Helper proteins for increased secretion

Abstract

Disclosed herein is a nucleic acid expression system comprising (i) a polynucleotide comprising a payload sequence, and (ii) at least one polynucleotide that encodes one or more helper polypeptides. Methods of making and using the disclosed nucleotide expression system, polypeptides, and compositions are also provided.

Description

Attorney Docket: 2012611-0187 HELPER PROTEINS FOR INCREASED SECRETION CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 707,967 filed on October 16, 2024, the entire contents of which are hereby incorporated by reference in its 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 4, 2025 is named 2012611-0187, and is 137,851 bytes in size. BACKGROUND

[0003] Biomanufacturing relies on the secretion of high amounts of biological therapeutics from live cells for their production. Improvements in protein secretion could enable greater production of biologics for less cost, smaller vaccine doses, and simpler experimental procedures for protein studies. SUMMARY

[0004] The present disclosure recognizes certain challenges with achieving meaningful expression and / or secretion levels (e.g., therapeutically meaningful levels) of exogenous polypeptides from mammalian cells. For example, the present disclosure recognizes that obtaining a desired level of expression and / or secretion of an exogenous polypeptide from a mammalian cell in which the exogenous polypeptide is encoded by an exogenous nucleic acid, e.g., a DNA or RNA therapeutic, depends on a variety of cellular factors and cellular machinery involved in protein translation, folding and / or trafficking. Furthermore, expression and / or secretion of large polypeptides or polypeptides that are not generally encountered by cellular machinery (such as fusion polypeptides or mutant polypeptides) is even more challenging at least due to the additional burdens placed on cellular translation, folding and / or trafficking machinery by such polypeptides. These challenges are particularly evident in the fields of biomanufacturing and RNA therapeutics, which rely on protein secretion from cells that are delivered a particular RNA therapeutic.

[0005] Accordingly, the present disclosure provides solutions to address this challenge of low secretion and / or expression of exogenous polypeptides from mammalian cells. Disclosed herein are certain insights and technologies for increasing secretion of polypeptides encoded by an exogenous nucleic acid, e.g., a DNA or RNA therapeutic. Technologies provided herein include nucleic acid expression systems that increase secretion and / or expression of polypeptides from mammalian cells, e.g., by reducing and / or eliminating stressors imposed on cellular translation, folding and / or trafficking machinery by the expression of such polypeptides. The present disclosure further provides the insight that increased levels polypeptide secretion and / or expression from cells can be beneficial to a number of processes, including the manufacture of polypeptides (e.g., therapeutic polypeptides) and expression of polypeptides in mammalian cells, tissues or subjects following delivery of polynucleotides encoding such polypeptides. Page 1 of 117 12869933v1Attorney Docket: 2012611-0187

[0006] Provided herein is a nucleic acid expression system comprising: (i) a polynucleotide comprising a payload sequence, and (ii) at least one polynucleotide that encodes one or more helper polypeptides. In some embodiments, one or more helper polypeptides comprise: (a) a chaperone or ER guardian polypeptide, or a fragment or variant thereof, (b) an unfolded protein response (UPR) inhibitor polypeptide, or a fragment or variant thereof, (c) an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof, (d) a SNARE polypeptide, or a fragment or variant thereof, (e) a ubiquitin protein ligase polypeptide, or a fragment or variant thereof, (f) an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof, (g) a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof, or (h) any combination of (a)-(g). In some embodiments, a fragment or variant thereof is a functional fragment, a functional variant, or a functional fragment variant thereof. Also provided herein are compositions comprising a nucleic acid expression system disclosed herein, and methods of administering a nucleic acid expression system disclosed herein, and cells comprising the same.

[0007] In some embodiments, one or more helper polypeptides described herein is or comprises a chaperone or ER guardian polypeptide, or a fragment or variant thereof. In some embodiments, a chaperone or ER guardian polypeptide described herein comprises a TAPT1 polypeptide, chaperone or a fragment or variant thereof. In some embodiments, a TAPT1 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, a TAPT1 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 50, or a codon optimized version thereof.

[0008] In some embodiments, a chaperone or ER guardian polypeptide described herein is or comprises a heat shock protein (HSP) polypeptide, or a fragment or variant thereof. In some embodiments, a HSP polypeptide comprises a HSP70 family member polypeptide, or a fragment or variant thereof. In some embodiments, a HSP70 family member polypeptide comprises a HSPA13 polypeptide, or a fragment or variant thereof. In some embodiments, a HSP polypeptide comprises a HSP40 family member polypeptide, or a fragment or variant thereof. In some embodiments, a HSP40 family member polypeptide comprises a DNAJC8 polypeptide, or a fragment or variant thereof.

[0009] In some embodiments, one or more helper polypeptides described herein is or comprises a UPR inhibitor polypeptide, or a fragment or variant thereof. In some embodiments, a UPR inhibitor polypeptide described herein comprises a K3L polypeptide, or a fragment or variant thereof. In some embodiments, a K3L polypeptide described herein comprises a K3L_H47R polypeptide. In some embodiments, a K3L polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, a K3L polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 52, or a codon optimized version thereof.

[0010] In some embodiments, one or more helper polypeptides described herein is or comprises a UPR inhibitor polypeptide, or a fragment or variant thereof. In some embodiments, a UPR inhibitor polypeptide described herein comprises a gamma 134.5 polypeptide, or a fragment or variant thereof. In some embodiments, a gamma 134.5 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, a gamma 134.5 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 53, or a codon optimized version thereof. Page 2 of 117 12869933v1Attorney Docket: 2012611-0187

[0011] In some embodiments, one or more helper polypeptides described herein is or comprises a GADD34 polypeptide, or a fragment or variant thereof. In some embodiments, a GADD34 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, a GADD34 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 54, or a codon optimized version thereof.

[0012] In some embodiments, a IRE1b polypeptide described herein is or comprises a IRE1b_delCTD polypeptide. In some embodiments, a IRE1b polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, a IRE1b polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 51, or a codon optimized version thereof.

[0013] In some embodiments, one or more helper polypeptides described herein is or comprises an ERAD polypeptide or a fragment or variant thereof. In some embodiments, an ERAD polypeptide described herein comprises a Derlin-1 polypeptide or a fragment or variant thereof, or a JKAMP polypeptide, or a fragment or variant thereof. In some embodiments, a Derlin-1 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16. In some embodiments, a Derlin-1 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 55, or a codon optimized version thereof.

[0014] In some embodiments, one or more helper polypeptides described herein is or comprises a SNARE polypeptide or a fragment or variant thereof. In some embodiments, a SNARE polypeptide described herein comprises a GosR1 polypeptide or a fragment or variant thereof, a Stx5 polypeptide or a fragment or variant thereof, a Bet1 polypeptide or a fragment or variant thereof, or any combination thereof. In some embodiments, a SNARE polypeptide described herein comprises a GosR1 polypeptide or a fragment or variant thereof, a Stx5 polypeptide or a fragment or variant thereof, and a Bet1 polypeptide or a fragment or variant thereof. In some embodiments, a GosR1 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, a GosR1 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 56, or a codon optimized version thereof. In some embodiments, a Stx5 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, a Stx5 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 57, or a codon optimized version thereof. In some embodiments, a Bet1 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, a Bet1 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 58, or a codon optimized version thereof.

[0015] In some embodiments, one or more helper polypeptides described herein is or comprises a ubiquitin protein ligase polypeptide or a fragment or variant thereof. In some embodiments, ubiquitin protein ligase polypeptides described herein comprises: a NEDD4L polypeptide or a fragment or variant thereof. In some embodiments, a NEDD4L polypeptide described herein comprises one or more mutations, wherein the one or more mutations comprise Y679C, Q694H, E893K, R897Q, or any combination thereof. In some embodiments, a NEDD4L Page 3 of 117 12869933v1Attorney Docket: 2012611-0187 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 20, 21, 22, 23, or 24. In some embodiments, a NEDD4L polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of any one of SEQ ID NOs: 59, 60, 61, 62, or 63,or a codon optimized version thereof.

[0016] In some embodiments, one or more helper polypeptides described herein is or comprises an E3 ubiquitin ligase substrate polypeptide or a fragment or variant thereof. In some embodiments, an E3 ubiquitin ligase substrate polypeptide described herein comprises: a RIPK2 polypeptide or a fragment or variant thereof, an ERBB4 polypeptide or a fragment or variant thereof, an AKT1 polypeptide or a fragment or variant thereof, a Notch1 polypeptide or a fragment or variant thereof, a NEMO polypeptide or a fragment or variant thereof, or any combination thereof. In some embodiments, an E3 ubiquitin ligase substrate polypeptide described herein comprises: a RIPK2 polypeptide or a fragment or variant thereof, an ERBB4 polypeptide or a fragment or variant thereof, an AKT1 polypeptide or a fragment or variant thereof, a Notch1 polypeptide or a fragment or variant thereof, and a NEMO polypeptide or a fragment or variant thereof. In some embodiments, a RIPK2 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, a RIPK2 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 64, or a codon optimized version thereof. In some embodiments, a ERBB4 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, a ERBB4 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 65, or a codon optimized version thereof. In some embodiments, an AKT1 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 27. In some embodiments, an AKT1 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 66, or a codon optimized version thereof. In some embodiments, a Notch1 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 28. In some embodiments, a Notch1 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 67, or a codon optimized version thereof. In some embodiments, a NEMO polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 29. In some embodiments, a NEMO polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 68, or a codon optimized version thereof.

[0017] In some embodiments, one or more helper polypeptides described herein is or comprises polypeptide upregulated during plasma cell differentiation or a fragment or variant thereof. In some embodiments, a polypeptide upregulated during plasma cell differentiation described herein comprises: a GJB2 polypeptide or a fragment or variant thereof, a NDFIP2 polypeptide or a fragment or variant thereof, a CD300A polypeptide or a fragment or variant thereof, a XCR1 polypeptide or a fragment or variant thereof, a STBD1 polypeptide or a fragment or variant thereof, a EDA2R polypeptide or a fragment or variant thereof, a TMEM200A polypeptide or a fragment or variant thereof, a GPR114 polypeptide or a fragment or variant thereof, a DPAGT1 polypeptide or a fragment or variant thereof, a CCR2 polypeptide or a fragment or variant thereof, or any combination thereof. In some embodiments, a GJB2 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, a GJB2 polypeptide described herein is encoded by a nucleic acid sequence Page 4 of 117 12869933v1Attorney Docket: 2012611-0187 having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 69, or a codon optimized version thereof. In some embodiments, a NDFIP2 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, a NDFIP2 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 70, or a codon optimized version thereof. In some embodiments, a CD300A polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, a CD300A polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 71, or a codon optimized version thereof. In some embodiments, a XCR1 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 33. In some embodiments, a XCR1 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 72, or a codon optimized version thereof. In some embodiments, a STBD1 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 34. In some embodiments, a STBD1 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 73, or a codon optimized version thereof. In some embodiments, a EDA2R polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, a EDA2R polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 74, or a codon optimized version thereof. In some embodiments, a TMEM200A polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 36. In some embodiments, a TMEM200A polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 75, or a codon optimized version thereof. In some embodiments, a GPR114 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 37. In some embodiments, a GPR114 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 76, or a codon optimized version thereof. In some embodiments, a DPAGT1 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 38. In some embodiments, a DPAGT1 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 77, or a codon optimized version thereof. In some embodiments, a CCR2 polypeptide described herein comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 39. In some embodiments, a CCR2 polypeptide described herein is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 78, or a codon optimized version thereof.

[0018] In some embodiments, one or more helper polypeptides described herein is or comprises one or more UPR inhibitor polypeptides, and one or more additional helper polypeptides. In some embodiments, one or more helper polypeptides described herein comprises is other than a UPR inhibitor polypeptide. In some embodiments, one or more helper polypeptides described herein comprises any one of: (a) a chaperone or ER guardian polypeptide, or a fragment or variant thereof, or (c) an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof, (d) a SNARE polypeptide, or a fragment or variant thereof, (e) a ubiquitin protein ligase polypeptide, or a fragment or variant thereof, (f) an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof, (g) a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof. Page 5 of 117 12869933v1Attorney Docket: 2012611-0187

[0019] In some embodiments, at least one polynucleotide that comprises a sequence encoding a helper polypeptide increases expression and / or secretion of a payload polypeptide encoded by the polynucleotide comprising a payload sequence. In some embodiments, a nucleic acid expression system described herein is characterized in that when administered to a cell, tissue, or subject, increased payload expression and / or secretion is observed as compared to expression and / or secretion of a payload in a cell, tissue or subject administered an otherwise similar polynucleotide comprising a payload sequence without at least one polynucleotide comprising a sequence encoding a helper polypeptide.

[0020] In some embodiments, increased payload expression described herein comprises at least 2-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least-500 fold, at least 600-fold, at least 700-fold, or at least 800-fold increased expression of the payload.

[0021] In some embodiments, increased payload secretion described herein comprises at least 2-fold, at least 10- fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, or at least 500-fold increased secretion of the payload.

[0022] In some embodiments, increased secretion and / or expression described herein comprises increased trafficking through a secretory pathway, increased proper folding of the polypeptide, reduced degradation of the polypeptide, reduced aggregation of the polypeptide, or any combination thereof.

[0023] In some embodiments, a polynucleotide described herein comprising a payload sequence is DNA. In some embodiments, at least one polynucleotide comprising a sequence that encodes a helper polypeptide is DNA.

[0024] In some embodiments, a polynucleotide described herein comprising a payload sequence is RNA. In some embodiments, at least one polynucleotide comprising a sequence that encodes a helper polypeptide is RNA.

[0025] In some embodiments, a polynucleotide is a polyribonucleotide comprising one or more modified ribonucleotides. In some embodiments, one or more ribonucleotides comprise: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0026] In some embodiments, one or more modified ribonucleotides have: a 5’ monophosphate, a 5’ diphosphate, or a 5’ triphosphate. In some embodiments, one or more modified ribonucleotides comprises a 5’ triphosphate.

[0027] In some embodiments, one or more modified ribonucleotides comprise a nucleoside comprising an acetyl group. In some embodiments, a nucleoside is N4-acetylcytidine. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 9A. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 9B 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 9B.

[0028] In some embodiments, a nucleoside has a structure of: Page 6 of 117 12869933v1Attorney Docket: 2012611-0187 O H3C NH O .

[0029] In some embodiments, one or more have the structure of:O H3C NH O .

[0030] In some embodiments, one orhave the structure of: O H3CNH O .

[0031] In some embodiments, one orhave the structure of: O H3CNH O .

[0032] In some embodiments, acomprises one or more modified ribonucleotides that have the following structure: O O ,Page 7 of 117 12869933v1Attorney Docket: 2012611-0187 wherein indicates the position of attachment to an adjacent ribonucleotide.

[0033] 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 of

[0034] 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 of

[0035] In some embodiments, a polyribonucleotide comprises cytidine nucleosides, and at least 5% of cytidine nucleosides in a polyribonucleotide comprise N4-acetylcytidine.

[0036] In some embodiments, a polyribonucleotide comprises cytidine nucleosides, and less than 100% of cytidine nucleosides in the polyribonucleotide comprise N4-acetylcytidine.

[0037] 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.

[0038] In some embodiments, one or more modified ribonucleotides comprise a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9A. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9B when incorporated in a polynucleotide (e.g., polyribonucleotide). In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprise one or more modified ribonucleotides that have a structure provided in Table 9B. Page 8 of 117 12869933v1Attorney Docket: 2012611-0187

[0039] In some embodiments, a nucleoside has a structure of: OH O NH O .

[0040] In some embodiments, one or more have a structure of:OH O NH O .

[0041] In some embodiments, one orhave a structure of: OH O N O .

[0042] In some embodiments, one orhave a structure of: OH O NH O .

[0043] In some embodiments, acomprises one or more modified ribonucleotides that have the following structure: OH O O ,wherein indicates the position of Page 9 of 117 12869933v1Attorney Docket: 2012611-0187

[0044] 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 of

[0045] 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 of

[0046] In some embodiments, a polyribonucleotide comprises uridine nucleosides and at least 5% of uridine nucleosides in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0047] In some embodiments, a polyribonucleotide comprises uridine nucleosides and less than 100% of uridine nucleosides in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0048] 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.

[0049] In some embodiments, a polyribonucleotide comprises uridine nucleosides and more than 60% of uridine nucleosides in a polyribonucleotide comprise 5-hydroxymethyluridine.

[0050] In some embodiments, one or more modified ribonucleotides comprise: 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’- Page 10 of 117 12869933v1Attorney Docket: 2012611-0187 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. 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.

[0051] In some embodiments, one or more modified ribonucleotides have a structure of: X O ,

[0052] (a) wherein R is aand

[0053] (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).

[0054] In some embodiments, one or more modified ribonucleotides comprise: (i) a 2’-O-acetylated ribose and (ii) an adenine nucleobase. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9C. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9D 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 9D.

[0055] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: H2N N ,wherein indicates the position of

[0056] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 11 of 117 12869933v1Attorney Docket: 2012611-0187 H2N N N N , wherein indicates the position of

[0057] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: H N N N N , wherein indicates the position of

[0058] In some embodiments, one or more modified ribonucleotides comprise: (i) a 2’-O-acetylated ribose and (ii) a guanine nucleobase. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 9D 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 9D.

[0059] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NH , wherein indicates the position of

[0060] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 12 of 117 12869933v1Attorney Docket: 2012611-0187 O NH N NH N , wherein indicates the position of

[0061] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NH N NH , wherein indicates the position of

[0062] In some embodiments, one or more modified ribonucleotides comprise: (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 have a structure provided in Table 9C. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9D 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 9D.

[0063] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: NH2N O , wherein indicates the position of

[0064] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 13 of 117 12869933v1Attorney Docket: 2012611-0187 NH2N O , wherein indicates the position of

[0065] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: NH2N O , wherein indicates the position of

[0066] In some embodiments, one or more modified ribonucleotides comprising a 2’-O-acetylated ribose comprise a N4-acetylcytosine nucleobase. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9C. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9D 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 9D.

[0067] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NH O , wherein indicates the position of

[0068] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 14 of 117 12869933v1Attorney Docket: 2012611-0187 O H3C NH O , wherein indicates the position of

[0069] 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 of

[0070] In some embodiments, one or more modified ribonucleotides comprising a 2’-O-acetylated ribose comprise a uracil nucleobase (e.g., uracil or 5-hydroxymethyluracil). In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9C. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9D 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 9D.

[0071] In In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NH O ,wherein indicates the position of

[0072] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 15 of 117 12869933v1Attorney Docket: 2012611-0187 O N O , wherein indicates the position of

[0073] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NH O , wherein indicates the position

[0074] In some embodiments, one or more modified ribonucleotides comprising a 2’-O-acetylated ribose comprise a 5-hydroxymethyluracil nucleobase. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9C. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9D 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 9D.

[0075] 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 of

[0076] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 16 of 117 12869933v1Attorney Docket: 2012611-0187 OH O NH O , wherein indicates the position of

[0077] 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 of

[0078] In some embodiments, one or more modified ribonucleotides comprising a 2’-O-acetylated ribose comprise a N1-methylpseudouracil nucleobase. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9C. In some embodiments, one or more modified ribonucleotides have a structure provided in Table 9D 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 9D.

[0079] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NNHO , wherein indicates the position of

[0080] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 17 of 117 12869933v1Attorney Docket: 2012611-0187 O NNH, wherein indicates the position of

[0081] In some embodiments, a polynucleotide (e.g., polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NNHO , wherein indicates the position of

[0082] In some embodiments of any of the polyribonucleotides disclosed herein, at least 5% of ribose moieties are acetylated (e.g., 2’-O-acetylated).

[0083] In some embodiments of any of the polyribonucleotides disclosed herein, about 5% to about 99% of ribose moieties are acetylated (e.g., 2’-O-acetylated).

[0084] 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.

[0085] 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.

[0086] 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.

[0087] In some embodiments of any of the polyribonucleotides disclosed herein, a polyribonucleotide comprises a coding region. 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.

[0088] In some embodiments, a polynucleotide comprising a payload sequence described herein comprises a coding region. In some embodiments, a coding region described herein encodes a payload.

[0089] In some embodiments, a payload described herein is or comprises a polypeptide.

[0090] In some embodiments, a payload described herein is or comprises an RNA payload.

[0091] In some embodiments, a payload described herein is or comprises a fusion polypeptide. In some embodiments, a polynucleotide encodes a fusion polypeptide payload. In some embodiments, a fusion polypeptide Page 18 of 117 12869933v1Attorney Docket: 2012611-0187 comprises (a) an antigen or a fragment or a variant thereof (e.g., an immunogenic fragment or immunogenic variant thereof), and (b) a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus. In some embodiments, an Sbi complement C3d-binding polypeptide comprises Sbi domain III and / or Sbi domain IV.

[0092] In some embodiments, a payload described herein comprises an antigen or a fragment thereof. In some embodiments, a payload described herein comprises an antigen or an immunogenic fragment thereof.

[0093] In some embodiments, a payload described herein comprises an antibody or a fragment thereof.

[0094] In some embodiments, a payload described herein comprises a secreted protein or a fragment thereof.

[0095] In some embodiments, a payload described herein comprises a cell-surface associated protein or a fragment thereof.

[0096] In some embodiments, a payload described herein comprises an intracellular protein or a fragment thereof.

