Compositions containing polyribonucleotides and their use

Abstract

This disclosure features compositions comprising a polyribonucleotide having one or more expression-enhancing elements or spacer elements. The polyribonucleotides described herein can enhance the stability of the polyribonucleotide and / or enhance the expression of the polynucleotide cargo encoded by the polyribonucleotide. In one embodiment, this disclosure provides a cyclic polyribonucleotide comprising, in the following order from 5' to 3', (a) a first post-cyclization element; (b) one or more expression-enhancing elements; (c) a polyribonucleotide cargo; and (d) a second post-cyclization element, wherein the first and second post-cyclization elements together form a cyclization junction.

Description

[Technical Field]

[0001] (Background technology) Polyribonucleotides are easily degraded within cells, hindering the persistence and potential expression of the therapeutic polypeptides they encode. This poses a significant obstacle to the potential therapeutic use of polyribonucleotides. Therefore, methods to enhance the stability of polyribonucleotides and improve the translation of their polypeptide cargoes remain necessary. [Overview of the project] [Means for solving the problem]

[0002] In one embodiment, the present disclosure provides a cyclic polyribonucleotide comprising, in the following order from 5' to 3', (a) a first post-cyclization element; (b) one or more expression-enhancing elements; (c) a polyribonucleotide cargo; and (d) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0003] In another embodiment, the present disclosure provides a cyclic polyribonucleotide comprising, in the following order from 5' to 3', (a) a first post-cyclization element; (b) a polyribonucleotide cargo; (c) one or more expression-enhancing elements; and (d) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0004] In some embodiments, one or more expression-enhancing elements comprise a fusion of a translation enhancer and a spacer element containing a poly(A) region. In some embodiments, the spacer element comprises ribonucleotides of length 5 to 200 (e.g., 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150). In some embodiments, one or more expression-enhancing elements comprise a translation enhancer. In some embodiments, the translation enhancer is derived from a plant virus. In some embodiments, the translation enhancer is derived from a mammalian virus. In some embodiments, the translation enhancer is a human translation enhancer. In some embodiments, the translational enhancer is selected from the group comprising BYDV-like elements (BTE), translational enhancer elements (TED), PMV / PEMV-like translational enhancers (PTE), type I structures (ISS), type Y structures (YSS), type t structures (TSS), dumbbell structures, viral RNA UTRs (including dengue, West Nile, Zika, and rotavirus), EMCV, CVB3, hepatitis B virus post-transcriptional regulatory elements, human genome fragments, histone mRNA sequences, cyclin D mRNA sequences, or eIF4g aptamer sequences. In some embodiments, the translational enhancer binds to eIF4g.

[0005] In some embodiments, the translation enhancer comprises 50 to 2000 ribonucleotides (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500).

[0006] In some embodiments, the translation enhancer comprises 60-800 ribonucleotides (e.g., 60-700, 60-600, 60-500, 60-400, 60-400, 60-300, 60-300, 60-200, 60-100, 100-800, 200-800, 300-800, 400-800, 500-800, 600-800, 700-800, and 200-500).

[0007] In some embodiments, cyclic polyribonucleotides exhibit at least twice the stability compared to polyribonucleotides without a translational enhancer. In some embodiments, cyclic polyribonucleotides can be detected at least twice as long after administration to a subject compared to polyribonucleotides without a translational enhancer. In some embodiments, cyclic polyribonucleotides comprise a polynucleotide cargo encoding a polypeptide having at least four times greater expression compared to polyribonucleotides without a translational enhancer.

[0008] In some embodiments, one or more expression-enhancing elements comprise a fusion of a stability element and a spacer element containing a poly(A) region. In some embodiments, the spacer element comprises ribonucleotides of length 5 to 200 (e.g., 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150). In some embodiments, the expression-enhancing element comprises a stability element. In some embodiments, the stability element is an untranslated region (UTR). In some embodiments, the stability element is a 3'UTR. In some embodiments, the stability element is a 5'UTR. In some embodiments, the UTR is derived from a gene encoding translocation-associated membrane protein 1 (TRAM1), transmembrane p24 transport protein 2 (TMED2), vesicle-associated membrane protein 3 (VAMP3), CXXC repeat-containing interactor of the PDZ3 domain (CRIP), adapter-associated protein complex 2 subunit alpha 2 (AP2A2), proteasome 26S subunit, non-ATPase 5 (PSMD5), glutathione peroxidase 4 (GPX4), or protein kinase AMP-activated non-catalytic subunit beta 1 (PRKAB1). In some embodiments, the UTR is derived from human beta-actin, DDB2, TP53I3, FcIgG, LSP1, AES, DRB4, or mitochondrial-encoded 12S rRNA. In some embodiments, the stability element comprises 50 to 2000 ribonucleotides (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500).In some embodiments, the stability element comprises 60-800 ribonucleotides (e.g., 60-700, 60-600, 60-500, 60-400, 60-400, 60-300, 60-300, 60-200, 60-100, 100-800, 200-800, 300-800, 400-800, 500-800, 600-800, 700-800, and 200-500). In some embodiments, the cyclic polyribonucleotide exhibits at least twice the stability compared to the polyribonucleotide without the stability element. In some embodiments, the cyclic polyribonucleotide can be detected at least twice as long after administration to a subject compared to the polyribonucleotide without the stability element. In some embodiments, the polynucleotide cargo encodes a polypeptide having at least twice the expression compared to the polyribonucleotide without the stability element.

[0009] In some embodiments, the cyclic polyribonucleotide further comprises one or more spacer elements containing a poly(A) region. In some embodiments, the spacer element has a ribonucleotide length of 50 to 500 (e.g., 50 to 450, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 50 to 200, 50 to 150, 50 to 100, 50 to 80, 55 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 110 to 500, 115 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500, 50 to 300, 50 to 200, 60 to 300, and 60 to 200). In some embodiments, the spacer element has a ribonucleotide length of 100 to 500 (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 500, 115 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500, 100 to 300, 100 to 200, 110 to 300, and 110 to 200). In some embodiments, the spacer element has a ribonucleotide length of 110 to 500 (e.g., 110 to 450, 110 to 400, 110 to 350, 110 to 300, 110 to 250, 110 to 200, 110 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500). In some embodiments, the spacer element has a ribonucleotide length of 120 to 500 (e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500).In some embodiments, the spacer element has a ribonucleotide length of 100 to 300 (e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 110 to 300, 115 to 300, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, 110 to 200, and 280 to 300). In some embodiments, the spacer has a ribonucleotide length of 10 to 200 (e.g., 10 to 175, 10 to 150, 10 to 125, 10 to 100, 10 to 75, 10 to 50, 10 to 25, 25 to 200, 50 to 200, 75 to 200, 100 to 200, 150 to 200, 175 to 200, and 25 to 100). In some embodiments, the spacer element has a ribonucleotide length of about 120. In some embodiments, the spacer element has a ribonucleotide length of about 50.

[0010] In another embodiment, the Disclosure provides a cyclic polyribonucleotide comprising (a) a first post-cyclization element; (b) a first expression-enhancing element; (c) a polyribonucleotide cargo; (d) a second expression-enhancing element; and (e) a second post-cyclization element in the following order from 5' to 3', wherein the first and second post-cyclization elements together form a cyclization junction. In some embodiments, the first expression-enhancing element comprises a fusion of a translation enhancer and a spacer element. In some embodiments, the spacer element comprises ribonucleotides of length 5 to 200 (e.g., 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150). In some embodiments, the second expression-enhancing element comprises a fusion of a translation enhancer and a spacer element. In some embodiments, the spacer element comprises ribonucleotides of length 5 to 200 (e.g., ribonucleotide lengths of 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150). In some embodiments, the first expression-enhancing element comprises a translational enhancer. In some embodiments, the second expression-enhancing element comprises a translational enhancer. In some embodiments, the translational enhancer is derived from a plant virus. In some embodiments, the translational enhancer is derived from a mammalian virus. In some embodiments, the translational enhancer is a human translational enhancer. In some embodiments, the translational enhancer is selected from the group comprising BTE, TED, PTE, ISS, YSS, TSS, dumbbell-shaped structures, viral RNA UTR (including dengue fever, West Nile, Zika, and rotavirus), EMCV, CVB3, hepatitis B virus posttranscriptional regulatory elements, human genome fragments, histone mRNA sequences, cyclin D mRNA sequences, or eIF4g aptamer sequences. In some embodiments, the translational enhancer binds to eIF4g.In some embodiments, the translation enhancer comprises 50 to 2000 ribonucleotides (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500). In some embodiments, the translation enhancer comprises 60-800 ribonucleotides (e.g., 60-700, 60-600, 60-500, 60-400, 60-400, 60-300, 60-300, 60-200, 60-100, 100-800, 200-800, 300-800, 400-800, 500-800, 600-800, 700-800, and 200-500). In some embodiments, the cyclic polyribonucleotide exhibits at least twice the stability compared to polyribonucleotide without the translation enhancer. In some embodiments, the cyclic polyribonucleotide can be detected at least twice as long after administration to a subject compared to polyribonucleotide without the translation enhancer. In some embodiments, the cyclic polyribonucleotide comprises a polynucleotide cargo encoding a polypeptide having at least four times greater expression compared to a polyribonucleotide without a translational enhancer.

[0011] In some embodiments, the first expression-enhancing element comprises a fusion of a stability element and a spacer element containing a poly(A) region. In some embodiments, the spacer element comprises ribonucleotides of length 5 to 200 (e.g., 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150). In some embodiments, the first expression-enhancing element comprises a stability element. In some embodiments, the spacer element comprises ribonucleotides of length 5 to 200 (e.g., 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150). In some embodiments, the second expression-enhancing element comprises a stability element. In some embodiments, the stability element is a UTR. In some embodiments, the UTR is derived from a gene encoding TRAM1, TMED2, VAMP3, CRIP, AP2A2, proteasome 26S subunit, PSMD5, GPX4, or PRKAB1. In some embodiments, the UTR is derived from human beta-actin, DDB2, TP53I3, FcIgG, LSP1, AES, DRB4, or mitochondrial-encoded 12S rRNA. In some embodiments, the stability element is a 3' UTR. In some embodiments, the stability element is a 5' UTR. In some embodiments, the stability element comprises 50 to 2000 ribonucleotides (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500).In some embodiments, the stability element comprises 60-800 ribonucleotides (e.g., 60-700, 60-600, 60-500, 60-400, 60-400, 60-300, 60-300, 60-200, 60-100, 100-800, 200-800, 300-800, 400-800, 500-800, 600-800, 700-800, and 200-500). In some embodiments, the cyclic polyribonucleotide exhibits at least twice the stability compared to the polyribonucleotide without the stability element. In some embodiments, the cyclic polyribonucleotide can be detected at least twice as long after administration to a subject compared to the polyribonucleotide without the stability element. In some embodiments, the polynucleotide cargo encodes a polypeptide having at least twice the expression compared to the polyribonucleotide without the stability element.

[0012] In some embodiments, the cyclic polynucleotide further comprises one or more spacer elements comprising a polyA region. In some embodiments, the spacer element has a length of 100 to 500 (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides. In some embodiments, the spacer element has a length of 120 to 500 (e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500) ribonucleotides. In some embodiments, the spacer element has a length of 100 to 300 (e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300) ribonucleotides.

[0013] In another aspect, the present disclosure provides a cyclic polynucleotide comprising, in the following order from 5' to 3': (a) a first post-cyclization element; (b) a spacer element; (c) a polynucleotide cargo; (d) an expression enhancing element; and (e) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0014] In another aspect, the present disclosure provides a cyclic polynucleotide comprising, in the following order from 5' to 3': (a) a first post-cyclization element; (b) an expression enhancing element; (c) a polynucleotide cargo; (d) a spacer element; and (e) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0015] In another aspect, the present disclosure provides a circular polynucleotide comprising, in the 5' to 3' order: (a) a first post-cyclization element; (b) a first expression enhancing element; (c) a polynucleotide cargo; (d) a second expression enhancing element; and (e) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0016] In another aspect, the present disclosure provides a circular polynucleotide comprising, in the 5' to 3' order: (a) a first post-cyclization element; (b) a first spacer element having a length of at least 100 ribonucleotides; (c) a polynucleotide cargo; (d) a second spacer element; and (e) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0017] In another aspect, the present disclosure provides a circular polynucleotide comprising, in the 5' to 3' order: (a) a first post-cyclization element; (b) a first spacer element; (c) a polynucleotide cargo; (d) a second spacer element having a length of at least 100 ribonucleotides; and (e) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0018] In some embodiments, the first spacer element has a ribonucleotide length of 50 to 500 (e.g., 50 to 450, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 50 to 200, 50 to 150, 50 to 100, 50 to 80, 55 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 110 to 500, 115 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500, 50 to 300, 50 to 200, 60 to 300, and 60 to 200). In some embodiments, the first spacer element has a ribonucleotide length of 100-500 (e.g., 100-450, 100-400, 100-350, 100-300, 100-250, 100-200, 100-150, 110-450, 110-400, 110-350, 110-300, 110-250, 110-200, 110-150, 120-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, and 450-500). In some embodiments, the second spacer element has a ribonucleotide length of 50 to 500 (e.g., 50 to 450, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 50 to 200, 50 to 150, 50 to 100, 50 to 80, 55 to 500, 60 to 500, 70 to 500, 80 to 500, 90 to 500, 110 to 500, 115 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500, 50 to 300, 50 to 200, 60 to 300, and 60 to 200). In some embodiments, the second spacer element has a ribonucleotide length of 100-500 (e.g., 100-450, 100-400, 100-350, 100-300, 100-250, 100-200, 100-150, 110-450, 110-400, 110-350, 110-300, 110-250, 110-200, 110-150, 120-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, and 450-500).In some embodiments, the first spacer element and the second spacer element each contain 110-500 ribonucleotides (e.g., 110-450, 110-400, 110-350, 110-300, 110-250, 110-200, 110-150, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, and 450-500). In some embodiments, the first spacer element and the second spacer element each contain 120 to 500 ribonucleotides (e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500). In some embodiments, the first spacer element and the second spacer element each have a ribonucleotide length of 150 to 500 (e.g., 150 to 450, 150 to 400, 150 to 350, 150 and 300, 150 and 250, 150 and 200, 200 and 500, 250 and 500, 300 and 500, 350 and 500, 400 and 500, or 450 and 500). In some embodiments, the first spacer element and the second spacer element each have a ribonucleotide length of 200 to 500 (e.g., 200 to 450, 200 to 400, 200 to 350, 200 to 300, 200 to 250, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 500). In some embodiments, the first spacer element and the second spacer element each have a ribonucleotide length of 100 to 300 (e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300).In some embodiments, the first spacer element and the second spacer element each have a ribonucleotide length of 100 to 200 (for example, 100 to 190, 100 to 180, 100 to 170, 100 to 160, 100 to 150, 100 to 140, 100 to 130, 100 to 120, 100 to 110, 110 to 200, 120 to 200, 130 to 200, 140 to 200, 150 to 200, 160 to 200, 170 to 200, 180 to 200, and 90 to 200). In some embodiments, the first spacer element and the second spacer element each have a ribonucleotide length of 110 to 300 (for example, 110 to 280, 110 to 260, 110 to 240, 110 to 220, 110 to 200, 110 to 180, 110 to 160, 110 to 140, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300). In some embodiments, the first spacer element and the second spacer element each have a ribonucleotide length of 120 to 300 (for example, 120 to 280, 120 to 260, 120 to 240, 120 to 220, 120 to 200, 120 to 180, 120 to 160, 120 to 140, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300). In some embodiments, the first spacer element and the second spacer element are each 150-300 (for example, 150-290, 150-280, 150-270, 150-260, 150-250, 150-240, 150-230, 150-220, 150-210, 150-200, 150-190, 150-1) The ribonucleotides have lengths of 80, 150-170, 150-160, 160-300, 170-300, 180-300, 190-300, 200-300, 210-300, 220-300, 230-300, 240-300, 250-300, 260-300, 270-300, 280-300, and 290-300.

[0019] In some embodiments, the difference between the length of the first spacer element and the length of the second spacer element is 0 to 100 nucleotides (e.g., 0 and 10, 0 and 20, 0 and 30, 0 and 40, 0 and 50, 0 to 60, 0 to 70, 0 to 80, 0 to 90, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 5 to 10, 5 to 20, 10 to 30, or 5 to 50). In some embodiments, the difference between the length of the first spacer element and the length of the second spacer element is 0 to 50 nucleotides (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50). In some embodiments, the first and second spacer elements have the same number of ribonucleotide lengths.

[0020] In some embodiments, the first or second spacer element is (i) a poly-A region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) adenosine residues or cytosine residues. (iii) A poly-AC region containing a group; (iii) A poly-AU region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) adenosine or uridine residues; or (iv) A poly-AG region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) adenosine or guanosine residues.

[0021] In some embodiments, cyclic polyribonucleotides exhibit at least twice the stability compared to polyribonucleotides without a first or second spacer element. In some embodiments, cyclic polyribonucleotides can be detected at least twice as long after administration to a subject compared to polyribonucleotides without a first or second spacer element. In some embodiments, the polynucleotide cargo encodes a polypeptide, which has at least four times greater expression compared to polyribonucleotides without a first or second spacer element.

[0022] In some embodiments, the polyribonucleotide cargo includes an expression sequence. In some embodiments, the polyribonucleotide cargo includes an IRES operably ligated to the expression sequence. In some embodiments, the expression sequence further includes a 3' untranslated region or a 5' untranslated region. In some embodiments, the expression sequence encodes a polypeptide.

[0023] In some embodiments, the polyribonucleotide comprises 500 to 20,000 ribonucleotides (e.g., 500 to 15,000, 500 to 10,000, 500 to 5,000, 500 to 1,000, 1,000 to 20,000, 5,000 to 20,000, 10,000 to 20,000, and 15,000 to 20,000). In some embodiments, the polyribonucleotides include 2,000 and 20,000 ribonucleotides (e.g., 2,000-5,000, 2,000-7,500, 2,000-10,000, 2,000-12,500, 2,000-15,000, 2,000-17,500, 5,000-20,000, 7,500-20,000, 10,000-20,000, 12,500-20,000, 15,000-20,000, and 17,500-20,000).

[0024] In some embodiments, the cyclization junction includes a splice junction. In some embodiments, the first post-cyclization element includes a first exon fragment, and the second post-cyclization element includes a second exon fragment, with the first and second exon fragments linked by a splice junction. In some embodiments, the cyclization junction includes an oligonucleotide sprint that hybridizes to the first and second post-cyclization elements. In some embodiments, the oligonucleotide sprint is a DNA sprint or an RNA sprint. In some embodiments, the first post-cyclization element includes a region that can hybridize to a first region of the oligonucleotide sprint, and the second post-cyclization element includes a region that can hybridize to a second region of the oligonucleotide sprint.

