Methods of delivering naked polyribonucleotides

Abstract

The present disclosure provides methods of delivering naked polyribonucleotides. The present disclosure also provides polyribonucleotides that can be useful in such methods and kits comprising the same.

Description

Attorney Docket No.: 2012611-0182 METHODS OF DELIVERING NAKED POLYRIBONUCLEOTIDES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No.63 / 646,773 filed on May 13, 2024, the entire contents of which is hereby incorporated in its entirety. BACKGROUND

[0002] The past decade has seen large technological progress in the use of polyribonucleotides as a therapeutic modality. However, to fully realize the potential of RNA therapeutics, there is still much progress to be made on further improvements, particularly for methods of delivering RNAs, such as the ability to deliver naked RNAs. SUMMARY

[0003] The present disclosure recognizes that the use of naked polyribonucleotides as a therapeutic agent has been challenging (e.g., short durations of expression and variable expression levels from delivery of naked polyribonucleotides). Indeed, presently, therapeutic polyribonucleotides are delivered to subjects using some type of delivery vehicle (e.g., lipid nanoparticles) to address the shortcomings that have been observed with delivering naked polyribonucleotides. However, use of a delivery vehicle can add complications to the manufacture of polyribonucleotide therapeutics. Delivery vehicles can also introduce safety, efficacy, and regulatory hurdles. As such, lipid nanoparticles have not fully solved the problems encountered with polyribonucleotide administration and the ability to effectively deliver naked polyribonucleotides to subjects remains an unmet need.

[0004] Additionally, the present disclosure provides the recognition that the amount of polyribonucleotides that cells at an administration site (e.g., an injection site) are able to take up is saturable. In other words, cells at a single administration site are only able to take up a limited amount of polyribonucleotide, e.g., RNA. The limited amount of polyribonucleotides that cells at a single administration site can take up is often less than a dose, e.g., a full dose, required for a therapeutic effect, which results in wasted amounts of polyribonucleotide being administered and / or lower efficacy than expected due to a lower level of polyribonucleotide uptake. Thus, a Page 1 of 87 12767355v1Attorney Docket No.: 2012611-0182 problem in the field is how to deliver a dose, e.g., a full dose, of a polyribonucleotide to a subject without saturating the cells at the administration site. The present disclosure describes that administering polyribonucleotides to multiple administration sites such that each administration site receives less than a dose (e.g., such that the cumulative dose from each administration site makes up a total dose) increases the overall uptake of the administered polyribonucleotides.

[0005] The present disclosure further provides that both problems – e.g., delivery of naked polyribonucleotides and efficient polyribonucleotide uptake – can be solved using methods for administering one or more naked polyribonucleotides to a subject, and in particular, using a split- dosing approach. In such an approach, a dose of one or more naked polyribonucleotides is administered to a subject via administration of at least two or more subdoses at two or more administration sites on the subject such that the subject receives the dose of the naked polyribonucleotide. For example, if a dose of an RNA is 3 µg is to be administered to a subject, it can be administered to a subject as three 1 µg-subdoses, wherein each subdose is administered to a different administration site on the subject. A split-dosing approach as provided herein can be beneficial because of: (i) increased expression of a payload; (ii) increased presence (e.g., increased persistence and / or bioavailability) of a payload; or (iii) both (i) and (ii), as compared to expression and / or presence of a payload delivered by an otherwise similar polyribonucleotide administered in a single dose or at a single site. Also provided herein are compositions comprising a naked polyribonucleotide for administration in a split-dosing approach disclosed herein, and kits comprising the same.

[0006] Provided herein is a method for administering two or more subdoses of a naked polyribonucleotide to a subject at two or more administration sites on the subject so that the subject receives a dose of the naked polyribonucleotide. In some embodiments, administration of two or more subdoses is performed in 30 minutes or less.

[0007] This disclosure also provides a kit comprising two or more subdoses of a naked polyribonucleotide formulated for administration to a subject. In some embodiments of a kit provided herein, two or more subdoses together make up a dose. Further provided herein is a kit comprising a dose of a naked polyribonucleotide formulated for administration to a subject.

[0008] In some embodiments of a kit provided herein, a kit further comprises a multi-needle injector. In some embodiments, a multi-needle injector comprises: (i) one or more cavities, and Page 2 of 87 12767355v1Attorney Docket No.: 2012611-0182 (ii) two or more needles, and wherein one or more cavities are configured to hold a dose of a naked polyribonucleotide, wherein one or more cavities are connected to two or more needles so that a dose of naked polyribonucleotide can traverse from one or more cavities to the two or more needles, and wherein two or more needles are each configured to deliver a subdose of a naked polyribonucleotide to a subject so that a subject receives a dose of a naked polyribonucleotide.

[0009] Among other things, provided herein is a multi-needle injector comprising: (i) one or more cavities, (ii) two or more needles, and (iii) a dose of a naked polyribonucleotide formulated for administration to a subject. In some embodiments, one or more cavities are configured to hold a dose of a naked polyribonucleotide. In some embodiments, one or more cavities are connected to two or more needles so that a dose of naked polyribonucleotide can traverse from one or more cavities to two or more needles. In some embodiments, two or more needles are each configured to deliver a subdose of a naked polyribonucleotide to a subject so that a subject receives a dose of a naked polyribonucleotide.

[0010] In some embodiments of any of the methods or kits provided herein, two or more subdoses together make up a dose.

[0011] In some embodiments of any of the methods or kits provided herein, a subdose comprises about 5% to about 95% of a dose, about 5% to about 90%, about 5% to about 80%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 95%, about 20% to about 95%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, or about 90% to about 95% of a dose.

[0012] In some embodiments, a subdose comprises about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90 %, or about 95% of a dose.

[0013] In some embodiments, a subdose comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at Page 3 of 87 12767355v1Attorney Docket No.: 2012611-0182 least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90 %, or at least 95% of a dose.

[0014] In some embodiments of any of the methods or kits provided herein, the subject is administered three or more subdoses, four or more subdoses, five or more subdoses, six or more subdoses, seven or more subdoses, eight or more subdoses, nine or more subdoses or ten or more subdoses.

[0015] In some embodiments of any of the methods or kits provided herein, each subdose of the two or more subdoses are the same. In some embodiments, each subdose of the two or more subdoses are different.

[0016] In some embodiments of any of the methods or kits provided herein, two or more administration sites are within a specified distance of each other. In some embodiments, a specified distance is about 2 square inches to about 30 square inches, about 2 square inches to about 25 square inches, about 2 square inches to about 24 inches, about 2 square inches to about 15 square inches, about 2 square inches to about 10 square inches, about 2 square inches to about 5 square inches, about 5 square inches to about 30 square inches, about 10 square inches to about 30 square inches, about 15 square inches to about 30 square inches, about 20 square inches to about 30 square inches, about 25 to about 30 square inches of each other.

[0017] In some embodiments, a specified distance is about 2 square inches, about 3 square inches, about 4 square inches, about 5 square inches, about 6 square inches, about 7 square inches, about 8 square inches, about 9 square inches, about 10 square inches, about 11 square inches, about 12 square inches, about 13 square inches, about 14 square inches, about 15 square inches , about 16 square inches, about 17 square inches, about 18 square inches, about 19 square inches, about 20 square inches, about 21 square inches, about 22 square inches, about 23 square inches, about 24 square inches, about 25 square inches, about 26 square inches, about 27 square inches, about 28 square inches, about 29 square inches, about 30 square inches of each other.

[0018] In some embodiments, a specified distance is at least 2 square inches, at least 3 square inches, at least 4 square inches, at least 5 square inches, at least 6 square inches, at least 7 square inches, at least 8 square inches, at least 9 square inches, at least 10 square inches, at least 11 square inches, at least 12 square inches, at least 13 square inches, at least 14 square inches, at least 15 square inches, at least 16 square inches, at least 17 square inches, at least 18 square Page 4 of 87 12767355v1Attorney Docket No.: 2012611-0182 inches, at least 19 square inches, at least 20 square inches, at least 21 square inches, at least 22 square inches, at least 23 square inches, at least 24 square inches, at least 25 square inches, at least 26 square inches, at least 27 square inches, at least 28 square inches, at least 29 square inches, at least 30 square inches of each other.

[0019] In some embodiments, a specified distance is about 1mm radius to about 50 mm radius, about 1 mm radius to about 45 mm radius, about 1 mm radius to about 40 mm radius, about 1 mm radius to about 35 mm radius, about 1 mm radius to about 30 mm radius, about 1 mm radius to about 25 mm radius, about 1 mm radius to about 20 mm radius, about 1 mm radius to about 15 mm radius, about 1 mm radius to about 10 mm radius. About 1 mm radius to about 5 mm radius, about 5 mm radius to about 50 mm radius, about 10 mm radius to about 50 mm radius, about 15 mm radius to about 50 mm radius, about 20 mm radius to about 50 mm radius, about 25 mm radius to about 50 mm radius, about 30 mm radius to about 50 mm radius, about 35 mm radius to about 50 mm radius, about 35 mm radius to about 50 mm radius, about 40 mm radius to about 50 mm radius, about 45 mm radius to about 50 mm radius of each other.

[0020] In some embodiments, a specified distance is about 1 mm radius, about 5 mm radius,about 10 mm radius, about 15 mm radius ̧about 20 mm radius ̧about 25 mm radius¸ about 30mm radius, about 35 mm radius ̧about 40 mm radius, about 45 mm radius, about 50 mm radiusof each other. In some embodiments, a specified distance is at least 1 mm radius, at least 5 mmradius, at least 10 mm radius, at least 15 mm radius ̧at least 20 mm radius¸ at least 25 mmradius ̧at least 30 mm radius, at least 35 mm radius ̧at least 40 mm radius, at least 45 mmradius, at least 50 mm radius of each other.

[0021] In some embodiments of any of the methods or kits provided herein, the two or more administration sites are in the same tissue. In some embodiments, two or more administration sites are in different tissue.

[0022] In some embodiments, the two or more administration sites are in muscle tissue. In some embodiments, a muscle tissue comprises striated muscle, non-striated muscle, or both. In some embodiments, a muscle tissue comprises skeletal muscle, smooth muscle, cardiac muscle, or any combination thereof. In some embodiments, skeletal muscle comprises a Type I muscle, a Type II muscle, or both. Page 5 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0023] In some embodiments of any of the methods or kits provided herein, two or more subdoses are administered at two or more administration sites at substantially the same time. In some embodiments, two or more subdoses are administered at two or more administration sites simultaneously. In some embodiments, two or more subdoses are administered at two or more administration sites sequentially. In some embodiments, two or more subdoses are administered in about 25 minutes to about 1 second, about 25 minutes to about 30 seconds, about 25 minutes to about 1 minute, about 25 minutes to about 2 minutes, about 25 minutes to about 5 minutes, about 25 minutes to about 10 minutes, about 25 minutes to about 15 minutes, about 25 minutes to about 20 minutes, about 20 minutes to about 1 second, about 15 minutes to about 1 second, about 10 minutes to about 1 second, about 5 minutes to about 1 second, about 2 minutes to about 1 second, about 1 minute to about 1 second, about 30 seconds to about 1 second of each other.

[0024] In some embodiments, two or more subdoses are administered about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 2 minutes, about 1 minute, about 45 seconds, about 30 seconds, about 15 seconds, about 5 seconds, or about 1 second of each other. In some embodiments, two or more subdoses are administered at least 20 minutes, at least 15 minutes, at least 10 minutes, at least 5 minutes, at least 2 minutes, at least 1 minute, at least 45 seconds, at least 30 seconds, at least 15 seconds, at least 5 seconds, or at least 1 second of each other.

[0025] In some embodiments of any of the methods or kits provided herein, a naked polyribonucleotide disclosed herein is linear.

[0026] In some embodiments of any of the methods or kits provided herein, a naked polyribonucleotide is circular.

[0027] In some embodiments, a naked polyribonucleotide comprises naturally occurring polyribonucleotides. In some embodiments, a naked polyribonucleotide comprises only naturally occurring polyribonucleotides.

[0028] In some embodiments, a naked polyribonucleotide does not comprise one or more modified ribonucleotides. Page 6 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0029] In some embodiments, a polynucleotide is a naked polyribonucleotide comprising one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0030] In some embodiments, one or more modified ribonucleotides has: a 5’ monophosphate; a 5’ diphosphate; or a 5’ triphosphate. In some embodiments, one or more modified ribonucleotides comprises a 5’ triphosphate.

[0031] In some embodiments, one or more modified ribonucleotides comprises a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3A. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3B when incorporated in a naked polynucleotide (e.g., naked polyribonucleotide). In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 3B.

[0032] In some embodiments, the nucleoside has a structure of: O H3C NH O .

[0033] In some embodiments, one orhas the structure of: O H3C NH O .

[0034] In some embodiments, one or has the structure of:Page 7 of 87 12767355v1Attorney Docket No.: 2012611-0182 O .

[0035] In some embodiments, one or more modified ribonucleotides has the structure of: O O .

[0036] In some embodiments, a naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NH O , wherein indicates the positionadjacent ribonucleotide.

[0037] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O H3CNH O , wherein indicates the ribonucleotide.Page 8 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0038] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O O ,wherein indicates the position of attachment to an adjacent ribonucleotide.

[0039] In some embodiments, a naked polyribonucleotide comprises cytidine nucleosides, and at least 5% of cytidine nucleosides in a polyribonucleotide comprise N4-acetylcytidine.

[0040] In some embodiments, a naked polyribonucleotide comprises cytidine nucleosides, and less than 100% of cytidine nucleosides in the naked polyribonucleotide comprise N4- acetylcytidine.

[0041] In some embodiments, a naked polyribonucleotide comprises cytidine nucleosides, and at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of cytidine nucleosides in a naked polyribonucleotide comprise N4-acetylcytidine.

[0042] In some embodiments, one or more modified ribonucleotides comprises a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3A. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3B when incorporated in a naked polynucleotide (e.g., naked polyribonucleotide). In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 3B.