[0097] Among other things, the present disclosure provides one or more compositions comprising a nucleic acid expression system comprising (i) a polynucleotide comprising a payload sequence, and (ii) at least one polynucleotide that encodes one or more helper polypeptides. In some embodiments, (i) and (ii) of a nucleic acid expression system as described herein are in separate compositions. In some embodiments, (i) and (ii) of a nucleic acid expression system as described herein are administered separately. In some embodiments, (i) and (ii) of a nucleic acid expression system as described herein are administered at substantially the same time. In some embodiments, in a nucleotide expression system as described herein, (i) is administered followed by (ii). In some embodiments, in a nucleotide expression system as described herein, (ii) is administered followed by (i). In some embodiments, (i) and (ii) of a nucleic acid expression system as described herein are in the same composition.

[0098] In some embodiments, a composition as described herein is a pharmaceutical composition. In some embodiments, a pharmaceutical composition described herein comprises one or more pharmaceutically acceptable excipients. In some embodiments, a pharmaceutical composition described herein is formulated for intramuscular, intradermal, or intravenous delivery.

[0099] In some embodiments, a set of polypeptides is encoded by a nucleic acid expression system described herein.

[0100] In some embodiments, a cell comprises a nucleic acid expression system as described herein. In some embodiments, a cell described herein is in vivo. In some embodiments, a cell described herein is ex vivo. In some embodiments, a nucleotide acid expression system described herein. In some embodiments, a cell described herein expresses one or more polypeptides encoded by the nucleic acid expression system.

[0101] The present disclosure, among other things, provides a method of delivering a nucleic acid expression system nucleic acid expression system described herein, or a composition comprising the same, to a cell, tissue, or subject. In some embodiments, delivering comprises administering the nucleic acid expression system or a composition comprising the same to the cell, tissue or subject.

[0102] In some embodiments, a method disclosed herein is a treatment method. Page 19 of 117 12869933v1Attorney Docket: 2012611-0187

[0103] In some embodiments, a method disclosed herein is a prevention method.

[0104] In some embodiments, a method is a method to stimulate an immune response. In some embodiments, a method is an antibody therapy method. In some embodiments, a method is an immune-modulation method. In some embodiments, a method is a vaccination method. In some embodiments, a method is a gene therapy method. In some embodiments, a method is a cell therapy engineering method. In some embodiments a method is an immunotherapy method. In some embodiments a method is a protein replacement therapy method. In some embodiments a method is a chemotherapeutic method.

[0105] In some embodiments, a subject is a human.

[0106] The present disclosure, among other things, also provides a method of stimulating an immune response to an antigen, comprising delivering a nucleic acid expression system described herein to a cell, tissue or subject. In some embodiments, a subject is human.

[0107] These, and other aspects encompassed by the present disclosure, are described in more detail below and in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] FIGS.1A-1G are graphs depicting secretion of an exemplary payload (e.g., NanoLuc-RBD_var protein) from HEK293T cells transfected with exemplary helper proteins. NanoL-RBD_var protein secretion was measured by screening for NanoLuc activity using the Nano-Glo Luciferase Assay System (Promega).

[0109] FIGs.2A-2D are graphs depicting secretion of an exemplary payload (e.g., NanoLuc-RBD_var protein) from HEK293T cells transfected with combinations of exemplary helper proteins. NanoL-RBD_var protein secretion was measured by screening for NanoLuc activity using the Nano-Glo Luciferase Assay System (Promega).

[0110] FIG.3 is a graph depicting secretion of exemplary payloads from HEK293T cells transfected with combinations of exemplary helper proteins. The exemplary payloads tested were: wild type SARS-CoV-2 Spike RBD with a Spike signal peptide (Ssp-RBD), RBD_var with Ssp, RBD_var with a sequence derived from the yeast prepro-α- factor PPa, RBD_var with an engineered prepro-α-factor sequence PPa_v2, an anti-CD3 x anti-gp75 bi-specific T cell engager (BiTE), and an anti-CD16 x IL-15 x anti-gp75 tri-specific natural killer cell engager (TriKE). Payload protein secretion was measured by screening for NanoLuc activity using the Nano-Glo Luciferase Assay System (Promega). CERTAIN DEFINITIONS

[0111] 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. Page 20 of 117 12869933v1Attorney Docket: 2012611-0187

[0112] 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.

[0113] 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 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.

[0114] Delivery / contacting: As used interchangeably herein, the term “delivery,” “delivering,” or “contacting” refers to introduction of a polynucleotide (e.g., as described herein) or a fusion polypeptide (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) or a fusion polypeptide (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) or a fusion polypeptide (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) or a fusion polypeptide (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) or a fusion polypeptide (e.g., as described herein) can be introduced into a target cell in a subject by administering a polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) to a subject. Page 21 of 117 12869933v1Attorney Docket: 2012611-0187

[0115] 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.

[0116] 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.

[0117] 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. In some embodiments, a fragment is a functional fragment (e.g., a fragment that maintains one or more functions of a parent polynucleotide or polypeptide.

[0118] 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, Page 22 of 117 12869933v1Attorney Docket: 2012611-0187 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, 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.

[0119] 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 Page 23 of 117 12869933v1Attorney Docket: 2012611-0187 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.

[0120] 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.

[0121] 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.

[0122] 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 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. In some Page 24 of 117 12869933v1Attorney Docket: 2012611-0187 embodiments, a variant is a functional variant. In some embodiments, a variant is a variant of a fragment (e.g., a polynucleotide fragment or a polypeptide fragment).

[0123] 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. Certain 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

[0124] The efficiency of protein secretion from living cells is an important factor in the efficacy and / or utility of many treatment modalities that utilize cellular machinery for the secretion and / or expression of therapeutic polypeptides. For example, biomanufacturing of biological therapeutics (such as peptide-based therapeutics) often requires secretion of high amounts of polypeptides from live cells. As another example, RNA therapeutics that encode polypeptide payloads can rely on the secretion of such polypeptide payloads from cells of individuals administered the RNA therapeutic. Therefore, enhancements in polypeptide secretion technologies would be beneficial to lower the costs of biologics (e.g., RNA therapeutics) and would also allow for the administration of such biologics at lower doses.

[0125] Protein sequences have evolved over long time scales under many different selective pressures. Without wishing to be bound by any particular theory, one force guiding protein sequence evolution is optimal expression level in a cell, which may be influenced by factors affecting protein translation, folding, and / or trafficking. Additionally, large polypeptides or polypeptides that are not typically encountered by cellular machinery (such as fusion polypeptide or mutated polypeptides) may not be efficiently funneled into the cellular translation, folding, and / or trafficking machinery resulting in lower secretion and / or expression. Additionally, due to inefficient and / or improper processing of such polypeptides, cellular responses such as ER stress responses and unfolded protein responses which decrease the rates of polypeptide processing, may also be triggered. Accordingly, a person of ordinary skill in the art will understand upon reading this disclosure that it can be difficult to express and / or secrete polypeptides at meaningful levels (e.g., therapeutic levels) from cells.

[0126] Certain strategies have been employed to boost polypeptide secretion from live cells, including expression boosters such as: (1) codon optimization, (2) use of particular promoters, (3) addition of N-terminal signal peptide sequences that can increase translocation to the ER, (4) engineering of protein sequences to enhance trafficking through the ER and Golgi, and (5) cell engineering strategies to direct more cellular resources to expression of the protein of interest. However, such efforts (which focus on understanding selective pressures and rules governing protein sequence evolution) have not produced generalizable solutions to address the problem of secreting and / or expressing polypeptides at desired levels, e.g., at therapeutically meaningful levels and to have therapeutic utility. Page 25 of 117 12869933v1Attorney Docket: 2012611-0187

[0127] Among other things, the present disclosure provides certain insights and technologies relating to improved polypeptide secretion and / or expression from cells. In particular, provided herein is the insight that helper proteins can enhance the secretion and / or expression of polypeptides from cells, e.g., mammalian cells. Helper proteins disclosed herein can modulate the function of cellular pathways involved in protein expression and / or secretion. Specifically, helper proteins disclosed herein can: (1) promote proper folding of nascent polypeptides by preventing their degradation, e.g., reducing ER associated degradation (ERAD), (2) reduce ER stress related to accumulation of unfolded and / or misfolded proteins, e.g., by preventing the activation of the unfolded protein response, (3) increase polypeptide secretion by modulating secretory pathways, and / or (4) modulate the activity of the ubiquitin- proteasome pathway.

[0128] This disclosure further recognizes that introducing one or more helper proteins into a cell can increase the secretion and / or expression of a polypeptide payload in the cell, e.g., where the polypeptide payload is encoded by a polynucleotide introduced into said cell. Provided herein are nucleic acid expression systems that can be used to do so. As shown in Examples 2 and 3, co-expression of helper proteins and polypeptide payloads (encoded from separate RNAs co-delivered to cells) enhanced the expression and / or secretion of all polypeptide payloads that were evaluated. The observed enhancement in expression and / or secretion was irrespective of polypeptide payload size or sequence.

[0129] Without wishing to be bound by any particular theory, helper proteins disclosed herein when co-expressed with a payload can enhance expression and / or secretion of payloads through one or a combination of the following mechanisms: (1) helper protein function itself enhances expression and / or secretion of proteins (including a payload); (2) helper protein interacts with other proteins or networks of proteins that function to enhance expression and / or secretion of payload; (3) helper protein impacts the overall kinetics of the cellular secretory system; and / or (4) helper protein serves as an inhibitor, decoy, or pseudo-substrate in one or more pathways that negatively impact protein expression and / or secretion.

[0130] Accordingly, the present disclosure provides, among other things, a solution to enhance protein expression and / or secretion with a nucleic acid expression system comprising: (i) a polynucleotide comprising a payload sequence, and (ii) at least one polynucleotide that encodes one or more helper polypeptides. In some embodiments, one or more helper polypeptides comprise : (a) a chaperone or ER guardian polypeptide, or a fragment or variant thereof, (b) an unfolded protein response (UPR) inhibitor polypeptide, or a fragment or variant thereof, (c) an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof, (d) a SNARE polypeptide, or a fragment or variant thereof, (e) a ubiquitin protein ligase polypeptide, or a fragment or variant thereof, (f) an E3 ubiquitin ligase substrate polypeptide , or a fragment or variant thereof, (g) a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof, or (h) any combination of (a)-(g). Also provided herein are methods of making and using nucleic acid expression systems and compositions comprising the same, e.g., to increase secretion and / or expression of a payload including for therapeutic purposes. Page 26 of 117 12869933v1Attorney Docket: 2012611-0187 Helper Proteins

[0131] Among other things, provided herein are helper polypeptides that can be used to increase secretion and / or expression of payloads when co-delivered to a cell. Without wishing to be bound by theory, helper proteins may enhance expression and / or secretion through one or any combination of the following mechanisms: in some embodiments, a helper protein function may enhance expression and / or secretion of a polypeptide; in some embodiments, a helper protein may interact with other proteins or networks of proteins that function to enhance expression and / or secretion of a polypeptide; in some embodiments, a helper protein may coordinate with cell surface receptors to enhance expression and / or secretion of a polypeptide; and / or in some embodiments, a helper protein may serve as an inhibitor, decoy, or pseudo-substrate in one or more pathways that negatively impact protein expression and / or secretion.

[0132] Also provided herein are nucleic acid expression systems comprising: (i) a polynucleotide comprising a payload sequence, and (ii) at least one polynucleotide that encodes one or more helper polypeptides. Exemplary helper polypeptides disclosed herein include but are not limited to: (a) a chaperone or ER guardian polypeptide or a fragment or variant thereof, (b) an unfolded protein response (UPR) inhibitor polypeptide or a fragment or variant thereof, (c) an ER associated degradation (ERAD) polypeptide or a fragment or variant thereof, (d) a SNARE polypeptide or a fragment or variant thereof, (e) a ubiquitin protein ligase polypeptide or a fragment or variant thereof, (f) an E3 ubiquitin ligase substrate polypeptide or a fragment or variant thereof, (g) a polypeptide upregulated during plasma cell differentiation or a fragment or variant thereof, or (h) any combination of (a)-(g). In some embodiments, a fragment or variant thereof is a functional fragment, a functional variant, or a functional fragment variant thereof. Chaperone Proteins and ER guardian polypeptides

[0133] Chaperone proteins are proteins that can improve exogenous protein expression by aiding nascent polypeptides in proper folding and / or protect folding peptides from degradation. Among other things, provided herein is the insight that co-expression of chaperone protein(s) with a payload can improve secretion and / or expression of a payload polypeptide.

[0134] 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. incorporated herein by reference in its entirety). This complex acts as a chaperone protein, protecting folding polypeptides from degradation (Zhang et al. incorporated herein by reference in its entirety). 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.

[0135] Heat shock proteins (HSPs) are also known to play a role in ensuring the proper folding or refolding of proteins. There are several families of HSPs including HSP90, HSP70, HSP40, HSP60, large HSPs and small HSPs, as disclosed in Hu, Chen et al. “Heat shock proteins: Biological functions, pathological roles, and therapeutic Page 27 of 117 12869933v1Attorney Docket: 2012611-0187 opportunities.” MedComm vol. 3,3 e161. 2 Aug. 2022, doi:10.1002 / mco2.161,their contents of which is hereby incorporated by reference. Without wishing to be bound by any particular theory, chaperone proteins such as TAPT1

[0137] In some embodiments, a helper protein is or comprises a chaperone protein or a fragment or variant thereof, and / or an ER guardian protein, or a fragment or variant thereof. In some embodiments, a helper protein is or comprises a chaperone protein, or a fragment or variant thereof. In some embodiments, a helper protein is or comprises an ER guardian protein, or a fragment or variant thereof.

[0138] In some embodiments, a chaperone protein is or comprises TAPT1, or a variant or fragment thereof.

[0139] In some embodiments, a TAPT1 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: 11. In some embodiments, a TAPT1 polypeptide comprises the amino acid sequence of SEQ ID NO: 11.

[0140] In some embodiments, a TAPT1 polynucleotide comprises a nucleic 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 nucleic acid sequence of SEQ ID NO: 50. In some embodiments, a TAPT1 polynucleotide comprises the sequence of SEQ ID NO: 50.

[0141] In some embodiments, a chaperone protein is or comprises a heat shock protein, or a fragment or variant thereof.

[0142] In some embodiments, a chaperon protein is or comprises a HSP70 family member, or a variant or fragment thereof. In some embodiments, a chaperone protein is or comprises HSPA13, or a variant or fragment thereof.

[0143] In some embodiments, a chaperon protein is or comprises a HSP40 family member, or a variant or fragment thereof. In some embodiments, a chaperone protein is or comprises DNAJC8, or a variant or fragment thereof. Table 1: Exemplary chaperone or ER guardian polypeptide sequence. Exemplary Amino Acid Sequence (SEQ ID NO:) Helper Protein T F LF S EI I G GPage 28 of 117 12869933v1Attorney Docket: 2012611-0187 Table 2: Exemplary chaperone or ER guardian polynucleotide sequence Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Protein TAPT1: ATGGCTGGAGTTGGAGATGCTGCTGCTCCTGGAGAAGGCGGAGGAGGCGGAGTGGACGGTCCTCA C C A AC GC C A TA G G AA G A C G T G G CT C G A A TUnfolded Protein Response Inhibitors

[0144] When a cell experiences stress, specifically ER stress related to the accumulation of unfolded and / or misfolded proteins, the unfolded protein response (herein “UPR”) can be activated (Park et al., incorporated herein by reference in its entirety). To express a protein of interest in cells, the expression system is often engineered so that the protein is expressed at levels significantly higher than what is physiologically relevant for the cell, often by orders of magnitude. Under such circumstances, ER stress and UPR induction are highly likely. Furthermore, if the protein being expressed is a mutant, synthetic, or other difficult-to-express proteins, such proteins may spend more time in unfolded states and present additional burdens to ER functions. Thus, in such situations, activation of the UPR can negatively impact protein expression and / or secretion.

[0145] One function of the UPR is to phosphorylate eIF2α via PERK, attenuating translation. In some embodiments, this functions to reduce the protein burden in the ER, but also reduces the expression of exogenous proteins. In some embodiments, UPR pathways also upregulate ERAD pathway components, resulting in enhanced protein retrotranslocation and degradation, similarly reducing the burden on the ER and reducing exogenous protein expression. In some embodiments, UPR induces expression of pro-apoptotic pathways when ER stress is too severe or prolonged, which can lead to toxicity, cell death, and cessation of protein expression.

[0146] In the IRE1 pathway of the UPR, 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 (Park et al. Page 29 of 117 12869933v1Attorney Docket: 2012611-0187 incorporated herein by reference in its entirety). Under conditions of ER stress, IRE1 oligomerizes and the kinase domain mediates autophosphorylation, activating the nuclease domain RNase activity. The IRE1 nuclease domain splices XBP1 mRNA, which is then translated into an active transcription factor that upregulates the expression of UPR target genes. In some embodiments, the IRE1 nuclease domain also mediates degradation of various mRNAs containing recognition sequences in a process named regulated IRE1-dependent decay (RIDD).

[0147] There are two mammalian IRE1 homologs, IRE1α and IRE1β (Grey et al. incorporated herein by reference in its entirety). IRE1α is ubiquitously expressed, and IRE1β is a close paralogue that may have distinct enzymatic activity. Without wishing to be bound by theory, in some embodiments, IRE1β negatively regulates IRE1α in response to ER stress, acting as a dominant-negative suppressor of IRE1α activity. Further without wishing to be bound by theory, in some embodiments, a dominant-negative effect of IRE1β does not require an IRE1β enzymatic activity, e.g., kinase and / or nuclease activity. In some embodiments, an IRE1β enzymatic activity is associated with one or more domains at the C terminal domain of IRE1β. In some embodiments, IRE1β or a variant or fragment thereof binds to, e.g., forms a complex with, IRE1α or a variant or fragment thereof. In some embodiments, an IRE1β variant, e.g., an IRE1β without one or more enzymatic activities (e.g., an IRE1β having a mutation and / or deletion at a C terminal domain), binds to, e.g., forms a complex with, IRE1α or a variant or fragment thereof. In some embodiments, a complex comprising IRE1β or a variant or fragment thereof and IRE1α or a variant or fragment thereof has an impaired ability to induce XBP1 splicing and / or UPR signaling. In some embodiments, IRE1β or a variant or fragment thereof retains RIDD activity that may be enhanced over that of IRE1α or a variant or fragment thereof.

[0148] Separately, it has been observed that mutant IRE1 proteins lacking the cytosolic effector domains can also exhibit dominant-negative effects on UPR induction (Yoshida et al. incorporated herein by reference in its entirety). Without wishing to be bound by theory, IRE1β mutant protein lacking an effector domain may exhibit IRE1α- sequestering dominant-negative effects on UPR signaling, while also having dominant-negative effects on RIDD.

[0149] Among other things, provided herein is the insight that co-expression of a UPR inhibitor with a payload can improve secretion and / or expression of a payload polypeptide. In some embodiments, provided herein are technologies that modulate the UPR by targeting one or more UPR signaling pathways that may regulate transcriptional and translational programs to ultimately relieve ER stress, e.g., PERK, IRE1, and / or ATF6.

[0150] In some embodiments, a helper protein attenuates IRE1 activity. In some embodiments, a helper protein is or comprises IRE1β lacking C-terminal effector domains (also referred to as IRE1b_delCTD herein). In some embodiments, a helper protein is or comprises IRE1β with an inactivating point mutation in the kinase domain (IRE1b_K548A).

[0151] In some embodiments, a helper protein comprises an unfolded protein response (UPR) inhibitor polypeptide, or a fragment or variant thereof. In some embodiments, a UPR inhibitor polypeptide comprises a K3L polypeptide, or a fragment or variant thereof, a gamma 134.5 polypeptide, or a fragment or variant thereof, a GADD34 polypeptide, or a fragment or variant thereof, or an IRE1b polypeptide, or a fragment or variant thereof.

[0152] In some embodiments, IRE1b_delCTD 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 Page 30 of 117 12869933v1Attorney Docket: 2012611-0187 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, IRE1b_delCTD polypeptide comprises the amino acid sequence of SEQ ID NO: 12.

[0153] In some embodiments, a IRE1b_delCTD polypeptide is encoded by 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 nucleotide sequence of SEQ ID NO: 51. In some embodiments, a IRE1b_delCTD polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 51.

[0154] Another context in which the cellular stress and UPR responses are often triggered is in viral infections (Mulvey et al. incorporated herein by reference in its entirety). As a result, many viruses have evolved to express proteins that modulate and inhibit the UPR, thereby attenuating the cell’s attempt to shut down viral protein production, allowing the virus to replicate. Among other things, the present disclosure provides the insight that co- expressing a viral modulator of the UPR with a payload can improve secretion and / or expression of a payload polypeptide.

[0155] In some embodiments, a helper protein is or comprises a viral inhibitor. In some embodiments, a viral inhibitor of the UPR is or comprises vaccinia virus K3L protein. In some embodiments, K3L functions as an eIF2α pseudo-substrate, attenuating PERK-mediated phosphorylation of eIF2α and translation inhibition.

[0156] In some embodiments, a viral inhibitor is or comprises a hyperactive variant of K3L, K3L_H47R. In some embodiments, a K3L_H47R 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: 13. In some embodiments, a K3L_H47R polypeptide comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, a K3L_H47R 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 nucleotide sequence of SEQ ID NO: 52. In some embodiments, a K3L_H47R polynucleotide comprises the sequence of SEQ ID NO: 52.

