOs: 227, 228, 229, or 230, wherein the variant of SEQ ID NO: 227 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 20-710 of SEQ ID NO: 227; the variant of SEQ ID NO: 228 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 23-713 of SEQ ID NO: 228; the variant of SEQ ID NO: 229 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-707 of SEQ ID NO: 229; or the variant of SEQ ID NO: 230 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-707 of SEQ ID NO: 230; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 231; or a variant of SEQ ID NO: 231, wherein the variant of SEQ ID NO: 231 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-449 of SEQ ID NO: 231;
(ii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 232, 233, 234, or 235; or a variant of any one of SEQ ID NOs: 232, 233, 234, or 235, wherein the variant of SEQ ID NO: 232 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-678 of SEQ ID NO: 232; the variant of SEQ ID NO: 233 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 23-681 of SEQ ID NO: 233; the variant of SEQ ID NO: 234 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 23-678 of SEQ ID NO: 234; or the variant of SEQ ID NO: 235 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 23-678 of SEQ ID NO: 235; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 236; or a variant of SEQ ID NO: 236, wherein the variant of SEQ ID NO: 236 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-437 of SEQ ID NO: 236;
(iii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 237, 238, or 239; or a variant of any one of SEQ ID NOs: 237, 238, or 239, wherein the variant of SEQ ID NO: 237 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-605 of SEQ ID NO: 237; the variant of SEQ ID NO: 238 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 21-606 of SEQ ID NO: 238; or the variant of SEQ ID NO: 239 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-605 of SEQ ID NO: 239; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 240; or a variant of SEQ ID NO: 240, wherein the variant of SEQ ID NO: 240 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-83 of SEQ ID NO: 240;
(iv) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 241; or a variant of SEQ ID NO: 241, wherein the variant of SEQ ID NO: 241 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-34 of SEQ ID NO: 241 ; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 242; or a variant of any one of SEQ ID NO: 242, wherein the variant of SEQ ID NO: 242 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 100-593 of SEQ ID NO: 242.
(v) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 243; or a variant of SEQ ID NO: 243, wherein the variant of SEQ ID NO: 243 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 30-45 of SEQ ID NO: 243; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 244; or a variant of SEQ ID NO: 244, wherein the variant of SEQ ID NO: 244 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 100-677 of SEQ ID NO: 244; and
(vi) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 245, 246, or 247; or a variant of any one of SEQ ID NOs: 245, 246, or 247, wherein the variant of SEQ ID NO: 245 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 29—171 of SEQ ID NO: 245; the variant of SEQ ID NO: 246 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 30—171 of SEQ ID NO: 246; or the variant of SEQ ID NO: 247 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 29-171 of SEQ ID NO: 247; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 248; or a variant of SEQ ID NO: 248, wherein the variant of SEQ ID NO: 248 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-91 of SEQ ID NO: 248.
Embodiment 25. The composition of Embodiment 16, wherein the virus is an alphavirus, and wherein a combination of the L region and the R region satisfies one of the following: (i) the L region comprises a nucleotide sequence set forth as 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 nucleotide positions selected from the group consisting of positions 60-274 of SEQ ID NO: 249; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 250 or 251; or a variant of SEQ ID NO: 250 or 251, wherein the variant of SEQ ID NO: 250 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-183 of SEQ ID NO: 250; or the variant of SEQ ID NO: 251 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-375 of SEQ ID NO: 251;
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 255; or a variant of SEQ ID NO: 255, wherein the variant of SEQ ID NO: 255 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-35 of SEQ ID NO: 255; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 256 or 257; or a variant of SEQ ID NO: 256 or 257, wherein the variant of SEQ ID NO: 256 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 600-273 of SEQ ID NO: 256; or the variant of SEQ ID NO: 257 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 60-377 of SEQ ID NO: 257; and
(iii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 261 ; or a variant of SEQ ID NO: 261, wherein the variant of SEQ ID NO: 261 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 60-215 of SEQ ID NO: 261; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 262 or 263; or a variant of SEQ ID NO: 262 or 263, wherein the variant of SEQ ID NO: 262 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 60-166 of SEQ
ID NO: 262; or the variant of SEQ ID NO: 263 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 60-379 of SEQ ID NO: 263.
Embodiment 26. The composition of any one of Embodiments 2 to 15, wherein the coding sequence within the encrypted RNA is in a sense orientation.
