Compositions comprising ire-1 binding sequences and uses thereof

Abstract

Disclosed herein are polynucleotides that can sustain payload expression and / or induce payload expression during cellular stress. Also provided herein are compositions comprising the disclosed polynucleotides, and methods of making and using the same.

Description

Attorney Docket: 2012611-0180 COMPOSITIONS COMPRISING IRE-1 BINDING SEQUENCES AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 669,897 filed on July 11, 2024, and U.S. Provisional Patent Application No. 63 / 707,867 filed on October 16, 2024, the entire contents of which are hereby incorporated by reference in their entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on July 11, 2025, is named 2012611-0180, and is 61,419 bytes in size. BACKGROUND

[0003] The past decade has seen large technological progress in the use of polyribonucleotides as a therapeutic modality. However, to fully realize the potential of RNA therapeutics, there is still much progress to be made on further improvements particularly for delivering RNAs at therapeutic doses. SUMMARY

[0004] The present disclosure recognizes that providing sustained and meaningful expression of polynucleotides and / or polypeptides encoded by polynucleotides has been challenging. Indeed, delivery of polynucleotides at levels that provide meaningful expression (e.g., therapeutic levels) of encoded polynucleotides and / or polypeptides, which can be higher than levels of polynucleotides and / or polypeptides expressed physiologically, often overwhelms a cell and induces stress responses (such as ER stress), which in turn activates the unfolded protein response (UPR). Expression of encoded polypeptides at high levels can also result in increased protein misfolding.

[0005] Accordingly, the present disclosure provides a solution to this problem with polynucleotide constructs disclosed herein that can sustain payload expression and / or induce payload expression in the presence of cellular stress. Using polynucleotide constructs disclosed herein, sustained and / or increased expression of a payload (e.g., a polyribonucleotide or a polypeptide encoded by a polynucleotide) can be achieved even when high doses of a polynucleotide is administered to a cell, tissue of subject. Polynucleotides disclosed herein comprise sequence elements referred to as Genetic Activation with Responsive Yield (GARY) sequence elements, which include features of components involved in the UPR. In some embodiments, a polynucleotide comprising a GARY sequence element provides sustained and / or continued expression of polynucleotides by utilizing the unfolded stress response (UPR) pathway and / or by preventing RNA degradation. Also provided herein are compositions for administering polynucleotides disclosed herein, as well as methods of using and making polynucleotides disclosed herein. Page 1 of 84 12871572v1Attorney Docket: 2012611-0180

[0006] Provided herein, among other things, is a polynucleotide comprising: (i) an Inositol-requiring Enzyme 1 (IRE-1) binding sequence, (ii) an intervening sequence, (iii) an AUG translation initiation codon, and (iv) a payload sequence encoding a payload.

[0007] In some embodiments, an intervening sequence is located between an IRE1-binding sequence and a payload sequence.

[0008] In some embodiments, a polynucleotide (e.g., a GARYon polynucleotide) comprises in 5’ to 3’ order: (a) an IRE-1 binding sequence, (b) an intervening sequence, (c) an AUG translation initiation codon, and (d) a payload sequence. In some embodiments, a polynucleotide further comprises a 5’ UTR. In some embodiments, a 5’ UTR comprises (a) an IRE-1 binding sequence, and (b) an intervening sequence. In some embodiments, a 5’ UTR does not comprise (c) an AUG translation initiation codon.

[0009] In some embodiments, a polynucleotide (e.g., a GARYon polynucleotide) is characterized such that when administered to a cell, tissue or subject, expression of a payload is observed at similar levels under physiological conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

[0010] In some embodiments, a polynucleotide (e.g., a GARYon polynucleotide) is characterized such that when administered to a cell, tissue or subject, increased expression of a payload is observed under cellular stress conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

[0011] In some embodiments, expression of a payload under cellular stress conditions is similar to or greater than expression of the payload under physiological conditions.

[0012] In some embodiments, a polynucleotide (e.g., a GARYswitch polynucleotide) comprises in 5’ to 3’ order: (a) an AUG translation initiation codon, (b) an IRE-1 binding sequence, (c) an intervening sequence, and (d) a payload sequence.

[0013] In some embodiments, a polynucleotide (e.g., a GARYswitch polynucleotide) further comprises a 5’ UTR. In some embodiments, a 5’ UTR comprises does not comprise (a) an AUG translation initiation codon, (b) an IRE-1 binding sequence, and (c) an intervening sequence.

[0014] In some embodiments, a polynucleotide (e.g., a GARYswitch polynucleotide) is characterized such that when administered to a cell, tissue or subject, minimal (e.g., low), expression of a payload is observed under physiological conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

[0015] In some embodiments, a polynucleotide (e.g., a GARYswitch polynucleotide) is characterized such that when administered to a cell, tissue or subject, increased expression of a payload is observed under cellular stress conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

[0016] In some embodiments of any of the polynucleotides disclosed herein, an intervening sequence comprises one or more UAG, UAA and / or UGA codons and / or one or more linker sequences. In some embodiments, an intervening sequence comprises any two of the following codons: UAG, UAA or UGA. In some embodiments, an intervening sequence comprises UAG, UAA and UGA codons.

[0017] In some embodiments of any of the polynucleotides disclosed herein, a polynucleotide further comprises a 5’ cap, a 3’ UTR and / or a polyA tail. Page 2 of 84 12871572v1Attorney Docket: 2012611-0180

[0018] In some embodiments of any of the polynucleotides disclosed herein, an IRE-1 binding sequence comprises a sequence that is recognized by a nuclease domain of IRE-1. In some embodiments, an IRE-1 binding sequence comprises: (i) a CUGCAG sequence, (ii) a CCGCAG sequence, or (iii) (i) and (ii).

[0019] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence or a fragment or variant thereof. In some embodiments, an XBP1 sequence comprises the nucleotide sequence provided in SEQ ID NO: 5 or 30, or a sequence with at least 85% identity thereto. In some embodiments, an XBP1 sequence comprises the nucleotide sequence provided in SEQ ID NO: 6 or 31, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises the nucleotide sequence provided in SEQ ID NO: 5 or 30, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises the nucleotide sequence provided in SEQ ID NO: 6 or 31, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises the nucleotide sequence provided in SEQ ID NO: 25 or 32, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises the nucleotide sequence provided in SEQ ID NO: 26 or 33, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises the nucleotide sequence provided in SEQ ID NO: 27 or 34, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises the nucleotide sequence provided in SEQ ID NO: 28 or 35, or a sequence with at least 85% identity thereto.

[0020] In some embodiments, an IRE-1 binding sequence comprises the nucleotide sequence provided in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49 or a sequence with at least 85% identity to any one of the foregoing.

[0021] In some embodiments, a fragment or variant thereof is a functional fragment, a functional variant, or a functional fragment variant thereof.

[0022] This disclosure also provides a composition comprising a polynucleotide disclosed herein. In some embodiments, a composition is a pharmaceutical composition. In some embodiments, a pharmaceutical composition comprises one or more pharmaceutically acceptable excipients. In some embodiments, a pharmaceutical composition is formulated for intramuscular, intradermal, intravenous, subcutaneous, mucosal, inhaled, or intranodal delivery.

[0023] Further provided herein is a cell comprising a polynucleotide disclosed herein. In some embodiments, a cell is in vivo. In some embodiments, a cell is ex vivo. In some embodiments, a polynucleotide is introduced into a cell. In some embodiments, a cell expresses one or more polypeptides encoded by a polynucleotide.

[0024] Among other things, this disclosure provides a method of delivering a polynucleotide disclosed herein or a composition comprising a polynucleotide. In some embodiments, delivering comprises administering a polynucleotide or a composition to a cell, tissue or subject.

[0025] In some embodiments, a method is a treatment method. In some embodiments, a method is: (i) a method to stimulate an immune response, (ii) an antibody therapy method, (iii) an immune-modulation method, (iv) a vaccination method, (v) a gene therapy method, (vi) a cell therapy engineering method, (vii) an immunotherapy method, (viii) a protein replacement therapy method, (ix) a chemotherapeutic method, or (x) any combination of (i)- (ix). Page 3 of 84 12871572v1Attorney Docket: 2012611-0180

[0026] Also provided herein is a method for stimulating an immune response to an antigen, comprising delivering a polynucleotide disclosed herein or a composition comprising a polynucleotide, to a cell, tissue or subject.

[0027] In some embodiments, a subject is a human.

[0028] In some embodiments of any of the polynucleotides, compositions, or methods disclosed herein, cellular stress response is endoplasmic-reticulum (ER) stress. In some embodiments, ER stress comprises the unfolded protein response (UPR). In some embodiments, UPR activates IRE-1 nuclease activity.

[0029] In some embodiments of any of the polynucleotides, compositions, or methods disclosed herein, physiological conditions refers to a cellular condition in which a cell is not under ER stress.

[0030] In some embodiments of any of the polynucleotides, compositions, or methods disclosed herein, a polynucleotide is or comprises a polyribonucleotide.

[0031] In some embodiments, a polyribonucleotide comprises naturally occurring polyribonucleotides.

[0032] In some embodiments, a polynucleotide is a polyribonucleotide comprising one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0033] In some embodiments, one or more modified ribonucleotides has: a 5’ monophosphate, a 5’ diphosphate, or a 5’ triphosphate. In some embodiments, one or more modified ribonucleotides comprises a 5’ triphosphate.

[0034] In some embodiments, one or more modified ribonucleotides comprises a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2A. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2B when incorporated in a polynucleotide (e.g., polyribonucleotide). In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 2B.

[0035] In some embodiments, the nucleoside has a structure of: O H3C NH O .

[0036] In some embodiments, one or more modified ribonucleotides has the structure of: Page 4 of 84 12871572v1Attorney Docket: 2012611-0180 O H3C NH N O .

[0037] In some embodiments, one or mo des has the structure of:O H3C NH O .

[0038] In some embodiments, one or mes has the structure of: O H3CNH O .

[0039] In some embodiments, a poly. ., tide) comprises one or more modified ribonucleotides that have the following structure: O H3C NH O , wherein indicates the position of attachment to an a jacent ribonucleotide.

[0040] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 5 of 84 12871572v1Attorney Docket: 2012611-0180 O H3C NH N O , wherein indicates the position of at nucleotide.

[0041] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O H3C NH O , wherein indicates the position ofcleotide.

[0042] In some embodiments, a polyribonucleotide comprises cytidine nucleosides, and at least 5% of cytidine nucleosides in a polyribonucleotide comprise N4-acetylcytidine.

[0043] In some embodiments, a polyribonucleotide comprises cytidine nucleosides, and less than 100% of cytidine nucleosides in the polyribonucleotide comprise N4-acetylcytidine.

[0044] In some embodiments, a polyribonucleotide comprises cytidine nucleosides, and at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of cytidine nucleosides in a polyribonucleotide comprise N4-acetylcytidine.

[0045] In some embodiments, one or more modified ribonucleotides comprises a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2A. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2B when incorporated in a polynucleotide (e.g., polyribonucleotide). In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 2B.

[0046] In some embodiments, the nucleoside has a structure of: Page 6 of 84 12871572v1Attorney Docket: 2012611-0180 OH O NH O .

[0047] In some embodiments, one or more eotides has a structure of:OH O NH O .

[0048] In some embodiments, one or moreotides has a structure of: OH O N O .

[0049] In some embodiments, one or mes has a structure of: OH O NH O .

[0050] In some embodiments, a polynuceo e (e.g., poy o uceotide) comprises one or more modified ribonucleotides that have the following structure: OH O NH O , wherein indicates the position of attachment to an adjacent ribonucleotide.

[0051] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 7 of 84 12871572v1Attorney Docket: 2012611-0180 OH O NH N O , wherein indicates the position of att onucleotide.

[0052] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: OH O NH O , wherein indicates the position ofcleotide.

[0053] In some embodiments, a polyribonucleotide comprises uridine nucleosides and at least 5% of uridine nucleosides in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0054] In some embodiments, a polyribonucleotide comprises uridine nucleosides and less than 100% of uridine nucleosides in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0055] In some embodiments, a polyribonucleotide comprises uridine nucleosides and at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of uridine nucleosides in a polyribonucleotide comprise 5-hydroxymethyluridine.

[0056] In some embodiments, a polyribonucleotide comprises uridine nucleosides and more than 60% of uridine nucleosides in a polyribonucleotide comprise 5-hydroxymethyluridine.

[0057] In some embodiments, one or more modified ribonucleotides comprises: N4-acetylcytidine, 5- hydroxymethyluridine, N1-methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5- aza-uridine, 2-thio-uridine (s2U), 5-methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3- methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6-diaminopurine, 2- amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (m1 I), wyosine (imG), methylwyosine (mimG), 5- hydroxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2’-O-methyluridine, 2’-O-methyl-5-methyluridine, 2’- fluoro-2’-deoxyuridine, 2’-amino-2’-deoxyuridine, 2’-azido-2’-deoxyuridine, 4-thiouridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5- fluorouridine, pseudouridine, 2’-O-methyl-pseudouridine, N1-hydroxypseudouridine, 2’-O-methyl-N1- Page 8 of 84 12871572v1Attorney Docket: 2012611-0180 methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, ara-uridine, N6-methyladenosine, 2- aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 8-oxoadenosine, thienoguanosine, 7-deazaguanosine, 8- oxoguanosine, 6-O-methylguanine, or any combination thereof. In some embodiments, one or more modified ribonucleotides comprises a nucleoside comprising a ribose moiety comprising an acetyl group, wherein the ribose is 2’-O-acetylated.

[0058] In some embodiments, one or more modified ribonucleotide has a structure of: X R O O 3 ,

[0059] (a) wherein R is a monophosphate,triphosphate, and

[0060] (b) wherein X is an adenine nucleobase, a guanine nucleobase, a cytosine nucleobase (e.g., N4- acetylcytosine), or a uracil nucleobase (e.g., 5-hydroxymethyluracil).

[0061] In some embodiments, one or more modified ribonucleotides comprises: (i) a 2’-O-acetylated ribose and (ii) an adenine nucleobase. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2D when incorporated in a polynucleotide. In some embodiments, a polynucleotide (e.g., a polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 2D.

[0062] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: H2N N N N , wherein indicates the position of attachment to an adjacent ribonucleotide.

[0063] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 9 of 84 12871572v1Attorney Docket: 2012611-0180 H2N N N N , wherein indicates the position of at nucleotide.

[0064] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: H2N N N N , wherein indicates the position of aucleotide.

[0065] In some embodiments, one or more modified ribonucleotides comprises: (i) a 2’-O-acetylated ribose and (ii) a guanine nucleobase. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2D when incorporated in a polynucleotide. In some embodiments, a polynucleotide (e.g., a polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 2D.

[0066] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NH N H2, wherein indicates the position of attachment to an adjacent ribonucleotide.

[0067] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 10 of 84 12871572v1Attorney Docket: 2012611-0180 O NH N NH N2, wherein indicates the position of a ucleotide.

[0068] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NH N NH N2, wherein indicates the position ofcleotide.

[0069] In some embodiments, one or more modified ribonucleotides comprises: (i) a 2’-O-acetylated ribose and (ii) a cytosine nucleobase (e.g., cytosine or N4-acetylcytosine). In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2D when incorporated in a polynucleotide. In some embodiments, a polynucleotide (e.g., a polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 2D.

[0070] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: NH2N O O 3 , wherein indicates the position of attachment to an adjacent ribonucleotide.

[0071] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 11 of 84 12871572v1Attorney Docket: 2012611-0180 NH2N O O 3 , wherein indicates the position of at nucleotide.

[0072] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: NH2N O O 3 , wherein indicates the position ofcleotide.

[0073] In some embodiments, one or more modified ribonucleotides comprising a 2’-O-acetylated ribose comprises a N4-acetylcytosine nucleobase. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2D when incorporated in a polynucleotide. In some embodiments, a polynucleotide (e.g., a polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 2D.

[0074] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O H3C NH O O 3 , wherein indicates the position of attachment to an adjacent ribonucleotide.

[0075] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 12 of 84 12871572v1Attorney Docket: 2012611-0180 O H3CNH N O O 3 , wherein indicates the position of at nucleotide.

[0076] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O H3C NH O O 3 , wherein indicates the position ofucleotide.

[0077] In some embodiments, one or more modified ribonucleotides comprising a 2’-O-acetylated ribose comprises a uracil nucleobase (e.g., uracil or 5-hydroxymethyluracil). In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2D when incorporated in a polynucleotide. In some embodiments, a polynucleotide (e.g., a polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 2D.

[0078] In In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NH O O 3 ,wherein indicates the position of attachment to an adjacent ribonucleotide.

[0079] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 13 of 84 12871572v1Attorney Docket: 2012611-0180 O N O O 3 , wherein indicates the position of at nucleotide.

[0080] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NH O O 3 , wherein indicates the position ofcleotide.

[0081] In some embodiments, one or more modified ribonucleotides comprising a 2’-O-acetylated ribose comprises a 5-hydroxymethyluracil nucleobase. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2D when incorporated in a polynucleotide. In some embodiments, a polynucleotide (e.g., a polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 2D.

[0082] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: OH O NH O O 3 , wherein indicates the position of attachment to an adjacent ribonucleotide.

[0083] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 14 of 84 12871572v1Attorney Docket: 2012611-0180 OH O NH O O 3 , wherein indicates the position of at nucleotide.

[0084] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: OH O NH O O 3 , wherein indicates the position ofcleotide.

[0085] In some embodiments, one or more modified ribonucleotides comprising a 2’-O-acetylated ribose comprises a N1-methylpseudouracil nucleobase. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 2D when incorporated in a polynucleotide. In some embodiments, a polynucleotide (e.g., a polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 2D.

[0086] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NNHO O , wherein indicates the position of attachment to an adjacent ribonucleotide.

[0087] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 15 of 84 12871572v1Attorney Docket: 2012611-0180 O NNHO O , wherein indicates the position of at nucleotide.

[0088] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NNHO O , wherein indicates the position ofcleotide.

[0089] In some embodiments of any of the polyribonucleotides disclosed herein, at least 5% of ribose moieties are acetylated (2’-O-acetylated).

[0090] In some embodiments of any of the polyribonucleotides disclosed herein, about 5% to about 99% of ribose moieties are acetylated (2’-O-acetylated).

[0091] In some embodiments of any of the polyribonucleotides disclosed herein, a polyribonucleotide comprises a cap structure and a cap structure does not comprise a 2’-O-acetylated ribose.

[0092] In some embodiments of any of the polyribonucleotides disclosed herein, a polyribonucleotide comprises a cap structure and a cap structure comprises a 2’-O-acetylated ribose.

[0093] In some embodiments of any of the polyribonucleotides disclosed herein, a polyribonucleotide further comprises one or more ribonucleotides that does not comprise a 2’-O acetylated ribose.

[0094] In some embodiments, a polyribonucleotide is or comprises an RNA oligo, a messenger RNA (mRNA), a gRNA, an inhibitory RNA, an miRNA or siRNA, an antisense oligonucleotide, or any combination thereof.

[0095] In some embodiments of any of the polynucleotides, compositions, or methods disclosed herein, a polynucleotide comprises a coding region.

[0096] In some embodiments, a coding region encodes a payload. In some embodiments, a payload is or comprises a polypeptide.