[0157] In some embodiments, a viral inhibitor of the UPR is or comprises herpes simplex virus type 1 γ134.5 (also referred to as g134.5), a protein that contains a domain homologous to a domain of GADD34, a cellular protein that attenuates the PERK pathway of the UPR (Cheng at al.). Specifically, both γ134.5 and GADD34 recruit protein phosphatase 1 and de-phosphorylate eIF2α, rescuing translation.

[0158] In some embodiments, a γ134.5 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: 14. In some embodiments, a γ134.5 polypeptide comprises the amino acid sequence of SEQ ID NO: 14.

[0159] In some embodiments, a γ134.5 polynucleotide comprises 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 nucleotide sequence of SEQ ID NO: 53. In some embodiments, a γ134.5 polynucleotide comprises the sequence of SEQ ID NO: 53.

[0160] In some embodiments, a viral inhibitor is or comprises GADD34. Page 31 of 117 12869933v1Attorney Docket: 2012611-0187

[0161] In some embodiments, a GADD34 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: 15. In some embodiments, a GADD34 polypeptide comprises the amino acid sequence of SEQ ID NO: 15.

[0162] In some embodiments, a GADD34 polynucleotide comprises 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 nucleotide sequence of SEQ ID NO: 54. In some embodiments, a GADD34 polynucleotide comprises the sequence of SEQ ID NO: 54. Table 3A: Exemplary UPR inhibitor polypeptide sequences Exemplary Amino Acid Sequence (SEQ ID NO:) Helper Protein IRE1b delCTD MASAVRGSRPWPRLGLQLQFAALLLGTLSPQVHTLRPENLLLVSTLDGSLHALSKQTGDLKWTLRDDPVIE G L T S TP Y P A D AI Q A G D S K SE LTable 3B: Exemplary UPR inhibitor polynucleotide sequences Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Protein T T G C CT A G TA C C A12869933v1Attorney Docket: 2012611-0187 Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Protein GACACACTGCATTTTCTCGCTCTGCGGTGGGGGCACATACGCTTACCAGCATCCGGGCCAAGGGATAC G C A A A G A TA A T C G C G G T G G T T G C T T T G G T A G C C G T G C C T T TG AC A A C A A C C GA C A CPage 33 of 117 12869933v1Attorney Docket: 2012611-0187 Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Protein CTAGCAGAAGTTCAGCGGCTGCTGCCGCCGCCCTTGATTTGAGCGGGCGTAGGGGCTAATGATAG- ssoca e egra a on ro ens

[0163] ER-associated degradation (herein “ERAD”) is an important pathway in ER quality control, mediating the retro-translocation of misfolded or unfolded proteins from the ER to the cytosol, where such proteins are degraded by the ubiquitin-proteasome system. ER stress-induced pre-emptive quality control (ERpQC) includes rerouting of nascent ER-targeted proteins from the cytosol to the ubiquitin-proteasome system by blocking translocation into the ER. Derlin-1 sits in the ER membrane and is centrally involved in both processes (Kadowaki et el., Ye et al., each of which is incorporated herein by reference in its entirety).

[0164] Among other things, provided herein is the insight that co-expression of an ERAD polypeptide, or a fragment or variant thereof with a payload can improve secretion and / or expression of a payload polypeptide. Without wishing to be bound by any particular theory, co-delivery of an ERAD protein enhances payload protein secretion by (1) enhancing ERAD and / or ERpQC, alleviating ER stress, and / or enhancing cellular and ER functions, and / or (2) functioning as a decoy for other ERAD and / or ERpQC machinery.

[0165] An exemplary ERAD polypeptide sequence is provided in Table 4A. An exemplary ERAD polynucleotide sequence is provided in Table 4B.

[0166] In some embodiments, a helper protein is or comprises an ERAD polypeptide, or a fragment or variant thereof. In some embodiments, an ERAD polypeptide is or comprises Derlin-1, or a fragment or variant thereof. In some embodiments, an ERAD polypeptide is or comprises JKAMP, or a fragment or variant thereof.

[0167] In some embodiments, a Derlin-1 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: 16. In some embodiments, a Derlin-1 polypeptide comprises the amino acid sequence of SEQ ID NO: 16.

[0168] In some embodiments, a Derlin-1 polynucleotide comprises a nucleic 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 nucleotide sequence of SEQ ID NO: 55. In some embodiments, a Derlin-1 polynucleotide comprises the sequence of SEQ ID NO: 55. Table 4A: Exemplary ERAD polypeptide sequence Exemplary Helper Amino Acid Sequence (SEQ ID NO:) Protein T Q TPTable 4B: Exemplary ERAD polynucleotide sequence Page 34 of 117 12869933v1Attorney Docket: 2012611-0187 Exemplary Helper Amino Acid Sequence (SEQ ID NO:) Protein Derlin-1 ATGTCTGATATCGGGGATTGGTTCAGGAGCATTCCTGCTATAACACGATACTGGTTTGCCGCCA C G A T A A AC T T G :ER-to-Golgi Trafficking SNARE Proteins

[0169] Anterograde trafficking of secretory proteins proceeds from the ER to the ER-Golgi intermediate compartment (ERGIC) and on to the Golgi (Linders et al. incorporated herein by reference in its entirety). In many embodiments, trafficking steps are mediated via vesicles coated with COP protein complexes. In some embodiments, vesicles also contain soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins, which are responsible for most intracellular organelle trafficking fusion events. SNARE proteins are long alpha-helical proteins containing SNARE motifs defined by the type and position of their central residue into four groups: R, Qa, Qb, and Qc. A functional SNARE complex is typically made up of three or four SNARE proteins containing all four SNARE motifs, which form a coiled-coil bundle on the vesicle surface. Qa-SNARE Stx5 is an essential component of transport vesicles headed to the ERGIC and Golgi, and can form complexes with the Qb-SNARE GosR1 and the Qc- SNARE Bet1.

[0170] Among other things, provided herein is the insight that co-expression of a SNARE polypeptide or a fragment or variant thereof with a payload can improve secretion and / or expression of a payload polypeptide. Exemplary SNARE polypeptide sequences are provided in Table 5A. Exemplary nucleotide sequences encoding SNARE polypeptides are provided in Table 5B.

[0171] In some embodiments, a SNARE protein is or comprises SEC22A. In some embodiments, a SNARE protein is or comprises SEC22C. In some embodiments, SNARE protein is or comprises STXB6. In some embodiments, a SNARE protein is or comprises GOSR2. In some embodiments, a SNARE protein is or comprises SNAB. In some embodiments, a SNARE protein is or comprises SNAG.

[0172] In some embodiments, a SNARE protein is or comprises GosR1. In some embodiments, a GosR1 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: 17. In some embodiments, a GosR1 polypeptide comprises the amino acid sequence of SEQ ID NO: 17.

[0173] In some embodiments, a GosR1 polynucleotide comprises 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 Page 35 of 117 12869933v1Attorney Docket: 2012611-0187 97%, at least 98%, at least 99% or 100% identity to the nucleotide sequence of SEQ ID NO: 56. In some embodiments, a GosR1 polynucleotide comprises the sequence of SEQ ID NO: 56.

[0174] In some embodiments, a SNARE protein is or comprises Stx5. In some embodiments, a Stx5 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: 18. In some embodiments, a Stx5 polypeptide comprises the amino acid sequence of SEQ ID NO: 18.

[0175] In some embodiments, a Stx5 polynucleotide comprises 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 nucleotide sequence of SEQ ID NO: 57. In some embodiments, a Stx5 polynucleotide comprises the sequence of SEQ ID NO: 57.

[0176] In some embodiments, a SNARE protein is or comprises BET1. In some embodiments, a BET1 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: 19. In some embodiments, a BET1 polypeptide comprises the amino acid sequence of SEQ ID NO: 19.

[0177] In some embodiments, a BET1 polynucleotide comprises 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 nucleotide sequence of SEQ ID NO: 58. In some embodiments, a BET1 polynucleotide comprises the sequence of SEQ ID NO: 58. Table 5A: Exemplary SNARE polypeptide sequences Exemplary Amino Acid Sequence (SEQ ID NO:) Hel er I K SL R QI P K STPage 36 of 117 12869933v1Attorney Docket: 2012611-0187 Table 5B: Exemplary SNARE polynucleotide sequences Exemplary Nucleotide Sequence (SEQ ID NO:) Helper P i G G C G C T G G C G A G G C G C G T G T C G G T A G A T CC TUbiquitin Protein Ligases

[0178] Among other things, provided herein is the insight that co-expression of a ubiquitin protein ligase polypeptide, or a fragment or variant thereof with a payload can improve secretion and / or expression of a payload polypeptide. Exemplary ubiquitin protein ligase polypeptide sequences are provided in Table 6A. Exemplary nucleotide sequences encoding ubiquitin protein ligases polypeptides are provided in Table 6B.

[0179] In some embodiments, a helper protein is or comprises a ubiquitin ligase polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a NEDD4 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a NEDD4L polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises an ITCH polypeptide, or a fragment or Page 37 of 117 12869933v1Attorney Docket: 2012611-0187 variant thereof. In some embodiments, a ubiquitin ligase is or comprises a WWP1 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a WWP2 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a SMURF1 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a SMURF2 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a RNF5 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a TRAF1 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a TRAF2 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a TRAF3 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a TRAF6 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a TRAF7 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a UBE3A polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a CUL1 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a CUL2 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a CUL3 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a MARCH-II polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a MARCH-III polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a MARCH-VI polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a AMFR polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a HERC3 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a TRIM63 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a TRIM21 polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin ligase is or comprises a BIRC3 polypeptide, or a fragment or variant thereof.

[0180] In some embodiments, a helper protein is or comprises NEDD4L. In some embodiments, a NEDD4L 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 NEDD4Lnucleotide comprises 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 nucleotide sequence of SEQ ID NO: 59.

[0181] In some embodiments, a helper protein is or comprises a gain-of-function NEDD4L mutant. In some embodiments, a gain of function-of function NEDD4L mutant is NEDD4L_Y679C. In some embodiments, a NEDD4L_Y679C 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: 21. In some embodiments, a NEDD4L_Y679C nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 60. Page 38 of 117 12869933v1Attorney Docket: 2012611-0187

[0182] In some embodiments, a gain of function-of function NEDD4L mutant is or comprises NEDD4L_Q694H. In some embodiments, a NEDD4L_Q694H 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: 22. In some embodiments, a NEDD4L_Q694H nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 61.

[0183] In some embodiments, a gain of function-of function NEDD4L mutant is or comprises NEDD4L_E893K. In some embodiments, a NEDD4L_E893K 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: 23. In some embodiments, a NEDD4L_E893K nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 62.

[0184] In some embodiments, a gain of function-of function NEDD4L mutant is or comprises NEDD4L_R897Q. In some embodiments, a NEDD4L_R897Q 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: 24. In some embodiments, a NEDD4L_R897Q nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 63.

[0185] The NEDD4L N-terminus contains one C2 and four WW domains that mediate protein-protein interactions and determine substrate specificity. In some embodiments, the C-terminus consists of a HECT domain (homologous to the E6-AP carboxyl terminus), which is responsible for interacting with an E2 ubiquitin conjugating enzyme and catalytical transferring ubiquitin to the target substrate. In some embodiments, the HECT domain is proposed to interact with the N-terminal C2 and WW domains to position NEDD4L in an inactive conformation. In some embodiments, a conformational change to the active conformation of NEDD4L allows for interaction with and ubiquitination of substrates, as well as auto-ubiquitination to regulate its own protein levels.

[0186] In some embodiments, mutations in NEDD4L have been identified in patients that exhibit the neurodevelopmental disorder periventricular nodular heterotopia (PNH), characterized by neuronal migration abnormalities, specifically bilateral ectopic nodules of grey matter lining the lateral ventricles, and heterogenous clinical presentation, with seizures and learning difficulties being the most common clinical features; as well as bilateral syndactyly, cleft palate, and neurodevelopmental delay (Broix et al. incorporated herein by reference in its entirety). In some embodiments, mutations reside in the HECT domain of NEDD4L. In some embodiments, mutations deregulate signaling pathways including mTORC1, Akt, Smad2 / 3, TGF-beta, and PI3K. In some embodiments, mutations also enhance ubiquitin ligase activity, presumably by mediating conformational changes that promote formation of the active conformation, leading to constitutive activation. Page 39 of 117 12869933v1Attorney Docket: 2012611-0187

[0187] Among other things, the present disclosure provides the insight that co-expression of wild-type NEDD4L and gain-of function NEDD4L mutant proteins with a payload can improve secretion and / or expression of a payload polypeptide. Without wishing to be bound by any particular theory, enhanced ubiquitin ligase activity from co- expression of mutant NEDD4L proteins may result in increased ubiquitination of one or more of the following sets of proteins: (1) NEDD4L target proteins, (2) an expanded set of proteins beyond NEDD4L targets, including proteins that have lower affinity for NEDD4L than its canonical targets, and / or (3) misfolded or slowly folding payload proteins. Further without wishing to be bound by any particular theory in some embodiments, functional outcomes of such ubiquitination may be enhanced degradation of proteins generally, which may alleviate ER stress and free up cellular resources, and / or alterations in cellular programs due to specific protein degradation, which may redirect resources towards protein secretion or otherwise positively impact protein secretion. Table 6A: Exemplary ubiquitin-protein ligase polypeptide sequences Exemplary Amino Acid Sequence (SEQ ID NO:) Helper L R A S LI G N L I KR P Y L R A S LI G N L FI KR P Y L R A S LIage o 12869933v1Attorney Docket: 2012611-0187 Exemplary Amino Acid Sequence (SEQ ID NO:) Helper Pr t in G N L I KR P Y L R A S LI G N L I KR P PY L R A S LI G N L I KR P YTable 6B: Exemplary ubiquitin-protein ligase polynucleotide sequences disclosed herein Exemplary Nucleotide Sequence (SEQ ID NO:) Helper G A A G G T CC A12869933v1Attorney Docket: 2012611-0187 Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Protein A C G C GT C C C G G A A T A A AT C A T G G CT T T G A GT G G AC G C C G G AT AT A A A G A A G G TG A A G C G A A A12869933v1Attorney Docket: 2012611-0187 Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Protein A C AT G A T C A TA A T A A C T C C C A A G G A G G T T G G A G C T T C A A C C G C C A A C T G G T T AT T A A12869933v1Attorney Docket: 2012611-0187 Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Protein T CT C T A GA A G T A G A TT A G A A G G C AA C T G C A A A A T T G C T C A T C A G AT T C T T CC T CA G TT A TG A12869933v1Attorney Docket: 2012611-0187 Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Protein T C G C A G C T T C CT A A G G C A T A G C G T A TC A G A A CT T T A G T GCE3 Ubiquitin Ligase Substrate Polypeptides

[0188] Proteins that are substrates of E3 ubiquitin-protein ligases may also play important roles in signaling pathways associated with protein secretion and / or expression and may improve protein secretion. Without wishing to be bound by theory, a helper protein that functions in signaling pathways can impact protein expression and / or secretion more than a potential decoy effect on the ubiquitin-proteasome system. Among other things, provided herein is the insight that co-expression of an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof with a payload can improve secretion and / or expression of a payload polypeptide. Exemplary E3 ubiquitin ligase substrate polypeptide sequences are provided in Table 7A. Nucleotide sequences encoding exemplary E3 ubiquitin ligase substrate polypeptides are provided in Table 7B. Page 45 of 117 12869933v1Attorney Docket: 2012611-0187

[0189] In many embodiments, E3 ubiquitin ligase substrate polypeptides can play roles in multifunctional and / or intersecting pathways. Without wishing to be bound by any particular theory, these proteins may positively regulate pathways that promote anabolism, survival, and growth, which may enhance protein expression and / or secretion.

[0190] In some embodiments, a helper protein is or comprises an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof. In some embodiments, an E3 ubiquitin ligase substrate polypeptide is or comprises: a RIPK2 polypeptide or a fragment or variant thereof, an ERBB4 polypeptide or a fragment or variant thereof, an AKT1 polypeptide or a fragment or variant thereof, a Notch1 polypeptide or a fragment or variant thereof, a NEMO polypeptide or a fragment or variant thereof, or any combination thereof.

[0191] In some embodiments, a helper protein is Receptor-interacting serine / threonine-protein kinase 2 (“RIPK2”). In some embodiments, RIPK2 is a substrate of the E3 ubiquitin-protein ligase ITCH. In some embodiments, RIPK2 is a peripheral membrane protein that functions in signaling pathways in innate and adaptive immune responses, including the pro-inflammatory NOD and NF-κB pathways (Boyle et al. incorporated herein by reference in its entirety). In some embodiments, RIPK2 may play a role in autophagy. In some embodiments, a RIPK2 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: 25. In some embodiments, a RIPK2 nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 64.

[0192] In some embodiments, a helper protein is or comprises Receptor tyrosine-protein kinase ERBB4 (also known as HER4). In some embodiments, ERBB4 is a substrate of the E3 ubiquitin-protein ligase WWP1. In some embodiments, ERBB4 is a transmembrane protein from the epidermal growth factor receptor (EGFR) family that can either promote cell survival and proliferation, or drive growth inhibition and apoptosis, depending on context (Lucas et al., incorporated herein by reference in its entirety). In some embodiments, a ERBB4 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: 26. In some embodiments, a ERBB4 nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 65.

[0193] In some embodiments, a helper protein is or comprises AKT1. In some embodiments, AKT1, a RAC (Rho family)-alpha serine / threonine-protein kinase, is or comprises a substrate of the E3 ubiquitin-protein ligase TRAF6. In some embodiments, AKT1 is implicated in the regulation of many cellular processes including metabolism, proliferation, cell survival, growth, protein synthesis, protein turnover, and membrane dynamics (Duggal et al. incorporated herein by reference in its entirety). In some embodiments, AKT activity is mediated through the phosphorylation of over 100 proposed substrates, including proteins of the phosphatidylinositol-3-kinase (PI3K) / AKT / mTOR and NF-κB signaling pathways. In some embodiments, a AKT1 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 Page 46 of 117 12869933v1Attorney Docket: 2012611-0187 sequence of SEQ ID NO: 27. In some embodiments, a AKT1 nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 66.

[0194] In some embodiments, a helper protein is or comprises Notch 1. In some embodiments, Notch1 is a substrate of the E3 ubiquitin-protein ligase ITCH. In some embodiments, Notch 1 is a transmembrane protein receptor that plays a role in the highly conserved Notch signaling pathway, which promotes cellular proliferation and differentiation (Zhou et al. incorporated herein by reference in its entirety). In some embodiments, Notch signaling is dysregulated in many cancers and diseases. In some embodiments, Notch 1 consists of an N-terminal extracellular domain that binds its ligands, a transmembrane domain, and a C-terminal cytoplasmic domain that mediates protein- protein interactions essential to its signaling functions. In some embodiments, a Notch1 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: 28. In some embodiments, a Notch1 nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 67.

[0195] In some embodiments, a helper protein is or comprises NEMO. In some embodiments, NEMO is an NF-κB modulator. In some embodiments, NEMO is a substrate of the E3 ubiquitin-protein ligase TRAF6. In some embodiments, NEMO is a γ regulatory subunit of an inhibitor of the NF-κB kinase IKK complex, which activates the NF-κB signaling pathway (Liu et al. incorporated herein by reference in its entirety). In some embodiments, NF-κB signaling pathway drives cell survival and proliferation and inhibits apoptosis. In some embodiments, NEMO binds to polyubiquitin chains, and through this mechanism interacts with RIPK2 (Abbott et al. incorporated herein by reference in its entirety). In some embodiments, a NEMO 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: 29. In some embodiments, a NEMO nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 68. Table 7A: Exemplary E3 ubiquitin ligase substrate polypeptide sequences Exemplary Amino Acid Sequence (SEQ ID NO:) R Q S E S DI YPage 47 of 117 12869933v1Attorney Docket: 2012611-0187 Exemplary Amino Acid Sequence (SEQ ID NO:) Helper Pr t in R VY G VK N G P N GL W H PI F FD R LP ) T V TH G EI DE D V EA N M LA T R Q TI S IL Q R E EPage 48 of 117 12869933v1Attorney Docket: 2012611-0187 Table 7B: Exemplary E3 ubiquitin ligase substrate polynucleotide sequences Exemplary Nucleotide Sequence (SEQ ID NO:) Helper P i C A T C G C G G C A AT C A AA CT T A T G T G A A G G C T T C C G A T C G G G T C A T C A A C T A G C C12869933v1Attorney Docket: 2012611-0187 Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Pr t in G A C G G T G C A C G T A G C T T TC A A A A G C C G G CT G G C C G A CA A C C G A C G A T T C A T C C A12869933v1Attorney Docket: 2012611-0187 Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Pr t in G G GC C G C C C G A G C A C G T T A T A G T A C G A G T T A A T A TC GC A A C G C C T T G G A AA A C T T12869933v1Attorney Docket: 2012611-0187 Proteins Upregulated During Plasma Cell Differentiation

[0196] Among other things, provided herein is the insight that co-expression of a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof with a payload can improve secretion and / or expression of a payload polypeptide. As known in the field, plasma cells are a subset of B lymphocytes that secrete antibodies at rates up to 10,000 antibodies per second (Hibi and Dosch et al. incorporated herein by reference in its entirety). Amino acid sequences of exemplary polypeptides upregulated during plasma cell differentiation disclosed herein are provided in Table 8A. Exemplary nucleotide sequences encoding polypeptides upregulated during plasma cell differentiation are provided in Table 8B.