[0025] In one embodiment, the present disclosure provides a linear polyribonucleotide comprising, in the following order from 5' to 3', (a) a first post-cyclization element; (b) one or more expression-enhancing elements; (c) a polyribonucleotide cargo; and (d) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0026] In one embodiment, the present disclosure provides a linear polyribonucleotide comprising, in the following order from 5' to 3', (a) a first post-cyclization element; (b) a polyribonucleotide cargo; (c) one or more expression-enhancing elements; and (d) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0027] In some embodiments, one or more expression-enhancing elements comprise a fusion of a translation enhancer and a spacer element containing a poly(A) region. In some embodiments, the spacer element comprises ribonucleotides of length 5 to 200 (e.g., 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150). In some embodiments, one or more expression-enhancing elements comprise a translation enhancer. In some embodiments, the translation enhancer is derived from a plant virus. In some embodiments, the translation enhancer is derived from a mammalian virus. In some embodiments, the translation enhancer is a human translation enhancer. In some embodiments, the translational enhancer is selected from the group comprising BTE, TED, PTE, ISS, YSS, TSS, dumbbell-shaped structures, viral RNA UTR (including dengue fever, West Nile, Zika, and rotavirus), EMCV, CVB3, hepatitis B virus posttranscriptional regulatory elements, human genome fragments, histone mRNA sequences, cyclin D mRNA sequences, or eIF4g aptamer sequences. In some embodiments, the translational enhancer binds to eIF4g. In some embodiments, the translation enhancer comprises 50 to 2000 ribonucleotides (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500). In some embodiments, the translation enhancer comprises 60-800 ribonucleotides (e.g., 60-700, 60-600, 60-500, 60-400, 60-400, 60-300, 60-300, 60-200, 60-100, 100-800, 200-800, 300-800, 400-800, 500-800, 600-800, 700-800, and 200-500).

[0028] In some embodiments, linear polyribonucleotides exhibit at least twice the stability compared to polyribonucleotides without translational enhancers. In some embodiments, linear polyribonucleotides can be detected at least twice as long after administration to a subject compared to polyribonucleotides without translational enhancers. In some embodiments, linear polyribonucleotides are polynucleotide cargoes encoding polypeptides with at least four times greater expression compared to polyribonucleotides without translational enhancers.

[0029] In some embodiments, one or more expression-enhancing elements comprise a fusion of a stability element and a spacer element containing a poly(A) region. In some embodiments, the spacer element comprises ribonucleotides of length 5 to 200 (e.g., 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150). In some embodiments, the expression-enhancing element comprises a stability element. In some embodiments, the stability element is a UTR. In some embodiments, the UTR is derived from a gene encoding TRAM1, TMED2, VAMP3, CRIP, AP2A2, proteasome 26S subunit, PSMD5, GPX4, or PRKAB1. In some embodiments, the UTR is derived from human beta-actin, DDB2, TP53I3, FcIgG, LSP1, AES, DRB4, or mitochondrial-encoded 12S rRNA. In some embodiments, the stabilizing element is 3'UTR. In some embodiments, the stabilizing element is 5'UTR.

[0030] In some embodiments, the stability element comprises 50 to 2000 ribonucleotides (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500). In some embodiments, the stability element comprises 60-800 ribonucleotides (e.g., 60-700, 60-600, 60-500, 60-400, 60-400, 60-300, 60-300, 60-200, 60-100, 100-800, 200-800, 300-800, 400-800, 500-800, 600-800, 700-800, and 200-500).

[0031] In some embodiments, linear polyribonucleotides exhibit at least twice the stability compared to polyribonucleotides without a stability element. In some embodiments, linear polyribonucleotides can be detected at least twice as long after administration to a subject compared to polyribonucleotides without a stability element. In some embodiments, polynucleotide cargoes encode polypeptides with at least twice the expression compared to polyribonucleotides without a stability element.

[0032] In some embodiments, the linear polyribonucleotide further comprises one or more spacer elements containing a poly-A region. In some embodiments, the spacer element has a ribonucleotide length of 100 to 500 (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 500, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500). In some embodiments, the spacer element has a ribonucleotide length of 110 to 500 (e.g., 110 to 450, 110 to 400, 110 to 350, 110 to 300, 110 to 250, 110 to 200, 110 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500). In some embodiments, the spacer element has a ribonucleotide length of 120 to 500 (e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500). In some embodiments, the spacer element has a ribonucleotide length of 100 to 300 (e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 110 to 300, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300).

[0033] In other embodiments, the Disclosure provides a linear polyribonucleotide comprising (a) a first post-cyclization element; (b) a first expression-enhancing element; (c) a polyribonucleotide cargo; (d) a second expression-enhancing element; and (e) a second post-cyclization element in the following order from 5' to 3', wherein the first and second post-cyclization elements together form a cyclization junction. In some embodiments, the first expression-enhancing element comprises a fusion of a translation enhancer and a spacer element. In some embodiments, the spacer element comprises ribonucleotides of length 5 to 200 (e.g., 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150). In some embodiments, the second expression-enhancing element comprises a fusion of a translation enhancer and a spacer element. In some embodiments, the spacer element comprises ribonucleotides of length 5 to 200 (e.g., ribonucleotide lengths of 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150). In some embodiments, the first expression-enhancing element comprises a translation enhancer. In some embodiments, the second expression-enhancing element comprises a translation enhancer. In some embodiments, the translation enhancer is derived from a plant virus. In some embodiments, the translation enhancer is derived from a mammalian virus. In some embodiments, the translation enhancer is a human translation enhancer. In some embodiments, the translational enhancer is selected from the group comprising BTE, TED, PTE, ISS, YSS, TSS, dumbbell-shaped structures, viral RNA UTR (including dengue fever, West Nile, Zika, and rotavirus), EMCV, CVB3, hepatitis B virus posttranscriptional regulatory elements, human genome fragments, histone mRNA sequences, cyclin D mRNA sequences, or eIF4g aptamer sequences. In some embodiments, the translational enhancer binds to eIF4g.

[0034] In some embodiments, the translation enhancer comprises 50 to 2000 ribonucleotides (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500). In some embodiments, the translation enhancer comprises 60-800 ribonucleotides (e.g., 60-700, 60-600, 60-500, 60-400, 60-400, 60-300, 60-300, 60-200, 60-100, 100-800, 200-800, 300-800, 400-800, 500-800, 600-800, 700-800, and 200-500).

[0035] In some embodiments, linear polyribonucleotides exhibit at least twice the stability compared to polyribonucleotides without translational enhancers. In some embodiments, linear polyribonucleotides can be detected at least twice as long after administration to a subject compared to polyribonucleotides without translational enhancers. In some embodiments, linear polyribonucleotides are polynucleotide cargoes encoding polypeptides with at least four times greater expression compared to polyribonucleotides without translational enhancers.

[0036] In some embodiments, the first expression-enhancing element comprises a fusion of a stability element and a spacer element containing a poly(A) region. In some embodiments, the spacer element comprises ribonucleotides of length 5 to 200 (e.g., 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150). In some embodiments, the first expression-enhancing element comprises a stability element. In some embodiments, the second expression-enhancing element comprises a fusion of a stability element and a spacer element. In some embodiments, the spacer element comprises ribonucleotides of length 5 to 200 (e.g., 5 to 150, 5 to 100, 5 to 50, 5 to 20, 20 to 200, 50 to 200, 100 to 200, 150 to 200, and 50 to 150). In some embodiments, the second expression-enhancing element comprises a stability element. In some embodiments, the stability element is the UTR. In some embodiments, the UTR is derived from a gene encoding TRAM1, TMED2, VAMP3, CRIP, AP2A2, proteasome 26S subunit, PSMD5, GPX4, or PRKAB1. In some embodiments, the UTR is derived from human beta-actin, DDB2, TP53I3, FcIgG, LSP1, AES, DRB4, or mitochondrial-encoded 12S rRNA. In some embodiments, the stability element is the 3'UTR. In some embodiments, the stability element is the 5'UTR.

[0037] In some embodiments, the stability element comprises 50 to 2000 ribonucleotides (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 50 to 1250, 50 to 1250, 50 to 750, 100 to 2000, 500 to 2000, 750 to 2000, 1000 to 2000, 1250 to 2000, 1500 to 2000, 1750 to 2000, 100 to 1000, and 500 to 1500). In some embodiments, the stability element comprises 60-800 ribonucleotides (e.g., 60-700, 60-600, 60-500, 60-400, 60-400, 60-300, 60-300, 60-200, 60-100, 100-800, 200-800, 300-800, 400-800, 500-800, 600-800, 700-800, and 200-500).

[0038] In some embodiments, linear polyribonucleotides exhibit at least twice the stability compared to polyribonucleotides without a stability element. In some embodiments, linear polyribonucleotides can be detected at least twice as long after administration to a subject compared to polyribonucleotides without a stability element. In some embodiments, polynucleotide cargoes encode polypeptides with at least twice the expression compared to polyribonucleotides without a stability element.

[0039] In some embodiments, the linear polyribonucleotide further comprises one or more spacer elements containing a poly-A region. In some embodiments, the spacer element has a ribonucleotide length of 100 to 500 (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 120 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500). In some embodiments, the spacer element has a ribonucleotide length of 120 to 500 (e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500). In some embodiments, the spacer element has a ribonucleotide length of 100 to 300 (e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300).

[0040] In other embodiments, the Disclosure provides a linear polyribonucleotide comprising, in the following order from 5' to 3', (a) a first post-cyclization element; (b) a spacer element; (c) a polyribonucleotide cargo; (d) an expression-enhancing element; and (e) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0041] In other embodiments, the present disclosure provides a linear polyribonucleotide comprising, in the following order from 5' to 3', (a) a first post-cyclization element; (b) an expression-enhancing element; (c) a polyribonucleotide cargo; (d) a spacer element; and (e) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0042] In other embodiments, the present disclosure provides a linear polyribonucleotide comprising, in the following order from 5' to 3': (a) a first post-cyclization element; (b) a first expression-enhancing element; (c) a polyribonucleotide cargo; (d) a second expression-enhancing element; and (e) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0043] In other embodiments, the present disclosure provides a linear polyribonucleotide comprising, in the following order from 5' to 3': (a) a first post-cyclization element; (b) a first spacer element having a length of at least 100 ribonucleotides; (c) a polyribonucleotide cargo; (d) a second spacer element; and (e) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0044] In other embodiments, the present disclosure provides a linear polyribonucleotide comprising, in the following order from 5' to 3': (a) a first post-cyclization element; (b) a first spacer element having a length of at least 100 ribonucleotides; (c) a polyribonucleotide cargo; (d) a second spacer element; and (e) a second post-cyclization element, wherein the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

[0045] In other embodiments, the Disclosure provides a cyclic polyribonucleotide comprising, in the following order from 5' to 3': (a) a first post-cyclization element; (b) a first spacer element; (c) a polyribonucleotide cargo; (d) a second spacer element having a length of at least 100 ribonucleotides; and (e) a second post-cyclization element, wherein the first and second post-cyclization elements together form a cyclization junction.

[0046] In some embodiments, the first spacer element has a ribonucleotide length of 100-500 (e.g., 100-450, 100-400, 100-350, 100-300, 100-250, 100-200, 100-150, 110-500, 110-450, 110-400, 110-350, 110-300, 110-250, 110-200, 110-150, 120-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, and 450-500). In some embodiments, the first spacer element has a length between 100 and 300 (e.g., 100-250, 100-200, 100-150, 110-300, 120-300, 150-300, 200-300, and 250-300). In some embodiments, the second spacer element has a ribonucleotide length of 100-500 (e.g., 100-450, 100-400, 100-350, 100-300, 100-250, 100-200, 100-150, 110-500, 120-500, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, and 450-500). In some embodiments, the first spacer element and the second spacer element each contain 110-500 ribonucleotides (e.g., 110-450, 110-400, 110-350, 110-300, 110-250, 110-200, 110-150, 150-500, 200-500, 250-500, 300-500, 350-500, 400-500, and 450-500). In some embodiments, the first spacer element and the second spacer element each contain 120 to 500 ribonucleotides (e.g., 120 to 450, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500).In some embodiments, the first spacer element and the second spacer element each have a ribonucleotide length of 150 to 500 (e.g., 150 to 450, 150 to 400, 150 to 350, 150 and 300, 150 and 250, 150 and 200, 200 and 500, 250 and 500, 300 and 500, 350 and 500, 400 and 500, or 450 and 500). In some embodiments, the first spacer element and the second spacer element each have a ribonucleotide length of 200 to 500 (e.g., 200 to 450, 200 to 400, 200 to 350, 200 to 300, 200 to 250, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 500). In some embodiments, the first spacer element and the second spacer element each have a ribonucleotide length of 100 to 300 (e.g., 100 to 280, 100 to 260, 100 to 240, 100 to 220, 100 to 200, 100 to 180, 100 to 160, 100 to 140, 100 to 120, 120 to 300, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300). In some embodiments, the first spacer element and the second spacer element each have a ribonucleotide length of 100 to 200 (for example, 100 to 190, 100 to 180, 100 to 170, 100 to 160, 100 to 150, 100 to 140, 100 to 130, 100 to 120, 100 to 110, 110 to 200, 120 to 200, 130 to 200, 140 to 200, 150 to 200, 160 to 200, 170 to 200, 180 to 200, and 90 to 200). In some embodiments, the first spacer element and the second spacer element each have a ribonucleotide length of 110 to 300 (for example, 110 to 280, 110 to 260, 110 to 240, 110 to 220, 110 to 200, 110 to 180, 110 to 160, 110 to 140, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300).In some embodiments, the first spacer element and the second spacer element each have a ribonucleotide length of 120 to 300 (for example, 120 to 280, 120 to 260, 120 to 240, 120 to 220, 120 to 200, 120 to 180, 120 to 160, 120 to 140, 140 to 300, 160 to 300, 180 to 300, 200 to 300, 220 to 300, 240 to 300, 260 to 300, and 280 to 300). In some embodiments, the first spacer element and the second spacer element are each 150-300 (for example, 150-290, 150-280, 150-270, 150-260, 150-250, 150-240, 150-230, 150-220, 150-210, 150-200, 150-190, 150-1) The ribonucleotides have lengths of 80, 150-170, 150-160, 160-300, 170-300, 180-300, 190-300, 200-300, 210-300, 220-300, 230-300, 240-300, 250-300, 260-300, 270-300, 280-300, and 290-300. In some embodiments, the difference between the length of the first spacer element and the length of the second spacer element is 0 to 100 nucleotides (e.g., 0 and 10, 0 and 20, 0 and 30, 0 and 40, 0 and 50, 0 to 60, 0 to 70, 0 to 80, 0 to 90, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 5 to 10, 5 to 20, 10 to 30, or 5 to 50). In some embodiments, the difference between the length of the first spacer element and the length of the second spacer element is 0 to 50 nucleotides (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50). In some embodiments, the first and second spacer elements have the same number of ribonucleotide lengths.

[0047] In some embodiments, the first or second spacer element is (i) a poly-A region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) adenosine residues or cytosine residues. (iii) A poly-AC region containing a group; (iii) A poly-AU region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) adenosine or uridine residues; or (iv) A poly-AG region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) adenosine or guanosine residues.

[0048] In some embodiments, the polyribonucleotide exhibits at least twice the stability compared to a polyribonucleotide without a first or second spacer element. In some embodiments, the polyribonucleotide can be detected at least twice as long after administration to a subject compared to a polyribonucleotide without a first or second spacer element. In some embodiments, the polynucleotide cargo encodes a polypeptide, which has at least four times greater expression compared to a polyribonucleotide without a first or second spacer element.

[0049] In some embodiments, the polyribonucleotide cargo includes an expression sequence. In some embodiments, the polyribonucleotide cargo includes an IRES operably ligated to the expression sequence. In some embodiments, the expression sequence further includes a 3' untranslated region or a 5' untranslated region. In some embodiments, the expression sequence encodes a polypeptide.

[0050] In some embodiments, the polyribonucleotide comprises 500 to 20,000 ribonucleotides (e.g., 500 to 15,000, 500 to 10,000, 500 to 5,000, 500 to 1,000, 1,000 to 20,000, 5,000 to 20,000, 10,000 to 20,000, and 15,000 to 20,000). In some embodiments, the polyribonucleotides include 2,000 and 20,000 ribonucleotides (e.g., 2,000-5,000, 2,000-7,500, 2,000-10,000, 2,000-12,500, 2,000-15,000, 2,000-17,500, 5,000-20,000, 7,500-20,000, 10,000-20,000, 12,500-20,000, 15,000-20,000, and 17,500-20,000).

[0051] In some embodiments, the cyclization junction includes a splice junction. In some embodiments, the first post-cyclization element includes a first exon fragment, and the second post-cyclization element includes a second exon fragment, with the first and second exon fragments linked by a splice junction. In some embodiments, the cyclization junction includes an oligonucleotide sprint that hybridizes to the first and second post-cyclization elements. In some embodiments, the oligonucleotide sprint is a DNA sprint or an RNA sprint. In some embodiments, the first post-cyclization element includes a region that can hybridize to a first region of the oligonucleotide sprint, and the second post-cyclization element includes a region that can hybridize to a second region of the oligonucleotide sprint. In some embodiments, the first cyclization element includes a first catalytic intron fragment, a first splice site dinucleotide, and a first exon fragment. The second cyclization element includes a second catalytic intron fragment, a second splice site dinucleotide, and a second exon fragment. In some embodiments, a first catalytic intron fragment and a second catalytic intron fragment can be self-spliced ​​together, thereby covalently linking the first and second exon regions to produce a cyclic polyribonucleotide. In some embodiments, the first and second catalytic intron fragments are derived from the cyanobacterial Anabaena pre-tRNA-Leu gene or Tetrahymena pre-rRNA. In some embodiments, the first cyclization element includes a region that can hybridize to a first region of the oligonucleotide sprint, and the second cyclization element includes a region that can hybridize to a second region of the oligonucleotide sprint. In another embodiment, the present disclosure provides a DNA vector comprising an RNA polymerase promoter operably linked to a sequence encoding any one of the linear polyribonucleotides described herein.

[0052] In another aspect, the Disclosure provides a cyclic polyribonucleotide produced from any one of the linear polyribonucleotides or from the DNA vectors described herein. In another aspect, the Disclosure provides a pharmaceutical composition comprising any one of the linear polyribonucleotides, cyclic polyribonucleotides, or DNA vectors described herein and a pharmaceutically acceptable excipient.

[0053] In another aspect, the Disclosure provides a method for expressing a polypeptide in cells or subjects, comprising providing the cells or subjects with one of the following: a linear polyribonucleotide, a cyclic polyribonucleotide, a DNA vector, or a pharmaceutical composition as described herein.

[0054] In another aspect, the Disclosure provides a method for generating a cyclic polyribonucleotide from any one of the linear polyribonucleotides described herein, comprising providing the linear polyribonucleotide under conditions suitable for self-splicing of the linear polyribonucleotide to generate the cyclic polyribonucleotide.

[0055] definition To facilitate understanding of this disclosure, several terms are defined below. Terms as defined herein have meanings that are generally understood by those skilled in the art relating to this disclosure. Terms such as “a,” “an,” and “the” are not intended to refer to only a single entity, but include general classes from which specific examples can be used for illustrative purposes. The term “or” is used to mean “and / or” unless it is expressly indicated that it refers to only substitutes or that the substitutes are mutually exclusive, but this disclosure supports the definitions that refer to only substitutes and “and / or.” Terms herein are used to describe specific embodiments, but their use should not be construed as limiting unless outlined in the claims.