[0097] In some embodiments, a polyribonucleotide comprises an RNA payload. Page 16 of 84 12871572v1Attorney Docket: 2012611-0180

[0098] In some embodiments, a payload is an antigen, an enzyme, an antibody, a secreted protein, a cell surface protein, or a helper protein, or a variant or fragment of any of the foregoing.

[0099] In some embodiments, a fragment or variant thereof is a functional fragment, a functional variant, or a functional fragment variant thereof.

[0100] This disclosure further provides a polypeptide encoded by a polynucleotide of disclosed herein.

[0101] These, and other aspects encompassed by the present disclosure, are described in more detail below and in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] FIG.1 shows a schematic of an exemplary GARYon construct. The exemplary construct comprises, in order from 5’ to 3’the following sequences or elements: a 5’ cap, a 5’ UTR, a splice site sequence, e.g., an XBP1 splice site, a nucleotide spacer element, a linker, a start codon immediately preceding a sequence encoding a Nanoluciferase (NanoLuc) payload, a 3’ UTR, and a poly-A tail. In the exemplary GARYswitch construct, the splice site and nucleotide spacer which can comprise one or more stop codons is part of the 5’ UTR.

[0103] FIG.2 shows a schematic of an exemplary GARYswitch construct. The exemplary construct comprises, in order from 5’ to 3’the following sequences or elements: a 5’ cap, a 5’ UTR, a start codon preceding a splice site sequence, e.g., an XBP1 splice site, a nucleotide spacer, a linker, a sequence encoding a Nanoluciferase (NanoLuc) payload, a 3’ UTR element, and a poly-A tail. In the exemplary GARYswitch construct, the splice site and nucleotide spacer which can comprise one or more stop codons is part of the ORF.

[0104] FIG.3 shows Nanoluciferase (NanoLuc) expression from cells transfected with control NanoLuc-RNA, GARYon-NanoLuc RNA, or GARYswitch-NanoLuc RNA normalized to untransfected cells. The cells were either treated with 5 uM Thapsigargin or left untreated.

[0105] FIG.4 shows NanoLuc expression in HEK293T cells in which one of the following have been delivered: (1) an RNA encoding an exemplary mutated antigen fused to NanoLuc (control), (2) an RNA encoding an exemplary mutated antigen fused to NanoLuc and a GARYon RNA encoding an exemplary helper protein (GARYon tapt1) or (3) an RNA encoding an exemplary mutated antigen fused to NanoLuc and a GARYswitch RNA encoding an exemplary helper protein (GARYswitch tapt1). RNA encoding an exemplary mutated antigen fused to Nanoluc was administered to the cells in doses ranging from 0 nanograms to 5000 nanograms per well. A fixed amount of each GARY RNA was delivered. The shown NanoLuc expression level was normalized to a NanoLuc expression level in untreated cells (negative control).

[0106] FIGS.5A-5C are graphs showing total flux from bioluminescence imaging of animals injected with a 0.1 ug dose (FIG.5A), 1ug dose (FIG.5B) or 10ug dose (FIG.5C) of RNA encoding luciferase (1) without GARY elements, (2) in a GARYon construct, or (3) in a GARYswitch construct. Each animal received the indicated dose of RNA per intramuscular injection in the right quadricep at time points 6, 24, 48, and 72 hours. N= 5 female balb / c mice per condition. The total flux was normalized to control animals administered a buffer control. Page 17 of 84 12871572v1Attorney Docket: 2012611-0180

[0107] FIGS.6A-6B shows Nanoluciferase (NanoLuc) expression from cells transfected with exemplary GARYswitch constructs. FIG.6A shows NanoLuc expression from cells transfected with control NanoLuc RNA, GARYswitch NanoLuc RNA, GARYswitch_gallus NanoLuc RNA, or GARYswitch_xenopus NanoLuc RNA normalized to untransfected cells. Cells were treated with 5 uM Thapsigargin or left untreated. FIG.6B shows NanoLuc expression from cells transfected with control NanoLuc RNA, GARYswitch NanoLuc RNA, or GARYswitch_2 NanoLuc RNA normalized to untransfected cells. Cells were treated with 5 uM Thapsigargin or left untreated. CERTAIN DEFINITIONS

[0108] About or approximately: As used herein, the terms “about” and “approximately,” when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” or “approximately” in that context. For example, in some embodiments, the term “about” or “approximately” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0109] Administering: As used herein, the term “administering” or “administration” typically refers to administration of a composition to a subject to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0110] Comparable: As used herein, the term “comparable” refers to two or more agents (e.g., entities or set(s) of conditions), situations, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena Page 18 of 84 12871572v1Attorney Docket: 2012611-0180 observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0111] Delivery / contacting: As used interchangeably herein, the term “delivery,” “delivering,” or “contacting” refers to introduction of a polynucleotide (e.g., as described herein) into a target cell. A target cell can be cultured in vitro or ex vivo or be present in a subject (in vivo). Methods of introducing a polynucleotide (e.g., as described herein) into a target cell can vary with in vitro, ex vivo, or in vivo applications. In some embodiments, a polynucleotide (e.g., as described herein) can be introduced into a target cell in a cell culture by in vitro transfection. In some embodiments, a polynucleotide (e.g., as described herein) can be introduced into a target cell via delivery vehicles (e.g., nanoparticles, liposomes, and / or complexation with a cell-penetrating agent). In some embodiments, a polynucleotide (e.g., as described herein) can be introduced into a target cell in a subject by administering a polynucleotide (e.g., as described herein) to a subject.

[0112] Encode: As used herein, the term “encode” or “encoding” refers to a first molecule that is produced by a second molecule, wherein the sequence information of the second molecule determines the sequence of the first molecule. For example, a first molecule can have a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids which are determined by the sequence of the second molecule (e.g., a polynucleotide). For example, a DNA molecule (e.g., a second molecule) can encode an RNA molecule (e.g., a first molecule, for example by a transcription process that includes a DNA-dependent RNA polymerase enzyme) or a polypeptide (e.g., a first molecule for example by a transcription and a translation process). An RNA molecule (e.g., a second molecule) can encode a polypeptide (e.g., a first molecule for example by a translation process). Thus, a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system.

[0113] Functional: As used herein, the term “functional” is used to refer to a form or fragment of an entity that exhibits a particular property and / or activity.

[0114] Fragment: A “fragment” of a material or entity as described herein has a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a fragment comprises a polynucleotide fragment. In some embodiments, a fragment comprises a polypeptide fragment. In some embodiments, a polynucleotide fragment or a polypeptide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) as found in the whole polynucleotide or whole polypeptide. In some embodiments, a polynucleotide fragment or a polypeptide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polynucleotide or whole polypeptide. The whole polypeptide or whole polynucleotide may in some embodiments be referred to as the “parent” of the polynucleotide fragment or polypeptide fragment. Page 19 of 84 12871572v1Attorney Docket: 2012611-0180

[0115] Nucleic acid / Oligonucleotide / Polynucleotide: As used herein, the terms “nucleic acid” and “polynucleotide” and “oligonucleotide” are used interchangeably, and refer to a polymer of 3 nucleotides or more. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. In some embodiments, a nucleic acid comprises messenger RNA (mRNA). In some embodiments, a nucleic acid is single stranded. In some embodiments, a nucleic acid is double stranded. In some embodiments, a nucleic acid comprises both single and double stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5’-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural nucleosides (e.g., adenosine, cytidine, guanosine, uridine, thymidine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, deoxyuridine). In some embodiments, a nucleic acid comprises one or more, or all, non-natural nucleotides. In some embodiments, a non-natural nucleoside comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, a nucleoside comprising a methylated base, a nucleoside comprising an intercalated base, and combinations thereof). In some embodiments, a non-natural nucleoside comprises a modified nucleoside, e.g., as described herein. In some embodiments, a non- natural nucleoside comprises N4-acetylcytidine, 5-hydroxymethyluridine and / or N1-methylpseudouridine. In some embodiments, a non-natural nucleoside comprises one or more modified sugars (e.g., 2’-fluororibose, ribose, 2’- deoxyribose, arabinose, and hexose) as compared to those in natural nucleosides. In some embodiments, a non- natural nucleoside comprises a 2’-O-acetylated ribose. As used herein, “adenosine nucleosides” encompasses a natural adenosine nucleoside, as well as modified adenosine nucleosides. Accordingly, when a percentage of adenosine nucleosides is provided herein, the percentage is based on a total number of natural and modified adenosine nucleosides. Similarly, “cytidine nucleosides” encompasses a natural cytidine nucleoside, as well as modified cytidine nucleosides (e.g., N4-acetylcytidine or 2’-O-acetylated N4-acetylcytidine); “guanosine nucleosides” encompasses a natural guanosine nucleoside, as well as modified guanosine nucleosides; and “uridine nucleosides” encompasses a natural uridine nucleoside, as well as modified uridine nucleosides (e.g., 5-hydroxymethyluridine or 2’-O-acetylated 5-hydroxymethyluridine). Further, as used herein, “adenine nucleobase” encompasses a natural adenine nucleobase, as well as modified adenine nucleobase; “cytosine nucleobase” encompasses a natural cytosine nucleobase, as well as modified cytosine nucleobase (e.g., N4-acetylcytosine); “guanine nucleobase” encompasses a natural guanine nucleobase, as well as modified guanine nucleobase; and “uracil nucleobase” encompasses a natural uracil nucleobase, as well as modified uracil nucleobase (e.g., 5-hydroxymethyluracil). In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, Page 20 of 84 12871572v1Attorney Docket: 2012611-0180 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 nucleotides long. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0116] Polypeptide: The term “polypeptide”, as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids or more. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional, biologically active, or characteristic fragments, portions or domains (e.g., fragments, portions, or domains retaining at least one activity) of such complete polypeptides. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, polypeptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

[0117] RNA oligonucleotide: As used herein, the term “RNA oligonucleotide” refers to an oligonucleotide of ribonucleotides. In some embodiments, an RNA oligonucleotide is single stranded. In some embodiments, an RNA oligonucleotide is double stranded. In some embodiments, an RNA oligonucleotide comprises both single and double stranded portions. In some embodiments, an RNA oligonucleotide can comprise a backbone structure as described in the definition of “Nucleic acid / Oligonucleotide” above. An RNA oligonucleotide can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA) oligonucleotide. In some embodiments an RNA oligonucleotide can comprise at its 3’ end a poly(A) region. In some embodiments an RNA oligonucleotide comprises at its 5’ end a cap structure, e.g., for recognizing and attachment of an RNA to a ribosome to initiate translation. In some embodiments, a polynucleotide comprises an RNA oligonucleotide. When a number of ribonucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of ribonucleotides on a single strand.

[0118] Subject: As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0119] Variant: As used herein, the term “variant” refers to an entity that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a Page 21 of 84 12871572v1Attorney Docket: 2012611-0180 reference entity is based on its degree of structural identity with the reference entity. For example, a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, a variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide shows a reduced level of one or more biological activities as compared with the reference polypeptide.

[0120] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, e.g., RNA synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated herein by reference for any purpose. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0121] RNA-based gene therapies have emerged as a promising therapeutic modality, e.g., due to their ability to manipulate gene expression and / or produce therapeutic proteins, offering the potential to target a wide range of diseases. However, the potential of RNA-based technologies as a therapeutic intervention is hampered by challenges in obtaining meaningful expression levels (e.g., therapeutic levels) of payloads even with the delivery of high doses of RNA encoding said payloads to target cells.

[0122] When an exogenous polypeptide is being expressed in live cells, e.g., for therapeutic purposes, the polypeptide is typically expressed at levels significantly higher than what is physiologically relevant for the particular cell, and often by orders of magnitude. Under such circumstances, endoplasmic reticulum (ER) stress and unfolded protein response (UPR) induction are highly likely to occur. ER stress can be related to the accumulation of unfolded and / or misfolded proteins and activates the UPR. The UPR consists of three distinct signaling pathways that regulate transcriptional and translational programs to relieve ER stress: PERK, ATF6, and IRE1. See, for example, Park et al., “Roles of XBP1s in Transcriptional Regulation of Target Genes,” Biomedicines 2021, 9, 791, which is incorporated herein by reference in its entirety.

[0123] In addition to stress induced by delivery of high doses of RNA, mutant, synthetic, fusion, and other difficult-to-express polypeptide payloads encoded by delivered polynucleotides may spend more time in unfolded states and present additional burdens to ER functions. UPR effector functions mediated by IRE1 that negatively impact mRNA levels and protein expression are particularly problematic in achieving sustained and / or high levels of payload expression under such circumstances. Page 22 of 84 12871572v1Attorney Docket: 2012611-0180

[0124] The IRE1 branch of the UPR has effects on cellular mRNA. IRE1 sits in the ER membrane and functions as a UPR sensor. It contains an N-terminal luminal receptor domain and C-terminal cytosolic kinase and nuclease effector domains. Under conditions of ER stress, IRE1 oligomerizes and the kinase domain mediates autophosphorylation, activating the nuclease domain RNase activity. The IRE1 nuclease domain carries out at least two functions: (1) the IRE1 nuclease domain recognizes a double stem loop structure present in XBP1 mRNA and splices the mRNA, removing 26 nucleotides and resulting in a frame shift, which allows the mRNA to be translated into an active transcription factor that upregulates the expression of UPR target genes; and (2) the IRE1 nuclease domain also mediates widespread degradation of various mRNAs containing recognition sequences in a process named regulated IRE1-dependent decay (RIDD), resulting in a global decrease in protein translation.

[0125] A person of skill in the art will appreciate upon reading this disclosure that polynucleotides which include features (e.g., sequence elements) of components involved in the UPR such as sequences that can be bound and / or spliced by IRE-1 can be beneficial in providing sustained and / or induced expression of payloads encoded by the polynucleotides even under cellular stress conditions.

[0126] Among other things, the present disclosure provides certain insights and technologies relating to improved polynucleotides for achieving meaningful expression levels (e.g., therapeutic levels) of a payload. In particular, provided herein is the insight that polynucleotides comprising features (e.g., sequence elements) that are responsive to the IRE1 branch of the UPR can achieve sustained payload expression and / or induced payload expression under cellular stress. Such polynucleotides as disclosed herein comprise GARY (Genetic Activation with Responsive Yield) sequence elements. Specifically, polynucleotides comprising GARY sequence elements comprise a double stem loop structure similar such as the double stem loop structure in XBP1 RNA that can be recognized and spliced by IRE1. Provided herein are two types of polynucleotides comprising GARY sequence elements: (1) GARYon polynucleotides which allow for maintenance of payload expression under cellular stress conditions, e.g., by preventing degradation via RIDD; and (2) GARYswitch polynucleotides which allow for an induction of expression of a payload under stress (e.g., ER stress). In some embodiments, a splicing event also protects GARYswitch RNA from degradation via RIDD.

[0127] Accordingly, the present disclosure is the first to recognize that polynucleotides disclosed herein can achieve sustained payload expression and / or induced payload expression even when delivered at high doses under conditions that likely induce cellular stress. In many embodiments, polynucleotides provided herein are useful for delivering therapeutic levels of payloads to cells.

[0128] The present disclosure provides, among other things, compositions comprising disclosed polynucleotides (e.g., comprising a GARY sequence element) and methods of using the same, e.g., to achieve desired payload expression levels even in the presence of cellular stress. GARY Circuits

[0129] This disclosure provide polynucleotides comprising sequence elements that allow for sustained payload expression under cellular stress and / or induced payload expression under cellular stress. In some embodiments, a polynucleotide provided herein comprises a GARY sequence element. Page 23 of 84 12871572v1Attorney Docket: 2012611-0180

[0130] In some embodiments, a polynucleotide comprises (i) an Inositol-requiring Enzyme 1 (IRE-1) binding sequence, (ii) an intervening sequence, (iii) an AUG translation initiation codon, and (iv) a payload sequence encoding a payload. In some embodiments, an intervening sequence is located between an IRE1-binding sequence and a payload sequence. Exemplary polynucleotide sequences disclosed herein are provided in Table 1.

[0131] In some embodiments, an intervening sequence comprises one or more UAG, UAA and / or UGA codons and / or one or more linker sequences.

[0132] In some embodiments, an intervening sequence comprises any two of the following codons: UAG, UAA or UGA.

[0133] In some embodiments, an intervening sequence comprises UAG, UAA and UGA codons.

[0134] In some embodiments, a polynucleotide further comprises a 5’ cap, a 3’ UTR and / or a polyA tail. GARYon polynucleotides

[0135] In some embodiments, a polynucleotide disclosed herein is a GARYon polynucleotide. In some embodiments, in a GARYon polynucleotide, a double stem loop splice site is located in a 5’ UTR of an RNA, upstream of an open reading frame (ORF) comprising a sequence encoding a payload. See FIG.1. Under physiological conditions (e.g., in the absence of cellular stress), the ORF is translated, and protein expression occurs at levels similar to control RNA (e.g., without a double stem loop splice site). Upon UPR induction, expression from control RNA drops, e.g., due to RNA degradation via RIDD. However, GARYon polynucleotides can be recognized by the activated IRE1 nuclease, and the double stem loop in the UTR can be spliced. Thus, the result is not degradation of GARYon polynucleotides but a polynucleotide with an untouched ORF which can continue to be translated, allowing for maintenance of payload (e.g., protein) expression. In some embodiments, a splicing event also protects GARYon polynucleotides from degradation via RIDD.

[0136] In some embodiments, a GARYon polynucleotide comprises in 5’ to 3’ order: (a) the IRE-1 binding sequence, (b) the intervening sequence, (c) the AUG translation initiation codon, and (d) the payload sequence.

[0137] In some embodiments, a polynucleotide further comprises a 5’ UTR.

[0138] In some embodiments, a 5’ UTR comprises (a) the IRE-1 binding sequence, and (b) the intervening sequence.

[0139] In some embodiments, a 5’ UTR does not comprise (c) the AUG translation initiation codon.

[0140] In some embodiments, a polynucleotide is characterized such that when administered to a cell, tissue or subject, expression of the payload is observed at similar levels under physiological conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

[0141] In some embodiments, a polynucleotide is characterized such that when administered to a cell, tissue or subject, increased expression of the payload is observed under cellular stress conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence. In some embodiments, payload expression is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at Page 24 of 84 12871572v1Attorney Docket: 2012611-0180 least 80%, at least 90% or at least 95%. In some embodiments, payload expression is increased by about 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold.

[0142] In some embodiments, expression of a payload under cellular stress conditions is similar to or greater than expression of the payload under physiological conditions.

[0143] In some embodiments, cellular stress response is endoplasmic-reticulum (ER) stress. In some embodiments, ER stress comprises the unfolded protein response (UPR). In some embodiments, UPR activates IRE-1 nuclease activity.

[0144] In some embodiments, physiological conditions refers to a cellular condition in which a cell is not under ER stress.

[0145] In some embodiments, a GARYon polynucleotide is unspliced.

[0146] In some embodiments, a GARYon polynucleotide is spliced.

[0147] In some embodiments, a GARYon polynucleotide has a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polynucleotide sequence of SEQ ID NO:1.