[0197] In some embodiments, a helper protein is or comprises GJB2. In some embodiments, GJB2 is multi-pass membrane protein that sits in the plasma membrane and serves as a structural component of gap junctions. In some embodiments, a GJB2 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: 30. In some embodiments, a GJB2 nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 69.

[0198] In some embodiments, a helper protein is or comprises NDFIP2. In some embodiments, NDFIP2, or NEDD4 family-interacting protein 2, activates HECT domain-containing E3 ubiquitin ligase proteins that mediate the degradation of protein substrates through the ubiquitin-proteasome system. Without wishing to be bound by theory, one possibility is NDFIP2 enhances protein expression and secretion via modulation of the ubiquitin-proteasome protein degradation pathway and potentially the ERAD pathway, either through enhancing its protein degradation functions and alleviating cellular stress, or by functioning as a decoy and inhibiting ubiquitin ligase proteins. In some embodiments, a NDFIP2 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: 31. In some embodiments, a NDFIP2 polypeptide comprises 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 nucleotide sequence of SEQ ID NO: 70.

[0199] In some embodiments, a helper protein is or comprises CD300A / CLM8. In some embodiments, CD300A / CLM8 is an inhibitory receptor glycoprotein expressed on the surface of leukocytes. In some embodiment, a CD300A 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: 32. In some embodiments, a CD300A nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 71.

[0200] In some embodiments, a helper protein is or comprises XCR1. In some embodiments, XCR1 is a cell surface-expressed chemokine receptor. In some embodiments, a XCR1 polypeptide comprises an amino acid Page 52 of 117 12869933v1Attorney Docket: 2012611-0187 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: 33. In some embodiments, a XCR1 nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 72.

[0201] In some embodiments, a helper protein is or comprises STBD1. In some embodiments, STBD1 is an ER membrane protein that acts as a cargo receptor for glycogen that delivers its cargo into the autophagic glycophagy pathway, resulting in the transport of glycogen to lysosomes, and its degradation in the lysosome. In some embodiments, STBD1 interacts with a number of proteins that modulate ubiquitin ligase activity. In some embodiments, STBD1 interacts with GABARAP, a ubiquitin-like modifier that plays a role in reticulophagy, the turnover and remodeling of the ER, particularly under stress conditions. Without wishing to be bound by theory, these interactions may mediate STBD1’s enhancement of payload expression, either through promoting or inhibiting these pathways.

[0202] In some embodiments, a STBD1 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: 34.In some embodiments, a STBD1 nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 73.

[0203] In some embodiments, a helper protein is or comprises EDA2R, or tumor necrosis factor receptor superfamily member 27, a cell surface cytokine receptor. EDA2R has been associated with muscle protein degradation via ubiquitin ligase induction in cancer patients suffering from cachexia (von Renesse and Mirtschink). Without wishing to be bound by theory, associations with protein degradation pathways may mediate EDA2R’s protein expression enhancement effect. In some embodiments, EDA2R activates NFκB and JNK pathways through TRAF3 and TRAF6, which possesses E3 ubiquitin ligase activity. In some embodiments, a EDA2R 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: 35. In some embodiments, a EDA2R nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 74.

[0204] In some embodiments, a helper protein is or comprises TMEM200A, a transmembrane protein of unknown function. In some embodiments, TMEM200A has been associated with gastric cancer, and in one study, co- expression and gene set enrichment analyses indicated that it may be associated with tumor invasion and metastasis, as well as tumor immune cell infiltration and immune checkpoint expression, suggesting it may be an adhesion molecule (Deng et al., incorporated herein by reference in its entirety). In some embodiments, a TMEM200A 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 Page 53 of 117 12869933v1Attorney Docket: 2012611-0187 100% identity to the amino acid sequence of SEQ ID NO: 36. In some embodiments, a TMEM200A nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 75.

[0205] In some embodiments, a helper protein is or comprises GPR114. In some embodiments, GPR114 is an adhesion G-protein coupled receptor. In some embodiments, GPR114 has seven transmembrane helices. In some embodiments, GPR114 is localized to a cellular plasma membrane. In some embodiments, a GPR114 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: 37. In some embodiments, a GPR114 nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 76.

[0206] In some embodiments, a helper protein is or comprises DPAGT1. In some embodiments, DPAGT1 is an enzyme involved in N-linked protein glycosylation. In some embodiments, DPAGT1 possesses ten transmembrane helices. In some embodiments DPAGT1 is localized to a cellular ER membrane. Without wishing to be bound by theory, modulation of helper proteins involved in glycosylation pathways could significantly impact protein expression and secretion. In some embodiments, a DPAGTI 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: 38. In some embodiments, a DPAGTI nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 77.

[0207] In some embodiments, a helper protein is or comprises CCR2. In some embodiments, CCR2 is a cell surface chemokine receptor. In some embodiments, CCR2 has seven transmembrane proteins. In some embodiments, a CCR2 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: 39. In some embodiments, a CCR2 nucleotide comprises 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 nucleotide sequence of SEQ ID NO: 78. Table 8A: Amino acid sequences of exemplary polypeptides upregulated during plasma cell differentiation Exemplary Amino Acid Sequence (SEQ ID NO:) H APage 54 of 117 12869933v1Attorney Docket: 2012611-0187 Exemplary Amino Acid Sequence (SEQ ID NO:) Helper Pr t in K G D S W N TT K S L M IF R T E V G W V VS D EV GI EN D CV LS P TI N Q LN VL E I P P MI12869933v1Attorney Docket: 2012611-0187 Exemplary Amino Acid Sequence (SEQ ID NO:) Helper Pr t in I LT CL T T IExemplary Nucleotide Sequence (SEQ ID NO:) Helper T G T G A T C T T C A T CC C C A G A G G C T C GT T A A T C A C C G12869933v1Attorney Docket: 2012611-0187 Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Pr t in TC C AT G AA G C G TT A A G A T T C C G T C T C A C C G C G T G C G G C T G C CT TC C G G G C AC ATg 12869933v1Attorney Docket: 2012611-0187 Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Pr t in A G G A G T A C A CA G T G G TA T A G G C A C T G C C TC T T CC T T G A T A G T C C A T C A A G T G C C12869933v1Attorney Docket: 2012611-0187 Exemplary Nucleotide Sequence (SEQ ID NO:) Helper Pr t in AA T A T C TT T C A C C T GT G A G G G G G ACombinations of helper proteins

[0208] In some embodiments, provided herein is a nucleic acid expression system or a composition comprising the same comprising: (i) a polynucleotide comprising a payload sequence, and (ii) at least one polynucleotide that encodes one or more helper polypeptides chosen from: (a) a chaperone or ER guardian polypeptide, or a fragment or variant thereof, (b) an unfolded protein response (UPR) inhibitor polypeptide, or a fragment or variant thereof, (c) an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof, (d) a SNARE polypeptide, or a fragment or variant thereof, (e) a ubiquitin protein ligase polypeptide, or a fragment or variant thereof, (f) an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof, (g) a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof, (h) any of (a)-(g).

[0209] In some embodiments, a nucleic acid expression system or a composition comprising the same comprises a polynucleotide encoding one or more helper polypeptides comprising a chaperone or ER guardian polypeptide, or a fragment or variant thereof. In some embodiments, a chaperone or ER guardian polypeptide comprises a polypeptide provided in Table 1 or a polypeptide encoded by a nucleic sequence provided in Table 2. In some embodiments, a nucleic acid expression system or a composition comprising the same further comprises one or more additional polynucleotides encoding one or more additional helper polypeptides. In some embodiments, one or more additional helper polypeptides comprise: an unfolded protein response (UPR) inhibitor polypeptide, or a fragment or variant thereof, an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof, a SNARE polypeptide, Page 59 of 117 12869933v1Attorney Docket: 2012611-0187 or a fragment or variant thereof, a ubiquitin protein ligase polypeptide, or a fragment or variant thereof, an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof, a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof. In some embodiments, a polynucleotide encoding a chaperone or ER guardian polypeptide and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in one composition. In some embodiments, a polynucleotide encoding a chaperone or ER guardian polypeptide and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in separate compositions. In some embodiments, a polynucleotide encoding a chaperone or ER guardian polypeptide and the one or more additional polynucleotides are in the same nucleic acid construct.

[0210] In some embodiments, a nucleic acid expression system or a composition comprising the same comprises a polynucleotide encoding one or more helper polypeptides comprising an unfolded protein response inhibitor, or a fragment or variant thereof. In some embodiments, an unfolded protein response inhibitor polypeptide comprises a polypeptide provided in Table 3A or a polypeptide encoded by a nucleic sequence provided in Table 3B. In some embodiments, a nucleic acid expression system or a composition comprising the same further comprises one or more additional polynucleotides encoding one or more additional helper polypeptides. In some embodiments, one or more additional helper polypeptides comprise: an chaperone or ER guardian polypeptide, or a fragment or variant thereof, an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof, a SNARE polypeptide, or a fragment or variant thereof, a ubiquitin protein ligase polypeptide, or a fragment or variant thereof, an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof, a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof. In some embodiments, a polynucleotide encoding an unfolded protein response inhibitor polypeptide and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in one composition. In some embodiments, a polynucleotide encoding an unfolded protein response inhibitor polypeptide and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in separate compositions. In some embodiments, a polynucleotide encoding an unfolded protein response inhibitor polypeptide and the one or more additional polynucleotides are in the same nucleic acid construct.

[0211] In some embodiments, a nucleic acid expression system or a composition comprising the same comprises a polynucleotide encoding one or more helper polypeptides comprising an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof. In some embodiments, an ER associated degradation (ERAD) polypeptide comprises a polypeptide provided in Table 4A or a polypeptide encoded by a nucleic sequence provided in Table 4B. In some embodiments, a nucleic acid expression system or a composition comprising the same further comprises one or more additional polynucleotides encoding one or more additional helper polypeptides. In some embodiments, one or more additional helper polypeptides comprise: an chaperone or ER guardian polypeptide, or a fragment or variant thereof, an unfolded protein response inhibitor, or a fragment or variant thereof, a SNARE polypeptide, or a fragment or variant thereof, a ubiquitin protein ligase polypeptide, or a fragment or variant thereof, an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof, a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof. In some embodiments, a polynucleotide encoding an ER associated degradation (ERAD) polypeptide and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in one composition. In some embodiments, a polynucleotide encoding an ER associated degradation (ERAD) polypeptide and the one or more additional polynucleotides encoding one or more Page 60 of 117 12869933v1Attorney Docket: 2012611-0187 additional helper polypeptides are in separate compositions. In some embodiments, a polynucleotide encoding an ER associated degradation (ERAD) polypeptide and the one or more additional polynucleotides are in the same nucleic acid construct.

[0212] In some embodiments, a nucleic acid expression system or a composition comprising the same comprises a polynucleotide encoding one or more helper polypeptides comprising a SNARE polypeptide, or a fragment or variant thereof. In some embodiments, a SNARE polypeptide comprises a polypeptide provided in Table 5A or a polypeptide encoded by a nucleic sequence provided in Table 5B. In some embodiments, a nucleic acid expression system or a composition comprising the same further comprises one or more additional polynucleotides encoding one or more additional helper polypeptides. In some embodiments, one or more additional helper polypeptides comprise: an chaperone or ER guardian polypeptide, or a fragment or variant thereof, an unfolded protein response inhibitor, or a fragment or variant thereof, an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof, a ubiquitin protein ligase polypeptide, or a fragment or variant thereof, an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof, a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof. In some embodiments, a polynucleotide encoding a SNARE polypeptide and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in one composition. In some embodiments, a polynucleotide encoding a SNARE polypeptide and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in separate compositions. In some embodiments, a polynucleotide encoding a SNARE polypeptide and the one or more additional polynucleotides are in the same nucleic acid construct.

[0213] In some embodiments, a nucleic acid expression system or a composition comprising the same comprises a polynucleotide encoding one or more helper polypeptides comprising a ubiquitin protein ligase polypeptide, or a fragment or variant thereof. In some embodiments, a ubiquitin protein ligase polypeptide comprises a polypeptide provided in Table 6A or a polypeptide encoded by a nucleic sequence provided in Table 6B. In some embodiments, a nucleic acid expression system or a composition comprising the same further comprises one or more additional polynucleotides encoding one or more additional helper polypeptides. In some embodiments, one or more additional helper polypeptides comprise: an chaperone or ER guardian polypeptide, or a fragment or variant thereof, an unfolded protein response inhibitor, or a fragment or variant thereof, an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof, a SNARE polypeptide, or a fragment or variant thereof, an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof, a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof. In some embodiments, a polynucleotide encoding a ubiquitin protein ligase polypeptide and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in one composition. In some embodiments, a polynucleotide encoding a ubiquitin protein ligase polypeptide and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in separate compositions. In some embodiments, a polynucleotide encoding a ubiquitin protein ligase polypeptide and the one or more additional polynucleotides are in the same nucleic acid construct.

[0214] In some embodiments, a nucleic acid expression system or a composition comprising the same comprises a polynucleotide encoding one or more helper polypeptides comprising an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof. In some embodiments, an E3 ubiquitin ligase substrate polypeptide comprises a polypeptide provided in Table 7A or a polypeptide encoded by a nucleic sequence provided in Table 7B. In some Page 61 of 117 12869933v1Attorney Docket: 2012611-0187 embodiments, a nucleic acid expression system or a composition comprising the same further comprises one or more additional polynucleotides encoding one or more additional helper polypeptides. In some embodiments, one or more additional helper polypeptides comprise: an chaperone or ER guardian polypeptide, or a fragment or variant thereof, an unfolded protein response inhibitor, or a fragment or variant thereof, an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof, a SNARE polypeptide, or a fragment or variant thereof, a ubiquitin protein ligase polypeptide, or a fragment or variant thereof, a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof. In some embodiments, a polynucleotide encoding an E3 ubiquitin ligase substrate polypeptide and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in one composition. In some embodiments, a polynucleotide encoding an E3 ubiquitin ligase substrate polypeptide and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in separate compositions. In some embodiments, a polynucleotide encoding an E3 ubiquitin ligase substrate polypeptide and the one or more additional polynucleotides are in the same nucleic acid construct.

[0215] In some embodiments, a nucleic acid expression system or a composition comprising the same comprises a polynucleotide encoding one or more helper polypeptides comprising a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof. In some embodiments, a polypeptide upregulated during plasma cell differentiation comprises a polypeptide provided in Table 8A or a polypeptide encoded by a nucleic sequence provided in Table 8B. In some embodiments, a nucleic acid expression system or a composition comprising the same further comprises one or more additional polynucleotides encoding one or more additional helper polypeptides. In some embodiments, one or more additional helper polypeptides comprise: an chaperone or ER guardian polypeptide, or a fragment or variant thereof, an unfolded protein response inhibitor, or a fragment or variant thereof, an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof, a SNARE polypeptide, or a fragment or variant thereof, a ubiquitin protein ligase polypeptide, or a fragment or variant thereof, an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof. In some embodiments, a polynucleotide a polypeptide upregulated during plasma cell differentiation and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in one composition. In some embodiments, a polynucleotide encoding a polypeptide upregulated during plasma cell differentiation and the one or more additional polynucleotides encoding one or more additional helper polypeptides are in separate compositions. In some embodiments, a polynucleotide encoding a polypeptide upregulated during plasma cell differentiation and the one or more additional polynucleotides are in the same nucleic acid construct. Characterization of a nucleic acid expression system

[0216] Nucleic acid expression systems disclosed herein comprise (i) a polynucleotide comprising a payload sequence, and (ii) at least one polynucleotide that encodes one or more helper polypeptides disclosed herein. In some embodiments of a nucleic acid expression system disclosed herein, a helper polypeptide expressed from the at least one polynucleotide comprising a sequence that encodes a helper polypeptide increases expression and / or secretion of a payload polypeptide encoded by a polynucleotide comprising a payload sequence.

[0217] In some embodiments, a nucleic acid expression system described herein is characterized in that when administered to a cell, tissue, or subject, increased payload expression and / or secretion is observed as compared to Page 62 of 117 12869933v1Attorney Docket: 2012611-0187 payload expression and / or secretion a cell, tissue, or subject administered an otherwise similar polynucleotide comprising a payload sequence without at least one polynucleotide comprising a sequence encoding a helper polypeptide.

[0218] In some embodiments, increased payload expression comprises at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, least 30-fold, at least 45-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, at least 95-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, or at least 800-fold increased expression of a payload.

[0219] In some embodiments, increased payload expression comprises about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, least 30-fold, about 45-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100- fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, or about 800- fold increased expression of a payload.

[0220] In some embodiments, increased payload expression comprises about 2-fold to about 800-fold, about 2- fold to about 700-fold, about 2-fold to about 600-fold, about 2-fold to about 500-fold, about 2-fold to about 400-fold, about 2-fold to about 300-fold, about 2-fold to about 200-fold, about 2-fold to about 100-fold, about 2-fold to about 90-fold, about 2-fold to about 80-fold, about 2-fold to about 70-fold, about 2-fold to about 60-fold, about 2-fold to about 50-fold, about 2-fold to about 40-fold, about 2-fold to about 30-fold, about 2-fold to about 20-fold, about 2- fold to about 10-fold, about 2-fold to about 5-fold increased expression.

[0221] In some embodiments, increased payload secretion comprises at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, or at least 500-fold increased secretion of a payload.

[0222] In some embodiments, increased payload secretion comprises about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 50-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, or about 500-fold increased secretion of a payload.

[0223] In some embodiments, increased payload secretion comprises about 2-fold to about 500-fold, about 2-fold to about 400-fold, about 2-fold to about 300-fold, about 2-fold to about 200-fold, about 2-fold to about 100-fold, about 2-fold to about 50-fold, about 2-fold to about 10-fold, about 2-fold to about 9-fold, about 2-fold to about 8- fold, about 2-fold to about 7-fold, about 2-fold to about 6-fold, about 2-fold to about 5-fold, about 2-fold to about 4- fold, about 2-fold to about 3-fold increased secretion of a payload.

[0224] In some embodiments, increased secretion and / or expression of a polypeptide described herein comprises increased trafficking through a secretory pathway, increased proper folding of a polypeptide, reduced degradation of a polypeptide, and / or reduced aggregation of a polypeptide. Page 63 of 117 12869933v1Attorney Docket: 2012611-0187 Modified ribonucleotides: Ac4C and 5-hmU

[0225] 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.

[0226] 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.

[0227] In some embodiments, a polyribonucleotide comprises one or more modified nucleosides or nucleobases.

[0228] In some embodiments, a modified ribonucleotides comprises an N4-acetylcytidine nucleoside. In some embodiments, the nucleoside has the following structure: O H3C NH O .

[0229] In some embodiments, a modifieda 5-hydroxymethyluridine nucleoside. In some embodiments, the nucleoside has the following structure: OH O NH O .

[0230] In some embodiments, a modifiedstructure provided in Table 9A. In some embodiments, a modified ribonucleotide has a structure provided in Table 9B 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 9B. Page 64 of 117 12869933v1:etahpsohpirtenidityclyteca-4N :etahpsohpN N H OniO N H iddO O C OirH H O O H e H3OunliyO dihtyOtclHe:eO mtH7yOPO ytOxa OP hO 1e Ho pO1cr sHfa- OPO dOyo4hOPO oHh-p5iO5N dH6ega:PetahpsohponOeonidH O H N miHrN e N N niO Ouly:OheO N H t O t HadC OHi3 eHhOtO H Oy pmc sl yoy xstOhOoeHe r pHcdPd O O oPi O Oat y n-o h oO O4 -elH HN 5mc7 u8 n1 o0 bi-r1d1ei6 fi2 d :1 o e0 nmi2dy: irrt a:l ueO OlepHkyniN Nhcm Htde io eN N H N H OtxyO OD E m 1Ocl y v:H O Oy y xC3H H OtA3e o 3O Oe r9Hn 9c dr 9aey-lo 6hO Ot4b-8tH HN 5a 2AT 1:etahpsohpirtenidityclyteca-4N :etahpsohpidH Or)eulO endiOyit diO-htto PO e:O ee- lycclOmyta OPO7uyt-xnohO1ec OPOorp-1adsbOo OPOfoi -yr 4hhy -piO.6lN 5 d e6odpietgoaa:oe el Pt ta cn ih und pes oao btirhroptpornOenOni Hecoocm H NdN NiNan HrjieO Ouly:O N HdOannidC OheehitOtt3aH Onw yHe hOap Oo( ctslmeytOyxsO O oote -hO dir p-netcaPOe- d oPOm o Oyl 4hn-oOhcc N 5mat utnafoobirn78odiet1 i0if s-io1d :p1o6me2yneih1r2 adt0 ir s:l :OulOepe tatemneidiH NyhHkN NtN cicHeO N HdxEtoy nc O O OmOiD:y BlyCyxH t O O13ve 9ecH OorO33nel ab- dOyh :9rot4Oe9t6t aN- T5o8A N21Attorney Docket: 2012611-0187

[0231] 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.

[0232] 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.

[0233] 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

[0234] 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 67 of 117 12869933v1Attorney Docket: 2012611-0187

[0235] 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.

[0236] 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 ,

[0237] (a) wherein R is aand

[0238] (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).

[0239] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and an adenine nucleobase.

[0240] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a guanine nucleobase.

[0241] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a cytosine nucleobase.

[0242] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a N4- acetylcytidine nucleoside.

[0243] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a uracil nucleobase.

[0244] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a 5- hydroxymethyluridine nucleoside.

[0245] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a N1- methylpseudouracil nucleobase.