[0043] In some embodiments, the nucleoside has a structure of: Page 9 of 87 12767355v1Attorney Docket No.: 2012611-0182 O .

[0044] In some embodiments, one or more modified ribonucleotides has a structure of: OH O O .

[0045] In some embodiments, one or more has a structure of: OH O O .

[0046] In some embodiments, onehas a structure of: OH O NH O .

[0047] In some embodiments, anaked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: OH O NH N O , wherein indicates the position ribonucleotide.12767355v1Attorney Docket No.: 2012611-0182

[0048] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: OH O O ,wherein indicates the position of attachment to an adjacent ribonucleotide.

[0049] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: OH O O , wherein indicates theribonucleotide.

[0050] In some embodiments, a naked polyribonucleotide comprises uridine nucleosides and at least 5% of uridine nucleosides in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0051] In some embodiments, a naked polyribonucleotide comprises uridine nucleosides and less than 100% of uridine nucleosides in the naked polyribonucleotide comprise 5- hydroxymethyluridine.

[0052] In some embodiments, a naked polyribonucleotide comprises uridine nucleosides and at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of uridine nucleosides in a naked polyribonucleotide comprise 5- hydroxymethyluridine. Page 11 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0053] In some embodiments, a naked polyribonucleotide comprises uridine nucleosides and more than 60% of uridine nucleosides in a polyribonucleotide comprise 5-hydroxymethyluridine.

[0054] In some embodiments, one or more modified ribonucleotides comprises: N4- acetylcytidine, 5-hydroxymethyluridine, N1-methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5-methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (m1 I), wyosine (imG), methylwyosine (mimG), 5- hydroxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-methoxycytidine, 5- propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5- methyluridine, 2’-O-methyluridine, 2’-O-methyl-5-methyluridine, 2’-fluoro-2’-deoxyuridine, 2’- amino-2’-deoxyuridine, 2’-azido-2’-deoxyuridine, 4-thiouridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5- bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2’-O-methyl-pseudouridine, N1- hydroxypseudouridine, 2’-O-methyl-N1-methylpseudouridine, N1-ethylpseudouridine, N1- hydroxymethylpseudouridine, ara-uridine, N6-methyladenosine, 2-aminoadenosine, 3- methyladenosine, 7-deazaadenosine, 8-oxoadenosine, thienoguanosine, 7-deazaguanosine, 8- oxoguanosine, 6-O-methylguanine, or any combination thereof. In some embodiments, one or more modified ribonucleotides comprises a nucleoside comprising a ribose moiety comprising an acetyl group, wherein the ribose is 2’-O-acetylated.

[0055] In some embodiments, one or more modified ribonucleotide has a structure of: X R O , (a) wherein R is a monophosphate, a and(b) wherein X is an adenine nucleobase, a guanine nucleobase, a cytosine nucleobase (e.g., N4- acetylcytosine), or a uracil nucleobase (e.g., 5-hydroxymethyluracil). Page 12 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0056] In some embodiments, one or more modified ribonucleotides comprises: (i) a 2’-O- acetylated ribose and (ii) an adenine nucleobase. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3D when incorporated in a naked polynucleotide. In some embodiments, a naked polynucleotide (e.g., a naked polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 3D.

[0057] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: H2N N , wherein indicates the positionribonucleotide.

[0058] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: H2N N N N , wherein indicates theribonucleotide.

[0059] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 13 of 87 12767355v1Attorney Docket No.: 2012611-0182 N N ,wherein indicates the position of attachment to an adjacent ribonucleotide.

[0060] In some embodiments, one or more modified ribonucleotides comprises: (i) a 2’-O- acetylated ribose and (ii) a guanine nucleobase. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3D when incorporated in a naked polynucleotide. In some embodiments, a naked polynucleotide (e.g., a naked polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 3D.

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

[0062] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NH N NNH2, wherein indicates the ribonucleotide.Page 14 of 87 12767355v1Attorney Docket No.: 2012611-0182

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

[0064] In some embodiments, one or more modified ribonucleotides comprises: (i) a 2’-O- acetylated ribose and (ii) a cytosine nucleobase (e.g., cytosine or N4-acetylcytosine). In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3D when incorporated in a polynucleotide. In some embodiments, a naked polynucleotide (e.g., a naked polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 3D.

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

[0066] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 15 of 87 12767355v1Attorney Docket No.: 2012611-0182 O ,wherein indicates the position of attachment to an adjacent ribonucleotide.

[0067] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: NH2O , wherein indicates theribonucleotide.

[0068] In some embodiments, one or more modified ribonucleotides comprising a 2’-O- acetylated ribose comprises a N4-acetylcytosine nucleobase. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3D when incorporated in a polynucleotide. In some embodiments, a naked polynucleotide (e.g., a naked polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 3D.

[0069] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O H3CNH N O , wherein indicates the position adjacent ribonucleotide.12767355v1Attorney Docket No.: 2012611-0182

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

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

[0072] In some embodiments, one or more modified ribonucleotides comprising a 2’-O- acetylated ribose comprises a uracil nucleobase (e.g., uracil or 5-hydroxymethyluracil). In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3D when incorporated in a polynucleotide. In some embodiments, a polynucleotide (e.g., a polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 3D.

[0073] In In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: Page 17 of 87 12767355v1Attorney Docket No.: 2012611-0182 O ,wherein indicates the position of attachment to an adjacent ribonucleotide.

[0074] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O O , wherein indicates theribonucleotide.

[0075] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NH O , wherein indicates theribonucleotide.

[0076] In some embodiments, one or more modified ribonucleotides comprising a 2’-O- acetylated ribose comprises a 5-hydroxymethyluracil nucleobase. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3D when incorporated in a naked polynucleotide. In some embodiments, a naked polynucleotide (e.g., a naked polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 3D. Page 18 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0077] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: OH O O ,wherein indicates the position of attachment to an adjacent ribonucleotide.

[0078] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: OH O O , wherein indicates theribonucleotide.

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

[0080] In some embodiments, one or more modified ribonucleotides comprising a 2’-O- acetylated ribose comprises a N1-methylpseudouracil nucleobase. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3C. In some embodiments, one or more modified ribonucleotides has a structure provided in Table 3D when incorporated in a Page 19 of 87 12767355v1Attorney Docket No.: 2012611-0182 polynucleotide. In some embodiments, a naked polynucleotide (e.g., a naked polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 3D.

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

[0082] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NNHO , wherein indicates theribonucleotide.

[0083] In some embodiments, a naked polynucleotide (e.g., naked polyribonucleotide) comprises one or more modified ribonucleotides that have the following structure: O NNHO , wherein indicates the ribonucleotide.

[0084] In some embodiments of any of the naked polyribonucleotides disclosed herein, at least 5% of ribose moieties are acetylated (2’-O-acetylated). Page 20 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0085] In some embodiments of any of the naked polyribonucleotides disclosed herein, about 5% to about 99% of ribose moieties are acetylated (2’-O-acetylated).

[0086] In some embodiments of any of the naked polyribonucleotides disclosed herein, a polyribonucleotide comprises a cap structure and a cap structure does not comprise a 2’-O- acetylated ribose.

[0087] In some embodiments of any of the naked polyribonucleotides disclosed herein, a polyribonucleotide comprises a cap structure and a cap structure comprises a 2’-O-acetylated ribose.

[0088] In some embodiments of any of the naked polyribonucleotides disclosed herein, a polyribonucleotide further comprises one or more ribonucleotides that does not comprise a 2’-O acetylated ribose.

[0089] In some embodiments of any of the naked polyribonucleotides disclosed herein, a polyribonucleotide comprises a coding region. In some embodiments, a payload disclosed herein comprises a polypeptide. In some embodiments, a naked polyribonucleotide disclosed herein comprises an RNA payload.

[0090] In some embodiments, a polyribonucleotide disclosed herein is in a composition. In some embodiments, a composition disclosed herein is a pharmaceutical composition. In some embodiments, a pharmaceutical composition disclosed herein comprises one or more pharmaceutically acceptable excipients.

[0091] In some embodiments, administration of the two or more subdoses provides more consistent expression of the payload as compared to expression of a payload delivered by an otherwise similar polyribonucleotide administered in a single dose or at a single site. In some embodiments, more consistent expression comprises substantially the same expression or higher expression of a payload at two or more administration sites.

[0092] In some embodiments, expression of a payload at an administration site can be determined by obtaining a sample from an administration site and evaluating expression of a payload.

[0093] In some embodiments, a method is a treatment method. Page 21 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0094] In some embodiments, a method is a prevention method.

[0095] In some embodiments, a method is a method to stimulate an immune response.

[0096] In some embodiments, a method is an antibody therapy method.

[0097] In some embodiments, a method is an immune-modulation method.

[0098] In some embodiments, a method is a vaccination method.

[0099] In some embodiments, a method is a gene therapy method.

[0100] In some embodiments, a method is a cell therapy engineering method.

[0101] In some embodiments a method is an immunotherapy method.

[0102] In some embodiments a method is a protein replacement therapy method.

[0103] In some embodiments a method is a chemotherapeutic method.

[0104] In some embodiments, a subject is a human.

[0105] These, and other aspects encompassed by the present disclosure, are described in more detail below and in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] FIG.1 shows a graph of in vivo bioluminescence data after delivery of 5 ug of unmodified naked linear RNA. Bars represent mean ± s.e.m. n=5.

[0107] FIG.2 shows a graph of in vivo bioluminescence data after a single intramuscular injection of naked Luciferase (Luc2) RNA. Intramuscular injection of naked Luciferase RNA resulted in sustained expression of the protein an injection. A boost at Day 21 increased the overall signal luciferase signal in vivo. Bars represent mean ± s.e.m. n=5.

[0108] FIG.3 shows a graph of Luciferase expression from multiple injection methods with naked Luciferase RNA. Intramuscular injection of naked Luciferase RNA through different methods of delivery resulted in similar signal overall. For the single injection groups, animals received one intramuscular (IM) RNA administration of 5 ug in the left lateral muscle. For the split injection groups, animals received two separate IM RNA administrations of 2.5 ug each in the same left lateral muscle. Bars represent mean ± s.e.m. n=5 for mice administered Ringers Page 22 of 87 12767355v1Attorney Docket No.: 2012611-0182 Lactated-buffered solution (RL; buffer) and n=20 for mice administered single injection and split injection of Luciferase RNA.

[0109] FIG.4 shows a bar graph of standard deviation (SD) of Luciferase signal over time. The split injection method (right bar for each time point) reduced variability in signal between mice when compared to a single injection method (middle bar for each time point). The differences in SD was determined by single tailed F test with exact p value shown.

[0110] FIG.5 shows a boxplot with whiskers of total Luciferase expression (as shown by “Total Flux”) from the different injection groups (split injection – right bar for each timepoint; single injection – left bar for each timepoint). Splitting the dose for delivery at multiple administration sites resulted in lower variability between mice, lower number of outliers, and a mean closer to the median. The differences were determined by a single tailed F test with exact p value shown. CERTAIN DEFINITIONS

[0111] About or approximately: As used herein, the terms “about” and “approximately,” when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” or “approximately” in that context. For example, in some embodiments, the term “about” or “approximately” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0112] Administering: As used herein, the term “administering” or “administration” typically refers to administration of a composition to a subject to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, Page 23 of 87 12767355v1Attorney Docket No.: 2012611-0182 intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0113] Delivery / contacting: As used interchangeably herein, the term “delivery,” “delivering,” or “contacting” refers to introduction of a polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) into a target cell. A target cell can be cultured in vitro or ex vivo or be present in a subject (in vivo). Methods of introducing a polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) into a target cell can vary with in vitro, ex vivo, or in vivo applications. In some embodiments, a polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) can be introduced into a target cell in a cell culture by in vitro transfection. In some embodiments, a polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) can be introduced into a target cell in a subject by administering a polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) to a subject.

[0114] Encode: As used herein, the team “encode” or “encoding” refers to a first molecule that is produced by a second molecule, wherein the sequence information of the second molecule determines the sequence of the first molecule. For example, a first molecule can have a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids which are determined by the sequence of the second molecule (e.g., a polynucleotide). For example, a DNA molecule (e.g., a second molecule) can encode an RNA molecule (e.g., a first molecule, for example by a transcription process that includes a DNA-dependent RNA polymerase enzyme) or a polypeptide (e.g., a first molecule for example by a transcription and a translation process). An RNA molecule (e.g., a second molecule) can encode a polypeptide (e.g., a first molecule for example by a translation process). Thus, a gene, a cDNA, or an RNA molecule encodes a Page 24 of 87 12767355v1Attorney Docket No.: 2012611-0182 polypeptide if transcription and translation of RNA corresponding to that gene or cDNA, or translation of the RNA, produces the polypeptide in a cell or other biological system.

[0115] Functional: As used herein, the term “functional” is used to refer to a form or fragment of an entity that exhibits a particular property and / or activity.

[0116] Fragment: A “fragment” of a material or entity as described herein has a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a fragment comprises a polynucleotide fragment. In some embodiments, a fragment comprises a polypeptide fragment. In some embodiments, a polynucleotide fragment or a polypeptide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) as found in the whole polynucleotide or whole polypeptide. In some embodiments, a polynucleotide fragment or a polypeptide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polynucleotide or whole polypeptide. The whole polypeptide or whole polynucleotide may in some embodiments be referred to as the “parent” of the polynucleotide fragment or polypeptide fragment.

[0117] Naked RNA: As used herein, the term “naked RNA” refers to a polyribonucleotide that is: (i) not encapsulated in a delivery vehicle or carrier; (ii) not complexed with a delivery vehicle or carrier; or (iii) not conjugated to a delivery vehicle or carrier. In some embodiments, a delivery vehicle or carrier comprises: a vector (e.g., a viral vector or a non-viral vector), a nanoparticle (e.g., a lipid nanoparticle, a polymeric nanoparticle, a silica nanoparticle, a gold nanoparticle, a magnetic nanoparticle), a lipid-based carrier (e.g., a liposome), a virus-like particle, a quantum dot, a peptide (e.g., a protamine), a polymer (e.g., a cationic polymer), a glycan-based conjugate (e.g., a GalNac conjugate), or any combination thereof.