[0056] As used herein, any value provided within a range of values ​​includes both the upper and lower limits, as well as any value that falls within the upper and lower limits.

[0057] As used herein, the term "approximately" refers to a value within ±10% of the listed values.

[0058] As used herein, the term “between” refers to all values ​​greater than or equal to an initial value and less than or equal to an endpoint such that the range of values ​​between two values ​​includes the endpoint of the range. For example, 1 and 5 refers to all values ​​greater than or equal to 1 and less than or equal to 5 such that the endpoints of 1 and 5 are included in the intended range.

[0059] As used herein, the term “carrier” refers to a compound, composition, reagent, or molecule that facilitates the transport or delivery of a composition (e.g., a cyclic polyribonucleotide) into a cell by covalent modification of a cyclic polyribonucleotide, partial or complete encapsulation, or a combination thereof. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-type materials), nanoparticles (e.g., nanoparticles that encapsulate or covalently link cyclic polyribonucleotides, thereby binding to the cyclic polyribonucleotide), liposomes, fusosomes, exosomes, protein carriers (e.g., proteins covalently bound to cyclic polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents).

[0060] As used herein, the terms “circRNA,” “cyclic polyribonucleotide,” “cyclic RNA,” and “cyclic polyribonucleotide molecule” are interchangeable and mean a polyribonucleotide molecule having a structure without free ends (i.e., no free 3' and / or 5' ends), such as a polyribonucleotide molecule that forms a cyclic or endless structure via covalent bonds (e.g., covalently closed) or non-covalent bonds. A cyclic polyribonucleotide may, for example, be a covalently closed polyribonucleotide.

[0061] As used herein, the term "cyclization efficiency" is a measure of the resulting cyclic polyribonucleotide versus its acyclic starting material.

[0062] As used herein, the terms “disease,” “disorder,” and “condition” refer, respectively, to a suboptimal health condition, such as one that is diagnosed or treated by a medical professional, or one that is typically diagnosed or treated by a medical professional.

[0063] As used herein, the term “expression-enhancing element” refers to a component of a polyribonucleotide construct that increases the expression of a polypeptide cargo encoded by the polyribonucleotide compared to a polyribonucleotide construct lacking an expression element. For example, in embodiments, the expression-enhancing element is a translation enhancer or a stability element. In embodiments, the expression-enhancing element increases the expression of the polypeptide cargo by increasing the length of time it persists before degradation, or by increasing the concentration of the polypeptide cargo expressed over a period of time.

[0064] As used herein, the term “expression sequence” refers to a nucleic acid sequence that encodes a product, such as a polypeptide. An exemplary expression sequence encoding a polypeptide may contain a group of nucleotide triads called “codons,” each of which may encode an amino acid.

[0065] As used herein, the term “fragment” with respect to a polypeptide or nucleic acid sequence means a contiguous portion less than the entire sequence of a polypeptide or nucleic acid. For example, a polypeptide fragment means a contiguous portion less than the entire fraction of a sequence such as those disclosed herein (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the total length). It is understood that all of this disclosure is intended to refer to the fragments of all polypeptides disclosed herein.

[0066] As used herein, the term “fusion” refers to a single continuous molecule comprising two or more distinct elements. For example, fusion elements described herein may include a translation enhancer fused (e.g., joined or connected) to a stability element, a translation enhancer fused (e.g., joined or connected) to a spacer element, or a stability element fused (e.g., joined or connected) to a spacer element. The fusion element may have functional properties derived from each of the original elements. In some embodiments, the translation enhancer is connected to a spacer element at its 5' end. In some embodiments, the translation enhancer is connected to a spacer element at its 3' end. In some embodiments, the stability element is connected to a spacer element at its 5' end. In some embodiments, the stability element is connected to a spacer element at its 3' end. In some embodiments, the translation enhancer is coupled to a stability element at its 5' end. In some embodiments, the translation enhancer is coupled to a translation stability element at its 3' end.

[0067] As used herein, the term "GC content" refers to the proportion of guanine (G) and cytosine (C) in a nucleic acid sequence. The formula for calculating GC content is (G+C) / (A+G+C+U)x100% (for RNA) or (G+C) / (A+G+C+T)x100% (for DNA). Similarly, the term "uridine content" refers to the percentage of uridine (U) in a nucleic acid sequence. The formula for calculating uridine content is U / (A+G+C+U)x100%. Similarly, the term "thymidine content" refers to the percentage of thymidine (T) in a nucleic acid sequence. The formula for calculating thymidine content is T / (A+G+C+T)x100%.

[0068] "Heterogeneous" means occurring in circumstances other than those in which it naturally occurs. A "heterogeneous" polynucleotide sequence indicates that the polynucleotide sequence is used in a way different from how it is found in the natural genome. For example, a "heterogeneous promoter" is used to drive the transcription of a sequence that is not naturally transcribed by that promoter. Therefore, "heterogeneous promoter" sequences are often included in expression constructs by recombinant nucleic acid technology. The term "heterogeneous" is also used to refer to a given sequence that is positioned in relation to another sequence in a way that is not naturally occurring. For example, heterogeneous coding or non-coding nucleotide sequences are commonly inserted into the genome by genome transformation technology, resulting in a genetically modified or recombinant genome.

[0069] As used herein, the term “impurity” refers to an undesirable substance present in a composition, for example, a pharmaceutical composition described herein. In some embodiments, the impurity is a process-related impurity. In some embodiments, the impurity is a product-related substance other than the desired product in the final composition, for example, an active drug component other than those described herein, such as a cyclic polyribonucleotide. As used herein, the term “process-related impurity” refers to a substance used, present, or produced in the manufacture of an undesirable composition, preparation, or product other than the linear polyribonucleotide described herein. In some embodiments, the process-related impurity is an enzyme used in the synthesis or cyclization of the polyribonucleotide. As used herein, the term “product-related substance” refers to a substance or by-product produced during the synthesis of a composition, preparation, or product, or any intermediate thereof. In some embodiments, the product-related substance is a deoxyribonucleotide fragment. In some embodiments, the product-related substance is a deoxyribonucleotide monomer. In some embodiments, the product-related substance consists of one or more derivatives or fragments of polyribonucleotides described herein, for example, fragments of 10, 9, 8, 7, 6, 5, or 4 ribonucleic acid, monoribonucleic acid, diribonucleic acid, or triribonucleic acid.

[0070] As used herein, “increased adaptability” or “enhanced adaptability” of the subject means any desirable change in the physiological or any activity of the subject organism as a result of administration of the peptide or polypeptide described herein, including, but not limited to, one or more of the following desired effects: (1) an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more in biotic or abiotic stress tolerance; (2) an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more in yield or biomass; (3) an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% in flowering time. (4) Increases of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more in resistance to pests or pathogens; (4) Increases of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more in resistance to herbicides; (5) Increases of approximately 10%, 20%, 30%, 40%, or 50% in the population of the target organism (e.g., an agriculturally important insect) (6) An increase of 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more in the reproductive rate of the target organism (e.g., insects, e.g., honeybees or silkworms) of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more; (7) An increase of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in the mobility of the target organism (e.g., insects, e.g., honeybees or silkworms) of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% (8) An increase of 99%, 100% or more in the body weight of the organism in question (e.g., insects, e.g., honeybees or silkworms) by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more; (9) An increase of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more in the metabolic rate or activity of the organism in question (e.g., insects, e.g., honeybees or silkworms);(10) An increase of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more in pollination by the target organism (e.g., insects, e.g., bees or silkworms) (e.g., the number of plants pollinated in a given amount of time); (11) An increase of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more in the production of by-products of the target organism (e.g., insects, e.g., bees or silkworms) (e.g., honey from bees or silk from silkworms); (12) An increase of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more in the production of by-products of the target organism (e.g., insects, e.g., bees or silkworms) (e.g., protein, (13) an increase of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more in the nutrient content of fatty acids or amino acids; or an increase of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more in the resistance of the target organism to pesticides (e.g., neonicotinoids (e.g., imidacloprid) or organophosphate insecticides (e.g., phosphorothioates, e.g., fenitrothion)); or an increase of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more; or an increase in the health of the target organism, such as humans or non-human animals, or a reduction in disease. The increase in host fitness can be determined by comparing it to the target organism that has not been administered polyribonucleotides. Conversely, "decreased adaptability" of the subject means any undesirable change in the physiological or any activity of the subject organism as a result of administration of the peptide or polypeptide described herein, and includes, but is not limited to, one or more of the following intended effects: (1) a decrease of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more in tolerance to biological or abiotic stress; (2) a decrease of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more in yield or biomass; (3) a modification of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more in altered flowering time;(4) A decrease of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more in resistance to pests or pathogens; (4) A decrease of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more in resistance to herbicides; (5) A decrease of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more in the population of the target organism (e.g., an agriculturally important insect). (6) A decrease in the reproductive rate of the target organism (e.g., insects, e.g., bees or silkworms) by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more; (7) A decrease in the motility of the target organism (e.g., insects, e.g., bees or silkworms) by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more; (8) A decrease in the body weight of the target organism (e.g., insects, e.g., bees or silkworms) by approximately 10%, 20%, 30%, 40%, 50% (9) A decrease of 60%, 70%, 80%, 90%, 95%, 99%, 100% or more in the metabolic rate or activity of the target organism (e.g., insects, e.g., bees or silkworms) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (10) A decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% in pollination by the target organism (e.g., insects, e.g., bees or silkworms) (e.g., the number of plants pollinated in a given time); (11) A reduction of 100% or more in the production of by-products (e.g., honey from honey or silk from silkworms) of the subject organism (e.g., insects, e.g., bees or silkworms) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more; (12) A reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more in the nutrient content of the subject organism (e.g., insects) (e.g., agriculturally important insects);or (13) a reduction of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or more in the resistance of the target organism to pesticides (e.g., insects, e.g., bees or silkworms); (14) a decrease in the health of the target organism, e.g., humans or non-human animals, or a reduction in disease. The reduction in host fitness can be determined by comparing it to the target organism that has not been administered polyribonucleotides. It will be apparent to those skilled in the art that specific changes in the physiology, phenotype, or activity of the target, e.g., alteration of flowering time in plants, can be considered to increase or decrease the fitness of the target, depending on the circumstances (e.g., to adapt to changes in climate or other environmental conditions). For example, a delay in flowering time (e.g., approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% fewer plants in a population flowering on a given calendar day) may be a beneficial adaptation to a later or slower spring time, and therefore is thought to increase the fitness of the plants. Conversely, the same delay in flowering time in earlier or warmer spring conditions can be thought to decrease the fitness of the plants.

[0071] As used herein, the terms “linear RNA,” “linear polyribonucleotide,” and “linear polyribonucleotide molecule” are interchangeable and mean a monoribonucleotide molecule or polyribonucleotide molecule having 5' and 3' ends. One or both of the 5' and 3' ends may be free ends or may be bound to another. In some embodiments, the linear RNA has a modified or degraded (e.g., 5' end protector or 3' end protector) 5' or 3' end. In some embodiments, the linear RNA has a non-covalently bonded 5' or 3' end. The linear RNA includes RNA that has not been cyclized (e.g., pre-cyclized) and can be used as a starting material for cyclization.

[0072] As used herein, the term “linear counterpart” refers to a polyribonucleotide molecule (and its fragments) having the same or similar nucleotide sequence as a cyclic polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage of sequence similarity between them) and having two free ends (i.e., a non-cyclized version (and its fragments) of the cyclized polyribonucleotide). In some embodiments, a linear counterpart (e.g., a pre-cyclized version) is a polyribonucleotide molecule (and its fragments) having the same or similar nucleotide sequence as a cyclic polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage of sequence similarity between them) and the same or similar nucleic acid modifications and having two free ends (i.e., a non-cyclized version (and its fragments) of the cyclized polyribonucleotide). In some embodiments, the linear counterpart is a polyribonucleotide molecule (and its fragments) having the same or similar nucleotide sequence as the cyclic polyribonucleotide (e.g., sequence similarity of 100%, 95%, 90%, 85%, 80%, 75%, or any percentage in between) and having different nucleic acid modifications or no nucleic acid modifications, and having two free ends (i.e., an uncyclized version (and its fragments) of the cyclized polyribonucleotide). In some embodiments, the fragment of the polyribonucleotide molecule that is the linear counterpart is any portion of the polyribonucleotide molecule that is shorter than the linear counterpart polyribonucleotide molecule. In some embodiments, the linear counterpart further comprises a 5' cap. In some embodiments, the linear counterpart further comprises a polyadenosine tail. In some embodiments, the linear counterpart further comprises a 3'UTR. In some embodiments, the linear counterpart further comprises a 5'UTR.

[0073] As used herein, the term “modified ribonucleotide” means a nucleotide having at least one modification to a sugar, nucleic acid base, or nucleoside bond.

[0074] The term “pharmaceutical composition” is also intended to disclose that the cyclic polyribonucleotides contained within the pharmaceutical composition may be used for therapeutic purposes in the human or animal body (e.g., veterinary use). Therefore, it is equivalent to “cyclic polyribonucleotides for therapeutic use.”

[0075] The term "poly-A based spacer element" or "poly-A spacer element" refers to a spacer element comprising an untranslated continuous region of a nucleic acid molecule at least 4 nucleotides in length, and consisting of one or more individual adenine (A) residues combined with one or more (A), thymine (T), cytosine (C), guanine (G), or uracil (U) residues. For example, in some embodiments, the poly-A spacer element is a poly-A region, which may be a sequence of adenine residues. In other embodiments, the poly-A spacer element is a poly-AT region, which is a combination of adenine and thymine residues. In other embodiments, the poly-A spacer element is a poly-AU region, which may be a combination of adenine and uracil residues. In some embodiments, the poly-A spacer element is a poly-AG region, which is a combination of adenine and guanine residues. Poly-A based spacer elements contain 50% to 100% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 70% to 90%, or 60% to 80%) adenine residues.

[0076] As used herein, the term “polynucleotide” means a molecule comprising one or more nucleic acid subunits or nucleotides and can be used interchangeably with “nucleic acid” or “nucleotide.” A polynucleotide may contain one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) or their variants. A nucleotide may contain a nucleoside and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphate (PO3) groups. A nucleotide may contain a nucleic acid base, a 5-carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. A ribonucleotide is a nucleotide in which the sugar is ribose. Polyribonucleotide or ribonucleic acid, or RNA, may refer to a macromolecule comprising multiple ribonucleotides polymerized via phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose. In some examples, a polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or their derivatives or variants. To give a few examples, polynucleotides include small interfering RNA (siRNA), microRNA (miRNA), plasmid DNA (pDNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), and antisense RNA (asRNA), and encompass both nucleotide sequences and any structural embodiment thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, etc. In some examples, polynucleotide molecules are cyclic. Polynucleotides can have a variety of lengths. Nucleic acid molecules can have lengths of at least about 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 1 kilobase (kb), 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 50 kb, or more. Polynucleotides can be isolated from cells or tissues. As implemented herein, polynucleotide sequences may include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA analogs.

[0077] "Polydeoxyribonucleotide," "deoxyribonucleic acid," and "DNA" refer to macromolecules containing multiple deoxyribonucleotides polymerized via phosphodiester bonds. A nucleotide may be a nucleoside monophosphate or a nucleoside polyphosphate. A nucleotide refers to a deoxyribonucleoside polyphosphate, e.g., deoxyribonucleoside triphosphate (dNTP), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP), and deoxythymidine triphosphate (dTTP) dNTPs, which may include a detectable tag, e.g., a luminescent tag or marker (e.g., a fluorophore). A nucleotide may contain any subunits that can be incorporated into a growing nucleic acid chain. Such subunits may be A, C, G, T, or U, or any other subunit specific to one or more complementary A, C, G, T, or U, or complementary to purines (i.e., A or G, or their variants) or pyrimidines (i.e., C, T, or U, or their variants).

[0078] Polynucleotides, such as polyribonucleotides or polydeoxyribonucleotides, may contain one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, nucleotide analogs, and / or modified nucleotides. Examples of modified nucleotides include diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, and 5-methylaminomethyl Examples include uracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mansylkeosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, wibutoxycin, pseudouracil, keosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, 3-(3-amino-3-carboxypropyl)uridine 2, 6-diaminopurine, etc. In some cases, nucleotides may have modifications to their phosphate moiety, including modifications to the triphosphate moiety. Non-limiting examples of such modifications include modifications with longer phosphate chains (e.g., phosphate chains with 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and thiol moieties (e.g., alpha-thiotriphosphates and beta-thiotriphosphates).Nucleic acid molecules can also be modified in the base moiety (e.g., in one or more atoms typically available to form hydrogen bonds with complementary nucleotides and / or one or more atoms typically unable to form hydrogen bonds with complementary nucleotides), the sugar moiety, or the phosphate backbone. Nucleic acid molecules can also contain amine-modifying groups such as amino-1-dUTP (aa-dUTP) and aminohexacylamide-dCTP (aha-dCTP) to enable covalent bonding of amine-reactive moieties such as N-hydroxysuccinimide esters (NHS). Substitutes for standard DNA base pairs or RNA base pairs in oligonucleotides of this disclosure can provide higher density in bits / cubic mm, higher safety (resistant to accidental or intentional synthesis of natural toxins), easier identification in photoprogrammed polymerases, or lower secondary structure. Such alternative base pairs compatible with natural and mutant polymerases for de novo and / or amplified synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. 2012 Jul;8(7):612-4, which are incorporated herein by reference for all purposes.

[0079] As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) most frequently linked together by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologues, paralogs, fragments, and other equivalents, variants, and analogs of the above. Polypeptides may be monomolecules or multimolecular complexes such as dimers, trimers, or tetramers. They may also include single-chain or multi-chain polypeptides such as antibodies or insulin, and may associate or link. Most commonly, disulfide bonds are found in multi-chain polypeptides. The term polypeptide may also be applied to amino acid polymers, in which one or more amino acid residues are artificial chemical analogs of corresponding natural amino acids.

[0080] As used herein, the term “polyribonucleotide cargo” includes any sequence comprising at least one polyribonucleotide. In embodiments, the polyribonucleotide cargo comprises one or more expression sequences, each expression sequence encoding a polypeptide. In embodiments, the polyribonucleotide cargo comprises one or more non-coding sequences, such as polyribonucleotides having regulatory or catalytic functions. In embodiments, the polyribonucleotide cargo comprises a combination of an expression sequence and a non-coding sequence. In embodiments, the polyribonucleotide cargo comprises one or more polyribonucleotide sequences described herein, for example, one or more regulatory elements, internal ribosome entry site (IRES) elements, or spacer elements.

[0081] As used herein, the term “preventive” means reducing the likelihood of developing a disease, disorder, or condition, or reducing the severity or frequency of symptoms of a subsequent disease or disorder. The polyribonucleotides described herein may be administered to subjects at higher risk of developing a disease or disorder compared to members of the general population in order to prevent the development of a disease or condition, or to reduce its severity. The polyribonucleotides described herein may be administered prophylactically (for example, before any symptoms or manifestation of a disease or disorder).