[0246] In some embodiments, a modified ribonucleotide comprises a structure provided in Table 9C. In some embodiments, a modified ribonucleotide comprises a structure provided in Table 9D 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 9D. Page 68 of 117 12869933v1iil-l--’iil-lyetOH PO l e etH teah yOtP cpHecmahO O 1y pO1a-s xHfOohOPOa- osOrohOPO o-’p d p2idO9H -’2yhidO H6egeaPnidireunliydi httyeclO3m yyH O t O H N HxO3e N NoN H c C a:eOrOdy :O N C -4 teO ahC3O HhO- tNaH lhO O 5 H ypsHlpO ste o yt se c hpO HeacoahdpO it- OonOPO -H oe- oOOo-nOPO’’oO7 lc 2m H2m H81 u0 n- o1 bi1r6de:2 if e1 indi0odi2tmy: ctl :eyrytek alenipcOdca- N O H3 iruH O l N O3ome4N H x N N -yH CDy ElO5l htO N C :ytO On CC OyeHt ee 3eyH1mO O Hvr 9 ceaH Oc33o l-aO-xOoO r9tOd 9t bO6a-’2H -’AT 2yhH821enidiruodOuesplyhtem-1-l ey tt aechap-soO-h’p2irtenidiruoduespOlyhtem-1-lyetteachap-sOo-h’p2idenidir hu po sd ou he pspH O OolnyNoH O3O N O3htm H 2 H N O H O O N CeN N CeeOetNniOnmidd:O Oi etO -1-al hHirHtpsOulOy achO H lpsOy y yt t to oe e eO O Oh hc c cH H Hp pa a aP P Po O O O O o O O- - -n nO O Oo o-O - O - O’ ’ ’H H H2m2 2menidiruoduesply ::h eteO O OnOe iH H3 3nO Oi2dN N H N Him dHt-iC CryN N N O O1- cuO O Ol l l1Ny y yvt t tO O O3H H He e e3c c cO O Oa a a 9- - - 9O O O 6O O O- - -’ ’ ’8H H H2 2 221:etahpH sNohO pirtenisonauglyteca- O-’2O O HdN e N C O nisN OeniO o O HsH n OoO anu: eO glyettOdeaHaOlyOH PO7hOPt 1c psOecO1aHaOHf- OohOPO -PO o-’p2iOO-’O1dH2H7egaPH N H eNniN eNNO O HniNO2H3son :eNCsatN Oon :eH N CetN O uagl hO p HdaytOahO slpH ysO ecoh tp OecoaHahpO - Oo - o OH nOPOP -’oOO-nO2m H’o2mOH7810-116:22eH n N:12ie0sHno N iNsN3 oN:t naOOHneN O 2 H3ekcuoglN Cdy OalH N C NyN O1Dty eO HtOeOvc cHeaO3n a3- -9r 9otO-’OOt-’O62 H2H8A21iil-l--’ii)ledi ecto a-rel 4OulO N:e O- O -7tP5 ylht :ee O- c P1unltO1oyteahOytfbcPe map yhOoirya-s-cxp- P2Oo O OalO- osoh ro O O7p-’pi O- dhOed’ yp2 2 higdaaPotni detear niodpirtoycclO y H N O H3ennH O i N O H3iteN N C nca-:etOdi Or :O N C eh 4uletO NahC H 3 O w ( O - ls5 yhatO O p H sl hepO eyte osi hOytdcp-ecmyohO toa- o OPOa x p- P el O-n-c’oOo-rodnO OO’ yoO u2m2 hm nobi7r81 de:0if e-indi1d1o i6t2my1 ycl :0 ryte2 a enli:t pcOdmairO -k 4H N O H3 ulH N Oe eN NyH3C - O N CcxoE NlyO O5l ht:tOyt eODCmH O O1vy D9ecaH3OecayexO33nr el- O- or 99ottba -’OO2-’d2yO6h8A T21:etaO hpsohpirtenidirulyteca- O-’2:etahpsO ohpidenidirulyteca- O-’2N H NNH O N O3O H 2 N O H3eO O H3C H CniN C e:O N niOeeni :N N Oson :N O deitraOdit taOeyhataO uhOu hO lytps cly pO sglecohOtytposopO-ec hO O a- oPp-ec hPpO- PO-nOa- o O OaOoOnO -oOo’ O -nO 2 m’-2m’o2m7810-121H6N2 :1:e0enH2:OeOinsN:tniHoNO d N O H3idi 2 N O H3nO H3ekiH CcruO N CtyN N CaoOcOuN lyly glN ODty eOtOytO1Oe eve cac-aOcO3n-a3-9rotOOOOOO9t-’2 -’ 62-’A 2 821N Henisooned:al ey tt aechap-soO-h’p2irtN2H N C O N O N OenOeOisOnio dOniredO -uoO 1d-7al:-yetOP1Oa OPO -lh Oyueet tt 1es ahOfoc psO-eaPO cpal p- oso O- y PO.4e7OhpO - iOh- -tehd’pOdiit e’2 o g2mdelacPunobirtNnNeN O3OceH HajniH2N CeN O ndsoN Oii Oa:dn r NOne et adaOl hO -uoet1aOoa p-dhpOtys lyu tte ohO -tes snceloOepah--pm oOPO ca-yhpOto OP -nOhncO’oO -’e oOatt2m2m mafonoitisopeh:teni NeNnsiesdtonO3 irH OaOcieN2HdaH N C -uoNdlNyO1ldOni-tOyuteN O1ecOesO pvacalO3y3- - :9O- OOh-t’OE9’e2mT62O8N21Attorney Docket: 2012611-0187

[0247] In some embodiments, at least 5% of ribose moieties of a polyribonucleotide are 2’-O-acetylated.

[0248] In some embodiments, about 5% to about 99% of the ribose moieties of a polyribonucleotide are 2’-O- acetylated.

[0249] In some embodiments, a polyribonucleotide disclosed herein comprises a cap structure and the cap structure does not comprise a 2’-O-acetylated ribose.

[0250] In some embodiments, a polyribonucleotide disclosed herein comprises a cap structure and the cap structure comprises a 2’-O-acetylated ribose.

[0251] In some embodiments, a polyribonucleotide disclosed herein further comprises one or more ribonucleotides that does not comprise a 2’-O acetylated ribose. Cap structures

[0252] In some embodiments of any of the polyribonucleotides disclosed herein, a polyribonucleotide comprises a cap structure.

[0253] 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.

[0254] 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')).

[0255] In some embodiments of a polyribonucleotide disclosed herein, the 5’ end of the polyribonucleotide comprises a compound of formula I: HO OR2R1Page 75 of 117 12869933v1Attorney Docket: 2012611-0187 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.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] As defined generally above for formula I, R2is hydrogen or -CH3. In some embodiments, R2is hydrogen. In some embodiments, R2is - CH3.

[0262] As defined generally above for formula I, X is O or S. In some embodiments, X is O. In some embodiments, X is S.

[0263] 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 76 of 117 12869933v1Attorney Docket: 2012611-0187 HO OHage o 12869933v1Attorney Docket: 2012611-0187 I-cPage 78 of 117 12869933v1Attorney Docket: 2012611-0187 I-f or a salt thereof, wherein each of B and B is as defined above and described herein.

[0264] In some embodiments, a compound of formula I is selected from HO OHNH2N N O O O or a salt thereof.

[0265] In some embodiments, a polyribonucleotide disclosed herein comprises at the 5’ end am7GpppN1pN2-OH cap with the following structure: Page 79 of 117 12869933v1Attorney Docket: 2012611-0187 O HN N 2 .

[0266] In somea compound with the following structure: O HN 2 , wherein

[0267] In some embodiments, a polyribonucleotide disclosed herein comprises am7GpppN1(2’-OMe)pN2-OH cap with the following structure: O 2 .Page 80 of 117 12869933v1Attorney Docket: 2012611-0187

[0268] In some embodiments, a polyribonucleotide disclosed herein comprises at the 5’ end a compound with the following structure: O HN N 2 , wherein

[0269] In some embodiments, a polyribonucleotide disclosed herein comprises am7GpppN1(2’-OMe)pN2(2’-OMe)-OH cap with the following structure: O HN 2 .

[0270] In somea compound with the following structure: Page 81 of 117 12869933v1Attorney Docket: 2012611-0187 O HN N 2 wherein

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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

[0275] Among other things, the present disclosure provides systems, compositions, and methods useful for expression and / or secretion of a payload (e.g., a polypeptide payload). A payload sequence is generally a sequence of interest (e.g., comprising a sequence that encodes a target payload, such as a target peptide or polypeptide) that is desired to be introduced into a cell, tissue, organ, organism, and / or system comprising cells. Page 82 of 117 12869933v1Attorney Docket: 2012611-0187

[0276] In some embodiments, a payload sequence comprises a sequence that encodes a single target peptide or polypeptide. For example, in some embodiments, a payload sequence encodes a target polypeptide (e.g., an enzyme, cytokine, antibody, receptor, etc.). In some embodiments, a payload sequence comprises a sequence that encodes a plurality of (e.g., at least 2, at least 3, at least 4, at least 5, or above) target peptides or polypeptides. In some embodiments, a payload sequence comprises a sequence that encodes a fusion polypeptide and / or a chimeric polypeptide, e.g., a payload sequence encoding at least a portion of two or more peptides or polypeptides. In some embodiments, a payload sequence comprises a sequence that encodes two or more polypeptides in the same oligonucleotide (e.g., two or more polypeptides are that controlled by the same or different regulatory elements).

[0277] In some embodiments, a polyribonucleotide comprising a payload sequence is a single-stranded RNA (ssRNA) oligonucleotide. In some embodiments, a polyribonucleotide comprising a payload sequence is a double- stranded RNA (dsRNA) oligonucleotide. In some embodiments, a polyribonucleotide comprising a payload sequence is a circularized RNA (circRNA) oligonucleotide.

[0278] In some embodiments, a polyribonucleotide comprising a payload sequence is a synthetic RNAs. Synthetic RNAs can be produced by any methods known in the art. For example, in some embodiments synthetic RNAs can be produced, e.g., by in vitro transcription of a DNA template.

[0279] In some embodiments, a polyribonucleotide comprising a payload sequence comprises a coding region. In some embodiments, a coding region encodes a payload. In some embodiments, a polyribonucleotide comprising a payload sequence further comprise an additional element, including, but not limited to, spacers, binding motifs, etc. In some embodiments, a polyribonucleotide comprising a payload sequence comprises one or more of: a target- encoding region, a gene regulatory element, and a transcription terminator. Non-limiting examples of gene regulatory elements include promoters, transcriptional activators, enhancers, and polyadenylation signals.

[0280] In some embodiments, a coding region encodes a gene product. In some embodiments, such a gene product is an RNA. In some embodiments, a coding region encodes a polypeptide (such as a protein, such as a glycoprotein). In some embodiments, a coding region encodes a fusion polypeptide and / or a chimeric polypeptide. In some embodiments a coding region encodes one gene product. In some embodiments, a coding region encodes more than one gene product (e.g., 2, 3, 4, 5, 6, 7 or more gene products). In some embodiments, a coding region encodes a regulatory RNA (e.g., a siRNA, microRNA, etc.).

[0281] In some embodiments, a payload sequence comprises one or more aptamer- or polypeptide-binding domains (e.g., transcription factor binding domains).

[0282] A payload sequence can be of any length, for example, between 2 and 100,000,000 nucleotides in length (or any integer value therebetween). In some embodiments, a payload sequence comprises at least 20 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, at least 550 nucleotides, at least 600 nucleotides, at least 650 nucleotides, at least 700 nucleotides, at least 750 nucleotides, at least 800 nucleotides, at least 850 nucleotides, at least 900 nucleotides, at least 950 nucleotides, at least 1000 nucleotides, at least 1100 nucleotides, at least 1200 nucleotides, at least 1300 nucleotides, at least 1400 nucleotides, at least 1500 nucleotides, at least 1600 nucleotides, Page 83 of 117 12869933v1Attorney Docket: 2012611-0187 at least 1700 nucleotides, at least 1800 nucleotides, at least 2000 nucleotides, at least 2500 nucleotides, at least 3000 nucleotides, at least 3000 nucleotides, at least 4000 nucleotides, at least 5000 nucleotides, at least 6000 nucleotides, at least 7000 nucleotides, at least 8000 nucleotides, at least 9000 nucleotides, at least 10,000 nucleotides, at least 11,000 nucleotides, at least 12,000 nucleotides, at least 13,000 nucleotides, at least 14,000 nucleotides, at least 15,000 nucleotides, at least 16,000 nucleotides, at least 17,000 nucleotides, at least 18,000 nucleotides, at least 19,000 nucleotides, at least 20,000 nucleotides, at least 21,000 nucleotides, at least 22,000 nucleotides, at least 23,000 nucleotides, at least 24,000 nucleotides, or at least 25,000 nucleotides.

[0283] In some embodiments, a polynucleotide comprising a payload sequence is between 50 and 25,000 nucleotides in length, between 100 and 20,000 nucleotides in length, between 500 and 10,000 nucleotides in length, between 1,000 and 8,000 nucleotides in length, and / or between 2,000 and 5,000 nucleotides in length.

[0284] In some embodiments, a payload is or comprises an antigen or a fragment or variant thereof. In some embodiments, an antigen comprises a cancer antigen, a viral antigen, a bacterial antigen or a fungal antigen or a fragment or variant of any of the foregoing.

[0285] In some embodiments, an antigen is a mutated antigen. In some embodiments, an antigen comprises a mutated cancer antigen, a mutated viral antigen, a mutated bacterial antigen or a mutated fungal antigen. In some embodiments of a mutated antigen comprises one or more mutation(s) that can avoid immune tolerance. In some embodiments, a mutated antigen may be expressed intracellularly for MHC presentation for T cell stimulation. In some embodiments, a mutated antigen may be secreted for stimulation of antibody production. In some embodiments, a mutated antigen is introduced to focus immune responses on certain epitopes. In some embodiments, a mutated antigen is intracellular. In some embodiments, a mutated antigen is secreted.

[0286] In some embodiments, a payload is or comprises an antigen comprising one or more epitopes from an antigen of interest to induce a broadly neutralizing immune response. In some embodiments, an antigen comprises fragments from one or more viral strains, bacterial strains and / or fungal strains. In some embodiments, an antigen comprises fragments from one or more cancer antigens. In some embodiments, an antigen comprises fragments from one or more mutated antigens.

[0287] In some embodiments, a payload is or comprises an antibody or a fragment thereof. In some embodiments, a payload is or comprises an antigen binding domain from an antibody. In some embodiments, a payload is or comprises an scFv, a Fab fragment, a single chain antibody domain (e.g., a VHH, a nanobody, a VNAR). In some embodiments, a payload is or comprises an antigen binding domain fused to a half-life extender (e.g., an Fc domain or albumin). In some embodiments, a payload is or comprises a single chain antibody fusion (e.g., bispecific T cell engager, e.g., BiTE polypeptides). In some embodiments, a payload is or comprises a single chain antibody fusion with cytokines (e.g., tri-specific natural killer cell engager, e.g., TriKe polypeptides).

[0288] In some embodiments, a payload sequence is or comprises a mutant transmembrane, membrane- associated, and / or a secreted costimulatory polypeptide.

[0289] In some embodiments, a payload is or comprises a secreted protein or a fragment thereof. Table 10: Exemplary payload amino acid sequences disclosed herein. Page 84 of 117 12869933v1Attorney Docket: 2012611-0187 Exemplary payload Amino Acid Sequence (SEQ ID NO: ) SARS-CoV-2 Spike secretion MFVFLVLLPLVSSAA (SEQ ID NO: 1) peptide (Ssp): G F F NS P AA NS P G A K G F R EV DI L R G E V K M QP E QP P D E K E SA L D V W W G Y APayloads comprising Sbi domains Page 85 of 117 12869933v1Attorney Docket: 2012611-0187

[0290] 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 a fragment or a variant thereof, and (b) a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus. In some 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.

[0291] 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 byaureus can as an an 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.

[0293] 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.

[0294] 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 variant thereof.

[0295] S. aureus Sbi is referenced by Gene ID: 3919725 and NCBI Ref No.: NC_007795.1. A polynucleotide sequence of Sbi is provided herein as SEQ ID NO: 79: ATGAAAAATAAATATATCTCGAAGTTGCTAGTTGGGGCAGCAACAATTACGTTAGCTACAATGATTTCAAATGGGGAAGCAAAAG CGAGTGAAAACACGCAACAAACTTCAACTAAGCACCAAACAACTCAAAACAACTACGTAACAGATCAACAAAAAGCTTTTTATCAA GTATTACATCTAAAAGGTATCACAGAAGAACAACGTAACCAATACATCAAAACATTACGCGAACACCCAGAACGTGCACAAGAAG TATTCTCTGAATCACTTAAAGACAGCAAGAACCCAGACCGACGTGTTGCACAACAAAACGCTTTTTACAATGTTCTTAAAAATGA TAACTTAACTGAACAAGAAAAAAATAATTACATTGCACAAATTAAAGAAAACCCTGATAGAAGCCAACAAGTTTGGGTAGAATCA GTACAATCTTCTAAAGCTAAAGAACGTCAAAATATTGAAAATGCGGATAAAGCAATTAAAGATTTCCAAGATAACAAAGCACCAC ACGATAAATCAGCAGCATATGAAGCTAACTCAAAATTACCTAAAGATTTACGTGATAAAAACAACCGCTTTGTAGAAAAAGTTTC AATTGAAAAAGCAATCGTTCGTCATGATGAGCGTGTGAAATCAGCAAATGATGCAATCTCAAAATTAAATGAAAAAGATTCAATT GAAAACAGACGTTTAGCACAACGTGAAGTTAACAAAGCACCTATGGATGTAAAAGAGCATTTACAGAAACAATTAGACGCATTAG TTGCTCAAAAAGATGCTGAAAAGAAAGTGGCGCCAAAAGTTGAGGCTCCTCAAATTCAATCACCACAAATTGAAAAACCTAAAGT AGAATCACCAAAAGTTGAAGTCCCTCAAATTCAATCACCAAAAGTTGAGGTTCCTCAATCTAAATTATTAGGTTACTACCAATCAT TAAAAGATTCATTTAACTATGGTTACAAGTATTTAACAGATACTTATAAAAGCTATAAAGAAAAATATGATACAGCAAAGTACTAC TATAATACGTACTATAAATACAAAGGTGCGATTGATCAAACAGTATTAACAGTACTAGGTAGTGGTTCTAAATCTTACATCCAAC CATTGAAAGTTGATGATAAAAACGGCTACTTAGCTAAATCATATGCACAAGTAAGAAACTATGTAACTGAGTCAATCAATACTGG TAAAGTATTATATACTTTCTACCAAAACCCAACATTAGTAAAAACAGCTATTAAAGCTCAAGAAACTGCATCATCAATCAAAAATA CATTAAGTAATTTATTATCATTCTGGAAATAA

[0296] A polypeptide sequence of Sbi is provided herein as SEQ ID NO: 80. Page 86 of 117 12869933v1Attorney Docket: 2012611-0187 MKNKYISKLLVGAATITLATMISNGEAKASENTQQTSTKHQTTQNNYVTDQQKAFYQVLHLKGITEEQRNQYIKTLREHPERAQEVF SESLKDSKNPDRRVAQQNAFYNVLKNDNLTEQEKNNYIAQIKENPDRSQQVWVESVQSSKAKERQNIENADKAIKDFQDNKAPHD KSAAYEANSKLPKDLRDKNNRFVEKVSIEKAIVRHDERVKSANDAISKLNEKDSIENRRLAQREVNKAPMDVKEHLQKQLDALVAQK DAEKKVAPKVEAPQIQSPQIEKPKVESPKVEVPQIQSPKVEVPQSKLLGYYQSLKDSFNYGYKYLTDTYKSYKEKYDTAKYYYNTYYKY KGAIDQTVLTVLGSGSKSYIQPLKVDDKNGYLAKSYAQVRNYVTESINTGKVLYTFYQNPTLVKTAIKAQETASSIKNTLSNLLSFWK

[0297] In some embodiments, a domain III of Sbi from S. aureus strain Mu50 is provided herein as SEQ ID NO: 81: IENADKAIKDFQDNKAPHDKSAAYEANSKLPKDLRDKNNRFV (SEQ ID NO: 81).

[0298] In some embodiments, a domain IV of Sbi from S. aureus strain Mu50 is provided herein as SEQ ID NO: 82: EKVSIEKAIVRHDERVKSANDAISKLNEKDSIENRRLAQREVNKAPMDVKEHLQKQLD (SEQ ID NO: 82).

[0299] In some embodiments, a Sbi complement C3d binding polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 100% identity to the amino acid sequence of SEQ ID NO: 81. In some embodiments, a Sbi complement C3d binding polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 100% identity to the amino acid sequence of SEQ ID NO: 82. 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: 80.

[0300] In some embodiments, a Sbi complement C3d binding polypeptide is encoded by a polynucleotide which encodes an amino acid having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 100% identity to the amino acid sequence of SEQ ID NO: 81. In some embodiments, a Sbi complement C3d binding polypeptide is encoded by a polynucleotide which encodes an amino acid having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 100% identity to the amino acid sequence of SEQ ID NO: 82. In some embodiments, a Sbi complement C3d binding polypeptide is encoded by a polynucleotide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 100% identity to the nucleotide sequence of SEQ ID NO: 79. 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.

