Polynucleotides containing a modified 3'-region

Abstract

This disclosure relates to the modified 3' region elements that confer improved therapeutic properties including, but not limited to, increased stability, increased expression, and / or a reduced innate immune response when introduced into a population of cells, even in the absence of a 5'-cap. The disclosure also provides polynucleotides (e.g., RNA molecules) containing modified 3' regions. In some embodiments of the disclosure, the modified 3' regions contain modified adenosines (e.g., 2'-0-methoxy- adenosine, 2'-fluoro-adenosine, or locked nucleic acid (LNA)-adenosine) and / or a modified thymidine (e.g., inverted deoxythymidine). In some embodiments of the disclosure, the modified 3' region may contain one or more modified internucleoside linkages (e.g., a phosphorothioate linkage) or modified nucleobase (e.g., N1-methyl-pseudouridine). The polynucleotides of the disclosure may be used to express a desired polypeptide in a subject, such as a subject having a disease or condition associated with a deficiency in the corresponding endogenous polypeptide.

Description

[0001] POLYNUCLEOTIDES CONTAINING A MODIFIED 3’-REGION BACKGROUND OF THE INVENTION

[0002] The use of exogenous nucleic acids has become a particularly effective strategy for delivering polypeptides of interest to a target cell, tissue, or organism, as a single protein-encoding nucleic acid can be translated to yield multiple copies of a desired polypeptide, allowing the administration of a small quantity of nucleic acid to achieve high levels of protein expression. However, there is a need for additional compositions and methods for improving nucleic acid stability in order to further enhance the therapeutic potential of this class of compounds.

[0003] SUMMARY OF THE INVENTION

[0004] The present disclosure provides, inter alia, polynucleotides including a modified 3’ region (e.g., containing an alternative nucleobase, sugar, or backbone) attached to the 3’ region of mRNA encoding a polynucleotide to be expressed.

[0005] In particular, the inventors have identified that mRNA containing one or more of the modified 3’ regions disclosed herein exhibit improved therapeutic properties including, but not limited to, increased stability and / or increased expression, when introduced into a population of cells, all of which may be achieved in a polynucleotide with or without a 5’-cap. The modified 3’ region may include, for example, one or more modifications selected from (i) at least one modified sugar (e.g., at least one modified ribose), and / or (ii) at least one modified internucleoside linkage (e.g., at least one phosphorothioate), and / or (Hi) a modified terminal group (e.g., a modified phosphate or an inverted nucleoside).

[0006] In a first aspect, the disclosure provides a polynucleotide encoding a polypeptide of interest. The polynucleotide includes, in the 5’ to 3’ direction:

[0007] (a) optionally, a 5’-cap structure;

[0008] (b) a 5’-untranslated region (UTR);

[0009] (c) an open reading frame (ORF) encoding the polypeptide of interest;

[0010] (d) a3’-UTR;

[0011] (e) a poly-A region;

[0012] (f) a linker; and

[0013] (g) a modified 3’ region comprising the structure of Formula I, in the 5’-to-3’ direction:

[0014] (A’) - (X1 - B) - (X2 - C)a - (X3 - D)b - (X4 - Z)c

[0015] Formula I

[0016] wherein:

[0017] A’ is a poly-A region (e.g., 20 adenosines in length);

[0018] B is a bridged nucleic acid (BNA) (e.g., a locked nucleic acid (LNA));

[0019] C is a BNA;

[0020] D is a BNA;

[0021] Z is an inverted nucleoside;

[0022] wherein each of x1, x2, x3, and x4 is a phosphodiester internucleoside linkage; and

[0023] wherein each of a, b, and c is, independently, 0 or 1;

[0024] further wherein: when c is 0, i) each of a and b is 1, ii) one of a or b is 1, or iii) both a and b is 0; and when c is 1, i) each of a and b is 0, ii) one of a or b is 1, or iii) both of a and b is 1.

[0025] In some embodiments, the polypeptide of interest encoded by the ORF of (c) corresponds to an antigen associated with a disease.

[0026] In some embodiments, the 5’-cap structure of (a) is present. In some embodiments, the 5’-cap structure of (a) is absent.

[0027] In some embodiments, the poly-A region of (e) is from 1 to 200 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200) nucleosides in length. In some embodiments, the poly-A region of (e) is from 10 to 200 nucleosides in length. In some embodiments, the poly-A region of (e) is from 50 to 150 nucleosides in length. In some embodiments, the poly-A region of (e) is 100 nucleosides in length.

[0028] In some embodiments, the BNA of B, C, and D, if present, is independently selected from one of LNA-adenosine, LNA-thymidine, LNA-guanosine, LNA-cytidine, LNA-(5-methyl-cytidine), LNA-uridine, BNA-NC-adenosine, BNA-NC-thymidine, BNA-NC-guanosine, BNA-NC-cytidine, BNA-NC-(5-methyl-cytidine), BNA-NC-uridine, ENA-adenosine, ENA-thymidine, ENA-guanosine, ENA-cytidine, ENA-(5-methyl-cytidine), ENA-uridine, constrained ethyl nucleic acid-adenosine ((A)AcEt), constrained ethyl nucleic acid-guanosine ((G)AcEt), constrained ethyl nucleic acid-thymidine ((T)AcEt), constrained ethyl nucleic acid-cytidine ((C)AcEt), constrained ethyl nucleic acid-(5-methyl-cytidine), or constrained ethyl nucleic acid-uridine ((U)AcEt). In some embodiments, the BNA of B, C, and D, if present, is LNA-adenosine. In some embodiments, the LNA-adenosine of B, C, and / or D has the structure of:

[0029]

[0030] In some embodiments, the inverted nucleoside of Z, if present, is an inverted deoxythymidine. In some embodiments, the inverted deoxythymidine of Z has the structure of:

[0031]

[0032] Inverted deoxythymidine.

[0033] In some embodiments, the linker comprises an Xbal scar (5’-UCUAG-3’).

[0034] In some embodiments, the modified 3’ region polynucleotide of (g) has the structure of Formula V:

[0035]

[0036] Formula V

[0037] or a pharmaceutically acceptable salt thereof.

[0038] In some embodiments, the modified 3’ region polynucleotide of (g) has the structure of Formula VI:

[0039]

[0040] Formula VI

[0041] or a pharmaceutically acceptable salt thereof.

[0042] In some embodiments, the disclosure provides a polynucleotide encoding a polypeptide of interest. The polynucleotide includes, in the 5’ to 3’ direction:

[0043] (a) optionally, a 5’-cap structure;

[0044] (b) a 5’-untranslated region (UTR);

[0045] (c) an open reading frame (ORF) encoding the polypeptide of interest;

[0046] (d) a 3’-UTR;

[0047] (e) a poly-A region;

[0048] (f) a linker; and

[0049] (g) a modified 3’ region comprising the structure of Formula II, in the 5’-to-3’ direction:

[0050] (A’) - (X1 - B)a - (X2 - C)b - (X3 - D)c - (X4 - E)d - (X5 - Z)e

[0051] Formula II

[0052] wherein:

[0053] A’ is a poly-A region;

[0054] B is a 2’-modified nucleoside or a BNA (e.g., LNA, ENA, BNA-NO, or AcEt);

[0055] C is a poly-A region;

[0056] D is a BNA (e.g., LNA, ENA, BNA-NC, or AcEt);

[0057] E is a BNA (e.g., LNA, ENA, BNA-NC, or AcEt);

[0058] Z is an inverted nucleoside;

[0059] wherein each of x1, x2, x3, x4, and x5 is, independently, an internucleoside linkage; and wherein each of a, b, c, d, and e is, independently, 0 or 1;

[0060] further wherein: when b is 0, (i) each of a, c, and d is 1 and e is 0, or (ii) each of a and e is 1 and each of c and d is 0; and

[0061] when b is 1, each of a, c, and e is 1, and d is 0.

[0062] In some embodiments, the 5’-cap structure of (a) is present. In some embodiments, the 5’-cap structure of (a) is absent.

[0063] In some embodiments, the polypeptide of interest encoded by the ORF of (c) corresponds to an antigen associated with a disease.

[0064] In some embodiments, the adenosines of the poly-A region of (e) are unmodified adenosines. In some embodiments, the poly-A region of (e) is from 1 to 200 (e.g., from 5 to 200, from 1 to 150, from 10 to 200, from 25 to 200, from 25 to 150, from 50 to 150, from 10 to 100, from 75 to 150, from 75 to 125, from 15 to 50, or from 15 to 25) nucleosides in length. In some embodiments, the poly-A region of (e) is from 50 to 150 nucleosides in length. In some embodiments, the poly-A region of (e) is 100 nucleosides in length.

[0065] In some embodiments, the linker of (f) has the structure of Formula III, in the 5’ to 3’ direction:

[0066]

[0067] Formula III

[0068] wherein A’ is the poly-A region of (e);

[0069] B’ is the 3’ modified region of (g);

[0070] n is from 0 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50);

[0071] X is each, independently, O or S; and

[0072] D is a nucleoside, each independently chosen from an adenosine, a modified adenosine, thymidine, a modified thymidine, a cytidine, a modified cytidine, a uridine, a modified uridine, a guanosine, or a modified guanosine.

[0073] In some embodiments, the linker of (f) is an Xbal scar. In some embodiments, the linker of (f) is a polynucleotide with the sequence 5’-UCUAG-3’.

[0074] In some embodiments, the linker is formed during the ligation of the modified 3’ region of B’. In some embodiments, the linker is a ligation sequence.

[0075] In some embodiments, each adenosine of the poly-A region A’ of Formula II of (g) is an unmodified adenosine. In some embodiments, the poly-A region A’ of Formula II is from 1 to 200 (e.g., from 5 to 200, from 1 to 150, from 10 to 200, from 25 to 200, from 25 to 150, from 50 to 150, from 10 to 100, from 75 to 150, from 75 to 125, from 15 to 50, from 15 to 25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200) nucleosides in length. In some embodiments, the poly-A region A’ of Formula II is from 15 to 25 nucleosides in length. In some embodiments, the poly-A region A’ of Formula II is 20 nucleosides in length.

[0076] In some embodiments, the B of Formula II of (g), if present, is selected from 2’-fluoro-adenosine and 2’-0-methyl-adenosine. In some embodiments B is 2’-fluoro-adenosine. In some embodiments B is 2’-0-methyl-adenosine.

[0077] In some embodiments, the BNA of B, D, and E of Formula II of (g), if present, is selected from one of LNA-adenosine, LNA-thymidine, LNA-guanosine, LNA-cytidine, LNA-(5-methyl-cytidine), LNA-uridine, BNA-NC-adenosine, BNA-NC-thymidine, BNA-NC-guanosine, BNA-NC-cytidine, BNA-NC-(5-methyl-cytidine), BNA-NC-uridine, ENA-adenosine, ENA-thymidine, ENA-guanosine, ENA-cytidine, ENA-(5-methyl-cytidine), ENA-uridine, constrained ethyl nucleic acid-adenosine ((A)AcEt), constrained ethyl nucleic acid-guanosine ((G)AcEt), constrained ethyl nucleic acid-thymidine ((T)AcEt), constrained ethyl nucleic acid-cytidine ((C)AcEt), constrained ethyl nucleic acid-(5-methyl-cytidine) ((m5C)AcEt), or constrained ethyl nucleic acid-uridine ((U)AcEt). In some embodiments, the BNA of B, D, and E of Formula II of (g), if present, is each LNA-adenosine. In some embodiments, B is LNA-adenosine. In some embodiments, D is LNA-adenosine. In some embodiments, E is LNA-adenosine. In some embodiments, B, D, and E are all LNA-adenosine.

[0078] In some embodiments, the inverted nucleoside Z of Formula II of (g), if present, is an inverted deoxythymidine.

[0079] In some embodiments, each of x1, x2, x3, x4, and x5 of Formula II of (g), if present, is a phosphoryl guanidine linkage. In some embodiments, each of x1, x2, x3, x4, and x5 of Formula II of (g), if present, is a phosphodiester internucleoside linkage. In some embodiments, x1 is a phosphodiester internucleoside linkage. In some embodiments, x2 is a phosphodiester internucleoside linkage. In some embodiments, x3 is a phosphodiester internucleoside linkage. In some embodiments, x4 is a phosphodiester internucleoside linkage. In some embodiments, x5 is a phosphodiester internucleoside linkage.

[0080] In some embodiments, the C of Formula II of (g), if present, is a poly-A region. In some embodiments, the poly-A region C is from 1 to 200 (e.g., from 5 to 200, from 1 to 150, from 10 to 200, from 25 to 200, from 25 to 150, from 50 to 150, from 10 to 100, from 75 to 150, from 75 to 125, from 15 to 50, from 15 to 25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200) nucleosides in length. In some embodiments, the poly-A region of C is from 15 to 50 nucleosides in length. In some embodiments, the poly-A region of C is 20 nucleosides in length. In some embodiments, each adenosine of the C poly-A region is an unmodified adenosine ribonucleotide. In some embodiments, the A’ and C of Formula II of (g) are poly-A regions of 20 nucleosides in length.

[0081] In some embodiments, each of a, b, c, d, and e of Formula II of (g) is, independently, 0 or 1. In some embodiments, a is 0. In some embodiments, a is 1. In some embodiments, b is 0. In some embodiments, b is 1. In some embodiments, c is 0. In some embodiments, c is 1. In some embodiments, d is 0. In some embodiments, d is 1. In some embodiments, e is 0. In some embodiments, e is 1. In some embodiments, a, b, c, and e is 1, and d is 0. In some embodiments, a, c, d, and e is 1, and b is 0. In some embodiments, a and e is 1, and b, c, and d is 0.

[0082] In some embodiments, the modified 3’ region polynucleotide of (g) has the structure of Formula IV, in the 5’-to-3’ direction:

[0083] -(A)20-o-Am-o-(A)20-o-Ai_NA-o-idT

[0084] Formula IV

[0085] wherein A represents an unmodified adenosine ribonucleoside;

[0086] Am represents a 2’-0-methyl-adenosine;

[0087] ALNA represents an LNA-adenosine;

[0088] idT represents an inverted deoxythymidine; and

[0089] each o represents a phosphodiester or phosphorothioate internucleoside linkage.

[0090] In some embodiments, the modified 3’ region polynucleotide of (g) has the structure of Formula VII, in the 5’-to-3’ direction:

[0091] -(A)20-o-Af-o-(A)20-o-Ai_NA-o-idT

[0092] Formula VII

[0093] wherein A represents an unmodified adenosine ribonucleoside;

[0094] Af represents a 2’-fluoro-adenosine;

[0095] ALNA represents an LNA-adenosine;

[0096] idT represents an inverted deoxythymidine; and

[0097] each o represents a phosphodiester or phosphorothioate internucleoside linkage.

[0098] In some embodiments, the modified 3’ region polynucleotide of (g) has the structure of Formula VIII, in the 5’-to-3’ direction:

[0099] -(A)20-O-ALNA-O-ALNA-O-ALNA

[0100] Formula VIII

[0101] wherein A represents an unmodified adenosine ribonucleoside;

[0102] ALNA represents an LNA-adenosine; and

[0103] each o represents a phosphodiester or phosphorothioate internucleoside linkage.

[0104] In some embodiments, the modified 3’ region polynucleotide of (g) has the structure of Formula IX, in the 5’-to-3’ direction: -(A)20-o-Ai_NA-o-idT

[0105] Formula IX

[0106] wherein A represents an unmodified adenosine ribonucleoside;

[0107] ALNA represents an LNA-adenosine;

[0108] idT represents an inverted deoxythymidine; and

[0109] each o represents a phosphodiester or phosphorothioate internucleoside linkage.

[0110] In some embodiments, the modified 3’ region contains at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) terminal ALNA.

[0111] In some embodiments, the modified 3’ region contains a sequence of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) ALNA immediately preceding the terminal inverted deoxythymidine.

[0112] In some embodiments, the modified 3’ region is a 3’-stabilizi ng region.

[0113] In some embodiments, the modified 3’ region contains a BNA (e.g., LNA), which are modified RNA nucleosides that contain bridging groups, typically, but not limited to, between the 2’ and 4’ position of the ribose sugar backbone, which confer a fixed Cs-endo sugar puckering conformation. In some embodiments, the BNA (e.g., LNA) containing a bridge between the 2’ and 4’ positions has the Formula XI:

[0114]

[0115] Formula XI

[0116] wherein each R1is, independently, -C(R2)2-, -0(0)-, -0-, -S-, or -NR3-, wherein each R2is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl, and each R3is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl;

[0117] n is at least one (e.g., 1, 2, 3, 4, 5, 6, or 7);

[0118] X is one of -CR12-, -0(0)-, -0-, -S-, or -NR2-, wherein each R1is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl, and each R2is, independently, H, alkyl, heteroalkyl, aryl or heteroaryl; and the base is an adenine, a modified adenine, a cytosine, a modified cytosine, a uracil, a modified uracil, a guanine, a modified guanine, a thymine, or a modified thymine.

[0119] In some embodiments, the BNA is one of:

[0120]

[0121] LNA-adenosine LNA-thymidine LNA-guanosine LNA-cytidine

[0122]

[0123] LNA-(5-methyl-cytidine) LNA-uridine ENA-(5-methyl-cytidine) ENA-uridine

[0124]

[0125] BNA-NC-(5-methyl-cytidine) BNA-NC-uridine AcEt-(5-methyl-cytidine) AcEt-uridine In some embodiments, the modified 3’ region includes a plurality of adenosines. In some embodiments, all of the nucleosides of the modified 3’ region are adenosines. In some embodiments, the modified 3’ region includes at least one (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten) alternative nucleoside (e.g., an L-nucleoside such as L-adenosine, 2’-0-methyl-adenosine, alpha-thio-2’-0-methyl-adenosine, 2’-fluoro-adenosine, arabino-adenosine, hexitol-adenosine, PNA-adenosine, LNA-adenosine (ALNA), LNA-thymidine (TLNA), LNA-guanosine (GLNA), LNA-cytidine (CLNA), LNA-(5-methyl-cytidine) ((m5C)LNA), LNA-uridine (ULNA), BNA-NC-adenosine (ABNA-NC), BNA-NC-thymidine (TBNA-NC), BNA-NC-guanosine (GBNA-NC), BNA-NC-cytidine (CBNA-NC), BNA-NC-(5-methyl-cytidine) ((m5C)BNA-NC), BNA-NC-uridine (UBNA-NC), ENA-adenosine (AENA), ENA-thymidine (TENA), ENA-guanosine (GENA), ENA-cytidine (CENA), ENA-(5-methyl-cytidine) ((m5C)ENA), ENA-uridine (UENA), constrained ethyl nucleic acid-adenosine ((A)AcEt), constrained ethyl nucleic acid-guanosine ((G)AcEt), constrained ethyl nucleic acid-thymidine ((T)AcEt), constrained ethyl nucleic acid-cytidine ((C)AcEt), constrained ethyl nucleic acid-(5-methyl-cytidine) ((m5C)AcEt), constrained ethyl nucleic acid-uridine ((U)AcEt), extended-adenosine (exA), extended-thymidine (exT), extended-guanosine (exG), extended-cytidine (exC), extended-(5-methyl-cytidine) (ex(m5C)), extended-uridine (exU), inverted LNA-adenosine (IALNA), inverted LNA-thymidine (ITLNA), inverted LNA-guanosine (IGLNA), inverted LNA-cytidine (ICLNA), inverted LNA-(5-methyl-cytidine) (i(m5C)LNA), inverted LNA-uridine (IULNA), inverted BNA-NC-adenosine (iABNA-NC), inverted BNA-NC-thymidine (iTBNA-NC), inverted BNA-NC-guanosine (iGBNA-NC), inverted BNA-NC-cytidine (iCBNA-NC), inverted BNA-NC-(5-methyl-cytidine) (i(m5C)BNA-NC), inverted BNA-NC-uridine (iUBNA-NC), inverted ENA-adenosine (iAENA), inverted ENA-thymidine (iTENA), inverted ENA-guanosine (iGENA), inverted ENA-cytidine (iCENA), inverted ENA-(5-methyl-cytidine) (i(m5C)ENA), inverted ENA-uridine (iUENA), inverted constrained ethyl nucleic acid-adenosine (i(A)AcEt), inverted constrained ethyl nucleic acidguanosine (i(G)AcEt), inverted constrained ethyl nucleic acid-thymidine (i(T)AcEt), inverted constrained ethyl nucleic acid-cytidine (i(C)AcEt), inverted constrained ethyl nucleic acid-(5-methyl-cytidine) (i(m5C)AcEt), inverted constrained ethyl nucleic acid-uridine (i(U)AcEt), inverted extended-adenosine (iexA), inverted extended-thymidine (iexT), inverted extended-guanosine (iexG), inverted extended-cytidine (iexC), inverted extended-(5-methyl-cytidine) (iex(m5C)), inverted extended-uridine (iexU)or inverted deoxythymidine). In some embodiments, the alternative nucleoside is L-adenosine, 2’-O-methyl-adenosine, 2’-fluoro-adenosine, LNA-adenosine, LNA-thymidine, LNA-(5-methyl-cytidine) ((m5C)LNA), ENA-adenosine, BNA-NC-adenosine, extended-adenosine, or inverted deoxythymidine. In some embodiments, the modified 3’ region includes a plurality of alternative nucleosides. In some embodiments, all of the nucleotides in the modified 3’ region are alternative nucleosides. In some embodiments, the modified 3’ region includes at least two (e.g., at least three) different alternative nucleosides. In some embodiments, at least one alternative nucleoside is 2’-0-methyl-adenosine. In some embodiments, at least one alternative nucleoside is inverted deoxythymidine. In some embodiments, at least one alternative nucleoside is 2’-0-methyl-adenosine, and at least one alternative nucleoside is inverted deoxythymidine. In some embodiments, at least one alternative nucleoside (e.g., at least one, at least two, or at least three) is LNA-adenosine. In some embodiments, at least one alternative nucleoside (e.g., at least one, at least two, or at least three) is LNA-thymidine. In some embodiments, at least one alternative nucleoside (e.g., at least one, at least two, at least three) is LNA-(5-methyl-cytidine) ((m5C)LNA). In some embodiments, at least one alternative nucleoside (e.g., at least one, at least two, or at least three) is ENA-adenosine. In some embodiments, at least one alternative nucleoside (e.g., at least one, at least two, or at least three) is BNA-NC-adenosine. In some embodiments, at least one alternative nucleoside (e.g., at least one, at least two, or at least three) is extended-adenosine. In some embodiments, at least one alternative nucleoside is LNA-adenosine, and at least one alternative nucleoside is inverted deoxythymidine. In some embodiments, at least one alternative nucleoside is LNA-thymidine, and at least one alternative nucleoside is inverted deoxythymidine. In some embodiments, at least one alternative nucleoside is 2’-0-methyl-adenosine, and at least one alternative nucleoside is LNA-adenosine. In some embodiments, at least one alternative nucleoside is 2’-0-methyl-adenosine, at least one alternative nucleoside is LNA-adenosine, and at least one is inverted deoxythymidine. In some embodiments, at least one alternative nucleoside is 2’-fluoro-adenosine. In some embodiments, at least one alternative nucleoside is 2’-fluoro-adenosine, and at least one is inverted deoxythymidine. In some embodiments, at least one alternative nucleoside is 2’-fluoro-adenosine, at least one is LNA-adenosine, and at least one is inverted deoxythymidine. In some embodiments, at least one alternative nucleoside is LNA-thymidine. In some embodiments, at least one alternative nucleoside is ENA-adenosine. In some embodiments, at least one alternative nucleoside is BNA-NC-adenosine. In some embodiments, at least one alternative nucleoside is extended-adenosine, at least one alternative nucleoside is LNA-adenosine, and at least one alternative nucleoside is inverted deoxythymidine. In some embodiments, at least one alternative nucleoside is extended-adenosine and at least one alternative nucleoside is LNA-adenosine.

[0126] In some embodiments, the modified 3’ region includes at least one (e.g., at least two) poly-A region. In some embodiments, all the adenosines in the poly-A region are unmodified adenosines. In some embodiments, at least one adenosine in the poly-A region is a modified adenosine. In some embodiments, the poly-A region is between about 1 to about 50 (e.g., 1 to 45, 5 to 40, 10 to 30, 15 to 25, or 5, 10, 15, 20, 25, 30, or 35) nucleosides. In some embodiments, the modified region includes the structure of Formula X:

[0127]

[0128] Formula X

[0129] or a salt thereof;

[0130] wherein each X is, independently, O or S; and

[0131] A represents adenine and T represents thymine.

[0132] In some embodiments, all of the plurality of alternative nucleosides are the same (e.g., all of the alternative nucleosides are L-adenosine). In some embodiments, the modified region includes 10 nucleosides. In some embodiments, the modified region includes 11 nucleosides. In some embodiments, the modified region includes 22 nucleosides. In some embodiments, the modified region includes 23 nucleosides. In some embodiments, the modified region includes 43 nucleosides.

[0133] In some embodiments, the linker is a polynucleotide of Formula III:

[0134]

[0135] Formula III

[0136] wherein A’ is the polynucleotide that contains:

[0137] (a) optionally, a 5’-cap structure;

[0138] (b) a 5’-untranslated region (UTR) (e.g., a 5’-UTR including a Kozak sequence);

[0139] (c) an open reading frame (ORF) encoding a polypeptide of interest;

[0140] (d) a 3’-UTR; and

[0141] (e) a poly-A region;

[0142] B’ is the modified 3’ region;

[0143] each X is, independently, O or S;

[0144] n is at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10); and

[0145] D is a nucleoside independently chosen from an adenosine, a modified adenosine, a thymidine, a modified thymidine, a cytidine, a modified cytidine, a uridine, a modified uridine, a guanosine, or a modified guanosine.

[0146] In some embodiments, the 5’-cap structure of (a) is present. In some embodiments, the 5’-cap structure of (a) is absent.

[0147] In some embodiments the linker is a polynucleotide with the sequence 5’-(Xbal scar)-(A)2o-Af-3’: wherein Af represents a 2’-fluoro-adenosine; and

[0148] Xbal scar represents (5’-UCUAG-3’).

[0149] In some embodiments the linker is a polynucleotide with the sequence 5’-(Xbal scar)-(A)2o-Am-3’: wherein Am represents a 2’-0-methoxy-adenosine; and

[0150] Xbal scar represents (5’-UCUAG-3’).

[0151] In some embodiments, the linker is a polynucleotide with the sequence 5’-UCUAG-3’, also known as an Xbal scar.

[0152] In some embodiments, the linker is attached to the 3’-terminus of A’ and the 5’-terminus of B’. In some embodiments, the linker is formed during the ligation of the modified 3’ region of B’. In some embodiments, the linker is a ligation sequence.

[0153] In some embodiments, the modified 3’ region includes at least one L-nucleoside (e.g., L-adenosine).

[0154] In some embodiments, the 5’-terminus of the modified 3’ region is ligated to the 3’-terminus of the 3’-UTR.

[0155] In some embodiments, the 5’-terminus of the modified 3’ region is ligated to the 3’-terminus of the poly-A region.

[0156] In some embodiments, the alternative nucleosides are 2’-0-methyl-adenosines, or 2’-fluoro-adenosine. In some embodiments, the polynucleotide includes a plurality of alternative nucleosides at the 3’-terminus. In some embodiments, the plurality of alternative nucleosides includes at least two different nucleosides. In some embodiments, the plurality of alternative nucleosides are all the same nucleoside. In some embodiments, all of the nucleosides in the modified 3’ region are alternative nucleosides. In some embodiments, the modified region includes 10 nucleosides. In some embodiments, the modified region includes 11 nucleosides. In some embodiments, the stabilizing region includes 22 nucleosides. In some embodiments, the stabilizing region includes 23 nucleosides. In some embodiments, the stabilizing region includes 43 nucleosides.

[0157] In another aspect, the present invention provides a polynucleotide encoding a polypeptide of interest, wherein said polynucleotide comprises, in the 5’-to-3’ direction:

[0158] (a) optionally, a 5’-cap structure;

[0159] (b) a 5’-untranslated region (UTR);

[0160] (c) an open reading frame (ORF) encoding the polypeptide of interest;

[0161] (d) a 3’-UTR;

[0162] (e) a poly-A region;

[0163] (f) a linker; and

[0164] (g) a modified 3’ region having the structure of Formula XII, in the 5’-to-3’ direction:

[0165] (A’) - (X1 - B) - (X2 - C)a - (X3 - D)b - (X4 - E)c - (X5 - F)d - (X6 - Z)e

[0166] Formula XII

[0167] wherein:

[0168] A’ is a poly-A region comprising from 10 to 100 (e.g., from 10 to 100, from 15 to 100, from 20 to 100, from 25 to 100, from 30 to 100, from 35 to 100, from 40 to 100, from 45 to 100, from 50 to 100, from 55 to 100, from 60 to 100, from 65 to 100, from 70 to 100, from 75 to 100, from 80 to 100, from 85 to 100, from 90 to 100, from 95 to 100, from 15 to 95, from 20 to 90, from 25 to 85, from 30 to 80, from 35 to 75, from 40 to 70, from 45 to 65, from 50 to 60, or from 55 to 55, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) nucleosides;

[0169] B is a bridged nucleic acid (BNA);

[0170] C is a BNA;

[0171] D is a BNA;

[0172] E is a BNA;

[0173] F is a BNA;

[0174] Z is an inverted nucleoside;

[0175] wherein each of xi, X2, X3, X4, xs, and xe is, independently, an internucleoside linkage; and wherein each of a, b, c, d, and e is, independently, 0 or 1;

[0176] wherein:

[0177] each BNA, independently, has the structure of Formula XI: ase

[0178]

[0179] Formula XI

[0180] wherein:

[0181] each R1is, independently, -C(R2)2-, -C(O)-, -0-, -S-, or -NR3-;

[0182] each R2is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl;

[0183] each R3is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl;

[0184] n is 1 or 2;

[0185] X is one of -C(R4)2-, -C(O)-, -O-, -S-, or -NR5-;

[0186] each R4is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl;

[0187] each R5is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl; and

[0188] the base is a nucleobase,

[0189] further wherein:

[0190] when e is 0, i) one of a, b, c, or d is 1 or ii) two of a, b, c, or d, is 1; and

[0191] when e is 1, i) each of a, b, c, or d is 0 or ii) one of a, b, c, or d is 1.

[0192] In some embodiments, the 5’-cap structure of (a) is present. In some embodiments, the 5’-cap structure of (a) is absent.

[0193] In some embodiments, each of xi, x2, xs, X4, xs, and xe is, independently, a phosphodiester linkage. In some embodiments, each of x1, x2, x3, x4, x5, and x6is, independently, a phosphoryl guanidine linkage.

[0194] In some embodiments, the nucleobase is selected from one of an adenine, a modified adenine, a thymine, a modified thymine, a guanine, a modified guanine, a cytosine, a modified cytosine, a uracil, or a modified uracil.

[0195] In some embodiments, each R2is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-Cs aryl, or C3-C7 heteroaryl. In some embodiments, each R3is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-Cs aryl, or C3-C7 heteroaryl. In some embodiments, each R4is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, 04-Cs aryl, or C3-C7 heteroaryl. In some embodiments, each R5is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, 04-Cs aryl, or C3-C7 heteroaryl.