[0082] As used herein, the terms “purify,” “refining,” and “purify” mean removing impurities (e.g., process-related impurities (e.g., enzymes), process-related substances (e.g., deoxyribonucleotide fragments, deoxyribonucleotide monomers)) or by-products (e.g., linear RNA)) from a sample containing a mixture of circular RNA and linear RNA among other substances to produce a composition containing an enriched population of circular RNA with reduced levels of impurities (e.g., process-related impurities (e.g., enzymes), process-related substances (e.g., deoxyribonucleotide fragments, deoxyribonucleotide monomers)) or by-products (e.g., linear RNA) compared to the original mixture, or one or more steps or processes in which linear RNA or substances are reduced by 40% or more by mass (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% or more) compared to the starting mixture.

[0083] As used herein, the terms “pure” and “purity” refer to the degree to which an analyte (e.g., circular RNA) is isolated and free from other components. In relation to nucleic acids (e.g., polyribonucleotides), the purity of isolated nucleic acids (e.g., circular RNA) can be expressed in relation to a population of nucleic acids that is free from any contaminants, impurities, or by-products (e.g., linear RNA and other substances). For example, the purity of a population of circular RNA can be determined by the total mass of the isolated material, which indicates how much of the population is circular RNA, for example, using pure circular RNA as a reference. The levels of purity seen herein may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, greater than 95%, or greater than 99% (w / w). In some embodiments, the levels of contaminants, impurities, or by-products are less than or equal to about 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (w / w). Purity can be determined by detecting the level of a specific analyte (e.g., circular RNA) or a specific impurity or by-product (e.g., linear RNA) using gel electrophoresis, spectrophotometry (e.g., NanoDrop by ThermoFisher Scientific), or other techniques suitable for measuring the purity of a nucleic acid population and calculating the proportion of the analyte to the total nucleic acid content (w / w) (e.g., determined by assays known in the art).

[0084] As used herein, the phrase "substantially free of one or more impurities or by-products" refers to the characteristics of a sample, such as a sample containing an enriched population of circular RNA, that does not contain one or more impurities or by-products (e.g., one or more impurities or by-products disclosed herein) or contains a minimum amount of one or more impurities or by-products. The minimum amount of one or more impurities or by-products may be 20% (w / w) or less (e.g., 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w / w) or less). In another example, if one or more impurities or by-products are present in amounts less than 15% (w / w) (e.g., 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w / w) or less), the sample or enriched population of circular RNA is substantially free of one or more impurities or by-products. In another example, if one or more impurities or by-products are present in amounts less than 10% (w / w) (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (w / w) or less), the sample or enriched population of circular RNA is substantially free of one or more impurities or by-products. In another example, if one or more impurities or by-products are present in amounts less than 5% (w / w) (e.g., 4%, 3%, 2%, 1% (w / w) or less), the sample or enriched population of circular RNA is substantially free of one or more impurities or by-products. In yet another example, if one or more impurities or by-products are present in amounts less than 1% (0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% (w / w) or less), the sample or enriched population of circular RNA is substantially free of one or more impurities or by-products.

[0085] As used herein, the term “regulatory element” refers to a portion of a nucleic acid sequence or the like that modifies the expression of an expression sequence within a cyclic polyribonucleotide.

[0086] As used herein, the term “replica element” refers to sequences and / or motifs that are useful for replication or that initiate transcription of a cyclic polyribonucleotide.

[0087] As used herein, the term “RNA equivalent” refers to an RNA sequence that is the RNA equivalent of a DNA sequence. Thus, an RNA equivalent of a DNA sequence refers to a DNA sequence in which each thymidine (T) residue is replaced by a uridine (U) residue. This disclosure is particularly intended to show that any of these DNA sequences may be converted into a corresponding RNA sequence and included in the RNA molecules described herein.

[0088] As used herein, the term “sequence identity” is determined by the alignment of two peptide or nucleotide sequences using a global or local alignment algorithm. Sequences may be referred to as “substantially identical” or “essentially similar” when they share at least a certain minimum percentage of sequence identity (for example, when optimally aligned by the program GAP or BESTFIT using default parameters). GAP uses Needleman and Wunsch’s global alignment algorithm to align two sequences across their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, GAP default parameters are used with a gap generation penalty of 50 (nucleotides) / 8 (protein) and a gap elongation penalty of 3 (nucleotides) / 2 (protein). For nucleotides, the default scoring matrix used is the nwsgapdna.cmp scoring matrix, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS89, 915-919). Sequence alignment and scores for sequence identity percentage can be determined using computer programs such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or EmbossWin version 2.10.0 (using the "needle" program). Alternatively or additionally, percentage identity can be determined by searching databases using algorithms such as FASTA and BLAST. Sequence identity refers to sequence identity across the entire length of the sequence.

[0089] A "signal sequence" refers to a polypeptide sequence, for example, one between 10 and 45 amino acids in length, located at the N-terminus of the polypeptide sequence of a newly synthesized protein that targets the polypeptide sequence in the secretory pathway.

[0090] As used herein, the term “spacer element” refers to any contiguous nucleotide sequence (e.g., one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Spacer elements may be present between any of the nucleic acid elements described herein. Spacer elements may also be present within the nucleic acid elements described herein.

[0091] As used herein, the term “stability element” refers to a polyribonucleotide element that increases the stability of a polyribonucleotide compared to a polyribonucleotide lacking a stability element. Stability elements can enhance the stability of a polyribonucleotide so that it degrades more slowly.

[0092] As used herein, the term “subject” refers to living organisms such as animals, plants, or microorganisms. In embodiments, the subject is a vertebrate (e.g., mammals, birds, fish, reptiles, or amphibians). In embodiments, the subject is a human. In embodiments, the subject is a non-human mammal. In embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., monkeys, apes), an ungulate (e.g., cattle, buffalo, bison, sheep, goats, pigs, camels, llamas, alpacas, deer, horses, donkeys), a carnivore (e.g., dogs, cats), a rodent (e.g., rats, mice), or a lagomorph (e.g., rabbits). In embodiments, the subject is birds such as members of the order Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleognatha (e.g., ostriches, emus), Columbiformes (e.g., domestic pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the subject is invertebrates, such as arthropods (e.g., insects, spiders, crustaceans), nematodes, annelids, helminths, or mollusks. In embodiments, the subject is invertebrates or invertebrate invertebrates that parasitize vertebrate hosts, agricultural pests, or invertebrates. In embodiments, the subject is plants, such as angiosperms (which may be dicotyledonous or monocotyledonous) or gymnosperms (e.g., conifers, buckwheat, ginkgo), ferns, cotton, hemlock, or mosses. In the embodiments, the target is eukaryotic algae (unicellular or multicellular). In the embodiments, the target is agricultural and horticulturally important plants such as crop plants, fruit-producing plants and trees, vegetables, trees, and ornamental plants including flowers, shrubs, trees, ground cover and turfgrass.

[0093] As used herein, the term “translation enhancer” refers to a polyribonucleotide element that enables translation of mRNA via ribosome recruitment, direct interaction with translation initiation factors, or interaction with RNA-binding proteins. Polyribonucleotides containing translation enhancers may exhibit increased stability and / or increased expression of the polyribonucleotide cargo compared, for example, with polyribonucleotides lacking translation enhancers.

[0094] As used herein, the term “termination element” refers to a portion of a cyclic polyribonucleotide, such as a nucleic acid sequence, that terminates the translation of an expression sequence.

[0095] As used herein, the term “total ribonucleotide molecules” means the total amount of any ribonucleotide molecules, including linear polyribonucleotide molecules, cyclic polyribonucleotide molecules, monomeric ribonucleotides, other polyribonucleotide molecules, their fragments, and their modified variant forms, as measured by the total mass of ribonucleotide molecules.

[0096] As used herein, the term “translation efficiency” refers to the rate or amount of protein or peptide production from a ribonucleotide transcript. In some embodiments, translation efficiency can be expressed as the amount of protein or peptide produced per given amount of protein or peptide-encoding transcript over a given period of time in a given translation system, for example, an in vitro translation system such as rabbit reticulocyte lysate, or an in vivo translation system such as eukaryotic or prokaryotic cells.

[0097] As used herein, the term “translation initiation sequence” refers to a nucleic acid sequence that initiates translation of an expression sequence in a cyclic polyribonucleotide.

[0098] As used herein, the terms “to treat” and “to treat” refer to the preventive or therapeutic treatment of a disease or condition in the subject. The effects of treatment may include reversing, alleviating, reducing the severity, curing, inhibiting progression, reducing the likelihood of recurrence of one or more symptoms or symptoms of a disease or condition or a disease or condition, compared to the state of the disease or condition or a disease or condition in the absence of the therapeutic treatment, stabilizing the state of the disease or condition (i.e., preventing exacerbation), or preventing the spread of the disease or condition.

[0099] As used herein, “mutant” refers to a polypeptide that, compared to the parent or wild-type polypeptide, has at least one change, e.g., substitution, insertion, deletion, and / or fusion, at one or more residue positions. The variant forms may include 1 to 10, 10 to 20, 20 to 50, 50 to 100, or more changes. [Brief explanation of the drawing]

[0100] [Figure 1A] This is a schematic diagram of exemplary linear and cyclic polyribonucleotide constructs having spacer elements and / or expression-enhancing elements. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 1F] Same as above. [Figure 2] This table shows the cyclization efficiency (%circRNA) of single-spacer or double-spacer design constructs having spacer elements of 50-300 nucleotides in length, where the constructs encode gluc polypeptide or human erythropoietin. [Figure 3] (Figure 3A) A table of translational enhancers including motifs and sequences. (Figure 3B) A table showing the cyclization efficiency (%circRNA) of single-design and dual-design constructs encoding Gluc polypeptides, which have spacer elements and / or translational enhancers. [Figure 4] (Figure 4A) A bar graph showing the expression of Gluc at 24 and 48 hours post-transfection in HeLa cells transfected with a circular RNA encoding a Gluc polypeptide that has a spacer element. (Figure 4B) A graph showing the expression of EPO at 24 and 48 hours post-transfection in HeLa cells transfected with a circular RNA encoding human erythropoietin that has a spacer element. [Figure 5]This bar graph shows the expression of Gluc 24 hours after transfection in HeLa cells transfected with circular RNA encoding Gluc polypeptide, either with a single-spacer design or a double-spacer design. [Figure 6] (Figure 6A) A bar graph showing the expression of Gluc 4, 24, and 48 hours after transfection in HeLa cells transfected with circular RNA encoding Gluc polypeptide, which has a double spacer design. (Figure 6B) A bar graph showing the expression of EPO 4, 24, and 48 hours after transfection in HeLa cells transfected with circular RNA encoding human erythropoietin, which has a double spacer design. [Figure 7] (Figure 7A) A bar graph showing the expression of Gluc in A549 cells transfected with a circular RNA having a spacer element and a translational enhancer, encoding a Gluc polypeptide. (Figure 7B) A bar graph showing the expression of EPO 24 hours after transfection in A549 cells transfected with a circular RNA having a spacer element and a translational enhancer, encoding human erythropoietin. [Figure 8] This bar graph shows the expression of EPO on day 1 and day 2 after transfection in HEK cells transfected with a cyclic polyribonucleotide encoding human erythropoietin with a translational enhancer. [Figure 9] This bar graph shows the expression of EPO on day 1 and day 2 after transfection in HeLa cells transfected with a cyclic polyribonucleotide that has a translational enhancer and encodes human erythropoietin. [Figure 10] This bar graph shows the in vivo expression of EPO encoded by cyclic polyribonucleotides containing translational enhancers. [Figure 11]This graph shows the expression of EPO 24 hours after transfection in A549 cells transfected with a cyclic polyribonucleotide encoding human erythropoietin having a spacer element and / or translational enhancer and containing a CVB3 IRES. [Figure 12] This graph shows the expression of EPO 24 hours after transfection in A549 cells transfected with a cyclic polyribonucleotide encoding human erythropoietin having a spacer element and / or translational enhancer and EMCV IRES. [Figure 13] This graph shows the expression of EPO in A549 cells transfected with a cyclic polyribonucleotide that has multiple spacer elements and multiple translational enhancers and encodes human erythropoietin and SARS-CoV-2 RBD polypeptide, 24 hours after cell transfection. [Figure 14] This graph shows the time course of EGFPd2 expression in A549 cells transfected with a circular RNA encoding the EGFPd2 polypeptide, which contains a spacer element and a translational enhancer. Figure 14A shows the time course of EGFPd2 expression. Figure 14B shows the area under the curve from 0 to 24 hours. [Figure 15] This bar graph shows the expression of EPO 24 hours after transfection in A549 cells transfected with a circular RNA encoding human erythropoietin, which has a spacer element and a translational enhancer. [Figure 16] This bar graph shows the expression of EPO 24 hours after transfection in A549 cells transfected with a circular RNA encoding human erythropoietin, which has a spacer element and a translational enhancer. [Figure 17] This bar graph shows HiBiT expression 24 hours after transfection in HEK cells transfected with a circular RNA encoding the factor IX-albumin-HiBiT polypeptide, which contains a spacer element and a translational enhancer. [Figure 18] This bar graph shows the expression of Gluc in HeLa cells 48 hours after transfection, transfected with different concentrations of circular RNA encoding a Gluc polypeptide, each containing a spacer element and a minimized or full-length eIF4g aptamer translational enhancer. [Figure 19] This bar graph shows CFTR expression in HEK293T cells transfected with one of 12(12) different circular RNAs encoding CFTR. [Figure 20] This bar graph shows polypeptide expression detected on the cell surface of HEK293T cells transfected with one of 13(13) different circular RNAs encoding polypeptides. [Figure 21] This bar graph shows polypeptide expression detected on the cell surface of HEK293T cells transfected with one of four different circular RNAs encoding polypeptides. [Figure 22] This graph shows the relative amount of cyclic polyribonucleotides present over time for a cyclic polyribonucleotide encoding an EGFPd2 polypeptide that has a 5' spacer element and a 3' RNA stability element and a modified CVB3 IRES. [Figure 23A] This is a series of bar graphs showing the concentration of the polypeptide factor IX-HiBiT, which is encoded by a cyclic polyribonucleotide having a 5' spacer element and a 3' translation enhancer, and encoding factor IX-HiBiT, 24 hours after transfection. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 24A] This graph shows the time course of expression data for EGFPd2 encoded by a cyclic polyribonucleotide that has a spacer element and / or translational enhancer and encodes a modified CVB3 IRES. Figure 24A shows EGFPd2 expression over time, and Figure 24B shows the area under the curve from 0 to 24 hours. [Figure 24B] Same as above. [Figure 25A] This graph shows the time course of expression data for EGFPd2, which is encoded by a cyclic polyribonucleotide that has a 5' translation enhancer and a 3' spacer element and a modified CVB3 IRES. Figure 25A shows the time course of EGFPd2 expression, and Figure 25B shows the area under the curve from 0 to 24 hours. [Figure 25B] Same as above. [Figure 26A] This bar graph shows the concentration of polypeptide B-HiBiT, encoded by a cyclic polyribonucleotide having a 5' spacer element and a 3' translation enhancer, and encoding polypeptide B-HiBiT having either EV69 IRES (Figure 26A) or a modified CVB3 IRES (Figure 26B), 24 hours after transfection. [Figure 26B] Same as above. [Figure 27] This is a bar graph showing the concentration of polypeptide B-HiBiT, which is encoded by a cyclic polyribonucleotide having multiple translation enhancers or multiple spacer elements. [Figure 28] This shows the expression of polypeptide G 24 hours after transfection, encoded by a cyclic polyribonucleotide that encodes polypeptide G having a spacer element and / or translational enhancer and a modified CVB3 IRES. [Figure 29A] The bar graphs show the area under the curve for GFPd2 (Figure 29A) and the concentration of polypeptide B-HiBiT (Figure 29B), where the polypeptide is encoded by a cyclic polyribonucleotide with a single-spacer design or a double-spacer design. [Figure 29B] Same as above. [Figure 30A] This is a bar graph showing the area under the curve for GFPd2 encoded by cyclic polyribonucleotides with a single-spacer design or a double-spacer design. [Figure 30B] Same as above. [Figure 31A]This is a bar graph showing the area under the curve for polypeptide B-HiBiT, encoded by cyclic polyribonucleotides having a single-spacer design or a double-spacer design. [Figure 31B] Same as above. [Figure 32A] This bar graph shows the area under the curve for GFPd2 encoded by a cyclic polyribonucleotide having a spacer element and / or a translational enhancer, where the translational enhancer is FcIgG (Figure 32A), TP53I3 (Figure 32B), LSP1 (Figure 32C), or histone 4E (Figure 32D). [Figure 32B] Same as above. [Figure 32C] Same as above. [Figure 32D] Same as above. [Figure 33A] This bar graph shows the concentration of polypeptide B-HiBiT encoded by a cyclic polyribonucleotide having a spacer element and / or a translational enhancer, where the translational enhancer is FcIgG (Figure 33A), TP53I3 (Figure 33B), LSP1 (Figure 33C), or histone 4E (Figure 33D). [Figure 33B] Same as above. [Figure 33C] Same as above. [Figure 33D] Same as above. [Figure 34A] This bar graph shows the area under the curve for GFPd2 encoded by cyclic polyribonucleotides with translational enhancers, where the translational enhancers are TBR16 (Figure 34A), 12S (Figure 34B), TRAM1 (Figure 34C), and GPX4 (Figure 34D). [Figure 34B] Same as above. [Figure 34C] Same as above. [Figure 34D] Same as above. [Figure 35A] This bar graph shows the concentrations of polypeptide B-HiBiT encoded by cyclic polyribonucleotides with translational enhancers, namely TBR16 (Figure 35A), 12S (Figure 35B), TRAM1 (Figure 35C), and GPX4 (Figure 35D). [Figure 35B] Same as above. [Figure 35C] Same as above. [Figure 35D] Same as above. [Modes for carrying out the invention]

[0101] This disclosure features compositions comprising polyribonucleotides having one or more expression-enhancing elements or spacer elements. The polyribonucleotides described herein are particularly useful in increasing the stability and / or expression of polynucleotide cargoes encoded by the polyribonucleotides (e.g., encoding genes or proteins).

[0102] This disclosure provides a polyribonucleotide comprising a first post-cyclization element; a first expression-enhancing element having a length of at least 100 ribonucleotides; a polyribonucleotide cargo; a second expression-enhancing element; and a second post-cyclization element, wherein the first and second post-cyclization elements together form a cyclization junction. This disclosure also provides a polyribonucleotide comprising a first post-cyclization element; a first spacer element having a length of at least 100 ribonucleotides; a polyribonucleotide cargo; a second spacer element; and a second post-cyclization element, wherein the first and second post-cyclization elements together form a cyclization junction. This disclosure also provides a polyribonucleotide comprising a first post-cyclization element; a first spacer element; a polyribonucleotide cargo; a second spacer element having a length of at least 100 ribonucleotides; and a second post-cyclization element, wherein the first and second post-cyclization elements together form a cyclization junction.