[0118] Nucleic acid / Oligonucleotide / Polynucleotide: As used herein, the terms “nucleic acid” and “polynucleotide” and “oligonucleotide” are used interchangeably, and refer to a polymer of 3 Page 25 of 87 12767355v1Attorney Docket No.: 2012611-0182 nucleotides or more. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. In some embodiments, a nucleic acid comprises messenger RNA (mRNA). In some embodiments, a nucleic acid is single stranded. In some embodiments, a nucleic acid is double stranded. In some embodiments, a nucleic acid comprises both single and double stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5’-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural nucleosides (e.g., adenosine, cytidine, guanosine, uridine, thymidine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, deoxyuridine). In some embodiments, a nucleic acid comprises one or more, or all, non-natural nucleosides. In some embodiments, a non-natural nucleoside comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O- methylguanine, 2-thiocytidine, a nucleoside comprising a methylated base, a nucleoside comprising an intercalated base, and combinations thereof). In some embodiments, a non-natural nucleoside comprises a modified nucleoside, e.g., as described herein. In some embodiments, a non-natural nucleoside comprises N4-acetylcytidine, 5-hydroxymethyluridine and / or N1- methylpseudouridine. In some embodiments, a non-natural nucleoside comprises one or more modified sugars (e.g., 2’-fluororibose, ribose, 2’-deoxyribose, arabinose, and hexose) as compared to those in natural nucleosides. In some embodiments, a non-natural nucleoside comprises a 2’-O-acetylated ribose. As used herein, “adenosine nucleosides” encompasses a natural adenosine nucleoside, as well as modified adenosine nucleosides. Accordingly, when a percentage of adenosine nucleosides is provided herein, the percentage is based on a total number of natural and modified adenosine nucleosides. Similarly, “cytidine nucleosides” encompasses a natural cytidine nucleoside, as well as modified cytidine nucleosides (e.g., N4- acetylcytidine or 2’-O-acetylated N4-acetylcytidine); “guanosine nucleosides” encompasses a Page 26 of 87 12767355v1Attorney Docket No.: 2012611-0182 natural guanosine nucleoside, as well as modified guanosine nucleosides; and “uridine nucleosides” encompasses a natural uridine nucleoside, as well as modified uridine nucleosides (e.g., 5-hydroxymethyluridine or 2’-O-acetylated 5-hydroxymethyluridine). Further, as used herein, “adenine nucleobase” encompasses a natural adenine nucleobase, as well as modified adenine nucleobase; “cytosine nucleobase” encompasses a natural cytosine nucleobase, as well as modified cytosine nucleobase (e.g., N4-acetylcytosine); “guanine nucleobase” encompasses a natural guanine nucleobase, as well as modified guanine nucleobase; and “uracil nucleobase” encompasses a natural uracil nucleobase, as well as modified uracil nucleobase (e.g., 5- hydroxymethyluracil). In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 nucleotides long. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0119] Polypeptide: The term “polypeptide”, as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids or more. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional, biologically active, or characteristic fragments, portions or domains (e.g., fragments, portions, or domains retaining at least one activity) of such complete polypeptides. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, Page 27 of 87 12767355v1Attorney Docket No.: 2012611-0182 polypeptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

[0120] RNA oligonucleotide: As used herein, the term “RNA oligonucleotide” refers to an oligonucleotide of ribonucleotides. In some embodiments, an RNA oligonucleotide is single stranded. In some embodiments, an RNA oligonucleotide is double stranded. In some embodiments, an RNA oligonucleotide comprises both single and double stranded portions. In some embodiments, an RNA oligonucleotide can comprise a backbone structure as described in the definition of “Nucleic acid / Oligonucleotide” above. An RNA oligonucleotide can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA) oligonucleotide. In some embodiments an RNA oligonucleotide can comprise at its 3’ end a poly(A) region. In some embodiments an RNA oligonucleotide comprises at its 5’ end a cap structure, e.g., for recognizing and attachment of an RNA to a ribosome to initiate translation. In some embodiments, a polynucleotide comprises an RNA oligonucleotide. When a number of ribonucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of ribonucleotides on a single strand.

[0121] Subject: As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0122] Variant: As used herein, the term “variant” refers to an entity that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity. For example, a variant polypeptide Page 28 of 87 12767355v1Attorney Docket No.: 2012611-0182 may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, a variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide shows a reduced level of one or more biological activities as compared with the reference polypeptide.

[0123] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, e.g., RNA synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated herein by reference for any purpose. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0124] RNA-based gene therapies have emerged as a promising therapeutic modality, e.g., due to their ability to manipulate gene expression and / or produce therapeutic proteins, offering the potential to target a wide range of diseases with known genetic targets, including infectious diseases, cancers, immune diseases, and neurological disorders. See, for example: Roberts, T.C. et al., “Advances in oligonucleotide drug delivery” Nat Rev Drug Discov (2020) vol.19, pp. 673–694; and Paunovska, K. et al., “Drug delivery systems for RNA therapeutics,” Nat Rev Genet (2022), vol.23, pp.265–280, the entire contents of each of which is incorporated herein by reference. However, the ability to use RNA-based technologies as a targeted therapeutic Page 29 of 87 12767355v1Attorney Docket No.: 2012611-0182 intervention is hampered without the capacity to efficiently deliver therapeutic RNA to target cells.

[0125] As will be appreciated by one with ordinary skill in the pertinent art upon reading this disclosure, irrespective of the therapeutic mechanism of action of an RNA-based gene therapy, safely delivering naked RNA (e.g., RNA that is unencapsulated, uncomplexed and / or unconjugated to a delivery vehicle or carrier) to a cell is challenging. Without wishing to be bound to any particular theory, naked RNA is thought to be quickly degraded by extracellular RNases and / or is not internalized into cells efficiently, which may result in low uptake, expression or persistence within a cell. See for example, Probst J, et al., “Spontaneous cellular uptake of exogenous messenger RNA in vivo is nucleic acid-specific, saturable and ion dependent;” Gene Ther.2007 (doi: 10.1038 / sj.gt.3302964); and Wolff JA et al., “Direct gene transfer into mouse muscle in vivo,” Science.1990 (doi: 10.1126 / science.1690918), the entire contents of each of which is incorporated herein by reference. Additionally, it has been recently suggested that delivery of naked RNA in vivo is challenging at least due to the expected presence of extracellular RNAses, e.g., in the bloodstream, which function to reduce or limit RNA induced systemic inflammation (Castellano M et al., “Ribonuclease activity undermines immune sensing of naked extracellular RNA.” bioRxiv 2024.04.23.590771, the entire contents of which is incorporated herein by reference). This report showed that efficient delivery of naked RNA into a cell, both in vivo and in vitro, could only be achieved in the presence of ribonuclease inhibitors.

[0126] It has been shown that some fraction of naked RNA that is delivered to cells with a single injection administered to a single administration site can be internalized by muscle cells in vivo and translated into protein; however, the resultant protein expression was observed for a very limited amount of time, with low expression and with significant variability in expression level of the resultant protein among different injection sites, all of which received a single bolus of naked RNA (Wolff 1990). It was later demonstrated that the total mass of RNA that can be taken up by cells is easily saturated, which limits the maximum dose of RNA that can be delivered (Probst 2007). These reports indicate that delivering naked RNA to cells is challenging and that the dose of RNA that is taken up by cells at an administration site is saturable. Page 30 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0127] To address the problem of delivering naked RNA to cells, various delivery vehicles or carriers have been developed in recent years for delivering RNA, such as lipid-based carriers, polymers, protein derivatives, and chemical conjugates. Without wishing to be bound by theory, lipid and polymer-based methods are thought to protect RNA from nuclease and accordingly facilitate cellular uptake. See, for example, Nitika et al., “The Delivery of mRNA Vaccines for Therapeutics,” Life (Basel), 2022, doi: 10.3390 / life12081254; and Hajj, K. et al., “Tools for translation: non-viral materials for therapeutic mRNA delivery,” Nat Rev Mater 2, 17056 (2017), the entire contents of each of which is incorporated herein by reference. However, there are disadvantages to using lipid-based delivery vehicles such as LNPs due to cost and manufacturing challenges.

[0128] Among other things, the present disclosure provides certain insights and technologies relating to improved methods of delivering naked polyribonucleotides. In particular, the present disclosure recognizes that naked polyribonucleotides can be successfully delivered to cells in vivo and can be particularly useful as therapeutic modality.

[0129] The present disclosure provides, among other things, methods and kits that address the limitations of prior methods. In many embodiments the present disclosure provides methods and kits that utilize a split-dosing approach in which a dose of naked polyribonucleotides is split into two or more subdoses and administered to a subject at two or more sites. Furthermore, the present disclosure is the first to recognize that using a split-dosing approach can successfully deliver a dose of naked polyribonucleotides to cells in vivo, resulting in long-term, persistent and / or durable expression of a payload (e.g., RNA payload encoded by a naked polyribonucleotide or polypeptide payload encoded by a naked polyribonucleotide).

[0130] In particular, provided herein is the insight that delivery (e.g., intramuscular delivery) of naked polyribonucleotides using a split-dosing approach disclosed herein to two or more administration sites, results in increased expression of a payload and / or reduced variability in payload expression as compared to delivery of naked polyribonucleotides in one dose to one tissue site (e.g., one muscle tissue site). Without wishing to be bound by any particular theory, delivering naked polyribonucleotides using a split-dosing approach disclosed herein to two or more administration sites: (i) spreads the dose of naked polyribonucleotides across a larger surface area of a target tissue (e.g., muscle tissue) and / or (ii) delivers the dose of naked Page 31 of 87 12767355v1Attorney Docket No.: 2012611-0182 polyribonucleotides to a larger volume of target tissue (e.g., muscle tissue), and prevents saturation of RNA uptake mechanisms that have been observed with single injection delivery of naked RNA.

[0131] The present disclosure also provides the insight that the use of ribonuclease inhibitors is not necessary for in vivo delivery of naked polyribonucleotides to a cell or tissue to provide long- term, persistent and / or durable payload expression. This is in contrast to the findings disclosed in Castellano M. et al., (2024).

[0132] As shown in Example 1, delivery of two doses of naked polyribonucleotides led to sustained expression of an exemplary payload with lower variability among animals. Furthermore, Example 2 demonstrates that delivery of naked polyribonucleotides using a split- dosing approach, whereby a dose of naked polyribonucleotides is divided into two or more subdoses delivered to two or more injection sites, may improve the delivery of naked polyribonucleotides and reduce variability of expression over time.

[0133] Accordingly, split-dosing administration methods disclosed herein allow for delivery (e.g., intramuscular delivery) of naked polyribonucleotides in vivo, such that consistent and / or long-lasting levels of payload (e.g., RNA payload encoded by a naked polyribonucleotide or polypeptide payload encoded by a naked polyribonucleotide) expression can be achieved, thus supporting the development of naked polyribonucleotides as a therapeutic modality. Method of Delivering Naked Polyribonucleotides

[0134] The present disclosure provides methods for administering a naked polyribonucleotide to a subject in a split-dosing approach wherein a dose is administered in at least two or more subdoses at two or more administration sites on a subject such that the subject receives a dose (e.g., a total dose) of the naked polyribonucleotide. A split-dosing approach as provided herein can be beneficial to (i) increase expression of a payload; (ii) increase presence (e.g., increase persistence / bioavailability) of a payload; or (iii) both (i) and (ii), as compared to expression and / or presence of a payload delivered by an otherwise similar polyribonucleotide administered in a single dose or at a single site. Page 32 of 87 12767355v1Attorney Docket No.: 2012611-0182 Split-dosing and administration sites in a subject

[0135] Provided herein is a method for administering two or more subdoses of a naked polyribonucleotide to a subject at two or more administration sites on the subject so that the subject receives a dose of the naked polyribonucleotide, and wherein administration of two or more subdoses is performed in 30 minutes or less.

[0136] In some embodiments of any of the methods provided herein, two or more subdoses together make up a dose.

[0137] In some embodiments of any of the methods provided herein, a subdose comprises about 5% to about 95%, about 5% to about 90%, about 5% to about 80%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 95%, about 20% to about 95%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, or about 90% to about 95% of a dose.

[0138] In some embodiments, a subdose comprises about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of a dose.

[0139] In some embodiments, a subdose comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90 %, or at least 95% of a dose.

[0140] In some embodiments of any of the methods or kits provided herein, the subject is administered three or more subdoses, four or more subdoses, five or more subdoses, six or more subdoses, seven or more subdoses, eight or more subdoses, nine or more subdoses or ten or more subdoses. Page 33 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0141] In some embodiments of any of the methods or kits provided herein, each subdose of the two or more subdoses are the same. In some embodiments, each subdose of the two or more subdoses is different.

[0142] Administration sites as described herein can be located within an area, e.g., a specified area, on a subject. An area on a subject can be measured by appropriate units or dimensions, for example, an area defined by, e.g., square inches, a cross-section length, a radius, or a diameter. In some embodiments, two or more administration sites are within a specified distance of each other. In some embodiments, two or more administration sites are within an area defined by, e.g., square inches or a radius.