[0196] In some embodiments, the poly-A region of A’ is from 10 to 30 (e.g., from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, or from 15 to 25, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In some embodiments, the poly-A region of A’ is 15 nucleosides in length. In some embodiments, the poly-A region of A’ is 20 nucleosides in length.

[0197] In some embodiments, the poly-A region of (e) is from 1 to 200 (e.g., from 5 to 200, from 1 to 150, from 10 to 200, from 25 to 200, from 25 to 150, from 50 to 150, from 10 to 100, from 75 to 150, from 75 to 125, from 15 to 50, from 15 to 25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200) nucleosides in length. In some embodiments, the poly-A region of (e) is from 10 to 200 nucleosides in length. In some embodiments, the poly-A region of (e) is from 50 to 150 nucleosides in length. In some embodiments, the poly-A region of (e) is 100 nucleosides in length.

[0198] In some embodiments, the BNA has the structure of Formula XXXIV:

[0199] '4UVbase

[0200]

[0201] O JW IW I

[0202] Formula XXXIV

[0203] wherein:

[0204] n is 1 or 2;

[0205] X is one of -C(R4)2-, -C(O)-, -O-, -S-, or -NR5-;

[0206] each R4is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl;

[0207] each R5is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl; and

[0208] the base is a nucleobase.

[0209] In some embodiments, each R4is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-Cs aryl, or C3-C7 heteroaryl. In some embodiments, each R5is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-Cs aryl, or C3-C7 heteroaryl.

[0210] In some embodiments, the nucleobase is selected from one of an adenine, a modified adenine, a thymine, a modified thymine, a guanine, a modified guanine, a cytosine, a modified cytosine, a uracil, or a modified uracil.

[0211] In some embodiments, the BNA has the structure of Formula XXXV:

[0212]

[0213] Formula XXXV

[0214] wherein: n is 1 or 2;

[0215] each R1is, independently, -C(R2)2-, -C(O)-, -O-, -S-, or -NR3-;

[0216] each R3is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl;

[0217] each R4is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl; and

[0218] the base is a nucleobase.

[0219] In some embodiments, each R4is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-Cs aryl, or C3-C7 heteroaryl. In some embodiments, each R5is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-Cs aryl, or C3-C7 heteroaryl.

[0220] In some embodiments, the nucleobase is selected from one of an adenine, a modified adenine, a thymine, a modified thymine, a guanine, a modified guanine, a cytosine, a modified cytosine, a uracil, or a modified uracil.

[0221] In some embodiments, the BNA, if present, is one of LNA-adenosine (ALNA), LNA-thymidine (TLNA), LNA-guanosine (GLNA), LNA-cytidine (CLNA), LNA-(5-methyl-cytidine) ((m5C)LNA), LNA-uridine (ULNA), BNA-NC-adenosine (ABNA-NC), BNA-NC-thymidine (TBNA-NC), BNA-NC-guanosine (GBNA-NC), BNA-NC-cytidine (CBNA-NC), BNA-NC-(5-methyl-cytidine) ((m5C)BNA-NC), BNA-NC-uridine (UBNA-NC), ENA-adenosine (AENA), ENA-thymidine (TENA), ENA-guanosine (GENA), ENA-cytidine (CENA), ENA-(5-methyl-cytidine) ((m5C)ENA), ENA-uridine (UENA), constrained ethyl nucleic acid-adenosine ((A)AcEt), constrained ethyl nucleic acid-guanosine ((G)AcEt), constrained ethyl nucleic acid-thymidine ((T)AcEt), constrained ethyl nucleic acid-cytidine ((C)AcEt), constrained ethyl nucleic acid-(5-methyl-cytidine) ((m5C)AcEt), or constrained ethyl nucleic acid-uridine ((U)AcEt). In some embodiments, the BNA is LNA-adenosine. In some embodiments, the BNA is LNA-thymidine. In some embodiments, the BNA is ENA-adenosine. In some embodiments, the BNA is BNA-NC-adenosine.

[0222] In some embodiments, the inverted nucleoside of Z, if present, is inverted deoxythymidine. In some embodiments, the linker includes an Xbal scar. In some embodiments, the Xbal scar has the sequence of 5’-UCUAG-3’.

[0223] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XII has the structure of Formula XIV:

[0224]

[0225] Formula XIV

[0226] or a pharmaceutically acceptable salt thereof.

[0227] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XII has the structure of Formula XV:

[0228]

[0229] Formula XV

[0230] or a pharmaceutically acceptable salt thereof.

[0231] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XII has the structure of Formula XVI:

[0232]

[0233] Formula XVI

[0234] or a pharmaceutically acceptable salt thereof.

[0235] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XII has the structure of Formula XVII:

[0236]

[0237] Formula XVII

[0238] or a pharmaceutically acceptable salt thereof.

[0239] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XII has the structure of Formula XVIII:

[0240]

[0241] Formula XVIII

[0242] or a pharmaceutically acceptable salt thereof.

[0243] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XII has the structure of Formula XIX:

[0244]

[0245] Formula XIX

[0246] or a pharmaceutically acceptable salt thereof.

[0247] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XII has the structure of Formula XX:

[0248]

[0249] Formula XX

[0250] or a pharmaceutically acceptable salt thereof.

[0251] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XII has the structure of Formula XXI:

[0252]

[0253] Formula XXI

[0254] or a pharmaceutically acceptable salt thereof.

[0255] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XII has the structure of Formula XXXI:

[0256]

[0257] Formula XXXI

[0258] or a pharmaceutically acceptable salt thereof.

[0259] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XII has the structure of Formula XXXII:

[0260]

[0261] Formula XXXII

[0262] or a pharmaceutically acceptable salt thereof.

[0263] In another aspect, the present invention provides a polynucleotide encoding a polypeptide of interest, wherein said polynucleotide comprises, in the 5’-to-3’ direction:

[0264] (a) optionally, a 5’-cap structure;

[0265] (b) a 5’-untranslated region (UTR);

[0266] (c) an open reading frame (ORF) encoding the polypeptide of interest;

[0267] (d) a 3’-UTR;

[0268] (e) a poly-A region;

[0269] (f) a linker; and

[0270] (g) a modified 3’ region having the structure of Formula XXII, in the 5’-to-3’ direction:

[0271] (A’) - (X1 - B) - (X2 - C)a - (X3 - D)b - (X4 - E)c - (X5 - F)d - (X6 - G)e - (X7 - Z)f Formula XXII

[0272] wherein:

[0273] A’ is a poly-A region comprising from 10 to 100 (e.g., from 10 to 100, from 15 to 100, from 20 to 100, from 25 to 100, from 30 to 100, from 35 to 100, from 40 to 100, from 45 to 100, from 50 to 100, from 55 to 100, from 60 to 100, from 65 to 100, from 70 to 100, from 75 to 100, from 80 to 100, from 85 to 100, from 90 to 100, from 95 to 100, from 15 to 95, from 20 to 90, from 25 to 85, from 30 to 80, from 35 to 75, from 40 to 70, from 45 to 65, from 50 to 60, or from 55 to 55, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) nucleosides;

[0274] B is an extended nucleic acid;

[0275] C is an extended nucleic acid;

[0276] D is an extended nucleic acid;

[0277] E is a LNA;

[0278] F is a LNA;

[0279] G is a LNA;

[0280] Z is an inverted nucleoside;

[0281] wherein each of xi, X2, xs, X4, xs, xe, and x? is a internucleoside linkage; and

[0282] wherein each of a, b, c, d, e, and f is, independently, 0 or 1;

[0283] wherein:

[0284] each extended nucleic acid (ex(base)), independently, has the structure of Formula XXIII:

[0285]

[0286] Formula XXIII,

[0287] wherein the base is a nucleobase,

[0288] further wherein:

[0289] when f is 0, i) each of a, b, c, d, and e is 1 or ii) each of a and b is 0 and each of c, d, and e is 1; and

[0290] when f is 1, i) each of a and b is 1 and one of c, d, or e is 1 or ii) each of a and b is 0 and one of c, d, or e is 1.

[0291] In some embodiments, the 5’-cap structure of (a) is present. In some embodiments, the 5’-cap structure of (a) is absent.

[0292] In some embodiments, each of xi, X2, X3, X4, xs, xe, and x?, is, independently, a phosphodiester linkage or a phosphoryl guanidine linkage. In some embodiments, each of xi, X2, X3, X4, xs, xe, and X7 is a phosphodiester linkage. In some embodiments, each of xi, X2, X3, X4, xs, xe, and X7 is a phosphoryl guanidine linkage.

[0293] In some embodiments, the poly-A region of (e) is from 1 to 200 (e.g., from 5 to 200, from 1 to 150, from 10 to 200, from 25 to 200, from 25 to 150, from 50 to 150, from 10 to 100, from 75 to 150, from 75 to 125, from 15 to 50, from 15 to 25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200) nucleosides in length. In some embodiments, the poly-A region of (e) is from 10 to 200 nucleosides in length. In some embodiments, the poly-A region of (e) is from 50 to 150 nucleosides in length. In some embodiments, the poly-A region of (e) is 100 nucleosides in length.

[0294] In some embodiments, the poly-A region of A’ is from 10 to 30 (e.g., from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, or from 15 to 25, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In some embodiments, the poly-A region of A’ is 15 nucleosides in length. In some embodiments, the poly-A region of A’ is 20 nucleosides in length.

[0295] In some embodiments, the nucleobase is selected from one of an adenine, a modified adenine, a thymine, a modified thymine, a guanine, a modified guanine, a cytosine, a modified cytosine, a uracil, or a modified uracil.

[0296] In some embodiments, the BNA of E, F, and G, if present, is one of LNA-adenosine (ALNA), LNA-thymidine (TLNA), LNA-guanosine (GLNA), LNA-cytidine (CLNA), LNA-(5-methyl-cytidine) ((m5C)LNA), LNA-uridine (ULNA), BNA-NC-adenosine (ABNA-NC), BNA-NC-thymidine (TBNA-NC), BNA-NC-guanosine (GBNA-NC), BNA-NC-cytidine (CBNA-NC), BNA-NC-(5-methyl-cytidine) ((m5C)BNA-NC), BNA-NC-uridine (UBNA-NC), ENA-adenosine (AENA), ENA-thymidine (TENA), ENA-guanosine (GENA), ENA-cytidine (CENA), ENA-(5-methyl-cytidine) ((m5C)ENA), ENA-uridine (UENA), constrained ethyl nucleic acid-adenosine ((A)AcEt), constrained ethyl nucleic acid-guanosine ((G)AcEt), constrained ethyl nucleic acid-thymidine ((T)AcEt), constrained ethyl nucleic acid-cytidine ((C)AcEt), constrained ethyl nucleic acid-(5-methyl-cytidine) ((m5C)AcEt), or constrained ethyl nucleic acid-uridine ((U)AcEt). In some embodiments, the BNA of E, F, and G, if present, is each is LNA-adenosine.

[0297] In some embodiments, the extended nucleic acid is an extended nucleic acid-adenosine (exA, extended-adenosine). In some embodiments, the extended nucleic acid is an extended nucleic acid-thymidine (exT, extended-thymidine). In some embodiments, the extended nucleic acid is an extended nucleic acid-guanosine (exG, extended-guanosine). In some embodiments, the extended nucleic acid is an extended nucleic acid-cytidine (exC, extended-cytidine). In some embodiments, the extended nucleic acid is an extended nucleic acid-(5-methyl-cytidine) (ex(m5C), extended-(5-methyl-cytidine)). In some embodiments, the extended nucleic acid is an extended nucleic acid-uridine (exU, extended-uridine).

[0298] In some embodiments, extended-adenosine (exA), extended-thymidine (exT), extended-guanosine (exG), extended-cytidine (exC), extended-(5-methyl-cytidine), and extended-uridine (exU) have the structure of:

[0299]

[0300] Extended-(5-methyl-cytidine) Extended-uridine

[0301] In some embodiments, the inverted nucleoside of Z, if present, is inverted deoxythymidine. In some embodiments, the linker includes an Xbal scar. In some embodiments, the Xbal scar has the sequence of 5’-UCUAG-3’.

[0302] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XXII has the structure of Formula XXIV:

[0303]

[0304] Formula XXIV

[0305] or a pharmaceutically acceptable salt thereof.

[0306] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XXII has the structure of Formula XXV:

[0307]

[0308] Formula XXV

[0309] or a pharmaceutically acceptable salt thereof.

[0310] In some embodiments, the modified 3’ region polynucleotide of (g) of Formula XXII has the structure of Formula XXVI:

[0311]

[0312] Formula XXVI

[0313] or a pharmaceutically acceptable salt thereof.

[0314] In another aspect, the present invention provides a polynucleotide encoding a polypeptide of interest, wherein said polynucleotide comprises, in the 5’-to-3’ direction:

[0315] (a) optionally, a 5’-cap structure;

[0316] (b) a 5’-untranslated region (UTR);

[0317] (c) an open reading frame (ORF) encoding the polypeptide of interest;

[0318] (d) a 3’-UTR;

[0319] (e) a poly-A region;

[0320] (f) a linker; and

[0321] (g) a modified 3’ region comprising the structure of Formula XXVII, in the 5’-to-3’ direction: (A’) - (xi - B) - (x2- C) - (x3- D)

[0322] Formula XXVII

[0323] wherein:

[0324] A’ is a poly-A region comprising from 10 to 100 (e.g., from 10 to 100, from 15 to 100, from 20 to 100, from 25 to 100, from 30 to 100, from 35 to 100, from 40 to 100, from 45 to 100, from 50 to 100, from 55 to 100, from 60 to 100, from 65 to 100, from 70 to 100, from 75 to 100, from 80 to 100, from 85 to 100, from 90 to 100, from 95 to 100, from 15 to 95, from 20 to 90, from 25 to 85, from 30 to 80, from 35 to 75, from 40 to 70, from 45 to 65, from 50 to 60, or from 55 to 55, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) nucleosides, wherein 5 or (e.g., 5, 4, 3, 2, 1, or 0) less 3’ adenosines are L-adenosines;

[0325] B is a BNA;

[0326] C is a BNA;

[0327] D is a BNA; and

[0328] wherein each of x1, x2, and x3, is a internucleoside linkage.

[0329] In some embodiments, the 5’-cap structure of (a) is present. In some embodiments, the 5’-cap structure of (a) is absent.

[0330] In some embodiments, each of xi, X2, and X3 is, independently, a phosphodiester linkage or a phosphoryl guanidine linkage. In some embodiments, each of xi, X2, and X3 is a phosphodiester linkage. In some embodiments, each of xi, X2, and X3 is a phosphoryl guanidine linkage.

[0331] In some embodiments, the poly-A region of (e) is from 1 to 200 (e.g., from 5 to 200, from 1 to 150, from 10 to 200, from 25 to 200, from 25 to 150, from 50 to 150, from 10 to 100, from 75 to 150, from 75 to 125, from 15 to 50, from 15 to 25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200) nucleosides in length. In some embodiments, the poly-A region of (e) is from 10 to 200 nucleosides in length. In some embodiments, the poly-A region of (e) is from 50 to 150 nucleosides in length. In some embodiments, the poly-A region of (e) is 100 nucleosides in length.

[0332] In some embodiments, the poly-A region of A’ is from 10 to 30 (e.g., from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, or from 15 to 25, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In some embodiments, the poly-A region of A’ is 15 nucleosides in length. In some embodiments, the poly-A region of A’ is 20 nucleosides in length. In some embodiments, the nucleobase is selected from one of an adenine, a modified adenine, a thymine, a modified thymine, a guanine, a modified guanine, a cytosine, a modified cytosine, a uracil, or a modified uracil.

[0333] In some embodiments, the BNA of B, C, and D, if present, is one of LNA-adenosine (ALNA), LNA-thymidine (TLNA), LNA-guanosine (GLNA), LNA-cytidine (CLNA), LNA-(5-methyl-cytidine) ((m5C)LNA), LNA-uridine (ULNA), BNA-NC-adenosine (ABNA-NC), BNA-NC-thymidine (TBNA-NC), BNA-NC-guanosine (GBNA-NC), BNA-NC-cytidine (CBNA-NC), BNA-NC-(5-methyl-cytidine) ((m5C)BNA-NC), BNA-NC-uridine (UBNA-NC), ENA-adenosine (AENA), ENA-thymidine (TENA), ENA-guanosine (GENA), ENA-cytidine (CENA), ENA-(5-methyl-cytidine) ((m5C)ENA), ENA-uridine (UENA), constrained ethyl nucleic acid-adenosine ((A)AcEt), constrained ethyl nucleic acid-guanosine ((G)AcEt), constrained ethyl nucleic acid-thymidine ((T)AcEt), constrained ethyl nucleic acid-cytidine ((C)AcEt), constrained ethyl nucleic acid-(5-methyl-cytidine) ((m5C)AcEt), or constrained ethyl nucleic acid-uridine ((U)AcEt). In some embodiments, the BNA of B, C, and D, if present, is each is LNA-adenosine.

[0334] In some embodiments, the linker includes an Xbal scar. In some embodiments, the Xbal scar has the sequence of 5’-UCUAG-3’.

[0335] In another aspect, the present invention provides a polynucleotide encoding a polypeptide of interest, wherein said polynucleotide comprises, in the 5’-to-3’ direction:

[0336] (a) optionally, a 5’-cap structure;

[0337] (b) a 5’-untranslated region (UTR);

[0338] (c) an open reading frame (ORF) encoding the polypeptide of interest;

[0339] (d) a 3’-UTR;

[0340] (e) a poly-A region;

[0341] (f) a linker; and

[0342] (g) a modified 3’ region comprising the structure of Formula XXVIII, in the 5’-to-3’ direction:

[0343] (A’) - (X1 - Z) - (X2 - B)a - (X3 - C)b- (X4 - D)c

[0344] Formula XXVIII

[0345] wherein:

[0346] A’ is a poly-A region comprising from 10 to 100 (e.g., from 10 to 100, from 15 to 100, from 20 to 100, from 25 to 100, from 30 to 100, from 35 to 100, from 40 to 100, from 45 to 100, from 50 to 100, from 55 to 100, from 60 to 100, from 65 to 100, from 70 to 100, from 75 to 100, from 80 to 100, from 85 to 100, from 90 to 100, from 95 to 100, from 15 to 95, from 20 to 90, from 25 to 85, from 30 to 80, from 35 to 75, from 40 to 70, from 45 to 65, from 50 to 60, or from 55 to 55, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) nucleosides;

[0347] B is a BNA;

[0348] C is a BNA;

[0349] D is a BNA;

[0350] Z is an inverted nucleoside;

[0351] wherein each of x1, x2, x3, and x4 is a internucleoside linkage; and wherein each of a, b, and c is, independently, 0 or 1.

[0352] In some embodiments, the 5’-cap structure of (a) is present. In some embodiments, the 5’-cap structure of (a) is absent.

[0353] In some embodiments, each of xi, X2, xs, and X4, is, independently, a phosphodiester linkage or a phosphoryl guanidine linkage. In some embodiments, each of xi, X2, X3, and X4 is a phosphodiester linkage. In some embodiments, each of xi, X2, X3, and X4 is a phosphoryl guanidine linkage.

[0354] In some embodiments, the poly-A region of (e) is from 1 to 200 (e.g., from 5 to 200, from 1 to 150, from 10 to 200, from 25 to 200, from 25 to 150, from 50 to 150, from 10 to 100, from 75 to 150, from 75 to 125, from 15 to 50, from 15 to 25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200) nucleosides in length. In some embodiments, the poly-A region of (e) is from 10 to 200 nucleosides in length. In some embodiments, the poly-A region of (e) is from 50 to 150 nucleosides in length. In some embodiments, the poly-A region of (e) is 100 nucleosides in length.

[0355] In some embodiments, the poly-A region of A’ is from 10 to 30 (e.g., from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, or from 15 to 25, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In some embodiments, the poly-A region of A’ is 15 nucleosides in length. In some embodiments, the poly-A region of A’ is 20 nucleosides in length.

[0356] In some embodiments, the nucleobase is selected from one of an adenine, a modified adenine, a thymine, a modified thymine, a guanine, a modified guanine, a cytosine, a modified cytosine, a uracil, or a modified uracil.

[0357] In some embodiments, the BNA of B, C, and D, if present, is each one of LNA-adenosine (ALNA), LNA-thymidine (TLNA), LNA-guanosine (GLNA), LNA-cytidine (CLNA), LNA-(5-methyl-cytidine) ((m5C)LNA), LNA-uridine (ULNA), BNA-NC-adenosine (ABNA-NC), BNA-NC-thymidine (TBNA-NC), BNA-NC-guanosine (GBNA-NC), BNA-NC-cytidine (CBNA-NC), BNA-NC-(5-methyl-cytidine) ((m5C)BNA-NC), BNA-NC-uridine (UBNA-NC), ENA-adenosine (AENA), ENA-thymidine (TENA), ENA-guanosine (GENA), ENA-cytidine (CENA), ENA-(5-methyl-cytidine) ((m5C)ENA), ENA-uridine (UENA), constrained ethyl nucleic acid-adenosine ((A)AcEt), constrained ethyl nucleic acid-guanosine ((G)AcEt), constrained ethyl nucleic acid-thymidine ((T)AcEt), constrained ethyl nucleic acid-cytidine ((C)AcEt), constrained ethyl nucleic acid-(5-methyl-cytidine) ((m5C)AcEt), or constrained ethyl nucleic acid-uridine ((U)AcEt). In some embodiments, the BNA of B, C, and D, if present, is each is LNA-adenosine.

[0358] In some embodiments, the inverted nucleoside of Z is inverted deoxythymidine.

[0359] In some embodiments, the linker includes an Xbal scar. In some embodiments, the Xbal scar has the sequence of 5’-UCUAG-3’. In another aspect, the present invention provides a polynucleotide encoding a polypeptide of interest, wherein said polynucleotide comprises, in the 5’-to-3’ direction:

[0360] (a) optionally, a 5’-cap structure;

[0361] (b) a 5’-untranslated region (UTR);

[0362] (c) an open reading frame (ORF) encoding the polypeptide of interest;

[0363] (d) a 3’-UTR;

[0364] (e) a poly-A region;

[0365] (f) a linker; and

[0366] (g) a modified 3’ region comprising the structure of Formula XXIX, in the 5’-to-3’ direction:

[0367] (A’) - (X1 - W) - (x2- X)a- (x3- Y)b

[0368] Formula XXIX

[0369] wherein:

[0370] A’ is a poly-A region comprising from 10 to 100 (e.g., from 10 to 100, from 15 to 100, from 20 to 100, from 25 to 100, from 30 to 100, from 35 to 100, from 40 to 100, from 45 to 100, from 50 to 100, from 55 to 100, from 60 to 100, from 65 to 100, from 70 to 100, from 75 to 100, from 80 to 100, from 85 to 100, from 90 to 100, from 95 to 100, from 15 to 95, from 20 to 90, from 25 to 85, from 30 to 80, from 35 to 75, from 40 to 70, from 45 to 65, from 50 to 60, or from 55 to 55, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) nucleosides;

[0371] W is an inverted BNA;

[0372] X is an inverted BNA;

[0373] Y is an inverted BNA;

[0374] wherein each of x1, x2, and x3 is a internucleoside linkage; and

[0375] wherein each of a and b is, independently, 0 or 1;

[0376] wherein each inverted BNA, independently, has the structure of Formula XXX:

[0377]

[0378] Formula XXX

[0379] wherein:

[0380] each R1is, independently, -C(R2)2-, -C(O)-, -O-, -S-, or -NR3-;

[0381] each R2is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl;

[0382] each R3is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl;

[0383] n is 1 or 2;

[0384] X is one of -C(R4)2-, -C(O)-, -O-, -S-, or -NR5-;

[0385] each R4is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl; each R5is, independently, H, alkyl, heteroalkyl, aryl, or heteroaryl; and

[0386] the base is a nucleobase.

[0387] In some embodiments, each R2is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, 04-Cs aryl, or C3-C7 heteroaryl. In some embodiments, each R3is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, 04-Cs aryl, or C3-C7 heteroaryl. In some embodiments, each R4is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-Cs aryl, or C3-C7 heteroaryl. In some embodiments, each R5is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-Cs aryl, or C3-C7 heteroaryl.

[0388] In some embodiments, the 5’-cap structure of (a) is present. In some embodiments, the 5’-cap structure of (a) is absent.

[0389] In some embodiments, each of x1, x2, x3, and x4, is, independently, a phosphodiester linkage or a phosphoryl guanidine linkage. In some embodiments, each of x1, x2, x3, and x4 is a phosphodiester linkage. In some embodiments, each of x1, x2, x3, and x4 is a phosphoryl guanidine linkage.

[0390] In some embodiments, the poly-A region of (e) is from 1 to 200 (e.g., from 5 to 200, from 1 to 150, from 10 to 200, from 25 to 200, from 25 to 150, from 50 to 150, from 10 to 100, from 75 to 150, from 75 to 125, from 15 to 50, from 15 to 25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200) nucleosides in length. In some embodiments, the poly-A region of (e) is from 10 to 200 nucleosides in length. In some embodiments, the poly-A region of (e) is from 50 to 150 nucleosides in length. In some embodiments, the poly-A region of (e) is 100 nucleosides in length.

[0391] In some embodiments, the poly-A region of A’ is from 10 to 30 (e.g., from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, or from 15 to 25, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In some embodiments, the poly-A region of A’ is 15 nucleosides in length. In some embodiments, the poly-A region of A’ is 20 nucleosides in length.

[0392] In some embodiments, the nucleobase is selected from one of an adenine, a modified adenine, a thymine, a modified thymine, a guanine, a modified guanine, a cytosine, a modified cytosine, a uracil, or a modified uracil.

[0393] In some embodiments, the inverted BNA of W, X, and Y, if present, is each one of inverted LNA-adenosine (IALNA), inverted LNA-thymidine (iTLNA), inverted LNA-guanosine (iGLNA), inverted LNA-cytidine (iCLNA), inverted LNA-(5-methyl-cytidine) (i(m5C)LNA), inverted LNA-uridine (IULNA), inverted BNA-NC-adenosine (iABNA-NC), inverted BNA-NC-thymidine (iTBNA-NC), inverted BNA-NC-guanosine (iGBNA-NC), inverted BNA-NC-cytidine (iCBNA-NC), inverted BNA-NC-(5-methyl-cytidine) (i(m5C)BNA-NC), inverted BNA-NC-uridine (iUBNA-NC), inverted ENA-adenosine (iAENA), inverted ENA-thymidine (iTENA), inverted ENA-guanosine (iGENA), inverted ENA-cytidine (iCENA), inverted ENA-(5-methyl-cytidine) (i(m5C)ENA), inverted ENA-uridine (iUENA), inverted constrained ethyl nucleic acid-adenosine (i(A)AcEt), inverted constrained ethyl nucleic acid-guanosine (i(G)AcEt), inverted constrained ethyl nucleic acid-thymidine (i(T)AcEt), inverted constrained ethyl nucleic acid-cytidine (i(C)AcEt), inverted constrained ethyl nucleic acid-(5-methyl-cytidine) (i(m5C)AcEt), or inverted constrained ethyl nucleic acid-uridine (i(U)AcEt). In some embodiments, the BNA of B, C, and D, if present, is each is inverted LNA-adenosine (iALNA). In some embodiments, the BNA of B, C, and D, if present, is each is inverted LNA-thymidine (iTLNA). In some embodiments, the BNA of B, C, and D, if present, is each is inverted BNA-NC-adenosine (iABNA-NC). In some embodiments, the BNA of B, C, and D, if present, is each is inverted BNA-NC-thymidine (iTBNA-NC).

[0394] In some embodiments, the inverted LNA-adenosine has the structure of:

[0395]

[0396] Inverted LNA-adenosine.

[0397] In some embodiments, the inverted LNA-thymidine has the structure of:

[0398]

[0399] Inverted LNA-thymidine.

[0400] In some embodiments, the inverted BNA-NC-adenosine has the structure of:

[0401]

[0402] Inverted BNA-NC-adenosine.

[0403] In some embodiments, the inverted BNA-NC-adenosine has the structure of:

[0404]

[0405] Inverted BNA-NC-thymidine.

[0406] In some embodiments, the linker includes an Xbal scar. In some embodiments, the Xbal scar has the sequence of 5’-UCUAG-3’.

[0407] In some embodiments of any of the foregoing polynucleotides, the 5’-UTR includes a Kozak sequence.

[0408] In some embodiments of any of the foregoing polynucleotides, the modified 3’ region includes the 3’-terminus of the polynucleotide.

[0409] In some embodiments of any of the foregoing polynucleotides, the modified 3’ region includes at least one non-nucleoside. In some embodiments, at least one non-nucleoside is at the 5’-terminus, the 3’-terminus, or at an internal position of the modified 3’ region.

[0410] In some embodiments of any of the foregoing polynucleotides, at least one of the ORF, the 5’-UTR, the 3’-UTR, the 5’-cap structure, and / or the modified 3’ region includes at least one alternative (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten) nucleoside (e.g., any alternative nucleoside described herein). Alternative nucleosides include a modified adenosine, a modified thymidine, a modified uridine (a 5-substituted uridine, e.g., 5-methoxy-uridine, a 1 -substituted pseudouridine, e.g., N1 -methylpseudouridine), a modified cytidine (a 5-substituted cytidine, e.g., 5-methyl-cytidine), or a modified guanosine.

[0411] In some embodiments of any of the foregoing polynucleotides, the poly-A region, if present, includes at least one alternative (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten) nucleoside (e.g., any alternative nucleoside described herein such a 5-substituted uridine, e.g., 5-methoxy-uridine, a 1 -substituted pseudouridine, or a 5-substituted cytidine, e.g., 5-methyl-cytidine).

[0412] In some embodiments of any of the foregoing polynucleotides, the poly-A region, if present, includes from about 20 to about 400 nucleosides (e.g., 1 to 10, 5 to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50, 45 to 65, 50 to 70, 60 to 70, 65 to 85, 70 to 90, 85 to 105, 90 to 110, 105 to 135, 120 to 150, 130 to 170, 150 to 200 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, or 400). In some embodiments of any of the foregoing polynucleotides, the poly-A region, if present, includes 64 nucleosides. In some embodiments of any of the foregoing polynucleotides, the poly-A region, if present, includes a polyadenylation signal.

[0413] In some embodiments, the polypeptide of interest, encoded by the ORF in any of the foregoing polynucleotides, is expressed in a subject by administering the polynucleotide to the subject. In some embodiments, the polypeptide of interest, encoded by the ORF in any of the foregoing polynucleotides, is expressed in a subject by administering the polynucleotide to the subject prophylactically. In some embodiments, the prophylactic administration of the polynucleotide encoding the polypeptide of interest serves to treat the subject at risk of developing a disease. In some embodiments, the subject is a human. In some embodiments, the polypeptide of interest, encoded by the ORF in any of the foregoing polynucleotides, is expressed in a cell or population of cells, by administering the polynucleotide to the cell or population of cells.