[0301] 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%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 100% identity to the amino acid sequence of SEQ ID NO: 81. In some embodiments, an Sbi domain III comprises the amino acid sequence of SEQ ID NO: 81.

[0302] 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%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 100% identity to the amino acid sequence of SEQ ID NO: 82. In some embodiments, an Sbi domain IV comprises the amino acid sequence of SEQ ID NO: 82. Page 87 of 117 12869933v1Attorney Docket: 2012611-0187

[0303] 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%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 100% identity to the amino acid sequence of SEQ ID NO: 81. In some embodiments, an Sbi domain III comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, an Sbi domain IV comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 100% identity to the amino acid sequence of SEQ ID NO: 82. In some embodiments, an Sbi domain IV comprises the amino acid sequence of SEQ ID NO: 82. Compositions and pharmaceutical compositions

[0304] Among other things, provided herein are compositions comprising polynucleotides disclosed herein.

[0305] In some embodiments, a composition is a pharmaceutical composition.

[0306] 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.

[0307] 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. Formulations

[0308] 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.

[0309] 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.

[0310] 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.

[0311] 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. Page 88 of 117 12869933v1Attorney Docket: 2012611-0187

[0312] 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.

[0313] 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

[0314] 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

[0315] The disclosure provides, among other things, methods for using a nucleic acid expression system disclosed herein, a polynucleotide disclosed herein, or a composition comprising the same.

[0316] In some embodiments, provided herein is a method of administering a nucleic acid expression system disclosed herein, a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject.

[0317] In some embodiments, provided herein is a vaccination method comprising administering a nucleic acid expression system disclosed herein, a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject.

[0318] In some embodiments, disclosed herein is a gene therapy method comprising administering a nucleic acid expression system disclosed herein, 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.

[0319] In some embodiments, provided herein is a method for stimulating an immune response comprising administering a nucleic acid expression system disclosed herein, a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject.

[0320] In some embodiments, also provided herein is a cell therapy engineering method comprising administering a nucleic acid expression system disclosed herein, a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject.

[0321] In some embodiments, provided herein is an immunotherapy method comprising administering a nucleic acid expression system disclosed herein, 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.

[0322] In some embodiments, disclosed herein is a protein replacement therapy method comprising administering a nucleic acid expression system disclosed herein, 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. Page 89 of 117 12869933v1Attorney Docket: 2012611-0187

[0323] In some embodiments, provided herein is a chemotherapeutic method comprising administering a nucleic acid expression system disclosed herein, a polynucleotide disclosed herein or a composition comprising a polynucleotide disclosed herein to a cell, tissue or subject. Kits

[0324] 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

[0325] Embodiment 1. A nucleic acid expression system comprising: (i) a polynucleotide comprising a payload sequence, and (ii) at least one polynucleotide that encodes one or more helper polypeptides that comprise: (a) a chaperone or ER guardian polypeptide, or a fragment or variant thereof, (b) an unfolded protein response (UPR) inhibitor polypeptide, or a fragment or variant thereof, (c) an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof, (d) a SNARE polypeptide, or a fragment or variant thereof, (e) a ubiquitin protein ligase polypeptide, or a fragment or variant thereof, (f) an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof, (g) a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof, or (h) any combination of (a)-(g).

[0326] Embodiment 2. The nucleic acid expression system of embodiment 1, wherein the one or more helper polypeptides comprise a chaperone or ER guardian polypeptide, or a fragment or variant thereof.

[0327] Embodiment 3. The nucleic acid expression system of embodiment 2, wherein the chaperone or ER guardian polypeptide comprises a TAPT1 polypeptide, chaperone or a fragment or variant thereof.

[0328] Embodiment 4. The nucleic acid expression system of embodiment 3, wherein the TAPT1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 11.

[0329] Embodiment 5. The nucleic acid expression system of embodiment 3 or 4, wherein the TAPT1 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 50, or a codon optimized version thereof.

[0330] Embodiment 6. The nucleic acid expression system of embodiment 2, wherein the chaperone or ER guardian polypeptide comprises a heat shock protein 70 (HSP70) family member or a fragment or variant thereof.

[0331] Embodiment 7. The nucleic acid expression system of embodiment 6, wherein the HSP70 family member comprises HSPA13 or a fragment or variant thereof.

[0332] Embodiment 8. The nucleic acid expression system of embodiment 2, wherein the chaperone or ER guardian polypeptide comprises a heat shock protein 40 (HSP40) family member or a fragment or variant thereof.

[0333] Embodiment 9. The nucleic acid expression system of embodiment 8, wherein the HSP40 family member comprises DNAJC8 or a fragment or variant thereof. Page 90 of 117 12869933v1Attorney Docket: 2012611-0187

[0334] Embodiment 10. The nucleic acid expression system of any one of the preceding embodiments, wherein the one or more helper polypeptides comprise a UPR inhibitor polypeptide, or a fragment or variant thereof.

[0335] Embodiment 11. The nucleic acid expression system of embodiment 10, wherein the UPR inhibitor polypeptide comprises: a K3L polypeptide, or a fragment or variant thereof, a gamma 134.5 polypeptide, or a fragment or variant thereof, a GADD34 polypeptide, or a fragment or variant thereof, or an IRE1b polypeptide, or a fragment or variant thereof, or any combination thereof.

[0336] Embodiment 12. The nucleic acid expression system of embodiment 11, wherein the K3L polypeptide comprises a K3L_H47R polypeptide.

[0337] Embodiment 13. The nucleic acid expression system of embodiment 12, wherein the K3L polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 12.

[0338] Embodiment 14. The nucleic acid expression system of embodiment 12 or 13, wherein the K3L polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 52, or a codon optimized version thereof.

[0339] Embodiment 15. The nucleic acid expression system of embodiment 11, wherein the gamma 134.5 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 13.

[0340] Embodiment 16. The nucleic acid expression system of embodiment 11 or 15, wherein the gamma 134.5 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 53, or a codon optimized version thereof.

[0341] Embodiment 17. The nucleic acid expression system of embodiment 11, wherein the GADD34 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 14.

[0342] Embodiment 18. The nucleic acid expression system of embodiment 11 or 17, wherein the GADD34 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 54, or a codon optimized version thereof.

[0343] Embodiment 19. The nucleic acid expression system of embodiment 11, wherein the IRE1b polypeptide comprises a IRE1b_delCTD polypeptide or an IRE1b_K548A mutation.

[0344] Embodiment 20. The nucleic acid expression system of embodiment 11 or 19, wherein the IRE1b polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 15.

[0345] Embodiment 21. The nucleic acid expression system any one of embodiments 11, or 19-20, wherein the IRE1b polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 51, or a codon optimized version thereof.

[0346] Embodiment 22. The nucleic acid expression system of any one of the preceding embodiments, wherein the one or more helper polypeptides comprise an ERAD polypeptide or a fragment or variant thereof.

[0347] Embodiment 23. The nucleic acid expression system of embodiment 22, wherein the ERAD polypeptide comprises a Derlin-1 polypeptide or a fragment or variant thereof, or a JKAMP polypeptide or a fragment or variant thereof. Page 91 of 117 12869933v1Attorney Docket: 2012611-0187

[0348] Embodiment 24. The nucleic acid expression system of embodiment 23, wherein the Derlin-1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 16.

[0349] Embodiment 25. The nucleic acid expression system of embodiment 23 or 24, wherein the Derlin-1 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 55, or a codon optimized version thereof.

[0350] Embodiment 26. The nucleic acid expression system of any one of the preceding embodiments, wherein the one or more helper polypeptides comprise a SNARE polypeptide or a fragment or variant thereof.

[0351] Embodiment 27. The nucleic acid expression system of embodiment 26, wherein the SNARE polypeptide comprises a GosR1 polypeptide or a fragment or variant thereof, a Stx5 polypeptide or a fragment or variant thereof, a Bet1 polypeptide or a fragment or variant thereof, a SEC22A polypeptide or a fragment or variant thereof, a SEC22C polypeptide or a fragment or variant thereof, a STXB6 polypeptide or a fragment or variant thereof, a GOSR2 polypeptide or a fragment or variant thereof, or any combination thereof.

[0352] Embodiment 28. The nucleic acid expression system of embodiment 26, wherein the SNARE polypeptide comprises a GosR1 polypeptide or a fragment or variant thereof, a Stx5 polypeptide or a fragment or variant thereof, and a Bet1 polypeptide or a fragment or variant thereof.

[0353] Embodiment 29. The nucleic acid expression system of embodiment 27 or 28, wherein the GosR1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 17.

[0354] Embodiment 30. The nucleic acid expression system of any one of embodiments 27-29, wherein the GosR1 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 56, or a codon optimized version thereof.

[0355] Embodiment 31. The nucleic acid expression system of embodiment 27 or 28, wherein the Stx5 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 18.

[0356] Embodiment 32. The nucleic acid expression system of any one of embodiments 27-28 and 31, wherein the Stx5 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 57, or a codon optimized version thereof.

[0357] Embodiment 33. The nucleic acid expression system of embodiment 27 or 28, wherein the Bet1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 19.

[0358] Embodiment 34. The nucleic acid expression system of any one of embodiments 27-28 and 33, wherein the Bet1 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 58, or a codon optimized version thereof.

[0359] Embodiment 35. The nucleic acid expression system of any one of the preceding embodiments, wherein the one or more helper polypeptides comprise a ubiquitin protein ligase polypeptide or a fragment or variant thereof.

[0360] Embodiment 36. The nucleic acid expression system of embodiment 35, wherein ubiquitin protein ligase polypeptide comprises: a NEDD4L polypeptide or a fragment or variant thereof, a NEDD4 polypeptide or a fragment or variant thereof an ITCH polypeptide or a fragment or variant thereof, a WWP1 polypeptide or a fragment or Page 92 of 117 12869933v1Attorney Docket: 2012611-0187 variant thereof, a WWP2 polypeptide or a fragment or variant thereof, a SMURF1 polypeptide or a fragment or variant thereof, a SMURF2 polypeptide or a fragment or variant thereof, a RNF5 polypeptide or a fragment or variant thereof, a TRAF1 polypeptide or a fragment or variant thereof, a TRAF2 polypeptide or a fragment or variant thereof, a TRAF3 polypeptide or a fragment or variant thereof, a TRAF6 polypeptide or a fragment or variant thereof, a TRAF7 polypeptide or a fragment or variant thereof, a UBE3A polypeptide or a fragment or variant thereof, a CUL1 polypeptide or a fragment or variant thereof, a CUL2 polypeptide or a fragment or variant thereof, a CUL3 polypeptide or a fragment or variant thereof, a MARCH-II polypeptide or a fragment or variant thereof, a MARCH-III polypeptide or a fragment or variant thereof, a MARCH-VI polypeptide or a fragment or variant thereof, a AMFR polypeptide or a fragment or variant thereof, a HERC3 polypeptide or a fragment or variant thereof, a TRIM63 polypeptide or a fragment or variant thereof, a TRIM21 polypeptide or a fragment or variant thereof, a BIRC3 polypeptide or a fragment or variant thereof.

[0361] Embodiment 37. The nucleic acid expression system of embodiment 36, wherein the NEDD4L polypeptide comprises one or more mutations, wherein the one or more mutations comprise Y679C, Q694H, E893K, R897Q, or any combination thereof.

[0362] Embodiment 38. The nucleic acid expression system of embodiment 37, wherein the NEDD4L polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 20, 21, 22, 23, or 24.

[0363] Embodiment 39. The nucleic acid expression system of embodiment 37 or 38, wherein the NEDD4L polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of any one of SEQ ID NOs: 59, 60, 61, 62, or 63, or a codon optimized version of any of the foregoing.

[0364] Embodiment 40. The nucleic acid expression system of any one of the preceding embodiments, wherein the one or more helper polypeptides comprise or a fragment or variant thereof. E3 ubiquitin ligase substrate polypeptide

[0365] Embodiment 41. The nucleic acid expression system of embodiment 40, wherein the E3 ubiquitin ligase substrate polypeptide comprises: a RIPK2 polypeptide or a fragment or variant thereof, an ERBB4 polypeptide or a fragment or variant thereof, an AKT1 polypeptide or a fragment or variant thereof, a Notch1 polypeptide or a fragment or variant thereof, a NEMO polypeptide or a fragment or variant thereof, or any combination thereof.

[0366] Embodiment 42. The nucleic acid expression system of embodiment 41, wherein the E3 ubiquitin ligase substrate polypeptide comprises: a RIPK2 polypeptide or a fragment or variant thereof, an ERBB4 polypeptide or a fragment or variant thereof, an AKT1 polypeptide or a fragment or variant thereof, a Notch1 polypeptide or a fragment or variant thereof, and a NEMO polypeptide or a fragment or variant thereof.

[0367] Embodiment 43. The nucleic acid expression system of embodiment 42, wherein the RIPK2 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 25.

[0368] Embodiment 44. The nucleic acid expression system of embodiment 42 or 43, wherein the RIPK2 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 64, or a codon optimized version thereof. Page 93 of 117 12869933v1Attorney Docket: 2012611-0187

[0369] Embodiment 45. The nucleic acid expression system of embodiment 42, wherein the ERBB4 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 26.

[0370] Embodiment 46. The nucleic acid expression system of embodiment 42 or 45, wherein the ERBB4 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 65, or a codon optimized version thereof.

[0371] Embodiment 47. The nucleic acid expression system of embodiment 42, wherein the AKT1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 27.

[0372] Embodiment 48. The nucleic acid expression system of embodiment 42 or 47, wherein the AKT1 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 66, or a codon optimized version thereof.

[0373] Embodiment 49. The nucleic acid expression system of embodiment 42, wherein the Notch1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 28.

[0374] Embodiment 50. The nucleic acid expression system of embodiment 42 or 49, wherein the Notch1 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO:67, or a codon optimized version thereof.

[0375] Embodiment 51. The nucleic acid expression system of embodiment 42, wherein the NEMO polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 29.

[0376] Embodiment 52. The nucleic acid expression system of embodiment 42 or 51, wherein the NEMO polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 68, or a codon optimized version thereof.

[0377] Embodiment 53. The nucleic acid expression system of any one of the preceding embodiments, wherein the one or more helper polypeptides comprise polypeptide upregulated during plasma cell differentiation or a fragment or variant thereof.

[0378] Embodiment 54. The nucleic acid expression system of embodiment 53, wherein the polypeptide upregulated during plasma cell differentiation comprises: a GJB2 polypeptide or a fragment or variant thereof, a NDFIP2 polypeptide or a fragment or variant thereof, a CD300A polypeptide or a fragment or variant thereof, a XCR1 polypeptide or a fragment or variant thereof, a STBD1 polypeptide or a fragment or variant thereof, a EDA2R polypeptide or a fragment or variant thereof, a TMEM200A polypeptide or a fragment or variant thereof, a GPR114 polypeptide or a fragment or variant thereof, a DPAGT1 polypeptide or a fragment or variant thereof, a CCR2 polypeptide or a fragment or variant thereof, or any combination thereof.

[0379] Embodiment 55. The nucleic acid expression system of embodiment 54, wherein the polypeptide upregulated during plasma cell differentiation comprises: a GJB2 polypeptide or a fragment or variant thereof, a NDFIP2 polypeptide or a fragment or variant thereof, a CD300A polypeptide or a fragment or variant thereof, a XCR1 polypeptide or a fragment or variant thereof, a STBD1 polypeptide or a fragment or variant thereof, a EDA2R polypeptide or a fragment or variant thereof, a TMEM200A polypeptide or a fragment or variant thereof, a GPR114 Page 94 of 117 12869933v1Attorney Docket: 2012611-0187 polypeptide or a fragment or variant thereof, a DPAGT1 polypeptide or a fragment or variant thereof, and a CCR2 polypeptide or a fragment or variant thereof.

[0380] Embodiment 56. The nucleic acid expression system of embodiment 54 or 55, wherein the GJB2 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 30.

[0381] Embodiment 57. The nucleic acid expression system of any one of embodiments 54-55 and 56, wherein the GJB2 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 69, or a codon optimized version thereof.

[0382] Embodiment 58. The nucleic acid expression system of embodiment 54 or 55, wherein the NDFIP2 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 31.

[0383] Embodiment 59. The nucleic acid expression system of any one of embodiments 54-55 and 58, wherein the NDFIP2 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 70, or a codon optimized version thereof.

[0384] Embodiment 60. The nucleic acid expression system of embodiment 54 or 55, wherein the CD300A polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 32.

[0385] Embodiment 61. The nucleic acid expression system of any one of embodiments 54-55 and 60, wherein the CD300A polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 71, or a codon optimized version thereof.

[0386] Embodiment 62. The nucleic acid expression system of embodiment 54 or 55, wherein the XCR1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 33.

[0387] Embodiment 63. The nucleic acid expression system of any one of embodiments 54-55 and 62, wherein the XCR1 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 72, or a codon optimized version thereof.

[0388] Embodiment 64. The nucleic acid expression system of embodiment 54 or 55, wherein the STBD1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 34.

[0389] Embodiment 65. The nucleic acid expression system of any one of embodiments 54-55 and 64, wherein the STBD1 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 73, or a codon optimized version thereof.

[0390] Embodiment 66. The nucleic acid expression system of embodiment 54 or 55, wherein the EDA2R polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 35.

[0391] Embodiment 67. The nucleic acid expression system of any one of embodiments 54-55 and 66, wherein the EDA2R polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 74, or a codon optimized version thereof.

[0392] Embodiment 68. The nucleic acid expression system of embodiment 54 or 55, wherein the TMEM200A polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 36. Page 95 of 117 12869933v1Attorney Docket: 2012611-0187

[0393] Embodiment 69. The nucleic acid expression system of any one of embodiments 54-55 and 68, wherein the TMEM200A polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 75, or a codon optimized version thereof.

[0394] Embodiment 70. The nucleic acid expression system of embodiment 54 or 55, wherein the GPR114 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 37.

[0395] Embodiment 71. The nucleic acid expression system of any one of embodiments 54-55 and 70, wherein the GPR114 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 76, or a codon optimized version thereof.

[0396] Embodiment 72. The nucleic acid expression system of embodiment 54 or 55, wherein the DPAGT1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 38.

[0397] Embodiment 73. The nucleic acid expression system of any one of embodiments 54-55 and 72, wherein the DPAGT1 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 77, or a codon optimized version thereof.

[0398] Embodiment 74. The nucleic acid expression system of embodiment 54 or 55, wherein the CCR2 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 39.

[0399] Embodiment 75. The nucleic acid expression system of any one of embodiments 54-55 and 74, wherein the CCR2 polypeptide is encoded by a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 78, or a codon optimized version thereof.

[0400] Embodiment 76. The nucleic acid expression system of embodiment 1, wherein the one or more helper polypeptides comprise one or more UPR inhibitor polypeptides, and one or more additional helper polypeptides.

[0401] Embodiment 77. The nucleic acid expression system of embodiment 76, wherein the one or more additional helper polypeptides is other than a UPR inhibitor polypeptide.

[0402] Embodiment 78. The nucleic acid expression system of embodiment 77, wherein the one or more additional helper polypeptides comprises any one of (a), or (c)-(g) specified in embodiment 1.

[0403] Embodiment 79. The nucleic acid expression system of any one of the preceding embodiments, wherein the at least one polynucleotide comprising a sequence that encodes a helper polypeptide increases expression and / or secretion of a payload polypeptide encoded by the polynucleotide comprising a payload sequence.

[0404] Embodiment 80. The nucleic acid expression system of any one of the preceding embodiments, wherein the nucleic acid expression system is characterized in that when administered to a cell, tissue, or subject, increased payload expression and / or secretion is observed as compared to payload expression and / or secretion in a cell, tissue, or subject administered an otherwise similar polynucleotide comprising a payload sequence without at least one polynucleotide comprising a sequence encoding a helper polypeptide.

[0405] Embodiment 81. The nucleic acid expression system of any one of the preceding embodiments, wherein increased payload expression comprises at least 2-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least Page 96 of 117 12869933v1Attorney Docket: 2012611-0187 200-fold, at least 300-fold, at least 400-fold at least-500 fold, at least 600-fold, at least 700-fold, or at least 800-fold increased expression of the payload.

[0406] Embodiment 82. The nucleic acid expression system of any one of the preceding embodiments, wherein increased payload secretion comprises at least 2-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 200- fold, at least 300-fold, at least 400-fold, or at least 500-fold increased secretion of the payload.

[0407] Embodiment 83. The nucleic acid expression system of embodiment 82, wherein increased secretion and / or expression comprises increased trafficking through a secretory pathway, increased proper folding of the polypeptide, reduced degradation of the polypeptide, or reduced aggregation of the polypeptide.

[0408] Embodiment 84. The nucleic acid expression system of any one of the preceding embodiments, wherein the polynucleotide comprising a payload sequence and / or the at least one polynucleotide comprising a sequence that encodes a helper polypeptide is a DNA.

[0409] Embodiment 85. The nucleic acid expression system of any one of embodiments 1-83, wherein the polynucleotide comprising a payload sequence and / or the at least one polynucleotide comprising a sequence that encodes a helper polypeptide is an RNA.

[0410] Embodiment 86. The nucleic acid expression system of embodiment 85, wherein the at least one polynucleotide is a polyribonucleotide comprising one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0411] Embodiment 87. The nucleic acid expression system of embodiment 86, wherein the one or more modified ribonucleotides comprise modified ribonucleotides that comprise a N4-acetylcytidine nucleoside.