[0148] In some embodiments, a GARYon polynucleotide has a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polynucleotide sequence of SEQ ID NO: 2. GARYswitch polynucleotides

[0149] In some embodiments, a polynucleotide disclosed herein is a GARYswitch polynucleotide. In some embodiments, in a GARYswitch polynucleotide, a double stem loop splice site along with one or more UAG, UAA and / or UGA codons are located within an ORF, in between the start codon and a sequence encoding a payload. See FIG.2. Under physiological conditions (e.g., in the absence of cellular stress), a ribosome will translate the start codon and the sequence encoded by the splice site, and then translation will be terminated at the stop codons, typically resulting in minimal expression (e.g., no detectable expression) of a payload; in some embodiments, low levels of expression of a payload can be observed due to stop codon readthrough and ribosome frame shifting. Upon UPR induction, the double stem loop in a GARYswitch RNA construct is spliced by IRE1, resulting in a frame shift that shifts the stop codons out of frame, and shifts the sequence encoding a payload in frame. The result is an induction of expression of a payload under stress (e.g., ER stress). In some embodiments, a splicing event also protects GARYswitch RNA from degradation via RIDD.

[0150] In some embodiments, a GARYswitch polynucleotide comprises in 5’ to 3’ order: (a) a AUG translation initiation codon, (b) an IRE-1 binding sequence, (c) a intervening sequence, and (d) a payload sequence.

[0151] In some embodiments, a polynucleotide further comprises a 5’ UTR.

[0152] In some embodiments, a 5’ UTR comprises does not comprise (a) the AUG translation initiation codon, (b) the IRE-1 binding sequence, and (c) the intervening sequence. Page 25 of 84 12871572v1Attorney Docket: 2012611-0180

[0153] In some embodiments, a polynucleotide is characterized such that when administered to a cell, tissue or subject, minimal (e.g., low), expression of the payload is observed under physiological conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

[0154] In some embodiments, a polynucleotide is characterized such that when administered to a cell, tissue or subject, increased expression of the payload is observed under cellular stress conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence. In some embodiments, payload expression is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. In some embodiments, payload expression is increased by about 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold.

[0155] In some embodiments, cellular stress response is endoplasmic-reticulum (ER) stress. In some embodiments, ER stress comprises the unfolded protein response (UPR). In some embodiments, UPR activates IRE-1 nuclease activity.

[0156] In some embodiments, physiological conditions refers to a cellular condition in which a cell is not under ER stress.

[0157] In some embodiments, a GARYswitch polynucleotide is unspliced.

[0158] In some embodiments, a GARYswitch polynucleotide is spliced.

[0159] In some embodiments, a GARYswitch polynucleotide has a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polynucleotide sequence of SEQ ID NO: 3.

[0160] In some embodiments, a GARYswitch polynucleotide has a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polynucleotide sequence of SEQ ID NO: 4.

[0161] In some embodiments, a GARYswitch polynucleotide has a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polynucleotide sequence of SEQ ID NO: 22.

[0162] In some embodiments, a GARYswitch polynucleotide has a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polynucleotide sequence of SEQ ID NO: 23.

[0163] In some embodiments, a GARYswitch polynucleotide has a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polynucleotide sequence of SEQ ID NO: 24.

[0164] Table 1: Sequences for exemplary polynucleotides comprising GARY sequence elements as disclosed herein. SEQ ID NO: Description Nucleotide Sequence E ID 1 ARY i h c g12871572v1Attorney Docket: 2012611-0180 SEQ ID NO: Description Nucleotide Sequence Nucleotide Sequence tgccaccATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGA Before Splicing CAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCA G C T C T A C G t tc c A A T T A A A G G C G ct c A G T TC C G A T G C C G AA a a A A G T A G G G G G A A caPage 27 of 84 12871572v1Attorney Docket: 2012611-0180 SEQ ID NO: Description Nucleotide Sequence cccgaatggagtctctaagctacataataccaacttacactttacaaaatgttgtcccccaaaa tgtagccattcgtatctgctcctaataaaaagaaagtttcttcacattct G A A C C A A C G T A G C g g G T A C C A A C G T A G C g g C G A A T T A A A G G C G ct clower case correspond to an untranslated region (UTR); nucleotide sequences in UPPER CASE correspond to an open reading frame, sequences in bold correspond to an IRE1 binding sequence, and nucleotide sequences in italics correspond to an intervening sequence. IRE-1 binding sequence Page 28 of 84 12871572v1Attorney Docket: 2012611-0180

[0166] Polynucleotides provided herein comprise one or more sequence elements that allow for expression of a payload under cellular stress conditions. In some embodiments, a sequence element that allows for expression of a payload under cellular stress conditions is an XBP1 sequence, or a fragment or a variant thereof, e.g., which can be bound and / or spliced by IRE-1. In some embodiments, a fragment or variant thereof is a functional fragment, a functional variant, or a functional fragment variant thereof.

[0167] In some embodiments, a polynucleotide disclosed herein comprising a GARY sequence element comprises an IRE-1 binding sequence.

[0168] In some embodiments, an IRE-1 binding sequence comprises a sequence that is recognized by a nuclease domain of IRE-1, e.g., a nuclease domain of activated IRE-1. As disclosed herein, IRE-1 can be activated by the unfolded protein response (UPR).

[0169] In some embodiments, an IRE-1 binding sequence is a polynucleotide sequence.

[0170] In some embodiments, an IRE-1 binding sequence is a polyribonucleotide sequence.

[0171] IRE-1 binding sequences are conserved among metazoan species (see, for example, Figure EV1 in Peschek J et al. “Conformational RNA zipper promotes intron ejection during non-conventional XBP1 mRNA splicing. EMBO Rep. 2015 Dec;16(12):1688-98. doi: 10.15252 / embr.201540955, which is incorporated herein by reference in its entirety). For example, as shown in FIG. 1A of Peschek 2015, XBP1 RNAs from different species have conserved residues that can be cleaved by IRE-1.

[0172] In some embodiments, an IRE-1 binding sequence is or comprises a CUGCAG sequence.

[0173] In some embodiments, an IRE-1 binding sequence is or comprises a CCGCAG sequence.

[0174] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence or a fragment or variant thereof.

[0175] Exemplary XBP1 sequences are provided below:

[0176] CAGAGACCGCAGCACTCAGTGATGATGCTCCTCTGCAGCAGGAGCA (SEQ ID NO: 5)

[0177] CAGAGACCGCAGCACUCAGUGAUGAUGCUCCUCUGCAGCAGGAGCA (SEQ ID NO: 30)

[0178] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 5 or 30, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 5 or 30.

[0179] ATGGGTCTGCTGAGTCCGCAGCACTCAGTAATAGTGCACCTCTGCAGCAGGGTGCAGGCCC (SEQ ID NO: 6)

[0180] AUGGGUCUGCUGAGUCCGCAGCACUCAGUAAUAGUGCACCUCUGCAGCAGGGUGCAGGCCC (SEQ ID NO: 31)

[0181] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 6 or 31, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 6 or 31. Page 29 of 84 12871572v1Attorney Docket: 2012611-0180

[0182] GGAGAGGAGAGTCCGCAGCACTCTCTAATAGTCCACCTCTGCAGCAGGGAGGAGGA (SEQ ID NO: 25)

[0183] GGAGAGGAGAGUCCGCAGCACUCUCUAAUAGUCCACCUCUGCAGCAGGGAGGAGGA (SEQ ID NO: 32)

[0184] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 25 or 32, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 25 or 32.

[0185] CGGGTCCGCTGAGTCCGCAGCACTCAGTAGTAGTGCCCCTCTGCAGCAGGAGCAGGCCCAGATGTTAGTAATAG (SEQ ID NO: 26)

[0186] CGGGUCCGCUGAGUCCGCAGCACUCAGUAGUAGUGCCCCUCUGCAGCAGGAGCAGGCCCAGAUGUUAGUAAUAG (SEQ ID NO: 33)

[0187] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 26 or 33, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 26 or 33.

[0188] CGGGTCCGCTGAGTCCGCAGCACTCAGTAGTAGTGTTCCTCTGCAGCAGGTGCAGGCCCAGCTGTTAGTAATAG (SEQ ID NO: 27)

[0189] CGGGUCCGCUGAGUCCGCAGCACUCAGUAGUAGUGUUCCUCUGCAGCAGGUGCAGGCCCAGCUGUUAGUAAUAG (SEQ ID NO: 34)

[0190] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 27 or 34, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 27 or 34.

[0191] GGGTCTGCTGAGTCCGCAGCACTCTAGTAGCGTGCACCTCTGCAGCAGGTGCAGGCCCATAGTAATAG (SEQ ID NO: 28)

[0192] GGGUCUGCUGAGUCCGCAGCACUCUAGUAGCGUGCACCUCUGCAGCAGGUGCAGGCCCAUAGUAAUAG (SEQ ID NO: 35)

[0193] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 28 or 35, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 28 or 35.

[0194] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence from one or more of the following species: Drosophila melanogaster, Caenorhabditis elegans, Canis lupus familiaris, Oryctolagus cuniculus, Xenopus laevis, Takifugu rubipres, Danio rerio, Saimiri boliviensis, Mus musculus, Homo sapiens, Pteropus Alecto, Mesocricetus auratus, Tursiops truncates, Nomascus leucogenys, Gorilla gorilla gorilla, Ovis aries, Macaca mulatta, Bos taurus, Papio Anubis, Pan paniscus, Pongo abelii, Pan troglodytes, Cricetus griseus, Sus scrofa, Rattus Page 30 of 84 12871572v1Attorney Docket: 2012611-0180 norvegicus, Gallus gallus, Pseudopodoces humilis, Zonotrichia albicollis, Chrysemis picta belli, Melopsittacus undulatus, Ficedula albicollis, Anas platyrhynchos, Alligator mississippiensis, and / or Alligator sinensis.

[0195] In some embodiments, an XBP1 sequence comprises one or more modifications, e.g., substitutions, deletions and / or insertions, to a naturally occurring XBP1 sequence. In some embodiments, one or more modifications comprises mutating one or more nucleotides to generate one or more stop codons, e.g., one or more internal stop codons in an XBP1 sequence.

[0196] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 36, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 36. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 36. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 36 to generate one or more stop codons.

[0197] CCACCAACCUUGGAUCUGCAGCAUCCAAAGCUGACCCUCUGCCGCAGGGUAUACAACAG (SEQ ID NO: 36)

[0198] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 37, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 37. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 37. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 37 to generate one or more stop codons.

[0199] GCCGUGCCUUUGAAUCAGCAGCAUUCAUUAAUGAGCCUCAGCAGUGGGAACAGGCCCGA (SEQ ID NO: 37)

[0200] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 38, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 38. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 38. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 38 to generate one or more stop codons.

[0201] CCGGGUCUGCUGAGUCCGCAGCACUCAGACUACGUGCAUCUGCAGCAGGUGCAGGCCCAG (SEQ ID NO: 38)

[0202] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 40, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 40. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 40. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 40 to generate one or more stop codons. Page 31 of 84 12871572v1Attorney Docket: 2012611-0180

[0203] CCGGGUCCGCUGAGUCCGCAGCACUCAGACUAUGUGCCCCUCUGCAGCAGGAGCAGGCCCAG (SEQ ID NO: 40)

[0204] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 41, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 41. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 41. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 41 to generate one or more stop codons.

[0205] UCGGGUCUUCUGAGUCCGCAGCACUCAGGCUACGUGUGCCUCCGCAGCAGGUGCAGGCCCAG (SEQ ID NO: 41)

[0206] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 42, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 42. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 42. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 42 to generate one or more stop codons.

[0207] CCGGGUCUUCUGAGUCCGCAGCACUCAGGCUACGUGUGCCUCCGCAGCAGGUGCAGGCCCAG (SEQ ID NO: 42)

[0208] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 43, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 43. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 43. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 43 to generate one or more stop codons.

[0209] CCGGGUCUGCUGAGUCCGCAGCACUCAGACUACGUGCACCUCUGCAGCAGGUGCAGGCCCA (SEQ ID NO: 43)

[0210] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 44, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 44. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 44. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 44 to generate one or more stop codons.

[0211] CCGGGUCUGCUGAGUCCGCAGCACUCAGACUAUGUGCACCUCUGCAGCAGGUGCAGGCCCAG (SEQ ID NO: 44)

[0212] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 45, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 Page 32 of 84 12871572v1Attorney Docket: 2012611-0180 sequence provided in SEQ ID NO: 45. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 45. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 45 to generate one or more stop codons.

[0213] CCGGGUCUGCUGAGUCCGCAGCACUCAGACUACGUGCACCUCUGCAGCAGGUGCAGGCCCAG (SEQ ID NO: 45)

[0214] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 46, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 46. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 46. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 46 to generate one or more stop codons.

[0215] CCGGGUCUGCUGAGUCCGCAGCACUCAGACUACGUGCGCCUCUGCAGCAGGUGCAGGCCCAG (SEQ ID NO: 46)

[0216] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 47, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 47. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 47. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 47 to generate one or more stop codons.

[0217] CCGGGUCCGCUGAGUCCGCAGCACUCAGACUACGUGUUCCUCUGCAGCAGGUGCAGGCCCAG (SEQ ID NO: 47)

[0218] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 48, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 48. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 48. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 48 to generate one or more stop codons.

[0219] CCGGGUCGGCUGAGUCCGCAGCACUCAGACUACGUGCUCCUCUGCAGCAGGCGCAGGCCCAG (SEQ ID NO: 48)

[0220] In some embodiments, an IRE-1 binding sequence comprises an XBP1 sequence comprising the nucleotide sequence provided in SEQ ID NO: 49, or a sequence with at least 85% identity thereto. In some embodiments, an IRE-1 binding sequence comprises at least 5, 10, 15, 20, or 50 contiguous nucleotide sequences of an XBP1 sequence provided in SEQ ID NO: 49. In some embodiments, an XBP1 sequence comprises a nucleotide sequence having one or more modifications, e.g., substitutions, deletions and / or insertions, relative to SEQ ID NO: 49. In some embodiments, one or more modifications comprises mutating one or more nucleotides in SEQ ID NO: 49 to generate one or more stop codons. Page 33 of 84 12871572v1Attorney Docket: 2012611-0180

[0221] CCGGGUCCGCUGAGUCCGCAGCACUCAGACUACGUGCUCCUCUGCAGCAGGCGCAGGCCCAG (SEQ ID NO: 49)

[0222] In some embodiments, a polynucleotide comprises a sequence that forms a double stem loop structure. In some embodiments, a double stem loop structure is similar to a double stem loop structure found in XBP1 mRNA. In some embodiments, a double stem loop structure may be recognized and spliced by IRE1.

[0223] In some embodiments, IRE-1 binds to an IRE-1 binding sequence under cellular stress conditions. In some embodiments, an IRE-1 binding sequence is part of a larger sequence that can form a double stem loop structure.

[0224] In some embodiments, recognition by IRE-1 comprises binding of IRE-1 to an IRE-1 binding sequence. In some embodiments, binding of IRE-1 to an IRE-1 binding sequence results in splicing of a sequence comprising an IRE-1 binding sequence. In some embodiments, splicing removes one or more nucleotides in a polynucleotide, e.g., in an IRE-1 binding sequence. In some embodiments, splicing results in a frameshift in a sequence downstream to a sequence that is spliced.

[0225] In some embodiments, splicing occurs in the cytoplasm.

[0226] In some embodiments, splicing prevents degradation of a polynucleotide, e.g., via RIDD.

[0227] In some embodiments, splicing allows for translation of a sequence encoded by a polynucleotide.

[0228] In some embodiments, a frameshift is in an intervening sequence.

[0229] In some embodiments when a polynucleotide comprises a GARYon sequence element, a frameshift results in the one or more UAG, UAA and / or UGA codons being out of frame such that the one or more UAG, UAA and / or UGA codons are not recognized by one or more release factors. In some embodiments, a frameshift results in a payload expression sequence being in frame for translation such that a payload sequence is translated into a polypeptide. Modified ribonucleotides: Ac4C and 5-hmU

[0230] Polynucleotides disclosed herein can be a polyribonucleotide which further comprises one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0231] In some embodiments, a polynucleotide is a polyribonucleotide. In some embodiments, a polyribonucleotide comprises one or more modified ribonucleotides. In some embodiments, a polyribonucleotide comprises a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0232] In some embodiments, a polyribonucleotide comprises one or more modified nucleosides or nucleobases.

[0233] In some embodiments, a modified ribonucleotides comprises an N4-acetylcytidine nucleoside. In some embodiments, the nucleoside has the following structure: Page 34 of 84 12871572v1Attorney Docket: 2012611-0180 O H3C NH N O .

[0234] In some embodiments, a modified rib prises a 5-hydroxymethyluridine nucleoside. In someembodiments, the nucleoside has the following structure: OH O NH O .

[0235] In some embodiments, a modified riba structure provided in Table 2A. In some embodiments, a modified ribonucleotide has a structure provided in Table 2B when incorporated in a polynucleotide. In some embodiments, a polynucleotide (e.g., a polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 2B. Page 35 of 84 12871572v1:etahpsohpirtenidityclyteca-4N :etahpsohN N H pnO idO OidC O HirH O O H e3OulO niHydihtOte:O ycHelyOPOmytH a4thOPO8eOxO c HorpHfoa- OPO dsyo OPO 64 N O Hhh-p5iO3d Hega:PetahpsohpoO noH Nenidi HO m N N H Or NenOui Oly:eO N H diC t3HhttaO H O y H OemhpO H c O sleytOy sHxdoohO ietcaOPOrdpo OH POoe- l4OyHhn-oOcN 5mHunobi0r81 d0 ei-fi1d1o:62meni10yr2 adi:l :Orut p enlemkeiH O d NyN i HhcxN N HteoEtyO N H clO OmyO1D:yO H OvyeAtC3HxoO H27n2ecH OrOrad51oetltb -y4OhO7AaT NH -5H821:etahpsohpirtenidityclyteca-4N :etahpsohpidH3OruO l O )eendiiOyhtdoitelycc O-tO PO e:e- lmyta OPO 48 uytO -xn eohpO -foo c OPOrbdso OPO 7iar-y4Oyhh-piO. 3lN 5 d eeodpitgaa: o Potetelcn i ah und pets obaoirhrtoppornOenooNiH Oneccnm H N N HdiNarjdi eO Oul :O N H OanneiC Oyhehdi ttH O OnawtH3OeahOo(ycslmp teytOy soOtneO-xPorhp- PeditcoaOe- dyonO Omhlc4Oh oOcuN-5mattnafoobin0r8oi1 detis0i-fio1dp1o:6 e eh2mni1dt0yr ir s2 al :OuOet:t p enliNyhHaNciemke diHcx tN N HtO N HdnoE ycO Oem Oi1D: lyytC3OyxH O Ov2eBn2ecH OorO7d5r 1oetl a-ytb4OhO:e 7AaT N-5 to8N21Attorney Docket: 2012611-0180

[0237] In some embodiments, a polyribonucleotide comprises cytidine nucleosides. In some embodiments, at least 5% of cytidine nucleosides in a polyribonucleotide comprise N4-acetylcytidine. In some embodiments, less than 100% of cytidine nucleosides in a polyribonucleotide comprise N4-acetylcytidine. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of cytidine nucleosides in a polyribonucleotide comprise N4- acetylcytidine.

[0238] In some embodiments, a polyribonucleotide comprises uridine nucleosides. In some embodiments, at least 5% of uridine residues in a polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, less than 100% of uridine nucleosides in a polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of uridine nucleosides in a polyribonucleotide comprise 5- hydroxymethyluridine. In some embodiments, more than 60% of uridine nucleosides in a polyribonucleotide comprise 5-hydroxymethyluridine.