[0414] In some embodiments, a pharmaceutical composition includes any of the foregoing polynucleotides and a pharmaceutically acceptable carrier. In some embodiments, the polypeptide of interest, encoded by the ORF in any of the foregoing polynucleotides, is expressed in a subject by administering the pharmaceutical composition to the subject. In some embodiments, the polypeptide of interest, encoded by the ORF in any of the foregoing polynucleotides, is expressed in a subject by administering the pharmaceutical composition to the subject prophylactically. In some embodiments, the prophylactic administration of the pharmaceutical composition serves to treat the subject at risk of developing a disease. In some embodiments, the subject is a human. In some embodiments, the polypeptide of interest, encoded by the ORF in any of the foregoing polynucleotides, is expressed in a cell or population of cells, by administering the pharmaceutical composition to the cell or population of cells.

[0415] In another aspect, the present invention provides a method of expressing a polypeptide of interest in a subject, wherein the method includes administering to the subject any one of the polynucleotides disclosed herein or the pharmaceutical compositions disclosed herein including any one of the polynucleotides disclosed herein. In some embodiments, the subject is a human.

[0416] In another aspect, the present invention provides a method of prophylactically treating a subject at risk of developing a disease, and the method includes administering to the subject the any one of the polynucleotides disclosed herein or the pharmaceutical compositions disclosed herein including any one of the polynucleotides disclosed herein, wherein the polypeptide of interest encoded by the ORF corresponds to an antigen associated with said disease. In some embodiments, the subject is a human.

[0417] In another aspect, the present invention provides a method of expressing a polypeptide of interest in a cell or population of cells, the method including providing to the cell or population of cells any one of the polynucleotides disclosed herein or the pharmaceutical compositions disclosed herein including any one of the polynucleotides disclosed herein.

[0418] In another aspect, the present invention provides a kit including any one of the polynucleotides disclosed herein or the pharmaceutical compositions disclosed herein including any one of the polynucleotides disclosed herein, wherein the package insert instructs a user of the kit to perform any one of the methods disclosed herein.

[0419] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 provides three graphs comparing the effect of modified 3’ regions on mRNA stability, in two separate cell lines, HEK293, Hep3B, and in human PBMCs (Peripheral Blood Mononuclear Cells). mRNA stability was measured for mRNA constructs containing an ORF encoding degron Green Fluorescent protein (degGFP) with three different modified 3’ regions. One construct contained a 100 nucleotide poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a twenty unmodified adenosines followed by an inverted deoxythymidine at the 3’ terminus (A20_idT) ligated to the 3’ terminus of the 5’-A100-UCUAG-3’ (A100-Xbal scar) sequence; and another construct contained twenty unmodified adenosines followed by a LNA-adenosine followed by an inverted deoxythymidine at the 3’ terminus (A20_LidT or LidT) ligated to the 3’ terminus of the 5’-A100-UCUAG-3’ (A100-Xbal scar) sequence. HEK293 and Hep3B cells were transfected using electroporation with 800 ng of degGFP mRNA, whereas hPBMCs were transfected with 4μg of degGFP mRNA. The three mRNA constructs in each cell line were assessed for stability at multiple time points: 2 hours, 6 hours, 8 hours, 24 hours, and 48 hours using RT- qPCR.

[0420] FIG.2A provides a graph comparing the in vivo expression levels of modified Luciferase (NpiLUC) reporter in groups of Balb / c mice over the course of 7 days. Each mouse group was injected with 0.5 mg / kg of mRNA contained an open reading frame (ORF) encoding for NpiLUC and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT (idT) ligated to the tail of the 100-residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a Azo-ALNA-idT (LidT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Balb / c mice, was included as well (Tris sucrose). FIG. 2A also includes a quantitative analysis at the 4-day mark, comparing luciferase activity in the A100, idT, and LidT mice groups. Luciferase activity is monitored via whole body imaging. Data represents mean ± SD, with statistical significance indicated by asterisks (*-p < 0.05, **-p < 0.01, ***-p < 0.001, ****-p < 0.0001).

[0421] FIG.2B provides a graph comparing the in vivo expression levels of human erythropoietin (hEpo) in groups of Balb / c mice over the course of 12 days. Each mouse group was injected with 0.5 mg / kg of mRNA contained an open reading frame (ORF) encoding for hEpo and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT (idT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a Azo-ALNA-idT (LidT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). FIG. 2B also includes a quantitative analysis at the 5-day mark, comparing hEPO levels within the serum, using enzyme-linked immunosorbent assay (ELISA), in the A100, idT, and LidT mice groups. Data represents mean ± SD, with statistical significance indicated by asterisks (*-p < 0.05, **-p < 0.01, ***-p < 0.001, ****-p < 0.0001).

[0422] FIG.2C provides two graphs comparing the in vivo expression levels of degron Green Lantern (degGLantern) expression levels in groups of 15 Balb / c mice over the course of 3 days. Two subgroups of cells were monitored via spleen flow cytometry, monocytes and dendritic cells (DCs). Each mouse group was injected with 0.5 mg / kg of mRNA contained an open reading frame (ORF) encoding for degGLantern and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT (idT) ligated to the tail of the 100-residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a Azo-ALNA-idT (LidT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Balb / c mice, was included as well (Tris sucrose). Data represents mean ± SD, with statistical significance indicated by asterisks (*-p < 0.05, **-p < 0.01, ***-p < 0.001, ****-p < 0.0001).

[0423] FIG.3A provides a graph comparing the in vivo expression levels of a protein of interest encoded by ORF1 in groups of Balb / c mice over the course of 4 days. The expression levels of the protein were monitored via a western blot of the spleens of mice injected with the mRNA. Each mouse group was dosed with 1 mg / kg of mRNA containing the ORF and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT (idT) ligated to the tail of the 100-residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a A20-ALNA-idT (LidT) ligated to the tail of the 100-residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Balb / c mice, was included as well (Tris sucrose). FIG. 3A also includes a quantitative analysis at the 4-day mark, comparing the ORF1 encoded protein levels within the spleen, using western blots, in the Tris sucrose, A100, idT, and LidT mice groups. Data represents mean ± SD, with statistical significance indicated by asterisks (*-p < 0.05, **-p < 0.01, ***-p < 0.001, ****-p < 0.0001).

[0424] FIG.3B provides a graph comparing the in vivo concentration levels of a substrate molecule, which is consumed by the protein of interest encoded by ORF2, in groups of Balb / c mice, over the course of 7 days. The concentration levels of the substrate were monitored via a mass spectrometry (Agilent 6470 LC-MS) of mice injected with the mRNA. Each mouse group was dosed with 1 mg / kg of mRNA containing the ORF and only differed in the structure of the modified 3’ region. One construct contained a Azo-idT (idT) ligated to the tail of the 100-residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a A20-ALNA-idT (LidT) ligated to the tail of the 100-residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Balb / c mice, was included as well (Tris sucrose). FIG. 3B also includes a quantitative analysis of the total normalized concentration of the substrate of ORF2 over the course of 7 days, for the tris sucrose, idT, and LidT mice groups. Data represents mean ± SD, with statistical significance indicated by asterisks (*-p < 0.05, **-p < 0.01, ***-p < 0.001, ****-p < 0.0001).

[0425] FIG.4A provides two graphs showing a comparison of protein expression levels and mRNA stability for HEK293 cell line. Protein expression and mRNA stability were measured using mRNA containing an ORF with degGFP (degron Green Fluorescent protein, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT (idT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-ALNA-idT (LidT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a AZO-ALNA-ALNA-ALNA (3XALNA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the HEK293 cells, was included as well (Cells). Fluorescence intensity was measured over 46 hours to assess protein expression levels associated with each of the modified 3’ regions (A100, idT, LidT, 3xLNA), IncuCyte® (Sartorius). mRNA stability was assessed by qPCR, post transfection, over the course of 72 hours. Data represents mean ± SD.

[0426] FIG.4B provides two graphs showing a comparison of protein expression levels and mRNA stability for Hep3B cell line. Protein expression and mRNA stability were measured using mRNA containing an ORF encoding degGFP (degron Green Fluorescent protein, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT (idT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-ALNA-idT (LidT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a AZO-ALNA- ALNA- ALNA (3XALNA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3B cells, was included as well (Cells). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 46 hours to assess protein expression levels associated with each of the modified 3’ regions (A100, idT, LidT, 3xALNA), IncuCyte® (Sartorius). mRNA stability was assessed by qPCR, post electroporation, over the course of 72 hours. Data represents mean ± SD.

[0427] FIG. 5 provides a graph comparing the in vivo expression levels of a ligated polypeptide structure of interest in groups of C57 / BL6 mice over the course of 22 days. Each mouse group was injected with 0.5 mg / kg of two mRNAs containing an open reading frame (ORF) encoding for one of the polypeptides of interest. Each set of mRNAs that encoded for the same ORF only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT (idT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-ALNA-idT (LidT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a AZO-ALNA-ALNA-ALNA (3XLNA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). Each of the strands of mRNAs provided to a mouse, each encoding one of the polypeptides of interest, contained the same modified 3’ region. The two polypeptides of interest were allowed to covalently bond and the concentrations of the resulting ligated polypeptide structure was measured using ELISA.

[0428] FIG.6 provides a graph comparing the in vivo expression levels of modified Luciferase (NpiLUC) reporter in groups of Balb / c mice over the course of 7 days. Each mouse group was injected with 0.5 mg / kg of mRNA contained an open reading frame (ORF) encoding for NpiLUO and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT (idT) ligated to the tail of the 100-residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A20-ALNA-idT (LidT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a AZO-ALNA-ALNA-ALNA (3XLN A) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Balb / c mice, was included as well (Tris sucrose). FIG. 6 also includes quantitative analyses at the 4-day and 7-day marks, comparing total luciferase activity and relative Luciferase activity, to the maximum value, in the A100, idT, LidT, and 3xLNA mice groups. Luciferase activity is monitored via whole body imaging. Data represents mean ± SD, with statistical significance indicated by asterisks (*-p < 0.05, **-p < 0.01, ***-p < 0.001, ****-p < 0.0001).

[0429] FIG. 7 provides two graphs comparing the in vivo expression levels of degron Green Lantern (degGLantern) expression levels in groups of 15 Balb / c mice over the course of 3 days. Two subgroups of cells were monitored via spleen flow cytometry, monocytes and dendritic cells. Each mouse group was injected with 0.5 mg / kg of mRNA contained an open reading frame (ORF) encoding for degGLantern and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT (idT) ligated to the tail of the 100-residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A20-ALNA-idT (LidT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a AZO-ALNA-ALNA-ALNA (3xLNA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Balb / c mice, was included as well (Tris sucrose). FIG. 7 also includes quantitative analyses at the 1-day mark, comparing percentage of cells expressing degron Green Lantern and mean fluorescence intensity (MFI) of degron Green Lantern, in the A100, idT, LidT, and 3xLNA mice groups for monocytes and dendritic cells. The data was acquired by flow cytometry of the monocytes and dendritic cells extracted from the spleens of the mice. Data represents mean ± SD, with statistical significance indicated by asterisks (*-p < 0.05, **-p < 0.01, ***-p < 0.001, ****-p < 0.0001).

[0430] FIG. 8 provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degGFP (degron Green Fluorescent protein, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-ALNA- Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-Ai_NA-Azo-Am-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-Af-Azo-ALNA-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a Azo-Am-Azo-Ai_NA-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3B cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 72 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0431] FIG. 9 provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL), in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-Ai_NA-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-2XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A20-5XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a AIS-5XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3B cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 72 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0432] FIG. 10 provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL), in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XTLNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-2XTLNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A20-5XTLNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-5XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XGLNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a AZO-5XGLNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3B cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 88 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0433] FIG. 11 provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL), in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2O-3XCLNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a A2o-3x(5-methyl-cytidine)LNA (A2O-3X(m5C)LNA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3B cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 78 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0434] FIG. 12 provides a graph comparing the protein expression levels of the provided mRNA sequences in HEK293 cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL), in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-(abasic)i_NA (Azo-(ab)LNA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-(abasic)i_NA-idT (Azo-(ab)LNA-idT) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-2x(abasic)i_NA (A2o-2x(ab)i_NA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a A2o-3x(abasic)i_NA (A2o-3x(ab)i_NA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the HEK293 cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 78 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0435] FIG. 13A-13C provides three graphs comparing the protein expression levels of the provided mRNA sequences in HEK293 cells, Hep3B cells, and monocytes. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-LidT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-3xAENA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a Azo-2xAENA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the HEK293 cells, HEP3B cells, and monocytes was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 78 hours for HEK293 cells (FIG. 13A) and HEP3B cells (FIG. 13B) and over 40 hours for monocytes (FIG. 13C) to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0436] FIG. 14A-14C provides three graphs comparing the protein expression levels of the provided mRNA sequences in HEK293 cells, Hep3B cells, and monocytes. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-LidT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-3xABNA-NC ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a Azo-2xABNA-NC ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the HEK293 cells, HEP3B cells, and monocytes was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 72 hours for HEK293 cells (FIG.

[0437] 14A), over 88 hours for and HEP3B cells (FIG. 14B), and over 40 hours for monocytes (FIG. 14C) to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0438] FIG. 15 provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-5XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a Azo-5xABNA-NC ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3b cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 72 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0439] FIG. 16 provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-3x(A)AcEt ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a Azo-2x(A)AcEt ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3b cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 88 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0440] FIG. 17 provides a graph comparing the mRNA stability of the provided mRNA sequences in HEK293 cells. mRNA stability was measured by qPCR using mRNA containing an ORF encoding degron Green Lantern (degGL) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a A20-3xAENA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). RNAs were delivered in the cell using electroporation and mRNA stability was measure over 96 hours for each of the mRNA containing the modified 3’ regions. Data represents mean ± SD.

[0441] FIG. 18 provides a graph comparing the mRNA stability of the provided mRNA sequences in HEK293 cells. mRNA stability was measured by qPCR using mRNA containing an ORF encoding degron Green Lantern (degGL) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a A20-2xABNA-NC ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). RNAs were delivered in the cell using electroporation and mRNA stability was measure over 96 hours for each of the mRNA containing the modified 3’ regions. Data represents mean ± SD.

[0442] FIG. 19 provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-LidT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-2xAi_NA-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-5XALNA-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a Ai5-5xAi_NA-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3b cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 72 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0443] FIG. 20 provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-LidT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-Ti_NA-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a Azo-Gi_NA-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3b cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 88 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0444] FIG. 21 A provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-LidT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-exA-LidT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a Azo-3x(exA)-LidT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3b cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 72 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0445] FIG. 21 B provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-LidT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Azo-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a AZO-6XA-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a A2o-3x(exA)-3xAi_NA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3b cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 72 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0446] FIG. 22 provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a Azo-LidT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2O-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-idT-Ai_NA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a A20-idT-3xAi_NA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3b cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 72 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0447] FIG. 23 provides a graph comparing the protein expression levels of the provided mRNA sequences in HEK293 cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a A2o-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2O-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-Ai_NA-2xA-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a A2o-2xAi_NA-A-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the HEK293 cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 88 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0448] FIG. 24 provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a A2o-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-LidT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’- UCUAG-3’); another construct contained a A2O-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A20-Ai_NA-(phosphoryl guanidine)-Ai_NA (A2O-ALNA-(PG)-ALNA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a A20-3x[(phosphoryl guanidine)-Ai_NA] (A2O-3X(PG-ALNA)) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3b cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 72 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0449] FIG. 25 provides a graph comparing the protein expression levels of the provided mRNA sequences in Hep3b cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a A2o-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-LidT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2O-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a Ai8-2x(L-adenosine)-3xAi_NA (A -2X(LA)-3XALNA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-(L-adenosine)-2xAi_NA (A2O-(LA)-2XALNA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a Ais-5x(L-adenosine)-3xAi_NA (AIS-5X(LA)-3XALNA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the Hep3b cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 72 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0450] FIG. 26 provides a graph comparing the protein expression levels of the provided mRNA sequences in HEK293 cells. Protein expression was measured using mRNA containing an ORF encoding degron Green Lantern (degGL, in order to facilitate fast degradation of the reporter protein) and only differed in the structure of the modified 3’ region. One construct contained a 100-residue long poly-A region followed by a terminal Xbal scar (5’-UCUAG-3’) sequence (A100); another construct contained a A2o-idT ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2O-3XALNA ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-3x(inverted-Ai_NA) (A2o-3xiAi_NA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A20-2x(inverted-Ai_NA) (A2o-2xiAi_NA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-(inverted-Ai_NA) (A2o-iAi_NA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-(inverted-TLNA) (A2o-iTi_NA) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-3x(inverted-ABNA-NC) (A2o-3xiABNA-NC) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-2x(inverted- ABNA-NC) (A2o-2xiABNA-NC) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); another construct contained a A2o-(inverted-ABNA-NC) (A2o-iABNA-NC) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’); and another construct contained a A2o-(inverted-TBNA-NC) (A2o-iTBNA-NC) ligated to the tail of the 100 residue poly-A region through an Xbal scar (5’-UCUAG-3’). A negative control, in which no RNA was provided to the HEK293 cells, was included as well (no RNA). RNAs were delivered in the cell using lipofectamine and Fluorescence intensity was measured over 88 hours to assess protein expression levels associated with each of the modified 3’ regions, IncuCyte® (Sartorius). Data represents mean ± SD.

[0451] DETAILED DESCRIPTION OF THE INVENTION

[0452] The present disclosure provides, inter alia, polynucleotides that exhibit improved therapeutic properties including, but not limited to, increased stability, increased expression, and / or a reduced innate immune response when introduced into a population of cells.

[0453] In particular, the inventors have identified that mRNA containing a modified 3’ region, terminating in specific sequences, may be particularly effective for use in therapeutic compositions. These modified regions may include, for example, one or more modification selected from (i) at least one modified sugar (e.g., at least one modified ribose), and / or (ii) at least one modified internucleoside linkage (e.g., at least one phosphorothioate), and / or (Hi) a modified terminal group (e.g., a modified phosphate or an inverted nucleoside). The modifications of the 3’ region may include any of the modifications mentioned in the sections that follow. These modifications may be installed into the nucleic acid molecules of the disclosure by any of a variety of methods described herein (e.g., co-transcriptionally or by way of ligation). The modification of the or 3’-end of the nucleic acid molecule (e.g., RNA) of the disclosure may have a beneficial impact on (i) the stability of the nucleic acid molecule, (ii) the immunogenicity of the nucleic acid molecule, and / or (Hi) extracellular and intracellular interactions of the nucleic acid molecule. These beneficial improvements may lead to an increased output of expressed protein.

[0454] Preferably, the alternative polynucleotides are substantially nontoxic and non mutagenic.

[0455] The compositions and methods described herein can be used, in vivo and in vitro, both extracellularly and intracellularly, as well as in assays such as cell free assays.

[0456] In another aspect, the present disclosure provides compositions including a polynucleotide as described herein. In some embodiments, the composition is a reaction mixture. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a cell culture.

[0457] It is further appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

[0458] 1. Modified 3’ Region

[0459] In some embodiments, the polynucleotides of the invention include a modified 3’ region including one or more nucleosides (e.g., 1 to 500 nucleosides such as 1 to 200, 1 to 400, 1 to 10, 5 to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50, 45 to 65, 50 to 70, 65 to 85, 70 to 90, 85 to 105, 90 to 110, 105 to 135, 120 to 150, 130 to 170, 150 to 200 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleosides). In some embodiments, the modified 3’ region contains one or more alternative nucleosides having an alternative nucleobase, sugar, or backbone (e.g., a 2’-deoxynucleoside, a 3’-deoxynucleoside, a 2’,3’-dideoxynucleoside, a 2’-O-methylnucleoside, a 3’-0-methylnucleoside, a 3’-0-ethyl-nucleoside, 3’-arabinoside, an L-nucleoside, alpha-thio-2’-0-methyl-adenosine, 2’-fluoro-adenosine, arabino-adenosine, hexitol-adenosine, LNA-adenosine (ALNA), LNA-thymidine (TLNA), LNA-guanosine (GLNA), LNA-cytidine (CLNA), LNA-(5-methyl-cytidine) ((m5C)LNA), LNA-uridine (ULNA) ENA-adenosine (AENA), ENA-thymidine (TENA), ENA-guanosine (GENA), ENA-cytidine (CENA), ENA-(5-methyl-cytidine) ((m5C)ENA), ENA-uridine (UENA), BNA-NC-adenosine (ABNA-NC), BNA-NC-thymidine (TBNA-NC), BNA-NC-guanosine (GBNA-NC), BNA-NC-cytidine (CBNA-NC), BNA-NC-(5-methyl-cytidine) ((m5C)BNA-NC), BNA-NC-uridine (UBNA-NC), constrained ethyl nucleic acid-adenosine ((A)AcEt), constrained ethyl nucleic acid-thymidine ((T)AcEt), constrained ethyl nucleic acid-guanosine ((G)AcEt), constrained ethyl nucleic acid-cytidine ((C)AcEt), constrained ethyl nucleic acid-(5-methyl-cytidine) ((m5C)AcEt), constrained ethyl nucleic acid-uridine ((U)AcEt), extended-adenosine (exA), extended-thymidine (exT), extended-guanosine (exG), extended-cytidine (exC), extended-(5-methyl-cytidine) (ex(m5C)), extended-uridine (exU), PNA-adenosine, inverted deoxythymidine, or 3’-azido-2’,3’-dideoxyadenosine). In some embodiments, the modified 3’ region includes a plurality of alternative nucleosides. In some embodiments, the modified 3’ region includes at least one non-nucleoside (e.g., an abasic ribose) at the 5’-terminus, the 3’-terminus, or at an internal position of the modified 3’ region.

[0460] In some embodiments, the 3’-stabilizing region consists of one nucleoside (e.g., a 2’-deoxynucleoside, a 3’-deoxynucleoside, a 2’,3’-dideoxynucleoside, a 2’-0-methylnucleoside, a 3’-O-methylnucleoside, a 3’-0-ethyl-nucleoside, 3’-arabinoside, an L-nucleoside, alpha-thio-2’-0-methyl-adenosine, 2’-fluoro-adenosine, arabino-adenosine, hexitol-adenosine, LNA-adenosine (ALNA), LNA-thymidine (TLNA), LNA-guanosine (GLNA), LNA-cytidine (CLNA), LNA-(5-methyl-cytidine) ((m5C)LNA), LNA-uridine (ULNA) ENA-adenosine (AENA), ENA-thymidine (TENA), ENA-guanosine (GENA), ENA-cytidine (CENA), ENA-(5-methyl-cytidine) ((m5C)ENA), ENA-uridine (UENA) BNA-NC-adenosine (ABNA-NC), BNA-NC-thymidine (TBNA-NC), BNA-NC-guanosine (GBNA-NC), BNA-NC-cytidine (CBNA-NC), BNA-NC-(5-methyl-cytidine) ((m5C)BNA-NC), BNA-NC-uridine (UBNA-NC), constrained ethyl nucleic acid-adenosine ((A)AcEt), constrained ethyl nucleic acid-thymidine ((T)AcEt), constrained ethyl nucleic acidguanosine ((G)AcEt), constrained ethyl nucleic acid-cytidine ((C)AcEt), constrained ethyl nucleic acid-(5-methyl-cytidine) ((m5C)AcEt), constrained ethyl nucleic acid-uridine ((U)AcEt), extended-adenosine (exA), extended-thymidine (exT), extended-guanosine (exG), extended-cytidine (exC), extended-(5-methyl-cytidine) (ex(m5C)), extended-uridine (exU), PNA-adenosine, inverted deoxythymidine, or 3’-azido-2’,3’-dideoxyadenosine).

[0461] In some embodiments, the modified 3’ region includes a plurality of adenosines. In some embodiments, all of the nucleosides of the modified 3’ region are adenosines. In some embodiments, the modified 3’ region includes at least one (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten) alternative nucleosides (e.g., an L-nucleoside such as L-adenosine, 2’-0-methyl-adenosine, alpha-thio-2’-0-methyl-adenosine, 2’-fluoro-adenosine, arabino-adenosine, hexitol-adenosine, LNA-adenosine (ALNA), LNA-thymidine (TLNA), LNA-guanosine (GLNA), LNA-cytidine (CLNA), LNA-(5-methyl-cytidine) ((m5C)LNA), LNA-uridine (ULNA) ENA- adenosine (AENA), ENA-thymidine (TENA), ENA-guanosine (GENA), ENA-cytidine (CENA), ENA-(5-methyl-cytidine) ((m5C)ENA), ENA-uridine (UENA) BNA-NC-adenosine (ABNA-NC), BNA-NC-thymidine (TBNA-NC), BNA-NC-guanosine (GBNA-NC), BNA-NC-cytidine (CBNA-NC), BNA-NC-(5-methyl-cytidine) ((m5C)BNA-NC), BNA-NC-uridine (UBNA-NC), constrained ethyl nucleic acid-adenosine ((A)AcEt), constrained ethyl nucleic acid-thymidine ((T)AcEt), constrained ethyl nucleic acid-guanosine ((G)AcEt), constrained ethyl nucleic acid-cytidine ((C)AcEt), constrained ethyl nucleic acid-(5-methyl-cytidine) ((m5C)AcEt), constrained ethyl nucleic acid-uridine ((U)AcEt), extended-adenosine (exA), extended-thymidine (exT), extended-guanosine (exG), extended-cytidine (exC), extended-(5-methyl-cytidine) (ex(m5C)), extended-uridine (exU), PNA-adenosine, or inverted deoxythymidine). In some embodiments, the alternative nucleoside is an L-adenosine, a 2’-0-methyl-adenosine, or an inverted deoxythymidine. In some embodiments, the modified 3’ region includes a plurality of alternative nucleosides. In some embodiments, all of the nucleotides in the modified 3’region are alternative nucleosides. In some embodiments, the modified 3’ region includes at least two different alternative nucleosides. In some embodiments, at least one alternative nucleoside is 2’-0-methyl-adenosine. In some embodiments, at least one alternative nucleoside is inverted deoxythymidine. In some embodiments, at least one alternative nucleoside is 2’-0-methyl-adenosine, and at least one alternative nucleoside is inverted deoxythymidine. In some embodiments, at least one alternative nucleoside is a (base)-LNA, wherein the base is a nucleobase. In some embodiments, the nucleobase is chosen from an adenine, a modified adenine, a thymine, a modified thymine, a cytosine, a modified cytosine, a uracil, a modified uracil, a guanine, or a modified guanine. In some embodiments, at least one alternative nucleoside is (adenosine)-LNA (ALNA). In some embodiments, at least one alternative nucleoside is (thymidine)-LNA (TLNA). In some embodiments, at least one alternative nucleoside is (guanosine)-LNA (GLNA). In some embodiments, at least one alternative nucleoside is (cytidine)-LNA (GLNA). In some embodiments, at least one alternative nucleoside is (5-methyl-cytidine)-LNA ((m5C)LNA). In some embodiments, at least one alternative nucleoside is (uridine)-LNA (ULNA). In some embodiments, at least one alternative nucleoside is (base)-ENA, wherein the base is a nucleobase. In some embodiments, the nucleobase is chosen from an adenine, a modified adenine, a thymine, a modified thymine, a cytosine, a modified cytosine, a uracil, a modified uracil, a guanine, or a modified guanine. In some embodiments, at least one alternative nucleoside is (adenosine)-ENA (AENA). In some embodiments, at least one alternative nucleoside is (thymidine)-ENA (TENA). In some embodiments, at least one alternative nucleoside is (guanosine)-ENA (GENA). In some embodiments, at least one alternative nucleoside is (cytidine)-ENA (CENA). In some embodiments, at least one alternative nucleoside is (5-methyl-cytidine)-ENA ((m5C)ENA). In some embodiments, at least one alternative nucleoside is (uridine)-ENA (UENA). In some embodiments, at least one alternative nucleoside is (base)-BNA-NC, wherein the base is a nucleobase. In some embodiments, the nucleobase is chosen from an adenine, a modified adenine, a thymine, a modified thymine, a uracil, a modified uracil, a cytosine, a modified cytosine, a guanine, or a modified guanine. In some embodiments, at least one alternative nucleoside is (adenosine)-BNA-NC (ABNA-NC). In some embodiments, at least one alternative nucleoside is (thymidine)-BNA-NC (TBNA-NC). In some embodiments, at least one alternative nucleoside is (guanosine)-BNA-NC (GBNA-NC). In some embodiments, at least one alternative nucleoside is (cytidine)-BNA-NC (CBNA-NC). In some embodiments, at least one alternative nucleoside is (5-methyl-cytidine)-BNA-NC ((m5C)BNA-NC). In some embodiments, at least one alternative nucleoside is (uridine)-BNA-NC (UBNA-NC). In some embodiments, at least one alternative nucleoside is extended-adenosine (exA). In some embodiments, at least one alternative nucleoside is extended-thymidine (exT). In some embodiments, at least one alternative nucleoside is extended-guanosine (exG). In some embodiments, at least one alternative nucleoside is extended-cytidine (exC). In some embodiments, at least one alternative nucleoside is extended-(5-methyl-cytidine) (ex(m5C)). In some embodiments, at least one alternative nucleoside is extended-uridine (exU). In some embodiments, at least one alternative nucleoside is constrained ethyl nucleic acid-(base) (AcEt-base). In some embodiments, at least one alternative nucleoside is AcEt-adenosine ((A)AcEt). In some embodiments, at least one alternative nucleoside is AcEt-thymidine ((T)AcEt). In some embodiments, at least one alternative nucleoside is AcEt-guanosine ((G)AcEt). In some embodiments, at least one alternative nucleoside is AcEt-cytidine ((C)AcEt). In some embodiments, at least one alternative nucleoside is AcEt-(5-methyl-cytidine) ((m5C)AcEt). In some embodiments, at least one alternative nucleoside is AcEt-uridine ((U)AcEt).

[0462] In some embodiments, the modified 3’ region includes poly-A regions. In some embodiments the poly-A regions are about 1 to about 200 (e.g., from 5 to 200, from 1 to 150, from 10 to 200, from 25 to 200, from 25 to 150, from 50 to 150, from 10 to 100, from 75 to 150, from 75 to 125, from 15 to 50, or from 15 to 25, or about 20) nucleosides in length. In some embodiments, the adenosines of the poly-A regions of the modified 3’ region are unmodified adenosines.

[0463] In some embodiments, the modified region includes the structure of Formula X:

[0464]

[0465] or a salt thereof;

[0466] wherein X is, O or S; and

[0467] A represents adenine and T represents thymine.

[0468] In some embodiments, all of the plurality of alternative nucleosides are the same (e.g., all of the alternative nucleosides are L-adenosine). In some embodiments, the modified 3’ region includes ten nucleosides. In some embodiments, the modified 3’ region includes eleven nucleosides. In some embodiments, the modified 3’ region comprises at least five L-adenosines (e.g., at least ten L-adenosines, or at least twenty L-adenosines). In some embodiments, the modified 3’ region consists of five L-adenosines. In some embodiments, the modified 3’ region consists of ten L-adenosines. In some embodiments, the modified 3’ region consists of twenty L-adenosines.