[0412] Embodiment 88. The nucleic acid expression system of embodiment 86, wherein the one or more modified ribonucleotides comprise modified ribonucleotides that comprise a 5-hydroxymethyluridine nucleoside.

[0413] Embodiment 89. The nucleic acid expression system of embodiment 86, 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. Page 97 of 117 12869933v1Attorney Docket: 2012611-0187

[0414] Embodiment 90. The nucleic acid expression system of any one of embodiments 86-89, wherein the one or more modified ribonucleotides comprise modified ribonucleotides that comprise a 2’-O-acetylated ribose.

[0415] Embodiment 91. The nucleic acid expression system of any one of embodiments 85-90, wherein the polyribonucleotide comprises a cap structure and the cap structure does not comprise a 2’-O-acetylated ribose.

[0416] Embodiment 92. The nucleic acid expression system of any one of embodiments 85-90, wherein the polyribonucleotide comprises a cap structure and the cap structure comprises a 2’-O-acetylated ribose.

[0417] Embodiment 93. The nucleic acid expression system of any one of the preceding embodiments, wherein the polynucleotide comprising a payload sequence comprises a coding region.

[0418] Embodiment 94. The nucleic acid expression system of embodiment 93, wherein the coding region encodes a payload.

[0419] Embodiment 95. The nucleic acid expression system of embodiment 93 or 94, wherein the payload is or comprises a polypeptide.

[0420] Embodiment 96. The nucleic acid expression system of embodiment 95, wherein the payload comprises an antigen or a fragment thereof.

[0421] Embodiment 97. The nucleic acid expression system of embodiment 95, wherein the payload comprises an antibody or a fragment thereof.

[0422] Embodiment 98. The nucleic acid expression system of embodiment 95, wherein the payload comprises a secreted protein or a fragment thereof.

[0423] Embodiment 99. The nucleic acid expression system of embodiment 95, wherein the payload comprises a cell-surface associated protein or a fragment thereof.

[0424] Embodiment 100. The nucleic acid expression system of embodiment 95, wherein the payload comprises an intracellular protein or a fragment thereof.

[0425] Embodiment 101. The nucleic acid expression system of embodiment 93 or 94, wherein the payload comprises an RNA payload.

[0426] Embodiment 102. One or more compositions comprising a nucleic acid expression system of any one of the preceding embodiments.

[0427] Embodiment 103. The composition of embodiment 102, wherein (i) and (ii) are in separate compositions.

[0428] Embodiment 104. The composition of embodiment 102 or 103, wherein (i) and (ii) are administered separately.

[0429] Embodiment 105. The composition of embodiment 102 or 103, wherein (i) and (ii) are administered at substantially the same time.

[0430] Embodiment 106. The composition of any one of embodiments 102-104, wherein (i) is administered first followed by (ii). Page 98 of 117 12869933v1Attorney Docket: 2012611-0187

[0431] Embodiment 107. The composition of any one of embodiments 102-104, wherein (ii) is administered first followed by (i).

[0432] Embodiment 108. The composition of embodiment 102, wherein (i) and (ii) are in the same composition.

[0433] Embodiment 109. The composition of any one of embodiments 102-108, wherein the composition is a pharmaceutical composition.

[0434] Embodiment 110. The composition of embodiment 109, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0435] Embodiment 111. The composition of embodiment 110, wherein the pharmaceutical composition is formulated for intramuscular, intradermal, or intravenous delivery.

[0436] Embodiment 112. A set of polypeptides encoded by the nucleic acid expression system of any one of embodiments 1-101.

[0437] Embodiment 113. A cell comprising a nucleic acid expression system of any one of embodiments 1-101.

[0438] Embodiment 114. The cell of embodiment 113, wherein the cell is in vivo.

[0439] Embodiment 115. The cell of embodiment 113, wherein the cell is ex vivo.

[0440] Embodiment 116. The cell of any one of embodiments 113-115, wherein the nucleic acid expression system is introduced into the cell.

[0441] Embodiment 117. The cell of any one of embodiments 113-116, wherein the cell expresses one or more polypeptides encoded by the nucleic acid expression system.

[0442] Embodiment 118. A method of delivering a nucleic acid expression system of any one of embodiments 1- 101, or a composition of any one of embodiments 102-111 to a cell, tissue or subject.

[0443] Embodiment 119. The method of embodiment 118, wherein delivering comprises administering the nucleic acid expression system or the composition comprising to the cell, tissue or subject.

[0444] Embodiment 120. The method of embodiment 118 or 119, wherein the method is a treatment method or a prevention method.

[0445] Embodiment 121. The method of any one of embodiments 118-120, wherein the method comprises: (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).

[0446] Embodiment 122. The method of any one of clams 118-121, wherein the subject is a human.

[0447] Embodiment 123. A method of stimulating an immune response to an antigen, comprising delivering a nucleic acid expression system of any one of embodiments 1-101, or a composition of any one of embodiments 102- 111 to a cell, tissue or subject. Page 99 of 117 12869933v1Attorney Docket: 2012611-0187

[0448] Embodiment 124. The method of embodiment 123, wherein the subject is a human.

[0449] Embodiment 125. A composition comprising a nucleic acid expression system of any one of embodiments 1-101, or the composition of any one of embodiments 102-111, for use in delivering the nucleic acid expression system to a cell, tissue, or subject.

[0450] Embodiment 126. A composition comprising a nucleic acid expression system of any one of embodiments 1-101, or the composition of any one of embodiments 102-111, for use in stimulating an immune response to an antigen.

[0451] Embodiment 127. The composition for use of embodiment 126, wherein the use comprise delivering the nucleic acid expression system to a cell, tissue, or subject.

[0452] Embodiment 128. The composition for use of embodiment 125 or 127, wherein delivering comprises administering.

[0453] Embodiment 129. The composition for use of any one of embodiments 125, and 127-128, wherein the subject is a human.

[0454] Embodiment 130. The composition for use of any one of embodiments 125-129, 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).

[0455] Embodiment 131. Use of a nucleic acid expression system according to any one of embodiments 1-101, or a composition according to any one of embodiments 102-111, in the preparation of a medicament for delivering the nucleic acid expression system or composition to a cell, tissue, or subject.

[0456] Embodiment 132. Use of a nucleic acid expression system according to any one of embodiments 1-101, or a composition according to any one of embodiments 102-111, in the preparation of a medicament for stimulating an immune response to an antigen.

[0457] Embodiment 133. The use of embodiment 132, further comprising delivering the nucleic acid expression system to a cell, tissue, or subject.

[0458] Embodiment 134. The use of embodiment 131 or 133, wherein delivering comprises administering.

[0459] Embodiment 135. The use of any one of embodiments 131 and 133-134, wherein the subject is a human.

[0460] Embodiment 136. The use of any one of embodiments 131-135, 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 100 of 117 12869933v1Attorney Docket: 2012611-0187 EXEMPLIFICATION Example 1: Methods and Materials used in Example 2 and Example 3 Disclosed Herein DNA Vectors

[0461] Primers used in generation and validation of DNA vectors are described in Table 11. Table 11: Exemplary Primer Nucleotide Sequences Exemplary Primer Nucleotide Sequence (SEQ ID NO:) NLuc_colony_forward CGAGACAGAGAAGACTCTTGCGTTTCTGATAGG (SEQ ID NO: 40) NLuc colony reverse GAGAGCTCGCCTGCAGGAATTGG (SEQ ID NO: 41) T G C T C A C

[0462] Wild type SARS-CoV-2 Spike protein receptor binding domain (RBD) and a mutant variant were ordered as gBlocks (Integrated DNA Technologies) with an N-terminal Spike secretion peptide (Ssp) and cloned into the NanoLuc protein fusion vector pNLF1-C (Promega) using NEBuilder HIFI DNA Assembly Master Mix (New England Biolabs). This master mix comprises enzymes and buffers that facilitate quick and efficient Gibson assembly of two or more pieces of DNA. A pNLF1-C vector was first linearized by double digesting it with restriction enzymes EcoRI-HF and Nhel-HF. The digested vector was then purified using QIAquick Gel Extraction Kit (Qiagen) and concentrated using the Zymo DNA Clean and Concentrator-5 (Zymoresearch). gBlock constructs were then inserted into the linearized vector by mixing, in a 1.5 ml tube, 50 ng of the purified vector, a two-fold molar excess of insert, 10 µl of the master mix, and an appropriate volume of nuclease-free water to make a 20 µl reaction. The reaction was then incubated at 50°C for 15 minutes. The ligation reaction was diluted 1:4 and 2 µl of the diluted reaction was then used to transform One Shot TOP10 Chemically Competent E. coli cells (ThermoFisher Scientific) by heat shock at 42°C for 30 seconds. Next, the cells were streaked on Ampicillin agar plates and incubated for 24 hours at 37°C. The next day, colonies were screened for uptake of the chimeric vector using colony PCR. Colonies were first suspended in 100 µl of water, and 1 µl of that mixture was mixed with 1 µl each of 10 µM forward and reverse primers (NLuc_colony_forward and NLuc_colony_reverse), 10 µl of 2x Platinum SuperFi PCR Master Mix (ThermoFisher Scientific), and 17 µL nuclease-free water in PCR tubes.

[0463] The following PCR program was carried out in an Eppendorf Mastercycler: 98°C for 5 min.; 30 cycles of 98°C for 10 sec., 62°C for 10 sec., 72°C for 30 sec.; 72°C for 5 min. The PCR products of positive colonies Page 101 of 117 12869933v1Attorney Docket: 2012611-0187 were sent out to Genewiz for sequencing using the NLuc_seq primer to confirm the insertion of the constructs. Meanwhile, positive colonies were grown for 18 hours in a solution of LB and Ampicillin by shaking in an incubator at 37°C. After 18 hours, chimeric plasmids from one positive colony per construct were extracted by miniprepping using the NucleoSpin Plasmid Mini Kit (Macherey-Nagel). Plasmids were sent out for sequencing again to ensure that the right constructs were inserted and that no mutations were introduced during replication in E. coli.

[0464] A PPa sequence from yeast prepro-α-factor was added to a RBD variant in pNLF1-C using restriction-free cloning with a gBlock megaprimer (IDT) encoding the PPa sequence with 50 bp homology on either end. Homology arms were designed to insert the PPa sequence between the Kozak sequence and the RBD variant, replacing the Spike secretion peptide. 27.2 µL water, 10 µL 5x Phusion HF Buffer, 1 µL 10 mM dNTPs, 20 ng vector in 1 µL water, 10 µL PPa megaprimer at 10 ng / µL, and 0.8 µL Phusion DNA Polymerase (New England BioLabs) were mixed together in a PCR tube on ice. The tube was placed in a thermal cycler and the following PCR program was run: 95°C for 30 sec.; 25 cycles of 95°C for 30 sec., 60°C for 1 min., and 72°C for 5 min.; and 72°C for 7 min. 10 µL of this reaction were mixed with 34 µL water, 5 µL 10x CutSmart Buffer, and 1 µL DpnI (20 units / µL) (New England BioLabs) in PCR tubes on ice. The tube was placed in a thermal cycler and incubated at 37°C for 1 hour to digest template DNA. The reaction was diluted 1:4 in water and transformed into TOP10 cells (Thermo Fisher Scientific) and plated on LB agar plates containing ampicillin (Teknova) as described above. The following day colony PCR was performed as described above using NLuc_Nterm_cPCR_F and NLuc_Nterm_cPCR_R primers to identify colonies containing the PPa insert. PCR products were sequenced by Genewiz using the NLuc_seq primer. Colonies containing inserts were grown up in LB media containing ampicillin overnight and miniprepped as described above.

[0465] To insert a PPa_v2 optimized sequence upstream of the RBD variant, a new vector was constructed consisting of the RBD variant in pNLF1-C with an upstream cloning site. pNLF1-C was digested using NheI and EcoRI as described above. A gBlock (IDT) containing a Kozak sequence, an NheI cut site, a 6 bp spacer, an EcoRI cut site, and the RBD variant was cloned into the digested vector using NEBuilder HiFi DNA Assembly Master Mix as described above. The homology arms of the gBlock were designed to remove the NheI and EcoRI restriction sites from the pNLF1-C plasmid, so the cut sites in the gBlock are unique in the new vector, named pSP. The vector was digested using NheI and EcoRI as described above. PPa_v2 was ordered from IDT as an eBlock containing 20 bp homology arms to pSP to enable in-frame cloning with the RBD variant and was cloned into digested pSP using NEBuilder HiFi DNA Assembly Master Mix as described above. Colony PCR was performed using NLuc_Nterm_cPCR_F and NLuc_Nterm_cPCR_R primers.

[0466] An anti-CD3 x anti-gp75 bi-specific T cell engager (BiTE) with Ssp was ordered from IDT as a clonal gene in IDT Vector Amp Blunt. The sequence was amplified by PCR using BiTE_forward and BiTE_reverse primers. An anti-CD16 x IL-15 x anti-gp75 tri-specific natural killer cell engager (TriKE) with Ssp was ordered from IDT as a Hi-Fi gBlock. The sequence was amplified by PCR using TriKE_forward and TriKE_reverse primers. For PCR of BiTE and TriKE constructs, 25.0 µL water, 10 µL 5x Herculase II Reaction Buffer, 0.5 µL 25 mM dNTPs, 1.25 µL 10 µM forward primer, 1.25 µL 10 µM reverse primer, 0.5 uL BiTE or TriKE construct at 10 ng / µL, 1 µL Herculase II Fusion DNA Polymerase (Agilent), 0.5 µL DMSO, and 10 µL 5M betaine were mixed together in PCR tubes on ice. Tubes were transferred to a thermal cycler and the following PCR program was run: 98°C for 4 min.; 30 cycles of 98°C for 20 sec., 68°C for 20 sec., 72°C for 1 min.; 72°C for 3 min. 8 µL of these BiTE PCR product were mixed with Page 102 of 117 12869933v1Attorney Docket: 2012611-0187 1 µL 10x CutSmart Buffer and 1 µL DpnI (20 units / µL) (New England BioLabs) in a PCR tube on ice. The tube was placed in a thermal cycler and incubated at 37°C for 30 min. to digest template DNA and at 80°C for 20 min. to heat inactivate the enzyme. PCR products were cloned into pNLF1-C using NEBuilder HiFi DNA Assembly Master Mix as described above.

[0467] A select group of helper proteins were selected for screening. Candidate helper proteins ordered from Twist included: TAPT1, K3L_H47R, g134.5, GADD34, IRE1b_delCTD, Derlin-1, GosR1, Stx5, Bet1, NEDD4L, NEDD4L_Y679C, NEDD4L_Q694H, NEDD4L_E893K, NEDD4L_R897Q, RIPK2, ERBB4, AKT1, Notch1, NEMO, GJB2, NDFIP2, CD300A, XCR1, STBD1, EDA2R, TMEM200A, GPR114, DPAGT1, and CCR2. All candidate helper proteins were ordered as clonal genes that were codon optimized and cloned into pTwist CMV BG WPRE Neo expression plasmid, inserted at EcoRI / XbaI sites, with added 5’ Kozak sequence (GCCGCCACC) and 3' 3x stop codons (TAATGATAG). Plasmids were delivered dried down and were re-suspended in nuclease-free water to a concentration of 100 ng / µL. Secretion Screen 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 resuspended to a concentration of 1e5 cells / ml. Next, 100 µl (1e4 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 transfected with 20 ng of plasmid DNA (e.g., 10 ng RBD plasmid + 10 ng helper plasmid(s) total) using Lipofectamine 3000 Transfection Reagent (ThermoFisher Scientific). After transfection, cells were incubated at 37°C and 5% CO2for 72 hours. At 72 hours, cell supernatants were screened for NanoLuc activity using the Nano-Glo Luciferase Assay System (Promega). 10 µL supernatant was diluted in 90 µL nuclease-free water in white 96-well plates. 200 µL Nano-Glo Luciferase Assay Substrate were diluted in 10 mL Nano-Glo Luciferase Assay Buffer, and 100 µL were added.Example 2: Identification of Helper Proteins that Increase Payload Secretion and / or Expression

[0468] This Example describes the identification of helper proteins that improve secretion and / or expression of an exemplary payload. The methods used in this Example are described in Example 1.

[0469] Candidate helper proteins selected for screening were chosen from pathways that have known roles in protein expression and / or secretion.

[0470] A variant form of SARS-CoV-2 Spike receptor binding domain (“RBD”) antigen was previously generated to focus immune response on neutralizing epitopes and high-risk variants. Expression of this RBD variant in HEK293T cells is approximately 100-fold lower than wild type RBD. This model protein was used as an exemplary paylaod to identify helper proteins that, when co-delivered, rescue protein expression.

[0471] An exemplary chaperone protein tested for impact on protein secretion was TAPT1, due to its ability to complex with Slp1 and protect folding polypeptides from degradation. As shown in FIG.1A, co-expression of TAPT1 enhanced secretion of RBD_var by 25x in HEK293T cells. Without wishing to be bound by any particular Page 103 of 117 12869933v1Attorney Docket: 2012611-0187 theory, this data suggests that expression of TAPT1 is beneficial for the expression of mutant, synthetic, and otherwise difficult-to-express proteins which may spend more time in the unfolded state and thus be more prone to ER-associated degradation than their wild type, native, and / or less complex counterparts.

[0472] Next, exemplary UPR protein inhibitors, K3L_H47R, g134.5, GADD34, and IRE1b_delCTD were tested for impact on protein secretion. Co-expression of K3L_H47R, γ134.5, GADD34, and IRE1b_delCTD enhanced secretion of RBD_var by 120x, 44x, 32x, and 46x, respectively, in HEK293T cells (FIG.1B). This data suggests that co-expression of modulators that attenuate the UPR allow enhanced expression and subsequent secretion of exogenous proteins that would otherwise trigger the UPR and result in decreased protein expression.

[0473] Co-expression of ER-associated degradation (“ERAD”) protein Derlin-1 (DERL1) also enhanced secretion of RBD_var by 17x in HEK293T cells (FIG.1C). This data suggests that co-expression of Derlin-1 may enhance protein secretion by either (1) enhancing ERAD and / or, ER stress-induced pre-emptive quality control (“ERpQC”), alleviating ER stress and enhancing cellular and ER functions, or (2) functioning as a decoy for other ERAD and / or ERpQC machinery, effectively inhibiting these processes and protecting RBD_var from degradation.

[0474] Multiple instances in which co-expression of helper proteins involved in anterograde trafficking of secretory proteins proceeding from the ER to the ER-Golgi compartment improved secretion of RBD_var were also observed. Specifically, co-expression of SNARE proteins GosR1, Stx5, or Bet1 enhanced secretion of RBD_var by 19x, 16x, or 16x, respectively, in HEK293T cells (FIG.1D).

[0475] Potential inhibitors of the ubiquitin-proteasome system, and functional proteins that play critical roles in ubiquitination were also screened. While strategies aimed at inhibiting the ubiquitin-proteasome system generally did not enhance protein secretion appreciably, co-expression of functional members of the pathway generally improved protein secretion, including both wild type and gain-of-function mutant proteins. Specifically, co- expression of the HECT domain-containing E3 ubiquitin-protein ligase NEDD4L enhanced secretion of RBD_var by 4x in HEK293T cells (FIG.1E). Furthermore, co-expression of NEDD4L containing patient-derived mutations Q694H, R897Q, E893K, or Y679C enhanced secretion of RBD_var by 283x, 151x, 72x, and 16x, respectively, in HEK293T cells (FIG.1E).

[0476] One additional group of proteins that was screened was E3 ubiquitin ligase substrates. The five proteins were: RIPK2, ERBB4, AKT1, Notch1, and NEMO. Co-expression of RIPK2, ERBB4, AKT1, Notch1, and NEMO enhanced secretion of RBD_var by 16x, 10x, 6x, 4x, and 3x in HEK293T cells, respectively (FIG.1F).

[0477] The above listed groups of proteins were chosen from pathways known to have a role in protein expression and / or secretion. Next, an unbiased approach was taken. Utilizing a published total RNA-sequencing dataset from an ex vivo model (Kassambara, Alboukadel et al. “RNA-sequencing data-driven dissection of human plasma cell differentiation reveals new potential transcription regulators.” Leukemia vol. 35,5 (2021): 1451-1462. doi:10.1038 / s41375-021-01234-0), about 100 top upregulated during plasma cell differentiation were screened for their ability to enhance payload expression and / or secretion. Genes were ordered from a vendor and screened as described for other exemplary helper proteins. Based on the screen, ten genes were selected that, when co- expressed, exhibited the greatest enhancement of secretion of RBD_var in HEK293T: GJB2 (28x enhanced expression), NDFIP2 (25x enhanced secretion), CD300A / CLM8 (24x enhanced secretion), XCR1 (23x enhanced Page 104 of 117 12869933v1Attorney Docket: 2012611-0187 secretion), STBD1 (18x enhanced secretion), EDA2R (16x enhanced secretion), TMEM200A (12x enhanced secretion), GPR114 (10x enhanced secretion), DPAGT1 (10x enhanced secretion), and CCR (9x enhanced secretion) (FIG.1G).

[0478] These results collectively suggest that helper proteins disclosed herein when co-expressed with a payload can enhance expression and / or secretion of payloads through one or a combination of the following mechanisms: (1) helper protein function itself enhances expression and / or secretion of proteins (including a payload); (2) helper protein interacts with other proteins or networks of proteins that function to enhance expression and / or secretion of payload; (3) helper protein impacts the overall kinetics of the cellular secretory system; (4) helper protein serves as an inhibitor, decoy, or pseudo-substrate in one or more pathways that negatively impact protein expression and / or secretion.