Embodiment 27. The composition of Embodiment 26, wherein the virus is a sarbecovirus, and wherein a combination of the L region and the R region satisfies one of the following:
(i) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, or 67; or a variant of any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, or 67, wherein the variant comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1426-1493 of any one of SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, or 67; and the R region comprises the nucleotide sequence set forth SEQ ID NO: 129: or a vaiaant of SEQ ID NO: 129, wherein the variant of SEQ ID NO: 129 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 129;
(ii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 68, 69, 70, 71, 72, 73, 74, 75, 76, or 77; or a variant of any one of SEQ ID NO: 68, 69, 70, 71, 72, 73, 74, 75, 76, or 77, wherein the variant of SEQ ID NO: 68 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 68; the valiant of SEQ ID NO: 69 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 69; the variant of SEQ ID NO: 70 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1446-1513 of SEQ ID NO: 70; the variant of SEQ ID NO: 71 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1455-1522 of SEQ ID NO: 71; the variant of SEQ ID NO: 72 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1462-1529 of SEQ ID NO: 72; the variant of SEQ ID NO: 73 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1469-1536 of SEQ ID NO: 73: the variant of SEQ ID NO: 74 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1485-1552 of SEQ ID NO: 74; the variant of SEQ ID NO: 75 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1686-1753 of SEQ ID NO: 75; the variant of SEQ ID NO: 76 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1704—1771 of SEQ ID NO: 76; or the variant of SEQ ID NO: 77 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1720-1787 of SEQ ID NO: 77; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 130: or a variant of SEQ ID NO: 130, wherein the variant of SEQ ID NO: 130 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130;
(iii) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 78, 79, 80, 81, 82, 83, 85, 86, 87, or 88; or a variant of any one of SEQ ID NOs: 78, 79, 80, 81, 82, 83, 85, 86, 87, or 88, wherein the variant of SEQ ID NO: 78 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1734—1801 of SEQ ID NO: 78; the variant of SEQ ID NO: 79 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1687-1754 of SEQ ID NO: 79; the variant of SEQ ID NO: 80 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1695-1762 of SEQ ID NO: 80; the variant of SEQ ID NO: 81 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 81; the variant of SEQ ID NO: 82 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1443—1510 of SEQ ID NO: 82: the variant of SEQ ID NO: 83 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1459-1526 of SEQ ID NO: 83; the variant of SEQ ID NO: 85 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 85; the variant of SEQ ID NO: 86 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1434-1501 of SEQ ID NO: 86; the variant of SEQ ID NO: 87 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1435-1502 of SEQ ID NO: 87; or the variant of SEQ ID NO: 88 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1463-1530 of SEQ ID NO: 88; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 130; or a variant of SEQ ID NO: 130, wherein the variant of SEQ ID NO: 130 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130;
(iv) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 89, 90, 91 , 92, 96, 104, 105, 106, 107, or 108; or a variant of any one of SEQ ID NOs: 89, 90, 91, 92, 96, 104, 105, 106, 107, or 108, wherein the variant of SEQ ID NO: 89 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1466-1533 of SEQ ID NO: 89; the variant of SEQ ID NO: 90 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1425-1492 of SEQ ID NO: 90; the variant of SEQ ID NO: 91 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1425-1492 of SEQ ID NO: 91; the variant of SEQ ID NO: 92 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1425-1492 of SEQ ID NO: 92: the variant of SEQ ID NO: 96 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-769 or 1471-1471 of SEQ ID NO: 96; the variant of SEQ ID NO: 104 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1446- 1513 of SEQ ID NO: 104; the variant of SEQ ID NO: 105 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1455- 1522 of SEQ ID NO: 105; the variant of SEQ ID NO: 106 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1462- 1529 of SEQ ID NO: 106; the variant of SEQ ID NO: 107 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 39-789 or 1469— 1536 of SEQ ID NO: 107; or the variant of SEQ ID NO: 108 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 89-839 or 1485- 1552 of SEQ ID NO: 108; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 130; or a variant of SEQ ID NO: 130, wherein the variant of SEQ ID NO: 130 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130;
(v) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 109, 110, 11 1, 112, 113, 114, 115, 116, 117, or 118; or a variant of any one of SEQ ID NOs: 109, 110, 111, 112, 113, 114, 115, 116, 117, or 118, wherein the variant of SEQ ID NO: 109 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1686— 1753 of SEQ ID NO: 109; the variant of SEQ ID NO: 110 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1704-