[0143] In some embodiments of any of the methods or kits provided herein, two or more administration sites are within a specified distance of each other or a specified area. In some embodiments, a specified area is about two square inches to about thirty square inches, about two square inches to about twenty five square inches, about two square inches to about twenty square inches, about two square inches to about fifteen square inches, about two square inches to about ten square inches, about two square inches to about five square inches, about five square inches to about thirty square inches, about ten square inches to about thirty square inches, about fifteen square inches to about thirty square inches, about twenty square inches to about thirty square inches, or about twenty five to about thirty square inches of each other. In some embodiments, a specified area is about 2 square inches, about 3 square inches, about 4 square inches, about 5 square inches, about 6 square inches, about 7 square inches, about 8 square inches, about 9 square inches, about 10 square inches, about 11 square inches, about 12 square inches, about 13 square inches, about 14 square inches, about 15 square inches, about 16 square inches, about 17 square inches, about 18 square inches, about 19 square inches, about 20 square inches, about 21 square inches, about 22 square inches, about 23 square inches, about 24 square inches, about 25 square inches, about 26 square inches, about 27 square inches, about 28 square inches, about 29 square inches, or about 30 square inches of each other. In some embodiments, a specified distance is at least 2 square inches, at least 3 square inches, at least 4 square inches, at least 5 square inches, at least 6 square inches, at least 7 square inches, at least 8 square inches, at least 9 square inches, at least 10 square inches, at least 11 square inches, at least 12 square inches, at least 13 square inches, at least 14 square inches, at least 15 square inches , at least 16 square Page 34 of 87 12767355v1Attorney Docket No.: 2012611-0182 inches, at least 17 square inches, at least 18 square inches, at least 19 square inches, at least 20 square inches, at least 21 square inches, at least 22 square inches, at least 23 square inches, at least 24 square inches, at least 25 square inches, at least 26 square inches, at least 27 square inches, at least 28 square inches, at least 29 square inches, or at least 30 square inches of each other.

[0144] In some embodiments, a specified area has an about 1mm radius to about 50 mm radius, about 1 mm radius to about 45 mm radius, about 1 mm radius to about 40 mm radius, about 1 mm radius to about 35 mm radius, about 1 mm radius to about 30 mm radius, about 1 mm radius to about 25 mm radius, about 1 mm radius to about 20 mm radius, about 1 mm radius to about 15 mm radius, about 1 mm radius to about 10 mm radius. about 1 mm radius to about 5 mm radius, about 5 mm radius to about 50 mm radius, about 10 mm radius to about 50 mm radius, about 15 mm radius to about 50 mm radius, about 20 mm radius to about 50 mm radius, about 25 mm radius to about 50 mm radius, about 30 mm radius to about 50 mm radius, about 35 mm radius to about 50 mm radius, about 35 mm radius to about 50 mm radius, about 40 mm radius to about 50 mm radius, about 45 mm radius to about 50 mm radius of each other. In some embodiments, a specified area has an about 1 mm radius, about 5 mm radius, about 10 mm radius, about 15 mmradius ̧about 20 mm radius ̧about 25 mm radius ̧about 30 mm radius, about 35 mm radius ̧about 40 mm radius, about 45 mm radius, about 50 mm radius of each other. In some embodiments, a specified distance is at least 1 mm radius, at least 5 mm radius, at least 10 mmradius, at least 15 mm radius ̧at least 20 mm radius ̧at least 25 mm radius¸ at least 30 mmradius, at least 35 mm radius ̧at least 40 mm radius, at least 45 mm radius, or at least 50 mmradius of each other.

[0145] In some embodiments, two or more administration sites are within a specified area. In some embodiments, two or more administration sites are within an area having a 2 mm or more radius, 3 mm or more radius, 4 mm or more radius, 5 mm or more radius, 6 mm or more radius, 7 mm or more radius, 8 mm or more radius, 9 mm or more radius, 10 mm or more radius, 11 mm or more radius, 12 mm or more radius, 13 mm or more radius, 14 mm or more radius, 15 mm or more radius, 20 mm or more radius, 25 mm or more radius, 30 mm or more radius, 35 mm or more radius, 40 mm or more radius, 45 mm or more radius, or 55 mm or more radius. Page 35 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0146] In some embodiments, two or more administration sites are within an area having about a 2 mm radius, about a 3 mm radius, about a 4 mm radius, about a 5 mm radius, about a 6 mm radius, about a 7 mm radius, about a 8 mm radius, about a 9 mm radius, about a 10 mm radius, about a 11 mm radius, about a 12 mm radius, about a 13 mm radius, about a 14 mm radius, about a 15 mm radius, about a 20 mm radius, about a 25 mm radius, about a 30 mm radius, about a 35 mm radius, about a 40 mm radius, about a 45 mm radius, or about a 55 mm radius.

[0147] In some embodiments of any of the methods or kits provided herein, two or more subdoses are administered at two or more administration sites at substantially the same time. In some embodiments, two or more subdoses are administered at two or more administration sites simultaneously. In some embodiments, two or more subdoses are administered at two or more administration sites sequentially. In some embodiments, two or more subdoses are administered within about 25 minutes to about 1 second, about 25 minutes to about 30 seconds, about 25 minutes to about 1 minute, about 25 minutes to about 2 minutes, about 25 minutes to about 5 minutes, about 25 minutes to about 10 minutes, about 25 minutes to about 15 minutes, about 25 minutes to about 20 minutes, about 20 minutes to about 1 second, about 15 minutes to about 1 second, about 10 minutes to about 1 second, about 5 minutes to about 1 second, about 2 minutes to about 1 second, about 1 minute to about 1 second, about 30 seconds to about 1 second of each other.

[0148] In some embodiments, two or more subdoses are administered within about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 2 minutes, about 1 minute, about 45 seconds, about 30 seconds, about 15 seconds, about 5 seconds, or about 1 second of each other. In some embodiments, two or more subdoses are administered within at least 20 minutes, at least 15 minutes, at least 10 minutes, at least 5 minutes, at least 2 minutes, at least 1 minute, at least 45 seconds, at least 30 seconds, at least 15 seconds, at least 5 seconds, or at least 1 second of each other.

[0149] In some embodiments of any of the methods or kits provided herein, the two or more subdoses are administered to two or more administration sites in a subject. In some embodiments, two or more administration sites are in the same tissue. In some embodiments, two or more administration sites are in different tissue. Page 36 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0150] In some embodiments, the two or more administration sites are in muscle tissue. In some embodiments, a muscle tissue comprises striated muscle, non-striated muscle, or both. In some embodiments, a muscle tissue comprises skeletal muscle, smooth muscle, cardiac muscle, or any combination thereof. In some embodiments, skeletal muscle comprises a Type I muscle, a Type II muscle, or both.

[0151] In some embodiments, the two or more administration sites are in non-muscle tissue.

[0152] In some embodiments, the two or more administration sites are in the skin.

[0153] In some embodiments, the two or more administration sites are in adipose tissue.

[0154] In some embodiments, the two or more administration sites are in an organ.

[0155] In some embodiments, (i) administration of two or more subdoses is performed within less than 5 minutes; (ii) two or more administration sites are in muscle tissue; and / or (iii) two or more administrations sites are more than 2 mm radius from each other. In some embodiments, (i) administration of two or more subdoses is performed within less than 5 minutes; and (ii) two or more administration sites are in muscle tissue. In some embodiments, (i) administration of two or more subdoses is performed within less than 5 minutes; and (iii) two or more administrations sites are more than 2 mm radius from each other. In some embodiments, (ii) two or more administration sites are in muscle tissue; and (iii) two or more administrations sites are more than 2 mm radius from each other. In some embodiments, (i) administration of two or more subdoses is performed within less than 5 minutes; (ii) two or more administration sites are in muscle tissue; and (iii) two or more administrations sites are more than 2 mm radius from each other.

[0156] In some embodiments, delivery of a naked polyribonucleotide to a subject according to a split-dosing regimen disclosed herein does not require delivery of one or more ribonuclease inhibitors to the subject, e.g., prior to, concurrently with, or after, administration of a naked polyribonucleotide according to a split-dosing regimen disclosed herein.

[0157] In some embodiments of any of the methods disclosed, a naked polyribonucleotide can be administered into a cell (e.g., resulting in uptake of a naked polyribonucleotide and / or expression of a payload) without the delivery (e.g., before, concurrently with, or after) of one or more ribonuclease inhibitors. Page 37 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0158] In some embodiments of any of the methods disclosed herein, two or more subdoses of a naked polyribonucleotide are administered to a subject without the administration of one or more ribonuclease inhibitors.

[0159] In some embodiments, a subject who receives two or more subdoses of a naked polyribonucleotide at two or more administration sites has not been administered, is not being administered, or will not be administered one or more ribonuclease inhibitors. Characterization of methods of delivering naked polyribonucleotides

[0160] Methods provided herein can result in increased expression and / or increased presence of a payload encoded by a naked polyribonucleotide when administered to a subject. A naked polyribonucleotide administered according to methods disclosed herein can also result in improved delivery of the naked polyribonucleotide and reduce variability of expression of a payload over time. In some embodiments, administration of a naked polyribonucleotide according to methods disclosed herein results in spreading of a dose of a naked polyribonucleotide across a larger surface area and / or volume of tissue (e.g., muscle tissue), such that uptake of the naked polyribonucleotide is not limited by cellular RNA uptake mechanisms which may be saturated when a dose of a naked polyribonucleotide is administered as a single to dose to a single tissue site (e.g., to a limited surface area and / or volume of tissue). In some embodiments, administration of a naked polyribonucleotide according to methods disclosed herein results in enhanced uptake of naked polyribonucleotide compared to delivery of an otherwise similar naked polyribonucleotide delivered as a single dose to a single site.

[0161] In some embodiments of any of the methods disclosed herein, administration of two or more doses to a subject: (i) increases expression of a payload; (ii) increases presence (e.g., increased persistence / bioavailability) of a payload; (iii) both (i) and (ii), as compared to expression and / or presence of a payload delivered by an otherwise similar polyribonucleotide administered in a single dose or at a single site.

[0162] In some embodiments of any of the methods disclosed herein, increased expression of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% is detected. Page 38 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0163] In some embodiments of any of the methods disclosed herein, increased expression is detected for at least 1 hour, 2 hours, 4 hours, 12 hours, 2 hours, 2 days, 4 days, 1 week, 2 weeks, 1 month compared to expression of a payload delivered by an otherwise similar polyribonucleotide in a single dose and / or at a single site.

[0164] In some embodiments of any of the methods disclosed herein, increased presence of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% is detected.

[0165] In some embodiments of any of the methods disclosed herein, increased presence is detected for at least 1 hour, 2 hours, 4 hours, 12 hours, 2 hours, 2 days, 4 days, 1 week, 2 weeks, 1 month compared to persistence of a payload delivered by an otherwise similar polyribonucleotide in a single dose and / or at a single site.

[0166] In some embodiments of any of the methods disclosed herein, increased expression and / or presence is a result of reduced degradation.

[0167] In some embodiments of any of the methods disclosed herein, increased expression and / or presence is a result of increased half-life.

[0168] In some embodiments of any of the methods disclosed herein, administration of the two or more subdoses provides more consistent expression of the payload as compared to expression of a payload delivered by an otherwise similar polyribonucleotide administered in a single dose or at a single site. In some embodiments of any of the methods disclosed herein, more consistent expression comprises substantially the same expression from the two or more administration sites. In some embodiments of any of the methods disclosed herein, more consistent expression comprises substantially the same expression from the administration of the two or more doses at the two or more administration sites.

[0169] In some embodiments of any of the methods disclosed herein, a method is a treatment method.

[0170] In some embodiments of any of the methods disclosed herein, a method is a vaccination method.

[0171] In some embodiments of any of the methods disclosed herein, a method is: (i) a method to stimulate an immune response; (ii) an antibody therapy method; (iii) an immune-modulation Page 39 of 87 12767355v1Attorney Docket No.: 2012611-0182 method; (iv) a vaccination method; (v) a gene therapy method; (vi) a cell therapy engineering method; (vii) an immunotherapy method; (viii) a protein replacement therapy method; (ix) a chemotherapeutic method; or (x) any combination of (i)-(ix).

[0172] In some embodiments of any of the methods disclosed herein, a subject is a human. Naked polyribonucleotides

[0173] Naked polyribonucleotides disclosed herein can have one or more features as disclosed herein.

[0174] In some embodiments, a polyribonucleotide is or comprises a synthetic nucleic acid. Synthetic RNAs can be produced by any methods known in the art. For example, in some embodiments synthetic RNAs can be produced, e.g., by in vitro transcription of a DNA template. In some embodiments, a polyribonucleotide is or comprises a polyribonucleotide made by an in vitro transcription (IVT) reaction.

[0175] In some embodiments, a polyribonucleotide is or comprises a single-stranded RNA (ssRNA). In some embodiments, a polyribonucleotide is or comprises a double-stranded RNA (dsRNA). In some embodiments, a polyribonucleotide is or comprises a circularized RNA (circRNA). In some embodiments, a polyribonucleotide is or comprises a linear RNA.

[0176] In some embodiments, a polyribonucleotide is or comprises a viral RNA. In some embodiments, a polyribonucleotide is or comprises a non-viral RNA.

[0177] In some embodiments, a polyribonucleotide can be of any length, for example, between 2 and 100,000,000 nucleotides in length (or any integer value therebetween). In some embodiments, a polyribonucleotide comprises at least 20 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, at least 550 nucleotides, at least 600 nucleotides, at least 650 nucleotides, at least 700 nucleotides, at least 750 nucleotides, at least 800 nucleotides, at least 850 nucleotides, at least 900 nucleotides, at least 950 nucleotides, at least 1000 nucleotides, at least 1100 nucleotides, at least 1200 nucleotides, at least 1300 nucleotides, at least 1400 nucleotides, at least 1500 nucleotides, at least 1600 nucleotides, at least Page 40 of 87 12767355v1Attorney Docket No.: 2012611-0182 1700 nucleotides, at least 1800 nucleotides, at least 2000 nucleotides, at least 2500 nucleotides, at least 3000 nucleotides, at least 3000 nucleotides, at least 4000 nucleotides, at least 5000 nucleotides, at least 6000 nucleotides, at least 7000 nucleotides, at least 8000 nucleotides, at least 9000 nucleotides, at least 10,000 nucleotides, at least 11,000 nucleotides, at least 12,000 nucleotides, at least 13,000 nucleotides, at least 14,000 nucleotides, at least 15,000 nucleotides, at least 16,000 nucleotides, at least 17,000 nucleotides, at least 18,000 nucleotides, at least 19,000 nucleotides, at least 20,000 nucleotides, at least 21,000 nucleotides, at least 22,000 nucleotides, at least 23,000 nucleotides, at least 24,000 nucleotides, or at least 25,000 nucleotides.