[0469] Further examples of modified 3’ regions are known in the art, e.g., as described in International Patent Publication Nos. WG2013 / 103659, WG2017 / 049275, and WG2017 / 049286, the modified 3’ regions of which are herein incorporated by reference.

[0470] In some embodiments, the 5’-terminus of the modified 3’ region is ligated to the 3’-terminus of the poly-A region. In some embodiments of any of the foregoing polynucleotides, the modified 3’ region includes the 3’-terminus of the polynucleotide. In some embodiments, the modified 3’ tail is ligated to the remainder of the polynucleotide, e.g., at the 3’-terminus of the 3’-UTR or poly-A region via a phosphate linkage. In some embodiments, the phosphate linkage is a natural phosphate linkage. In some embodiments, the conjugation of the modified 3’ tail and the remainder of the polynucleotide is produced via enzymatic ligation. In some embodiments, the enzymatic ligation of the modified 3’ tail and the remainder of the polynucleotide utilizes polyethylene glycol to assist with ligation. In some embodiments, the enzymatic ligation of the modified 3’ tail and the remainder of the polynucleotide utilizes a DNA strand that is complementary to the two RNA strands to be ligated, to enhance reaction kinetics.

[0471] In preferred embodiments, the modified 3’ region is ligated to the poly-A region via a polynucleotide linker of the structure of Formula III:

[0472]

[0473] Formula III

[0474] wherein A’ is the poly-A region of the any of the aforementioned polynucleotides;

[0475] B’ is the modified 3’ region;

[0476] each X is, independently, O or S;

[0477] n is from 0 to 50 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50); and

[0478] D is a nucleoside independently chosen from an adenosine, a modified adenosine, a thymidine, a modified thymidine, a cytidine, a modified cytidine, a uridine, a modified uridine, a guanosine, or a modified guanosine (see section 2. Chemically modified Nucleic Acids for exemplary nucleosides).

[0479] In preferred embodiments, the polynucleotide linker has the sequence 5’-UCUAG-3’ also known as a Xbal scar, which is the remnant of the of the restriction site cleaved by the restriction endonuclease Xbal.

[0480] In some embodiments the linker is a polynucleotide with the sequence 5’-(Xbal scar)-(A)2o-Af-3’: wherein Af represents a 2’-fluoro-adenosine; and

[0481] Xbal scar represents (5’-UCUAG-3’).

[0482] In some embodiments the linker is a polynucleotide with the sequence 5’-(Xbal scar)-(A)2o-Am-3’: wherein Am represents a 2’-0-methoxy-adenosine; and

[0483] Xbal scar represents (5’-UCUAG-3’).

[0484] Another example of a linker includes a phosphodiester or phosphorothioate bond.

[0485] In preferred embodiments, the modified 3’ region has the structure of Formula II:

[0486] (A’) - (X1 - B)a - (X2 - C)b - (X3 - D)c - (X4 - E)d - (X5 - Z)e

[0487] Formula II

[0488] wherein:

[0489] A’ is a poly-A region;

[0490] B is a 2’-modified nucleoside or a bridged nucleic acid (BNA) (e.g., LNA);

[0491] C is a poly-A region;

[0492] D is a BNA (e.g., LNA); E is a BNA (e.g., LNA);

[0493] Z is an inverted nucleoside;

[0494] wherein each of xi, X2, xs, X4, and xs is, independently, an internucleoside linkage (e.g., phosphodiester linkage or phosphorothioate linkage); and

[0495] wherein each of a, b, c, d, and e is, independently, 0 or 1.

[0496] In preferred embodiments, each of xi, X2, X3, X4, and xs is, independently, if present, a phosphodiester linkage. In preferred embodiments, the BNA of B, D, and E are each independently, if present, LNA-adenosine. In preferred embodiments, the 2’-modified nucleoside of B, if present, is 2’-O-methyl-adenosine, or 2’-fluoro-adenosine. In preferred embodiments, the inverted nucleoside of Z, if present, is an inverted deoxythymidine. In preferred embodiments, the poly-A regions of A’ and C are each independently, if present, 20 unmodified adenosines.

[0497] In preferred embodiments, the modified 3’ region has the sequence, A20_ALNA_ALNA_ALNA, twenty unmodified adenosines followed by three LNA-adenosines at the 3’ terminus (3xLNA). In preferred embodiments, the A20_ALNA_ALNA_ALNA sequence can be described by Formula V:

[0498]

[0499] Formula V.

[0500] In preferred embodiments, the A20_ALNA_ALNA_ALNA sequence can also be described by Formula VIII:

[0501] -(A)20-O-ALNA-O-ALNA-O-ALNA

[0502] Formula VIII.

[0503] In preferred embodiments, the modified 3’ region has the sequence, A20_Ai_NA_idT, twenty unmodified adenosines followed by a LNA-adenosines, followed by an inverted deoxythymidine at the 3’ terminus (LidT). In preferred embodiments, the A20_Ai_NA_idT sequence can be described by Formula VI:

[0504]

[0505] Formula VI.

[0506] In preferred embodiments, the A20_Ai_NA_idT sequence can also be described by Formula IX:

[0507] -(A)20-o-Ai_NA-o-idT

[0508] Formula IX.

[0509] In preferred embodiments, the modified 3’ region has the sequence, A20_Am_A20_Ai_NA_idT, twenty unmodified adenosines followed by a 2’-0-methoxy-adenosine followed by twenty unmodified adenosines, followed by LNA-adenosine, followed by inverted deoxythymidine at the 3’ terminus.

[0510] In preferred embodiments, the modified 3’ region has the sequence, A20_Af_A20_Ai_NA_idT, twenty unmodified adenosines followed by a 2’-fluoro-adenosine followed by twenty unmodified adenosines, followed by LNA-adenosine, followed by inverted deoxythymidine at the 3’ terminus.

[0511] In preferred embodiments, the modified 3’ region has the structure of Formula XII:

[0512] (A’) - (X1 - B) - (X2 - C)a - (X3 - D)b - (X4 - E)c - (X5 - F)d - (X6 - Z)e

[0513] Formula XII

[0514] wherein:

[0515] A’ is a poly-A region comprising 10 to 100 nucleosides;

[0516] B is a bridged nucleic acid (BNA);

[0517] C is a BNA;

[0518] D is a BNA;

[0519] E is a BNA;

[0520] F is a BNA;

[0521] Z is an inverted nucleoside;

[0522] wherein each of xi, X2, X3, X4, xs, and xe is, independently, an internucleoside linkage (e.g., phosphodiester linkage or phosphorothioate linkage); and wherein each of a, b, c, d, and e is, independently, 0 or 1;

[0523] wherein:

[0524] each BNA, independently, has the structure of Formula XI:

[0525]

[0526] Formula XI

[0527] wherein:

[0528] each R1is, independently, -C(R2)2-, -C(O)-, -O-, -S-, or -NR3-;

[0529] each R2is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, 04-Cs aryl, or C3-C7 heteroaryl;

[0530] each R3is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, 04-Cs aryl, or C3-C7 heteroaryl;

[0531] n is 1 or 2;

[0532] X is one of -C(R4)2-, -C(O)-, -0-, -S-, or -NR5-;

[0533] each R4is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-0s aryl, or C3-O7 heteroaryl

[0534] each R5is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-0s aryl, or C3-O7 heteroaryl; and the base is a nucleobase.

[0535] In preferred embodiments, each of x1, x2, x3, x4, x5, and x6is, independently, if present, a phosphodiester linkage. In preferred embodiments, the BNA of B, C, D, E, or F are each, independently, if present, chosen from LNA-adenosine, LNA-thymidine, ENA-adenosine, and BNA-NC-adenosine. In preferred embodiments, the BNA of B, C, D, E, or F are each, independently, if present, LNA-adenosine. In preferred embodiments, the BNA of B, C, D, E, or F are each, independently, if present, LNA-thymidine. In preferred embodiments, the BNA of B, C, D, E, or F are each, independently, if present, LNA-cytidine. In preferred embodiments, the BNA of B, C, D, E, or F are each, independently, if present, LNA-(5-methyl-cytidine). In preferred embodiments, the BNA of B, C, D, E, or F are each, independently, if present, ENA-adenosine. In preferred embodiments, the BNA of B, C, D, E, or F are each, independently, if present, BNA-NC-adenosine. In preferred embodiments, the inverted nucleoside of Z, if present, is an inverted deoxythymidine. In preferred embodiments, the poly-A region of A’ is 20 unmodified adenosines.

[0536] In preferred embodiments, the modified 3’ region has the sequence, A20_ALNA_ALNA, twenty unmodified adenosines followed by two LNA-adenosines at the 3’ terminus (A2O-2XALNA). In preferred embodiments, the A2O-2XALNA sequence can be described by Formula XIV:

[0537]

[0538] Formula XIV.

[0539] In preferred embodiments, the modified 3’ region has the sequence, A20_AENA_AENA, twenty unmodified adenosines followed by two ENA-adenosines at the 3’ terminus (Azo-2xAENA). In preferred embodiments, the Azo_2xAENA sequence can be described by Formula XV:

[0540]

[0541] Formula XV.

[0542] In preferred embodiments, the modified 3’ region has the sequence, A20_AENA_AENA_AENA, twenty unmodified adenosines followed by three ENA-adenosines at the 3’ terminus (Azo-3xAENA). In preferred embodiments, the Azo-3xAENA sequence can be described by Formula XVI:

[0543]

[0544] Formula XVI.

[0545] In preferred embodiments, the modified 3’ region has the sequence, A20_ABNA-NC_ABNA-NC, twenty unmodified adenosines followed by two BNA-NC-adenosines at the 3’ terminus (A2o-2xABNA-NC). In preferred embodiments, the A2o-2xABNA-NC sequence can be described by Formula XVII:

[0546]

[0547] Formula XVII.

[0548] In preferred embodiments, the modified 3’ region has the sequence, A20_ABNA-NC_ABNA-NC_ABNA-NC, twenty unmodified adenosines followed by three BNA-NC-adenosines at the 3’ terminus (A2O-3XABNA-NC). In preferred embodiments, the A2o-3xABNA-NC sequence can be described by Formula XVIII:

[0549]

[0550] Formula XVIII.

[0551] In preferred embodiments, the modified 3’ region has the sequence, A20_TLNA_TLNA_TLNA, twenty unmodified adenosines followed by three LNA-thymidine at the 3’ terminus (A2O-3XTLNA). In preferred embodiments, the A2O-3XTLNA sequence can be described by Formula XIX:

[0552]

[0553] Formula XIX.

[0554] In preferred embodiments, the modified 3’ region has the sequence, A20_ALNA_Ai_NA_idT, twenty unmodified adenosines followed by two LNA-adenosines and an idT at the 3’ terminus (A2o-2xAi_NA-idT). In preferred embodiments, the A2o-2xAi_NA-idT sequence can be described by Formula XX:

[0555]

[0556] Formula XX.

[0557] In preferred embodiments, the modified 3’ region has the sequence, A20_Ti_NA_idT, twenty unmodified adenosines followed by a LNA-thymidine and an idT at the 3’ terminus (Azo-TLNA-idT). In preferred embodiments, the Azo-TLNA-idT sequence can be described by Formula XXI:

[0558]

[0559] Formula XXL

[0560] In preferred embodiments, the modified 3’ region has the sequence, A20_CLNA_CLNA _CLNA, twenty unmodified adenosines followed by three LNA-cytidines at the 3’ terminus (A20-3XCLNA). In preferred embodiments, the A2O-3XCLNA sequence can be described by Formula XXXI:

[0561]

[0562] Formula XXXI.

[0563] In preferred embodiments, the modified 3’ region has the sequence, A20_(m5C)LNA_(m5C)i_NA _(m5C)LNA, twenty unmodified adenosines followed by a LNA-(5-methyl-cytidine) at the 3’ terminus (A20-3X(m5C)LNA). In preferred embodiments, the A2O-3X(m5C)LNA sequence can be described by Formula XXXII:

[0564]

[0565] Formula XXXII.

[0566] In preferred embodiments, the modified 3’ region has the structure of Formula XXII:

[0567] (A’) - (X1 - B) - (X2 - C)a - (X3 - D)b - (X4 - E)c - (X5 - F)d - (X6 - G)e - (X7 - Z)f Formula XXII

[0568] wherein:

[0569] A’ is a poly-A region comprising 10 to 100 nucleosides;

[0570] B is an extended nucleic acid;

[0571] C is an extended nucleic acid;

[0572] D is an extended nucleic acid;

[0573] E is a LNA;

[0574] F is a LNA;

[0575] G is a LNA;

[0576] Z is an inverted nucleoside;

[0577] wherein each of xi, X2, X3, X4, xs, xe, and x? is a internucleoside linkage (e.g., phosphodiester linkage or phosphorothioate linkage); and

[0578] wherein each of a, b, c, d, e, and f is, independently, 0 or 1;

[0579] wherein:

[0580] each extended nucleic acid (ex(base)), independently, has the structure of Formula XXIII:

[0581]

[0582] Formula XXIII,

[0583] wherein the base is a nucleobase.

[0584] In preferred embodiments, each of xi, X2, X3, X4, xs, xe, and x? is, independently, if present, a phosphodiester linkage. In preferred embodiments, the extended nucleic acid of B, C, an D are each, independently, if present, extended-adenosine (exA). In preferred embodiments, the LNA of E, F, or G are each, independently, if present, LNA-adenosine. In preferred embodiments, the inverted nucleoside of Z, if present, is an inverted deoxythymidine. In preferred embodiments, the poly-A region of A’ is 20 unmodified adenosines.

[0585] In preferred embodiments, the modified 3’ region has the sequence, A20_exA_Ai_NA_idT, twenty unmodified adenosines followed by an extended-adenosine, a LNA-adenosines, and an idT at the 3’ terminus (A2o-exA-Ai_NA-idT). In preferred embodiments, the A2o-exA-Ai_NA-idT sequence can be described by Formula XXIV:

[0586]

[0587] Formula XXIV.

[0588] In preferred embodiments, the modified 3’ region has the sequence, A20_exA_exA_exA_Ai_NA_idT, twenty unmodified adenosines followed by three extended-adenosine, a LNA-adenosines, and an idT at the 3’ terminus (A2o-3x(exA)-Ai_NA-idT). In preferred embodiments, the A20-3x(exA)-Ai_NA-idT sequence can be described by Formula XXV:

[0589]

[0590] Formula XXV.

[0591] In preferred embodiments, the modified 3’ region has the sequence, A20_exA_exA_exA_Ai_NA_ALNA_Ai_NA, twenty unmodified adenosines followed by three extended-adenosine and three LNA-adenosines at the 3’ terminus (A2O-3X(6XA)-3XALNA). In preferred embodiments, the A2o-3x(exA)-3xAi_NA sequence can be described by Formula XXVI:

[0592]

[0593] Formula XXVI.

[0594] In preferred embodiments, the modified 3’ region is ligated to the 3’-end of a poly-A region presenting the sequence, 5’-UCUAG-3’.

[0595] In some embodiments, the modified 3’ region is also a 3’-stabilizing region. In some embodiments, the modified 3’ region is a 3’-stabi lizing region due to the increased half-life of the polynucleotide.

[0596] 2. Chemically Modified Nucleic Acids

[0597] The modified 3’ region elements of the disclosure, as well as the open reading frame, UTR, modified 5’-cap, and other elements of the nucleic acid constructs described herein, may have one or more chemical modifications. According to Aduri et al., (Aduri, R. et al., AMBER force field parameters for the naturally occurring modified nucleosides in RNA. Journal of Chemical Theory and Computation. 2006.

[0598] 3(4):1464-75), there are 107 naturally occurring nucleosides, including 1 -methyladenosine, 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine, 2-methyladenosine, 2-O-ribosylphosphate adenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-acetyladenosine, N6-glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, N6, N6-dimethyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6-hydroxynorvalylcarbamoyladenosine, 1,2-0-dimethyladenosine, N6,2-0-dimethyladenosine, 2-O-methyladenosine, N6, N6,0-2-trimethyladenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6-isopentenyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, 2-thiocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, lysidine, N4-acetyl-2-O-methylcytidine, 5-formyl-2-0-methylcytidine, 5,2-O-dimethylcytidine, 2-0-methylcytidine, N4,2-O-dimethylcytidine, N4, N4,2-O-trimethylcytidine, 1 -methylguanosine, N2,7-dimethylguanosine, N2-methylguanosine, 2-O-ribosylphosphate guanosine, 7-methylguanosine, under modified hydroxywybutosine, 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, N2, N2-dimethylguanosine, 4-demethylwyosine, epoxyqueuosine, hydroxywybutosine, isowyosine, N2, 7,2-0-trimethylguanosine, N2,2-0-dimethylguanosine, 1,2-0-dimethylguanosine, 2-0-methylguanosine, N2, N2,2-0-trimethylguanosine, N2, N2,7-trimethylguanosine, peroxywybutosine, galactosyl-queuosine, mannosyl-queuosine, queuosine, archaeosine, wybutosine, methylwyosine, wyosine, 2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 3-methyluridine, 4-thiouridine, 5-methyl-2-thiouridine, 5-methylaminomethyluridine, 5-carboxymethyluridine, 5-carboxymethylaminomethyluridine, 5-hydroxyuridine, 5-methyluridine, 5-taurinomethyluridine, 5-carbamoylmethyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, dihydrouridine, 5-methyldihydrouridine, 5-methylaminomethyl-2-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, 3,2-O-dimethyluridine, 5-carboxymethylaminomethyl-2-0-methyluridine, 5-carbamoylmethyl-2-0-methyluridine, 5-methoxycarbonylmethyl-2-0-methyluridine, 5-(isopentenylaminomethyl)-2-0-methyluridine, 5,2-O-dimethyluridine, 2-0-methyluridine, 2-thio-2-0-methyluridine, uridine 5-oxyacetic acid, 5-methoxycarbonylmethyluridine, uridine 5-oxyacetic acid methyl ester, 5-methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-taurinomethyl-2-thiouridine, pseudouridine, 1 -methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 1 -methylpseudouridine, 3-methylpseudouridine, 2-0-methylpseudouridine, inosine, 1 -methylinosine, 1,2-0-dimethylinosine, and 2-O-methylinosine. Each of these may be components of nucleic acids of the present invention.

[0599] a. Nucleosides containing modified sugars

[0600] The alternative nucleosides and nucleotides (e.g., building block molecules), which may be incorporated into a polynucleotide e.g., RNA or mRNA, as described herein, including in modified 3’ regions), can be altered on the sugar of the ribonucleic acid. For example, the 2’ hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2’-position include, but are not limited to, H, halo, optionally substituted C1-6 alkyl; optionally substituted C1-6 alkoxy; optionally substituted Ce-w aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted Ce-io aryloxy; optionally substituted Ce-io aryl-Ci-6 alkoxy, optionally substituted C1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), -©(CHzCHzOJnCHzCHzOR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA) in which the 2’-hydroxyl is connected by a C1-6 alkylene or C1-6 heteroalkylene bridge to the 4’-carbon of the same ribose sugar, where exemplary bridges included methylene, propylene, ether, or amino bridges; aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino as defined herein; and amino acid, as defined herein

[0601] Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with a-L-threofuranosyl-(3’->2’)), and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone). The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar.

[0602] In some embodiments, the sugars of the alternative nucleosides and nucleotides are modified in a way to create bridge nucleic acids (BNA), which are modified RNA nucleosides that contain bridging groups, typically, but not limited to, between the 2’ and 4’ position of the ribose sugar backbone, which confer a fixed Cs-endo sugar puckering conformation. The bridging groups contain at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) member. Each member of the bridging group may be -CR12-, -C(O)-, -O-, -S-, or -NR2-, wherein each R1is, independently, H, alkyl, heteroalkyl, aryl or heteroaryl, and each R2is, independently, H, alkyl, heteroalkyl, aryl or heteroaryl. In some embodiments, the BNA contains an adenine, a modified adenine, a cytosine, a modified cytosine, a uracil, a modified uracil, a guanine, a modified guanine, a thymine, or a modified thymine. In some embodiments the BNA is a LNA, which is a RNA nucleoside with an extra methylene (CH2) group forming bridging bonds between the 2’ oxygen and 4’ carbon of the ribose moiety, locking the ribose in the 3’-endo conformation. In some embodiments, the BNA is a ENA which is an RNA nucleoside with two extra methylene (CH2) groups forming a bridging bond between the 2’ oxygen and 4’ carbon of the ribose moiety, locking the ribose in the 3’-endo conformation. In some embodiments, the BNA is a BNA-NC, which is a RNA nucleoside with a (-CH2-N(CH3)-) bridging bond between the 2’ oxygen and 4’ carbon of the ribose moiety, locking the ribose in the 3’-endo conformation, wherein the nitrogen is attached to the 2’ oxygen. In some embodiments, the BNA is a AcEt, which is a RNA nucleoside with a (-CH(CH3)-) bridging bond between the 2’ oxygen and 4’ carbon of the ribose moiety, locking the ribose in the 3’-endo conformation. b. Alterations on the nucleobase

[0603] The present disclosure provides for modified nucleosides and nucleotides, which may be used, for example, in conjunction with any of the modified 3’ regions described herein. As described herein, “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). As described herein, “nucleotide” is defined as a nucleoside including a phosphate group.

[0604] Exemplary non-limiting alterations include an amino group, a thiol group, an alkyl group, a halo group, or any described herein. The modified nucleotides may be synthesized by any useful method, as described herein e.g., chemically, enzymatically, or recombinantly to include one or more alternative or alternative nucleosides).

[0605] In some embodiments, a nucleic acid of the invention e.g., an mRNA or an oligonucleotide) includes one or more 2’-0Me nucleotides, 2’-0-methoxyethyl nucleotides (2’-MOE nucleotides), 2’-F nucleotide, 2’-NH2 nucleotide, 2’fluoroarabino nucleotides (FANA nucleotides), locked nucleic acid nucleotides (LNA nucleotides), or 4’-S nucleotides.

[0606] The modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, and guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or alternative nucleotides including non-standard or alternative bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the alternative nucleotide inosine and adenine, cytosine, or uracil.

[0607] The modified nucleosides and nucleotides can include an modified nucleobase. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil.

[0608] Examples of nucleobase found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. These nucleobases can be altered or wholly replaced to provide polynucleotide molecules having enhanced properties (e.g., resistance to nucleases and stability), and these properties may manifest through disruption of the binding of a major groove binding partner.

[0609] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (i ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1 -carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (TITI5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), 1- taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1qj), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4qj), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3qj), 2-thio-1-methyl-pseudouridine, 1 -methyl-1 -deaza-pseudouridine, 2-thio-1 -methyl-1 -deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3qj), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2’-O-methyl-uridine (Um), 5,2’-O-dimethyl-uridine (m5Um), 2’-0-methyl-pseudouridine (qjm), 2-thio-2’-0-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2’-0-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2’-0-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2’-0-methyl-uridine (cmnm5Um), 3,2’-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2’-0-methyl-uridine (inm5Um), 1 -thio-uridine, thymidine, 2’-F-ara-uridine, 2’-F-uridine, 2’-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine.

[0610] In preferred embodiments, the nucleic acid is modified to contain 1 -methylpseudouridine (m1qj) in lieu of uridine at each instance.

[0611] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-th io- 1 -methyl-1 -deaza-pseudoisocytidine, 1 -methyl-1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1 -methyl-pseudoisocytidine, lysidine (kzC), a-thio-cytidine, 2’-O-methyl-cytidine (Cm), 5,2’-O-dimethyl-cytidine (m5Cm), N4-acetyl-2’-O-methyl-cytidine (ac4Cm), N4,2’-O-dimethyl-cytidine (m4Cm), 5-formyl-2’-0-methyl-cytidine (f5Cm), N4, N4,2’-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2’-F-ara-cytidine, 2’-F-cytidine, and 2’-OH-ara-cytidine.

[0612] In some embodiments, the alternative nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6, N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2- methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2’-0-methyl-adenosine (Am), N6,2’-O-dimethyl-adenosine (m6Am), N6, N6,2’-0-trimethyl-adenosine (m62Am), 1,2’-0-dimethyl-adenosine (m’Am), 2’-0-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido-adenosine, 2’-F-ara-adenosine, 2’-F-adenosine (At), 2’-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0613] In some embodiments, the alternative nucleobase is an alternative guanine. Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1-methyl-inosine (m1l), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (ozyW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza- 8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2, N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m27G), N2, N2,7-dimethyl-guanosine (m22'7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2, N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2’-0-methyl-guanosine (Gm), N2-methyl-2’-0-methyl-guanosine (m2Gm), N2, N2-dimethyl-2’-0-methyl-guanosine (m22Gm), 1-methyl-2’-0-methyl-guanosine (m1Gm), N2,7-dimethyl-2’-0-methyl-guanosine (m27Gm), 2’-0-methyl-inosine (Im), 1,2’-0-dimethyl-inosine (m1lm), 2’-O-ribosylguanosine (phosphate) (Gr(p)), 1 -thio-guanosine, 06-methyl-guanosine, 2’-F-ara-guanosine, and 2’-F-guanosine.

[0614] The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine, or pyrimidine analog. For example, the nucleobase can each be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In some embodiments, the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides are depicted using the shorthand A, G, C, T or U, each letter refers to the representative base and / or derivatives thereof (e.g., A includes adenine or adenine analogs (e.g., 7-deaza adenine)).

[0615] In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-methyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-trifluoromethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uracil, uracil, 5-hydroxymethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-bromo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-iodo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-methoxy-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-ethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-phenyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-ethnyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, N4-methyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-fluoro-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, N4-acetyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, pseudoisocytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-formyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-aminoallyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-carboxy-cytosine, and cytosine as the only uracils and cytosines.

[0616] In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-methyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-trifluoromethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-hydroxymethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-bromo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-iodo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-methoxy-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-ethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-phenyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-ethnyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, N4-methyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1 -methyl-pseudouracil, uracil, 5-fluoro-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, N4-acetyl- cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, pseudoisocytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-formyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-aminoallyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1 -methyl-pseudouracil, uracil, 5-carboxy-cytosine, and cytosine as the only uracils and cytosines.

[0617] In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-trifluoromethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-hydroxymethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-bromo-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-iodo-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-methoxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-ethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-phenyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-ethnyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, N4-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-fluoro-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, N4-acetyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, pseudoisocytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-formyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-am inoallyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uridine, uridine, 5-carboxy-cytidine, and cytidine as the only uridines and cytidines.

[0618] In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-trifluoromethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-hydroxymethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-bromo-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-iodo-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-methoxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-ethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-phenyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-eth nyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, N4-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1 -methyl-pseudouridine, uridine, 5-fluoro-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, N4-acetyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, pseudoisocytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-formyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-aminoallyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-carboxy-cytidine, and cytidine as the only uridines and cytidines.

[0619] In some embodiments, the polynucleotides of the invention contain the uracil of one of the nucleosides of Table 1 and uracil as the only uracils. In other embodiments, the polynucleotides of the invention contain a uridine of Table 1 and uridine as the only uridines.

[0620] Table 1. Exemplary modified uridine nucleosides

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628] In some embodiments, the polynucleotides of the invention contain the cytosine of one of the nucleosides of Table 2 and cytosine as the only cytosines. In other embodiments, the polynucleotides of the invention contain a cytidine of Table 2 and cytidine as the only cytidines.

[0629] Table 2. Exemplary modified cytidine nucleosides

[0630]

[0631]

[0632] c. Alterations on the internucleoside linkage

[0633] The alternative nucleotides, which may be incorporated into a polynucleotide molecule, can be altered on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be altered by replacing one or more of the oxygen atoms with a different substituent.

[0634] The alternative nucleosides and nucleotides can include the wholesale replacement of an unaltered phosphate moiety with another internucleoside linkage as described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be altered by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0635] The alternative nucleosides and nucleotides can include the replacement of one or more of the non-bridging oxygens with a borane moiety (BH3), sulfur (thio), methyl, ethyl and / or methoxy. As a nonlimiting example, two non-bridging oxygens at the same position (e.g., the alpha (a), beta (p) or gamma (y) position) can be replaced with a sulfur (thio) and a methoxy.

[0636] The replacement of one or more of the oxygen atoms at the a position of the phosphate moiety (e.g., a-thio phosphate) is provided to confer stability (such as against exonucleases and endonucleases) to RNA and DNA through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. While not wishing to be bound by theory, phosphorothioate linked polynucleotide molecules are expected to also reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.

[0637] In specific embodiments, an alternative nucleoside includes an alpha-thio-nucleoside (e.g., 5’-O-(1 -thiophosphate)-adenosine, 5’-O-(1 -thiophosphate)-cytidine (a-th io-cytid ine), 5’-O-(1 -thiophosphate)-guanosine, 5’-O-(1 -thiophosphate)-uridine, or 5’-O-(1 -thiophosphate)-pseudouridine).

[0638] In some embodiments, the internucleoside linkage is a phosphoryl guanidine (PG) linkage, having the structure of Formula XXXIII:

[0639]

[0640] Formula XXXIII.

[0641] Other internucleoside linkages that may be employed according to the present invention, including internucleoside linkages which do not contain a phosphorous atom, are described herein below.

[0642] d. Combinations of alternative sugars, nucleobases, and internucleoside linkages

[0643] The polynucleotides of the invention can include a combination of alterations to the sugar, the nucleobase, and / or the internucleoside linkage. These combinations can include any one or more alterations described herein.

[0644] 3. Poly-A Regions

[0645] In some embodiments, the polynucleotides of the present disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding a therapeutic polypeptide) further comprise a poly-A region. In some embodiments, terminal groups on the poly-A region can be incorporated for stabilization. In some embodiments, a poly-A region comprises des-3’ hydroxyl tails. In some embodiments, a terminal (e.g., 3’ terminal) poly-A region is also known as a poly-A tail.

[0646] During RNA processing, a long chain of adenine nucleotides (poly-A tail) can be added to a polynucleotide (e.g., an mRNA molecule) in order to increase stability. Immediately after transcription, the 3’ end of the transcript can be cleaved to free a 3’ hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long. In some embodiments, the poly-A tail is 100 nucleotides in length.

[0647] Poly-A tails can also be added after the construct is exported from the nucleus.

[0648] According to the present disclosure, terminal groups on the poly-A region can be incorporated for stabilization. Polynucleotides of the present disclosure can include des-3’ hydroxyl tails. They can also include structural moieties or 2’-O-methyl modifications as taught by Junjie Li, et al. (Current Biology, vol.

[0649] 15, 1501-1507, August 23, 2005), the contents of which are incorporated herein by reference in its entirety). The polynucleotides of the present disclosure can be designed to encode transcripts with alternative poly-A region structures including histone mRNA. According to Norbury, “Terminal uridylation has also been detected on human replication-dependent histone mRNAs. The turnover of these mRNAs is thought to be important for the prevention of potentially toxic histone accumulation following the completion or inhibition of chromosomal DNA replication. These mRNAs are distinguished by their lack of a 3' poly(A) tail, the function of which is instead assumed by a stable stem-loop structure and its cognate stem-loop binding protein (SLBP); the latter carries out the same functions as those of PABP on polyadenylated mRNAs” (Norbury, “Cytoplasmic RNA: a case of the tail wagging the dog,” Nature Reviews Molecular Cell Biology; AOP, published online 29 August 2013; doi:10.1038 / nrm3645), the contents of which are incorporated herein by reference in its entirety.