[0103] The polyribonucleotide constructs described herein may exhibit increased stability and potentially increase the length of time that polyribonucleotides can survive before degradation. Furthermore, the polyribonucleotide constructs described herein may exhibit increased expression of the polynucleotide cargo encoded by the polyribonucleotides. Each of the DNA sequences described herein includes an RNA equivalent sequence, as will be understood by those skilled in the art. Similarly, all RNA sequences described herein include a DNA equivalent sequence, as will be understood by those skilled in the art. The molecules, methods for producing them, and uses thereof are described in more detail below.

[0104] Cyclic polyribonucleotide element A cyclic polyribonucleotide may comprise a first post-cyclization element at 5' to 3'; one or more expression-enhancing elements; a polyribonucleotide cargo; and a second post-cyclization element. In some embodiments, the cyclic polyribonucleotide comprises a first post-cyclization element at 5' to 3'; a polyribonucleotide cargo; one or more expression-enhancing elements; and a second post-cyclization element.

[0105] In some embodiments, the cyclic polyribonucleotide comprises a first post-cyclization element from 5' to 3'; a first expression-enhancing element; a polyribonucleotide cargo; a second expression-enhancing element; and a second post-cyclization element.

[0106] In some embodiments, the cyclic polyribonucleotide comprises a first post-cyclization element from 5' to 3'; a spacer element; a polyribonucleotide cargo; an expression-enhancing element; and a second post-cyclization element.

[0107] In some embodiments, the cyclic polyribonucleotide comprises a first post-cyclization element from 5' to 3'; a first expression-enhancing element; a polyribonucleotide cargo; a second expression-enhancing element; and a second post-cyclization element. In some embodiments, the cyclic polyribonucleotide comprises a first post-cyclization element from 5' to 3'; a first spacer element having a length of at least 100 ribonucleotides; a polyribonucleotide cargo; a second spacer element; and a second post-cyclization element.

[0108] The cyclic polyribonucleotides disclosed herein include a first post-cyclization element; (b) a first spacer element having at least 100 ribonucleotides (e.g., at least 100, 120, 140, 160, 180, 200, 250, 300, 400, or 500 ribonucleotides). The cyclic polyribonucleotides disclosed herein also include a polyribonucleotide cargo and a second spacer element. In some embodiments, the second spacer element has a length of at least 100 ribonucleotides (e.g., at least 100, 120, 140, 160, 180, 200, 250, 300, 400, or 500 ribonucleotides). For example, the second spacer element may have a ribonucleotide length of 120 to 500 (e.g., 120 to 400, 120 to 300, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 400 to 500, or 350 to 500). In some embodiments, the second spacer element has a ribonucleotide length of 100-300 (e.g., 100-280, 100-260, 100-240, 100-220, 100-200, 100-180, 100-160, 100-140, 100-120, 120-300, 140-300, 160-300, 180-300, 200-300, 220-300, 240-300, 260-300, or 280-300). In some embodiments, the second spacer element has a ribonucleotide length of 200 to 500 (e.g., 200 to 450, 200 to 400, 200 to 350, 200 to 300, 200 to 250, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500, 300 to 500, or 300 to 400).

[0109] Expression-enhancing elements The polyribonucleotides described herein may comprise one or more expression-enhancing elements. In some embodiments, a cyclic or linear polyribonucleotide comprises 1 to 5 expression-enhancing elements (e.g., expression-enhancing elements 1, 2, 3, 4, or 5). For example, a cyclic or linear polyribonucleotide may comprise a first expression-enhancing element and a second expression-enhancing element. One or more expression-enhancing elements may comprise a translation enhancer, a stability element, a translation enhancer fused (e.g., conjugated, linked) to a spacer element, or a stability element fused (e.g., conjugated, linked) to a spacer element. In some embodiments, a cyclic or linear polyribonucleotide comprises a translation enhancer and a stability element.

[0110] The polyribonucleotides described herein may comprise combinations of elements in a 5' to 3' order, as shown in Table 1. One or more spacer elements may be included between any one of the elements shown in Table 1.

[0111] [Table 1-1]

[0112] [Table 1-2]

[0113] Translation Enhancer The polyribonucleotides described herein may comprise a first or second expression-enhancing element. The first expression-enhancing element may comprise a translation enhancer. The second enhancement element may comprise a translation enhancer. In some embodiments, the cyclic or linear polyribonucleotides described herein comprise one or more translation enhancers. In some embodiments, the translation enhancer may be fused to (e.g., joined to or connected to) a spacer element. The translation enhancer may be present between any of the nucleic acid elements described herein. The translation enhancer may also be present within the nucleic acid elements described herein. In some embodiments, the translation enhancer is a spacer element. In some embodiments, the translation enhancer is a stability element.

[0114] In some embodiments, the translational enhancer is derived from a gene encoding an RNA-binding protein. In some embodiments, the translational enhancer comprises a nucleic acid sequence containing a fragment derived from a gene encoding an RNA-binding protein. In some embodiments, the translational enhancer has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%<99%, or 100%) sequence identity with the nucleic acid sequence containing a fragment derived from a gene encoding an RNA-binding protein. In some embodiments, the translational enhancer is derived from a gene encoding a BYDV-like element (BTE), a translational enhancer element (TED), a PMV / PEMV-like translational enhancer (PTE), a type I structure (ISS), a type Y structure (YSS), a type t structure (TSS), a dumbbell structure, a viral RNA UTR (including dengue, West Nile, Zika, and rotavirus), EMCV, CVB3, a hepatitis B virus post-transcriptional regulatory element, a human genome fragment, a histone mRNA sequence, a cyclin D mRNA sequence, or an eIF4g aptamer sequence. In some embodiments, the translational enhancer element is derived from a plant virus. In some embodiments, the translational enhancer comprises a nucleic acid sequence containing a fragment derived from a plant virus. In some embodiments, the translational enhancer has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%<99%, or 100%) sequence identity with a nucleic acid sequence containing a fragment derived from a plant virus. In some embodiments, the plant virus is a barley yellow dwarf virus (BYDV)-like element (BTE) translational enhancer. Non-limiting examples of plant viruses having BTEs include BYDV, TNVD, OLV1, LWSV, SCNMV, CRSV, TBTV, GRV, OMMV, BBSV, RSDaV, and OPMV. The BTE binds to eIF4g with high affinity. In certain embodiments, the cyclic or linear polyribonucleotide contains the BTE sequence listed in Table 2.In some embodiments, the BTE includes sequences having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequences, sequence numbers 1-22, of Table 2.

[0115] In some embodiments, the translational enhancer element is derived from a plant virus containing TED. Non-limiting examples of plant viruses containing TED include STNV, PLPV, PCRPV, ELV, RrLDV, PelRSV, and CbMV. TED binds to eIF4F with high affinity. In certain embodiments, the cyclic or linear polyribonucleotide comprises the TED sequence listed in Table 3. In some embodiments, the TED comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequences in Table 3, SEQ ID NOs. 23-29.

[0116] In some embodiments, the translational enhancer element is derived from a plant virus containing a Panicum mosaic virus / pea spurium mosaic virus (PMV / PEMV)-like translational enhancer (PTE). Non-limiting examples of PTEs include plant viruses having SCV, PFBV, CarMV, HnRSV, PSNV, HCRSV, GaMV, CMMV, TPAV, JINRV, PEMV2, PMV, MCMV, BGLV, AdMV, and CLSV. The PTE binds to eIF4E with high affinity. In certain embodiments, the cyclic or linear polyribonucleotide comprises the PTE sequence listed in Table 4. In some embodiments, the PTE comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequences in Table 4, SEQ ID NOs. 30 to 46.

[0117] In some embodiments, the translational enhancer element is derived from a plant virus containing the anISS translational enhancer. Non-limiting examples of plant viruses having ISS include MNeSV, MNSV264, CBV, MWLMV, JCSMV, and GoMVA. The ISS is bound to eIF4E conjugated to eIF4G. In certain embodiments, the cyclic or linear polyribonucleotide comprises the TSS sequence described in Table 5. In some embodiments, the TSS comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequences in Table 5, SEQ ID NOs. 47-53.

[0118] In some embodiments, the translational enhancer element is derived from a plant virus containing the YSS translational enhancer. Non-limiting examples of plant viruses having YSS include TBSV, CIRV, CymRSV, CNV, AMCV, PNSV, GALV, PLCV, PeLV, and LNV. YSS binds to eIF4F. In certain embodiments, the cyclic or linear polyribonucleotide comprises the YSS sequence described in Table 6. In some embodiments, the YSS comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequences in Table 6, SEQ ID NOs. 54-63.

[0119] In some embodiments, the translational enhancer element is derived from a plant virus containing a TSS translational enhancer. Non-limiting examples of plant viruses having a TSS include TYMV, RCNM, TCV, and CCFV. The TSS binds to a 60s ribosomal subunit. In certain embodiments, the cyclic or linear polyribonucleotide comprises the TSS sequence listed in Table 7. In some embodiments, the TSS comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequences in Table 7, SEQ ID NOs. 64 to 67.

[0120] In some embodiments, the translational enhancer element is derived from a plant virus containing a dumbbell-shaped translational enhancer. Non-limiting examples of plant viruses having a dumbbell-shaped translational enhancer include CABYV-X. In certain embodiments, a cyclic or linear polyribonucleotide comprises a dumbbell-shaped translational enhancer sequence described in SEQ ID NO: 68 of Table 8. In some embodiments, the dumbbell-shaped enhancer comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequence in Table 8, SEQ ID NO: 68.

[0121] In some embodiments, the translational enhancer element is derived from a mammalian gene. In some embodiments, the translational enhancer includes a nucleic acid sequence containing a fragment derived from a mammalian gene. In some embodiments, the translational enhancer has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%<99%, or 100%) sequence identity with the nucleic acid sequence containing the fragment derived from a mammalian gene. The mammalian gene may, but is not limited to, a histone or cyclin D mRNA sequence that binds to eIF4E. In some embodiments, the translational enhancer element is derived from a synthetic sequence that includes, but is not limited to, an eIF4G aptamer that binds to eIF4G. In some embodiments, the translational enhancer element is derived from a viral sequence. Non-limiting examples of translational enhancers from viral sequences are HCV and DENV. In certain embodiments, the cyclic or linear polyribonucleotide comprises mammalian, synthetic, or viral translational enhancer sequences listed in Table 8. In some embodiments, the mammalian, synthetic, or viral translational enhancer comprises sequences having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequences in Table 8, SEQ ID NOs. 68-159, and 273-287.

[0122] In certain embodiments, the cyclic or linear polyribonucleotide comprises a translational enhancer element as described in Tables 2-8. In some embodiments, the translational enhancer comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequences in Tables 2-8.

[0123] In certain embodiments, the translation enhancer includes a nucleic acid sequence having at least 85% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with any one of the nucleic acid sequences SEQ ID NOs: 1-183, 255, 256, 273-287, 290-303, or 305-333. In some embodiments, the translation enhancer includes a nucleic acid sequence having at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with any one of the nucleic acid sequences SEQ ID NOs: 1-183, 255, 256, 273-287, 290-303, or 305-333. In some embodiments, the translation enhancer includes a nucleic acid sequence having at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with any one of the nucleic acid sequences SEQ ID NOs: 1-183, 255, 256, 273-287, 290-303, or 305-333. In some embodiments, the translation enhancer includes a nucleic acid sequence having one of the following nucleic acid sequences: SEQ ID NOs: 1-183, 255, 256, 273-287, 290-303, or 305-333.

[0124] In some embodiments, the translation enhancer element may be TBR16, 12S, TRAM1, or GPX4.

[0125] The translation enhancer element may be a 5'UTR or a 3'UTR. In some embodiments, the 5'UTR or 3'UTR is a human translation enhancer. In some embodiments, the 5'UTR or 3'UTR includes one or a portion of the 5'UTR or 3'UTRs listed in Table 9.

[0126] The translation enhancer element may have 20 to 750 ribonucleotides (e.g., 20 to 700, 20 to 650, 20 to 600, 20 to 550, 20 to 500, 20 to 450, 20 to 400, 20 to 350, 20 to 300, 20 to 250, 20 to 200, 20 to 150, 20 to 100, 20 to 50, 50 to 100, 50 to 750, 100 to 750, 150 to 750, 200 to 750, 250 to 750, 300 to 750, 350 to 750, 400 to 750, 450 to 750, 500 to 750, 550 to 750, 600 to 750, 650 to 750, 700 to 750 ribonucleotides). For example, a translation enhancer may have a length of 100 to 500 ribonucleotides (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 500 ribonucleotides).

[0127] [Table 2-1]

[0128] [Table 2-2]

[0129] [Table 2-3]

[0130] [Table 2-4]

[0131] Table 2-5

[0132] Table 3-1

[0133] Table 3-2

[0134] Table 4-1

[0135] Table 4-2

[0136] Table 4-3

[0137] Table 5

[0138] Table 6-1

[0139] Table 6-2

[0140] Table 7

[0141] [Table 8-1]

[0142] [Table 8-2]

[0143] [Table 8-3]

[0144] [Table 8-4]

[0145] [Table 8-5]

[0146] [Table 8-6]

[0147] [Table 8-7]

[0148] The expression-enhancing element may include a translation enhancer fused to a spacer element. The spacer element may be conjugated to the 5' end of the translation enhancer. In some embodiments, the spacer element may be conjugated to the 3' end of the translation enhancer.

[0149] Stability element The polyribonucleotides described herein may include a first expression-enhancing element, the first expression-enhancing element may include a stability element. The second expression-enhancing element may include a stability element. The second spacer element may include an expression-enhancing element. One or more expression-enhancing elements may be stability elements. In some embodiments, the stability element is a translation enhancer. In some embodiments, the stability element is a spacer element.

[0150] The stability element may be a 5'UTR or a 3'UTR. In some embodiments, the 5'UTR or 3'UTR is a human UTR. In some embodiments, the 5'UTR or 3'UTR includes one or a portion of the 5'UTR or 3'UTRs listed in Table 9 below. The 5'UTR or 3'UTR may be derived from a gene encoding TRAM1, TMED2, VAMP3, CRIP, AP2A2, PSMD5, GPX4, or PRKAB1. The 5'UTR or 3'UTR may be derived from human beta-actin, DDB2, TP53I3, FcIgG, LSP1, AES, DRB4, or mitochondrial-encoded 12S rRNA.

[0151] The stability element may contain 50 to 2000 ribonucleotides (e.g., 50 to 1500, 50 to 1000, 50 to 500, 50 to 100, 100 to 2000, 500 to 2000, 1000 to 2000, or 1500 to 2000). The stability element may include 20-750 ribonucleotides (e.g., 20-700, 20-650, 20-600, 20-550, 20-500, 20-450, 20-400, 20-350, 20-300, 20-250, 20-200, 20-150, 20-100, 20-50, 50-100, 50-750, 100-750, 150-750, 200-750, 250-750, 300-750, 350-750, 400-750, 450-750, 500-750, 550-750, 600-750, 650-750, and 700-750 ribonucleotides). For example, the stability element may have a length of 100 to 500 ribonucleotides (e.g., 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, or 450 to 500 ribonucleotides).

[0152] The expression-enhancing element may include a stability element fused to a spacer element. The spacer element may be conjugated to the 5' end of the stability element. In some embodiments, a spacer element that does not encode the expression-enhancing element may be conjugated to the 3' end of the stability element.

[0153] The stability element may be any known stability element. In some embodiments, the stability element has a nucleic acid sequence that has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the sequences listed in Table 9. In some embodiments, the stability element has a nucleic acid sequence that has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the sequences listed in Table 9. In some embodiments, the stability element has a nucleic acid sequence that has at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the sequences listed in Table 9. In some embodiments, the stability element has one nucleic acid sequence from the sequences listed in Table 9.

[0154] [Table 9-1]

[0155] [Table 9-2]

[0156] [Table 9-3]

[0157] [Table 9-4]

[0158] [Table 9-5]

[0159] [Table 9-6]

[0160] [Table 9-7]

[0161] [Table 9-8]

[0162] Non-translated areas In some embodiments, a circular or linear polyribonucleotide includes an untranslated region (UTR). In some embodiments, a stability element may include a UTR. In some embodiments, a translation enhancer may include a UTR. In some embodiments, the UTR is a 3' UTR. In some embodiments, the UTR is a 5' UTR. The UTR of a genomic region containing a gene may be transcribed but not translated. In some embodiments, the UTR may be located upstream of the translation initiation sequence of the expression sequence described herein. In some embodiments, the UTR may be located downstream of the expression sequence described herein. In some examples, one UTR for a first expression sequence is the same as, contiguous with, or overlaps with another UTR for a second expression sequence. In some embodiments, the intron is a human intron. In some embodiments, the intron is a full-length human intron, e.g., ZKSCAN1.

[0163] Exemplary untranslated areas are described in paragraphs

[0197] to

[0201] of the International Publication WO2019 / 118919, which are incorporated herein by reference in their entirety.

[0164] In some embodiments, the cyclic polyribonucleotide includes a poly-A sequence. Exemplary poly-A sequences are described in paragraphs

[0202] to

[0205] of International Publication WO2019 / 118919, which is incorporated in its entirety herein by reference. In some embodiments, the cyclic polyribonucleotide lacks a poly-A sequence.

[0165] In some embodiments, cyclic or linear polyribonucleotides contain UTRs having one or more elongated adenosine and uridine molecules embedded within them. These AU-rich signatures may increase the turnover rate of the expression product.

[0166] The introduction, removal, or modification of UTR AU-rich elements (AREs) may be useful in regulating the stability or immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response) of cyclic or linear polyribonucleotides. When manipulating specific cyclic polyribonucleotides, one or more copies of AREs can be introduced into the cyclic polyribonucleotide, and these copies of AREs can regulate the translation and / or production of the expression product. Similarly, AREs can be identified, removed, or manipulated into cyclic polyribonucleotides to regulate intracellular stability and thus affect the translation and production of the resulting protein.

[0167] It should be understood that any UTR from any gene can be incorporated into each adjacent region of a circular polyribonucleotide.

[0168] In some embodiments, cyclic polyribonucleotides lack a 5'-UTR and are eligible for protein expression from one or more expression sequences. In some embodiments, cyclic or linear polyribonucleotides lack a 3'-UTR and are eligible for protein expression from one or more expression sequences. In some embodiments, cyclic or linear polyribonucleotides lack a poly(A) sequence and are eligible for protein expression from one or more expression sequences. In some embodiments, cyclic or linear polyribonucleotides lack a termination element and are eligible for protein expression from one or more expression sequences. In some embodiments, cyclic or linear polyribonucleotides lack an internal ribosome entry site and are eligible for protein expression from one or more expression sequences. In some embodiments, cyclic or linear polyribonucleotides lack a cap and are eligible for protein expression from one or more expression sequences. In some embodiments, cyclic or linear polyribonucleotides lack a 5'-UTR, 3'-UTR, and IRES and are eligible for protein expression from one or more expression sequences. In some embodiments, the cyclic or linear polyribonucleotide includes one or more sequences from the following: sequences encoding one or more miRNAs, sequences encoding one or more replication proteins, sequences encoding exogenous genes, therapeutic agents, sequences encoding regulatory elements (e.g., translation modulators, e.g., translation enhancers or translation inhibitors), translation initiation sequences, one or more regulatory nucleic acids targeting endogenous genes (e.g., siRNA, lncRNA, shRNA), and sequences encoding therapeutic mRNA or proteins.