[0239] In some embodiments, a polyribonucleotide disclosed herein comprises one or more modified ribonucleotides comprises a nucleoside comprising: N4-acetylcytidine, 5-hydroxymethyluridine, N1- methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5-methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl- cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (m1 I), wyosine (imG), methylwyosine (mimG), 5-hydroxycytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5- methyluridine, 5,6-dihydro-5-methyluridine, 2’-O-methyluridine, 2’-O-methyl-5-methyluridine, 2’-fluoro-2’- deoxyuridine, 2’-amino-2’-deoxyuridine, 2’-azido-2’-deoxyuridine, 4-thiouridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5- fluorouridine, pseudouridine, 2’-O-methyl-pseudouridine, N1-hydroxypseudouridine, 2’-O-methyl-N1- methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, ara-uridine, N6-methyladenosine, 2- aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 8-oxoadenosine, thienoguanosine, 7-deazaguanosine, 8- oxoguanosine, 6-O-methylguanine, or any combination thereof. Modified ribose

[0240] Polynucleotides disclosed herein can be a polyribonucleotide which further comprises one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof. Page 38 of 84 12871572v1Attorney Docket: 2012611-0180

[0241] In some embodiments, a polynucleotide is a polyribonucleotide. In some embodiments, a polyribonucleotide comprises one or more modified ribonucleotides. In some embodiments, a modified ribonucleotide comprises a modified ribose.

[0242] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose. In some embodiments, a modified ribonucleotide comprises a structure of: X R O O 3 ,

[0243] (a) wherein R is a monophosphateriphosphate; and

[0244] (b) wherein X is an adenine nucleobase, a guanine nucleobase, a cytosine nucleobase (e.g., N4- acetylcytosine), or a uracil nucleobase (e.g., 5-hydroxymethyluracil).

[0245] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and an adenine nucleobase.

[0246] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a guanine nucleobase.

[0247] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a cytosine nucleobase.

[0248] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a N4- acetylcytidine nucleoside.

[0249] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a uracil nucleobase.

[0250] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a 5- hydroxymethyluridine nucleoside.

[0251] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a N1- methylpseudouracil nucleobase.

[0252] In some embodiments, a modified ribonucleotide comprises a structure provided in Table 2C. In some embodiments, a modified ribonucleotide comprises a structure provided in Table 2D when incorporated in a polynucleotide. In some embodiments, a polynucleotide (e.g., a polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 2D. Page 39 of 84 12871572v1a- Oo-hOPOa- ’piOOo-ro OPO’dh0ypiO42dH2 h dHegaePnidireunliydi httyclOem O yteH N O H3 yH x N O H3cN N CoN C -: rO a O Od:O 4etOyh etH O NahC3H -ahO H ly psH O5lpO s stee o yh Ote oO dca pHcahpH it- o OPO - o OPOoe Ol-noOO-noO c’2m H’2m H unobi0r81 d :0 eie-f1 indid16 o it2my1cl :0yryteeni2 a:lt pcaOdirH Oem - N O H3 uke4HlN O H3C -yCcxNN N5htO NoElyO OlyeO1D:yeCtecC3O HtecmyH O O Hv27n2aH Oa xoO5r el- O - Ord 1ottb -’O -’ yO78AaT 2H2 hH21enidiruodO uesplyhtem-1-l eyttaechap-soO-h’p2irtenidiruoduesO plyhtem-1-lyetteachap-sOo-h’p2idenidirhuposdouhepspOoO O l H O NnoH O32 O3yN H H N H htO m e O N CeC : N N e OnOniO m-et N1-aOiOdlhHdipOrHiOtetO yahH sul clpO ytytysecoO OtoO ah-pOo OHePO caOHePO cahpOH PO-n- - o’oOO-’OO-noO 2 m H2H’2m H eni08di1r0u-o1d1u6e2s1pl0 yh: :2:tt eH O OeneiH OeO O3 niO3km- Nc 1OdiN Hdr i 2 H N H O N CtyN N Co -lOulOclO1D ytN OytOytOvy ee cH OecHecH27n a aOaO5r - - - 1otOt-’OOH -’OOH -’O7H8A 2 2 221:etahpH soNhpO irtenisonauglyteca- O-’23 H N e O OHdN O niNCsN OeniO H o OsO n H Ooanu: edO gl eOaH ytteaHlPyOPO 48 chO O tpeOfa sO o HcaOH PO o-O-hOPO - 2’pi O O- O42d H’2HegaP2 H N H eN NNnNiO3enN O H3son :O H e Cis 2:HNC t NNOoneN O auagl hOetytpHdaO H s Oal hpsO eco yah t-pOecohO OoHa pnOPO -OoH nOPO-’o2O- o2O m H’m H0810-11622 :H1 e0 nN:2isHeni:to Nen N sNoN3O O H3nN O 2 HkauN CedH N CcoglyN OalN O1Dtyy eOteO Hv2e cHn acO7r -Oa 5tO- O1ot-’O H -’O7H8A 2 221cul etOPOte OPOfn ylt ayt eta oobierchapO sO- POecmayhO xps-3P4loOo O Oy - o -p -h’pOO-rodh ep Oga2id’2yhidaPotni detear niodpirtoyccO lnyO3H O3H N HeiteN N CniN O H d O N C nca :eO O iru:eO eh-w4 t(NahC OlH3O -5 ytH haO O thO sleytps lye ohOt eps-e myohO ditcpoa- o OPO ca x pn- oronO- POel O-’oOO-’d oO cu2m2yhm nobi0r81 d0 e:i-f eini1d1odi6t2my1c :0ylry eateni2l:t pcOdO3 irH O3ema-ke4H N HulN O H N N C -yhO N CcxoE NlO O5ltD:yt eO1C OytmH OvyeD ec 3Oec yO O27n2aHr -a-xor 51oetltbO-’OO- dO7AaT 2’2yh 821:etaO hpsohpirtenidirulyteca- O-’2:etahpsoO hpidenidirulyteca- O-’2N H N H O ONN O H3N32 O H3O O H C H CenC e O NeN Nin: sN e Oni :eOo:N O iditOdra itOnetO ul hOty a auaO ytps cl hytpO sgl hytpseoahOe ohOe oO c-p- Pcp-c hp- P Oo-nO OaOPOaO O’oO - Oo-n’oO - Oo-noO 2 m2m’2m0810-121H6N2 :1 eH02:e: n:nOeisNNiH N O H3 niO 2 N O H3 onO O H3tekdicrO N CdityH N N CauN C NoulOclOglO1D ytOytOytOvy ee cOecOecO27n a a a 5r - - - 1otOt-’OO- OO- O7A 2’2’2 821N H2enisooned:al eyttaechap-soO-h’p2irtN H2N C O N O e N O n O i OesOniO odniredal:O -uO4ye-1o-de O- PO8ttea OPO lchOyute t fo-es aphO -5ap-sOo OPO calypsOPO. 4-h- hto ed e’pOO- ehpOitg2id’2mid oael PcunobirtNnNe3 OcaeN O H H Ojni2CeNdsH Nniaon :N e OdiO reNOnaetaOutOodO -oal Otah1y-dh ttps lyuete psnecoahO -pO-esPO cpaloyhO pO- m POhcOo-n’oO - Oh-toenoOatt2m’2m mafono0i8ti1s0o-p11e6ht2 :1 e0 ne se2isNNni:dttoenN O H3 irH OacN Oidke2cdH N C -uoOnioalN O1y-lduN O1Dty eOyteOvOese cp 2n ac lr -a-yO7:5otOht E1t-’OO- eO7A 2’2mTO8N21Attorney Docket: 2012611-0180

[0253] In some embodiments, at least 5% of ribose moieties of a polyribonucleotide are 2’-O-acetylated.

[0254] In some embodiments, about 5% to about 99% of the ribose moieties of a polyribonucleotide are 2’-O- acetylated.

[0255] In some embodiments, a polyribonucleotide disclosed herein comprises a cap structure and the cap structure does not comprise a 2’-O-acetylated ribose.

[0256] In some embodiments, a polyribonucleotide disclosed herein comprises a cap structure and the cap structure comprises a 2’-O-acetylated ribose.

[0257] In some embodiments, a polyribonucleotide disclosed herein further comprises one or more ribonucleotides that does not comprise a 2’-O acetylated ribose. Cap structures

[0258] In some embodiments of any of the polyribonucleotides disclosed herein, a polyribonucleotide comprises a cap structure.

[0259] Prior versions of RNA capping have been described, e.g., in Decroly E et al. (2012) Nature Reviews 10: 51- 65; and in Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511–7526, the entire contents of each of which is hereby incorporated by reference. 5’ caps include a Cap-0 (also referred herein as “Cap0”), a Cap-1 (also referred herein as “Cap1”), or Cap-2 (also referred herein as “Cap2”). See, e.g., Figure 1 of Ramanathan A et al., and Figure 1 of Decroly E et al.

[0260] The term “5'-cap” as used herein refers to a structure found on the 5'-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5'-triphosphate linkage (also referred to as Gppp or G(5')ppp(5')).

[0261] In some embodiments of a polyribonucleotide disclosed herein, the 5’ end of the polyribonucleotide comprises a compound of formula I: HO OR2O R1Page 46 of 84 12871572v1Attorney Docket: 2012611-0180 I or a salt thereof, wherein: indicates the position at which the compound is attached to the polyribonucleotide; each of B1and B2is independently selected from a natural base and a modified base; R1is selected from hydrogen, C1-6alkyl, and -C(=O)CH3; R2is hydrogen or -CH3; and X is O or S.

[0262] As defined generally above for formula I, each of B1and B2is independently selected from a natural base and a modified base. In some embodiments, B1is a natural base. In some embodiments, B1is adenine. In some embodiments, B1is cytosine. In some embodiments, B1is guanine. In some embodiments, B1is uracil.

[0263] In some embodiments, B1is a modified base. In some embodiments, B1is selected from N1- methylpseudouracil, a pyridin-4-one, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil, 5-methyl cytosine, 5- aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine, 5-formyl-cytosine, N4-methyl-cytosine, a 2- amino-purine, a 2,6-diaminopurine, a 2-amino-6-halo-purine, a 6-halo-purine, hypoxanthine, 1-methyl-hypoxanthine, 4,6-dimethyl-3,3a,4,9a-tetrahydro-9H-imidazo[1,2-a]purin-9-one, 4,6,7-trimethyl-3,3a,4,9a-tetrahydro-9H- imidazo[1,2-a]purin-9-one, N4-acetylcytosine, and 5-hydroxymethyluracil.

[0264] In some embodiments, B2is a natural base. In some embodiments, B2is adenine. In someembodiments, B2is cytosine. In some embodiments, B2is guanine. In some embodiments, B2is uracil.

[0265] In some embodiments, B2is a modified base. In some embodiments, B2is selected from N1- methylpseudouracil, a pyridin-4-one, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil, 5-methyl cytosine, 5- aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine, 5-formyl-cytosine, N4-methyl-cytosine, a 2- amino-purine, a 2,6-diaminopurine, a 2-amino-6-halo-purine, a 6-halo-purine, hypoxanthine, 1-methyl-hypoxanthine, 4,6-dimethyl-3,3a,4,9a-tetrahydro-9H-imidazo[1,2-a]purin-9-one, 4,6,7-trimethyl-3,3a,4,9a-tetrahydro-9H- imidazo[1,2-a]purin-9-one, N4-acetylcytosine, and 5-hydroxymethyluracil.

[0266] As defined generally above for formula I, R1is selected from hydrogen, C1-6alkyl, and -C(=O)CH3. In some embodiments, R1is hydrogen. In some embodiments, R1is C1-6alkyl. In some embodiments, R1is -CH3. In some embodiments, R1is -C(=O)CH3.

[0267] As defined generally above for formula I, R2is hydrogen or -CH3. In some embodiments, R2is hydrogen. In some embodiments, R2is - CH3.

[0268] As defined generally above for formula I, X is O or S. In some embodiments, X is O. In some embodiments, X is S.

[0269] In some embodiments, the present disclosure provides a compound of any of formulae I-a, I-b, I-c, I-d, I- e, and I-f: Page 47 of 84 12871572v1Attorney Docket: 2012611-0180 HO OHPage 48 of 84 12871572v1Attorney Docket: 2012611-0180 I-c12871572v1Attorney Docket: 2012611-0180 or a salt thereof, wherein each of B1and B2is as defined above and described herein.

[0270] In some embodiments, a compound of formula I is selected from HO OHNH2N N O O O or a salt thereof.

[0271] In some embodiments, a polyribonucleotide disclosed herein comprises at the 5’ end am7GpppN1pN2-OH cap with the following structure: O HN e 2 .Page 50 of 84 12871572v1Attorney Docket: 2012611-0180

[0272] In some embodiments, a polyribonucleotide disclosed herein comprises at the 5’ end a compound with the following structure: O HN N B 1 e 2 , wherein inditide.

[0273] In some embodiments, a polyribonucleotide disclosed herein comprises am7GpppN1(2’-OMe)pN2-OH cap with the following structure: O HN e 2 .

[0274] In someembodiments, a polyribonucleotide disclosed herein comprises at the 5 end a compound with the following structure: Page 51 of 84 12871572v1Attorney Docket: 2012611-0180 O HN N Base 1 e 2 , wherein inditide.

[0275] In some embodiments, a polyribonucleotide disclosed herein comprises am7GpppN1(2’-OMe)pN2(2’-OMe)-OH cap with the following structure: O HN e 2 .

[0276] In some, p y p d a compound with the following structure: Page 52 of 84 12871572v1Attorney Docket: 2012611-0180 O HN N Base 1 e 2 wherein indi tide.

[0277] In some embodiments, for any of the cap structures disclosed herein, each of B1and B2is independently selected from a natural base and a modified base. In some embodiments, B1is a natural base. In some embodiments, B1is adenine. In some embodiments, B1is cytosine. In some embodiments, B1is guanine. In some embodiments, B1is uracil.

[0278] In some embodiments, B1is a modified base. In some embodiments, B1is selected from N1- methylpseudouracil, a pyridin-4-one, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil, 5-methyl cytosine, 5- aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine, 5-formyl-cytosine, N4-methyl-cytosine, a 2- amino-purine, a 2,6-diaminopurine, a 2-amino-6-halo-purine, a 6-halo-purine, hypoxanthine, 1-methyl-hypoxanthine, 4,6-dimethyl-3,3a,4,9a-tetrahydro-9H-imidazo[1,2-a]purin-9-one, 4,6,7-trimethyl-3,3a,4,9a-tetrahydro-9H- imidazo[1,2-a]purin-9-one, N4-acetylcytosine, and 5-hydroxymethyluracil.

[0279] In some embodiments, B2is a natural base. In some embodiments, B2is adenine. In some embodiments, B2is cytosine. In some embodiments, B2is guanine. In some embodiments, B2is uracil.

[0280] In some embodiments, B2is a modified base. In some embodiments, B2is selected from N1- methylpseudouracil, a pyridin-4-one, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil, 5-methyl cytosine, 5- aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine, 5-formyl-cytosine, N4-methyl-cytosine, a 2- amino-purine, a 2,6-diaminopurine, a 2-amino-6-halo-purine, a 6-halo-purine, hypoxanthine, 1-methyl-hypoxanthine, 4,6-dimethyl-3,3a,4,9a-tetrahydro-9H-imidazo[1,2-a]purin-9-one, 4,6,7-trimethyl-3,3a,4,9a-tetrahydro-9H- imidazo[1,2-a]purin-9-one, N4-acetylcytosine, and 5-hydroxymethyluracil. Payloads

[0281] Polynucleotides disclosed herein can comprise a sequence encoding one or more payloads.

[0282] In some embodiments, a polynucleotide comprises a coding region.

[0283] In some embodiments, a coding region encodes a payload.

[0284] In some embodiments, a payload is or comprises a polypeptide. Page 53 of 84 12871572v1Attorney Docket: 2012611-0180

[0285] In some embodiments, a polyribonucleotide comprises an RNA payload.

[0286] In some embodiments, a payload is an antigen, an enzyme, an antibody or a fragment thereof, a secreted protein, a cell surface protein, or a helper protein, or a fragment or variant of any of the foregoing. In some embodiments, a payload is an antigen, an enzyme, an antibody or a fragment thereof, a secreted protein, a cell surface protein, or a helper protein, or a functional fragment or functional variant of any of the foregoing.

[0287] In some embodiments, a payload is an antigen or a fragment or variant thereof. In some embodiments, a payload is an antigen or an immunogenic fragment or an immunogenic variant thereof.

[0288] In some embodiments, a payload is a helper protein. In some embodiments, a helper protein is a chaperone or ER guardian polypeptide or a fragment or variant thereof. In some embodiments, a helper protein is a chaperone or ER guardian polypeptide or a functional fragment or functional variant thereof.

[0289] A person of skill in the art will appreciate, upon reading this disclosure, that chaperone proteins improve exogenous protein expression by aiding nascent polypeptides in proper folding and protect folding peptides from degradation.

[0290] TAPT1 is the mammalian homolog of yeast Emp65, a multi-pass transmembrane protein that forms an ER membrane-associated complex with Slp1 (mammalian homolog SUCO) that is highly conserved, ubiquitously expressed, and essential (Friederichs et al.). This complex acts as a chaperone protein, protecting folding polypeptides from degradation (Zhang et al.). Specifically, TAPT1 sits in the ER membrane and interacts with SUCO, which projects into the ER lumen and binds nascent unfolded proteins via its SUN domain, protecting them from being shuttled into ER-associated degradation (ERAD) pathways, and providing time for them to fold properly. When this complex is knocked out, approximately 20-30% of newly synthesized proteins that could otherwise fold are degraded. Without wishing to be bound by theory, these proteins significantly influence proteostasis and the delicate balance between protein folding and degradation.

[0291] In some embodiments, a chaperone protein is or comprises TAPT1, or a variant or fragment thereof. In some embodiments, a chaperone protein is or comprises TAPT1, or a functional variant or functional fragment thereof. In some embodiments, a TAPT1 polypeptide has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 9.

[0292] SEQ ID NO: 9

[0293] MAGVGDAAAPGEGGGGGVDGPQRDGRGEAEQPGGSGGQGPPPAPQLTETLGFYESDRRRERRRGRTELSLLRFLSAE LTRGYFLEHNEAKYTERRERVYTCLRIPRELEKLMVFGIFLCLDAFLYVFTLLPLRVFLALFRLLTLPCYGLRDRRLLQPAQVCDILKGVI LVICYFMMHYVDYSMMYHLIRGQSVIKLYIIYNMLEVADRLFSSFGQDILDALYWTATEPKERKRAHIGVIPHFFMAVLYVFLHAILIM VQATTLNVAFNSHNKSLLTIMMSNNFVEIKGSVFKKFEKNNLFQMSNSDIKERFTNYVLLLIVCLRNMEQFSWNPDHLWVLFPDVCM VIASEIAVDIVKHAFITKFNDITADVYSEYRASLAFDLVSSRQKNAYTDYSDSVARRMGFIPLPLAVLLIRVVTSSIKVQGILSYACVILF YFGLISLKVLNSIVLLGKSCQYVKEAKMEEKLSNPPATCTPGKPSSKSQNKCKPSQGLSTEENLSASITKQPIHQKENIIPLLVTSNSDQ FLTTPDGDEKDITQDNSELKHRSSKKDLLEIDRFTICGNRID Page 54 of 84 12871572v1Attorney Docket: 2012611-0180

[0294] In some embodiments, a TAPT1 nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the nucleic acid sequence of SEQ ID NO: 10.