[0650] Unique poly-A region lengths provide certain advantages to the polynucleotides of the present disclosure. Generally, the length of a poly-A region, when present, is greater than 30 nucleotides in length. In some embodiments, the poly-A region is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides).

[0651] In some embodiments, the polynucleotide or region thereof includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

[0652] In some embodiments, the poly-A region is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design can be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate product expressed from the polynucleotides.

[0653] In this context, the poly-A region can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof. The poly-A region can also be designed as a fraction of the polynucleotides to which it belongs. In this context, the poly-A region can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A region. Further, engineered binding sites and conjugation of polynucleotides for Poly-A binding protein can enhance expression.

[0654] Additionally, multiple distinct polynucleotides can be linked together via the PABP (Poly-A binding protein) through the 3’-end using modified nucleotides at the 3’-terminus of the poly-A region. Transfection experiments can be conducted in relevant cell lines and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72hr and day 7 post-transfection.

[0655] In some embodiments, the polynucleotides of the present disclosure are designed to include a poly-A-G Quartet region. The G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In some embodiments, the G-quartet is incorporated at the end of the poly-A region. The resultant polynucleotide is assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the poly-A-G quartet results in protein production from an mRNA equivalent to at least 75% of that seen using a poly-A region of 120 nucleotides alone.

[0656] In some embodiments, the poly-A region comprises an alternative nucleoside, e.g., inverted deoxythymidine. Poly-A tails comprising an alternative nucleoside, e.g., inverted deoxythymidine, may be generated as described herein. For instance, mRNA constructs may be modified by enzymatic ligation, using polyethylene glycol, to stabilize the poly-A region. The polyethylene glycol enzymatic ligation may be performed using 0.5-1.5 mg / mL mRNA (5’-Cap1, 3’ A100), 50 mM Tris-HCI pH 7.5, 10 mM MgCl2, 1 mM TCEP, 1000 units / mL T4 RNA Ligase 1, 1 mM ATP, 20% w / v polyethylene glycol 8000, and 5:1 molar ratio of modifying oligo to mRNA. Modifying oligo has a sequence of 5’-phosphate-AAAAAAAAAAAAAAAAAAAA-(inverted deoxythymidine (idT) (SEQ ID NO: 1)) (see below). Ligation reactions are mixed and incubated at room temperature (~22°C) for, e.g., 4 hours. Stable tail mRNA is purified by, e.g., dT purification, reverse phase purification, hydroxyapatite purification, ultrafiltration into water, and sterile filtration. The resulting stable tail-containing mRNAs contain the following structure at the 3’end, starting with the poly-A region: Aioo-UCUAGAAAAAAAAAAAAAAAAAAAA-inverted deoxythymidine (SEQ ID NO: 2).

[0657] In some embodiments, the enzymatic ligation of the poly-A region to a modifying oligo may be accomplished by utilizing a strand of DNA as a splint. DNA splint enzymatic ligation may be performed using 3 pM mRNA (5’-Cap1, 3’ A100), 30 pM modifying oligo, 1x RNA Ligase Buffer, 9 pM DNA splint, 1 mM ATP, and 1 U / pM T4 RNA Ligase 1. Modifying oligo has a sequence of 5’-phosphate-AAAAAAAAAAAAAAAAAAAA-(inverted deoxythymidine (idT) (SEQ ID NO: 1)) (see below). Ligation reactions are mixed and incubated at 25°C for, e.g., 2 hours. Following the reaction, DNase I is added at 0.2 U / pM to digest the DNA splint. This step is incubated at 37°C for 15 minutes and quenched with EDTA. The samples are then purified using dT affinity resin, buffer exchanged into 2 mM sodium citrate, and sterile filtered. Final samples are measured for concentration by spectrophotometry and assessed for purity and ligation efficiency using HPLC methods. The resulting stable tail-containing mRNAs contain the following structure at the 3’end, starting with the poly-A region: A100-UCUAGAAAAAAAAAAAAAAAAAAAA-inverted deoxythymidine (SEQ ID NO: 2).

[0658] Modifying oligo to stabilize the 3’-tail (5’-phosphate-AAAAAAAAAAAAAAAAAAAA-(inverted deoxythymidine) (SEQ ID NO: 1)): 5'-phosphate-AAAAAAAAAAAAAAA

[0659] 3'-3' linkage -

[0660] Inverted

[0661] deoxythymidine -

[0662] (i T)

[0663]

[0664] In some embodiments, the polynucleotide of the present disclosure contains a non-terminal (e.g., non-3’ terminal) poly-A region.

[0665] In some embodiments, the adenosines of the poly-A region are all unmodified adenosine. In some embodiments, the internucleoside linkages of the poly-A region are all phosphodiester linkages.

[0666] In some embodiments, the poly-A region is from about 1 to about 200 residues long (e.g., 5 to 200, 10 to 100, 50 to 150, or 15 to 50). In some embodiments, the poly-A region is greater than 10 nucleotides in length (e.g., at least or greater than 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100).

[0667] In some embodiments, the polynucleotide of the present disclosure contains at least one (e.g., at least two, or at least three) poly-A regions.

[0668] 4. 5’-Caps

[0669] Although not required, the polynucleotides of the disclosure may contain a 5’-cap structure, if desired. It will be understood that in one embodiment, the nucleic acid molecules of the invention lack a 5’-cap.

[0670] The 5’-cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5’ proximal introns during mRNA splicing.

[0671] Endogenous mRNA molecules can be 5’-end capped generating a 5’-ppp (5’-triphosphate) linkage between a terminal guanosine cap residue and the 5’-terminal transcribed sense nucleotide of the mRNA molecule. This 5’-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5’ end of the mRNA can optionally also be 2’-O-methylated. 5’-decapping through hydrolysis and cleavage of the guanylate cap structure can target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[0672] In some embodiments, the mRNA is capable of being translated by ribosomal machinery without a 5’-cap. In some embodiments, the mRNA is resistant to exonucleases without a 5’-cap.

[0673] In some embodiments, the polynucleotides of the present disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding a therapeutic polypeptide) incorporate a cap moiety. In any of the embodiments disclosed herein, a 5’ terminal cap may terminate at the 3’-end with an A or G, even if not shown in the disclosure below. In some embodiments, polynucleotides of the present disclosure comprise a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5’-ppp-5’ phosphorodiester linkages, modified nucleotides can be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) can be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5’-ppp-5’-cap. Additional modified guanosine nucleotides can be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.

[0674] Additional modifications include, but are not limited to, 2’-0-methylation of the ribose sugars of 5’-terminal and / or 5’-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2’-hydroxyl group of the sugar ring. Multiple distinct 5’-cap structures can be used to generate the 5’-cap of a nucleic acid molecule, such as a polynucleotide that functions as an mRNA molecule. Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural ( / '.e., endogenous, wild-type or physiological) 5’-caps in their chemical structure, while retaining cap function. Cap analogs can be chemically ( / '.e., non-enzymatically) or enzymatically synthesized and / or linked to the polynucleotides of the present disclosure.

[0675] For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5’-5’-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3’-O-methyl group ( / '.e., N7,3’-0-dimethyl-guanosine-5’-triphosphate-5’-guanosine (m7G-3’mppp-G; which can equivalently be designated 3’ O-Me-m7G(5’)ppp(5’)G). The 3’-0 atom of the other, unmodified, guanine becomes linked to the 5’-terminal nucleotide of the capped polynucleotide. The N7- and 3’-O-methlyated guanine provides the terminal moiety of the capped polynucleotide.

[0676] Another exemplary cap is mCAP, which is similar to ARCA but has a 2’-O-methyl group on guanosine ( / '.e., N7,2’-0-dimethyl-guanosine-5’-triphosphate-5’-guanosine, m7Gm-ppp-G).

[0677] Another exemplary cap is m7G-ppp-Gm-A ( / '.e., N7,guanosine-5’-triphosphate-2’-0-dimethyl-guanosine-adenosine).

[0678] In some embodiments, the cap is a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog can be modified at different phosphate positions with a boranophosphate group or a phosphoroselenoate group such as the dinucleotide cap analogs described in U. S. Patent No. US 8,519,110, the contents of which are herein incorporated by reference in its entirety.

[0679] In some embodiments, the cap is a cap analog is a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog known in the art and / or described herein. Non-limiting examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)-G(5’)ppp(5’)G and a N7-(4-chlorophenoxyethyl)-m3’ °G(5’)ppp(5’)G cap analog (See, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al.

[0680] Bioorganic & Medicinal Chemistry 201321:4570-4574; the contents of which are herein incorporated by reference in its entirety). In some embodiments, a cap analog of the present disclosure is a 4-chloro / bromophenoxyethyl analog.

[0681] Polynucleotides of the present disclosure can also be capped post-manufacture (whether IVT or chemical synthesis), using enzymes, in order to generate more authentic 5’-cap structures. As used herein, the phrase “more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a “more authentic” feature is better representative of an endogenous, wild-type, natural or physiological cellular function and / or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5’-cap structures of the present disclosure are those that, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5’ endonucleases and / or reduced 5’decapping, as compared to synthetic 5-’cap structures known in the art (or to a wildtype, natural or physiological 5’-cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2’-O-methyltransferase enzyme can create a canonical 5’-5’-triphosphate linkage between the 5’-terminal nucleotide of a polynucleotide and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5’-terminal nucleotide of the mRNA contains a 2’-O-methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5’-cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5’)ppp(5’)N1pN2p (cap 0), 7mG(5’)ppp(5’)N1 mpNp (cap1), and 7mG(5’)-ppp(5’)N1 mpN2mp (cap 2).

[0682] As a non-limiting example, capping chimeric polynucleotides post-manufacture can be more efficient as nearly 100% of the chimeric polynucleotides can be capped. This is in contrast to -80% when a cap analog is linked to a chimeric polynucleotide in the course of an in vitro transcription reaction.

[0683] According to the present disclosure, 5’ terminal caps can include endogenous caps or cap analogs. According to the present disclosure, a 5’ terminal cap can comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2’fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0684] Also provided herein are exemplary caps including those that can be used in co-transcriptional capping methods for ribonucleic acid (RNA) synthesis, using RNA polymerase, e.g., wild type RNA polymerase or variants thereof, e.g., such as those variants described herein. In some embodiments, caps can be added when RNA is produced in a “one-pot” reaction, without the need for a separate capping reaction. Thus, the methods, in some embodiments, comprise reacting a polynucleotide template with an RNA polymerase variant, nucleoside triphosphates, and a cap analog under in vitro transcription reaction conditions to produce RNA transcript.

[0685] As used here the term “cap” includes the inverted G nucleotide and can comprise one or more additional nucleotides 3’ of the inverted G nucleotide, e.g., 1, 2, 3, or more nucleotides 3’ of the inverted G nucleotide and 5’ to the 5’ UTR, e.g., a 5’ UTR described herein.

[0686] Exemplary caps comprise a sequence of GG, GA, or GGA, wherein the underlined, italicized G is an in inverted G nucleotide followed by a 5’-5’-triphosphate group.

[0687] In some embodiments, a cap comprises a compound of Formula (C-l)

[0688]

[0689] ring Bi is a modified or unmodified Guanine;

[0690] ring B2 and ring B3 each independently is a nucleobase or a modified nucleobase;

[0691] X2 is O, S(O)P, NR24 or CR25R26 in which p is 0, 1, or 2;

[0692] Yo is O or CReR?;

[0693] Y1 is O, S(O)n, CReR?, or NRs, in which n is 0, 1, or 2;

[0694] each — is a single bond or absent, wherein when each — is a single bond, Yi is O, S(O)n, CR6R7, or NRs; and when each — is absent, Y1 is void;

[0695] Y2 is (OP(O)R4)m in which m is 0, 1, or 2, or -0-(CR4oR4i)u-Qo-(CR42R43)v-, in which Qo is a bond, O, S(O)r, NR44, or CR45R46, r is 0, 1, or 2, and each of u and v independently is 1, 2, 3 or 4;

[0696] each R2 and R2' independently is halo, LNA, or OR3;

[0697] each R3 independently is H, Ci-Ce alkyl, C2-C6 alkenyl, or C2-C6 alkynyl and R3, when being Ci-Ce alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, is optionally substituted with one or more of halo, OH and Ci-Ce alkoxyl that is optionally substituted with one or more OH or OC(O)-Ci-C6 alkyl;

[0698] each R4and R4' independently is H, halo, Ci-Ce alkyl, OH, SH, SeH, or BHs-;

[0699] each of Re, R7, and Rs, independently, is -Q1-T1, in which Qi is a bond or C1-C3 alkyl linker optionally substituted with one or more of halo, cyano, OH and Ci-Ce alkoxy, and T1 is H, halo, OH, COOH, cyano, or Rsi, in which Rsiis C1-C3 alkyl, C2-O6 alkenyl, C2-O6 alkynyl, Ci- Ce alkoxyl, C(O)O-Ci- Ce alkyl, Cs-Os cycloalkyl, Ce-Oio aryl, NR31R32, (NR3iR32R33)+, 4 to 12- membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and Rsi is optionally substituted with one or more substituents selected from the group consisting of halo, OH, oxo, Ci-Ce alkyl, COOH, C(O)O-Ci-C6 alkyl, cyano, Ci-Ce alkoxyl, NR31R32, (N R31 R32R33)+, C3-C8 cycloalkyl, Ce-Cio aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6- membered heteroaryl; each of R10, Rn, R12, R13 Ru, and R15, independently, is -Q2-T2, in which Q2 is a bond or C1-C3 alkyl linker optionally substituted with one or more of halo, cyano, OH and Ci-Ce alkoxy, and T2 is H, halo, OH, NH2, cyano, NO2, N3, Rs2, or ORS2, in which RS2 is Ci-Ce alkyl, C2-O6 alkenyl, C2-O6 alkynyl, C3-Cs cycloalkyl, Ce-Oio aryl, NHC(O)-CI-C6 alkyl, NR31R32, (NR3iR32R33)+, 4 to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and RS2 is optionally substituted with one or more substituents selected from the group consisting of halo, OH, oxo, Ci-Ce alkyl, COOH, C(O)O-Ci-C6 alkyl, cyano, Ci - Ce alkoxyl, NR31R32, (NRsiR32R33)+, C3-O8 cycloalkyl, Ce-Oio aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6- membered heteroaryl; or alternatively R12 together with Ru is oxo, or R13 together with R15 is oxo,

[0700] each of R20, R21, R22, and R23 independently is -Q3-T3, in which Q3 is a bond or C1-C3 alkyl linker optionally substituted with one or more of halo, cyano, OH and Ci-Ce alkoxy, and T3 is H, halo, OH, NH2, cyano, NO2, N3, Rss, or ORss, in which Rss is Ci-Ce alkyl, C2-O6 alkenyl, C2-O6 alkynyl, C3-O8 cycloalkyl, Ce-Oio aryl, NHC(O)-CI-C6 alkyl, mono-Ci-Ce alkylamino, di-Ci-Ce alkylamino, 4 to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and Rss is optionally substituted with one or more substituents selected from the group consisting of halo, OH, oxo, Ci-Ce alkyl, COOH, C(O)O-Ci-C6 alkyl, cyano, Ci-Ce alkoxyl, amino, mono-Ci-Ce alkylamino, di-Ci-Ce alkylamino, C3-C8 cycloalkyl, Ce-Cio aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl;

[0701] each of R24, R25, and R26 independently is H or Ci-Ce alkyl;

[0702] each of R27 and R28 independently is H or OR29; or R27 and R28 together form O-R30-O; each R29 independently is H, Ci-Ce alkyl, C2-C6 alkenyl, or C2-C6 alkynyl and R29, when being Ci-Ce alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, is optionally substituted with one or more of halo, OH and Ci-Ce alkoxyl that is optionally substituted with one or more OH or OC(O)-Ci-C6 alkyl;

[0703] R30 is Ci-Ce alkylene optionally substituted with one or more of halo, OH and Ci-Ce alkoxyl; each of R31, R32, and R33 is, independently, H, Ci-Ce alkyl, C3-C8 cycloalkyl, Ce-Cio aryl, 4 to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl;

[0704] each of R40, R41, R42, and R43 independently is H, halo, OH, cyano, N3, OP(O)R47R48, or Ci-Ce alkyl optionally substituted with one or more OP(O)R47R48, or one R41 and one R43, together with the carbon atoms to which they are attached and Qo, form C4-C10 cycloalkyl, 4- to 14-membered heterocycloalkyl, Ce-Cio aryl, or 5- to 14-membered heteroaryl, and each of the cycloalkyl, heterocycloalkyl, phenyl, or 5- to 6-membered heteroaryl is optionally substituted with one or more of OH, halo, cyano, N3, oxo, OP(O)R47R48, Ci-Ce alkyl, Ci-Ce haloalkyl, COOH, C(O)O-Ci-Ce alkyl, Ci-Ce alkoxyl, Ci-Ce haloalkoxyl, amino, mono-Ci-Ce alkylamino, and di-Ci-Ce alkylamino;

[0705] R44 is H, Ci-Ce alkyl, or an amine protecting group;

[0706] each of R45 and R46 independently is H, OP(O)R47R48, or Ci-Ce alkyl optionally substituted with one or more OP(O)R47R48, and

[0707] each of R47 and R48 is, independently, H, halo, Ci-Ce alkyl, OH, SH, SeH, or BHs-.

[0708] It should be understood that a cap analog, as provided herein, may include any of the cap analogs described in international publication WO 2017 / 066797, published on 20 April 2017, incorporated by reference herein in its entirety.

[0709] In some embodiments, the B2 middle position can be a non-ribose molecule, such as arabinose. In some embodiments R2 is ethyl-based. Thus, in some embodiments, a cap comprises the following structure:

[0710]

[0711] (C-ll) In other embodiments, a cap comprises the following structure:

[0712]

[0713] In yet other embodiments, a cap comprises the following structure:

[0714]

[0715] (C-IV) In still other embodiments, a cap comprises the following structure:

[0716]

[0717] (C-V)

[0718] In some embodiments, R is an alkyl (e.g., Ci-Ce alkyl). In some embodiments, R is a methyl group (e.g., Ci alkyl). In some embodiments, R is an ethyl group (e.g., C2 alkyl).

[0719] In some embodiments, a cap comprises a sequence selected from the following sequences: GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU. In some embodiments, a cap comprises GAA. In some embodiments, a cap comprises GAC. In some embodiments, a cap comprises GAG. In some embodiments, a cap comprises GAU. In some embodiments, a cap comprises GCA. In some embodiments, a cap comprises GCC. In some embodiments, a cap comprises GCG. In some embodiments, a cap comprises GCU. In some embodiments, a cap comprises GGA. In some embodiments, a cap comprises GGC. In some embodiments, a cap comprises GGG. In some embodiments, a cap comprises GGU. In some embodiments, a cap comprises GUA. In some embodiments, a cap comprises GUC. In some embodiments, a cap comprises GUG. In some embodiments, a cap comprises GUU.

[0720] In some embodiments, a cap comprises a sequence selected from the following sequences: m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GpppCpG, m7GpppCpU, m7GpppGpA, m7GpppGpC, m7GpppGpG, m7GpppGpU, m7GpppUpA, m7GpppUpC, m7GpppUpG, and m7GpppUpU.

[0721] In some embodiments, a cap comprises m7GpppApA. In some embodiments, a cap comprises m7GpppApC. In some embodiments, a cap comprises m7GpppApG. In some embodiments, a cap comprises m7GpppApU. In some embodiments, a cap comprises m7GpppCpA. In some embodiments, a cap comprises m7GpppCpC. In some embodiments, a cap comprises m7GpppCpG. In some embodiments, a cap comprises m7GpppCpU. In some embodiments, a cap comprises m7GpppGpA. In some embodiments, a cap comprises m7GpppGpC. In some embodiments, a cap comprises m7GpppGpG. In some embodiments, a cap comprises m7GpppGpU. In some embodiments, a cap comprises m7GpppUpA. In some embodiments, a cap comprises m7GpppUpC. In some embodiments, a cap comprises m7GpppUpG. In some embodiments, a cap comprises m7GpppUpU. A cap, in some embodiments, comprises a sequence selected from the following sequences: m7G3'OMepppapA, m7G3'OMepppapC, m7G3'OMepppapG, m7G3'OMepppapU, m7G3'OMepppcpA, m7G3'OMePPpCpC, m7G3'OMePPpCpG, m7G3'OMePPpCpU, m7G3'OMePPPgpA, m7G3'OMePPPgpC, m7G3'OMepppgpG, m7G3'OMepppgpU, m7G3'OMepppUpA, m7G3'OMepppUpC, m7G3'OMepppUpG, and m7G3'OMepppUpU.

[0722] in some embodiments, a cap comprises m7G3'OMepppApA. in some embodiments, a cap comprises m7G3'OMepppApC. in some embodiments, a cap comprises m7G3'OMepppApG. in some embodiments, a cap comprises m7G3'OMepppApU. in some embodiments, a cap comprises m7G3'OMepppCpA. in some embodiments, a cap comprises m7G3'OMepppCpC. in some embodiments, a cap comprises m7G3'OMepppCpG. in some embodiments, a cap comprises m7G3'OMepppCpU. in some embodiments, a cap comprises m7G3'OMepppGpA. in some embodiments, a cap comprises m7G3'OMepppGpC. in some embodiments, a cap comprises m7G3'OMepppGpG. in some embodiments, a cap comprises m7G3'OMepppGpU. in some embodiments, a cap comprises m7G3'OMepppUpA. in some embodiments, a cap comprises m7G3'OMepppUpC. in some embodiments, a cap comprises m7G3'OMepppUpG. in some embodiments, a cap comprises m7G3'OMepppUpU.

[0723] In some embodiments, a cap comprises a sequence selected from the following sequences: ITI7G3'oMePPpA2'oMepA, ITI7G3'oMePPpA2'oMepC, ITI7G3'oMePPpA2'oMepG, ITI7G3'oMePPpA2'oMepU, ITI7G3'oMePPpC2'oMepA, ITI7G3'oMePPpC2'oMepC, ITI7G3'oMePPpC2'oMepG, ITI7G3'oMePPpC2'oMepU, ITI7G3'oMePPpG2'oMepA, ITI7G3'oMePPpG2'oMepC, ITI7G3'oMePPpG2'oMepG, ITI7G3'oMePPpG2'oMepU, ITI7G3'oMePPpU2'oMepA, ITI7G3'OMePPpU2'OMepC, ITI7G3'OMePPpU2'OMepG, and m7G3'OMepppU2'OMepU.

[0724] In some embodiments, a cap comprises m7G3-0MepppA2-0MepA. in some embodiments, a cap comprises m7G3'OMepppA2'OMepC. in some embodiments, a cap comprises m7G3'OMepppA2'OMepG. in some embodiments, a cap comprises m7G3'OMepppA2'OMepU. in some embodiments, a cap comprises m7G3'OMepppC2'OMepA. in some embodiments, a cap comprises m7G3'OMepppC2'OMepC. in some embodiments, a cap comprises m7G3'OMepppC2'OMepG. in some embodiments, a cap comprises m7G3'OMepppC2'OMepU. in some embodiments, a cap comprises m7G3-0MepppG2-0MepA. in some embodiments, a cap comprises m7G3'OMepppG2'OMepC. in some embodiments, a cap comprises m7G3'OMepppG2'OMepG. in some embodiments, a cap comprises m7G3'OMepppG2'OMepU. in some embodiments, a cap comprises m7G3'OMepppU2'OMepA. in some embodiments, a cap comprises m7G3'OMepppU2'OMepC. in some embodiments, a cap comprises m7G3'OMepppU2'OMepG. in some embodiments, a cap comprises m7G3'OMepppU2'OMepU.

[0725] A cap, in still other embodiments, comprises a sequence selected from the following sequences: m7GpppA2'OMepA, m7GpppA2'OMepC, m7GpppA2'OMepG, m7GpppA2'OMepU, m7GpppC2'OMepA, m7GpppC2'OMepC, m7GpppC2'OMepG, m7GpppC2'OMepU, m7GpppG2'OMepA, m7GpppG2'OMepC, m7GpppG2'OMepG, m7GpppG2'OMepU, m7GpppU2'OMepA, m7GpppU2'OMepC, m7GpppU2'OMepG, and m7Gpppll2'OMepU.

[0726] In some embodiments, a cap comprises m7GpppA2'OMepA. In some embodiments, a cap comprises m7GpppA2'OMepC. In some embodiments, a cap comprises m7GpppA2'OMepG. In some embodiments, a cap comprises m7GpppA2'OMepU. In some embodiments, a cap comprises m7GpppC2'OMepA. In some embodiments, a cap comprises m7GpppC2'OMepC. In some embodiments, a cap comprises m7GpppC2'OMepG. In some embodiments, a trinucleotide cap comprises m7GpppC2'OMepU. In some embodiments, a cap comprises m7GpppG2'OMepA. In some embodiments, a cap comprises m7GpppG2'OMepC. In some embodiments, a cap comprises m7GpppG2'OMepG. In some embodiments, a cap comprises m7GpppG2'OMepU. In some embodiments, a cap comprises n GppplhoMepA. In some embodiments, a cap comprises n GppplhoMepC. In some embodiments, a cap comprises m7Gpppl)2'OMepG. In some embodiments, a cap comprises n GppplhoMepU.

[0727] In some embodiments, a cap comprises m7Gpppm6A20mepG. In some embodiments, a cap comprises m7Gpppe6A20mepG.

[0728] In some embodiments, a cap comprises GAG. In some embodiments, a cap comprises GCG. In some embodiments, a cap comprises GUG. In some embodiments, a cap comprises GGG.

[0729] In some embodiments, a cap comprises any one of the following structures:

[0730]

[0731] In some embodiments, the cap comprisesm7GpppNiN2N3, where Ni, N2, and N3 are optional (i.e., can be absent or one or more can be present) and are independently a natural, a modified, or an unnatural nucleoside base. In some embodiments,m7G is further methylated, e.g., at the 3’ position. In some embodiments, them7G comprises an O-methyl at the 3’ position. In some embodiments N1, N2, and Ns if present, optionally, are independently an adenine, a uracil, a guanidine, a thymine, or a cytosine. In some embodiments, one or more (or all) of Ni, N2, and Ns, if present, are methylated, e.g., at the 2’ position. In some embodiments, one or more (or all) of N1, N2, and Ns, if present have an O-methyl at the 2’ position.

[0732] In some embodiments, the cap comprises the following structure:

[0733]

[0734] (C-IX) wherein Bi, B2, and Bs are independently a natural, a modified, or an unnatural nucleoside based; and R1, R2, Rs, and R4 are independently OH or O-methyl. In some embodiments, Rs is O-methyl and R4 is OH. In some embodiments, Rs and R4 are O-methyl. In some embodiments, R4 is O-methyl. In some embodiments, R1 is OH, R2 is OH, Rs is O-methyl, and R4 is OH. In some embodiments, R1 is OH, R2 is OH, Rs is O-methyl, and R4 is O-methyl. In some embodiments, at least one of R1 and R2 is O-methyl, Rs is O-methyl, and R4 is OH. In some embodiments, at least one of R1 and R2 is O-methyl, Rs is O-methyl, and R4 is O-methyl.

[0735] In some embodiments, Bi, Bs, and Bs are natural nucleoside bases. In some embodiments, at least one of Bi, B2, and Bs is a modified or unnatural base. In some embodiments, at least one of Bi, B2, and Bs is N6-methyladenine. In some embodiments, Bi is adenine, cytosine, thymine, or uracil. In some embodiments, Bi is adenine, B2 is uracil, and Bs is adenine. In some embodiments, R1 and R2 are OH, Rs and R4 are O-methyl, Bi is adenine, B2 is uracil, and Bs is adenine.

[0736] In some embodiments the cap comprises a sequence selected from the following sequences: GAAA, GACA, GAGA, GAUA, GCAA, GCCA, GCGA, GCUA, GGAA, GGCA, GGGA, GGUA, GUCA, and GUUA. In some embodiments the cap comprises a sequence selected from the following sequences: GAAG, GACG, GAGG, GAUG, GCAG, GCCG, GCGG, GCUG, GGAG, GGCG, GGGG, GGUG, GUCG, GUGG, and GUUG. In some embodiments the cap comprises a sequence selected from the following sequences: GAAU, GACU, GAGU, GAUU, GCAU, GCCU, GCGU, GCUU, GGAU, GGCU, GGGU, GGUU, GUAU, GUCU, GUGU, and GUUU. In some embodiments the cap comprises a sequence selected from the following sequences: GAAC, GACC, GAGC, GAUC, GCAC, GCCC, GCGC, GCUC, GGAC, GGCC, GGGC, GGUC, GUAC, GUCC, GUGC, and GUUC.

[0737] A cap, in some embodiments, comprises a sequence selected from the following sequences: m7G3'OMepppApApN, m7G3'OMepppApCpN, m7G3'OMepppApGpN, m7G3'OMepppApllpN, m7G3'OMepppCpApN, m7G3'OMepppCpCpN, m7G3'OMepppCpGpN, m7G3'OMepppCpllpN, m7G3'OMepppGpApN, m7G3'OMepppGpCpN, m7G3'OMepppGpGpN, m7G3'OMepppGpllpN, m7G3'OMepppUpApN, m7G3'OMepppUpCpN, m7G3'OMepppUpGpN, and m7G3'OMepppUpUpN, where N is a natural, a modified, or an unnatural nucleoside base.

[0738] A cap, in some embodiments, comprises a sequence selected from the following sequences: m7G3'OMepppA2'OMepapN, ITI7G3'OMePPpA2'OMepCpN, ITI7G3'OMePPpA2'OMePgpN, ITI7G3'OMePPpA2'OMepupN, ITI7G3'OMePPpC2'OMepapN, ITI7G3'OMePPpC2'OMepCpN, m7G3'OMepppC2'OMepgpN, m7G3'OMepppC2'OMepupN, ITI7G3'OMePPpG2'OMepapN, ITI7G3'OMePPpG2'OMepCpN, m7G3'OMepppG2'OMepgpN, m7G3'OMepppG2'OMepupN, m7G3'OMePPpU2'OMepapN, m7G3'OMepppU2'OMepCpN, m7G3'OMepppU2'OMepGpN, and m7G3'OMepppU2'OMepUpN, where N is a natural, a modified, or an unnatural nucleoside base.

[0739] A cap, in some embodiments, comprises a sequence selected from the following sequences: m7GpppA2'OMepApN, m7GpppA2'OMepCpN, m7GpppA2'OMepGpN, m7GpppA2'OMepUpN, m7GpppC2'OMepApN, m7GpppC2'OMepCpN, m7GpppC2'OMepGpN, m7GpppC2'OMepUpN, m7GpppG2'OMepApN, m7GpppG2'OMepCpN, m7GpppG2'OMepGpN, m7GpppG2'OMepUpN, m7Gpppl)2'OMepApN, m7GpppU2'OMepCpN, m7Gpppl)2'OMepGpN, and m7Gpppl)2'OMepUpN, where N is a natural, a modified, or an unnatural nucleoside base.