[0178] In some embodiments, a polyribonucleotide is between 50 and 25,000 nucleotides in length, between 100 and 20,000 nucleotides in length, between 500 and 10,000 nucleotides in length, between 1,000 and 8,000 nucleotides in length, and / or between 2,000 and 5,000 nucleotides in length. Payloads and additional sequence elements

[0179] The present disclosure provides methods and kits useful for the delivery of polyribonucleotides that comprise a sequence encoding a payload.

[0180] In some embodiments, a payload is a polypeptide. In some embodiments, a payload is a fusion polypeptide and / or a chimeric polypeptide.

[0181] In some embodiments, a payload is a polyribonucleotide. In some embodiments, a payload is a regulatory RNA (e.g., a siRNA, microRNA, etc.).

[0182] In some embodiments, a polyribonucleotide encodes one payload (e.g., one polyribonucleotide or one polypeptide). In some embodiments, a polyribonucleotide encodes a plurality of payloads (e.g., a plurality of polyribonucleotide or a plurality of polypeptides).

[0183] In some embodiments, a polyribonucleotide comprising a sequence encoding a payload comprises a target-encoding region. In some embodiments, a target-encoding region encodes a gene product. In some embodiments, such a gene product is an RNA. In some embodiments, a target-encoding region encodes a regulatory RNA (e.g., a siRNA, microRNA, etc.). In some embodiments, a target-encoding region encodes a polypeptide (such as a protein, such as a Page 41 of 87 12767355v1Attorney Docket No.: 2012611-0182 glycoprotein). In some embodiments, a target-encoding region encodes a fusion polypeptide and / or a chimeric polypeptide. In some embodiments, a target-encoding region encodes one gene product. In some embodiments, a target-encoding region encodes more than one gene product (e.g., 2, 3, 4, 5, 6, 7 or more gene products).

[0184] In some embodiments, a polyribonucleotide comprising a sequence encoding a payload further comprises an additional element, including, but not limited to, spacers, binding motifs, etc. In some embodiments, a polyribonucleotide comprises one or more of: a target-encoding region, a gene regulatory element, and / or a transcription terminator. Non-limiting examples of gene regulatory elements include promoters, transcriptional activators, enhancers, and polyadenylation signals.

[0185] In some embodiments, a sequence encoding a payload comprises one or more aptamer- or polypeptide-binding domains (e.g., transcription factor binding domains).

[0186] In some embodiments, a sequence encoding a payload can be of any length, for example, between 2 and 100,000,000 nucleotides in length (or any integer value therebetween). In some embodiments, a sequence encoding a payload comprises at least 20 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, at least 550 nucleotides, at least 600 nucleotides, at least 650 nucleotides, at least 700 nucleotides, at least 750 nucleotides, at least 800 nucleotides, at least 850 nucleotides, at least 900 nucleotides, at least 950 nucleotides, at least 1000 nucleotides, at least 1100 nucleotides, at least 1200 nucleotides, at least 1300 nucleotides, at least 1400 nucleotides, at least 1500 nucleotides, at least 1600 nucleotides, at least 1700 nucleotides, at least 1800 nucleotides, at least 2000 nucleotides, at least 2500 nucleotides, at least 3000 nucleotides, at least 3000 nucleotides, at least 4000 nucleotides, at least 5000 nucleotides, at least 6000 nucleotides, at least 7000 nucleotides, at least 8000 nucleotides, at least 9000 nucleotides, at least 10,000 nucleotides, at least 11,000 nucleotides, at least 12,000 nucleotides, at least 13,000 nucleotides, at least 14,000 nucleotides, at least 15,000 nucleotides, at least 16,000 nucleotides, at least 17,000 nucleotides, at least 18,000 nucleotides, at least 19,000 nucleotides, at least 20,000 nucleotides, at least 21,000 nucleotides, at least 22,000 Page 42 of 87 12767355v1Attorney Docket No.: 2012611-0182 nucleotides, at least 23,000 nucleotides, at least 24,000 nucleotides, or at least 25,000 nucleotides.

[0187] In some embodiments, a sequence encoding a payload is between 50 and 25,000 nucleotides in length, between 100 and 20,000 nucleotides in length, between 500 and 10,000 nucleotides in length, between 1,000 and 8,000 nucleotides in length, and / or between 2,000 and 5,000 nucleotides in length. Modified ribose

[0188] Polynucleotides disclosed herein can be a polyribonucleotide which further comprises one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0189] In some embodiments, a polynucleotide is a polyribonucleotide (e.g., a naked polyribonucleotide).

[0190] In some embodiments, a polyribonucleotide (e.g., a naked polyribonucleotide) comprises one or more modified ribonucleotides.

[0191] In some embodiments, a modified ribonucleotide comprises a modified ribose. In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose. In some embodiments, a modified ribonucleotide comprises a structure of: X R O ,

[0192] (a) wherein R is a or a triphosphate; and

[0193] (b) wherein X is an adenine nucleobase or a variant thereof, a guanine nucleobase or a variant thereof, a cytosine nucleobase or a variant thereof (e.g., N4-acetylcytosine), or a uracil nucleobase or a variant thereof (e.g., 5-hydroxymethyluracil or N1-methylpseudouracil). Page 43 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0194] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and an adenine nucleobase or a variant thereof.

[0195] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a guanine nucleobase or a variant thereof.

[0196] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a cytosine nucleobase or a variant thereof.

[0197] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a N4-acetylcytsoine nucleobase.

[0198] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a uracil nucleobase or a variant thereof.

[0199] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a 5-hydroxymethyluracil nucleobase.

[0200] In some embodiments, a modified ribonucleotide comprises a 2’-O-acetylated ribose and a N1-methylpseudouracil nucleobase.

[0201] In some embodiments, a modified ribonucleotide comprises a structure provided in Table 3C. In some embodiments, a modified ribonucleotide comprises a structure provided in Table 3D when incorporated in a polynucleotide. In some embodiments, a polynucleotide (e.g., a polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 3D. Page 44 of 87 12767355v1O -’2yphidO H H4egaPeOniN O H3 dirH O O3H N Nul :e NH C N C - C H3O H5tO Oylhyt ate hO pH O O H O OesO cmOOayoxhpO H- oro OH P POOO-’dnyohm O H2H :enidirOu3-lyH O O H5Nl hyteN O H3C O N C Ote mO1O H Ocay-xH O O Hv5OoO5-rd37O H’2yhO6H721nidirH O OeHO3enuoetNnOid:et NO HiO O N Cdit:e2 O3tHNH N N C dahN O iru ahOyc ahO uespsO H OlytpsO H OlytpsO H O ployhe ohe ohhpOctoeno OH POa-poOcnOH POa-poO nOH POm m OO-’o2m OO- o2m O H H’H eni:d :ie ern niuioH O Odidr OitO duH NlN O H3ycl2 N O H3uesO N C H N N C p OytO OytlN y O HecO HeO c1Ov5htOa- OaH O e OO- 5-’OO-3’7O6m H2H2H721N OoO H O HnOedO al:etO Oyt aH P7OH POeOchp O OO8fO H OaP -so OH PO o7OOH -h’p2idO H4egaP2 H Neni NNsoNO3O3n :N2H He etH N C N C OdaN O OaHl hpO H OytesO OcohOHaPO -poO nOH POOOH -’o2m O H : 2eHnNisoNnNOeH3daNN O32 H N ClN OytH N C N O1O HecaO HvO - O5O537O - O6H’2H721aro -’eO- d pO pr 2ca’2yhigdaPocnineh enwOi(dse-:i O O3 HO3HNHru: NH di 4t eetN N C O -ly etO N C O oNellncyiahO C O5l ht ahH O tdi uetpsH3Oyt epsO O ncyo a- colhe mbiypoOc yoO - POa-xhopO ro O- POrO-t’enoOO-’dnyoO de 2cam2 hm ifidom:yeranilpdi m -re4: uxNeO -lyH O lniH N O H3C5l htN O H3E y dN NeO N C :t itO OytmO De3ca ycC3Oec yxH O O1v5el -lH Oab Oyt- OorO5d37a -’eO -2ca’ yO67T2 h21di :e2 tHNHoN N Cna:et NC tyclahO Oug aN O ytphO Olesy pO cot sahe oh-pO -ca-pO - OonOPO onOPO-’oOO-’oO 2 m2m ::eeni2 H ni soN di nH NN3tyO O3 auO H3H Ccl2 y H N HglO C t N N C e OytN N O ceaOcO1v-OaO5O--’OO5-3O72’26721onO i edO -r eu Odial: 1ye - otdOo 7tteaO -lPyueeta- OePO lc 8fchO O tapesh-sO Ouo-cpPa- lypsO-noPO ob05O-hO O’piOO- h’toehpiOirteg2 d 2mdneacPajdanesNe aniNnotoN O H3idH O N Otne:detH2alaNNC i O -ruh O1- oetnd aO h N Oem OhytpesOlyue psOcacot2ahesotp O -ca pl h tp O -af8- onOPO -yhtonOPO1O0-oOO-n-’o e oO12m’2m moit1 is6o2p10e2: : ht. en e soi nietNsto diacene NN-ru iOdkcdoalN O H3 1oH N OniD ytH2N C-ldN Oyt uey esO N O1e cnraOec-Oa-plO yv5ohO:E53ttO-’OO-’ teOT 7O67A2 2m N21Attorney Docket No.: 2012611-0182

[0202] In some embodiments, at least 5% of ribose moieties of a polyribonucleotide (e.g., a naked polyribonucleotide) are 2’-O-acetylated.

[0203] In some embodiments, about 5% to about 99% of the ribose moieties of a polyribonucleotide (e.g., a naked polyribonucleotide) are 2’-O-acetylated.

[0204] In some embodiments, a polyribonucleotide disclosed herein (e.g., a naked polyribonucleotide) comprises a cap structure and the cap structure does not comprise a 2’-O- acetylated ribose.

[0205] In some embodiments, a polyribonucleotide disclosed herein (e.g., a naked polyribonucleotide) comprises a cap structure and the cap structure comprises a 2’-O-acetylated ribose.

[0206] In some embodiments, a polyribonucleotide disclosed herein (e.g., a naked polyribonucleotide) further comprises one or more ribonucleotides that does not comprise a 2’-O acetylated ribose. Modified ribonucleotides: Ac4C and 5-hmU

[0207] Polynucleotides disclosed herein can be a polyribonucleotide which further comprises one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0208] In some embodiments, a polynucleotide is a polyribonucleotide (e.g., a naked polyribonucleotide). In some embodiments, a polyribonucleotide comprises one or more modified ribonucleotides. In some embodiments, a polyribonucleotide comprises a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0209] In some embodiments, a polyribonucleotide (e.g., a naked polyribonucleotide) comprises one or more modified nucleosides or nucleobases.

[0210] In some embodiments, a modified ribonucleotides comprises an N4-acetylcytidine nucleoside. In some embodiments, an N4-acetylcytidine nucleoside has the following structure: Page 51 of 87 12767355v1Attorney Docket No.: 2012611-0182 O .

[0211] In some embodiments, a modified ribonucleotide comprises a 5-hydroxymethyluridine nucleoside. In some embodiments, a 5-hydroxymethyluridine nucleoside has the following structure: OH O O .

[0212] In some embodiments, a has a structure provided in Table 3A. In some embodiments, a modified ribonucleotide has a structure provided in Table 3B when incorporated in a polynucleotide. In some embodiments, a polynucleotide (e.g., a polyribonucleotide, e.g., a naked polyribonucleotide) comprises one or more modified ribonucleotides that have a structure provided in Table 3B. Page 52 of 87 12767355v1ditCyc:leH3H OtO tem:etO ytaO ehHy acp OPOx hOH PO 78as-oOoHrp4hp O OsOfPdyohhp OH PO o3d5NidO H -5iO HegaPenidi e Orn: Nuly: HO se ididietH ttaN N H Oht etaNO N H eyhO Oe hO olcpC3HmpH O H cly steoH Oy shx oO O Oorh2u8ncapo1o nOH PO dpyoO O H PO0b--ir 4 oN m O Hhn-o15m O H1d6ei2 fi10do:2e:mn.io yr dNaitl :e ruepkmni ldyhc eitOo xEycH NteH O N N N HmDy:leAytO OyO N H O1C OxoH Ovnr3eelcaH3H OrdO H5yO53ottb -a4O Hh-7O6H7A T N521p yacl toa O- POmytaO O - Pit7t ytniehcpsOx hO oo O-oPOrpdsO o -el8c foda- h y hOPO un4et4 paiOh-piOo5rNd 5 dboir egpr taPoncencie ajnndeid ah wseO irnulHOa(eni :eH Ny:Notd diitt taN N H yhO Oht eO N HtC Oe tahO HnOeoelcl ps 3OmpsO O m cyuteoHyhx o hcnc pOorhOatoab-o- nOPO dpyo- nOPO taifr 4 oOh-oOodN m5mneiofiti idso:e onpmeyirdalp:ihe rtsemn uilytaexditOhtceHOidEyH NN: clN N Hm niByO OyO N H O13 teelcC3OxoH O Ovba- H OrdO553a4Oyh- O:et7o67T N5N21Attorney Docket No.: 2012611-0182

[0213] In some embodiments, a polyribonucleotide (e.g., a naked polyribonucleotide) comprises cytidine nucleosides. In some embodiments, at least 5% of cytidine nucleosides in a polyribonucleotide comprise N4-acetylcytidine. In some embodiments, less than 100% of cytidine nucleosides in a polyribonucleotide comprise N4-acetylcytidine. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of cytidine nucleosides in a polyribonucleotide comprise N4-acetylcytidine.

[0214] In some embodiments, a polyribonucleotide (e.g., a naked polyribonucleotide) comprises uridine nucleosides. In some embodiments, at least 5% of uridine residues in a polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, less than 100% of uridine nucleosides in a polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of uridine nucleosides in a polyribonucleotide comprise 5- hydroxymethyluridine. In some embodiments, more than 60% of uridine nucleosides in a polyribonucleotide comprise 5-hydroxymethyluridine.