[0295] SEQ ID NO: 10:

[0296] ATGGCTGGAGTTGGAGATGCTGCTGCTCCTGGAGAAGGCGGAGGAGGCGGAGTGGACGGTCCTCAACGGGATGGG CGCGGTGAAGCAGAACAACCTGGTGGTAGCGGCGGCCAGGGACCTCCTCCTGCACCCCAGCTGACCGAAACACTGGGATTCTAT GAAAGCGATAGGCGCCGTGAGAGGCGACGAGGGAGAACTGAGCTGTCCCTGCTCCGGTTTCTCTCAGCGGAACTTACCCGCGG TTACTTCTTAGAACATAATGAAGCTAAATATACTGAGCGGCGTGAGAGAGTTTATACTTGCCTGAGAATCCCACGCGAGCTCGAG AAACTGATGGTGTTTGGGATCTTTCTGTGTTTAGATGCATTCCTTTATGTATTCACCCTGCTCCCTCTTCGCGTGTTTCTTGCGC TGTTTCGGTTACTCACGCTGCCCTGCTACGGCCTTAGAGACCGGCGCCTGCTCCAGCCAGCCCAGGTTTGCGATATATTAAAAG GTGTGATTCTGGTCATCTGCTACTTTATGATGCACTATGTCGATTATTCTATGATGTATCATCTGATCCGGGGCCAAAGCGTCAT TAAACTGTACATAATATATAATATGCTGGAAGTGGCGGATAGACTGTTCTCCTCATTTGGGCAAGATATCTTGGACGCGCTGTAT TGGACGGCAACAGAACCCAAAGAGCGGAAACGCGCACATATCGGCGTGATACCCCATTTCTTCATGGCGGTACTTTACGTATTC CTGCATGCCATCTTAATTATGGTGCAGGCAACTACTTTAAACGTGGCCTTCAACAGCCATAACAAATCTCTCCTGACCATCATGA TGAGTAACAATTTTGTCGAGATTAAGGGGTCTGTGTTCAAGAAGTTCGAGAAGAACAATCTGTTTCAAATGTCAAATTCTGATAT AAAAGAACGCTTTACTAACTATGTCCTGTTACTGATAGTGTGTCTCCGGAACATGGAACAATTCAGCTGGAATCCAGATCATCTC TGGGTTTTATTTCCTGATGTATGCATGGTTATAGCATCCGAGATCGCCGTTGATATCGTTAAACATGCATTTATAACGAAATTCA ATGACATCACTGCTGACGTTTACTCCGAGTACCGCGCCAGCCTGGCGTTCGACTTAGTAAGTTCTCGGCAGAAGAACGCCTACA CTGACTACTCCGATTCCGTGGCAAGAAGGATGGGCTTCATTCCCCTTCCATTGGCTGTGTTGCTTATTAGAGTTGTGACCTCTTC AATCAAGGTGCAAGGTATCCTTAGTTACGCGTGTGTTATACTGTTTTACTTTGGCCTGATTAGTCTTAAAGTGTTAAATTCTATA GTTTTGCTTGGAAAGAGCTGCCAATATGTGAAAGAAGCCAAAATGGAAGAGAAACTTTCAAACCCACCCGCTACTTGCACACCTG GGAAGCCTTCAAGCAAATCTCAAAATAAGTGTAAGCCGTCTCAGGGCCTGTCTACAGAAGAGAACCTGAGCGCATCAATCACAAA ACAACCCATTCATCAAAAGGAGAACATAATCCCCCTGTTGGTGACATCAAATAGTGATCAATTCCTGACTACTCCCGACGGAGAC GAGAAGGATATAACCCAAGATAATTCCGAGCTGAAACACCGTAGTAGCAAGAAAGATCTGCTCGAGATTGACAGGTTTACTATTT GTGGAAATAGGATCGATTGATAATAA

[0297] In some embodiments, a chaperone protein is or comprises a heat shock protein (e.g., HSP70) family. In some embodiments, a chaperone protein is or comprises HSPA13, or a variant or fragment thereof. In some embodiments, a chaperone protein is or comprises HSPA13, or a functional variant or functional fragment thereof.

[0298] In some embodiments, a helper protein is a co-chaperone protein. In some embodiments, a co-chaperone protein described herein is a HSP40 family member. In some embodiments, a chaperone protein is or comprises DNAJC8, or a variant or fragment thereof. In some embodiments, a chaperone protein is or comprises DNAJC8, or a functional variant or functional fragment thereof. Payloads comprising Sbi domains

[0299] Among other things, the present disclosure provides payloads that are fusion polypeptides. In some embodiments, a polynucleotide encodes a fusion polypeptide payload. In some embodiments, a fusion polypeptide comprises (a) an antigen or an immunogenic fragment or an immunogenic variant thereof; and (b) a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus. In some Page 55 of 84 12871572v1Attorney Docket: 2012611-0180 embodiments, an Sbi complement C3d-binding polypeptide comprises Sbi domain III and / or Sbi domain IV. Exemplary SBi domain III and Sbi domain IV polypeptide sequences are provided herein.

[0300] S. aureus binder of immunoglobulin (Sbi) is an exemplary polypeptide which can bind complement C3d (as described in Clark et al. (2011) Mol Immunol. 48(4): 452–462, the entire contents of which is incorporated herein by reference). Sbi comprises two immunoglobulin binding domains (Domains I and II) and two complement C3d binding domains (Domains III and IV). Sbi domains III and IV can bind C3d (in native C3, iC3b and C3dg) and can result in fluid phase consumption of C3 via activation of the alternative pathway (see Clark et al 2011). It has also been shown that Sbi can be secreted and is involved in S. aureus immune evasion (Burman et al., 2008 J. Biol. Chem; 283:17579–17593).

[0301] Without wishing to be bound by theory, it is believed that in some embodiments, a complement C3d- binding polypeptide from Sbi of S. aureus can be used as an adjuvant to enhance and / or modulate an immune response from an antigen described herein. In some embodiments, the immune response is elicited by an antigen, disclosed herein. In some embodiments, the immune response is elicited by a component of Sbi of S. aureus.

[0302] Methods of generating and using a fusion polypeptide comprising an Sbi domain III and / or IV is described in US Patent No. 11,771,758, which is incorporated by reference in its entirety.

[0303] In some embodiments, any of the fusion polypeptides, fusion nucleotides, compositions, methods or uses disclosed herein, comprises a complement C3d-binding polypeptide of Sbi of S. aureus. In some embodiments, a Sbi complement C3d binding polypeptide comprises one or both of domain III and domain IV of Sbi, or a functional fragment or a functional variant thereof.

[0304] In some embodiments, a domain III of Sbi from S. aureus strain Mu50 is provided herein as SEQ ID NO: 20.

[0305] IENADKAIKDFQDNKAPHDKSAAYEANSKLPKDLRDKNNRFV

[0306] In some embodiments, a domain IV of Sbi from S. aureus strain Mu50 is provided herein as SQ ID NO: 21.

[0307] EKVSIEKAIVRHDERVKSANDAISKLNEKDSIENRRLAQREVNKAPMDVKEHLQKQLD

[0308] In some embodiments, a Sbi complement C3d binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, a Sbi complement C3d binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, a Sbi complement C3d binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 20.

[0309] In some embodiments, a Sbi complement C3d binding polypeptide is encoded by a polynucleotide which encodes an amino acid having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at Page 56 of 84 12871572v1Attorney Docket: 2012611-0180 least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, a Sbi complement C3d binding polypeptide is encoded by a polynucleotide which encodes an amino acid having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, a Sbi complement C3d binding polypeptide is encoded by a polynucleotide which encodes an amino acid having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 21. Due to degeneracy in the genetic code, those of ordinary skill in the art would understand that other DNA sequences (including codon-optimized sequences) could encode these polypeptides, as well as the others disclosed herein.

[0310] In some embodiments, a payload comprises a fusion polypeptide comprising an antigen or a fragment or variant thereof, and a complement C3d-binding polypeptide comprising an Sbi domain III. In some embodiments, an Sbi domain III comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, an Sbi domain III comprises the amino acid sequence of SEQ ID NO: 20.

[0311] In some embodiments, a payload comprises a fusion polypeptide comprising an antigen or a fragment or variant thereof, and a complement C3d-binding polypeptide comprising an Sbi domain IV. In some embodiments, an Sbi domain IV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, an Sbi domain IV comprises the amino acid sequence of SEQ ID NO: 21.

[0312] In some embodiments, a payload comprises a fusion polypeptide comprising an antigen or a fragment or variant thereof, and a complement C3d-binding polypeptide comprising an Sbi domain III and an Sbi domain IV. In some embodiments, an Sbi domain III comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, an Sbi domain III comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, an Sbi domain IV comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, an Sbi domain IV comprises the amino acid sequence of SEQ ID NO: 21. Compositions and pharmaceutical compositions

[0313] Among other things, provided herein are one or more compositions comprising polynucleotides disclosed herein.

[0314] In some embodiments, a composition is a pharmaceutical composition. Page 57 of 84 12871572v1Attorney Docket: 2012611-0180

[0315] Pharmaceutical compositions of the present disclosure may comprise a polynucleotide disclosed herein, or an expression vector comprising a polynucleotide. In some embodiments, a pharmaceutical composition may comprise a pharmaceutically acceptable excipient, a diluent, or a combination thereof. In some embodiments, a pharmaceutical composition may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose, or dextrans; mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; and preservatives.

[0316] In some embodiments, a pharmaceutical composition is formulated for administration according to any of the routes of administration disclosed herein. In some embodiments, a pharmaceutical composition is formulated for intramuscular, intradermal, intravenous, subcutaneous, mucosal, inhaled, or intranodal delivery.

[0317] In some embodiments, a composition disclosed herein comprises: (i) a first composition comprising a polynucleotide disclosed herein (e.g., a first polynucleotide comprising one or more elements of a GARY construct disclosed herein), and (ii) a second composition comprising a second polynucleotide (e.g., a second polynucleotide does not comprise one or more elements of GARY construct).

[0318] In some embodiments, a first composition and a second composition are in separate compositions.

[0319] In some embodiments, a first composition and a second composition are administered separately.

[0320] In some embodiments, a first composition and a second composition are administered at substantially the same time.

[0321] In some embodiments, a first composition is administered first followed by administration of a second composition.

[0322] In some embodiments, a second composition is administered first followed by administration of a first composition.

[0323] In some embodiments, a first composition and a second composition are in the same composition.

[0324] In some embodiments, a second polynucleotide does not comprise one or more elements of a GARY construct disclosed herein. In some embodiments, a second polynucleotide encodes a second payload. In some embodiments, a second payload is different than a payload encoded by a polynucleotide in a first composition (e.g., a polynucleotide having one or more elements of a GARY construct disclosed herein).

[0325] In some embodiments, a second payload is an antigen, an enzyme, an antibody, a secreted protein, a cell surface protein, or a variant or fragment of any of the foregoing.

[0326] In some embodiments, a second payload is not a helper protein.

[0327] In some embodiments, a second payload comprises a fusion polypeptide.

[0328] In some embodiments, a fusion polypeptide comprises a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of S. aureus.

[0329] In some embodiments, a Sbi complement C3d-binding polypeptide comprises an Sbi domain III polypeptide and / or an Sbi domain IV polypeptide. Page 58 of 84 12871572v1Attorney Docket: 2012611-0180

[0330] In some embodiments of compositions disclosed herein, (i) a first composition comprises a GARYon or GARYswitch polynucleotide, and (ii) a second composition comprises a second polynucleotide that does not comprise one or more elements of GARY construct. In some embodiments, a first composition comprising GARYon or GARYswitch polynucleotide encodes a helper protein, e.g., as disclosed herein. In some embodiments, a second composition comprises a second polynucleotide encoding an antigen, an enzyme, an antibody, a secreted protein, a cell surface protein, or a variant or fragment of any of the foregoing. Formulations

[0331] Polynucleotides disclosed herein can be polyribonucleotides. Among other things, provided herein are compositions comprising a polyribonucleotide disclosed herein, and formulations thereof. In some embodiments, a composition comprising a polyribonucleotide disclosed herein is formulated in a lipid nanoparticle (LNP) formulation.

[0332] In some embodiments, a polyribonucleotide disclosed herein encodes for a polypeptide. In some embodiments, a polyribonucleotide disclosed herein is or comprises a messenger RNA. In some embodiments, a composition comprising a polyribonucleotide comprising a messenger RNA is formulated in a lipid nanoparticle (LNP) formulation.

[0333] In some embodiments, a polyribonucleotide disclosed herein is or comprises a gRNA. In some embodiments, a composition comprising a polyribonucleotide comprising a gRNA is formulated in a lipid nanoparticle (LNP) formulation.

[0334] In some embodiments, a polyribonucleotide disclosed herein is or comprises an inhibitory RNA. In some embodiments, a composition comprising a polyribonucleotide comprising an inhibitory RNA is formulated in a lipid nanoparticle (LNP) formulation.

[0335] In some embodiments, a polyribonucleotide disclosed herein is or comprises an miRNA or siRNA. In some embodiments, a composition comprising a polyribonucleotide comprising a miRNA or siRNA is formulated in a lipid nanoparticle (LNP) formulation.

[0336] In some embodiments, a polyribonucleotide disclosed herein is or comprises an antisense oligonucleotide. In some embodiments, a composition comprising a polyribonucleotide comprising an antisense oligonucleotide is formulated in a lipid nanoparticle (LNP) formulation

[0337] In some embodiments, the disclosure provides an LNP formulation comprising a polyribonucleotide disclosed herein for use in a pharmaceutical composition, e.g., an immunogenic composition. Methods of using polynucleotides or compositions disclosed herein

[0338] The disclosure provides, among other things, methods for using a polynucleotide disclosed herein, or a composition comprising the same.

[0339] In some embodiments, provided herein is a method of administering a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject. Page 59 of 84 12871572v1Attorney Docket: 2012611-0180

[0340] In some embodiments, provided herein is a vaccination method comprising administering a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject.

[0341] In some embodiments, disclosed herein is a gene therapy method comprising administering a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject. In some embodiments, a gene therapy method comprises delivery of one or more components of a gene therapy, e.g., a guide RNA and / or a Cas polypeptide.

[0342] In some embodiments, provided herein is a method for stimulating an immune response comprising administering a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject.

[0343] In some embodiments, also provided herein is a cell therapy engineering method comprising administering a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject.

[0344] In some embodiments, provided herein is an immunotherapy method comprising administering a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject. In some embodiments, an immunotherapy method comprises delivery of an antibody therapy and / or an immune checkpoint therapy.

[0345] In some embodiments, disclosed herein is a protein replacement therapy method comprising administering a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject. In some embodiments, a protein replacement therapy comprises delivery of an enzyme replacement therapy.

[0346] In some embodiments, provided herein is a chemotherapeutic method comprising administering a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject. Kits

[0347] Another aspect of the present disclosure further provides a pharmaceutical pack or kit. In some embodiments, a kit can comprise a polynucleotide or a composition described herein. In some embodiment, kits may be used in any applicable method, e.g., methods as described herein. ENUMERATED EMBODIMENTS

[0348] Embodiment 1. A polynucleotide comprising: (i) an Inositol-requiring Enzyme 1 (IRE-1) binding sequence, (ii) an intervening sequence, (iii) an AUG translation initiation codon, and (iv) a payload sequence encoding a payload, wherein the intervening sequence is located between the IRE1-binding sequence and the payload sequence.

[0349] Embodiment 2. The polynucleotide of embodiment 1, wherein the polynucleotide comprises in 5’ to 3’ order: (a) the IRE-1 binding sequence, (b) the intervening sequence, (c) the AUG translation initiation codon, and (d) the payload sequence. Page 60 of 84 12871572v1Attorney Docket: 2012611-0180

[0350] Embodiment 3. The polynucleotide of embodiment 2, wherein the polynucleotide further comprises a 5’ UTR.

[0351] Embodiment 4. The polynucleotide of embodiment 3, wherein the 5’ UTR comprises (a) the IRE-1 binding sequence, and (b) the intervening sequence.

[0352] Embodiment 5. The polynucleotide of embodiment 3, wherein the 5’ UTR does not comprise (c) the AUG translation initiation codon.

[0353] Embodiment 6. The polynucleotide of any one of embodiments 2-5, wherein the polynucleotide is characterized such that when administered to a cell, tissue or subject, expression of the payload is observed at similar levels under physiological conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

[0354] Embodiment 7. The polynucleotide of any one of embodiments 2-6, wherein the polynucleotide is characterized such that when administered to a cell, tissue or subject, increased expression of the payload is observed under cellular stress conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

[0355] Embodiment 8. The polynucleotide of embodiment 7, wherein expression of the payload under cellular stress conditions is similar to or greater than expression of the payload under physiological conditions.

[0356] Embodiment 9. The polynucleotide of embodiment 1, wherein the polynucleotide comprises in 5’ to 3’ order: (a) the AUG translation initiation codon, (b) the IRE-1 binding sequence, (c) the intervening sequence, and (d) the payload sequence.

[0357] Embodiment 10. The polynucleotide of embodiment 9, wherein the polynucleotide further comprises a 5’ UTR.

[0358] Embodiment 11. The polynucleotide of embodiment 10, wherein the 5’ UTR comprises does not comprise (a) the AUG translation initiation codon, (b) the IRE-1 binding sequence, and (c) the intervening sequence.

[0359] Embodiment 12. The polynucleotide of any one of embodiments 9-11, wherein the polynucleotide is characterized such that when administered to a cell, tissue or subject, minimal (e.g., low), expression of the payload is observed under physiological conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

[0360] Embodiment 13. The polynucleotide of any one of embodiments 9-12, wherein the polynucleotide is characterized such that when administered to a cell, tissue or subject, increased expression of the payload is observed under cellular stress conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

[0361] Embodiment 14. The polynucleotide of any one of the preceding embodiments, wherein the intervening sequence comprises one or more UAG, UAA and / or UGA codons and / or one or more linker sequences.

[0362] Embodiment 15. The polynucleotide of embodiment 14, wherein the intervening sequence comprises any two of the following codons: UAG, UAA or UGA. Page 61 of 84 12871572v1Attorney Docket: 2012611-0180

[0363] Embodiment 16. The polynucleotide of embodiment 15, wherein the intervening sequence comprises UAG, UAA and UGA codons.

[0364] Embodiment 17. The polynucleotide of any one of the preceding embodiments, wherein the polynucleotide further comprises a 5’ cap, a 3’ UTR and / or a polyA tail.

[0365] Embodiment 18. The polynucleotide of any one of the preceding embodiments, wherein the IRE-1 binding sequence comprises a sequence that is recognized by a nuclease domain of IRE-1.

[0366] Embodiment 19. The polynucleotide of embodiment 18, wherein the IRE-1 binding sequence comprises: (i) a CUGCAG sequence, (ii) a CCGCAG sequence, or (iii) both (i) and (ii).

[0367] Embodiment 20. The polynucleotide of any one of embodiments 1-19, wherein the IRE-1 binding sequence comprises an XBP1 sequence or a fragment or variant thereof, optionally wherein the XBP1 sequence comprises the nucleotide sequence provided in SEQ ID NO: 5, SEQ ID NO: 30, SEQ ID NO: 6, SEQ ID NO: 31, or a sequence with at least 85% identity to any of the foregoing.