[0479] In summary, the data provided herein demonstrates the utility of co-delivering a payload and one or more helper proteins to increase the expression and / or secretion of the payload. In some embodiments, co- delivery comprises co-expression of a payload and one or more helper proteins. In some embodiments, co-delivery comprises co-administration of a payload and one or more helper proteins to a cell, tissue or subject. Example 3: Co-Expression of Multiple Helper Proteins Increased Payload Expression

[0480] This Example describes co-expression of two or more helper proteins in HEK293T cells, which result in increased secretion of exemplary payloads. The methods used in this Example are described in Example 1.

[0481] Co-expression of the UPR-modulating helper proteins K3L_H47R, γ134.5, GADD34, and IRE1b_delCTD enhanced secretion of RBD_var by 30x in HEK293T cells (FIG.2A, “UPR”). Interestingly, these proteins target multiple different steps in the UPR response, creating the potential to modulate the stress response at multiple points when expressed together.

[0482] Co-expression of the SNARE proteins GosR1, Stx5, and Bet1 enhanced secretion of RBD_var by 19x in HEK293T cells (FIG.2A, “SNAREs”). These three proteins are complementary SNARE molecules and together form complexes on vesicles trafficking from the ER to the ERGIC and Golgi; co-expression of all three may lead to enhanced formation of these SNARE complexes and thus anterograde transport of secretory protein cargo.

[0483] Co-expression of all nine proteins TAPT1, K3L_H47R, γ134.5, GADD34, IRE1b_delCTD, Derlin-1, GosR1, Stx5, and Bet1 enhanced secretion of RBD_var by 58x in HEK293T cells (FIG.2A, “TAPT1 + UPR + DERL1 + SNAREs”). Notably, these proteins synergized to enhance protein secretion, as co-expression of all four groups enhanced secretion of RBD_var greater than any one group alone.

[0484] Co-expression of the five proteins hypothesized to enhance protein secretion via their roles in signaling pathways RIPK2, ERBB4, AKT1, Notch 1, and NEMO enhanced secretion of RBD_var by 45x in HEK293T cells (FIG.2B, “SIG”). In some embodiments, RIPK2, ERBB4, AKT1, Notch 1, and NEMO are also referred to herein as E3 ubiquitin ligase substrate polypeptides. Co-expression of all five proteins enhanced secretion greater than any single protein alone, suggesting that these proteins act in synergy to increase secretion.

[0485] Next, co-expression of the ten helper proteins identified from plasma cell RNA-sequencing dataset with TAPT1, UPR modulators, Derlin-1, and the SNARE proteins (Figure 2C) was analyzed. When co-expressed, Page 105 of 117 12869933v1Attorney Docket: 2012611-0187 almost every combination of proteins enhanced secretion of RBD_var, with effect sizes ranging from 4x to 205x. The only exception to this pattern is the co-expression of TMEM200A and the SNARE proteins, which decreased expression of RBD_var by 30x. TMEM200A has been observed to interact with the SNARE protein GOSR1 (Huttlin, Edward L., et al. "Dual proteome-scale networks reveal cell-specific remodeling of the human interactome." Cell 184.11 (2021): 3022-3040). These data suggest that the TMEM200A-SNARE interaction negatively impacts protein secretion.

[0486] Effects on secretion of the exemplary payload (RBD_var) from co-expression of the helper proteins upregulated during plasma cell differentiation with TAPT1 and with DERL1 were mostly additive, with a few small synergistic interactions (FIG.2C). Co-expression of the helper proteins upregulated during plasma cell differentiation with SNARE proteins led to small synergistic interactions (FIG.2C). Effects on secretion of the exemplary payload (RBD_var) from co-expression of the helper proteins upregulated during plasma cell differentiation with the UPR proteins revealed large amounts of synergy between helper proteins, with secretion enhanced 2.2-7.5x greater than expected from the average of the two protein groups alone (FIG.2C).

[0487] Together, these data suggest that modulating the UPR to increase payload expression and / or secretion is a beneficial strategy which may synergize with the modulation mechanisms of other helper proteins. Without wishing to be bound by any particular theory, in some embodiments, modulating the UPR attenuates the cellular stress responses that typically decreases protein expression and can be triggered with the overexpression of proteins.

[0488] Next, co-expression of: (a) the mutant NEDD4L_Q694H protein, or (b) the signaling pathway proteins RIPK2, ERBB4, AKT1, Notch 1, and NEMO (“SIG”), with (c) TAPT1, UPR modulators, Derlin-1, SNARE proteins, and the proteins upregulated during plasma cell differentiation, was investigated (FIG.2D). When co- expressed, every combination of proteins enhanced secretionof RBD_var, with effect sizes ranging from 4x to 514x. NEDD4L_Q694H exhibited synergy with nine out of the fourteen helper protein groups (FIG.2D, center), and the signaling pathway proteins (“SIG”) exhibited synergy with all helper protein groups (FIG.2D, right, “SIG”). Co- delivery of the UPR modulator proteins provided the greatest enhancement of secretion of the exemplary payload for each group (NEDD4L_Q694H or SIG proteins), with secretion enhanced 180x when UPR modulator proteins were co- delivered with NEDD4L_Q694H and 514x when UPR modulator proteins were co-delivered with the signaling pathway proteins.

[0489] These data suggest that overall, helper proteins that target different cellular pathways generally work together to enhance protein expression and / or secretion. The data provided herein also demonstrates that co- expression of UPR-modulating proteins synergizes with other helper proteins, increases payload expression and / or secretion.

[0490] Next, the effect of the TAPT1 and UPR helper proteins on the secretion of additional exemplary polypeptide payloads from HEK293T cells was evaluated. The exemplary polypeptide payloads tested were: wild type SARS-CoV-2 Spike RBD with a Spike signal peptide (Ssp-RBD), RBD_var with Ssp, RBD_var with a sequence derived from the yeast prepro-α-factor PPa, RBD_var with an engineered prepro-α-factor sequence PPa_v2, an anti-CD3 x anti-gp75 bi-specific T cell engager (BiTE), and an anti-CD16 x IL-15 x anti-gp75 tri-specific natural killer cell engager (TriKE). PPa was taken directly from the wild type yeast prepro-alpha factor sequence. PPa_v2 was engineered for Page 106 of 117 12869933v1Attorney Docket: 2012611-0187 higher secretion, by (1) mutating the N-term of the pro sequence to contain 2 copies of a high-affinity binding site for the Surf4 ER cargo receptor, separated by a Gly-Ser linker, and (2) mutating the C-term yeast protease cleavage site to contain 3 copies of the human S1P protease cleavage site. While Ssp-RBD was secreted at a high level without the addition of helper proteins, there was a significant drop in secretion of the mutant Ssp-RBD_var. This drop in expression for RBD_var was partially rescued in the PPa-RBD_var sequence and fully rescued in the PPa_v2-RBD_var (FIG.3). The fusion BiTE and TriKE proteins were secreted at levels in between the wild type RBD and the mutant RBD_var proteins without co-expression of helper proteins (FIG.3).

[0491] Co-expression of TAPT1 or UPR helper proteins increased payload expression as compared to payload expression in the absence of helper protein (FIG.3). The largest increase in protein secretion was observed for the mutant Ssp-RBD_var protein, with co-expression of TAPT1 and UPR proteins enhancing secretion 20x and 53x, respectively. The next largest increase in protein secretion was observed for the triple fusion TriKE protein, with co-expression of TAPT1 and UPR proteins enhancing expression 8x and 7x, respectively. For the fusion BiTE protein, co-expression of TAPT1 and UPR proteins enhanced expression 2x and 3x, respectively. Co-expression of TAPT1 and UPR proteins did not appreciably affect secretion of PPa-RBD_var or PPa_v2-RBD_var.

[0492] Notably, when TAPT1 and UPR proteins were co-expressed, they acted synergistically to increase payload secretion for all tested payloads (FIG.3). Co-expression of TAPT1 and UPR proteins enhanced secretion of Ssp-RBD_var by 128x, of TriKE protein by 25x, of BiTE protein by 10x, of PPa-RBD_var by 6x, and of PPa_v2- RBD_var by 2x.

[0493] Together, these data introduce twenty-nine unique helper proteins that when co-expressed enhance the secretion of exemplary payloads. In some embodiments, helper proteins disclosed herein enhance secretion of payloads when co-expressed. In some embodiments, co-expression of helper proteins disclosed herein result in synergistic effects on the expression and / or secretion of payloads. Without wishing to be bound by theory, UPR-modulating helper proteins may act in synergy with other helper proteins by inhibiting cellular stress responses that can limit protein expression and secretion. REFERENCES

[0494] The following documents are incorporated herein in their entirety.

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[0516] Zhou B, Lin W, Long Y, Yang Y, Zhang H, Wu K, Chu Q. Signal Transduction and Targeted Therapy 7, 95, 2022. Page 108 of 117 12869933v1Attorney Docket: 2012611-0187 EQUIVALENTS

[0517] 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 109 of 117 12869933v1

Claims

Attorney Docket: 2012611-0187 CLAIMS 1. A nucleic acid expression system comprising: (i) a polynucleotide comprising a payload sequence, and (ii) at least one polynucleotide that encodes one or more helper polypeptides that comprise: (a) a chaperone or ER guardian polypeptide or a fragment or variant thereof, (b) an unfolded protein response (UPR) inhibitor polypeptide or a fragment or variant thereof, (c) an ER associated degradation (ERAD) polypeptide or a fragment or variant thereof, (d) a SNARE polypeptide or a fragment or variant thereof, (e) a ubiquitin protein ligase polypeptide or a fragment or variant thereof, (f) an E3 ubiquitin ligase substrate polypeptide or a fragment or variant thereof, (g) a polypeptide upregulated during plasma cell differentiation or a fragment or variant thereof, or (h) any combination of (a)-(g).

2. The nucleic acid expression system of claim 1, wherein the one or more helper polypeptides comprise a chaperone or ER guardian polypeptide or a fragment or variant thereof.

3. The nucleic acid expression system of claim 2, wherein the chaperone or ER guardian polypeptide comprises: (i) a TAPT1 polypeptide, chaperone or a fragment or variant thereof, (ii) a heat shock protein 70 (HSP70) family member or a fragment or variant thereof, optionally wherein the HSP70 family member comprises HSPA13 or a fragment or variant thereof, (iii) a heat shock protein 40 (HSP40) family member or a fragment or variant thereof, optionally wherein the HSP40 family member comprises DNAJC8 or a fragment or variant thereof, or (iv) any combination of (i)-(iii), 4. The nucleic acid expression system of any one of the preceding claims, wherein the one or more helper polypeptides comprise a UPR inhibitor polypeptide, or a fragment or variant thereof.

5. The nucleic acid expression system of claim 4, wherein the UPR inhibitor polypeptide comprises: a K3L polypeptide or a fragment or variant thereof, a gamma 134.5 polypeptide or a fragment or variant thereof, a GADD34 polypeptide or a fragment or variant thereof, or an IRE1b polypeptide or a fragment or variant thereof, or any combination thereof, optionally wherein: (i) the K3L polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 12, (ii) the gamma 134.5 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 13, Page 110 of 117 12869933v1Attorney Docket: 2012611-0187 (iii) the GADD34 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 14, and / or (iv) the IRE1b polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

15.

6. The nucleic acid expression system of any one of the preceding claims, wherein the one or more helper polypeptides comprise an ERAD polypeptide or a fragment or variant thereof.

7. The nucleic acid expression system of claim 6, wherein the ERAD polypeptide comprises a Derlin-1 polypeptide or a fragment or variant thereof, a JKAMP polypeptide or a fragment or variant thereof, or any combination thereof, optionally wherein the Derlin-1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

16.

8. The nucleic acid expression system of any one of the preceding claims, wherein the one or more helper polypeptides comprise a SNARE polypeptide or a fragment or variant thereof.

9. The nucleic acid expression system of claim 8, wherein the SNARE polypeptide comprises a GosR1 polypeptide or a fragment or variant thereof, a Stx5 polypeptide or a fragment or variant thereof, a Bet1 polypeptide or a fragment or variant thereof, a SEC22A polypeptide or a fragment or variant thereof, a SEC22C polypeptide or a fragment or variant thereof, a STXB6 polypeptide or a fragment or variant thereof, a GOSR2 polypeptide or a fragment or variant thereof, or any combination thereof, optionally wherein: (i) the GosR1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 17, (ii) the Stx5 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 18, and / or (iii) the Bet1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

19.

10. The nucleic acid expression system of any one of the preceding claims, wherein the one or more helper polypeptides comprise a ubiquitin protein ligase polypeptide or a fragment or variant thereof.

11. The nucleic acid expression system of claim 10, wherein ubiquitin protein ligase polypeptide comprises: a NEDD4L polypeptide or a fragment or variant thereof, an ITCH polypeptide or a fragment or variant thereof, a WWP1 polypeptide or a fragment or variant thereof, a WWP2 polypeptide or a fragment or variant thereof, a SMURF1 polypeptide or a fragment or variant thereof, a SMURF2 polypeptide or a fragment or variant thereof, a RNF5 polypeptide or a fragment or variant thereof, a TRAF1 polypeptide or a fragment or variant thereof, a TRAF2 polypeptide or a fragment or variant thereof, a TRAF3 polypeptide or a fragment or variant thereof, a TRAF6 polypeptide or a fragment or variant thereof, a TRAF7 polypeptide or a fragment or variant thereof, a UBE3A Page 111 of 117 12869933v1Attorney Docket: 2012611-0187 polypeptide or a fragment or variant thereof, a CUL1 polypeptide or a fragment or variant thereof, a CUL2 polypeptide or a fragment or variant thereof, a CUL3 polypeptide or a fragment or variant thereof, a MARCH-II polypeptide or a fragment or variant thereof, a MARCH-III polypeptide or a fragment or variant thereof, a MARCH-VI polypeptide or a fragment or variant thereof, a AMFR polypeptide or a fragment or variant thereof, a HERC3 polypeptide or a fragment or variant thereof, a TRIM63 polypeptide or a fragment or variant thereof, a TRIM21 polypeptide or a fragment or variant thereof, a BIRC3 polypeptide or a fragment or variant thereof, optionally wherein the NEDD4L polypeptide comprises: (i) one or more mutations, wherein the one or more mutations comprise Y679C, Q694H, E893K, R897Q, or any combination thereof, and / or (ii) a sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 20, 21, 22, 23, or 24.

12. The nucleic acid expression system of any one of the preceding claims, wherein the one or more helper polypeptides comprise E3 ubiquitin ligase substrate polypeptide or a fragment or variant thereof.

13. The nucleic acid expression system of claim 12, wherein the E3 ubiquitin ligase substrate polypeptide comprises: a RIPK2 polypeptide or a fragment or variant thereof, an ERBB4 polypeptide or a fragment or variant thereof, an AKT1 polypeptide or a fragment or variant thereof, a Notch1 polypeptide or a fragment or variant thereof, a NEMO polypeptide or a fragment or variant thereof, or any combination thereof, optionally wherein: (i) the RIPK2 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 25, (ii) the ERBB4 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 26, (iii) the AKT1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 27, (iv) the Notch1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 28, and / or (v) the NEMO polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

29.

14. The nucleic acid expression system of any one of the preceding claims, wherein the one or more helper polypeptides comprise polypeptide upregulated during plasma cell differentiation or a fragment or variant thereof.

15. The nucleic acid expression system of claim 14, wherein the polypeptide upregulated during plasma cell differentiation comprises: a GJB2 polypeptide or a fragment or variant thereof, a NDFIP2 polypeptide or a fragment or variant thereof, a CD300A polypeptide or a fragment or variant thereof, a XCR1 polypeptide or a fragment or variant thereof, a STBD1 polypeptide or a fragment or variant thereof, a EDA2R polypeptide or a fragment or variant thereof, a TMEM200A polypeptide or a fragment or variant thereof, a GPR114 polypeptide or a fragment or variant Page 112 of 117 12869933v1Attorney Docket: 2012611-0187 thereof, a DPAGT1 polypeptide or a fragment or variant thereof, a CCR2 polypeptide or a fragment or variant thereof, or any combination thereof, optionally wherein: (i) the GJB2 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 30, (ii) the NDFIP2 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 31, (iii) the CD300A polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 32, (iv) the XCR1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 33, (v) the STBD1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 34, (vi) the EDA2R polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 35, (vii) the TMEM200A polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 36, (viii) the GPR114 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 37, (ix) the DPAGT1 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 38, and / or (x) the CCR2 polypeptide comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

39.

16. The nucleic acid expression system of claim 1, wherein the one or more helper polypeptides comprise one or more UPR inhibitor polypeptides or variants or fragments thereof, and one or more additional helper polypeptides, optionally wherein the one or more additional helper polypeptides comprises: (a) a chaperone or ER guardian polypeptide, or a fragment or variant thereof, (b) an ER associated degradation (ERAD) polypeptide, or a fragment or variant thereof, (c) a SNARE polypeptide, or a fragment or variant thereof, (d) a ubiquitin protein ligase polypeptide, or a fragment or variant thereof, (e) an E3 ubiquitin ligase substrate polypeptide, or a fragment or variant thereof, or (f) a polypeptide upregulated during plasma cell differentiation, or a fragment or variant thereof.

17. The nucleic acid expression system of any one of the preceding claims, wherein the nucleic acid expression system is characterized in that when administered to a cell, tissue, or subject, increased payload expression and / or secretion is observed as compared to payload expression and / or secretion in a cell, tissue, or subject administered an otherwise similar polynucleotide comprising a payload sequence without at least one polynucleotide comprising a sequence encoding a helper polypeptide. Page 113 of 117 12869933v1Attorney Docket: 2012611-0187 18. The nucleic acid expression system of any one of the preceding claims, wherein increased payload expression and / or comprises at least 2-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold at least-500 fold, at least 600-fold, at least 700-fold, or at least 800-fold increased expression and / or secretion of the payload.

19. The nucleic acid expression system of claim 18 or 19, wherein increased secretion and / or expression comprises increased trafficking through a secretory pathway, increased proper folding of the polypeptide, reduced degradation of the polypeptide, or reduced aggregation of the polypeptide.

20. The nucleic acid expression system of any one of the preceding claims, wherein the polynucleotide comprising a payload sequence and / or the at least one polynucleotide comprising a sequence that encodes a helper polypeptide is a DNA or RNA.

21. The nucleic acid expression system of claim 20, wherein the at least one polynucleotide is a polyribonucleotide comprising 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.

22. The nucleic acid expression system of claim 21, wherein the one or more modified ribonucleotides comprise a 2’-O-acetylated ribose.

23. The nucleic acid expression system of claim 21 or 22, wherein the polyribonucleotide comprises a cap structure and the cap structure does not comprise a 2’-O-acetylated ribose.

24. The nucleic acid expression system of claim 21 or 22, wherein the polyribonucleotide comprises a cap structure and the cap structure comprises a 2’-O-acetylated ribose. Page 114 of 117 12869933v1Attorney Docket: 2012611-0187 25. The nucleic acid expression system of any one of the preceding claims, wherein the polynucleotide comprising a payload sequence comprises a coding region, optionally wherein the coding region encodes a payload.

26. The nucleic acid expression system of claim 25, wherein the payload comprises: (i) an antigen or a fragment thereof, (ii) an antibody or a fragment thereof, (iii) a secreted protein or a fragment thereof, (iv) a cell-surface associated protein or a fragment thereof, (v) an intracellular protein or a fragment thereof, or (vi) any combination of (i)–(v).

27. The nucleic acid expression system of claim 25, wherein the payload comprises an RNA payload.

28. One or more compositions comprising a nucleic acid expression system of any one of the preceding claims.

29. The composition of claim 28, wherein: (i) a polynucleotide comprising a payload sequence, and (ii) at least one polynucleotide that encodes one or more helper polypeptides, are in separate compositions.

30. The composition of claim 28, wherein the composition is a pharmaceutical composition, optionally wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

31. The composition of claim 30 wherein the pharmaceutical composition is formulated for intramuscular, intradermal, or intravenous delivery.

32. A set of polypeptides encoded by the nucleic acid expression system of any one of claims 1-27.

33. A cell comprising a nucleic acid expression system of any one of claims 1-27, optionally wherein the cell is in vivo or ex vivo.

34. A method of delivering a nucleic acid expression system of any one of claims 1-27, or a composition of any one of claims 28-31 to a cell, tissue or subject.

35. The method of claim 34, wherein the method is a treatment method or a prevention method.

36. The method of claim 34 or 35, wherein the method comprises: (i) a method to stimulate an immune response, (ii) an antibody therapy method, (iii) an immune-modulation method, (iv) a vaccination method, Page 115 of 117 12869933v1Attorney Docket: 2012611-0187 (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).

37. A method of stimulating an immune response to an antigen, comprising delivering a nucleic acid expression system of any one of claims 1-27, or a composition of any one of claims 28-31 to a cell, tissue or subject, optionally wherein the subject is a human.

38. The composition of any one of claims 28-31, for use in delivering the nucleic acid expression system to a cell, tissue, or subject.

39. Use of a nucleic acid expression system according to any one of claims 1-27, or a composition according to any one of claims 28-31, in the preparation of a medicament for delivering the nucleic acid expression system or composition to a cell, tissue, or subject.

40. The composition for use of claim 38, or the use of claim 39, 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 116 of 117 12869933v1

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