[0169] In some embodiments, cyclic or linear polyribonucleotides lack a 5'-UTR. In some embodiments, cyclic polyribonucleotides lack a 3'-UTR. In some embodiments, cyclic polyribonucleotides lack a poly(A) sequence. In some embodiments, cyclic or linear polyribonucleotides lack a termination element. In some embodiments, cyclic or linear polyribonucleotides lack an internal ribosome entry site. In some embodiments, cyclic or linear polyribonucleotides lack sensitivity to exonuclease degradation. In some embodiments, the fact that cyclic polyribonucleotides lack sensitivity to degradation may mean that cyclic polyribonucleotides are not degraded by exonucleases, or are degraded only to a limited extent in the presence of exonucleases, for example, to a degree equivalent to or similar to that in the absence of exonucleases. In some embodiments, cyclic polyribonucleotides are not degraded by exonucleases. In some embodiments, cyclic polyribonucleotides exhibit reduced degradation when exposed to exonucleases. In some embodiments, the cyclic polyribonucleotide lacks binding to a cap-binding protein. In some embodiments, the cyclic polyribonucleotide lacks a 5' cap.

[0170] Spacer element In some embodiments, a polyribonucleotide includes one or more spacer elements. For example, either a first or second expression-enhancing element may include a spacer element fused to a translational enhancer or a spacer element fused to a stability element. In some embodiments, the spacer may be fused to the 5' end of the translational enhancer or stability element. In some embodiments, the spacer may be fused to the 3' end of the translational enhancer or stability element. In some embodiments, the spacer element is a translational enhancer. Not all spacers are translational enhancers. In some embodiments, the spacer element is a stability element. Not all spacers are stability elements. A spacer element refers to any adjacent nucleotide sequence (e.g., one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Spacer elements may be present between any of the nucleic acid elements described herein. Spacer elements may also be present within the nucleic acid elements described herein. A cyclic polyribonucleotide may comprise a first post-cyclization element; a first spacer element having a length of at least 100 ribonucleotides; a polyribonucleotide cargo; a second spacer element; and a second post-cyclization element, wherein the first and second post-cyclization elements together form a cyclization junction.

[0171] In some embodiments, the spacer element has a ribonucleotide length of at least 100. The spacer element may have a ribonucleotide length of 100 to 500 (e.g., 100 to 400, 100 to 300, 100 to 200, 200 to 500, 300 to 500, 400 to 500, 200 to 400, or 200 to 300). For example, a spacer element without an expression-enhancing element may have a ribonucleotide length of 120 to 500 (e.g., 120 to 400, 120 to 300, 120 to 200, 120 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 400 to 500, or 350 to 500). In some embodiments, the spacer element contains 110 to 500 ribonucleotides (e.g., 110 to 400, 110 to 300, 110 to 200, 110 to 150, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 400 to 500, or 350 to 500). In some embodiments, the spacer element contains 100-300 ribonucleotides (e.g., 100-280, 100-260, 100-240, 100-220, 100-200, 100-180, 100-160, 100-140, 100-120, 110-300, 120-300, 140-300, 160-300, 180-300, 200-300, 220-300, 240-300, 260-300, or 280-300). In some embodiments, the spacer element has a ribonucleotide length of 200 to 500 (e.g., 200 to 450, 200 to 400, 200 to 350, 200 to 300, 200 to 250, 250 to 500, 300 to 500, 350 to 500, 400 to 500, 450 to 500, 300 to 500, or 300 to 400). In some embodiments, the spacer element has at least 50 ribonucleotides. For example, a spacer element may have 50 to 500 ribonucleotides (e.g., 50 to 100, 50 to 150, 50 to 200, 50 to 250, 50 to 300, 50 to 350, 50 to 400, 50 to 450, 100, 500, 150 to 500, 200, 500, 250 to 500, 300, 500, 350 to 500, 400 to 500, and 450 to 500).

[0172] In some embodiments, the spacer element has a ribonucleotide length of about 50 (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55). In some embodiments, the spacer element has a ribonucleotide length of about 80 (e.g., 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, or 88). In some embodiments, the spacer element has a ribonucleotide length of about 100 (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110). In some embodiments, the spacer element has a ribonucleotide length of about 120 (e.g., 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132). In some embodiments, the spacer element has a ribonucleotide length of about 150 (e.g., 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, or 165). In some embodiments, the spacer element has a ribonucleotide length of about 200 (e.g., 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, or 220).

[0173] In some embodiments, the polyribonucleotide includes a first spacer element and a second spacer element, the first and second spacers being of the same length. In some embodiments, the first and second spacer elements are of approximately the same length.

[0174] In some embodiments, the polyribonucleotide includes a first spacer element and a second spacer element, the first and second spacer elements being of different lengths. In some embodiments, the difference between the length of the first spacer element and the length of the second region is 0 to 100 (e.g., 0 to 95, 0 to 90, 0 to 85, 0 to 80, 0 to 75, 0 to 70, 0 to 65, 0 to 60, 0 to 55, 0 to 50, 0 to 45, 0 to 40, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, 5 to 100). The ranges are 10-100, 15-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100, 80-100, 85-100, 90-100, or 95-100, and they are nucleotides. In some embodiments, the difference between the length of the first spacer element and the length of the second spacer element is 0 to 50 nucleotides (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50). In some embodiments, the length of the first spacer is about 50 (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55) ribonucleotides, and the length of the second spacer is about 120 (e.g., 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132) ribonucleotides.In some embodiments, the length of the first spacer is approximately 120 (e.g., 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, or 132) ribonucleotides, and the length of the second spacer is approximately 50 (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55) ribonucleotides.

[0175] In some embodiments, the spacer element may be a poly-A spacer or a poly-A spacer element. In some embodiments, the first spacer is a poly-A spacer or a poly-A spacer element. In some embodiments, the second spacer is a poly-A spacer or a poly-A spacer element. In some embodiments, the first and second spacers may be poly-A spacers or poly-A spacer elements. The poly-A spacer or poly-A spacer element corresponds to the poly-A region described herein (e.g., poly-AC region, poly-AU region, poly-AG region, or poly-AT region).

[0176] The first spacer element may consist of a poly-A region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine residues. The first spacer element may consist of a poly-AC region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or cytosine residues. Adenosine and cytosine residues may be present in any ratio to each other. In some embodiments, the first spacer element may consist of a poly-AU region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or uridine residues. In some embodiments, the first spacer element may consist of a polyAG region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or guanosine residues. The adenosine and guanosine residues may be present in any ratio to each other. In some embodiments, the first spacer element may consist of a polyAT region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or thymidine residues. The adenosine and thymidine residues may be present in any ratio to each other. In some embodiments, the first spacer element includes a polyAT region containing 100 ribonucleotides and 150 ribonucleotides (e.g., 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 ribonucleotides).

[0177] The second spacer element may consist of a poly-A region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine residues. The second spacer element may consist of a poly-AC region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or cytosine residues. In some embodiments, the second spacer element may consist of a poly-AU region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or uridine residues. The adenosine and uridine residues may be present in any ratio to each other. In some embodiments, the second spacer element may consist of a polyAG region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or guanosine residues. The adenosine and guanosine residues may be present in any ratio to each other. In some embodiments, the first spacer element may consist of a polyAT region containing 80% to 100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) adenosine or thymidine residues. In some embodiments, the second spacer element includes a polyAT region containing 100 ribonucleotides and 150 ribonucleotides (e.g., 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 ribonucleotides). The adenosine and thymidine residues may be present in any ratio to each other.

[0178] In some embodiments, the spacer element sequence may be a poly-A region (e.g., polyAT, polyAU, polyAC, or polyAG) or a random sequence. In some embodiments, the spacer element may be referred to based on a combination of nucleotide content and length. For example, an AU120 spacer element may consist of a poly-AU region containing 80% to 100% adenosine or uridine residues and a length of 120 nucleotides.

[0179] In some embodiments, the spacer element has a nucleic acid sequence that has at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the sequences listed in Table 10. In some embodiments, the spacer element has a nucleic acid sequence that has at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the sequences listed in Table 10. In some embodiments, the spacer element has a nucleic acid sequence that has at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with any one of the sequences listed in Table 10. In some embodiments, the spacer element has one nucleic acid sequence from the sequences listed in Table 10.

[0180] [Table 10]

[0181] An exemplary arrangement of spacer elements is described in paragraphs

[0293] to

[0302] of the International Publication WO2019 / 118919, which is incorporated herein by reference in its entirety.

[0182] Internal ribosome entry site In some embodiments, the cyclic or linear polyribonucleotides described herein include one or more internal ribosome entry site (IRES) elements. In some embodiments, the IRESs are operably ligated to one or more expression sequences (for example, each IRES is operably ligated to one or more expression sequences, each of which may encode a polypeptide). In embodiments, the IRESs are located at the 5' end of a heterologous promoter and a coding sequence.

[0183] Suitable IRES elements for inclusion in polyribonucleotides include RNA sequences that can engage with eukaryotic ribosomes. In some embodiments, the IRES elements are at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.

[0184] In some embodiments, the IRES element is derived from the DNA of organisms including, but not limited to, viruses, mammals, and fruit flies. Such viral DNA may be derived from, but not limited to, picomavirus complementary DNA (cDNA) having encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, the IRES element is derived from the Antennapedia gene from the fruit fly (Drosophila melanogaster).

[0185] In some embodiments, the IRES sequence is used to detect Taura syndrome virus, triatomavirus, Tyler encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Roparodynum padivirus, reticuloendotheliosis virus, human poliovirus 1, Plautia stol enteric virus, Cashmere bee virus, human rhinovirus 2 (HRV-2), Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Homalodisca coagulata virus 1, Himeto P virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, Hectorpicalis picorna-like virus, Encephalomyocarditis virus (EMCV), Drosophila C virus, Crucifer Tobamo virus, Cricket paralysis virus, bovine viral diarrhea virus 1, Black Queen cell virus, Aphid lethal paralysis virus, avian encephalomyelitis virus (AEV), acute bee paralysis virus, Hibiscus chlorotin ring spot virus, swine cholera virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 alpha, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-l, mouse Rbm3, Drosophila, Campanula canis, Drosophila Ubx), human UNR, mouse UtrA, human VEGF-A, human XIAP, salivirus, cosavirus, parechovirus, fruit fly (Drosophila hairless), budding yeast (S. cerevisiae) TFIID, budding yeast (S.The IRES sequence is for cerevisiae)YAP1, human c-src, human FGF-1, simianpicomavirus, crwinklevirus, Aichi virus, black hemovirus, echovirus 11, an aptamer for eIF4G, or coxsackievirus B3 (CVB3) or coxsackievirus A (CVB1 / 2). In yet another embodiment, the IRES is the IRES sequence of coxsackievirus B3 (CVB3). In yet another embodiment, the IRES is the IRES sequence of encephalomyocarditis virus. In yet another embodiment, the IRES is the IRES sequence of Tyler's encephalomyelitis virus.

[0186] In some embodiments, the IRES sequence has a modified sequence compared to the wild-type IRES sequence. In some embodiments, if the last nucleotide of the wild-type IRES is not a cytosine nucleic acid residue, the last nucleotide of the wild-type IRES sequence is modified to be a cytosine residue. For example, the IRES sequence may be a CVB3 IRES sequence in which the terminal adenosine residue is modified to a cytosine residue. In some embodiments, the modified CVB3 IRES may have the following nucleic acid sequence: [ka]

[0187] In some embodiments, the IRES sequence is enterovirus 71 (EV17) IRES. In some embodiments, the terminal guanosine residue of the EV17 IRES sequence is modified with a cytosine residue. In some embodiments, the modified EV71 IRES may have the following nucleic acid sequence. [ka]

[0188] In some embodiments, the IRES sequence is a synthetic IRES. A "synthetic IRES" is an IRES modified compared to a wild-type IRES to modulate its structure and / or activity. For example, in some embodiments, an IRES modified to incorporate an aptamer sequence is a synthetic IRES. In some embodiments, the polyribonucleotide includes at least one IRES adjacent to at least one (e.g., 2, 3, 4, 5 or more) expression sequences. In some embodiments, the IRESs are adjacent to both sides of at least one (e.g., 2, 3, 4, 5 or more) expression sequences. In some embodiments, the polyribonucleotide includes one or more IRES sequences on one or both sides of each expression sequence, resulting in the separation of the resulting peptide and / or polypeptide. For example, the polyribonucleotide described herein may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence.

[0189] In some embodiments, the polyribonucleotides described herein include IRESs (e.g., IRESs operably ligated to a coding region). For example, polyribonucleotides are mentioned in Chen et al. Nature Biotechnology 41:262-272, 2023; Chen et al. Mol. Cell 81(20):4300-4318, 2021; Jopling et al. Oncogene 20:2664-2670, 2001; Baranick et al. PNAS 105(12):4733-4738, 2008; Lang et al. Molecular Biology of the Cell 13(5):1792-1801, 2002; Dorokhov et al. PNAS 99(8):5301-5306, 2002; Wang et al. Nucleic Acids Research 33(7):2248-2258, 2005; Petz et al. Nucleic Acids This may include any IRES as described in Research 35(8):2473-2482, 2007; Chen et al. Science 268:415-417, 1995; Fan et al. Nature Communication 13(1):3751-3765, 2022; and International Publication No. 2021 / 263124 and International Publication No. 2022 / 271965 (each incorporated herein by reference in its entirety).

[0190] signal sequence In some embodiments, polypeptides expressed from cyclic or linear polyribonucleotides disclosed herein include secretory proteins, such as proteins that naturally contain a signal sequence, or proteins that do not normally encode a signal sequence but have been modified to contain one. In some embodiments, the polypeptide includes a secretory signal. For example, the secretory signal may be a secretory signal that is naturally encoded for a secretory protein. In another example, the secretory signal may be a modified secretory signal for a secretory protein. In other embodiments, the polypeptide does not include a secretory signal.

[0191] In some embodiments, the polynucleotide encodes multiple copies of the same polypeptide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, at least one copy of the polypeptide includes a signal sequence and at least one copy of the polypeptide does not include a signal sequence. In some embodiments, the circular polynucleotide encodes multiple polypeptides, at least one of the multiple polypeptides includes a signal sequence, and at least one copy of the multiple polypeptides does not include a signal sequence.

[0192] In some embodiments, the signal sequence is the wild-type signal sequence that is present at the N-terminus of the corresponding wild-type polypeptide when expressed endogenously, for example. In some embodiments, the signal sequence is heterologous to the polypeptide and does not exist, for example, when the wild-type polypeptide is expressed endogenously. The polynucleotide sequence encoding the polypeptide can be modified to remove the nucleotide sequence encoding the wild-type signal sequence and / or add a sequence encoding a heterologous signal sequence.

[0193] The polypeptide encoded by a can include a signal sequence that directs the polypeptide into the secretory pathway. In some embodiments, the signal sequence can direct the polypeptide to be present in a particular organelle (e.g., endoplasmic reticulum, Golgi apparatus, or endosome). In some embodiments, the signal sequence directs the polypeptide to be secreted from the cell. In the case of a secreted protein, the signal sequence can be cleaved after secretion, resulting in a mature protein. In other embodiments, the signal sequence can be embedded in the membrane of the cell or a particular organelle, forming a transmembrane segment that anchors the protein to the membrane of the cell, endoplasmic reticulum, or Golgi apparatus. In certain embodiments, the signal sequence of a transmembrane protein is a short sequence at the N-terminus of the polypeptide. In other embodiments, the first transmembrane domain acts as the first signal sequence that targets the protein to the membrane.

[0194] In some embodiments, the secretion signal is a human interleukin-2 (IL-2) secretion signal. In some embodiments, the IL-2 secretion signal has an amino acid sequence with at least 90% sequence identity to MYRMQLLSCIALSLALVTNS (SEQ ID NO: 186). In some embodiments, the IL2 secretion signal has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 186. In some embodiments, the IL-2 secretion signal has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 186. In some embodiments, the IL-2 secretion signal has an amino acid sequence with 100% sequence identity to SEQ ID NO: 186.

[0195] In some embodiments, the secretion signal is a Gaussia luciferase secretion signal. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence with at least 90% sequence identity to MGVKVLFALICIAVAEAK (SEQ ID NO: 187). In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 187. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 187. In some embodiments, the Gaussia luciferase secretion signal has an amino acid sequence with 100% sequence identity to SEQ ID NO: 187.

[0196] In some embodiments, the secretion signal is an EPO (e.g., human EPO) secretion signal. In some embodiments, the EPO secretion signal has an amino acid sequence with at least 90% sequence identity to MGVHECPAWLWLLLSLLSLPLGLPVLGA (SEQ ID NO: 188). In some embodiments, the EPO secretion signal has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 188. In some embodiments, it is 88. In some embodiments, the EPO secretion signal has an amino acid sequence with 100% sequence identity to SEQ ID NO: 188.

[0197] In some embodiments, the secretion signal is the wild-type SARS-CoV-2 secretion signal. In some embodiments, the wild-type SARS-CoV-2 secretion signal has an amino acid sequence with at least 90% sequence identity to MFVFLVLLPLVSS (SEQ ID NO: 189). In some embodiments, the wild-type SARS-CoV-2 secretion signal has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 189. In some embodiments, the wild-type SARS-CoV-2 secretion signal has an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 189. In some embodiments, the wild-type SARS-CoV-2 secretion signal has an amino acid sequence with 100% sequence identity to SEQ ID NO: 189.

[0198] In some embodiments, the polypeptide encoded by the polyribonucleotide includes either a secretion signal sequence, a transmembrane insertion signal sequence, or does not include a signal sequence.

[0199] Adjustment element In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargoes of polyribonucleotides) include one or more regulatory elements. In some embodiments, the polyribonucleotide includes regulatory elements, for example, sequences that modify the expression of an expression sequence within the polyribonucleotide.

[0200] A regulatory element may include a sequence located adjacent to an expression sequence encoding an expression product. The regulatory element may be operably ligated to the adjacent sequence. The regulatory element may increase the amount of expressed product compared to the amount of expressed product in the absence of the regulatory element. A regulatory element can be used to increase the expression of one or more polypeptides encoded by polyribonucleotides. Similarly, a regulatory element may be used to decrease the expression of one or more polypeptides encoded by polyribonucleotides. In some embodiments, one regulatory element is used to increase the expression of a polypeptide, and another regulatory element is used to decrease the expression of another polypeptide on the same polyribonucleotide. Furthermore, one regulatory element can increase the amount of product expressed for multiple tandem-bound expression sequences. Thus, one regulatory element can enhance the expression of one or more expression sequences. For example, multiple regulatory elements can be used to differentially regulate the expression of different expression sequences.

[0201] In some embodiments, the regulatory element is a translation modulator. A translation modulator can regulate the translation of an expression sequence in a polyribonucleotide. The translation modulator may be a translation enhancer or a translation repressor. In some embodiments, the polyribonucleotide includes at least one translation modulator adjacent to at least one expression sequence. In some embodiments, the polyribonucleotide includes a translation modulator adjacent to each expression sequence. In some embodiments, the translation modulator is present on one or both sides of each expression sequence, resulting in the separation of the expression product, e.g., peptides and / or polypeptides.