[0368] Embodiment 21. The polynucleotide of any one of embodiments 1-19, wherein the IRE-1 binding sequence comprises the nucleotide sequence provided in SEQ ID NO: 25, SEQ ID NO: 32, SEQ ID NO: 26, SEQ ID NO: 33, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 28, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49 or a sequence with at least 85% identity to any of the foregoing.

[0369] Embodiment 22. The polynucleotide of any one of the preceding embodiments, wherein the cellular stress response is endoplasmic-reticulum (ER) stress.

[0370] Embodiment 23. The polynucleotide of embodiment 22, wherein ER stress comprises the unfolded protein response (UPR).

[0371] Embodiment 24. The polynucleotide of embodiment 23, wherein UPR activates IRE-1 nuclease activity.

[0372] Embodiment 25. The polynucleotide of any one of embodiments 6, 8, or 12, wherein physiological conditions refers to a cellular condition in which a cell is not under ER stress.

[0373] Embodiment 26. The polynucleotide of any one of the preceding embodiments, wherein the polynucleotide is or comprises a polyribonucleotide.

[0374] Embodiment 27. The polynucleotide of embodiment 26, wherein the polyribonucleotide comprises naturally occurring polyribonucleotides.

[0375] Embodiment 28. The polynucleotide of embodiment 26, wherein the polyribonucleotide comprises one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0376] Embodiment 29. The polynucleotide of embodiment 28, wherein the one or more modified ribonucleotides comprise modified ribonucleotides that comprise a 2’-O-acetylated ribose. Page 62 of 84 12871572v1Attorney Docket: 2012611-0180

[0377] Embodiment 30. The polynucleotide of embodiment 29, wherein the one or modified ribonucleotides comprise 2’-O-acetyl guanosine.

[0378] Embodiment 31. The polynucleotide of embodiment 29, wherein the one or modified ribonucleotides comprise 2’-O-acetyl adenosine.

[0379] Embodiment 32. The polynucleotide of embodiment 29, wherein the one or modified ribonucleotides comprise 2’-O-acetyl uridine.

[0380] Embodiment 33. The polynucleotide of embodiment 29, wherein the one or modified ribonucleotides comprise 2’-O-acetyl cytidine.

[0381] Embodiment 34. The polynucleotide of embodiment 29, wherein the one or modified ribonucleotides comprise 2’-O-acetyl N1-methylpseudouridine.

[0382] Embodiment 35. The polynucleotide of any one of embodiments 26-34, wherein the polyribonucleotide comprises a cap structure and the cap structure does not comprise a 2’-O-acetylated ribose.

[0383] Embodiment 36. The polynucleotide of any one of embodiments 26-34, wherein the polyribonucleotide comprises a cap structure and the cap structure comprises a 2’-O-acetylated ribose.

[0384] Embodiment 37. The polynucleotide of any one of embodiments 28-36, wherein the one or more modified ribonucleotides comprises a nucleoside comprising: N4-acetylcytidine, 5-hydroxymethyluridine, N1- methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5-methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl- cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (m1 I), wyosine (imG), methylwyosine (mimG), 5-hydroxycytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5- methyluridine, 5,6-dihydro-5-methyluridine, 2’-O-methyluridine, 2’-O-methyl-5-methyluridine, 2’-fluoro-2’- deoxyuridine, 2’-amino-2’-deoxyuridine, 2’-azido-2’-deoxyuridine, 4-thiouridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5- fluorouridine, pseudouridine, 2’-O-methyl-pseudouridine, N1-hydroxypseudouridine, 2’-O-methyl-N1- methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, ara-uridine, N6-methyladenosine, 2- aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 8-oxoadenosine, thienoguanosine, 7-deazaguanosine, 8- oxoguanosine, 6-O-methylguanine, or any combination thereof.

[0385] Embodiment 38. The polynucleotide of any one of embodiments 28-37, wherein the one or more modified ribonucleotides comprise modified ribonucleotides that comprise a N4-acetylcytidine nucleoside.

[0386] Embodiment 39. The polynucleotide of any one of embodiments 28-38, wherein the one or more modified ribonucleotides comprise modified ribonucleotides that comprise a 5-hydroxymethyluridine nucleoside.

[0387] Embodiment 40. The polynucleotide of any one of the preceding embodiments, wherein the polynucleotide comprises a coding region.

[0388] Embodiment 41. The polynucleotide of embodiment 40, wherein the coding region encodes a payload. Page 63 of 84 12871572v1Attorney Docket: 2012611-0180

[0389] Embodiment 42. The polynucleotide of embodiment 41, wherein the payload is or comprises a polypeptide.

[0390] Embodiment 43. The polynucleotide of embodiment 40, wherein the polyribonucleotide comprises an RNA payload.

[0391] Embodiment 44. The polynucleotide of any one of the preceding embodiments, wherein the payload is an antigen, an enzyme, an antibody, a secreted protein, a cell surface protein, a helper protein, or a variant or fragment of any of the foregoing.

[0392] Embodiment 45. The polynucleotide of clam 44, wherein the helper protein is a chaperone or ER guardian polypeptide, or a fragment or variant thereof.

[0393] Embodiment 46. One or more compositions comprising a polynucleotide of any one of the preceding embodiments.

[0394] Embodiment 47. The composition of embodiment 46, comprising: (i) a first composition comprising a polynucleotide of embodiments 45, and (ii) a second composition comprising a second polynucleotide.

[0395] Embodiment 48. The composition of embodiment 47, wherein the second polynucleotide encodes a second payload.

[0396] Embodiment 49. The composition of embodiment 48, wherein the second payload is different than the payload encoded by the polynucleotide in the first composition.

[0397] Embodiment 50. The composition of embodiment 48 or 49, wherein the second payload is an antigen, an enzyme, an antibody, a secreted protein, a cell surface protein, or a variant or fragment of any of the foregoing.

[0398] Embodiment 51. The composition of embodiment 48 or 49, wherein the second payload is not a helper protein.

[0399] Embodiment 52. The composition of any one of embodiments 48-51, wherein the second payload comprises a fusion polypeptide.

[0400] Embodiment 53. The composition of embodiment 52, wherein the fusion polypeptide comprises a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus.

[0401] Embodiment 54. The composition of embodiment 53, wherein the Sbi complement C3d-binding polypeptide comprises an Sbi domain III polypeptide and / or an Sbi domain IV polypeptide.

[0402] Embodiment 55. The composition of any one of embodiments 47-54, wherein (i) and (ii) are in separate compositions.

[0403] Embodiment 56. The composition of any one of embodiments 47-55, wherein (i) and (ii) are administered separately.

[0404] Embodiment 57. The composition of any one of embodiments 47-55, wherein (i) and (ii) are administered at substantially the same time. Page 64 of 84 12871572v1Attorney Docket: 2012611-0180

[0405] Embodiment 58. The composition of any one of embodiments 47-56, wherein (i) is administered first followed by (ii).

[0406] Embodiment 59. The composition of any one of embodiments 47-56, wherein (ii) is administered first followed by (i).

[0407] Embodiment 60. The composition of any one of embodiments 47-54, wherein (i) and (ii) are in the same composition.

[0408] Embodiment 61. The composition of any one of embodiments 46-60, wherein the composition is a pharmaceutical composition.

[0409] Embodiment 62. The composition of embodiment 61, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0410] Embodiment 63. The composition of embodiment 62, wherein the pharmaceutical composition is formulated for intramuscular, intradermal, intravenous, subcutaneous, mucosal, inhaled, or intranodal delivery.

[0411] Embodiment 64. A cell comprising a polynucleotide of any one of embodiments 1-45, or a composition of any one of embodiments 46-63.

[0412] Embodiment 65. The cell of embodiment 64, wherein the cell is in vivo.

[0413] Embodiment 66. The cell of embodiment 64, wherein the cell is ex vivo.

[0414] Embodiment 67. The cell of any one of embodiments 64-66, wherein the polynucleotide is introduced into the cell.

[0415] Embodiment 68. The cell of any one of embodiments 64-67, wherein the cell expresses one or more polypeptides encoded by the polynucleotide.

[0416] Embodiment 69. A method of delivering a composition of any one of embodiments 46-63 to a cell, tissue or subject.

[0417] Embodiment 70. The method of embodiment 69, wherein delivering comprises administering the composition comprising to the cell, tissue or subject.

[0418] Embodiment 71. The method of embodiment 69 or 70, wherein the method is a treatment method or a prevention method.

[0419] Embodiment 72. The method of any one of embodiments 69-71, wherein the method is:(i) a method to stimulate an immune response, (ii) an antibody therapy method, (iii) an immune-modulation method, (iv) a vaccination method, (v) a gene therapy method, (vi) a cell therapy engineering method, (vii) an immunotherapy method, (viii) a protein replacement therapy method, (ix) a chemotherapeutic method, or (x) a combination of (i)- (ix).

[0420] Embodiment 73. The method of any one of embodiments 69-72, wherein the subject is a human.

[0421] Embodiment 74. A method of stimulating an immune response to an antigen, comprising delivering a composition of any one of embodiments 46-63, to a cell, tissue or subject. Page 65 of 84 12871572v1Attorney Docket: 2012611-0180

[0422] Embodiment 75. The method of any one of embodiments 69-74, wherein the subject is a human.

[0423] Embodiment 76. A composition comprising a polynucleotide of any one of embodiments 1-45, or the composition of any one of embodiments 46-60, for use in delivering the polynucleotide to a cell, tissue, or subject.

[0424] Embodiment 77. A composition comprising a polynucleotide of any one of embodiments 1-45, or the composition of any one of embodiments 46-63, for use in stimulating an immune response to an antigen.

[0425] Embodiment 78. The composition for use of embodiment 77, wherein the use comprise delivering the polynucleotide to a cell, tissue, or subject.

[0426] Embodiment 79. The composition for use of embodiment 76 or 78, wherein delivering comprises administering.

[0427] Embodiment 80. The composition for use of any one of embodiments 76, and 77-79, wherein the subject is a human.

[0428] Embodiment 81. The composition for use of any one of embodiments 76-80, wherein the use is for: (i) stimulating an immune response, (ii) an antibody therapy, (iii) immune-modulation, (iv) vaccination, (v) gene therapy, (vi) cell therapy engineering, (vii) immunotherapy, (viii) protein replacement therapy, (ix) chemotherapy, or (x) any combination of (i)-(ix).

[0429] Embodiment 82. Use of a polynucleotide according to any one of embodiments 1-45, or a composition according to any one of embodiments 46-63, in the preparation of a medicament for delivering the polynucleotide or composition to a cell, tissue, or subject.

[0430] Embodiment 83. Use of a polynucleotide according to any one of embodiments 1-45, or a composition according to any one of embodiments 46-63, in the preparation of a medicament for stimulating an immune response to an antigen.

[0431] Embodiment 84. The use of embodiment 83, further comprising delivering the polynucleotide to a cell, tissue, or subject.

[0432] Embodiment 85. The use of embodiment 82 or 84, wherein delivering comprises administering.

[0433] Embodiment 86. The use of any one of embodiments 82 and 84-85, wherein the subject is a human.

[0434] Embodiment 87. The use of any one of embodiments 82-86, wherein the use is for: (i) stimulating an immune response, (ii) an antibody therapy, (iii) immune-modulation, (iv) vaccination, (v) gene therapy, (vi) cell therapy engineering, (vii) immunotherapy, (viii) protein replacement therapy, (ix) chemotherapy, or (x) any combination of (i)-(ix). EXEMPLIFICATION Example 1: Methods and Materials for Example 2 and Example 6.

[0435] The methods described in this Example relate to those used in Example 2 and Example 6. IVT (In Vitro Transcription) Template production Page 66 of 84 12871572v1Attorney Docket: 2012611-0180

[0436] Tested architectures were cloned in plasmids provided by IDT. The sequences of interest were amplified with primers to add a T7 promoter for IVT and a 120 nt polyA tail (SEQ ID NO: 29) into the DNA template. Amplification was carried out at an annealing temperature of 50°C in a 20 μL reaction consisting of 0.25 μM each primer “T7-AGG_fwd” and “120pA_rev,” 1X Herculase II buffer, 25 mM each dNTP, 15 ng NanoLuc plasmid (IDT), and 0.4 μL Herculase II enzyme. After incubation, the PCR reaction was subjected to treatment with 20U of Dpn1 enzyme (New England Biolabs) to digest template plasmid. Digested product was purified using the DNA Clean & Concentrator-25 kit (Zymo Research) and eluted into 30 μL nuclease free water.

[0437] Sequences of primers used were as follows: T7-AGG_fwd: gaattTAATA CGACTCACTA TAAGGcttgt tctttttgca gaagc (SEQ ID NO: 7) 120pA_rev: TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT agaatgtgaa gaaactttct ttttattag (SEQ ID NO: 8)

[0438] RNAs were synthesized in 20 μL IVT reactions consisting of 200 ng of the corresponding DNA amplified templates, 20 mM MgCl2, 7.5 mM each NTP, 7.5 mM CleanCap AG (TriLink) 1X HiScribe Transcription Buffer, and 2 uL HiScribe polymerase mix (New England Biolabs) and incubated at 37°C for 2 hours.

[0439] IVT products were then digested with 2U of DNase I (RNase-free) (New England Biolabs) and 20U CIAP (Promega) at 37°C for 5 minutes to degrade DNA template and remove residual 5’ triphosphates. Treated samples were purified using Monarch 500 ug RNA Clean Up kit (New England Biolabs) and eluted into 100 μL nuclease-free water. RNA Quantification

[0440] RNA concentration was determined using a NanoDrop OneC spectrophotometer (Thermo Scientific). Human Skeletal Myoblast Cell Culture Methods

[0441] Human Skeletal Myoblasts (Thermo Fisher Scientific) were cultured in DMEM (Thermo Fisher Scientific) supplemented with 2% Horse Serum (Thermo Fisher Scientific). Cells were thawed in a 37°C water bath and suspended in 7 mL of the differentiation media. Cells were then centrifuged at 180gx for 5 minutes, resuspended in 6 mL of differentiation media and centrifuged a final time at 180gx for 5 minutes. Cells were resuspended in 6 mL of differentiation media. Next, cell concentration was measured and 4.8e4 cells from the diluted cell solution were added to the wells of cell culture-treated 96-well plates. Cells were incubated at 37°C and 5% CO2 for 72 hours. At 72 hours, media was removed, cells were washed with 200 μL of PBS, and 200 μL of fresh differentiation media was added. Cells were transfected with 50 ng of synthesized modified RNA described above using Lipofectamine MessengerMAX Transfection Reagent (ThermoFisher) using a 1:1.5 μg:uL ratio of RNA:MessengerMAX. Transfections were performed in triplicate. Cells were treated with 5 μM of Thapsigargin (Abcam) 90 minutes after transfection. Then, cells were incubated at 37°C and 5% CO2 for 6 hours. Finally, nano-luciferase expression was determined using the Nano-Glo Luciferase Assay System (Promega). Page 67 of 84 12871572v1Attorney Docket: 2012611-0180 Example 2: Characterization of GARY constructs in HEK293T cells

[0442] This Example describes the design and characterization of two Genetic Activation with Responsive Yield (“GARY”) constructs in HEK293T cells. The two GARY constructs tested were: GARYon and GARYswitch. GARY constructs comprise a double stem loop structure similar to that found in XBP1 RNA, which can be recognized and spliced by IRE-1. IRE-1 is an unfolded protein response (UPR) sensor, which can: (1) splice XBP1 RNA promoting its translation, and (2) promote degradation of various RNA containing certain recognition sequences in a process named regulated IRE1-dependent decay (RIDD). The GARY constructs are responsive to IRE-1 splicing and were designed to either maintain protein expression under conditions of cellular stress, e.g., ER stress, (GARYon) or increase protein expression under conditions of cellular stress, e.g., ER stress, (GARYswitch).

[0443] GARYon. In this construct, a double stem loop splice site is located in a 5’ UTR of an RNA, upstream of an open reading frame (ORF) comprising a sequence encoding a payload. See FIG.1. Under physiological conditions (e.g., in the absence of cellular stress), the ORF is translated, and protein expression occurs at levels similar to control RNA (e.g., without a double stem loop splice site). Upon UPR induction, expression from control RNA drops, e.g., due to RNA degradation via RIDD. However, GARYon RNA constructs can be recognized by the activated IRE1 nuclease, and the double stem loop in the UTR can be spliced. Thus, the result is not degradation of GARYon RNA constructs but an RNA with an untouched ORF, which can continue to be translated, allowing for maintenance of protein expression. In some embodiments, a splicing event also protects GARYon RNA from degradation via RIDD.

[0444] An exemplary schematic of a GARYon construct pre-splice and post-splice is shown below: Pre-splice RNA: cap – 5’ UTR – splice site – AUG – ORF – 3’ UTR – pA tail Pre-splice protein: Methionine (Met) – payload Post-splice RNA: cap – 5’ UTR – *splice scar* – AUG – ORF – 3’ UTR – pA tail Post-splice protein: Met – payload

[0445] GARYsw itch. In this construct, a double stem loop splice site along with stop codons (e.g., 3 stopcodons) are located within an ORF, in between the start codon and a sequence encoding a payload. See FIG.2. Under physiological conditions (e.g., in the absence of cellular stress), a ribosome will translate the start codon and the sequence encoded by the splice site, and then translation will be terminated at the stop codons, typically resulting in minimal expression (e.g., no detectable expression) of a payload. Low levels of expression of a payload have been observed due to stop codon readthrough and ribosome frame shifting. Upon UPR induction, the double stem loop in a GARYswitch RNA construct is spliced by IRE1, resulting in a frame shift that shifts the stop codons out of frame, and shifts the sequence encoding a payload in frame. The result is an induction of expression of a payload under stress (e.g., ER stress). In some embodiments, a splicing event also protects GARYswitch RNA from degradation via RIDD.

[0446] An exemplary schematic of a GARYswitch construct pre-splice and post-splice is shown below: Pre-splice RNA: cap – 5’ UTR – AUG – splice site – stop – ORF – 3’ UTR – pA tail Page 68 of 84 12871572v1Attorney Docket: 2012611-0180 Pre-splice protein: Met – splice site AA Post-splice RNA: cap – 5’ UTR – AUG – *splice scar* – ORF – 3’ UTR – pA tail Post-splice protein: Met – splice scar AA – payload

[0447] A “GARYon” construct (SEQ ID NO: 1) was designed to promote expression of a payload under both unstressed conditions and stressed conditions e.g., ER stress conditions.

[0448] A “GARYswitch” (SEQ ID NO: 3) construct was designed to promote minimal expression of a payload under unstressed conditions, and increased expression of a payload under stressed conditions, e.g., ER stress conditions.

[0449] Each GARY construct was engineered to harbor a conserved XBP1 site recognized and spliced by IRE1 in ER stress conditions. IRE1 has been shown to interact with the conserved double-stem loop present in XBP1 RNA and splices the XBP1 RNA at an indicated conserved splice site under ER stress conditions. Each construct design was generated using ribonucleotides disclosed above and transfected into HEK293T cells.

[0450] Results: Delivery of GARYon-NanoLuc RNA resulted in similar protein expression under physiological conditions (e.g., no or low stress) and increased protein expression under induced ER stress (5 uM Thapsigargin treatment) as compared to control HEK293T cells that were untransfected (FIG.3). This result suggests that the exemplary GARYon construct allows for payload expression in the absence of cellular stress and sustains payload expression during ER stress, e.g., via recruitment of IRE-1 to the double stem loop structure and / or inhibition of degradation via RIDD.