[0740] A cap, in some embodiments, comprises a sequence selected from the following sequences: m7G3'OmePPP A2'OMepa2'OMepN, m7G3'OmePPpA2'OMepC2'OMepN, ITI7G3'OmePPP A2'OMepg2'OMepN, m7G3'OmePPP A2'OMepU2'OMepN, m7G3'OmepppC2'OMepa2'OMepN, m7G3'OmepppC2'OMepC2'OMepN, m7G3'OmePPpC2'OMePg2'OMepN, m7G3'OmepppC2'OMepU2'OMepN, m7G3'OmePPpG2'OMepa2'OMepN, m7G3'OmepppG2'OMepC2'OMepN, m7G3'OmePPpG2'OMepg2'OMepN, m7G3'OmePPpG2'OMepU2'OMepN, m7G3'OmepppU2'OMepa2'OMepN, m7G3'OmepppU2'OMepC2'OmepN, m7G3'OMepppU2'OMepG2'OMepN, and m7G3'OMepppU2'OMepU2'OMepN, where N is a natural, a modified, or an unnatural nucleoside base.

[0741] A cap, in some embodiments, comprises a sequence selected from the following sequences: m7GpppA2'OMepA2'OMepN, m7GpppA2'OMepC2'OMepN, m7GpppA2'OMepG2'OMepN, m7GpppA2'OMepU2'OMepN, m7GpppC2'OMepA2'OMepN, m7GpppC2'OMepC2'OMepN, m7GpppC2'OMepG2'OMepN, m7GpppC2'OMepU2'OMepN, m7GpppG2'OMepA2'OMepN, m7GpppG2'OMepC2'OMepN, m7GpppG2'OMepG2'OMepN, m7GpppG2'OMepU2'OMepN, m7GpppU2'OMepA2'OMepN, m7GpppU2'OMepC2'oMepN, m7GpppU2'OMepG2'OMepN, and m7GpppU2'OMepU2'OMepN, where N is a natural, a modified, or an unnatural nucleoside base.

[0742] In some embodiments, a cap comprises GGAG. In some embodiments, a cap comprises the following structure:

[0743]

[0744] 5. Lipid Nanoparticle (LNP) Compositions

[0745] The present disclosure provides LNP compositions that encapsulate a nucleic acid molecule (e.g., linear or circular RNA molecule) described herein. The LNPs of the disclosure may confer one or more advantageous properties. The lipid nanoparticle compositions described herein may be used for the delivery of therapeutic and / or prophylactic agents, e.g., mRNAs, to mammalian cells or organs. For example, the lipid nanoparticles described herein have little or no immunogenicity. For example, the lipid compounds disclosed herein have a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA). For example, a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent, e.g., mRNA, has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.

[0746] In some embodiments, the present application provides pharmaceutical compositions comprising: (a) a delivery agent comprising a lipid nanoparticle; and

[0747] (b) a polynucleotide comprising an IRES of the disclosure.

[0748] a. Lipid Nanoparticles

[0749] In some embodiments, polynucleotides of the present disclosure are included in a lipid nanoparticle (LNP). Lipid nanoparticles according to the present disclosure may comprise: (i) an ionizable lipid (e.g., an ionizable amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-modified lipid. In some embodiments, lipid nanoparticles according to the present disclosure further comprise one or more polynucleotides of the present disclosure (e.g., a linear or circular RNA encoding a therapeutic polypeptide, such as a therapeutic polypeptide disclosed herein).

[0750] The lipid nanoparticles according to the present disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551; PCT / US2015 / 027400; PCT / US2016 / 047406;

[0751] PCT / US2016000129; PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426;

[0752] PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491 all of which are incorporated by reference herein in their entirety.

[0753] In some embodiments, the lipid nanoparticle comprises an ionizable cationic lipid (e.g., an ionizable amino lipid) at a content of 20-60 mol.%, 25-60 mol.%, 30-60 mol.%, 35-60 mol.%, 40-60 mol.%, 45-60 mol.%, 20-55 mol.%, 25-55 mol.%, 30-55 mol.%, 35-55 mol.%, 40-55 mol.%, 45-55 mol.%, 20-50 mol.%, 25-50 mol.%, 30-50 mol.%, 35-50 mol.%, or 40-50 mol.%. For example, the lipid nanoparticle may comprise an ionizable cationic lipid (e.g., an ionizable amino lipid) at a content of 40-50 mol.%, 45-50 mol.%, 45-46 mol.%, 46-47 mol.%, 47-48 mol.%, 48-49 mol.%, or 49-50 mol.%, for example about 45 mol.%, about 45.5 mol.%, about 46 mol.%, about 46.5 mol.%, about 47 mol.%, about 47.5 mol.%, about 48 mol.%, about 48.5 mol.%, about 49 mol.%, or about 49.5 mol.% ionizable cationic lipid (e.g., an ionizable amino lipid).

[0754] In some embodiments, the lipid nanoparticle comprises a non-cationic helper lipid or phospholipid at a content of 5-25 mol.%. For example, the lipid nanoparticle may comprise a non-cationic helper lipid or phospholipid at a content of molar ratio of 5-25 mol.%, 5-20 mol.%, 5-15 mol.%, 10-25 mol.%, 10-20 mol.%, 10-15 mol.%, 5-6 mol.%, 6-7 mol.%, 7-8 mol.%, 8-9 mol.%, 9-10 mol.%, 10-11 mol.%, 11-12 mol.%, 12-13 mol.%, 13-14 mol.%, 14-15 mol.%, 10-14 mol.%, 10-13 mol.%, 10-12 mol.%, 10-11 mol.%, 9-15 mol.%, 9-14 mol.%, 9-13 mol.%, 9-12 mol.%, or 9-11 mol.% non-cationic lipid.

[0755] In some embodiments, the lipid nanoparticle comprises a sterol or other structural lipid at a content molar ratio of 25-55 mol.%, 25-50 mol.%, 25-45 mol.%, 25-40 mol.%, 25-35 mol.%, 30-55 mol.%, 30-50 mol.%, 30-45 mol.%, 30-40 mol.%, 30-35 mol.%, 35-55 mol.%, 35-50 mol.%, 35-45 mol.%, 35-40 mol.%, 25-30 mol.%, 30-35 mol.%, 25-28 mol.%, 28-30 mol.%, 30-33 mol.%, 35-38 mol.%, 38-40 mol.%, 36-40 mol.%, 37-40 mol.%, 38-40 mol.%, 38-39 mol.%, 36-40 mol.%, 37-40 mol.%, 36-39 mol.%, or 37-39 mol.%. For example, the lipid nanoparticle may comprise a sterol or other structural lipid at a content of about 30 mol.%, about 30.5 mol.%, about 31.0 mol.%, about 31.5 mol.%, about 32.0 mol.%, about 32.5 mol.%, about 33.0 mol.%, about 33.5 mol.%, about 34.0 mol.%, about 34.5 mol.%, about 35.0 mol.%, about 35.5 mol.%, about 36.0 mol.%, about 36.5 mol.%, about 37.0 mol.%, about 37.5 mol.%, about 38.0 mol.%, about 38.5 mol.%, about 39.0 mol.%, about 39.5 mol.%, about 40.0 mol.%, about 40.5 mol.%, about 41.0 mol.%, about 41.5 mol.%, about 42.0 mol.%, about 42.5 mol.%, about 43.0 mol.%, about 43.5 mol.%, about 44.0 mol.%, about 44.5 mol.%, or about 45.0 mol.%.

[0756] In some embodiments, the lipid nanoparticle comprises a PEG-modified lipid at a content of 0.5-15 mol.%, 1.0-15 mol.%, 1.5-15 mol.%, 2.0-15 mol.%, 2.5-15 mol.%, 3.0-15 mol.%, 3.5-15 mol.%, 4.0-15 mol.%, 4.5-15 mol.%, 5.0-15 mol.%, 10-15 mol.%, 0.5-10 mol.%, 0.5-5 mol.%, 0.5-4.5 mol.%, 0.5-4.0 mol.%, 0.5-3.5 mol.%, 0.5-3.0 mol.%, 0.5-2.5 mol.%, 0.5-2.0 mol.%, 0.5-1.5 mol.%, 0.5-1.0 mol.%, 1.0-10 mol.%, 1.0-5 mol.%, 1.0-4.5 mol.%, 1.0-4.0 mol.%, 1.0-3.5 mol.%, 1.0-3.0 mol.%, 1.0-2.5 mol.%, 1.0-2.0 mol.%, 1.0-1.5 mol.%, 1.5-5.0 mol.%, 1.5-4.5 mol.%, 1.5-4.0 mol.%, 1.5-3.5 mol.%, 1.5-3.0 mol.%, 1.5-2.5 mol.%, 1.5-2.0 mol.%, 2.0-5.0 mol.%, 2.0-4.5 mol.%, 2.0-4.0 mol.%, 2.0-3.5 mol.%, 2.0-3.0 mol.%, or 2.0- 2.5 mol.%. For example, the lipid nanoparticle may comprise a PEG-modified lipid at a content of a about 0.5 mol.%, about 1.0 mol.%, about 1.5 mol.%, about 2.0 mol.%, about 2.5 mol.%, about 3.0 mol.%, about 3.5 mol.%, about 4.0 mol.%, about 4.5 mol.%, about 5.0 mol.%, about 6.0 mol.%, about 7.0 mol.%, about 8.0 mol.%, about 9.0 mol.%, about 10.0 mol.%, or about 15.0 mol.%. In some embodiments, the lipid nanoparticle comprises: (i) 20 to 60 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 25 to 55 mol.% sterol or other structural lipid, (iii) 5 to 25 mol.% noncationic lipid (e.g., phospholipid), and (iv) 0.5 to 15 mol.% PEG-modified lipid.

[0757] In some embodiments, the lipid nanoparticle comprises: (i) 40 to 50 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 30 to 45 mol.% sterol or other structural lipid, (iii) 5 to 15 mol.% noncationic lipid (e.g., phospholipid), and (iv) 1 to 5 mol.% PEG-modified lipid.

[0758] In some embodiments, the lipid nanoparticle comprises: (i) 45 to 50 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 35 to 45 mol.% sterol or other structural lipid, (iii) 8 to 12 mol.% noncationic lipid (e.g., phospholipid), and (iv) 1.5 to 3.5 mol.% PEG-modified lipid.

[0759] In the following sections, “Compounds” numbered with an “I-” prefix e.g., “Compound 1-1,” “Compound I-2,” “Compound I-3,” “Compound l-VI,” etc., indicate specific ionizable lipid compounds. Likewise, compounds numbered with a “P-” prefix (e.g., “Compound P-l,” etc.) indicate a specific PEG-modified lipid compound.

[0760] b. Ionizable Amino Lipids

[0761] In some embodiments, the lipid nanoparticle of the present disclosure comprises an ionizable cationic lipid (e.g., an ionizable amino lipid).

[0762] In some embodiments, the ionizable lipid is a compound of Formula (IL*)

[0763]

[0764] or a salt thereof, wherein:

[0765] R1is -OH, -NRN-C4-W cycloalkenyl optionally substituted with one or more oxo or -N(RN’RN”); RNis H or C1-6 alkyl;

[0766] RN’ is H or C1-6 alkyl;

[0767] RN” is H or C1-6 alkyl;

[0768] o is 1, 2, 3, or 4;

[0769] n is 4, 5, 6, 7, or 8;

[0770] m is 4, 5, 6, 7, or 8;

[0771] M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2;

[0772] M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3;

[0773] R2aR2b

[0774] , A \xR2c

[0775] R2is or -(C1-6 alkylene)-(C3-8 cycloalkyl)-Ci-6 alkyl;

[0776] R2ais -H or C1-10 alkyl;

[0777] R2bis -H or C1-10 alkyl;

[0778] R2cis Ci-8 alkyl or C2-8 alkenyl;

[0779]

[0780] R3ais H or C1-10 alkyl;

[0781] R3bis H or C1-8 alkyl; and

[0782] R3cis C1-10 alkyl or O2-8 alkenyl.

[0783] In some embodiments, the ionizable lipid is of Formula (IL**-I):

[0784]

[0785] (IL**-I)

[0786] or a salt thereof, wherein:

[0787] R1is -OH;

[0788] 0 is 2, 3, or 4;

[0789] n is 4, 5, 6, 7, or 8;

[0790] M is -C(=O)-O-*, wherein * indicates attachment to R2;

[0791] m is 6, 7, or 8;

[0792] M’ is -C(=O)-O-*, wherein * indicates attachment to R3;

[0793] R2cis O4-8 alkyl;

[0794] R3ais O7-10 alkyl; and

[0795] R3cis O3-5 alkyl.

[0796] In some embodiments, the ionizable lipid is of Formula (IL**-III):

[0797]

[0798] (IL**-III)

[0799] or a salt thereof, wherein:

[0800] R1is NRN-04-IO cycloalkenyl optionally substituted with one or more oxo or -N(RN’RN”); RNis H;

[0801] RN’ is O1-2 alkyl;

[0802] RN’’ is H;

[0803] 0 is 2, 3, or 4;

[0804] n is 6, 7, or 8;

[0805] M is -C(=O)-O-*, wherein * indicates attachment to R2;

[0806] m is 6, 7, or 8;

[0807] M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2ais C7-10 alkyl;

[0808] R2cis C4-6 alkyl;

[0809] R3ais C1-3 alkyl; and

[0810] R3cis C4-6 alkyl.

[0811] In some embodiments, the ionizable lipid is of Formula (IL**-IV):

[0812]

[0813] (IL**-IV)

[0814] or a salt thereof, wherein:

[0815] R1is OH;

[0816] 0 is 2, 3, or 4;

[0817] n is 6, 7, or 8;

[0818] M is -C(=O)-O-*, wherein * indicates attachment to R2;

[0819] m is 6, 7, or 8;

[0820] M’ is -C(=O)-O-*, wherein * indicates attachment to R3;

[0821] R2bis C3-5 alkyl;

[0822] R2cis C2-4 alkyl;

[0823] R3ais C7-10 alkyl; and

[0824] R3cis O4-6 alkyl.

[0825] In some embodiments, the ionizable lipid is of Formula (IL*-I):

[0826]

[0827] (IL*-la)

[0828] or a salt thereof, wherein:

[0829] R1, 0, m, n, M, M’, R2c, and R3care as defined for variable IL*; and R3ais C1-8 alkyl.

[0830] In some embodiments, ionizable lipid is of Formula (IL*-la):

[0831]

[0832] or a salt thereof, wherein:

[0833] R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais Ci-8 alkyl.

[0834] In some embodiments, the ionizable lipid is of Formula (IL*-la’):

[0835]

[0836] or a salt thereof, wherein:

[0837] o, M, M’, R2cand R3care as defined for variable IL*; and

[0838] R3ais Ci-8 alkyl.

[0839] In some embodiments, the ionizable lipid is of Formula (IL*-lla):

[0840]

[0841] or a salt thereof, wherein:

[0842] R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais Ci-8 alkyl.

[0843] In some embodiments, the ionizable lipid is of Formula (IL*-H’):

[0844]

[0845] or a salt thereof, wherein:

[0846] o, M, M’, R2cand R3care as defined for variable IL*; and

[0847] R3ais Ci-8 alkyl.

[0848] In some embodiments, the ionizable lipid is of Formula (IL*-III):

[0849]

[0850] or a salt thereof, wherein:

[0851] R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;

[0852] R2ais a Ci-8 alkyl; and

[0853] R3ais Ci-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-llla):

[0854]

[0855] or a salt thereof, wherein:

[0856] R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;

[0857] R2ais a Ci-8 alkyl; and

[0858] R3ais Ci-8 alkyl.

[0859] In some embodiments, the ionizable lipid is of Formula (IL*-llla):

[0860]

[0861] or a salt thereof, wherein:

[0862] R1, o, M, M’, R2c, and R3care as defined for variable IL*;

[0863] R2ais a Ci-8 alkyl; and

[0864] R3ais Ci-8 alkyl.

[0865] In some embodiments, the ionizable lipid is of Formula (IL*-llla’):

[0866]

[0867] or a salt thereof, wherein:

[0868] R1, o, M, M’, R2c, and R3care as defined for variable IL*;

[0869] R2ais a Ci-8 alkyl; and

[0870] R3ais Ci-8 alkyl.

[0871] In some embodiments, the ionizable lipid is of Formula (IL*-lllb):

[0872]

[0873] or a salt thereof, wherein:

[0874] R1, o, M, M’, R2c, and R3care as defined for variable IL*;

[0875] R2ais a Ci-8 alkyl; and

[0876] R3ais Ci-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-lllb’):

[0877]

[0878] or a salt thereof, wherein:

[0879] R1, o, M, M’, R2c, and R3care as defined for variable IL*;

[0880] R2ais a Ci-8 alkyl; and

[0881] R3ais Ci-8 alkyl.

[0882] In some embodiments, the ionizable lipid is of Formula (IL*-IV):

[0883]

[0884] (IL*-IV)

[0885] or a salt thereof, wherein:

[0886] R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;

[0887] R2bis a Ci-8 alkyl; and

[0888] R3ais Ci-8 alkyl.

[0889] In some embodiments, the ionizable lipid is of Formula (IL*-IVa):

[0890]

[0891] or a salt thereof, wherein:

[0892] R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;

[0893] R2bis a Ci-8 alkyl; and

[0894] R3ais Ci-8 alkyl.

[0895] In some embodiments, the ionizable lipid is of Formula (IL*-lva’):

[0896]

[0897] or a salt thereof, wherein:

[0898] o, M, M’, R2c, and R3care as defined for variable IL*;

[0899] R2ais a Ci-8 alkyl; and

[0900] R3ais Ci-8 alkyl.

[0901] Variables o, R1, RN, RN’, RN” of Ionizable Lipid

[0902] In some embodiments of the ionizable lipid, o is 1.

[0903] In some embodiments of the ionizable lipid, o is 2.

[0904] In some embodiments of the ionizable lipid, o is 3.

[0905] In some embodiments of the ionizable lipid, o is 4.

[0906] In some embodiments of the ionizable lipid, R1is -OH.

[0907] In some embodiments of the ionizable lipid, RNis H.

[0908] In some embodiments of the ionizable lipid, RNis methyl.

[0909] In some embodiments of the ionizable lipid, RNis ethyl.

[0910] In some embodiments of the ionizable lipid, R1is -NRN-cyclobutenyl, wherein the cyclobutenyl is optionally substituted with one or more oxo or -N(RN’RN”).

[0911] In some embodiments of the ionizable lipid, RN’ is H.

[0912] In some embodiments of the ionizable lipid, RN’ is methyl.

[0913] In some embodiments of the ionizable lipid, RN’ is ethyl.

[0914] In some embodiments of the ionizable lipid, RN” is H.

[0915] In some embodiments of the ionizable lipid, RN” is methyl.

[0916] In some embodiments of the ionizable lipid, RN” is ethyl.

[0917] In some embodiments of the ionizable lipid, RN’ is H and RN” is methyl.

[0918] In some embodiments of the ionizable lipid,

[0919]

[0920] In some embodiments of the ionizable lipid,

[0921]

[0922] Variables m and n of the Ionizable Lipid

[0923] In some embodiments of the ionizable lipid, m is 4.

[0924] In some embodiments of the ionizable lipid, m is 5.

[0925] In some embodiments of the ionizable lipid, m is 6.

[0926] In some embodiments of the ionizable lipid, m is 7.

[0927] In some embodiments of the ionizable lipid, m is 8.

[0928] In some embodiments of the ionizable lipid, m is 4.

[0929] In some embodiments of the ionizable lipid, n is 5.

[0930] In some embodiments of the ionizable lipid, n is 6. In some embodiments of the ionizable lipid, n is 7.

[0931] In some embodiments of the ionizable lipid, n is 8.

[0932] In some embodiments of the ionizable lipid, n is 5 and m is 7.

[0933] In some embodiments of the ionizable lipid, n is 7 and m is 7.

[0934] In some embodiments of the ionizable lipid, m is 6 and n is 6.

[0935] Variables M and M’

[0936] In some embodiments of the ionizable lipid, M is -O-C(=O)-*, wherein * indicates attachment to R2.

[0937] In some embodiments of the ionizable lipid, M is -C(=O)-O-* wherein * indicates attachment to R2. In some embodiments of the ionizable lipid, M’ is -O-C(=O)-*, wherein * indicates attachment to R3.

[0938] In some embodiments of the ionizable lipid, M’ is -C(=O)-O-* wherein * indicates attachment to R3.

[0939] In some embodiments of the ionizable lipid, M is -O-C(=O)-*, wherein * indicates attachment to R2, and M’ is -C(=O)-O-* wherein * indicates attachment to R3

[0940] Variables R2, R2a, R2b, R2c

[0941] R2aR2b

[0942] In some embodiments of the ionizable lipid, R2is

[0943]

[0944] In some embodiments of the ionizable lipid, R2ais hydrogen.

[0945] In some embodiments of the ionizable lipid, R2ais methyl.

[0946] In some embodiments of the ionizable lipid, R2ais ethyl.

[0947] In some embodiments of the ionizable lipid, R2ais propyl.

[0948] In some embodiments of the ionizable lipid, R2ais butyl.

[0949] In some embodiments of the ionizable lipid, R2ais pentyl.

[0950] In some embodiments of the ionizable lipid, R2ais hexyl.

[0951] In some embodiments of the ionizable lipid, R2ais heptyl.

[0952] In some embodiments of the ionizable lipid, R2ais octyl.

[0953] In some embodiments of the ionizable lipid, R2bis hydrogen.

[0954] In some embodiments of the ionizable lipid, R2bis methyl.

[0955] In some embodiments of the ionizable lipid, R2bis ethyl.

[0956] In some embodiments of the ionizable lipid, R2bis propyl.

[0957] In some embodiments of the ionizable lipid, R2bis butyl.

[0958] In some embodiments of the ionizable lipid, R2bis pentyl.

[0959] In some embodiments of the ionizable lipid, R2bis hexyl.

[0960] In some embodiments of the ionizable lipid, R2bis heptyl.

[0961] In some embodiments of the ionizable lipid, R2bis octyl.

[0962] In some embodiments of the ionizable lipid, R2ais hydrogen and R2bis hydrogen.

[0963] In some embodiments of the ionizable lipid, R2ais hexyl and R2bis hydrogen.

[0964] In some embodiments of the ionizable lipid, R2ais octyl and R2bis hydrogen.

[0965] In some embodiments of the ionizable lipid, R2ais hydrogen and R2bis butyl. In some embodiments of the ionizable lipid, R2cis methyl.

[0966] In some embodiments of the ionizable lipid, R2cis ethyl.

[0967] In some embodiments of the ionizable lipid, R2cis propyl.

[0968] In some embodiments of the ionizable lipid, R2cis butyl.

[0969] In some embodiments of the ionizable lipid, R2cis pentyl.

[0970] In some embodiments of the ionizable lipid, R2cis hexyl.

[0971] In some embodiments of the ionizable lipid, R2cis heptyl.

[0972] In some embodiments of the ionizable lipid, R2cis octyl.

[0973] In some embodiments of the ionizable lipid, R2is -(C1-6 alkylene)-(C3-8 cycloalkyl)-Ci-6 alkyl. In some embodiments of the ionizable lipid, R2is -(C1-6 alkylene)-(cyclohexyl)-Ci-6 alkyl. In some embodiments of the ionizable lipid, R2is -(C1-6 alkylene)-(cyclopentyl)-Ci-6 alkyl.

[0974] Variables R3, R3a, R3b, and R3c

[0975]

[0976] In some embodiments of the ionizable lipid, R3is R3aR3b

[0977] In some embodiments of the ionizable lipid, R3ais hydrogen.

[0978] In some embodiments of the ionizable lipid, R3ais methyl.

[0979] In some embodiments of the ionizable lipid, R3ais ethyl.

[0980] In some embodiments of the ionizable lipid, R3ais propyl.

[0981] In some embodiments of the ionizable lipid, R3ais butyl.

[0982] In some embodiments of the ionizable lipid, R3ais pentyl.

[0983] In some embodiments of the ionizable lipid, R3ais hexyl.

[0984] In some embodiments of the ionizable lipid, R3ais heptyl.

[0985] In some embodiments of the ionizable lipid, R3ais octyl.

[0986] In some embodiments of the ionizable lipid, R3bis hydrogen.

[0987] In some embodiments of the ionizable lipid, R3bis methyl.

[0988] In some embodiments of the ionizable lipid, R3bis ethyl.

[0989] In some embodiments of the ionizable lipid, R3bis propyl.

[0990] In some embodiments of the ionizable lipid, R3bis butyl.

[0991] In some embodiments of the ionizable lipid, R3bis pentyl.

[0992] In some embodiments of the ionizable lipid, R3bis hexyl.

[0993] In some embodiments of the ionizable lipid, R3bis heptyl.

[0994] In some embodiments of the ionizable lipid, R3bis octyl.

[0995] In some embodiments of the ionizable lipid, R3ais octyl and R3bis hydrogen.

[0996] In some embodiments of the ionizable lipid, R3ais ethyl and R3bis hydrogen.

[0997] In some embodiments of the ionizable lipid, R3ais hexyl and R3bis hydrogen.

[0998] In some embodiments of the ionizable lipid, R3cis methyl.

[0999] In some embodiments of the ionizable lipid, R3cis ethyl.

[1000] In some embodiments of the ionizable lipid, R3cis propyl.

[1001] In some embodiments of the ionizable lipid, R3cis butyl.

[1002] In some embodiments of the ionizable lipid, R3cis pentyl. In some embodiments of the ionizable lipid, R3cis hexyl.

[1003] In some embodiments of the ionizable lipid, R3cis heptyl.

[1004] In some embodiments of the ionizable lipid, R3cis octyl.

[1005] It is understood that, for an ionizable lipid, variables o, R1, RN, RN’, RN’, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and R3ccan each be, where applicable, selected from the groups described herein, and any group described herein for any of variables o, R1, RN, RN’, RN’, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and R3ccan be combined, where applicable, with any group described herein for one or more of the remainder of variables o, R1, RN, RN’, RN’, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and R3c.

[1006] In some embodiments, the ionizable lipid is a compound selected from:

[1007]

[1008] In some embodiments, the ionizable lipid is

[1009]

[1010] In some embodiments, the ionizable lipid is

[1011]

[1012] In some embodiments, the ionizable lipid is

[1013]

[1014] In some embodiments, the ionizable lipid is

[1015]

[1016] Without wishing to be bound by theory, it is understood that an ionizable lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino)lipids. Lipids may also be zwitterionic, e.g., neutral molecules having both a positive and a negative charge.

[1017] c. Phospholipids

[1018] The lipid composition of the lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[1019] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

[1020] A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[1021] Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.

[1022] Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[1023] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.

[1024] In some embodiments, a phospholipid of the present disclosure comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPO), 1,2-di myristoyl-sn-gly cero-phosphocholine (DMPO), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1.2-diundecanoyl-sn-glycero-phosphocholine (DUPO), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPO), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1 -oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[1025] In certain embodiments, a phospholipid useful or potentially useful in the present disclosure is an analog or variant of DSPC. In certain embodiments, a phospholipid useful or potentially useful in the present disclosure is a compound of Formula (IV):

[1026]

[1027] (IV),

[1028] or a salt thereof, wherein:

[1029] each R1is, independently, optionally substituted alkyl; or optionally two R1are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R1are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl;

[1030] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[1031] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[1032] A is of the Formula:

[1033]

[1034] each instance of L2is, independently, a bond or optionally substituted C1-6 alkylene, wherein one methylene unit of the optionally substituted C1-6 alkylene is optionally replaced with O, N(RN), S, C(O), -C(O)N(RN), NRNC(O), C(O)O, 00(0), 00(0)0, OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN);

[1035] each instance of R2is, independently, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl; optionally wherein one or more methylene units of R2are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, 0(0), C(O)N(RN), -NRNC(O), NRNC(O)N(RN), 0(0)0, 00(0), 00(0)0, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(0), -0S(0), S(0)0, 0S(0)0, 0S(0)2, S(0)20, 0S(0)20, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), -OS(O)N(RN), N(RN)S(O)O, S(0)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or -N(RN)S(O)2O;

[1036] each instance of RNis, independently, hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

[1037] Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

[1038] p is 1 or 2;

[1039] provided that the compound is not of the Formula:

[1040]

[1041] wherein each instance of R2is, independently, unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[1042] In some embodiments, the phospholipids may be one or more of the phospholipids described in U. S. Application No. 62 / 520,530.

[1043] Phospholipid Head Modifications

[1044] In certain embodiments, a phospholipid useful or potentially useful in the present disclosure comprises a modified phospholipid head (e.g., a modified choline group). In certain embodiments, a phospholipid with a modified head is DSPC, or analog thereof, with a modified quaternary amine. For example, in embodiments of Formula (IV), at least one of R1is not methyl. In certain embodiments, at least one of R1is not hydrogen or methyl. In certain embodiments, the compound of Formula (IV) is of one of the following Formulae:

[1045]

[1046] or a salt thereof, wherein:

[1047] each t is, independently, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[1048] each u is, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

[1049] each v is, independently, 1, 2, or 3.

[1050] In certain embodiments, a compound of Formula (IV) is of Formula (IV-a):

[1051]

[1052] (IV-a),

[1053] or a salt thereof.

[1054] In certain embodiments, a phospholipid useful or potentially useful in the present disclosure comprises a cyclic moiety in place of the glyceride moiety. In certain embodiments, a phospholipid useful in the present disclosure is DSPC, or analog thereof, with a cyclic moiety in place of the glyceride moiety. In certain embodiments, the compound of Formula (IV) is of Formula (IV-b):

[1055]

[1056] (iv-b),

[1057] or a salt thereof.

[1058] Phospholipid Tail Modifications

[1059] In certain embodiments, a phospholipid useful or potentially useful in the present disclosure comprises a modified tail. In certain embodiments, a phospholipid useful or potentially useful in the present disclosure is DSPC, or analog thereof, with a modified tail. As described herein, a “modified tail” may be a tail with shorter or longer aliphatic chains, aliphatic chains with branching introduced, aliphatic chains with substituents introduced, aliphatic chains wherein one or more methylenes are replaced by cyclic or heteroatom groups, or any combination thereof. For example, in certain embodiments, the compound of (IV) is of Formula (IV-a), or a salt thereof, wherein at least one instance of R2is each instance of R2is optionally substituted C1-30 alkyl, wherein one or more methylene units of R2are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(0), C(O)N(RN), -NRNC(O), NRNC(O)N(RN), 0(0)0, 00(0), 00(0)0, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(0), -0S(0), S(0)0, 0S(0)0, 0S(0)2, S(0)20, 0S(0)20, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), -OS(O)N(RN), N(RN)S(O)O, S(0)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or -N(RN)S(O)2O.