[0215] In some embodiments, a polyribonucleotide disclosed herein (e.g., a naked polyribonucleotide) comprises one or more modified ribonucleotides comprises a nucleoside comprising: N4-acetylcytidine, 5-hydroxymethyluridine, N1-methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5-methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5- formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6-diaminopurine, 2-amino- 6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (m1 I), wyosine (imG), methylwyosine (mimG), 5-hydroxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5- methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6- dihydro-5-methyluridine, 2’-O-methyluridine, 2’-O-methyl-5-methyluridine, 2’-fluoro-2’- Page 55 of 87 12767355v1Attorney Docket No.: 2012611-0182 deoxyuridine, 2’-amino-2’-deoxyuridine, 2’-azido-2’-deoxyuridine, 4-thiouridine, 5- carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5- propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2’-O-methyl- pseudouridine, N1-hydroxypseudouridine, 2’-O-methyl-N1-methylpseudouridine, N1- ethylpseudouridine, N1-hydroxymethylpseudouridine, ara-uridine, N6-methyladenosine, 2- aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 8-oxoadenosine, thienoguanosine, 7- deazaguanosine, 8-oxoguanosine, 6-O-methylguanine, or any combination thereof. Cap structures

[0216] In some embodiments of any of the polyribonucleotides disclosed herein (e.g., a naked polyribonucleotide), a polyribonucleotide comprises a cap structure.

[0217] Prior versions of RNA capping have been described, e.g., in Decroly E et al. (2012) Nature Reviews 10: 51-65; and in Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511– 7526, the entire contents of each of which is hereby incorporated by reference.5’ caps include a Cap-0 (also referred herein as “Cap0”), a Cap-1 (also referred herein as “Cap1”), or Cap-2 (also referred herein as “Cap2”). See, e.g., Figure 1 of Ramanathan A et al., and Figure 1 of Decroly E et al.

[0218] The term “5'-cap” as used herein refers to a structure found on the 5'-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5'-triphosphate linkage (also referred to as Gppp or G(5')ppp(5')).

[0219] In some embodiments of a polyribonucleotide disclosed herein (e.g., a naked polyribonucleotide), the 5’ end of the polyribonucleotide comprises a compound of formula I: Page 56 of 87 12767355v1Attorney Docket No.: 2012611-0182 R1or a salt thereof, wherein: indicates the position at which the compound is attached to the polyribonucleotide; each of B1and B2is independently selected from a natural base and a modified base; R1is selected from hydrogen, C1-6alkyl, and -C(=O)CH3; R2is hydrogen or -CH3; and X is O or S.

[0220] As defined generally above for formula I, each of B1and B2is independently selected from a natural base and a modified base. In some embodiments, B1is a natural base. In some embodiments, B1is adenine. In some embodiments, B1is cytosine. In some embodiments, B1is guanine. In some embodiments, B1is uracil.

[0221] In some embodiments, B1is a modified base. In some embodiments, B1is selected from N1-methylpseudouracil, a pyridin-4-one, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio- uracil, 5-methyl cytosine, 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine, 5-formyl-cytosine, N4-methyl-cytosine, a 2-amino-purine, a 2,6-diaminopurine, a 2-amino-6- halo-purine, a 6-halo-purine, hypoxanthine, 1-methyl-hypoxanthine, 4,6-dimethyl-3,3a,4,9a- tetrahydro-9H-imidazo[1,2-a]purin-9-one, 4,6,7-trimethyl-3,3a,4,9a-tetrahydro-9H-imidazo[1,2- a]purin-9-one, N4-acetylcytosine, and 5-hydroxymethyluracil.

[0222] In some embodiments, B2is a natural base. In some embodiments, B2is adenine. In some embodiments, B2is cytosine. In some embodiments, B2is guanine. In some embodiments, B2is uracil. Page 57 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0223] In some embodiments, B2is a modified base. In some embodiments, B2is selected from N1-methylpseudouracil, a pyridin-4-one, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio- uracil, 5-methyl cytosine, 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine, 5-formyl-cytosine, N4-methyl-cytosine, a 2-amino-purine, a 2,6-diaminopurine, a 2-amino-6- halo-purine, a 6-halo-purine, hypoxanthine, 1-methyl-hypoxanthine, 4,6-dimethyl-3,3a,4,9a- tetrahydro-9H-imidazo[1,2-a]purin-9-one, 4,6,7-trimethyl-3,3a,4,9a-tetrahydro-9H-imidazo[1,2- a]purin-9-one, N4-acetylcytosine, and 5-hydroxymethyluracil.

[0224] As defined generally above for formula I, R1is selected from hydrogen, C1-6alkyl, and - C(=O)CH3. In some embodiments, R1is hydrogen. In some embodiments, R1is C1-6alkyl. In some embodiments, R1is -CH3. In some embodiments, R1is -C(=O)CH3.

[0225] As defined generally above for formula I, R2is hydrogen or -CH3. In some embodiments, R2is hydrogen. In some embodiments, R2is - CH3.

[0226] As defined generally above for formula I, X is O or S. In some embodiments, X is O. In some embodiments, X is S.

[0227] In some embodiments, the present disclosure provides a compound of any of formulae I- a, I-b, I-c, I-d, I-e, and I-f: HO OHPage 58 of 87 12767355v1Attorney Docket No.: 2012611-0182 12767355v1Attorney Docket No.: 2012611-0182or a salt thereof, wherein each of B1and B2is as defined above and described herein.

[0228] In some embodiments, a compound of formula I is selected from Page 60 of 87 12767355v1Attorney Docket No.: 2012611-0182 NHor a salt thereof.

[0229] In some embodiments, a polyribonucleotide disclosed herein (e.g., a naked polyribonucleotide) comprises at the 5’ end am7GpppN1pN2-OH cap with the following structure: O HN N 2 .

[0230] In somea Page 61 of 87 12767355v1Attorney Docket No.: 2012611-0182 2 ,wherein indicates the position at which the compound is attached to the polyribonucleotide.

[0231] In some embodiments, a polyribonucleotide disclosed herein (e.g., a naked polyribonucleotide) comprises am7GpppN1(2’-OMe)pN2-OH cap with the following structure: O 2 .

[0232] In somepolyribonucleotide) comprises at the 5’ end a compound with the following structure: Page 62 of 87 12767355v1Attorney Docket No.: 2012611-0182 2 ,wherein indicates the position at which the compound is attached to the polyribonucleotide.

[0233] In some embodiments, a polyribonucleotide disclosed herein (e.g., a naked polyribonucleotide) comprises am7GpppN1(2’-OMe)pN2(2’-OMe)-OH cap with the following structure: O 2 .

[0234] In somepolyribonucleotide) comprises at the 5’ end a compound with the following structure: Page 63 of 87 12767355v1Attorney Docket No.: 2012611-0182 2wherein polyribonucleotide.

[0235] In some embodiments, for any of the cap structures disclosed herein, each of B1and B2is independently selected from a natural base and a modified base. In some embodiments, B1is a natural base. In some embodiments, B1is adenine. In some embodiments, B1is cytosine. In some embodiments, B1is guanine. In some embodiments, B1is uracil.

[0236] In some embodiments, B1is a modified base. In some embodiments, B1is selected from N1-methylpseudouracil, a pyridin-4-one, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio- uracil, 5-methyl cytosine, 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine, 5-formyl-cytosine, N4-methyl-cytosine, a 2-amino-purine, a 2,6-diaminopurine, a 2-amino-6- halo-purine, a 6-halo-purine, hypoxanthine, 1-methyl-hypoxanthine, 4,6-dimethyl-3,3a,4,9a- tetrahydro-9H-imidazo[1,2-a]purin-9-one, 4,6,7-trimethyl-3,3a,4,9a-tetrahydro-9H-imidazo[1,2- a]purin-9-one, N4-acetylcytosine, and 5-hydroxymethyluracil.

[0237] In some embodiments, B2is a natural base. In some embodiments, B2is adenine. In some embodiments, B2is cytosine. In some embodiments, B2is guanine. In some embodiments, B2is uracil.

[0238] In some embodiments, B2is a modified base. In some embodiments, B2is selected from N1-methylpseudouracil, a pyridin-4-one, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio- uracil, 5-methyl cytosine, 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine, 5-formyl-cytosine, N4-methyl-cytosine, a 2-amino-purine, a 2,6-diaminopurine, a 2-amino-6- halo-purine, a 6-halo-purine, hypoxanthine, 1-methyl-hypoxanthine, 4,6-dimethyl-3,3a,4,9a- Page 64 of 87 12767355v1Attorney Docket No.: 2012611-0182 tetrahydro-9H-imidazo[1,2-a]purin-9-one, 4,6,7-trimethyl-3,3a,4,9a-tetrahydro-9H-imidazo[1,2- a]purin-9-one, N4-acetylcytosine, and 5-hydroxymethyluracil. Compositions and Pharmaceutical compositions

[0239] Naked polyribonucleotides disclosed herein may be provided in a composition. In some embodiments, a composition comprises one or more naked polyribonucleotides.

[0240] In some embodiments, one or more naked polyribonucleotides in a composition are the same (e.g., encoding the same payload).

[0241] In some embodiments, one or more naked polyribonucleotides in a composition are different (e.g., encoding different payloads).

[0242] In some embodiments, a composition comprising one or more naked polyribonucleotides for delivery according to any of the methods disclosed herein does not comprise one or more ribonuclease inhibitors.

[0243] Pharmaceutical compositions of the present disclosure may comprise a naked polyribonucleotide disclosed herein and a pharmaceutically acceptable excipient, a diluent, or any combination thereof. In some embodiments, a pharmaceutical composition may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose, or dextrans; mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; and preservatives.

[0244] In some embodiments, a pharmaceutical composition disclosed herein comprises a naked polyribonucleotide that is not encapsulated, not complexed and / or not conjugated to a delivery vehicle or carrier. In some embodiments, a pharmaceutical composition is formulated for administration according to any of the routes of administration disclosed herein. In some embodiments, a pharmaceutical composition is formulated for intramuscular administration, intradermal administration, intravenous administration, or subcutaneous administration. Page 65 of 87 12767355v1Attorney Docket No.: 2012611-0182 Kits and Multi-needle injectors

[0245] Also provided herein are kits comprising a naked polyribonucleotide disclosed herein. In some embodiments, a kit comprises two or more subdoses of a naked polyribonucleotide formulated for administration to a subject. In some embodiments, two or more subdoses together make up a dose.

[0246] In some embodiments, each subdose comprises about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of a dose.

[0247] In some embodiments, each subdose comprises about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 95%, about 15% to about 95%, about 20% to about 95%, about 25% to about 95%, about 30% to about 95%, about 35% to about 95%, about 40% to about 95%, about 45% to about 95%, about 50% to about 95%, about 55% to about 95%, about 60% to about 95%, about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 90% to about 95% of a dose.

[0248] In some embodiments, a subject is administered three or more subdoses, four or more subdoses, five or more subdoses, six or more subdoses, seven or more subdoses, or ten or more subdoses.

[0249] In some embodiments, each subdose of two or more subdoses are the same.

[0250] In some embodiments, each subdose of the two or more subdoses are different.

[0251] In some embodiments, a kit further comprises two or more injector needles. In some embodiments, two or more injector needles comprises a subdose of a naked polyribonucleotide.

[0252] This disclosure also provides a kit comprising: a dose of a naked polyribonucleotide formulated for administration to a subject. Page 66 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0253] In some embodiments, a kit further comprises a multi-needle injector.

[0254] In some embodiments, multi-needle injector comprises: (i) one or more cavities, and (ii) two or more needles, and wherein the one or more cavities are configured to hold the dose of the naked polyribonucleotide, wherein the one or more cavities are connected to the two or more needles so that the dose of naked polyribonucleotide can traverse from the one or more cavities to the two or more needles, and wherein the two or more needles are each configured to deliver a subdose of the naked polyribonucleotide to the subject so that the subject receives the dose of the naked polyribonucleotide.

[0255] Methods disclosed herein can be administered to a subject using a multi-needle injector. In some embodiments, a multi-needle injector comprises: (i) one or more cavities, (ii) two or more needles, and (iii) a dose of a naked polyribonucleotide formulated for administration to a subject. In some embodiments, one or more cavities are configured to hold a dose of a naked polyribonucleotide. In some embodiments, one or more cavities are connected to two or more needles so that a dose of naked polyribonucleotide can traverse from one or more cavities to the two or more needles. In some embodiments, two or more needles are each configured to deliver a subdose of a naked polyribonucleotide to a subject so that a subject receives a dose of a naked polyribonucleotide.

[0256] In some embodiments, administration of a dose of a naked polyribonucleotide disclosed herein with a multi-needle injector can be achieved in about 5 minutes, about 2 minutes, about 1 minute, about 45 seconds, about 30 seconds, about 15 seconds, about 5 seconds, or about 1 second of each other.

[0257] In some embodiments, two or more subdoses are administered at least 20 minutes, at least 15 minutes, at least 10 minutes, at least 5 minutes, at least 2 minutes, at least 1 minute, at least 45 seconds, at least 30 seconds, at least 15 seconds, at least 5 seconds, or at least 1 second of each other. Page 67 of 87 12767355v1Attorney Docket No.: 2012611-0182 ENUMERATED EMBODIMENTS

[0258] Embodiment 1. A method comprising: administering two or more subdoses of a naked polyribonucleotide to a subject at two or more administration sites on the subject so that the subject receives a dose of the naked polyribonucleotide, and wherein administration of the two or more subdoses is performed in 30 minutes or less.

[0259] Embodiment 2. The method of embodiment 1, wherein the two or more subdoses together make up the dose.

[0260] Embodiment 3. The method of embodiment 1 or 2, wherein each of the subdoses comprise about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 95% of the dose.

[0261] Embodiment 4. The method of embodiment 1 or 2, wherein each of the subdoses comprise about 5% to about 95% of the dose.