[0451] Delivery of GARYswitch-NanoLuc RNA resulted in low payload expression under physiological conditions (e.g., no or low stress) and induction of high levels of expression under induced ER stress (5 uM Thapsigargin treatment) as compared to control HEK293T cells that were untransfected (FIG.3). This result suggests that the exemplary GARYswitch construct is able to induce payload expression during ER stress, e.g., via recruitment of IRE-1 to the double stem loop structure and / or inhibition of degradation via RIDD. Without wishing to be bound by any particular theory, in some embodiments, minimal payload expression observed with GARYswitch construct without Thapsigargin treatment is likely due to stop codon readthrough and / or ribosome shifting.

[0452] Together, these results demonstrate the generation and characterization of polynucleotide constructs that are responsive to IRE-1 and can sustain payload expression under conditions of ER stress (GARYon) or increase payload expression under conditions of ER stress (GARYswitch). The data presented herein supports the development of GARY constructs for delivering payloads to cells (e.g., for delivering a therapeutic payload) to achieve sustained or increased payload expression under cellular stress conditions.

[0453] In some embodiments, delivery of a GARY construct (e.g., GARYon or GARYswitch construct) encoding a therapeutic payload can promote payload expression under cellular stress conditions. Page 69 of 84 12871572v1Attorney Docket: 2012611-0180 Example 3: Co-delivery of RNA encoding an exemplary payload and a GARY RNA construct encoding a helper protein increases payload expression

[0454] This example demonstrates that co-delivery of: (1) RNA encoding an exemplary payload (e.g., a mutated antigen (e.g., mutant gp75)) fused to a NanoLuc reporter and S. aureus second binding protein of immunoglobulin (Sbi) domains III and IV, and (2) RNA having a GARY construct encoding an exemplary helper protein, enhanced payload expression particularly when the payload was provided at high doses.

[0455] Results: The expression of mutant proteins (e.g., mutant antigens) can place additional burdens on cellular ER function (e.g., inducing ER stress) and trigger the unfolded protein response. This in turn can limit the amount of protein that may be produced and negatively impact cellular health. Thus, to express mutant proteins at high levels (e.g., at therapeutically meaningful levels) it would be helpful to reduce ER stress and / or avoid triggering the unfolded protein response, e.g., by co-expressing helper proteins (such as ER chaperone proteins) that can reduce and / or mitigate ER stress. Since GARYon constructs were able to sustain payload expression under conditions of ER stress and GARYswitch constructs were able to increase payload expression under conditions of ER stress (see Example 2 herein), the ability of GARY constructs encoding helper proteins to allow expression of mutant proteins was tested.

[0456] The exemplary antigen used for these experiments is a mutated version of Gp75 (also referred to as tyrosinase-related protein 1 (TYRP1)), a glycoprotein and an enzyme expressed in melanocytes that is thought to play a role in melanosome formation and melanin production. The exemplary mutated antigen was designed as a fusion protein consisting of 3 parts: (1) a mutant mouse gp75 protein, (2) domains III and IV of S. aureus second binding protein of immunoglobulin (Sbi), and (3) a luciferase protein reporter. RNA encoding the exemplary mutant antigen was either delivered alone to HEK293T cells or co-delivered with GARYon RNA encoding an exemplary helper protein (GARYon tapt1) or GARYswitch RNA encoding an exemplary helper protein (GARYswitch tapt1).

[0457] As shown in FIG.4, delivering increasing amounts of RNA encoding the exemplary mutated antigen to HEK293T cells resulted in increasing amounts of antigen protein expression as measured by luciferase luminescence but only at doses up to 500ng. When RNA encoding the exemplary mutated antigen was delivered at higher doses, expression of the exemplary antigen plummeted (see control). This data suggests that expression of the mutant antigen at higher levels induced cellular stress responses, e.g., including and induction of the UPR. In contrast, co- delivery of GARYon RNA or GARYswitch RNA encoding TAPT1 along with RNA encoding the exemplary mutated antigen rescued expression of antigen protein at higher doses (see GARYon tap1 and GARYswtich tapt1 data points).

[0458] This data shows that UPR induction promoted TAPT1 RNA splicing thus allowing TAPT1 protein expression. As TAPT1 is an exemplary helper protein that promotes proper protein folding and can reduce ER stress, increased TAPT1 expression allowed for increased antigen expression at high doses.

[0459] Taken together, this data supports the utility of expressing one or more helper proteins in a GARY construct to reduce ER stress and / or prevent induction of the UPR. This data further shows that high level of antigen (e.g., mutated antigen) expression can be achieved with the co-delivery of one or more helper proteins in a GARY construct. Page 70 of 84 12871572v1Attorney Docket: 2012611-0180 Example 4: In vivo payload expression from GARY constructs

[0460] This example describes in vivo expression of an exemplary payload encoded by a GARY construct. Delivery of RNA having a GARY construct encoding an exemplary payload resulted in enhanced payload expression.

[0461] Results: To investigate activities of GARY sequence elements in vivo, RNA encoding luciferase (Luc2) was delivered to mice in three different RNA contexts: (1) without any GARY sequence elements, (2) with GARYon elements, or (3) with GARYswitch elements. Mice were treated with three different doses of RNA spanning two orders of magnitude. At all three doses, treatment with Luc2 RNA without GARY sequence elements resulted in luciferase expression that decreased over time (FIGS.5A-5C). Treatment with GARYon Luc2 RNA resulted in enhanced expression at all time points and all doses. This data suggests that the unfolded protein response was induced by exogenous protein over-expression, and that the GARYon RNA sequence element responded to UPR induction and enabled enhanced protein expression despite the cellular state. The enhancement effect of GARYon Luc2 compared to regular Luc2 increased at higher doses and longer time points. This data suggests that as the UPR response increased with increasing protein loads, the expression advantage from the GARYon sequence element was enhanced.

[0462] At the 0.1 ug dose, treatment with GARYswitch Luc2 RNA resulted in a low level of expression, consistent with in vitro results under no- or low-stress conditions (FIG.5A). At higher doses, GARYswitch Luc2 expression increased over time (FIGS.5B and 5C). This data suggests that UPR induction with increasing protein loads led to GARYswitch RNA cleavage and increased protein expression. This is in contrast to the decrease in expression observed from Luc2 and GARYon Luc2 mRNA over time. At 1 ug and 10 ug doses, the expression from both Luc2 and GARYswitch Luc2 mRNAs was the same at 72 hours as compared to expression at 48 hours.

[0463] This data demonstrates in vivo payload expression from RNA having GARY constructs (e.g., GARYon or GARYswitch construct) encoding a payload. Payload expression was also increased under cellular stress conditions as evidenced by sustained expression at an increased level with GARYon constructs and increased expression over time with GARYswitch constructs. This data supports the utility of delivering RNA having GARY construct (e.g., GARYon or GARYswitch construct) encoding a therapeutic payload to achieve sustained and / or increased payload expression, e.g., under cellular stress conditions. Example 5: Materials and Methods for Examples 3 and 4

[0464] The materials and methods described in this Example relate to those used in Examples 3 and 4 above.

[0465] Random Mutagenesis of gp 75: mTYRP1-Sbi_gs was synthesized as a HiFi gBlock (IDT), PCR-amplified with gp75_pUC19_fwd / gp75_pUC19_rev using Q52x Master Mix (NEB) per the manufacturer’s instructions and cleaned up using QIAquick PCR Purification Kit (Qiagen). pUC19 (NEB) was double-digested with EcoRI-HF (NEB) and HindIII-HF (NEB), cleaned up using QIAquick PCR Purification Kit, and digested with DpnI. The amplicon was cloned into the digested pUC19 using Gibson Assembly Master Mix (NEB) per the manufacturer’s instructions. The assembled pUC19-mTYRP1-Sbi was cleaned up with QIAquick PCR Purification Kit, digested with BamHI-HF, cleaned up again, and cloned into OneShot TOP10 cells. The pUC19-mTYRP1-Sbi plasmid was purified using the ZymoPURE Plasmid Miniprep kit. Page 71 of 84 12871572v1Attorney Docket: 2012611-0180

[0466] mTYRP1 was mutagenized via error-prone PCR using the GeneMorph II EZClone Domain Mutagenesis Kit (Agilent) and gp75_mut_fwd / gp75_mut_rev, with the library cloned into XL10-Gold (Agilent) cells. The library was screened by sequencing, carried out by Priomordium Labs. pUC19-mTYRP1-290 was selected for further testing. mRNA was prepared from pUC19-mTYRP1-290 using the Unmodified In vitro transcription (IVT) Template production methods as described above.

[0467] The sequences of primers used were as follows:

[0468] gp75_pUC19_fwd: GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGcttgttctttttgcagaagc (SEQ ID NO: 11); and

[0469] gp75_pUC19_rev: AATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTagaatgtgaagaaactttctttttattag (SEQ ID NO: 12).

[0470] IVT (In Vitro Transcription) Template production: Tested architectures were cloned in plasmids provided by IDT. The sequences of interest were amplified with primers to add a T7 promoter for IVT and a 120 nt polyA tail (SEQ ID NO: 29) into the DNA template. Amplification was carried out at an annealing temperature of 50°C in a 20 Μl reaction consisting of 0.25 μM each primer “T7-AGG_fwd” and “120pA_rev,” 1X Herculase II buffer, 25 mM each dNTP, 15 ng NanoLuc plasmid (IDT), and 0.4 μL Herculase II enzyme. After incubation, the PCR reaction was subjected to treatment with 20U of Dpn1 enzyme (New England Biolabs) to digest template plasmid. Digested product was purified using the DNA Clean & Concentrator-25 kit (Zymo Research) and eluted into 30 μL nuclease free water.

[0471] Sequences of primers used were as follows:

[0472] T7-AGG_fwd: gaattTAATA CGACTCACTA TAAGGcttgt tctttttgca gaagc (SEQ ID NO: 13); and

[0473] 120pA_rev: TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTTTTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTTagaatgtgaa

[0474] RNAs were synthesized in 20 μL IVT reactions comprising 200 ng of the corresponding DNA amplified templates, 20 mM MgCl2, 7.5 mM each NTP, 7.5 mM CleanCap AG (TriLink) 1X HiScribe Transcription Buffer, and 2 uL HiScribe polymerase mix (New England Biolabs), which were incubated at 37°C for 2 hours. IVT products were then digested with 2U of DNase I (RNase-free) (New England Biolabs) and 20U CIAP (Promega) at 37°C for 5 minutes to degrade DNA template and remove residual 5’ triphosphates. Treated samples were purified using Monarch 500 ug RNA Clean Up kit (New England Biolabs) and eluted into 100 μL nuclease-free water.

[0475] RNA Quantification: RNA concentration was determined using a NanoDrop OneC spectrophotometer (Thermo Scientific).

[0476] HEK293T Cell Culture Methods: HEK293T cells (ATCC) were cultured in DMEM (Thermo Fisher Scientific) containing 10% FBS and 100 units / mL of penicillin and streptomycin. Once cells achieved ~90% confluence, they were harvested using TrypLE Express (Thermo Fisher Scientific), centrifuged at 200gx for 5 minutes, and Page 72 of 84 12871572v1Attorney Docket: 2012611-0180 resuspended to a concentration of 2.5e5 cells / ml. Next, 100 µl (2.5e4 cells) of the diluted cell solution were added to the wells of cell culture-treated 96-well plates. Cells were incubated in at 37°C and 5% CO2for 24 hours. At 24 hours, cells were co-transfected with the gp 75 mRNA and helper protein mRNA using Lipofectamine MessengerMAX Transfection Reagent (ThermoFisher Scientific). After transfection, cells were incubated at 37°C and 5% CO2for 72 hours. Finally, intracellular Nano Luciferase expression was determined using the Nano-Glo Luciferase Assay System (Promega).

[0477] Animal vaccination: All animal experiments were carried out in accordance with the guidelines set forth by Charles River Accelerator Development Lab (CRADL) and were approved by the CRADL Institutional Animal Care and Use committee. Female BALB / C mice (7-9 weeks old) were purchased from Charles River Laboratories and housed at CRADL. Mice were acclimated for at least 3 days before the initiation of a study. On Day 1, mice were injected in the right quadriceps with 50 μL RNA-LNP formulation with 0.1, 1, and 10 ug mRNA doses used as indicated in each graph. At the indicated time points (6, 24, 48, and 72 hours), whole body bioluminescence imaging includes local hair removal, intraperitoneal administration of D-luciferin (150 mg / kg, 100 μL) and Isoflurane anesthesia (3% induction, 2% maintained on nose cone) to prevent animal movement artifacts during imaging – all performed by the CRADL staff member. Supportive care for mice pre and post anesthesia (performed by CRADL staff) includes application of eye lubricant, maintenance of body temperature via a heated stage in the imager, and observation in the home cage during recovery. This procedure will take about 15 minutes for a group of 5 animals which was the group size of each condition.

[0478] Sequences for construct used in Examples 3 and 4:

[0479] mutant mouse gp75 – Sbi – luciferase MQFPRECANIEALRRGVCCPDLLPSSGPGTDPCGSSSGRGRCVAVIADSRPHSRHYPHNGKDDREAWPLRFFNRTCQCNDNFSGH NCGTCRPGWRGAACNQKILTVRRNLLDLSPEEKSHFVRALDMAKRTTHPQFVIATRRLEDILGPDGNTPQFENISVYNYFVWTHYYS VKKTFLGTGQESFGDVDFSHEGPAFLTWHRYHLLQLERDMQEMLQEPSFSLPYWNFATGKNVCDVCTDDLMGSRSNFDSTLISPNS VFSQWRVVCESLEEYGTLGTLCNSTEGGPIRRNPAGNVGRPAVQRLPEPQDVTQCLEVRVFDTPPFYSNSTDSFRNTVEGYSAPTGK YDPAVRSLHNLAHLFLNGTGGQTHLSPNDPIFVLLHTFTDAVFDEWLRRYNADISTFPLENAPIGHNSQYNMVPFWPPVTNTEMFVT APDNIGYAYEVQWPGQEFTVSEGGGGSGGGGSGGGGSIENADKAIKDFQDNKAPHDKSAAYEANSKLPKDLRDKNNRFVEKVSIEK AIVRHDERVKSANDAISKLNEKDSIENRRLAQREVNKAPMDVKEHLQKQLDSGGGSGGSGSMEDAKNIKKGPAPFYPLEDGTAGEQ LHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNEREL LNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTG LPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFS FFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTL GVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGV AGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV (SEQ ID NO: 15)

[0480] GARYon – tapt1 CttgttctttttgcagaagctcagaataaacgctcaactttggccaccCAGAGACCGCAGCACTCAGTGATGATGCTCCTCTGCAGCAGGAGCATG ATGATGAGGTGGAGGTGGAAGTGCCACCATGCTGCGGCCCCTGGCGAGGGAGGAGGAGGTGGCGTGGATGGCCCTCAGCGCG Page 73 of 84 12871572v1Attorney Docket: 2012611-0180 ACGGTAGGGGAGAGGCCGAGCAGCCTGGCGGCAGCGGTGGGCAGGGGCCTCCTCCTGCACCTCAGCTCACCGAGACCTTGGGC TTTTACGAAAGCGACCGGCGGAGGGAGCGGAGGCGCGGGCGGACAGAATTGAGTCTGCTCCGCTTTCTGTCCGCGGAGTTGAC CAGGGGATATTTTCTGGAGCACAATGAAGCTAAGTACACAGAGAGAAGAGAGCGAGTGTATACATGTCTTCGGATACCTCGGGA GCTGGAAAAGCTGATGGTCTTCGGAATCTTTCTCTGCTTGGATGCCTTCCTGTATGTGTTCACACTGCTGCCTCTTAGGGTGTTC CTGGCTCTTTTTAGGCTTCTGACTCTGCCTTGCTACGGTCTCCGCGACCGGAGACTCCTGCAACCTGCCCAGGTATGCGATATCC TTAAGGGTGTAATCCTGGTCATCTGCTATTTTATGATGCATTACGTGGACTACTCCATGATGTATCACTTGATCCGAGGCCAGTC TGTTATTAAGTTGTACATCATCTACAATATGCTGGAGGTGGCAGATAGGCTCTTTTCTAGCTTCGGACAGGACATCCTGGATGCA CTTTACTGGACTGCTACGGAACCTAAAGAGAGAAAACGCGCCCACATAGGCGTTATTCCTCATTTCTTCATGGCCGTGCTCTATG TCTTCCTGCACGCCATCCTGATCATGGTTCAAGCGACTACCCTGAATGTTGCATTCAACAGCCATAACAAGAGCCTGCTGACCAT TATGATGTCCAATAATTTCGTCGAGATAAAAGGGAGCGTCTTCAAAAAGTTTGAGAAAAACAACCTGTTCCAGATGTCCAATTCT GATATAAAGGAAAGATTCACCAATTACGTGCTCCTCCTGATTGTATGTCTGAGAAACATGGAACAGTTCAGCTGGAACCCTGATC ACCTGTGGGTTTTGTTTCCTGACGTTTGCATGGTCATTGCCAGTGAAATAGCAGTGGACATTGTCAAACACGCCTTCATTACCAA ATTTAACGACATCACCGCAGACGTGTATTCAGAGTACCGCGCCTCTCTGGCCTTTGACCTCGTAAGCTCCCGACAGAAGAACGCC TACACAGATTACTCCGATTCAGTGGCTAGACGGATGGGATTCATCCCTTTGCCTTTGGCCGTGTTGCTCATCAGAGTGGTGACC AGCAGCATCAAGGTCCAAGGCATACTGTCATATGCTTGCGTCATTCTGTTTTATTTCGGGCTCATCTCTCTCAAAGTGCTTAACA GTATTGTGCTGCTCGGCAAATCCTGCCAATACGTTAAGGAGGCAAAGATGGAAGAAAAGCTGTCAAACCCTCCTGCTACATGTA CTCCTGGGAAGCCTTCCTCAAAGTCCCAGAACAAATGTAAACCTAGTCAGGGGCTTTCCACAGAAGAGAATCTTTCTGCGAGTAT TACTAAGCAGCCTATCCATCAAAAGGAAAATATTATTCCTCTGCTTGTGACGTCAAACAGTGACCAGTTTCTCACAACCCCTGAC GGGGACGAGAAAGACATCACTCAGGATAATTCTGAGCTGAAACATCGCTCTAGTAAGAAGGACCTCCTGGAAATTGATAGGTTT ACGATTTGTGGCAATCGAATCGATaccagcctcaagaacacccgaatggagtctctaagctacataataccaacttacactttacaaaatgttgtcccccaaa atgtagccattcgtatctgctcctaataaaaagaaagtttcttcacattct (SEQ ID NO: 16)