[1060] In certain embodiments, the compound of Formula (IV) is of Formula (IV-c):

[1061]

[1062] (IV-c), or a salt thereof, wherein:

[1063] each x is, independently, an integer between 0-30, inclusive; and

[1064] each instance is G is, independently, selected from the group consisting of optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), NRNC(O)N(RN), 0(0)0, 00(0), -00(0)0, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), -NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(0), 0S(0), S(0)0, 0S(0)0, 0S(0)2, S(0)20, 0S(0)20, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(0)2, -N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O. Each possibility represents a separate embodiment of the present disclosure.

[1065] In certain embodiments, a phospholipid useful or potentially useful in the present disclosure comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, a phospholipid useful or potentially useful in the present disclosure is a compound of Formula (IV), wherein n is 1, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, a compound of Formula (IV) is of one of the following Formulae:

[1066]

[1067] or a salt thereof.

[1068] Hi. Alternative Lipids

[1069] In certain embodiments, a phospholipid useful or potentially useful in the present disclosure comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, a phospholipid is useful.

[1070] In certain embodiments, an alternative lipid is used in place of a phospholipid of the present disclosure.

[1071] In certain embodiments, an alternative lipid of the present disclosure is oleic acid.

[1072] In certain embodiments, the alternative lipid is one of the following:

[1073]

[1074]

[1075] d. Structural Lipids

[1076] The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids. As used herein, the term “structural lipid” refers to sterols and also to lipids containing sterol moieties.

[1077] Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol.

[1078] In some embodiments, the structural lipids may be one or more of the structural lipids described in U. S. Application No. 62 / 520,530.

[1079] e. Polyethylene Glycol (PEG)-Lipids

[1080] The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more polyethylene glycol (PEG) lipids.

[1081] As used herein, the term “PEG lipid” refers to polyethylene glycol (PEG)-modified lipids. Nonlimiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DPPE, or a PEG-DSPE lipid. In some embodiments, the PEG lipid includes, but not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

[1082] In one embodiment, the PEG lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.

[1083] In some embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about Cuto about C22, preferably from about Cuto about C16. In some embodiments, a PEG moiety, for example, an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In one embodiment, the PEG lipid is PEG2K-DMG.

[1084] In one embodiment, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG, PEG-DPPE, and PEG-DSPE.

[1085] PEG lipids are known in the art, such as those described in U. S. Patent No. 8158601 and International Publ. No. WO 2015 / 130584 A2, which are incorporated herein by reference in their entirety.

[1086] In general, some of the other lipid components (e.g., PEG lipids) of various formulae, described herein may be synthesized as described in International Patent Application No. PCT / US2016 / 000129, filed December 10, 2016, entitled “Compositions and Methods for Delivery of Therapeutic Agents,” which is incorporated by reference in its entirety.

[1087] The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DPPE, or a PEG-DSPE lipid.

[1088] In some embodiments, the PEG-modified lipids are a modified form of PEG DMG. PEG-DMG has the following structure:

[1089]

[1090] In some embodiments, the PEG-modified lipids are a modified form of PEG-DSG. PEG-DSG has the following structure:

[1091]

[1092] In one embodiment, PEG lipids useful in the present invention can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In some embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In some embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In some embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an -OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[1093] In some embodiments, a PEG lipid useful in the present invention is a compound of Formula (PL-I). Provided herein are compounds of Formula (PL-I):

[1094]

[1095] or salts thereof, wherein:

[1096] R3is -OR0;

[1097] R° is hydrogen, optionally substituted alkyl, or an oxygen-protecting group;

[1098] r is an integer between 1 and 150, inclusive;

[1099] L1is optionally substituted C1-10 alkylene, wherein at least one methylene of the optionally substituted C1-10 alkylene is, independently, replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(RN), S, 0(0), C(O)N(RN), NRNC(O), 0(0)0, 00(0), 00(0)0, OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN);

[1100] D is absent; or

[1101] D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[1102] A is of the formula:

[1103]

[1104] each instance of L2is, independently, a bond or optionally substituted C1-6 alkylene, wherein one methylene unit of the optionally substituted C1-6 alkylene is optionally replaced with O, N(RN), S, C(0), -C(O)N(RN), NRNC(O), 0(0)0, 00(0), 00(0)0, OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN);

[1105] each instance of R2is, independently, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl; optionally wherein one or more methylene units of R2are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(0), C(O)N(RN), -NRNC(O), NRNC(O)N(RN), 0(0)0, 00(0), 00(0)0, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(0), -0S(0), S(0)0, 0S(0)0, 0S(0)2, S(0)20, 0S(0)20, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), -OS(O)N(RN), N(RN)S(O)O, S(0)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or -N(RN)S(O)2O;

[1106] each instance of RNis, independently, hydrogen, optionally substituted alkyl, or a nitrogenprotecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

[1107] p is 1 or 2.

[1108] In some embodiments, the compound of Formula (PL-I) is a PEG-OH lipid (e.g., R3is -OR0, and R° is hydrogen). In some embodiments, the compound of Formula (PL-I) is of Formula (PL-I-OH):

[1109]

[1110] salt thereof.

[1111] In some embodiments, a PEG lipid useful in the present invention is a PEGylated fatty acid. In some embodiments, a PEG lipid useful in the present invention is a compound of Formula (PL-I I).

[1112] Provided herein are compounds of Formula (PL-II):

[1113]

[1114] (PL-II), or a salt thereof, wherein:

[1115] R3is-OR°;

[1116] R° is hydrogen, optionally substituted alkyl or an oxygen-protecting group;

[1117] r is an integer between 1 and 150, inclusive;

[1118] R5is optionally substituted C10-40 alkyl, optionally substituted C10-40 alkenyl, or optionally substituted C10-40 alkynyl; and optionally one or more methylene groups of R5are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), NRNC(O)N(RN), 0(0)0, 00(0), 00(0)0, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), -NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(0), 0S(0), S(0)0, 0S(0)0, 0S(0)2, S(0)20, 0S(0)20, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(0)2, -N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O; and

[1119] each instance of RNis, independently, hydrogen, optionally substituted alkyl, or a nitrogenprotecting group.

[1120] In some embodiments, the compound of Formula (PL-II) is of Formula (PL-II-OH):

[1121]

[1122] (PL-II-OH), or a salt thereof. In some embodiments, r is 35-55. In some embodiments, r is 45. In some embodiments, r is 104-124, In some embodiments, r is 114. In some embodiments, r is 68-88. In some embodiments, r is 78.

[1123] In yet other embodiments the compound of Formula (PL-II) is:

[1124]

[1125] salt thereof. In some embodiments, r is 1 -150. In some embodiments, r is about 35 to about 55. In some embodiments, r is 35-55. In some embodiments, r is 45. In some embodiments, r is 104-124, In some embodiments, r is 114. In some embodiments, r is 68-88. In some embodiments, r is 78. In one embodiment, the compound of Formula (PL-II) is

[1126]

[1127] In yet other embodiments, the PEG lipid is PEG1. PEG1 is a plurality of compounds of Formula (PL-01):

[1128]

[1129] salts thereof, wherein r is 1 -150. In some embodiments, r is about 35 to about 55. In some embodiments, r is 35-55. In some embodiments, r is 45. In some embodiments, r is 104-124, In some embodiments, r is 114. In some embodiments, r is 68-88. In some embodiments, r is 78.

[1130] In some embodiments, the PEG lipids may be one or more of the PEG lipids described in WO 2018 / 232357.

[1131] f. Other Lipid Composition Components

[1132] The lipid composition of a pharmaceutical composition disclosed herein can include one or more components in addition to those described above. For example, the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g., surfactants), or other components. For example, a permeability enhancer molecule can be a molecule described by U. S. Patent Application Publication No. 2005 / 0222064. Carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

[1133] A polymer can be included in and / or used to encapsulate or partially encapsulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form). A polymer can be biodegradable and / or biocompatible. A polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.

[1134] The ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt / wt).

[1135] In some embodiments, the ratio between the lipid composition and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt / wt). In some embodiments, the wt / wt ratio of the lipid composition to the polynucleotide encoding a therapeutic agent is about 20:1 or about 15:1.

[1136] In some embodiments, the pharmaceutical composition disclosed herein can contain more than one polypeptide. For example, a pharmaceutical composition disclosed herein can contain two or more polynucleotides e.g., RNA, e.g., mRNA). In some embodiments, the lipid nanoparticles described herein can comprise polynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios such as, but not limited to, 5:1 to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about 20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, from about 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 to about 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 to about 25:1, from about 10:1 to about 30:1, from about 10:1 to about 35:1, from about 10:1 to about 40:1, from about 10:1 to about 45:1, from about 10:1 to about 50:1, from about 10:1 to about 55:1, from about 10:1 to about 60:1, from about 10:1 to about 70:1, from about 15:1 to about 20:1, from about 15:1 to about 25:1,from about 15:1 to about 30:1, from about 15:1 to about 35:1, from about 15:1 to about 40:1, from about 15:1 to about 45:1, from about 15:1 to about 50:1, from about 15:1 to about 55:1, from about 15:1 to about 60:1 or from about 15:1 to about 70:1.

[1137] In some embodiments, the lipid nanoparticles described herein can comprise the polynucleotide in a concentration from approximately 0.1 mg / ml to 2 mg / ml such as, but not limited to, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2.0 mg / ml or greater than 2.0 mg / ml.

[1138] g. Nanoparticle Compositions

[1139] In some embodiments, the pharmaceutical compositions disclosed herein are Formulated as lipid nanoparticles (LNP). Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as compound as described herein, and (ii) a polynucleotide containing an IRES described herein and encoding a polypeptide of interest. In such nanoparticle composition, the lipid composition disclosed herein can encapsulate the polynucleotide containing the IRES and encoding the polypeptide.

[1140] Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[1141] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers can be functionalized and / or crosslinked to one another. Lipid bilayers can include one or more ligands, proteins, or channels.

[1142] In some embodiments, a lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable amino lipid, a PEG-modified lipid, a sterol and a structural lipid. In some embodiments, the LNP has a molar ratio of about 40-50% ionizable amino lipid; about 5-15% structural lipid; about 30-45% sterol; and about 1-5% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises 47-49 mol.% ionizable cationic lipid (e.g., ionizable amino lipid, e.g., Compound 1-1, Compound I-2, or Compound I-3), 10-12 mol.% non-cationic lipid (e.g., phospholipid, e.g., DSPC), 38-40 mol.% sterol (e.g., cholesterol) or other structural lipid, and 1-3 mol.% PEG-modified lipid (e.g., PEG-DMG or Compound P-l).

[1143] For instance, in some embodiments, the lipid nanoparticle (“LNP-1”) may comprise the following components at the following molar ratios:

[1144] (i) 45-50 mol.% Compound 1-1

[1145] (ii) 35-45 mol.% sterol (e.g., cholesterol);

[1146] (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and

[1147] (iv) 1.5-3.5 mol.% PEG-lipid (e.g., Compound P-l or PEG-DMG).

[1148] For instance, in some embodiments, the lipid nanoparticle (“LNP-1 A”) may comprise the following components at the following molar ratios:

[1149] (i) 45-50 mol.% Compound 1-1

[1150] (ii) 35-45 mol.% Cholesterol;

[1151] (iii) 8-12 mol.% DSPC; and

[1152] (iv) 1.5-3.5 mol.% PEG-DMG.

[1153] For instance, in some embodiments, the lipid nanoparticle (“LNP-1 B”) may comprise the following components at the following molar ratios:

[1154] (i) 45-50 mol.% Compound 1-1

[1155] (ii) 35-45 mol.% Cholesterol;

[1156] (iii) 8-12 mol.% DSPC; and

[1157] (iv) 1.5-3.5 mol.% Compound P-l.

[1158] In some embodiments, the lipid nanoparticle (“LNP-2”) may comprise the following:

[1159] (i) 45-50 mol.% Compound I-2;

[1160] (ii) 35-45 mol.% sterol (e.g., Cholesterol);

[1161] (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and

[1162] (iv) 1.5-3.5 mol.% PEG-lipid (e.g., Compound P-l or PEG-DMG).

[1163] In some embodiments, the lipid nanoparticle (“LNP-2A”) may comprise the following:

[1164] (i) 45-50 mol.% Compound I-2;

[1165] (ii) 35-45 mol.% Cholesterol;

[1166] (iii) 8-12 mol.% DSPC; and

[1167] (iv) 1.5-3.5 mol.% PEG-DMG.

[1168] For instance, in some embodiments, the lipid nanoparticle (“LNP-2B”) may comprise the following components at the following molar ratios:

[1169] (i) 45-50 mol.% Compound I-2;

[1170] (ii) 35-45 mol.% Cholesterol;

[1171] (iii) 8-12 mol.% DSPC; and

[1172] (iv) 1.5-3.5 mol.% Compound P-l.

[1173] In some embodiments, the lipid nanoparticle (“LNP-3”) may comprise the following:

[1174] (i) 45-50 mol.% Compound I-3;

[1175] (ii) 35-45 mol.% sterol (e.g., Cholesterol); (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and

[1176] (iv) 1.5-3.5 mol.% PEG-lipid (e.g., Compound P-l or PEG-DMG).

[1177] In some embodiments, the lipid nanoparticle (“LNP-3A”) may comprise the following:

[1178] (i) 45-50 mol.% Compound I-3;

[1179] (ii) 35-45 mol.% Cholesterol;

[1180] (iii) 8-12 mol.% DSPC; and

[1181] (iv) 1.5-3.5 mol.% PEG-DMG.

[1182] In some embodiments, the lipid nanoparticle (“LNP-3B”) may comprise the following:

[1183] (i) 45-50 mol.% Compound I-3;

[1184] (ii) 35-45 mol.% Cholesterol;

[1185] (iii) 8-12 mol.% DSPC; and

[1186] (iv) 1.5-3.5 mol.% Compound P-l.

[1187] In some embodiments, the LNP has a polydispersity value of less than 0.4. In some embodiments, the LNP has a net neutral charge at a neutral pH. In some embodiments, the LNP has a mean diameter of 50-150 nm. In some embodiments, the LNP has a mean diameter of 80-100 nm.

[1188] As generally defined herein, the term “lipid” refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, and prenol lipids. In some instances, the amphiphilic properties of some lipids lead them to form liposomes, vesicles, or membranes in aqueous media.

[1189] In some embodiments, a lipid nanoparticle (LNP) may comprise an ionizable amino lipid. As used herein, the term “ionizable amino lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, an ionizable amino lipid may be positively charged or negatively charged. An ionizable amino lipid may be positively charged, in which case it can be referred to as “cationic lipid”. In certain embodiments, an ionizable amino lipid molecule may comprise an amine group and can be referred to as an ionizable amino lipid. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc. The charged moiety may be anionic ( / '.e., negatively charged) or cationic ( / '.e., positively charged). Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium groups, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively- charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and / or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired.

[1190] It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge” or “partial positive charge” on a molecule. The terms “partial negative charge” and “partial positive charge” are given their ordinary meaning in the art. A “partial negative charge” may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way.

[1191] The ionizable amino lipid is sometimes referred to in the art as an “ionizable cationic lipid”. In some embodiments, the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure.

[1192] In addition to these, an ionizable amino lipid may also be a lipid including a cyclic amine group. In some embodiments, the ionizable amino lipid may be selected from, but not limited to, an ionizable amino lipid described in International Publication Nos. WO2013086354 and WO2013116126; the contents of each of which are herein incorporated by reference in their entirety.

[1193] In yet another embodiment, the ionizable amino lipid may be selected from, but not limited to, Formula CLI-CLXXXXII of US Patent No. 7,404,969; each of which is herein incorporated by reference in their entirety.

[1194] In some embodiments, the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety. In some embodiments, the lipid may be synthesized by methods known in the art and / or as described in International Publication Nos. WO2013086354; the contents of each of which are herein incorporated by reference in their entirety.

[1195] Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to measure zeta potentials. Dynamic light scattering can also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[1196] The size of the nanoparticles can help counter biological reactions such as, but not limited to, inflammation, or can increase the biological effect of the polynucleotide.

[1197] As used herein, “size” or “mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.

[1198] In some embodiments, a polynucleotide of the disclosure is formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and / or about 90 to about 100 nm. In some embodiments, the nanoparticles have a diameter from about 10 to 500 nm. In some embodiments, the nanoparticle has a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.

[1199] In some embodiments, the largest dimension of a nanoparticle composition is 1 pm or shorter (e.g., 1 pm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter).

[1200] A nanoparticle composition can be relatively homogenous. A polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition can have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle composition disclosed herein can be from about 0.10 to about 0.20.

[1201] The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a nanoparticle composition disclosed herein can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about 10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[1202] In some embodiments, the zeta potential of the lipid nanoparticles can be from about 0 mV to about 100 mV, from about 0 mV to about 90 mV, from about 0 mV to about 80 mV, from about 0 mV to about 70 mV, from about 0 mV to about 60 mV, from about 0 mV to about 50 mV, from about 0 mV to about 40 mV, from about 0 mV to about 30 mV, from about 0 mV to about 20 mV, from about 0 mV to about 10 mV, from about 10 mV to about 100 mV, from about 10 mV to about 90 mV, from about 10 mV to about 80 mV, from about 10 mV to about 70 mV, from about 10 mV to about 60 mV, from about 10 mV to about 50 mV, from about 10 mV to about 40 mV, from about 10 mV to about 30 mV, from about 10 mV to about 20 mV, from about 20 mV to about 100 mV, from about 20 mV to about 90 mV, from about 20 mV to about 80 mV, from about 20 mV to about 70 mV, from about 20 mV to about 60 mV, from about 20 mV to about 50 mV, from about 20 mV to about 40 mV, from about 20 mV to about 30 mV, from about 30 mV to about 100 mV, from about 30 mV to about 90 mV, from about 30 mV to about 80 mV, from about 30 mV to about 70 mV, from about 30 mV to about 60 mV, from about 30 mV to about 50 mV, from about 30 mV to about 40 mV, from about 40 mV to about 100 mV, from about 40 mV to about 90 mV, from about 40 mV to about 80 mV, from about 40 mV to about 70 mV, from about 40 mV to about 60 mV, and from about 40 mV to about 50 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be from about 10 mV to about 50 mV, from about 15 mV to about 45 mV, from about 20 mV to about 40 mV, and from about 25 mV to about 35 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be about 10 mV, about 20 mV, about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70 mV, about 80 mV, about 90 mV, and about 100 mV.

[1203] The term “encapsulation efficiency” of a polynucleotide describes the amount of the polynucleotide that is encapsulated by or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. As used herein, “encapsulation” can refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.

[1204] Encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of the polynucleotide in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents.

[1205] Fluorescence can be used to measure the amount of free polynucleotide in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a polynucleotide can be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[1206] The amount of a polynucleotide present in a pharmaceutical composition disclosed herein can depend on multiple factors such as the size of the polynucleotide, desired target and / or application, or other properties of the nanoparticle composition as well as on the properties of the polynucleotide.

[1207] For example, the amount of an mRNA useful in a nanoparticle composition can depend on the size (expressed as length, or molecular mass), sequence, and other characteristics of the mRNA. The relative amounts of a polynucleotide in a nanoparticle composition can also vary.

[1208] The relative amounts of the lipid composition and the polynucleotide present in a lipid nanoparticle composition of the present disclosure can be optimized according to considerations of efficacy and tolerability. For compositions including an mRNA as a polynucleotide, the N: P ratio can serve as a useful metric.

[1209] As the N: P ratio of a nanoparticle composition controls both expression and tolerability, nanoparticle compositions with low N: P ratios and strong expression are desirable. N: P ratios vary according to the ratio of lipids to RNA in a nanoparticle composition.

[1210] In general, a lower N: P ratio is preferred. The one or more RNA, lipids, and amounts thereof can be selected to provide an N: P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N: P ratio can be from about 2:1 to about 8:1. In other embodiments, the N: P ratio is from about 5:1 to about 8:1. In certain embodiments, the N: P ratio is between 5:1 and 6:1. In one specific aspect, the N: P ratio is about is about 5.67:1.

[1211] In addition to providing nanoparticle compositions, the present disclosure also provides methods of producing lipid nanoparticles comprising encapsulating a polynucleotide. Such method comprises using any of the pharmaceutical compositions disclosed herein and producing lipid nanoparticles in accordance with methods of production of lipid nanoparticles known in the art. See, e.g., Wang et al. (2015) “Delivery of oligonucleotides with lipid nanoparticles” Adv. Drug Deliv. Rev. 87:68-80; Silva et al. (2015) “Delivery Systems for Biopharmaceuticals. Part I: Nanoparticles and Microparticles” Curr. Pharm. Technol. 16: 940-954; Naseri et al. (2015) “Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: Structure, Preparation and Application” Adv. Pharm. Bull. 5:305-13; Silva et al. (2015) “Lipid nanoparticles for the delivery of biopharmaceuticals” Curr. Pharm. Biotechnol. 16:291-302, and references cited therein.

[1212] In some embodiments, the LNP formulations described herein can additionally comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in U. S. Pub. No. US20050222064, herein incorporated by reference in its entirety.

[1213] The LNP formulations can further contain a phosphate conjugate. The phosphate conjugate can increase in vivo circulation times and / or increase the targeted delivery of the nanoparticle. Phosphate conjugates can be made by the methods described in, e.g., Inti. Pub. No. WO2013033438 or U. S. Pub. No. US20130196948. The LNP formulation can also contain a polymer conjugate (e.g., a water-soluble conjugate) as described in, e.g., U. S. Pub. Nos. US20130059360, US20130196948, and

[1214] US20130072709. Each of the references is herein incorporated by reference in its entirety.

[1215] The LNP formulations can comprise a conjugate to enhance the delivery of nanoparticles of the present disclosure in a subject. Further, the conjugate can inhibit phagocytic clearance of the nanoparticles in a subject. In some embodiments, the conjugate can be a “self” peptide designed from the human membrane protein CD47 (e.g., the “self” particles described by Rodriguez et al, Science 2013339, 971-975, herein incorporated by reference in its entirety). As shown by Rodriguez et al., the self peptides delayed macrophage-mediated clearance of nanoparticles which enhanced delivery of the nanoparticles.

[1216] The LNP formulations can comprise a carbohydrate carrier. As a non-limiting example, the carbohydrate carrier can include, but is not limited to, an anhydride-modified phytoglycogen or glycogentype material, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin (e.g., Inti. Pub. No. W02012109121, herein incorporated by reference in its entirety).

[1217] The LNP formulations can be coated with a surfactant or polymer to improve the delivery of the particle. In some embodiments, the LNP can be coated with a hydrophilic coating such as, but not limited to, PEG coatings and / or coatings that have a neutral surface charge as described in U. S. Pub. No.

[1218] US20130183244, herein incorporated by reference in its entirety.

[1219] The LNP formulations can be engineered to alter the surface properties of particles so that the lipid nanoparticles can penetrate the mucosal barrier as described in U. S. Pat. No. 8,241,670 or Inti. Pub. No. WO2013110028, each of which is herein incorporated by reference in its entirety.

[1220] The LNP engineered to penetrate mucus can comprise a polymeric material (i.e., a polymeric core) and / or a polymer-vitamin conjugate and / or a tri-block co-polymer. The polymeric material can include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.

[1221] LNP engineered to penetrate mucus can also include surface altering agents such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin p4 dornase alfa, neltenexine, erdosteine) and various DNases including rhDNase.

[1222] In some embodiments, the mucus penetrating LNP can be a hypotonic formulation comprising a mucosal penetration enhancing coating. The formulation can be hypotonic for the epithelium to which it is being delivered. Non-limiting examples of hypotonic formulations can be found in, e.g., Inti. Pub. No. WO2013110028, herein incorporated by reference in its entirety.

[1223] In some embodiments, the polynucleotide described herein is Formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and nontargeted delivery of nucleic acids (Aleku et al. Cancer Res. 200868:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 201250:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 201023:334-344; Kaufmann et al. Microvasc Res 201080:286-293Weide et al. J Immunother. 200932:498-507; Weide et al. J Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl Acad Sci U S A. 2007 6;104:4095-4100; deFougerolles Hum Gene Ther. 2008 19:125-132; all of which are incorporated herein by reference in its entirety).

[1224] In some embodiments, the polynucleotides described herein are Formulated as a solid lipid nanoparticle (SLN), which can be spherical with an average diameter between 10 to 1000 nm. SLNs possess a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized with surfactants and / or emulsifiers. Exemplary SLNs can be those as described in Inti. Pub. No.

[1225] WG2013105101, herein incorporated by reference in its entirety.

[1226] In some embodiments, the polynucleotides described herein can be Formulated for controlled release and / or targeted delivery. As used herein, “controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome. In some embodiments, the polynucleotides can be encapsulated into a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term “encapsulate” means to enclose, surround or encase. As it relates to the formulation of the compounds of the present disclosure, encapsulation can be substantial, complete or partial. The term “substantially encapsulated” means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or greater than 99% of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded or encased within the delivery agent. “Partial encapsulation” or “partially encapsulate” means that less than 10, 10, 20, 30, 4050 or less of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded or encased within the delivery agent.

[1227] Advantageously, encapsulation can be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the present disclosure using fluorescence and / or electron micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, or greater than 99% of the pharmaceutical composition or compound of the present disclosure are encapsulated in the delivery agent.

[1228] In some embodiments, the polynucleotides described herein can be encapsulated in a therapeutic nanoparticle, referred to herein as “therapeutic nanoparticle polynucleotides.” Therapeutic nanoparticles can be Formulated by methods described in, e.g., Inti. Pub. Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, and WO2012054923; and U. S. Pub. Nos. US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286,

[1229] US20120288541, US20120140790, US20130123351 and US20130230567; and U. S. Pat. Nos.

[1230] 8,206,747, 8,293,276, 8,318,208 and 8,318,211, each of which is herein incorporated by reference in its entirety.

[1231] In some embodiments, the therapeutic nanoparticle polynucleotide can be Formulated for sustained release. As used herein, “sustained release” refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time can include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle of the polynucleotides described herein can be Formulated as disclosed in Inti. Pub. No. WO2010075072 and U. S. Pub. Nos. US20100216804, US20110217377, US20120201859 and US20130150295, each of which is herein incorporated by reference in their entirety.

[1232] In some embodiments, the therapeutic nanoparticle polynucleotide can be Formulated to be target specific, such as those described in Inti. Pub. Nos. WO2008121949, WO2010005726, WO2010005725, WO2011084521 and WO2011084518; and U. S. Pub. Nos. US20100069426, US20120004293 and US20100104655, each of which is herein incorporated by reference in its entirety.

[1233] The LNPs can be prepared using microfluidic mixers or micromixers. Exemplary microfluidic mixers can include, but are not limited to, a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and / or a staggered herringbone micromixer (SHM) (see Zhigaltsevet al., “Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing,” Langmuir 28:3633-40 (2012); Belliveau et al., “Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA,” Molecular Therapy-Nucleic Acids. 1:e37 (2012); Chen et al., “Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation,” J. Am. Chem. Soc. 134(16):6948-51 (2012); each of which is herein incorporated by reference in its entirety). Exemplary micromixers include Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM,) from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany. In some embodiments, methods of making LNP using SHM further comprise mixing at least two input streams wherein mixing occurs by microstructure-induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. This method can also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U. S. Pub. Nos. US20040262223 and US20120276209, each of which is incorporated herein by reference in their entirety.

[1234] In some embodiments, the polynucleotides described herein can be Formulated in lipid nanoparticles using microfluidic technology (see Whitesides, George M., “The Origins and the Future of Microfluidics,” Nature 442: 368-373 (2006); and Abraham et al., “Chaotic Mixer for Microchannels,” Science 295: 647-651 (2002); each of which is herein incorporated by reference in its entirety). In some embodiments, the polynucleotides can be Formulated in lipid nanoparticles using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.

[1235] In some embodiments, the polynucleotides described herein can be Formulated in lipid nanoparticles having a diameter from about 1 nm to about 100 nm such as, but not limited to, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and / or about 90 to about 100 nm.

[1236] In some embodiments, the lipid nanoparticles can have a diameter from about 10 to 500 nm. In some embodiments, the lipid nanoparticle can have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.

[1237] In some embodiments, the polynucleotides can be delivered using smaller LNPs. Such particles can comprise a diameter from below 0.1 pm up to 100 nm such as, but not limited to, less than 0.1 pm, less than 1.0 pm, less than 5pm, less than 10 pm, less than 15 urn, less than 20 urn, less than 25 urn, less than 30 urn, less than 35 urn, less than 40 urn, less than 50 urn, less than 55 urn, less than 60 urn, less than 65 urn, less than 70 urn, less than 75 urn, less than 80 urn, less than 85 urn, less than 90 urn, less than 95 urn, less than 100 urn, less than 125 urn, less than 150 urn, less than 175 urn, less than 200 urn, less than 225 urn, less than 250 urn, less than 275 urn, less than 300 urn, less than 325 urn, less than 350 urn, less than 375 urn, less than 400 urn, less than 425 urn, less than 450 urn, less than 475 urn, less than 500 urn, less than 525 urn, less than 550 urn, less than 575 urn, less than 600 urn, less than 625 urn, less than 650 urn, less than 675 urn, less than 700 urn, less than 725 urn, less than 750 urn, less than 775 um, less than 800 um, less than 825 um, less than 850 um, less than 875 um, less than 900 um, less than 925 um, less than 950 um, or less than 975 um.

[1238] The nanoparticles and microparticles described herein can be geometrically engineered to modulate macrophage and / or the immune response. The geometrically engineered particles can have varied shapes, sizes and / or surface charges to incorporate the polynucleotides described herein for targeted delivery such as, but not limited to, pulmonary delivery (see, e.g., Inti. Pub. No. WO2013082111, herein incorporated by reference in its entirety). Other physical features the geometrically engineering particles can include, but are not limited to, fenestrations, angled arms, asymmetry and surface roughness, charge that can alter the interactions with cells and tissues.

[1239] In some embodiment, the nanoparticles described herein are stealth nanoparticles or targetspecific stealth nanoparticles such as, but not limited to, those described in U. S. Pub. No.

[1240] US20130172406, herein incorporated by reference in its entirety. The stealth or target-specific stealth nanoparticles can comprise a polymeric matrix, which can comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polyesters, polyanhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates, polycyanoacrylates, or combinations thereof.

[1241] 6. Therapeutic Polypeptides

[1242] The polynucleotides described herein may encode a therapeutic polypeptide, such as a polypeptide that, when provided to a subject (e.g., a mammalian subject, such as a human), exerts a beneficial effect, such as the alleviation of one or more symptoms of a disease, diminishment of extent of a disease, stabilized (i.e., not worsening) state of a disease, delay or slowing of progression of a disease, or amelioration or palliation of a state of a disease. The disease may be one that is associated with a deficiency in an endogenous version of the polypeptide.

[1243] In some embodiments, the polypeptide encoded by the open reading frame is a secreted protein, (e.g., a cytokine, a growth factor, an enzyme, an immunomodulator, an antibody or antigen-binding fragment thereof, or a cell-penetrating peptide), an extracellular membrane-bound protein, an intracellular membrane-bound protein, a cytoplasmic protein, a cytoskeletal protein, or a nuclear protein.