[0262] Embodiment 5. The method of any one of the preceding embodiments, wherein the subject is administered three or more subdoses, four or more subdoses, five or more subdoses, six or more subdoses, seven or more subdoses, or ten or more subdoses.

[0263] Embodiment 6. The method of any one of the preceding embodiments, wherein each subdose of the two or more subdoses are the same.

[0264] Embodiment 7. The method of any one of embodiments 1-5 wherein each subdose of the two or more subdoses are different.

[0265] Embodiment 8 The method of any one of the preceding embodiments, wherein the two or more administration sites are within a specified distance of each other.

[0266] Embodiment 9. The method of embodiment 8, wherein the specified distance is about two square inches, about three square inches, about four square inches, about five square inches, about ten square inches, about twenty square inches, or about thirty square inches of each other.

[0267] Embodiment 10. The method of embodiment 8, wherein the specified distance is about two square inches to about thirty square inches of each other. Page 68 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0268] Embodiment 11. The method of embodiment 8, wherein the specified distance is about 1mm radius to about 50mm radius of each other.

[0269] Embodiment 12. The method of any one of the preceding embodiments, wherein the two or more administration sites are in the same tissue.

[0270] Embodiment 13. The method of any one of embodiments 1-11, wherein the two or more administration sites are in different tissue.

[0271] Embodiment 14. The method of any one of the preceding embodiments, wherein the two or more administration sites are in muscle tissue.

[0272] Embodiment 15. The method of embodiment 14, wherein the muscle tissue comprises striated muscle, non-striated muscle, or both.

[0273] Embodiment 16. The method of embodiment 14, wherein the muscle tissue comprises skeletal muscle, smooth muscle, cardiac muscle, or any combination thereof.

[0274] Embodiment 17. The method of embodiment 16, wherein the skeletal muscle comprises a Type I muscle, a Type II muscle, or both.

[0275] Embodiment 18. The method of any one of the preceding embodiments, wherein the two or more subdoses are administered at the two or more administration sites at substantially the same time.

[0276] Embodiment 19. The method of any one of embodiments 1-17, wherein the two or more subdoses are administered at the two or more administration sites simultaneously.

[0277] Embodiment 20. The method of any one of embodiments 1-17, wherein the two or more subdoses are administered at the two or more administration sites sequentially.

[0278] Embodiment 21. The method of any one of the preceding embodiments, wherein the two or more subdoses are administered in about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 2 minutes, about 1 minute, about 45 seconds, about 30 seconds, about 15 seconds or about 1 second.

[0279] Embodiment 22. The method of any one of the preceding embodiments, wherein the two or more subdoses are administered in about 25 minutes to about 1 second. Page 69 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0280] Embodiment 23. The method of any one of the preceding embodiments, wherein the two or more subdoses are administered using a multi-needle injector.

[0281] Embodiment 24. The method of any one of the preceding embodiments, wherein: (i) administration of the two or more subdoses is performed within less than 5 minutes; (ii) the two or more administration sites are in muscle tissue; and (iii) the two or more administrations sites are more than 2 mm radius from each other.

[0282] Embodiment 25. The method of any one of the preceding embodiments, wherein the polyribonucleotide is linear.

[0283] Embodiment 26. The method of any one of embodiments 1-24, wherein the polyribonucleotide is circular.

[0284] Embodiment 27. The method of any one of the preceding embodiments, wherein the polyribonucleotide comprises naturally occurring polyribonucleotides.

[0285] Embodiment 28. The method of any one of the preceding embodiments, wherein the polyribonucleotide comprises only naturally occurring polyribonucleotides.

[0286] Embodiment 29. The method of any one of embodiments 1-27, wherein the polyribonucleotide comprises one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0287] Embodiment 30. The method of embodiment 29, wherein the one or more modified ribonucleotides comprise modified ribonucleotides that comprise a 2’-O-acetylated ribose.

[0288] Embodiment 31. The method of embodiment 30, wherein the one or modified ribonucleotides comprise a 2’-O-acetyl ribose and a guanine nucleobase or a variant thereof.

[0289] Embodiment 32. The method of embodiment 30, wherein the one or modified ribonucleotides comprise a 2’-O-acetyl ribose and an adenosine nucleobase or a variant thereof.

[0290] Embodiment 33. The method of embodiment 30, wherein the one or modified ribonucleotides comprise a 2’-O-acetyl ribose and a uridine nucleobase or a variant thereof.

[0291] Embodiment 34. The method of embodiment 30, wherein the one or modified ribonucleotides comprise a 2’-O-acetyl ribose and a cytosine nucleobase or a variant thereof. Page 70 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0292] Embodiment 35. The method of embodiment 30, wherein the one or modified ribonucleotides comprise a 2’-O-acetyl ribose and a N1-methylpseudouracil nucleobase.

[0293] Embodiment 36. The method of embodiment 30, wherein the one or more modified ribonucleotides comprise a 2’-O acetyl ribose and a N4-acetylcytosine nucleobase.

[0294] Embodiment 37. The method of embodiment 30, wherein the one or more modified ribonucleotides comprise a 2’-O acetyl ribose and a 5-hydroxymethyuracil nucleobase.

[0295] Embodiment 38. The method of any one of embodiments 1-37, wherein the one or more modified ribonucleotides comprises a nucleoside comprising: N4-acetylcytidine, 5- hydroxymethyluridine, N1-methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6- aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5-methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4- methyl-cytidine (m4C), 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine, 6-halo- purine, inosine (I), 1-methyl-inosine (m1 I), wyosine (imG), methylwyosine (mimG), 5- hydroxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-methoxycytidine, 5- propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5- methyluridine, 2’-O-methyluridine, 2’-O-methyl-5-methyluridine, 2’-fluoro-2’-deoxyuridine, 2’- amino-2’-deoxyuridine, 2’-azido-2’-deoxyuridine, 4-thiouridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5- bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2’-O-methyl-pseudouridine, N1- hydroxypseudouridine, 2’-O-methyl-N1-methylpseudouridine, N1-ethylpseudouridine, N1- hydroxymethylpseudouridine, ara-uridine, N6-methyladenosine, 2-aminoadenosine, 3- methyladenosine, 7-deazaadenosine, 8-oxoadenosine, thienoguanosine, 7-deazaguanosine, 8- oxoguanosine, 6-O-methylguanine, or any combination thereof.

[0296] Embodiment 39. The method of any one of embodiments 1-38, wherein the polyribonucleotide comprises a cap structure and the cap structure does not comprise a 2’-O- acetylated ribose.

[0297] Embodiment 40. The method of any one of embodiments 1-38, wherein polyribonucleotide comprises a cap structure and the cap structure comprises a 2’-O-acetylated ribose. Page 71 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0298] Embodiment 41. The method of any one of the preceding embodiments, wherein the polyribonucleotide comprises a coding region.

[0299] Embodiment 42. The method of embodiment 41, wherein the coding region encodes a payload.

[0300] Embodiment 43. The method of embodiment 41, wherein the payload is or comprises a polypeptide.

[0301] Embodiment 44. The method of any one of embodiments 1-42, wherein the polyribonucleotide comprises an RNA payload.

[0302] Embodiment 45. The method of any one of the preceding embodiments, wherein the polyribonucleotide is in a composition.

[0303] Embodiment 46. The method of embodiment 45, wherein the composition is a pharmaceutical composition.

[0304] Embodiment 47. The method of embodiment 46, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0305] Embodiment 48. The method of any one of embodiments 42-47, wherein administration of the two or more doses to a subject: (i) increases expression of the payload; (ii) increases presence (e.g., increased persistence / bioavailability) of the payload; (iii) both (i) and (ii), as compared to expression and / or presence of a payload delivered by an otherwise similar polyribonucleotide administered in a single dose or at a single site.

[0306] Embodiment 49. The method of embodiment 48, wherein increased expression of at least 5% is detected.

[0307] Embodiment 50. The method of embodiment 48 or 49, wherein increased expression is detected for at least 1 hour, 2 hours, 4 hours, 12 hours, 2 hours, 2 days, 4 days, 1 week, 2 weeks, 1 month compared to expression of a payload delivered by an otherwise similar polyribonucleotide in a single dose and / or at a single site.

[0308] Embodiment 51. The method of embodiment 48, wherein increased presence of at least 5% is detected. Page 72 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0309] Embodiment 52. The method of embodiment 48 or 51, wherein increased presence is detected for at least 1 hour, 2 hours, 4 hours, 12 hours, 2 hours, 2 days, 4 days, 1 week, 2 weeks, 1 month compared to persistence of a payload delivered by an otherwise similar polyribonucleotide in a single dose and / or at a single site.

[0310] Embodiment 53. The method of any one of embodiments 48-52, wherein increased expression and / or presence is a result of reduced degradation.

[0311] Embodiment 54. The method of any one of embodiments 48-53, wherein increased expression and / or presence is a result of increased half-life.

[0312] Embodiment 55. The method of any one of the preceding embodiments, wherein administration of the two or more subdoses provides more consistent expression of the payload as compared to expression of a payload delivered by an otherwise similar polyribonucleotide administered in a single dose or at a single site.

[0313] Embodiment 56. The method of embodiment 55, wherein more consistent expression comprises substantially the same expression from the two or more administration sites.

[0314] Embodiment 57. The method of any one of the preceding embodiments, wherein the method is a treatment method.

[0315] Embodiment 58. The method of any one of embodiments 1-56, wherein the method is a vaccination method.

[0316] Embodiment 59. The method of any one of the preceding embodiments, wherein the method is: (i) a method to stimulate an immune response; (ii) an antibody therapy method; (iii) an immune-modulation method; (iv) a vaccination method; (v) a gene therapy method; (vi) a cell therapy engineering method; (vii) an immunotherapy method; (viii) a protein replacement therapy method; (ix) a chemotherapeutic method; or (x) any combination of (i)-(ix).

[0317] Embodiment 60. The method of any one of the preceding embodiments, wherein the subject is a human.

[0318] Embodiment 61. A kit comprising two or more subdoses of a naked polyribonucleotide formulated for administration to a subject. Page 73 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0319] Embodiment 62. The kit of embodiment 61, wherein the two or more subdoses together make up the dose.

[0320] Embodiment 63. The kit of embodiment 61 or 62, wherein each of the subdoses comprise about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the dose.

[0321] Embodiment 64. The kit of embodiment 61 or 62, wherein each of the subdoses comprise about 5% to about 95% of the dose.

[0322] Embodiment 65. The kit of any one of embodiments 61-64, wherein the subject is administered three or more subdoses, four or more subdoses, five or more subdoses, six or more subdoses, seven or more subdoses, or ten or more subdoses.

[0323] Embodiment 66. The kit of any one of embodiments 61-65, wherein each subdose of the two or more subdoses is the same.

[0324] Embodiment 67. The kit of any one of embodiments 61-65, wherein each subdose of the two or more subdoses is different.

[0325] Embodiment 68. The kit of any one of embodiments 61-67, wherein the kit further comprises two or more injector needles.

[0326] Embodiment 69. The kit of embodiment 68, wherein each of the two or more injector needles comprises a subdose of the naked polyribonucleotide.

[0327] Embodiment 70. A kit comprising a dose of a naked polyribonucleotide formulated for administration to a subject.

[0328] Embodiment 71. The kit of embodiment 70, wherein the kit further comprises a multi- needle injector.

[0329] Embodiment 72. The kit of embodiment 71, wherein the multi-needle injector comprises: (i) one or more cavities, and (ii) two or more needles, and wherein the one or more cavities are configured to hold the dose of the naked polyribonucleotide, wherein the one or more cavities are connected to the two or more needles so that the dose of naked polyribonucleotide can traverse from the one or more cavities to the two or more needles, and wherein the two or more needles Page 74 of 87 12767355v1Attorney Docket No.: 2012611-0182 are each configured to deliver a subdose of the naked polyribonucleotide to the subject so that the subject receives the dose of the naked polyribonucleotide.

[0330] Embodiment 73. A multi-needle injector comprising: (i) one or more cavities, (ii) two or more needles, and (iii) a dose of a naked polyribonucleotide formulated for administration to a subject, wherein the one or more cavities are configured to hold the dose of the naked polyribonucleotide, wherein the one or more cavities are connected to the two or more needles so that the dose of naked polyribonucleotide can traverse from the one or more cavities to the two or more needles, and wherein the two or more needles are each configured to deliver a subdose of the naked polyribonucleotide to the subject so that the subject receives the dose of the naked polyribonucleotide. EXEMPLIFICATION

[0331] The invention now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of some aspects and embodiments of the present invention, and is not intended to be limiting. Example 1: In Vivo Expression of Naked Polyribonucleotides

[0332] This Example describes the in vivo delivery and expression of naked polyribonucleotides. Methods:

[0333] In Vitro Transcription (IVT) Template Production: The Luc2 gene encoding an optimized version of firefly Luciferase (Promega) was amplified from pUCIDT-AMP (IDT). Amplification was carried out at an annealing temperature of 50°C in a 20 μL reaction consisting of 0.25 μM each primer T7-AGG+6A_fwd and 120pA_rev, 1X Herculase II buffer, 25 mM each dNTP, 30 ng pGL4.10[luc2] plasmid (Promega), 0.25M Betaine and 0.4 μL Herculase II enzyme. PCR reactions were treated with 40U Dpn1 (NEB) at 37°C for 30 min to digest plasmid template. PCR product was purified using a Ampure XP beads (Beckman Coulter) according to manufacturer’s protocol and eluted into 100 μL Nuclease free water. Page 75 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0334] The sequences of primers used were T7-AGG+6A_fwd (gaattTAATA CGACTCACTA TAAGGcttgt tctttttgca gaagc) (SEQ ID NO: 1) and 120pA_rev (TTTTTTTTTT TTTTTTTTTTTTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT agaatgtgaa gaaactttct ttttattag) (SEQ ID NO: 2).