[0481] GARYswitch – tapt1 CttgttctttttgcagaagctcagaataaacgctcaactttggccaccATGGGAGAGGAGAGTCCGCAGCACTCTCTAATAGTCCACCTCTGCAGC AGGGAGGAGGAAATAGTAATAGGTTGGAGGTGGAAGTTCTGCGGCCCCTGGCGAGGGAGGAGGAGGTGGCGTGGATGGCCCT CAGCGCGACGGTAGGGGAGAGGCCGAGCAGCCTGGCGGCAGCGGTGGGCAGGGGCCTCCTCCTGCACCTCAGCTCACCGAGAC CTTGGGCTTTTACGAAAGCGACCGGCGGAGGGAGCGGAGGCGCGGGCGGACAGAATTGAGTCTGCTCCGCTTTCTGTCCGCGG AGTTGACCAGGGGATATTTTCTGGAGCACAATGAAGCTAAGTACACAGAGAGAAGAGAGCGAGTGTATACATGTCTTCGGATAC CTCGGGAGCTGGAAAAGCTGATGGTCTTCGGAATCTTTCTCTGCTTGGATGCCTTCCTGTATGTGTTCACACTGCTGCCTCTTA GGGTGTTCCTGGCTCTTTTTAGGCTTCTGACTCTGCCTTGCTACGGTCTCCGCGACCGGAGACTCCTGCAACCTGCCCAGGTAT GCGATATCCTTAAGGGTGTAATCCTGGTCATCTGCTATTTTATGATGCATTACGTGGACTACTCCATGATGTATCACTTGATCCG AGGCCAGTCTGTTATTAAGTTGTACATCATCTACAATATGCTGGAGGTGGCAGATAGGCTCTTTTCTAGCTTCGGACAGGACAT CCTGGATGCACTTTACTGGACTGCTACGGAACCTAAAGAGAGAAAACGCGCCCACATAGGCGTTATTCCTCATTTCTTCATGGCC GTGCTCTATGTCTTCCTGCACGCCATCCTGATCATGGTTCAAGCGACTACCCTGAATGTTGCATTCAACAGCCATAACAAGAGCC TGCTGACCATTATGATGTCCAATAATTTCGTCGAGATAAAAGGGAGCGTCTTCAAAAAGTTTGAGAAAAACAACCTGTTCCAGAT GTCCAATTCTGATATAAAGGAAAGATTCACCAATTACGTGCTCCTCCTGATTGTATGTCTGAGAAACATGGAACAGTTCAGCTGG AACCCTGATCACCTGTGGGTTTTGTTTCCTGACGTTTGCATGGTCATTGCCAGTGAAATAGCAGTGGACATTGTCAAACACGCCT TCATTACCAAATTTAACGACATCACCGCAGACGTGTATTCAGAGTACCGCGCCTCTCTGGCCTTTGACCTCGTAAGCTCCCGACA GAAGAACGCCTACACAGATTACTCCGATTCAGTGGCTAGACGGATGGGATTCATCCCTTTGCCTTTGGCCGTGTTGCTCATCAG AGTGGTGACCAGCAGCATCAAGGTCCAAGGCATACTGTCATATGCTTGCGTCATTCTGTTTTATTTCGGGCTCATCTCTCTCAAA Page 74 of 84 12871572v1Attorney Docket: 2012611-0180 GTGCTTAACAGTATTGTGCTGCTCGGCAAATCCTGCCAATACGTTAAGGAGGCAAAGATGGAAGAAAAGCTGTCAAACCCTCCT GCTACATGTACTCCTGGGAAGCCTTCCTCAAAGTCCCAGAACAAATGTAAACCTAGTCAGGGGCTTTCCACAGAAGAGAATCTTT CTGCGAGTATTACTAAGCAGCCTATCCATCAAAAGGAAAATATTATTCCTCTGCTTGTGACGTCAAACAGTGACCAGTTTCTCAC AACCCCTGACGGGGACGAGAAAGACATCACTCAGGATAATTCTGAGCTGAAACATCGCTCTAGTAAGAAGGACCTCCTGGAAAT TGATAGGTTTACGATTTGTGGCAATCGAATCGATaccagcctcaagaacacccgaatggagtctctaagctacataataccaacttacactttacaaaat gttgtcccccaaaatgtagccattcgtatctgctcctaataaaaagaaagtttcttcacattct (SEQ ID NO: 17)

[0482] GARYon – luciferase CttgttctttttgcagaagctcagaataaacgctcaactttggccaccCAGAGACCGCAGCACTCAGTGATGATGCTCCTCTGCAGCAGGAGCATG ATGATGAGGTGGAGGTGGAAGTGCCACCATGgaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggc gagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatg agcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccct gttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagg gctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgt gacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattg cccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggt gccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaa gactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggc ggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctga tcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaacca gcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcg acatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatc ctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccat gaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggc aagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtgtaaaccagcctcaagaacacccgaatggagtctctaagcta cataataccaacttacactttacaaaatgttgtcccccaaaatgtagccattcgtatctgctcctaataaaaagaaagtttcttcacattct (SEQ ID NO: 18)

[0483] GARYswitch – luciferase CttgttctttttgcagaagctcagaataaacgctcaactttggccaccATGGGAGAGGAGAGTCCGCAGCACTCTCTAATAGTCCACCTCTGCAGC AGGGAGGAGGAAATAGTAATAGGTTGGAGGTGGAAGTTgaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacggg accgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtac ttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgtt gggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagc aagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgta caccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagta ccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctc agcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgca gcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgc cagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgc cattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtg Page 75 of 84 12871572v1Attorney Docket: 2012611-0180 tgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcac agcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactgg agagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggt aaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggact gaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtgtaaaccagcctcaagaacacccgaatggagtct ctaagctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccattcgtatctgctcctaataaaaagaaagtttcttcacattct (SEQ ID NO: 19)Example 6: Exemplary GARYsw itch constructs screening in HEK293T cells

[0484] This Example describes screening of exemplary GARYswitch constructs in HEK293T cells. As described in Example 2, “GARYswitch” constructs were designed to promote minimal expression of a payload under unstressed (e.g., physiological) conditions, and increased expression of a payload under stressed conditions, e.g., ER stress conditions. Methods used to assess exemplary payload expression with GARY switch construct described in this example are provided in Example 1. Species screen:

[0485] It has been reported that IRE-1 binding sequences (e.g., a IRE-1 binding sequence comprising a double stem loop splice site sequence) within XBP1 are conserved among metazoans (see, for example, Peschek J et al. “Conformational RNA zipper promotes intron ejection during non-conventional XBP1 mRNA splicing. EMBO Rep. 2015 Dec;16(12):1688-98. doi: 10.15252 / embr.201540955, which is incorporated herein by reference in its entirety). To test whether species-specific differences affect XBP1 RNA processing under induced ER stress conditions, the IRE-1 binding sequence of SEQ ID NO: 25 assessed in Example 2 (used in SEQ ID NO: 3) was substituted with exemplary IRE-1 binding sequences from other species (e.g., Gallus gallus (SEQ ID NO: 27) and Xenopus laevis (SEQ ID NO: 26)). Engineered stop codons between stem loops, as described in Example 2, were maintained. As shown in FIG.6A, HEK293T cells expressing GARYswitch NanoLuc RNA constructs comprising IRE-1 binding sequences from Gallus gallus (GARYswitch_gallus (SEQ ID NO: 23)) and Xenopus laevis (GARYswitch_xenopus (SEQ ID NO: 22)), showed low payload expression under physiological conditions (e.g., no or low stress) and induction of high levels of expression under induced ER stress (5 uM Thapsigargin treatment) compared to control HEK293T cells which did not have GARY constructs. Expression levels of the exemplary payload in cells expressing the GARYswitch_gallus or the GARYswitch_xenopus constructs, in the absence or presence of stress, was comparable to the exemplary payload expression level in cells expressing the GARYswitch construct having the IRE-1 binding sequence of SEQ ID NO: 25. These data show that GARYswitch constructs comprising IRE-1 binding sequences from other species such as Gallus gallus or Xenopus laevis induce payload expression during ER stress. Without wishing to be bound by any particular theory, the observed induction of payload expression upon ER stress is a result of recruitment of IRE-1 to the double stem loop structure of the IRE-1 binding sequences and / or inhibition of degradation of the IRE-1 binding sequences via RIDD. Page 76 of 84 12871572v1Attorney Docket: 2012611-0180 Secondary structure screen:

[0486] Stem loop structures in an RNA can be dynamic. To test the plasticity and / or stability of stem loop structures in IRE-1 binding sequences, 18 exemplary nucleotides were mutated in SEQ ID NO: 25 generating the exemplary sequence of SEQ ID NO: 28 (also referred to as “switch_2”) which was incorporated into the GARYswitch_2 construct (SEQ ID NO: 24). Particular nucleotides were chosen based on computational modeling predicting alternative secondary structure conformations in an IRE-1 binding sequence (see, for example, Figure EV5 in Peschek et al.). As shown in FIG.6B, HEK293T cells expressing a GARYswitch_2 (SEQ ID NO: 24) construct resulted in low payload expression under physiological conditions (e.g., no or low stress) and induction of high levels of payload expression under induced ER stress (5 uM Thapsigargin treatment) compared to control HEK293T cells which did not have GARY constructs. Expression levels of the exemplary payload in cells expressing GARYswitch_2, in the absence or presence of stress, was comparable to the exemplary payload expression level in cells expressing the GARYswitch construct having the IRE-1 binding sequence of SEQ ID NO: 25. Without wishing to be bound by any particular theory, these results suggest that more than one secondary structure (e.g., other than that formed by naturally occurring XBP1 RNA) may be recognized and / or cleaved by IRE-1.

[0487] Together, these results support development of GARYswitch constructs for delivering payloads to cells (e.g., for delivering a therapeutic payload) to achieve sustained or increased payload expression under cellular stress conditions. EQUIVALENTS

[0488] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Further, it should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the claims that follow. Page 77 of 84 12871572v1

Claims

1. Attorney Docket: 2012611-0180 CLAIMS What is claimed is:

1. A polynucleotide comprising: (i) an Inositol-requiring Enzyme 1 (IRE-1) binding sequence, (ii) an intervening sequence, (iii) an AUG translation initiation codon, and (iv) a payload sequence encoding a payload, wherein the intervening sequence is located between the IRE1-binding sequence and the payload sequence.

2. The polynucleotide of claim 1, wherein the polynucleotide comprises in 5’ to 3’ order: (a) the IRE-1 binding sequence, (b) the intervening sequence, (c) the AUG translation initiation codon, and (d) the payload sequence, optionally wherein the polynucleotide further comprises a 5’ UTR.

3. The polynucleotide of claim 2, wherein the 5’ UTR comprises (a) the IRE-1 binding sequence, and (b) the intervening sequence.

4. The polynucleotide of claim 2, wherein the 5’ UTR does not comprise (c) the AUG translation initiation codon.

5. The polynucleotide of any one of claims 2-4, wherein the polynucleotide is characterized such that when administered to a cell, tissue or subject, expression of the payload is observed at similar levels under physiological conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

6. The polynucleotide of any one of claims 2-5, wherein the polynucleotide is characterized such that when administered to a cell, tissue or subject, increased expression of the payload is observed under cellular stress conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence, optionally wherein expression of the payload under cellular stress conditions is similar to or greater than expression of the payload under physiological conditions.

7. The polynucleotide of claim 1, wherein the polynucleotide comprises in 5’ to 3’ order: (a) the AUG translation initiation codon, (b) the IRE-1 binding sequence, (c) the intervening sequence, and (d) the payload sequence, optionally wherein the polynucleotide further comprises a 5’ UTR. Page 78 of 84 12871572v1 Attorney Docket: 2012611-0180 8. The polynucleotide of claim 7, wherein the 5’ UTR comprises does not comprise (a) the AUG translation initiation codon, (b) the IRE-1 binding sequence, and (c) the intervening sequence.

9. The polynucleotide of claim 7 or 8, wherein the polynucleotide is characterized such that when administered to a cell, tissue or subject, minimal (e.g., low), expression of the payload is observed under physiological conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

10. The polynucleotide of any one of claims 7-9, wherein the polynucleotide is characterized such that when administered to a cell, tissue or subject, increased expression of the payload is observed under cellular stress conditions as compared to an otherwise similar polynucleotide without the IRE-1 binding sequence.

11. The polynucleotide of any one of the preceding claims, wherein the intervening sequence comprises one or more UAG, UAA and / or UGA codons and / or one or more linker sequences, optionally wherein the intervening sequence comprises any two of the following codons: UAG, UAA or UGA.

12. The polynucleotide of any one of the preceding claims, wherein the polynucleotide further comprises a 5’ cap, a 3’ UTR and / or a polyA tail.

13. The polynucleotide of any one of the preceding claims, wherein the IRE-1 binding sequence comprises a sequence that is recognized by a nuclease domain of IRE-1, optionally wherein the IRE-1 binding sequence comprises: (i) a CUGCAG sequence, (ii) a CCGCAG sequence, or (iii) (i) and (ii).

14. The polynucleotide of any one of the preceding claims, wherein the IRE-1 binding sequence comprises: (i) an XBP1 sequence or a fragment or variant thereof, optionally wherein the XBP1 sequence comprises the nucleotide sequence provided in: SEQ ID NO: 5 or a sequence with 85% to SEQ ID NO:5, SEQ ID NO: 6 or a sequence with at least 85% identity to SEQ ID NO: 6, or (ii) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49 or a sequence with at least 85% identity to any one of the foregoing SEQ ID NOs.

15. The polynucleotide of any one of claims 6-14, wherein the cellular stress response is endoplasmic-reticulum (ER) stress, optionally wherein ER stress comprises the unfolded protein response (UPR), further optionally wherein UPR activates IRE-1 nuclease activity. Page 79 of 84 12871572v1 Attorney Docket: 2012611-0180 16. The polynucleotide of any one of claims 5, 6, and 9, wherein physiological conditions refers to a cellular condition in which a cell is not under ER stress.

17. The polynucleotide of any one of the preceding claims, wherein the polynucleotide is or comprises a polyribonucleotide.

18. The polynucleotide of claim 17, wherein the polyribonucleotide comprises one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof, optionally wherein the one or more modified ribonucleotides comprises a nucleoside comprising: N4- acetylcytidine, 5-hydroxymethyluridine, N1-methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza- uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5-methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6- diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (m1 I), wyosine (imG), methylwyosine (mimG), 5-hydroxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-methoxycytidine, 5- propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2’-O-methyluridine, 2’-O-methyl-5-methyluridine, 2’-fluoro-2’-deoxyuridine, 2’-amino-2’-deoxyuridine, 2’-azido-2’-deoxyuridine, 4- thiouridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5- bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2’-O-methyl-pseudouridine, N1-hydroxypseudouridine, 2’-O-methyl-N1-methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, ara-uridine, N6- methyladenosine, 2-aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 8-oxoadenosine, thienoguanosine, 7- deazaguanosine, 8-oxoguanosine, 6-O-methylguanine, or any combination thereof.

19. The polynucleotide of claim 18, wherein the one or more modified ribonucleotides comprise modified ribonucleotides that comprise a 2’-O-acetylated ribose, optionally wherein the one or modified ribonucleotides comprise: (i)2’-O-acetyl guanosine, (ii) 2’-O-acetyl uridine, (iii) 2’-O-acetyl cytidine, (iv) 2’-O-acetyl N1-methylpseudouridine, or (v) any combination of (i) – (iv).

20. The polynucleotide of any one of claims 17-19, wherein the polyribonucleotide comprises a cap structure and the cap structure does not comprise a 2’-O-acetylated ribose.

21. The polynucleotide of any one of claims 17-19, wherein the polyribonucleotide comprises a cap structure and the cap structure comprises a 2’-O-acetylated ribose. Page 80 of 84 12871572v1 Attorney Docket: 2012611-0180 22. The polynucleotide of any one of the preceding claims, wherein the polynucleotide comprises a coding region.

23. The polynucleotide of claim 22, wherein the coding region encodes the payload.

24. The polynucleotide of claim 22 or 23, wherein the polynucleotide comprises an RNA payload.

25. The polynucleotide of any one of the preceding claims, wherein the payload is an antigen, an enzyme, an antibody, a secreted protein, a cell surface protein, a helper protein, or a variant or fragment of any of the foregoing, optionally wherein the helper protein is a chaperone or ER guardian polypeptide, or a fragment or variant thereof.

26. One or more compositions comprising a polynucleotide of any one of the preceding claims.

27. The composition of claim 26, comprising: (i) a first composition comprising a polynucleotide that encodes a payload, wherein the payload is an antigen, an enzyme, an antibody, a secreted protein, a cell surface protein, a helper protein, or a variant or fragment of any of the foregoing, optionally wherein the helper protein is a chaperone or ER guardian polypeptide, or a fragment or variant thereof, and (ii) a second composition comprising a second polynucleotide.

28. The composition of claim 27, wherein the second polynucleotide encodes a second payload, optionally wherein: (i) the second payload is different than the payload encoded by the polynucleotide in the first composition, (ii) the second payload is an antigen, an enzyme, an antibody, a secreted protein, a cell surface protein, or a variant or fragment of any of the foregoing, (iii) the second payload is not a helper protein, and / or (iv) the second payload comprises a fusion polypeptide.

29. The composition of claim 28, wherein the fusion polypeptide comprises a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus, optionally wherein the Sbi complement C3d-binding polypeptide comprises an Sbi domain III polypeptide and / or an Sbi domain IV polypeptide.

30. The composition of any one of claims 27-29, wherein (i) and (ii) are in separate compositions.

31. The composition of any one of claims 27-29, wherein (i) and (ii) are in the same composition. Page 81 of 84 12871572v1 Attorney Docket: 2012611-0180 32. The composition of any one of claims 26-31, wherein the composition is a pharmaceutical composition, optionally wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

33. The composition of claim 32, wherein the pharmaceutical composition is formulated for intramuscular, intradermal, intravenous, subcutaneous, mucosal, inhaled, or intranodal delivery.

34. A cell comprising a polynucleotide of any one of claims 1-25, or a composition of any one of claims 26-33, optionally wherein the cell is in vivo or ex vivo.

35. The cell of claim 34, wherein the cell expresses one or more polypeptides encoded by the polynucleotide.

36. A method of delivering a composition of any one of claims 26-33 to a cell, tissue or subject.

37. The method of claim 36, wherein delivering comprises administering the composition comprising to the cell, tissue or subject.

38. The method of claim 36 or 37, wherein the method is a treatment method.

39. The method of any one of claims 36-38, wherein the method is: (i) a method to stimulate an immune response, (ii) an antibody therapy method, (iii) an immune-modulation method, (iv) a vaccination method, (v) a gene therapy method, (vi) a cell therapy engineering method, (vii) an immunotherapy method, (viii) a protein replacement therapy method, (ix) a chemotherapeutic method, or (x) a combination of (i)-(ix).

40. A method of stimulating an immune response to an antigen, comprising delivering a composition of any one of claims 26-33, to a cell, tissue or subject.

41. The composition of any one of claims 26-33, for use in delivering the polynucleotide to a cell, tissue, or subject.

42. Use of a polynucleotide according to any one of claims 1-25, or a composition according to any one of claims 26-33, in the preparation of a medicament for delivering the nucleic acid expression system or composition to a cell, tissue, or subject. Page 82 of 84 12871572v1 Attorney Docket: 2012611-0180 43. The composition for use of claim 41, or the use of claim 42, wherein the use is for: (i) stimulating an immune response, (ii) an antibody therapy, (iii) immune-modulation, (iv) vaccination, (v) gene therapy, (vi) cell therapy engineering, (vii) immunotherapy, (viii) protein replacement therapy, (ix) chemotherapy, or (x) any combination of (i)-(ix). Page 83 of 84 12871572v1

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