[1244] In some embodiments, the polypeptide is a protein of the human proteome. For example, the polypeptide may have the amino acid sequence of any one of SEQ ID NOs: 769 to 1392 of PCT / US2013 / 030062; SEQ ID NOs: 884 to 1611 of PCT / US2013 / 030068; SEQ ID NOs: 1827 to 3497 of PCT / US2013 / 030064; SEQ ID NOs: 3858 to 7559 of PCT / US2013 / 030067; SEQ ID NOs: 4672 to 9187 of PCT / US2013 / 030066; SEQ ID NOs: 4704 to 9203 of PCT / US2013 / 030070; SEQ ID NOs: 8144 to 16131 of PCT / US2013 / 030059; SEQ ID NOs: 8922 to 17687 of PCT / US2013 / 030060; and SEQ ID NOs: 35608 to 45601 of PCT / US2013 / 030061, the disclosures of each of such applications are incorporated herein by reference in their entirety.

[1245] In some embodiments, the polypeptide has an amino acid sequence that is at least 70% identical to the amino acid sequence of any one of SEQ ID NOs: 769 to 1392 of PCT / US2013 / 030062; SEQ ID NOs: 884 to 1611 of PCT / US2013 / 030068; SEQ ID NOs: 1827 to 3497 of PCT / US2013 / 030064; SEQ ID NOs: 3858 to 7559 of PCT / US2013 / 030067; SEQ ID NOs: 4672 to 9187 of PCT / US2013 / 030066; SEQ ID NOs: 4704 to 9203 of PCT / US2013 / 030070; SEQ ID NOs: 8144 to 16131 of PCT / US2013 / 030059; SEQ ID NOs: 8922 to 17687 of PCT / US2013 / 030060; and SEQ ID NOs: 35608 to 45601 of

[1246] PCT / US2013 / 030061.

[1247] In some embodiments, the polypeptide has an amino acid sequence that is at least 75% identical to the amino acid sequence of any one of SEQ ID NOs: 769 to 1392 of PCT / US2013 / 030062; SEQ ID NOs: 884 to 1611 of PCT / US2013 / 030068; SEQ ID NOs: 1827 to 3497 of PCT / US2013 / 030064; SEQ ID NOs: 3858 to 7559 of PCT / US2013 / 030067; SEQ ID NOs: 4672 to 9187 of PCT / US2013 / 030066; SEQ ID NOs: 4704 to 9203 of PCT / US2013 / 030070; SEQ ID NOs: 8144 to 16131 of PCT / US2013 / 030059; SEQ ID NOs: 8922 to 17687 of PCT / US2013 / 030060; and SEQ ID NOs: 35608 to 45601 of

[1248] PCT / US2013 / 030061.

[1249] In some embodiments, the polypeptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 769 to 1392 of PCT / US2013 / 030062; SEQ ID NOs: 884 to 1611 of PCT / US2013 / 030068; SEQ ID NOs: 1827 to 3497 of PCT / US2013 / 030064; SEQ ID NOs: 3858 to 7559 of PCT / US2013 / 030067; SEQ ID NOs: 4672 to 9187 of PCT / US2013 / 030066; SEQ ID NOs: 4704 to 9203 of PCT / US2013 / 030070; SEQ ID NOs: 8144 to 16131 of PCT / US2013 / 030059; SEQ ID NOs: 8922 to 17687 of PCT / US2013 / 030060; and SEQ ID NOs: 35608 to 45601 of

[1250] PCT / US2013 / 030061.

[1251] In some embodiments, the polypeptide has an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 769 to 1392 of PCT / US2013 / 030062; SEQ ID NOs: 884 to 1611 of PCT / US2013 / 030068; SEQ ID NOs: 1827 to 3497 of PCT / US2013 / 030064; SEQ ID NOs: 3858 to 7559 of PCT / US2013 / 030067; SEQ ID NOs: 4672 to 9187 of PCT / US2013 / 030066; SEQ ID NOs: 4704 to 9203 of PCT / US2013 / 030070; SEQ ID NOs: 8144 to 16131 of PCT / US2013 / 030059; SEQ ID NOs: 8922 to 17687 of PCT / US2013 / 030060; and SEQ ID NOs: 35608 to 45601 of

[1252] PCT / US2013 / 030061.

[1253] In some embodiments, the polypeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 769 to 1392 of PCT / US2013 / 030062; SEQ ID NOs: 884 to 1611 of PCT / US2013 / 030068; SEQ ID NOs: 1827 to 3497 of PCT / US2013 / 030064; SEQ ID NOs: 3858 to 7559 of PCT / US2013 / 030067; SEQ ID NOs: 4672 to 9187 of PCT / US2013 / 030066; SEQ ID NOs: 4704 to 9203 of PCT / US2013 / 030070; SEQ ID NOs: 8144 to 16131 of PCT / US2013 / 030059; SEQ ID NOs: 8922 to 17687 of PCT / US2013 / 030060; and SEQ ID NOs: 35608 to 45601 of

[1254] PCT / US2013 / 030061.

[1255] In some embodiments, the polypeptide has an amino acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of any one of SEQ ID NOs: 769 to 1392 of PCT / US2013 / 030062; SEQ ID NOs: 884 to 1611 of PCT / US2013 / 030068; SEQ ID NOs: 1827 to 3497 of PCT / US2013 / 030064; SEQ ID NOs: 3858 to 7559 of PCT / US2013 / 030067; SEQ ID NOs: 4672 to 9187 of PCT / US2013 / 030066; SEQ ID NOs: 4704 to 9203 of PCT / US2013 / 030070; SEQ ID NOs: 8144 to 16131 of PCT / US2013 / 030059; SEQ ID NOs: 8922 to 17687 of PCT / US2013 / 030060; and SEQ ID NOs: 35608 to 45601 of PCT / US2013 / 030061.

[1256] In some embodiments, the polypeptide has an amino acid sequenc...

Claims

1. CLAIMS1. A polynucleotide encoding a polypeptide of interest, wherein said polynucleotide comprises, in the 5’-to-3’ direction:3.(a) a 5’-cap structure;4.(b) a 5’-untranslated region (UTR);5.(c) an open reading frame (ORF) encoding the polypeptide of interest;6.(d) a3’-UTR;7.(e) a poly-A region;8.(f) a linker; and9.(g) a modified 3’ region having the structure of Formula I, in the 5’-to-3’ direction:10.(A’) - (X1 - B) - (X2 - C)a - (X3 - D)b - (X4 - Z)c11.Formula I12.wherein:13.A’ is a 20 adenosine poly-A region;14.B is a locked nucleic acid (LN A);15.C is a LNA;16.D is a LNA;17.Z is an inverted nucleoside;18.wherein each of x1, x2, x3, and x4 is a phosphodiester internucleoside linkage; and19.wherein each of a, b, and c is, independently, 0 or 1;20.further wherein:21.when c is 0, each of a and b is 1; and22.when c is 1, i) each of a and b is 0, or ii) one of a or b is 1.

2. The polynucleotide of claim 1, wherein the poly-A region of (e) is from 1 to 200 nucleosides in length;24.optionally wherein the poly-A region of (e) is from 10 to 200 nucleosides in length; optionally wherein the poly-A region of (e) is from 50 to 150 nucleosides in length; and optionally wherein the poly-A region of (e) is from 100 nucleosides in length.

3. The polynucleotide of claim 1 or 2, wherein the LNA of B, C, and D, if present, is LNA-adenosine, wherein the LNA-adenosine has the structure of:

27. 29.LNA-adenosine.

4. The polynucleotide of any one of claims 1 -3, wherein the inverted nucleoside of Z, if present, is an inverted deoxythymidine, wherein the inverted deoxythymidine has the structure of:

31. 33.Inverted deoxythymidine.

5. The polynucleotide of any one of claims 1 -4, wherein the linker comprises an Xbal scar (5’-UCUAG-3’).

6. The polynucleotide of any one of claims 1 -5, wherein the modified 3’ region has the structure of Formula V:

37. 39.Formula V40.or a pharmaceutically acceptable salt thereof.

7. The polynucleotide of any one of claims 1 -5, wherein the modified 3’ region has the structure of Formula VI:

42. 44.Formula VI45.or a pharmaceutically acceptable salt thereof.

8. A polynucleotide encoding a polypeptide of interest, wherein said polynucleotide comprises, in the 5’-to-3’ direction:47.(a) a 5’-cap structure;48.(b) a 5’-untranslated region (UTR);49.(c) an open reading frame (ORF) encoding the polypeptide of interest;50.(d) a 3’-UTR;51.(e) a poly-A region;52.(f) a linker; and53.(g) a modified 3’ region having the structure of Formula II, in the 5’-to-3’ direction:54.(A’) - (X1 - B)a - (X2 - O)b - (X3 - D)c - (X4 - E)d - (X5 - Z)e55.Formula II56.wherein:57.A’ is a poly-A region;58.B is a 2’-modified nucleoside or a LNA;59.C is a poly-A region;60.D is a LNA;61.E is a LNA;62.Z is an inverted nucleoside; wherein each of xi, X2, xs, X4, and xs is, independently, an internucleoside linkage; and wherein each of a, b, c, d, and e is, independently, 0 or 1;63.further wherein:64.when b is 0, (i) each of a, c, and d is 1 and e is 0, or (ii) each of a and e is 1 and each of c and d is 0; and65.when b is 1, each of a, c, and e is 1, and d is 0.

9. The polynucleotide of claim 8, wherein the adenosines of the poly-A region of (e) are unmodified adenosines.

10. The polynucleotide of claim 9, wherein the poly-A region of (e) is from 1 to 200 nucleosides in length.

11. The polynucleotide of claim 10, wherein the poly-A region of (e) is from 10 to 200 nucleosides in length.

12. The polynucleotide of claim 11, wherein the poly-A region of (e) is from 50 to 150 nucleosides in length.

13. The polynucleotide of claim 12, wherein the poly-A region of (e) is 100 nucleosides in length.

14. The polynucleotide of any one of claims 8-13, wherein the linker of (f) comprises a polynucleotide of Formula III in the 5’-to-3’ direction:

76. 78.Formula III79.wherein A’ is the poly-A region of (e);80.B’ is the 3’ modified region of (g);81.n is 0 to 50;82.X is each, independently, O or S; and83.D is a nucleoside, each independently chosen from an adenosine, a modified adenosine, a cytidine, a modified cytidine, a uridine, a modified uridine, a guanosine, or a modified guanosine.

15. The polynucleotide of claim 14, wherein n is from 0 to 30.

16. The polynucleotide of claim 15, wherein n is 26.

17. The polynucleotide of claim 15, wherein n is 0 to 15.

18. The polynucleotide of claim 17, wherein n is 5 to 10.

19. The polynucleotide of claim 18, wherein n is 5.

20. The polynucleotide of claim 19, wherein the linker is an Xbal scar (5’-UCUAG-3’).

21. The polynucleotide of any one of claims 8-20, wherein:90.i) the 2’-modified nucleoside of B, if present, is selected from 2’-fluoro-adenosine and 2’-O-methyl-adenosine, wherein 2’-fluoro-adenosine has the structure of:

92. 94.2’-fluoro-adenosine; and95.the 2’-0-methyl-adenosine has the structure of:

97. 99.2’-O-methyl-adenosine; or100.ii) the LNA of B, D, and E, if present, is each LNA-adenosine having the structure of:

102.

22. The polynucleotide of any one of claims 8-21, wherein each of xi, X2, X3, X4, and xs, if present, is a phosphodiester internucleoside linkage.

23. The polynucleotide of any one of claims 8-22, wherein each adenosine of the A’ poly-A region is an unmodified adenosine ribonucleotide.

24. The polynucleotide of any one of claims 8-23, wherein the A’ poly-A region is from 1 to 200 nucleosides in length.

25. The polynucleotide of claim 24, wherein the A’ poly-A region is from 5 to 200 nucleosides in length.

26. The polynucleotide of claim 25, wherein the A’ poly-A region is from 10 to 100 nucleosides in length.

27. The polynucleotide of claim 26, wherein the A’ poly-A region is from 15 to 25 nucleosides in length.

28. The polynucleotide of claim 27, wherein the A’ poly-A region is 20 nucleosides in length.

29. The polynucleotide of any one of claims 8-28, wherein a is 0.

30. The polynucleotide of any one of claims 8-28, wherein a is 1.

31. The polynucleotide of claim 30, wherein B is LNA-adenosine.

32. The polynucleotide of claim 30, wherein B is 2’-fluoro-adenosine.

33. The polynucleotide of claim 30, wherein B is 2’-0-methyl-adenosine.

34. The polynucleotide of any one of claims 8-33, wherein each adenosine of the C poly-A region is an unmodified adenosine ribonucleotide.

35. The polynucleotide of any one of claims 8-34, wherein b is 0.

36. The polynucleotide of any one of claims 8-34, wherein b is 1.

37. The polynucleotide of claim 36, wherein the C poly-A region is from 1 to 200 nucleosides in length.

38. The polynucleotide of claim 37, wherein the C poly-A region is from 5 to 200 nucleosides in length.

39. The polynucleotide of claim 38, wherein the C poly-A region is from 10 to 100 nucleosides in length.

40. The polynucleotide of claim 39, wherein the C poly-A region is from 15 to 50 nucleosides in length.

41. The polynucleotide of claim 40, wherein the C poly-A region is 20 nucleosides in length.

42. The polynucleotide of any one of claims 8-41, wherein the poly-A regions of A’ and C are 20 nucleosides in length.

43. The polynucleotide of any one of claims 8-42, wherein c is 0.

44. The polynucleotide of any one of claims 8-42, wherein c is 1.

45. The polynucleotide of claim 44, wherein D is LNA-adenosine.

46. The polynucleotide of any one of claims 8-45, wherein d is 0.

47. The polynucleotide of any one of claims 8-45, wherein d is 1.

48. The polynucleotide of claim 47, wherein E is LNA-adenosine.

49. The polynucleotide of any one of claims 8-48, wherein e is 0.

50. The polynucleotide of any one of claims 8-48, wherein e is 1.

51. The polynucleotide of claim 50, wherein the Z is an inverted deoxythymidine having the structure of:

133. 135.Inverted deoxythymidine.

52. The polynucleotide of claim 8, wherein a, b, c, and e is 1, and d is 0.

53. The polynucleotide of claim 52, wherein the linker of (f) is a polynucleotide with the sequence 5’-UCUAG-3’.

54. The polynucleotide of claim 52 or 53, wherein each adenosine of the poly-A region of A’ is an unmodified adenosine; B is a 2’-modified nucleoside; each adenosine of the poly-A region of C is an unmodified adenosine; D is a LNA; and Z is an inverted nucleoside.

55. The polynucleotide of any one of claims 52-54, wherein the 2’-modified nucleoside is selected from 2’-fluoro-adenosine and 2’-0-methyl-adenosine, wherein 2’-fluoro-adenosine has the structure of:

140. 142.2 -fluoro-adenosine: and143.the 2’-0-methyl-adenosine has the structure of:

145. 147.2’-O-methyl-adenosine.

56. The polynucleotide of any one of claims 52-55, wherein the LNA is an LNA-adenosine having the structure of:

150. 152.LNA-adenosine.

57. The polynucleotide of any one of claims 52-56, wherein the inverted nucleoside is an inverted deoxythymidine having the structure of:

155. 157.Inverted deoxythymidine.

58. The polynucleotide of any one of claims 52-57, wherein the poly-A regions of A’ and C are 15-25 nucleosides in length.

59. The polynucleotide of any one of claims 8 and 52-58, wherein the modified 3’ region has the structure of Formula IV, in the 5’-to-3’ direction:159.-(A)20-o-Am-o-(A)20-o-Ai_NA-o-idT160.Formula IV161.wherein A represents an unmodified adenosine ribonucleoside;162.Am represents a 2’-0-methyl-adenosine;163.ALNA represents an LNA-adenosine;164.idT represents an inverted deoxythymidine; and165.each o represents a phosphodiester internucleoside linkage.

60. The polynucleotide any one of claims 8 and 52-58, wherein the modified 3’ region has the structure of Formula VII, in the 5’-to-3’ direction:167.-(A)20-o-Af-o-(A)20-o-Ai_NA-o-idT168.Formula VII169.wherein A represents an unmodified adenosine ribonucleoside;170.Af represents a 2’-fluoro-adenosine;171.ALNA represents an LNA-adenosine;172.idT represents an inverted deoxythymidine; and173.each o represents a phosphodiester internucleoside linkage.

61. The polynucleotide of claim 8, wherein a, c, and d is 1, and b and e is 0.

62. The polynucleotide of claim 61, wherein each adenosine of the poly-A region of A’ is an unmodified adenosine; B is a LNA; D is a LNA; E is a LNA; and Z is an inverted nucleoside.

63. The polynucleotide of claim 61 or 62, wherein the LNA is an LNA-adenosine.

64. The polynucleotide of any one of claims 61-63, wherein the inverted nucleoside is an inverted deoxythymidine.

65. The polynucleotide of any one of claims 61 -64, wherein the linker is a polynucleotide with the sequence 5’-UCUAG-3’ (Xbal scar).

66. The polynucleotide of any one of claims 61-65, wherein the linker is a polynucleotide having the sequence 5’-(Xbal scar)-(A)2o-Af-3’:180.wherein Af represents a 2’-fluoro-adenosine; and181.Xbal scar represents (5’-UCUAG-3’).

67. The polynucleotide of any one of claims 61-65, wherein the linker is a polynucleotide having the sequence 5’-(Xbal scar)-(A)2o-Am-3’:182.wherein Am represents a 2’-0-methyl-adenosine; and183.Xbal scar represents (5’-UCUAG-3’).

68. The polynucleotide any one of claims 61-67, wherein the poly-A regions of A’ is 15-25 nucleosides in length.

69. The polynucleotide of any one of claims 8 and 61 -68, wherein the modified 3’ region has the structure of Formula VIII, in the 5’-to-3’ direction:186.-(A)20-O-ALNA-O-ALNA-O-ALNA187.Formula VIII188.wherein A represents an unmodified adenosine ribonucleoside;189.ALNA represents an LNA-adenosine; and190.each o represents a phosphodiester internucleoside linkage.

70. The polynucleotide of claim 8, wherein a and e are 1, and b, c, and d is 0.

71. The polynucleotide of claim 70, wherein each adenosine of the poly-A region of A’ is an unmodified adenosine; B is a LNA; and Z is an inverted nucleoside.

72. The polynucleotide of claim 71, wherein the LNA is an LNA-adenosine.

73. The polynucleotide of claim 72, wherein the inverted nucleoside is an inverted deoxythymidine.

74. The polynucleotide of claim 73, wherein the poly-A regions of A’ is 15-25 nucleosides in length.

75. The polynucleotide of claim 74, wherein the linker is a polynucleotide with the sequence 5’- UCUAG-3’ (Xbal scar).

76. The polynucleotide of claim 74, wherein the linker is a polynucleotide having the sequence 5’- (Xbal scar)-(A)20-Af-3’:198.wherein Af represents a 2’-fluoro-adenosine; and199.Xbal scar represents (5’-UCUAG-3’).

77. The polynucleotide of claim 74, wherein the linker is a polynucleotide having the sequence 5’-(Xbal scar)-(A)2o-Am-3’:201.wherein Am represents a 2’-0-methyl-adenosine; and202.Xbal scar represents (5’-UCUAG-3’).

78. The polynucleotide of any one of claims 8 and 70-77, wherein the modified 3’ region has the structure of Formula IX, in the 5’-to-3’ direction:203.-(A)20-o-Ai_NA-o-idT204.Formula IX205.wherein A represents an unmodified adenosine ribonucleoside;206.ALNA represents an LNA-adenosine;207.idT represents an inverted deoxythymidine; and208.each o represents a phosphodiester internucleoside linkage.

79. A polynucleotide encoding a polypeptide of interest, wherein said polynucleotide comprises, in the 5’-to-3’ direction:210.(a) a 5’-cap structure;211.(b) a 5’-untranslated region (UTR);212.(c) an open reading frame (ORF) encoding the polypeptide of interest;213.(d) a 3’-UTR;214.(e) a poly-A region;215.(f) a linker; and216.(g) a modified 3’ region having the structure of Formula XII, in the 5’-to-3’ direction:217.(A’) - (X1 - B) - (X2 - C)a - (X3 - D)b - (X4 - E)c - (X5 - F)d - (X6 - Z)e218.Formula XII219.wherein:220.A’ is a poly-A region comprising 10 to 100 nucleosides;221.B is a bridged nucleic acid (BNA);222.C is a BNA;223.D is a BNA;224.E is a BNA;225.F is a BNA;226.Z is an inverted nucleoside;227.wherein each of xi, X2, X3, X4, xs, and xe is, independently, an internucleoside linkage; and wherein each of a, b, c, d, and e is, independently, 0 or 1;228.wherein:229.each BNA, independently, has the structure of Formula XI:230.base232. 234.O235.^ / W IW236.Formula XI wherein:237.each R1is, independently, -C(R2)2-, -C(0)-, -0-, -S-, or -NR3-;238.each R2is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, 04-Cs aryl, or C3-C7 heteroaryl;239.each R3is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, 04-Cs aryl, or C3-C7 heteroaryl;240.n is 1 or 2;241.X is one of -C(R4)2-, -C(O)-, -0-, -S-, or -NR5-;242.each R4is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-0s aryl, or C3-O7 heteroaryl;243.each R5is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-0s aryl, or C3-O7 heteroaryl; and244.the base is a nucleobase,245.further wherein:246.when e is 0, i) one of a, b, c, or d is 1 or ii) two of a, b, c, or d, is 1; and247.when e is 1, i) each of a, b, c, or d is 0 or ii) one of a, b, c, or d is 1.

80. The polynucleotide of claim 79, wherein the poly-A region of A’ is from 10 to 30 nucleosides in length.

81. The polynucleotide of claim 80, wherein the poly-A region of A’ is 15 nucleosides in length.

82. The polynucleotide of claim 80, wherein the poly-A region of A’ is 20 nucleosides in length.

83. The polynucleotide of any one of claims 79-82, wherein each of x1, x2, x3, x4, x5, and x6, if present, is a phosphodiester internucleoside linkage.

84. The polynucleotide of any one of claims 79-83, wherein the poly-A region of (e) is from 1 to 200 nucleosides in length;253.optionally wherein the poly-A region of (e) is from 10 to 200 nucleosides in length; optionally wherein the poly-A region of (e) is from 50 to 150 nucleosides in length; and optionally wherein the poly-A region of (e) is from 100 nucleosides in length.

85. The polynucleotide of any one of claims 79-84, wherein the BNA has the structure of Formula XXXIV:

255. 257.Formula XXXIV258.wherein:259.n is 1 or 2;260.X is one of -C(R4)2-, -C(O)-, -0-, -S-, or -NR5-;261.each R4is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-0s aryl, or C3-O7 heteroaryl;262.each R5is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, C4-0s aryl, or C3-O7 heteroaryl; and263.the base is a nucleobase.

86. The polynucleotide of any one of claims 79-84, wherein the BNA has the structure of Formula XXXV:

266. 268.Formula XXXV269.wherein:270.each R1is, independently, -C(R2)2-, -C(O)-, -O-, -S-, or -NR3-;271.each R2is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, 04-Cs aryl, or C3-C7 heteroaryl;272.each R3is, independently, H, Ci-Ce alkyl, Ci-Ce heteroalkyl, 04-Cs aryl, or C3-C7 heteroaryl;273.n is 1 or 2; and274.the base is a nucleobase.

87. The polynucleotide of any one of claims 79-86, wherein the BNA of B, C, D, E, and F, if present, is LNA-adenosine, LNA-thymidine, LNA-cytidine, LNA-(5-methyl-cytidine), ENA-adenosine, or BNA-NC-adenosine,276.wherein the LNA-adenosine has the structure of:

277.

278. LNA-adenosine;279.wherein the LNA-thymidine has the structure of:

281.

282. LNA-thymidine;283.wherein the LNA-cytidine has the structure of:

285.

286. LNA-cytidine;287.wherein the LNA-(5-methyl-cytidine) has the structure of:

289.

290. LNA-(5-methyl-cytidine); wherein the ENA-adenosine has the structure of:

292.

293. ENA-adenosine; and wherein the BNA-NC-adenosine has the structure of:

295. 297.BNA-NC-adenosine.

88. The polynucleotide of any one of claims 79-87, wherein the inverted nucleoside of Z, if present, is an inverted deoxythymidine, wherein the inverted deoxythymidine has the structure of:

300. 302.Inverted deoxythymidine.

89. The polynucleotide of any one of claims 79-88, wherein the linker comprises an Xbal scar (5’-UCUAG-3’).

90. The polynucleotide of claim 79, wherein the modified 3’ region has the structure of Formula XIV:

306. 308.Formula XIV309.or a pharmaceutically acceptable salt thereof.

91. The polynucleotide of claim 79, wherein the modified 3’ region has the structure of Formula XV:

311. 313.Formula XV314.or a pharmaceutically acceptable salt thereof.

92. The polynucleotide of claim 79, wherein the modified 3’ region has the structure of Formula XVI:

317. 319.Formula XVI320.or a pharmaceutically acceptable salt thereof.

93. The polynucleotide of claim 79, wherein the modified 3’ region has the structure of Formula XVII:

322. 324.Formula XVII325.or a pharmaceutically acceptable salt thereof.

94. The polynucleotide of claim 79, wherein the modified 3’ region has the structure of Formula XVIII:

328. 330.Formula XVIII331.or a pharmaceutically acceptable salt thereof.

5. The polynucleotide of claim 79, wherein the modified 3’ region has the structure of Formula XIX:

333. 335.Formula XIX336.or a pharmaceutically acceptable salt thereof.

96. The polynucleotide of claim 79, wherein the modified 3’ region has the structure of Formula XX338.

339.

7. The polynucleotide of claim 79, wherein the modified 3’ region has the structure of Formula XXI:

341. 343.Formula XXI344.or a pharmaceutically acceptable salt thereof.

8. The polynucleotide of claim 79, wherein the modified 3’ region has the structure of Formula XXXI:

346. 348.Formula XXXI349.or a pharmaceutically acceptable salt thereof.

99. The polynucleotide of claim 79, wherein the modified 3’ region has the structure of Formula XXXII:

351. 353.Formula XXXII354.or a pharmaceutically acceptable salt thereof.

100. A polynucleotide encoding a polypeptide of interest, wherein said polynucleotide comprises, in the 5’-to-3’ direction:356.(a) a 5’-cap structure;357.(b) a 5’-untranslated region (UTR);358.(c) an open reading frame (ORF) encoding the polypeptide of interest;359.(d) a 3’-UTR;360.(e) a poly-A region;361.(f) a linker; and362.(g) a modified 3’ region having the structure of Formula XXII, in the 5’-to-3’ direction:363.(A’) - (X1 - B) - (X2 - C)a - (X3 - D)b - (X4 - E)c - (X5 - F)d - (X6 - G)e - (X7 - Z)f Formula XXII364.wherein:365.A’ is a poly-A region comprising 10 to 100 nucleosides; B is an extended nucleic acid;366.C is an extended nucleic acid;367.D is an extended nucleic acid;368.E is a LNA;369.F is a LNA;370.G is a LNA;371.Z is an inverted nucleoside;372.wherein each of xi, X2, xs, X4, xs, xe, and x? is a phosphodiester internucleoside linkage; and wherein each of a, b, c, d, e, and f is, independently, 0 or 1;373.wherein:374.each extended nucleic acid (ex(base)), independently, has the structure of Formula XXIII:

376. 378.Formula XXIII,379.wherein the base is a nucleobase,380.further wherein:381.when f is 0, i) each of a, b, c, d, and e is 1 or ii) each of a and b is 0 and each of c, d, and e is 1; and382.when f is 1, i) each of a and b is 1 and one of c, d, or e is 1 or ii) each of a and b is 0 and one of c, d, or e is 1.

101. The polynucleotide of claim 100, wherein the poly-A region of A’ is from 10 to 30 nucleosides in length.

102. The polynucleotide of claim 101, wherein the poly-A region of A’ is 20 nucleosides in length.

103. The polynucleotide of any one of claims 100-102, wherein each of xi, X2, X3, X4, xs, xe, and x?, if present, is a phosphodiester internucleoside linkage.

104. The polynucleotide of any one of claims 100-103, wherein the poly-A region of (e) is from 1 to 200 nucleosides in length;387.optionally wherein the poly-A region of (e) is from 10 to 200 nucleosides in length;388.optionally wherein the poly-A region of (e) is from 50 to 150 nucleosides in length; and optionally wherein the poly-A region of (e) is from 100 nucleosides in length.

105. The polynucleotide of any one of claims 100-104, wherein the LNA of E, F, and G, if present, is a LNA-adenosine having the structure of:

390. 392.LNA-adenosine.

106. The polynucleotide of any one of claims 100-105, wherein the inverted nucleoside of Z, if present, is an inverted deoxythymidine having the structure of:

395. 397.Inverted deoxythymidine.

107. The polynucleotide of any one of claims 100-106, wherein the extended nucleic acid is an extended adenosine (exA) having the structure of:

400. 402.Extended adenosine.

108. The polynucleotide of any one of claims 100-107, wherein the linker comprises an Xbal scar (5’-UCUAG-3’).

109. The polynucleotide of claim 100, wherein the modified 3’ region comprises the structure of Formula XXIV:

405. 407.Formula XXIV408.or a pharmaceutically acceptable salt thereof.

110. The polynucleotide of claim 100, wherein the modified 3’ region comprises the structure of Formula XXV:

411.

412. Formula XXV413.or a pharmaceutically acceptable salt thereof.

111. The polynucleotide of claim 100, wherein the modified 3’ region comprises the structure of Formula XXVI:

417. 419.Formula XXVI420.or a pharmaceutically acceptable salt thereof.

112. The polynucleotide of any one of claims 1-111, wherein the polynucleotide is an RNA polynucleotide, optionally wherein the polynucleotide is an mRNA polynucleotide.

113. The polynucleotide of any one of claims 1-112, wherein each nucleoside in the ORF is, independently, selected from an adenosine, a modified adenosine, a uridine, a modified uridine, a guanosine, a modified guanosine, a cytidine, and a modified cytidine.

114. The polynucleotide of claim 113, wherein the modified uridine is a N1 -methylpseudouridine.

115. A pharmaceutical composition comprising the polynucleotide of any one of claims 1-114 and a pharmaceutically acceptable carrier.

116. A method of expressing a polypeptide of interest in a subject, the method comprising administering to the subject the polynucleotide of any one of claims 1-114 or the pharmaceutical composition of claim 115.

117. A method of prophylactical ly treating a subject at risk of developing a disease, the method comprising administering to the subject the polynucleotide of any one of claims 1 -114 or the pharmaceutical composition of claim 115, wherein the polypeptide of interest encoded by the ORF corresponds to an antigen associated with said disease.

118. The method of claim 116 or 117, wherein the subject is a human.

119. A method of expressing a polypeptide of interest in a cell or population of cells, the method comprising providing to the cell or population of cells the polynucleotide of any one of claims 1 -114 or the pharmaceutical composition of claim 115.

120. A kit comprising the polynucleotide of any one of claims 1 -114 or the pharmaceutical composition of claim 115 and a package insert, wherein the package insert instructs a user of the kit to perform the method of any one of claims 116-119.

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