[0202] In some embodiments, the regulatory element is a microRNA (miRNA) or a miRNA binding site.

[0203] In some embodiments, the regulatory elements provided herein include selective translation sequences. As used herein, the term “selective translation sequence” refers to a nucleic acid sequence that selectively initiates or activates the translation of a polyribonucleotide, for example, an expression sequence in a particular riboswitch aptazyme. The regulatory elements may also include selective degradation sequences. As used herein, the term “selective degradation sequence” refers to a nucleic acid sequence that initiates the degradation of a polyribonucleotide, or the expression product of a polyribonucleotide.

[0204] In some embodiments, the regulatory element is a translation modulator. A translation modulator can regulate the translation of an expression sequence in a polyribonucleotide. The translation modulator may be a translation enhancer or a translation repressor. In some embodiments, the translation initiation sequence can function as the regulatory element.

[0205] In some embodiments, the polyribonucleotide produces expression products in stoichiometric ratios. Rolling circle translation successively produces expression products in substantially equal ratios. In some embodiments, the polyribonucleotide has stoichiometric translation efficiency such that expression products are produced in substantially equivalent ratios. In some embodiments, the polyribonucleotide has stoichiometric translation efficiency of products from multiple expression sequences, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more expression sequences. In some embodiments, the polyribonucleotide produces expression products in substantially different ratios. For example, the translation efficiencies of multiple expression products may have ratios of 1:10,000; 1:7000, 1:5000, 1:1000, 1:700, 1:500, 1:100, 1:50, 1:10, 1:5, 1:4, 1:3 or 1:2. In some embodiments, the ratios of multiple expression products can be modified using modulating elements.

[0206] Further examples of adjustment elements are described in paragraphs

[0154] to

[0161] of the International Publication No. 2019 / 118919, which is incorporated herein by reference in its entirety.

[0207] Disconnected domain The cyclic or linear polyribonucleotides of this disclosure may include cleavage domains (e.g., stagger elements or cleavage sequences).

[0208] As used herein, the term “stagger element” refers to a portion, such as a nucleotide sequence, that induces ribosome rest during translation. In some embodiments, the stagger element is a non-conserved sequence of amino acids having a strong alpha-helical tendency, followed by a consensus sequence -D(V / I)ExNPGP (SEQ ID NO: 190), where x = any amino acid. In some embodiments, the stagger element may include a chemical portion such as glycerol, a non-nucleic acid linking portion, a chemical modification, a modified nucleic acid, or any combination thereof.

[0209] In some embodiments, the cyclic or linear polyribonucleotide includes at least one stagger element adjacent to the expression sequence. In some embodiments, the cyclic or linear polyribonucleotide includes a stagger element adjacent to each expression sequence. In some embodiments, the stagger element is present on one or both sides of each expression sequence, resulting in the separation of the expression product. In some embodiments, the stagger element is part of one or more expression sequences. In some embodiments, the cyclic or linear polyribonucleotide includes one or more expression sequences, each of which is separated from subsequent expression sequences by alternating elements on the cyclic or linear polyribonucleotide. In some embodiments, the stagger element prevents the formation of a single polypeptide from (a) two translations of a single expression sequence, or (b) one or more translations of two or more expression sequences. In some embodiments, the stagger element is a sequence distinct from one or more expression sequences. In some embodiments, the stagger element includes part of the expression sequences of one or more expression sequences.

[0210] Examples of stagger elements are given in paragraphs

[0172] to

[0175] of International Publication No. 2019 / 118919, which is incorporated herein by reference in its entirety.

[0211] To avoid the production of continuous expression products while maintaining rolling circle translation, stagger elements may be included to induce ribosome rest during translation. In some embodiments, the stagger element is located at the 3' end of at least one of one or more expression sequences. The stagger element may be configured to stall the ribosome during rolling circle translation of a cyclic or linear polyribonucleotide. The stagger element may include, but is not limited to, a 2A-like or CHYSEL (SEQ ID NO: 191) (cis-acting hydrolase element) sequence. In some embodiments, the stagger element encodes a sequence having a C-terminal consensus sequence X1X2X3EX5NPGP, where X1 is absent or G or H, X2 is absent or D or G, X3 is D or V or I or S or M, and X5 is any amino acid (SEQ ID NO: 192). Some non-restrictive examples of staggered elements include GDVESNPGP (sequence number 193), GDIEENPGP (sequence number 194), VEPNPGP (sequence number 195), IETNPGP (sequence number 196), GDIESNPGP (sequence number 197), GDVELNPGP (sequence number 198), GDIETNPGP (sequence number 199), GDVENPGP (sequence number 200), GDVEENPGP (sequence number 201), GDVEQNPGP (sequence number 202), IESNPGP (sequence number 203), GDIELNPGP (sequence number 204), HDIETTNPGP (sequence number 205), HDVETNPGP (sequence number 206), HDVEMNPGP (sequence number 207), GDMESNPGP (sequence number 208), GDVETNPGP (sequence number 209), GDIEQNPGP (sequence number 210), and DSEFNPGP (sequence number 211).

[0212] In some embodiments, the stagger elements described herein cleave expression products such as G and P of the consensus sequence described herein. As one non-limiting example, a cyclic or linear polyribonucleotide includes at least one stagger element for cleaving expression products. In some embodiments, a cyclic or linear polyribonucleotide includes a stagger element adjacent to at least one expression sequence. In some embodiments, a cyclic or linear polyribonucleotide includes alternating elements after each expression sequence. In some embodiments, a cyclic or linear polyribonucleotide includes stagger elements present on one or both sides of each expression sequence, resulting in the translation of individual peptides and / or polypeptides from each expression sequence.

[0213] In some embodiments, the stagger element comprises one or more modified or non-native nucleotides that induce ribosome rest during translation. Non-native nucleotides may include peptide nucleic acids (PNA), morpholino and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). Such examples are distinguished from naturally occurring DNA or RNA by alterations in the molecular skeleton. Exemplary modifications include sugars, nucleic acid bases, nucleoside intrabonding (e.g., to linked phosphates / phosphodiester bonds / phosphodiester skeletons), and any modifications to any combination thereof that can induce ribosome rest during translation. Some of the exemplary modifications provided herein are described elsewhere in this specification.

[0214] In some embodiments, staggered elements are present in other forms of cyclic or linear polyribonucleotides. For example, in some exemplary cyclic or linear polyribonucleotides, the staggered element includes a termination element of a first expression sequence in the cyclic or linear polyribonucleotide and a nucleotide spacer sequence that separates the termination element from the first translation initiation sequence of expression following the first expression sequence. In some examples, the first staggered element of the first expression sequence is located upstream (from 5') of the first translation initiation sequence of expression following the first expression sequence in the cyclic or linear polyribonucleotide. In some cases, the first expression sequence and the expression sequence following the first expression sequence are two distinct expression sequences in the cyclic or linear polyribonucleotide. The distance between the first staggered element and the first translation initiation sequence may allow for sequential translation of the first expression sequence and the subsequent expression sequence. In some embodiments, the first staggered element includes a termination element that separates the expression product of the first expression sequence from the expression product of the subsequent expression sequence, thereby creating discrete expression products. In some cases, a cyclic or linear polyribonucleotide containing a first stagger element upstream of the first translation initiation sequence of a subsequent sequence is translated sequentially, while a corresponding cyclic or linear polyribonucleotide containing a stagger element of a second expression sequence upstream of the second translation initiation sequence of a subsequent expression sequence is not translated sequentially. In some cases, a cyclic or linear polyribonucleotide has only one expression sequence, and the first expression sequence and its subsequent expression sequences are the same expression sequence. In some exemplary cyclic or linear polyribonucleotides, the staggered element includes a first termination element of the first expression sequence in the cyclic or linear polyribonucleotide and a nucleotide spacer sequence that separates the termination element from the downstream translation initiation sequence. In some such examples, the first stagger element is located upstream (from 5') of the first translation initiation sequence of the first expression sequence in the cyclic or linear polyribonucleotide.In some cases, the distance between the first stager element and the first translation start array allows for continuous translation of the first expression array and any subsequent expression arrays. In some embodiments, the first stager element separates one round of the expression product of the first expression array from the next round of the expression product of the first expression array, thereby creating discrete expression products. In some cases, a circular or linear polynucleotide comprising a first stager element upstream of the first translation start array of the first expression array in a circular or linear polynucleotide is translated continuously, and a corresponding circular or linear polynucleotide comprising a stager element upstream of the second translation start array of the second expression array in the corresponding circular or linear polynucleotide is not translated continuously. Optionally, the distance between the second stager element and the second translation start array is at least 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× greater than the distance between the first stager element and the first translation start in the corresponding circular or linear polynucleotide. In some cases, the distance between the first stager element and the first translation start is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt, or more. In some embodiments, the distance between the second stager element and the second translation start is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt, or greater than the distance between the first stager element and the first translation start. In some embodiments, the circular or linear polynucleotide comprises two or more expression arrays.

[0215] In some embodiments, multiple expression sequences encoded by a cyclic ribonucleotide can be separated from each expression sequence by an IRES. For example, a cyclic polyribonucleotide may include a first IRES operably ligated to a first expression sequence and a second IRES operably ligated to a second expression sequence. The IRES may be the same across all expression sequences. The IRES may differ across expression sequences.

[0216] In some embodiments, multiple expression sequences can be separated by a 2A self-cleaving peptide. For example, a cyclic polyribonucleotide may encode an IRES operably ligated to an open reading frame encoding the first expression sequence, 2A, and the second expression sequence. In some embodiments, 2A may have the sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 212).

[0217] In some embodiments, multiple expression sequences may be separated by a protease cleavage site (e.g., a Furin cleavage site). For example, a cyclic polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first expression sequence, a protease cleavage site (e.g., a Furin cleavage site), and a second expression sequence. In some embodiments, the Furin cleavage site may have the sequence GRLRR (SEQ ID NO: 213).

[0218] In some embodiments, multiple expression sequences may be separated by a 2A self-cleaving peptide and a protease cleavage site (e.g., a furin cleavage site). For example, a cyclic polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first expression sequence, 2A, a protease cleavage site (e.g., a furin cleavage site), and a second expression sequence. A cyclic polyribonucleotide may also encode an IRES operably linked to an open reading frame encoding a first expression sequence, a protease cleavage site (e.g., a furin cleavage site), 2A, and a second expression sequence. A tandem 2A and furin cleavage site may be referred to as furin 2A (including furin-2A or 2A-furin, arranged in either orientation).

[0219] Furthermore, multiple expression sequences encoded by cyclic ribonucleotides can be separated by both IRES and 2A sequences. For example, an IRES may be present in one expression sequence and a second expression sequence, while a 2A peptide may be present in a second expression sequence and a third expression sequence. The selection of a particular IRES or 2A self-cleaving peptide can be used to control the expression level of the expression sequence under the control of the IRES or 2A sequence. For example, depending on the selected IRES and / or 2A peptide, the expression on the polypeptide may be higher or lower.

[0220] In some embodiments, a circular or linear polyribonucleotide includes at least one cleavage sequence. In some embodiments, the cleavage sequence is adjacent to an expression sequence. In some embodiments, the cleavage sequence is located between two expression sequences. In some embodiments, the cleavage sequence is included within an expression sequence. In some embodiments, a circular or linear polyribonucleotide includes 2 to 10 cleavage sequences. In some embodiments, a circular or linear polyribonucleotide includes 2 to 5 cleavage sequences. In some embodiments, multiple cleavage sequences are located between multiple expression sequences. For example, a circular or linear polyribonucleotide may include three expression sequences and two cleavage sequences, such that a cleavage sequence is present between each expression sequence. In some embodiments, a circular or linear polyribonucleotide includes, for example, a cleavage sequence in sacrificial circRNA, cleavable circRNA, or self-cleaving circRNA. In some embodiments, the cyclic polyribonucleotide or linear polyribonucleotide contains two or more cleavage sequences, resulting in separation of the cyclic polyribonucleotide or linear polyribonucleotide into multiple products, such as miRNA, linear RNA, or smaller cyclic polyribonucleotides or linear polyribonucleotides.

[0221] In some embodiments, the cleavage sequence includes a ribozyme RNA sequence. Ribozymes (from ribonucleic acid enzymes, also called RNA enzymes or catalytic RNAs) are RNA molecules that catalyze chemical reactions. Many natural ribozymes catalyze either the hydrolysis of one phosphodiester bond of their own or the hydrolysis of a bond in another RNA, but they have also been found to catalyze the aminotransferase activity of ribosomes. Catalytic RNAs can be "evolved" by in vitro methods. Similar to the riboswitch activity described above, ribozymes and their reaction products can regulate gene expression. In some embodiments, catalytic RNAs or ribozymes can be placed within larger non-coding RNAs so that the ribozymes are present in many copies within the cell for the purpose of chemical transformation of the molecule from bulk volume. In some embodiments, both the aptamer and the ribozyme may be encoded in the same non-coding RNA.

[0222] In some embodiments, the cleavage sequence encodes a cleavable polypeptide linker. For example, a polyribonucleotide may encode two or more expression sequences encoded by a single open reading frame (ORF). For example, two or more expression sequences may be encoded by a single open reading frame, and their expression is controlled by an IRES. In some embodiments, the ORF further encodes a polypeptide linker, for example, such that the expression product of the ORF encodes two or more expression sequences separated by sequences encoding polypeptide linkers (e.g., linkers of 5-200 amino acids, 5-100 amino acids, 5-50 amino acids, 5-20 amino acids, 50-100 amino acids, or 50-200 amino acids). The polypeptide linker may include a cleavage site, for example, a protease (e.g., an endogenous protease in the subject after administration of the polyribonucleotide to the subject) that is recognized and cleaved. In such embodiments, a single expression product containing the amino acid sequences of two or more expression sequences is cleaved at expression, and as a result, the two or more expression sequences are separated after expression. Exemplary protease cleavage sites, such as amino acid sequences acting as protease cleavage sites recognized by metalloproteinases (e.g., matrix metalloproteinases (MMPs), e.g., one or more of MMP1-28), disintegrins and metalloproteinases (ADAMs, e.g., one or more of ADAM2, 7-12, 15, 17-23, 28-30, and 33), serine proteases, urokinase-type plasminogen activators, matryptases, cysteine ​​proteases, aspartate proteases, or cathepsin proteases, are known to those skilled in the art. In some embodiments, the protease is MMP9 or MMP2. In some embodiments, the protease is matryptase.

[0223] In some embodiments, the cyclic or linear polyribonucleotides described herein are sacrificial cyclic or linear polyribonucleotides, cleavable cyclic or linear polyribonucleotides, or self-cleaving cyclic or linear polyribonucleotides. The cyclic or linear polyribonucleotides can deliver cellular components, for example, RNA, lncRNA, lincRNA, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRNA. In some embodiments, the cyclic or linear polyribonucleotide includes miRNA separated by (i) a self-cleaving element; (ii) a cleavage recruitment site; (iii) a degradable linker; (iv) a chemical linker; and / or (v) a spacer element sequence. In some embodiments, the circRNA includes siRNA separated by (i) a self-cleaving element; (ii) a cleavage recruitment site (e.g., ADAR); (iii) a degradable linker (e.g., glycerol); (iv) a chemical linker; and / or (v) a spacer element sequence. Non-limiting examples of self-cleaving elements include hammerheads, splicing elements, hairpins, hepatitis delta virus (HDV), Varkud satellites (VS), and glmS ribozymes.

[0224] In some embodiments, the cyclic polyribonucleotide includes at least one stagger element adjacent to the expression sequence. In some embodiments, the cyclic polyribonucleotide includes a stagger element adjacent to each expression sequence. In some embodiments, the stagger element is present on one or both sides of each expression sequence, resulting in the separation of the expression product, e.g., peptides and / or polypeptides. In some embodiments, the stagger element is part of one or more expression sequences. In some embodiments, the cyclic polyribonucleotide includes one or more expression sequences, each of which is separated from subsequent expression sequences by a stagger element on the cyclic polyribonucleotide. In some embodiments, the stagger element prevents the formation of a single polypeptide from (a) two translations of a single expression sequence, or (b) one or more translations of two or more expression sequences. In some embodiments, the stagger element is a sequence distinct from one or more expression sequences. In some embodiments, the stagger element includes part of the expression sequences of one or more expression sequences.

[0225] Translation start sequence In some embodiments, the cyclic or linear polyribonucleotide encodes the expression sequence and includes a translation initiation sequence, e.g., a start codon. In some embodiments, the polyribonucleotide includes a translation initiation sequence operably linked to the expression sequence. In some embodiments, the translation initiation sequence includes a Kossack or Shine-Dalgarno sequence. In some embodiments, the translation initiation sequence includes a Kossack sequence. In some embodiments, the cyclic or linear polyribonucleotide includes a translation initiation sequence adjacent to the expression sequence, e.g., a Kossack sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In some embodiments, the translation initiation sequence, e.g., a Kossack sequence, is present on one or both sides of each expression sequence, resulting in the separation of the expression product. In some embodiments, the cyclic or linear polyribonucleotide includes at least one translation initiation sequence adjacent to the expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the cyclic or linear polyribonucleotide. In some embodiments, the translation initiation sequence is located within a single-stranded region of the cyclic or linear polyribonucleotide. Further examples of translation initiation sequences are provided in paragraphs

[0163] –

[0165] of International Publication No. 2019 / 118919, which is incorporated herein by reference in its entirety.

[0226] A cyclic or linear polyribonucleotide may contain two or more start codons, including, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 16, at least 60, or 60 or more start codons. Translation may begin at the first start codon or downstream of the first start codon.

[0227] In some embodiments, cyclic or linear polyribonucleotides may begin with a codon other than the first start codon, e.g., AUG. Translation of cyclic or linear polyribonucleotides may begin with alternative translation initiation sequences, such as ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AUA, ATT / AUU, TTG / UUG, etc. In some embodiments, translation begins with an alternative translation initiation sequence under selective conditions, e.g., under stress-induced conditions. As an unrestricted example, translation of a polyribonucleotide may begin with an alternative translation initiation sequence such as ACG. As another unrestricted example, polyribonucleotide translation may begin with an alternative translation initiation sequence CTG / CUG. As yet another unrestricted example, polyribonucleotide translation may begin with an alternative translation initiation sequence GTG / GUG. As another non-limiting example, polyribonucleotides can initiate translation with repeat-associated non-AUG(RAN) sequences, such as short stretches of repetitive RNA, including alternative translation initiation sequences such as CGG, GGGGCC, CAG, and CTG.

[0228] In some embodiments, translation is initiated by treatment with eukaryotic initiation factor 4A (eIF4A) using Locagrat (translation is suppressed by blocking 43S scanning, resulting in premature upstream translation initiation and reduced protein expression from transcripts having the RocA-eIF4A target sequence (see, for example, nature.com / articles / nature17978)).

[0229] Termination element In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargoes of polyribonucleotides) include at least one termination element. In some embodiments, the polyribonucleotide includes a termination element operably linked to an expression sequence. In some embodiments, the polynucleotide lacks a termination element.