[0335] IVT of Luciferase RNA for In Vivo Studies: Luciferase mRNA was synthesized in 20 μL IVT reactions consisting of 200 ng Luc2 T7 template, 20 mM MgCl2, 7.5 mM NTP, 7.5 mM CleanCap AG (TriLink), 1X HiScribe Transcription Buffer, and 2 uL HiScribe polymerase mix (NEB) and incubated at 37°C for 2 hours.

[0336] To degrade DNA template and remove rare immunogenic 5’ triphosphates from RNA transcripts that did not incorporate CleanCap AG. The IVT products were then digested in 22 μL reactions consisting of 1U of DNase I (RNase-free) (New England Biolabs) and 100U Calf Intestinal Alkaline Phosphotase (CIAP) (Promega) at 37°C for 5 minutes. Digested samples were then cleaned up using Monarch 500 μg RNA Clean Up kit (New England Biolabs) and eluted into 100 μL nuclease-free water.

[0337] RNA Quantification: RNA concentration was determined using a NanoDrop OneC spectrophotometer (Thermo Scientific).

[0338] RNA Dose Preparation and Administration: Animal experiments were carried out in accordance with the guidelines set forth by Charles River Accelerator Development Lab (CRADL, Cambridge, MA, USA) and were approved by the CRADL Institutional Animal Care and Use (IACUC) committee. Female BALB / C mice (7-9 weeks old) were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed at CRADL. Mice (n=5 per condition) were acclimated for at least 2 days before the initiation of the study.

[0339] Before injecting RNA into the mice, RNA was diluted using Ringers solution lactated- buffered (ThermoFisher) to a total dose of 5 μg in 50 uL and loaded into 1 mL syringes (BD syringes). For single injection groups, animals received one intramuscular (IM) RNA administration in the left lateral muscle. For split injection groups, animals received two separate IM RNA administrations of 2.5 μg each in the same left lateral muscle. Page 76 of 87 12767355v1Attorney Docket No.: 2012611-0182

[0340] Mice were imaged via whole body bioluminescence imaging at different time points following RNA injection. For whole body bioluminescence imaging, animals were injected with 200 μL of D-luciferin K+ salt (PerkinElmer122799) diluted to 15 mg / mL in PBS, via intraperitoneal (IP) injection, 10 minutes prior to the imaging time point. Three minutes prior to imaging, mice were placed under 3% isoflurane anesthesia in an induction chamber, then moved to isoflurane-delivering nosecones in the imaging chamber (IVIS-Spectrum Model 124262; Perkin Elmer, Waltham, MA) immediately prior to imaging. Mice were positioned on their left lateral (right side up) in the imaging chamber and were maintained on 3% isoflurane throughout imaging. Images were acquired using field of view D and continued to be exposed until 30,000 photons were collected or 1 min has passed, whichever occurred first. After imaging, animals were returned to their home cage for recovery. Results:

[0341] To facilitate the delivery of naked polyribonucleotides in vivo, naked Luciferase RNA was administered to mice. Sustained expression of Luciferase was observed for at least 1 week (FIG.1). Although Luciferase expression levels were sustained, high variability of Luciferase signal was observed among mice (n=5). In order to reduce the variability of Luciferase expression among animals, a second dose was administered on Day 21. A shown in FIG.2, administration of the second dose of naked polyribonucleotides resulted in a higher overall Luciferase signal and lower variability among mice (n=5).

[0342] These results demonstrate that delivery of two doses of naked polyribonucleotides led to sustained expression with lower variability among animals. This data suggests that administering naked polyribonucleotides in two or more doses can improve the delivery and / or expression of a payload (e.g., an RNA payload or a polypeptide payload). Example 2: A Novel Split-Dosing Approach to Deliver Naked Polyribonucleotides Results in Increased Expression with Lower Variability

[0343] This Example describes the delivery of naked polyribonucleotides using a split-dosing approach, whereby a dose of naked polyribonucleotides is divided into two or more subdoses and administered to animals at two or more administration sites. Page 77 of 87 12767355v1Attorney Docket No.: 2012611-0182 Methods:

[0344] To address the high variability of Luciferase signal among mice, an injection method of naked polyribonucleotides was developed. This approach included splitting a dose into multiple subdoses that were administered at multiple administration sites in the same muscle. The specific experimental methods used in this Example are described in Example 1. Results:

[0345] Delivery of naked polyribonucleotides resulted in a similar Luciferase signal in both groups (split injection and single injection group; see FIG.3), but with a statistically significant reduction in standard deviation of Luciferase signal among mice in the split injection group compared to the single injection group (FIG.4 and FIG.5). Reduction in the standard deviation was analyzed by a single tail F test, which showed a statistically significant difference between the single injection group and split injection group at 6 hours and 48 hours. No statistically significant difference was observed between the single injection group and split injection group at 24 hours. Without wishing to be bound by any particular theory, in some embodiments, this data may suggest that naked polyribonucleotides are taken up in cells by various mechanisms and / or kinetics. This novel delivery method also resulted in a lower number of outliers and a mean closer to the median for the split injection group (FIG.5).

[0346] Together, these results suggest that delivery of naked polyribonucleotides using a split- dosing approach, whereby a dose of naked polyribonucleotides is divided into two or more subdoses delivered to two or more injection sites, may improve the delivery of naked polyribonucleotides and reduce variability of expression over time. This data supports the development of the novel split-dosing approach described herein to deliver naked polyribonucleotides (e.g., RNA therapeutics) to achieve increased expression, sustained expression and / or controlled expression of a payload (e.g., an RNA payload or a polypeptide payload). Page 78 of 87 12767355v1Attorney Docket No.: 2012611-0182 EQUIVALENTS

[0347] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Further, it should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the claims that follow. Page 79 of 87 12767355v1

Claims

1. Attorney Docket No.: 2012611-0182 CLAIMS What is claimed is:

1. A method comprising: administering two or more subdoses of a naked polyribonucleotide to a subject at two or more administration sites on the subject so that the subject receives a dose of the naked polyribonucleotide, and wherein administration of the two or more subdoses is performed in 30 minutes or less.

2. The method of claim 1, wherein the two or more subdoses together make up the dose.

3. The method of claim 1 or 2, wherein each of the subdoses comprises about 5% to about 95% of the dose.

4. The method of any one of the preceding claims, wherein the subject is administered three or more subdoses, four or more subdoses, five or more subdoses, six or more subdoses, seven or more subdoses, or ten or more subdoses.

5. The method of any one of the preceding claims, wherein each subdose of the two or more subdoses is the same.

6. The method of any one of claims 1-5, wherein each subdose of the two or more subdoses is different.

7. The method of any one of the preceding claims, wherein the two or more administration sites are within a specified distance of each other.

8. The method of claim 7, wherein the specified distance is about two square inches, about three square inches, about four square inches, about five square inches, about ten square inches, about twenty square inches, or about thirty square inches of each other. Page 80 of 87 12767355v1 Attorney Docket No.: 2012611-0182 9. The method of claim 7, wherein the specified distance is about 1mm radius to about 50 mm radius of each other.

10. The method of any one of the preceding claims, wherein the two or more administration sites are in the same tissue.

11. The method of any one of claims 1-9, wherein the two or more administration sites are in different tissue.

12. The method of any one of the preceding claims, wherein the two or more administration sites are in muscle tissue.

13. The method of claim 12, wherein the muscle tissue comprises: (i) striated muscle, non-striated muscle, or both, (ii) skeletal muscle, smooth muscle, cardiac muscle, or any combination thereof, or (iii) both (i) and (ii).

14. The method of any one of the preceding claims, wherein the two or more subdoses are administered at the two or more administration sites at substantially the same time, optionally wherein the two or more subdoses are administered at the two or more administration sites simultaneously.

15. The method of any one of claims 1-13, wherein the two or more subdoses are administered at the two or more administration sites sequentially.

16. The method of any one of the preceding claims, wherein the two or more subdoses are administered in about 25 minutes to about 1 second.

17. The method of any one of the preceding claims, wherein the two or more subdoses are administered using a multi-needle injector. Page 81 of 87 12767355v1 Attorney Docket No.: 2012611-0182 18. The method of any one of the preceding claims, wherein: (i) administration of the two or more subdoses is performed within less than 5 minutes; (ii) the two or more administration sites are in muscle tissue; and / or (iii) the two or more administrations sites are within an area having a 2 mm or more radius.

19. The method of any one of the preceding claims, wherein the polyribonucleotide is linear.

20. The method of any one of claims 1-18, wherein the polyribonucleotide is circular.

21. The method of any one of the preceding claims, wherein the polyribonucleotide comprises one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

22. The method of claim 21, wherein the one or more modified ribonucleotides comprise modified ribonucleotides that comprise a 2’-O-acetylated ribose.

23. The method of claim 22, wherein the one or modified ribonucleotides comprise: (i) a 2’-O-acetyl ribose and a guanine nucleobase or a variant thereof, (ii) a 2’-O-acetyl ribose and an adenine nucleobase or a variant thereof, (iii) a 2’-O-acetyl ribose and an uracil nucleobase or a variant thereof, and / or (iv) a 2’-O-acetyl ribose and a cytosine nucleobase or a variant thereof.

24. The method of any one of claims 1-23, wherein the polyribonucleotide comprises a cap structure.

25. The method of any one of claims 1-24, wherein the one or more modified ribonucleotides comprises a nucleoside comprising: N4-acetylcytidine, 5-hydroxymethyluridine, N1- methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza- Page 82 of 87 12767355v1 Attorney Docket No.: 2012611-0182 uridine, 2-thio-uridine (s2U), 5-methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (m1 I), wyosine (imG), methylwyosine (mimG), 5-hydroxycytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-methoxycytidine, 5-propynylcytidine, 2- thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2’-O- methyluridine, 2’-O-methyl-5-methyluridine, 2’-fluoro-2’-deoxyuridine, 2’-amino-2’- deoxyuridine, 2’-azido-2’-deoxyuridine, 4-thiouridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5- bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2’-O-methyl-pseudouridine, N1- hydroxypseudouridine, 2’-O-methyl-N1-methylpseudouridine, N1-ethylpseudouridine, N1- hydroxymethylpseudouridine, ara-uridine, N6-methyladenosine, 2-aminoadenosine, 3- methyladenosine, 7-deazaadenosine, 8-oxoadenosine, thienoguanosine, 7-deazaguanosine, 8- oxoguanosine, 6-O-methylguanine, or any combination thereof.

26. The method of any one of the preceding claims, wherein the polyribonucleotide comprises a coding region, optionally wherein the coding region encodes a payload.

27. The method of claim 26, wherein the payload is or comprises a polypeptide.

28. The method of any one of claims 1-26, wherein the polyribonucleotide comprises an RNA payload.

29. The method of any one of the preceding claims, wherein the polyribonucleotide is in a composition, optionally wherein the composition is a pharmaceutical composition.

30. The method of any one of claims 26-29, wherein administration of the two or more doses to a subject: (i) increases expression of the payload; (ii) increases presence (e.g., increased persistence / bioavailability) of the payload; (iii) both (i) and (ii), Page 83 of 87 12767355v1 Attorney Docket No.: 2012611-0182 as compared to expression and / or presence of a payload delivered by an otherwise similar polyribonucleotide administered in a single dose or at a single site.

31. The method of claim 30, wherein increased expression and / or presence is detected for at least 1 hour, at least 2 hours, at least 4 hours, at least 12 hours, at least 2 hours, at least 2 days, at least 4 days, at least 1 week, at least 2 weeks, or at least 1 month compared to expression of a payload delivered by an otherwise similar polyribonucleotide in a single dose and / or at a single site.

32. The method of any one of claims 29-31, wherein increased expression and / or presence is a result of reduced degradation and / or a result of increased half-life.

33. The method of any one of the preceding claims, wherein administration of the two or more subdoses provides more consistent expression of the payload as compared to expression of a payload delivered by an otherwise similar polyribonucleotide administered in a single dose or at a single site, optionally wherein more consistent expression comprises substantially the same expression from the two or more administration sites.

34. The method of any one of the preceding claims, wherein the method is a treatment method, optionally wherein the method is a vaccination method.

35. The method of any one of the preceding claims, wherein the method is: (i) a method to stimulate an immune response; (ii) an antibody therapy method; (iii) an immune-modulation method; (iv) a vaccination method; (v) a gene therapy method; (vi) a cell therapy engineering method; (vii) an immunotherapy method; (viii) a protein replacement therapy method; (ix) a chemotherapeutic method; or Page 84 of 87 12767355v1 Attorney Docket No.: 2012611-0182 (x) any combination of (i)-(ix).

36. A kit comprising: two or more subdoses of a naked polyribonucleotide formulated for administration to a subject.

37. The kit of claim 36, wherein the kit further comprises two or more injector needles, optionally wherein each of the two or more injector needles comprises a subdose of the naked polyribonucleotide.

38. A kit comprising: a dose of a naked polyribonucleotide formulated for administration to a subject.

39. The kit of claim 38, wherein the kit further comprises a multi-needle injector comprising: (i) one or more cavities, and (ii) two or more needles, and wherein the one or more cavities are configured to hold the dose of the naked polyribonucleotide, wherein the one or more cavities are connected to the two or more needles so that the dose of naked polyribonucleotide can traverse from the one or more cavities to the two or more needles, and wherein the two or more needles are each configured to deliver a subdose of the naked polyribonucleotide to the subject so that the subject receives the dose of the naked polyribonucleotide.

40. A multi-needle injector comprising: (i) one or more cavities, (ii) two or more needles, and (iii) a dose of a naked polyribonucleotide formulated for administration to a subject, wherein the one or more cavities are configured to hold the dose of the naked polyribonucleotide, Page 85 of 87 12767355v1 Attorney Docket No.: 2012611-0182 wherein the one or more cavities are connected to the two or more needles so that the dose of naked polyribonucleotide can traverse from the one or more cavities to the two or more needles, and wherein the two or more needles are each configured to deliver a subdose of the naked polyribonucleotide to the subject so that the subject receives the dose of the naked polyribonucleotide. Page 86 of 87 12767355v1

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