1771 of SEQ ID NO: 110; the variant of SEQ ID NO: 111 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1720- 1787 of SEQ ID NO: 111; the variant of SEQ ID NO: 112 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1734- 1801 of SEQ ID NO: 112; the variant of SEQ ID NO: 113 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1687- 1754 of SEQ ID NO: 113; the variant of SEQ ID NO: 114 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 40-789 or 1695- 1762 of SEQ ID NO: 114; the variant of SEQ ID NO: 115 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434- 1501 of SEQ ID NO: 115; the variant of SEQ ID NO: 116 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434- 1501 of SEQ ID NO: 116; the variant of SEQ ID NO: 117 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 40-789 or 1434- 1501 of SEQ ID NO: 117; or the variant of SEQ ID NO: 118 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434- 1501 of SEQ ID NO: 118; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 130; or a variant of SEQ ID NO: 130, wherein the variant of SEQ ID NO: 130 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 20-320 of SEQ ID NO: 130; and
(vi) the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 126, or 127; or a variant of any one of SEQ ID NOs: 119, 120, 122, 123, 124, 125, 126, or 127, wherein the variant of SEQ ID NO: 119 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1443-
1510 of SEQ ID NO: 119; the variant of SEQ ID NO: 120 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 40-789 or 1459- 1526 of SEQ ID NO: 120: the variant of SEQ ID NO: 122 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434- 1501 of SEQ ID NO: 122; the variant of SEQ ID NO: 123 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1434- 1501 of SEQ ID NO: 123; the variant of SEQ ID NO: 124 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 40-789 or 1434- 1501 of SEQ ID NO: 124; the variant of SEQ ID NO: 125 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1463- 1530 of SEQ ID NO: 125; the variant of SEQ ID NO: 126 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 40-789 or 1466- 1533 of SEQ ID NO: 126; the variant of SEQ ID NO: 127 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 40-789 or 1425- 1492 of SEQ ID NO: 127; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 130; or a variant of SEQ ID NO: 130, wherein the variant of SEQ ID NO: 130 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 20-320 of SEQ ID NO: 130.
Embodiment 28. The composition of Embodiment 26, wherein the virus is a Respiratory Syncytial Virus (RSV), wherein the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 148, 149, 150, 151, or 152; or a variant of any one of SEQ ID NOs: 148, 149, 150, 151, or 152, wherein the variant of SEQ ID NO: 148 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-78 of SEQ ID NO: 148; the variant of SEQ ID NO: 149 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 15-33 of SEQ ID NO: 149; the variant of SEQ ID NO: 150 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-35 of SEQ ID NO: 150; the variant of SEQ ID NO: 151 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 18-36 of SEQ ID NO: 151 ; or the variant of SEQ ID NO: 152 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-38 of SEQ ID NO: 152; and wherein the R region comprises the nucleotide sequence set forth as SEQ ID NO: 154 or 155; or a variant of SEQ ID NO: 154 or 155, wherein the variant of SEQ ID NO: 154 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-207 of SEQ ID NO: 154; or the variant of SEQ ID NO: 155 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 15-32 of SEQ ID NO: 155.
Embodiment 29. The composition of Embodiment 26, wherein the virus is a parainfluenzavirus, and wherein a combination of the L region and the R region satisfies one of the following:
(i) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 180; or a variant of SEQ ID NO: 180, wherein the variant of SEQ ID NO: 180 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-136 of SEQ ID NO: 180; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 179; or a variant of SEQ ID NO: 179, wherein the variant of SEQ ID NO: 179 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17 -98 of SEQ ID NO: 179;
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 186; or a variant of SEQ ID NO: 186, wherein the variant of SEQ ID NO: 186 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 21-95 of SEQ ID NO: 186; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 185; or a variant of SEQ ID NO: 185, wherein the variant of SEQ ID NO: 185 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 21-93 of SEQ ID NO: 185.
Embodiment 30. The composition of Embodiment 26, wherein the virus is a nietapneumovirus, and wherein a combination of the L region and the R region satisfies one of the following:
(i) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 194; or a variant of SEQ ID NO: 194, wherein the variant of SEQ ID NO: 194 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-220 of SEQ ID NO: 194; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 192; or a variant of any one of SEQ ID NO: 192, wherein the variant of SEQ ID NO: 192 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-32 of SEQ ID NO: 192; and
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 193; or a variant of SEQ ID NO: 193, wherein the variant of SEQ ID NO: 193 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-220 of SEQ ID NO: 193; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 191 ; or a variant of SEQ ID NO: 191, wherein the variant of SEQ ID NO: 191 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-32 of SEQ ID NO: 191.
Embodiment 31. The composition of Embodiment 26, wherein the virus is a henipavirus, and wherein a combination of the L region and the R region satisfies one of the following: (i) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 203; or a variant of SEQ ID NO: 203, wherein the variant of SEQ ID NO: 203 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-77 of SEQ ID NO: 203; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 202: or a variant of SEQ ID NO: 202, wherein the variant of SEQ ID NO: 202 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-91 of SEQ ID NO: 202: and
(ii) the L region comprises a nucleotide sequence set forth as SEQ ID NO: 207; or a variant of SEQ ID NO: 207, wherein the variant of SEQ ID NO: 207 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-77 of SEQ ID NO: 207; and the R region comprises the nucleotide sequence set forth as SEQ ID NO: 208; or a variant of SEQ ID NO: 208, wherein the variant of SEQ ID NO: 208 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 17-91 of SEQ ID NO: 208.
Embodiment 32. The composition of Embodiment 26, wherein the virus is a hepadnavirus, wherein the L region comprises a nucleotide sequence set forth as any one of SEQ ID NOs: 212, 213, 214, 215, or 216; or a variant of any one of SEQ ID NOs: 212, 213, 214, 215 , or 216, wherein the variant of SEQ ID NO: 212 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 101—1326 of SEQ ID NO: 212; the variant of SEQ ID NO: 213 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-1291 of SEQ ID NO: 213; the variant of SEQ ID NO: 214 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-1325 of SEQ ID NO: 214; the variant of SEQ ID NO: 215 comprises a variation at one or more nucleotide positions selected from the group consi sting of positions 101-15 of SEQ
ID NO: 215: or the variant of SEQ ID NO: 216 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-211 of SEQ
ID NO: 216; and wherein the R region comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 217, 218, 219, or 220: or a variant of any one of SEQ ID NOs: 217, 218, 219, or 220, wherein the variant of SEQ ID NO: 217 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-754 of SEQ ID NO: 217; the variant of SEQ ID NO: 218 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-790 of SEQ ID NO: 218; the variant of SEQ ID NO: 219 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-892 of SEQ ID NO: 219; or the variant of SEQ ID NO: 220 comprises a variation at one or more nucleotide positions selected from the group consisting of positions 101-2309 of SEQ ID NO: 220.
Embodiment 33. The composition of any one of Embodiments 1-32, wherein the therapeutic polypeptide is a secreted polypeptide, optionally an antibody, or wherein the therapeutic polypeptide is 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-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, IL-12B, or IL-2, or wherein the therapeutic polypeptide is a caspase, or wherein the therapeutic polypeptide is an interferon, optionally an IFN-a, IFN-P, IFN- s, IEN-K, IFN-a), 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-01, IFN-s, IFN-K, IFN-col, IFN-y, IFN-X1 (IL28A), IFN- X2 (IL28B), IFN- Z3 (IL.29), or IFN- M.
Embodiment 34. The composition of any one of Embodiments 1 to 33 , wherein the coding sequence of the 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 35. The composition of any one of Embodiments 1 to 34, wherein the encrypted RNA or the target mRNA comprises a polyadenylation signal and/or a 3’ poly(A) tail, or wherein the encrypted RNA or the target mRNA is in a linear form or a covalently- closed circular’ form.
Embodiment 36. The composition of any one of Embodiments 1 to 34, wherein the composition comprises a lipid nanoparticle encapsulating the encrypted RNA and the target mRNA.
Embodiment 37. The composition of any one of Embodiments 1 to 36, wherein the RNA editor is (i) an endogenous ADAR, (ii) an exogenous ADAR, or (iii) an engineered ADAR, wherein the substrate comprises an adenosine to cytidine mispairing within the stop codon, and wherein the RNA editor converts the adenosine to cytidine mispairing into an inosine to cytidine pairing in the substrate.
Embodiment 38. The composition of Embodiment 37, wherein the RNA editor is selected from the group consisting of: ADAR2, ADAR1, ADAR1 pl 50, ADAR1 pl 10, ADAR2 R455G, ADAR2 S486T, ADAR2 T375G E488Q T490A, ADAR2 T375G, ADAR2 T375S, ADAR2 N473D, ADAR2 deaminase domain, ADAR2 T490S, ADAR2 T490A, MCP-ADAR2 deaminase domain, ADAR2 R455E, ADAR2 T375G T490A, ADAR2 E488Q, MCP-ADAR2 deaminase domain E488Q T490A, ADAR2 R510E, ADAR2 R455S, and ADAR2 V351 L.
Embodiment 39. The composition of any one of Embodiments 1 to 38, wherein the target mRNA comprises, in 5’ to 3’ order, the following operably linked elements: a 5’ UTR, an upstream coding sequence (uCDS), the edit tract, and a downstream coding sequence (dCDS) encoding the translation activator or the translation activator component.
Embodiment 40. The composition of any one of Embodiments 1 to 39, wherein the edit tract comprises two or more stop codons that prevent translation of the translation activator or the translation activator component encoded by the target mRNA.
Embodiment 41. The composition of Embodiment 39, wherein the edit tract and the dCDS are operably linked via (i) a ribosomal skipping sequence; (ii) a coding sequence encoding a protease cleavage site (CDS-PS); or (iii) a coding sequence encoding a flexible linker (CDS-FL).
Embodiment 42. The composition of any one of Embodiments 1-41, w'herein the guide RNA has a length of 200-10,000 nucleotides.
Embodiment 43. The composition of any one of Embodiments 1-42, wherein the guide RNA is (i) native to the cell; (ii) delivered to the cell; or (ii) exogenous to the cell and transcribed by the cell.
Embodiment 44. A method of modulating expression of a therapeutic polypeptide in a cell of a subject, comprising introducing the composition of any one of Embodiments 1-43 into the cell.
Embodiment 45. The method of Embodiment 44, 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 46. The method of Embodiment 44, further comprising introducing an amplification target mRNA into the cell, wherein the amplification target mRNA comprises an edit tract, and wherein a stop codon within the edit tract prevents translation of the RNA editor encoded by the amplification target mRNA; wherein an amplification guide RNA binds to the amplification target mRNA to form a double -stranded RNA complex as a substrate for the RNA editor, wherein the substrate comprises a mispairing within the stop codon, and wherein the RNA editor acts upon the substrate to convert the mispairing into a pairing, thereby allowing translation of the RNA editor from the amplification target mRNA.
Embodiment 47. The method of Embodiment 46, wherein the RNA editor is (i) an endogenous ADAR or (ii) an exogenous ADAR, or (iii) an engineered ADAR, wherein the substrate comprises an adenosine to cytidine mispairing within the stop codon, and wherein the RNA editor converts the adenosine to cytidine mispairing into an inosine to cytidine pairing in the substrate,
Embodiment 48. The lipid nanoparticle of Embodiment 47, wherein the RNA editor is selected from the group consisting of: ADAR2, AD ARI, AD ARI pl50, AD ARI pl 10, ADAR2 R455G, ADAR2 S486T, ADAR2 T375G E488Q T490A, ADAR2 T375G, ADAR2 T375S, ADAR2 N473D, ADAR2 deaminase domain, ADAR2 T490S, ADAR2 T490A, MCP-ADAR2 deaminase domain, ADAR2 R455E, ADAR2 T375G T490A, ADAR2 E488Q, MCP-ADAR2 deaminase domain E488Q T490A, ADAR2 R510E, ADAR2 R455S, and ADAR2 V351L.
Embodiment 49. The method of any one of Embodiments 46 to 48, wherein the amplification guide RNA is (i) native to the cell; (ii) delivered to the cell; or (iii) exogenous to the cell and transcribed by the cell.
Embodiment 50. The method of any one of Embodiments 46 to 49, wherein the amplification guide RNA has a length of 200-10,000 nucleotides. Embodiment 51. A composition comprising:
(a) an 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:
(b) an RNA editor;
(c) a guide RNA;
(d) two or more mRNA molecules, each of which encodes a portion of a translation activator or a portion of a translation activator component, wherein each portion is not functional, wherein delivering the composition into a cell results in (i) activation of the RNA editor by the guide RNA; (ii) a ligation of the two or more mRNA molecules by the RNA editor to form a mRNA product encoding the translation activator or the translation activator component; and (iii) translation of the translation activator or the translation activator component; and wherein the encrypted RNA contacts the translation activator, thereby resulting in translation of the therapeutic polypeptide,
Embodiment 52. The composition of Embodiment 51, wherein the RNA editor is eDisCas7-ll.