[0230] In some embodiments, the polyribonucleotide comprises one or more expression sequences, each expression sequence may or may not have a termination element. In some embodiments, the polyribonucleotide comprises one or more expression sequences, and the expression sequences lack a termination element so that the polyribonucleotide is translated sequentially. The exclusion of the termination element may result in rolling circle translation or sequential expression of the expression product.

[0231] In some embodiments, the cyclic polyribonucleotide comprises one or more expression sequences, each expression sequence may or may not have a termination element. In some embodiments, the cyclic polyribonucleotide comprises one or more expression sequences, and the expression sequences lack a termination element so that the cyclic polyribonucleotide is translated sequentially. The exclusion of the termination element can result in rolling circle translation or sequential expression of an expression product, such as a peptide or polypeptide, due to the absence of ribosome stalling or detachment. In such embodiments, rolling circle translation expresses a sequential expression product through each expression sequence. In some other embodiments, the termination element of the expression sequence may be part of a stagger element. In some embodiments, one or more expression sequences in the cyclic polyribonucleotide include a termination element. However, rolling circle translation or expression of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in the cyclic polyribonucleotide takes place. In such cases, the expression product may detach from the ribosome when the ribosome encounters a stop element, such as a stop codon, and terminates translation. In some embodiments, translation is completed while the ribosome, for example, at least one subunit of the ribosome, remains in contact with the cyclic polyribonucleotide.

[0232] In some embodiments, the cyclic polyribonucleotide includes termination elements at the end of one or more expression sequences. In some embodiments, one or more expression sequences include two or more consecutive termination elements. In such embodiments, translation is completed, and circular translation is completed. In some embodiments, the ribosome completely disengages from the cyclic polyribonucleotide. In some such embodiments, the production of subsequent expression sequences (e.g., second, third, fourth, fifth, etc.) in the cyclic polyribonucleotide may require the ribosome to reengage with the cyclic polyribonucleotide before translation initiation. Generally, the termination elements include an in-frame nucleotide triplet, e.g., UAA, UGA, UAG, that signals the termination of translation. In some embodiments, one or more termination elements in the cyclic polyribonucleotide are frameshifted termination elements, e.g., off-frame or -1 and +1 shifted leading frames (e.g., hidden stop regions), which can terminate translation. Frameshifted termination elements include nucleotide triples, TAA, TAG, and TGA appearing in the second and third reading frames of the expression sequence. Frameshifted termination elements can be important in preventing misreading of mRNA, which is often harmful to cells. In some embodiments, the termination element is a stop codon.

[0233] Further examples of terminal elements are described in paragraphs

[0169] to

[0170] of International Publication No. 2019 / 118919, which is incorporated herein by reference in its entirety.

[0234] Polyribonucleotide cargo In some embodiments, a cyclic polyribonucleotide encodes a polyribonucleotide cargo. A polyribonucleotide cargo described herein comprises any sequence containing at least one polyribonucleotide. In some embodiments, a polyribonucleotide cargo comprises an expression sequence, a non-coding sequence, or an expression sequence and a non-coding sequence. In some embodiments, a polyribonucleotide cargo comprises an expression sequence encoding a polypeptide. In some embodiments, a polyribonucleotide cargo comprises an IRES operably ligated to an expression sequence encoding a polypeptide. In some embodiments, a polyribonucleotide cargo comprises an expression sequence encoding a polypeptide having a biological effect on a subject.

[0235] The polyribonucleotide cargo may contain, for example, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

[0236] In some embodiments, the polyribonucleotide cargo comprises 1 to 20,000 nucleotides, 1 to 10,000 nucleotides, 1 to 5,000 nucleotides, 100 to 20,000 nucleotides, 100 to 10,000 nucleotides, 100 to 5,000 nucleotides, 500 to 20,000 nucleotides, 500 to 10,000 nucleotides, 500 to 5,000 nucleotides, 1,000 to 20,000 nucleotides, 1,000 to 10,000 nucleotides, or 1,000 to 5,000 nucleotides.

[0237] In the embodiments, the polyribonucleotide cargo comprises one or more expression (or coding) sequences, each expression (or coding) sequence encoding a polypeptide. In the embodiments, the polyribonucleotide cargo comprises one or more non-coding sequences. In the embodiments, the polyribonucleotide cargo consists entirely of non-coding sequences. In the embodiments, the polyribonucleotide cargo comprises a combination of expression (or coding) sequences and non-coding sequences.

[0238] In some embodiments, the GC content of the nucleic acid sequence encoding the polypeptide is at least 51% (e.g., at least 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%). In some embodiments, the GC content of the nucleic acid sequence encoding the polypeptide is at most 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 59%, or 60%. In some embodiments, the GC content of the nucleic acid sequence encoding the polypeptide is 51%-60%, 52%-60%, 53%-60%, 54%-60%, 55%-60%, 52%-58%, or 53%-58%.

[0239] In some embodiments, the uridine content (for RNA) or thymidine content (for DNA) of the nucleic acid sequence encoding the polypeptide exceeds 10% (e.g., 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%). In some embodiments, the uridine content (for RNA) or thymidine content (for DNA) of the nucleic acid sequence encoding the polypeptide is up to 30% (e.g., up to 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20%). In some embodiments, the uridine content (for RNA) or thymidine content (for DNA) of the nucleic acid sequence encoding the polypeptide is 20%-28%, 21%-26%, 10%-24%, 15%-24%, 20%-24%, 21%-24%, 22%-24%, 23%-24%, 10%-23%, 15%-23%, 20%-23%, 21%-23%, or 22%-23%.

[0240] The GC content of an expression sequence encoding a polypeptide refers to the GC content of an expression sequence that exclusively encodes the polypeptide without any other coding regions encoding peptides other than the polypeptide. Similarly, the uridine content or thymidine content of an expression sequence encoding a polypeptide refers to the uridine content of an expression sequence that exclusively encodes the polypeptide without any other coding regions encoding peptides other than the polypeptide. In some embodiments, the calculation of the GC content or uridine (or thymidine) content of an expression sequence encoding a polypeptide considers only a continuous nucleic acid sequence that starts in the 5' to 3' direction from the first nucleoside of the start codon of the open reading frame encoding the polypeptide to the last nucleoside of the stop codon of the same open reading frame. In other embodiments, the calculation of the GC content or uridine (or thymidine) content of an expression sequence encoding a polypeptide considers only a continuous nucleic acid sequence that starts in the 5' to 3' direction from the first nucleoside of the codon encoding the N-terminal amino acid residue of the polypeptide to the last nucleoside of the codon encoding the C-terminal amino acid residue of the polypeptide. In some embodiments, the nucleic acid sequence encoding the polypeptide has a uridine content of more than 20%.

[0241] In some embodiments, the uridine content of the nucleic acid sequence encoding the polypeptide exceeds 10% (e.g., 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or more than 25%). In some embodiments, the uridine content of the nucleic acid sequence encoding the polypeptide is up to 30% (e.g., up to 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20%). In some embodiments, the uridine content of the nucleic acid sequence encoding the polypeptide is 20%-28%, 21%-26%, 10%-24%, 15%-24%, 20%-24%, 21%-24%, 22%-24%, 23%-24%, 10%-23%, 15%-23%, 20%-23%, 21%-23%, or 22%-23%. In some embodiments, the nucleic acid sequence encoding the polypeptide has a uridine content of 20%-28%.

[0242] In some embodiments, polyribonucleotides prepared as described herein are used as effectors in therapy or agriculture. For example, cyclic polyribonucleotides prepared by the methods described herein (e.g., the cell-free methods described herein) can be administered to a subject (e.g., a pharmaceutical composition or an agricultural composition). In another example, cyclic polyribonucleotides prepared by the methods described herein (e.g., the cell-free methods described herein) can be delivered to cells.

[0243] In some embodiments, the polyribonucleotide includes any feature or any combination of features disclosed in International Publication No. 2019 / 118919, which is incorporated herein in its entirety by reference.

[0244] Polypeptide expression sequence In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargo of cyclic polyribonucleotides) comprise one or more expression (or coding) sequences, where each expression sequence codes for a polypeptide. In some embodiments, the cyclic polyribonucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more expression (or coding) sequences.

[0245] Each encoded polypeptide may be linear or branched. In various embodiments, the polypeptide may have a length of about 5 to about 40,000 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to about 20,000 amino acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, about 1,000 to about 2,500 amino acids, or any range in between.

[0246] In some embodiments, the polypeptide may have or be useful lengths of less than 40,000 amino acids, less than 35,000 amino acids, less than 30,000 amino acids, less than 25,000 amino acids, less than 20,000 amino acids, less than 15,000 amino acids, less than 10,000 amino acids, less than 9,000 amino acids, less than 8,000 amino acids, less than 7,000 amino acids, less than 6,000 amino acids, less than 5,000 amino acids, less than 4,000 amino acids, less than 3,000 amino acids, less than 2,500 amino acids, less than 2,000 amino acids, less than 1,500 amino acids, less than 1,000 amino acids, less than 900 amino acids, less than 800 amino acids, less than 700 amino acids, less than 600 amino acids, less than 500 amino acids, less than 400 amino acids, or less than 300 amino acids.

[0247] Polypeptides included herein may include naturally occurring polypeptides or polypeptides that do not exist naturally. In some embodiments, polypeptides are or include functional fragments or variants of reference polypeptides (e.g., enzymatically active fragments or variants of enzymes). For example, a polypeptide may be a functionally active variant of any of the polypeptides described herein, having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of a polypeptide described herein or a naturally occurring polypeptide. In some cases, polypeptides may have at least 50% identity with respect to the target protein (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more).

[0248] Some examples of polypeptides include, but are not limited to, fluorescent tags or markers, antigens, therapeutic polypeptides, or polypeptides for agricultural applications.

[0249] Therapeutic polypeptides may include hormones, neurotransmitters, growth factors, enzymes (e.g., oxidoreductase, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzymes, lysosomal enzymes, desaturases), cytokines, antigen-binding polypeptides (e.g., antigen-binding antibodies or antibody-like fragments, e.g., single-chain antibodies, nanobodies, or other Ig heavy or light chain-containing polypeptides), Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, interferons, interleukins, and thrombolytic agents.

[0250] Polypeptides for agricultural applications may include bacteriocins, lysines, antimicrobial polypeptides, antifungal polypeptides, nodule C-rich peptides, bacterial cell regulatory peptides, peptide toxins, pesticide polypeptides (e.g., insecticidal polypeptides or nematodic polypeptides), antigen-binding polypeptides (e.g., antigen-binding antibodies or antibody-like fragments, e.g., single-chain antibodies, nanobodies, or other Ig heavy or light chain-containing polypeptides), enzymes (e.g., nucleases, amylases, cellulases, peptidases, lipases, chitinases), peptide pheromones, and transcription factors.

[0251] In some embodiments, cyclic polyribonucleotides express non-human proteins.

[0252] In some embodiments, the cyclic polyribonucleotide expresses an antibody, such as an antibody fragment or a portion thereof. In some embodiments, the antibody expressed by the cyclic polyribonucleotide may be any isotype, such as IgA, IgD, IgE, IgG, or IgM. In some embodiments, the cyclic polyribonucleotide expresses a portion of an antibody, such as a light chain, heavy chain, Fc fragment, CDR (complementarity-determining region), Fv fragment, or Fab fragment, or further portions thereof. In some embodiments, the cyclic polyribonucleotide expresses one or more portions of an antibody. For example, the cyclic polyribonucleotide may contain two or more expression (or coding) sequences, each expressing a portion of an antibody, and the sum may constitute an antibody. In some cases, the cyclic polyribonucleotide may include one expression sequence encoding the heavy chain of an antibody and another expression sequence encoding the light chain of an antibody. In some cases, when the cyclic polyribonucleotide is expressed in a cell or cell-free environment, the light and heavy chains may undergo appropriate modifications, folding, or other post-translational modifications to form a functional antibody.

[0253] In the embodiment, the polypeptide comprises multiple polypeptides, for example, multiple copies of a single polypeptide sequence, or multiple different polypeptide sequences. In the embodiment, the multiple polypeptides are linked together by linker amino acids or spacer amino acids.

[0254] In embodiments, the polynucleotide cargo contains a sequence encoding a signal peptide. Many signal peptide sequences have been described; for example, the Tat (twin arginine translocation) signal sequence is typically an N-terminal peptide sequence containing the consensus SRRxFLK "twin arginine" motif, which helps translocate folded proteins containing such Tat signal peptides across the lipid bilayer. See, for example, the Signal Peptide Database, published at www[dot]signalpeptide[dot]de. Signal peptides are also useful for directing proteins to specific organelles. See, for example, proline, and experimentally determined and computationally predicted signal peptides disclosed in the Spdb signal peptide database, published at bic.nus.edu.sg / spdb.

[0255] In embodiments, the polynucleotide cargo includes a sequence encoding a cell-permeable peptide (CPP). Hundreds of CPP sequences have been described; see, for example, the publicly available database of cell-permeable peptides, CPPsite, at crdd[dot]osdd[dot]net / raghava / cppsite / . Examples of commonly used CPP sequences are polyarginine sequences that can be fused to the C-terminus of a CGI peptide, such as octoarginine or nonoarginine.

[0256] In embodiments, the polynucleotide cargo includes a sequence encoding a self-assembling peptide; see, for example, Miki et al. (2021) Nature Communications, 21:3412, DOI:10.1038 / s41467-021-23794-6.

[0257] In some embodiments, the expression (or coding) sequence includes a polyA sequence (e.g., the 3' end of the expression sequence). In some embodiments, the length of the polyA sequence is greater than 10 nucleotides. In one embodiment, the polyA sequence is longer than 15 nucleotides (for example, at least or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500 and 3,000 nucleotides or more). In some embodiments, the polyA sequence is designed in accordance with the description of the polyA sequence in sections

[0202] to

[0204] of International Publication No. 2019 / 118919, which is incorporated herein by reference in its entirety. In some embodiments, the expression sequence lacks the polyA sequence (e.g., the 3' end of the expression sequence).

[0258] In some embodiments, the cyclic polyribonucleotide contains poly(A), lacks poly(A), or has modified poly(A) to modulate one or more features of the cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide lacking poly(A) or having modified poly(A) improves one or more functional features, such as immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response), half-life, and / or expression efficiency.

[0259] Therapeutic polypeptides In some embodiments, the cyclic polyribonucleotides described herein (e.g., polyribonucleotide cargoes of cyclic polyribonucleotides) include at least one expression sequence encoding a therapeutic polypeptide. The therapeutic polypeptide is a polypeptide that provides some therapeutic benefit when administered to or expressed in a subject. Administration to or expression of the therapeutic polypeptide in a subject may be used to treat or prevent a disease, disorder, or condition or its symptoms. In some embodiments, the cyclic polyribonucleotide encodes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more therapeutic polypeptides.

[0260] In some embodiments, the cyclic polyribonucleotide contains an expression sequence encoding a therapeutic protein. The protein can treat a disease in a target that requires it. In some embodiments, the therapeutic protein can compensate for a mutated, underexpressed, or absent protein in a target that requires it. In some embodiments, the therapeutic protein can target, interact with, or bind to cells, tissues, or viruses in a target that require it.

[0261] Therapeutic polypeptides may be polypeptides that can be secreted from cells or that can be localized in the cytoplasm, nucleus, or membrane compartment of cells.

[0262] Therapeutic polypeptides include hormones, neurotransmitters, growth factors, enzymes (e.g., oxidoreductase, metabolic enzymes, mitochondrial enzymes, oxygenases, dehydrogenases, ATP-independent enzymes, lysosomal enzymes, desaturases), cytokines, transcription factors, antigen-binding polypeptides (e.g., antigen-binding antibodies or antibody-like fragments, e.g., single-chain antibodies, nanobodies, or other Ig heavy or light chain-containing polypeptides), Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, interferons, interleukins, thrombolytic agents, and antigens (e.g., tumor antigens, viral antigens, or bacterial antigens). These may be antigens, nucleases (e.g., Cas proteins, endonucleases such as Cas9), membrane proteins (e.g., chimeric antigen receptors (CARs), transmembrane receptors, G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), antigen receptors, ion channels, or membrane transporters), secretory proteins, gene-editing proteins (e.g., CRISPR-Cas, TALENs, or zinc fingers), or gene-writing proteins (see, for example, International Publication No. 2020 / 047124, which is incorporated herein in its entirety by reference).

[0263] In some embodiments, the therapeutic polypeptide is an antibody, such as a full-length antibody, an antibody fragment, or a portion thereof. In some embodiments, the antibody expressed by the cyclic polyribonucleotide may be any isotype, such as IgA, IgD, IgE, IgG, or IgM. In some embodiments, the cyclic polyribonucleotide expresses a portion of the antibody, such as the light chain, heavy chain, Fc fragment, CDR (complementarity-determining region), Fv fragment, or Fab fragment, or further portions thereof. In some embodiments, the cyclic polyribonucleotide expresses one or more portions of the antibody. For example, the cyclic polyribonucleotide may contain two or more expression sequences, each expressing a portion of the antibody, and the sum may constitute the antibody. In some cases, the cyclic polyribonucleotide may include one expression sequence encoding the heavy chain of the antibody and another expression sequence encoding the light chain of the antibody. When the cyclic polyribonucleotide is expressed intracellularly, the light and heavy chains may undergo appropriate modifications, folding, or other post-translational modifications to form a functional antibody.

[0264] In some embodiments, cyclic polyribonucleotides prepared as described herein are used as effectors in therapy or agriculture. For example, cyclic polyribonucleotides prepared by the methods described herein (e.g., the cell-free methods described herein) can be administered to a subject (e.g., a pharmaceutical composition or an agricultural composition). In embodiments, the subject is a vertebrate (e.g., mammals, birds, fish, reptiles, or amphibians). In embodiments, the subject is a human. In embodiments, the subject of the method is a non-human mammal. In embodiments, the subject is a non-human mammal such as a non-human primate (e.g., monkeys, apes), an ungulate (e.g., cattle, buffalo, sheep, goats, pigs, camels, llamas, alpacas, deer, horses, donkeys), a carnivore (e.g., dogs, cats), a rodent (e.g., rats, mice), or a rabbit (e.g., rabbits). In some embodiments, the subject is birds such as members of the order Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleognatha (e.g., ostriches, emus), Columbiformes (e.g., domestic pigeons, doves), or Psittaciformes (e.g., parrots). In some embodiments, the subject is invertebrates, such as arthropods (e.g., insects, spiders, crustaceans), nematodes, annelids, helminths, or mollusks. In some embodiments, the subject is invertebrates or invertebrate pests or invertebrates that parasitize vertebrate hosts. In some embodiments, the subject is plants, such as angiosperms (which may be dicotyledonous or monocotyledonous) or gymnosperms (e.g., conifers, buckwheat, ginkgo), ferns, cotton, hemlock, or mosses. In the embodiments, the target is eukaryotic algae (unicellular or multicellular). In the embodiments, the target is agricultural and horticulturally important plants such as crop plants, fruit-producing plants and trees, vegetables, trees, and ornamental plants including flowers, shrubs, trees, ground cover and turfgrass.

[0265] Secretory polypeptide effector In some embodiments, the cyclic polyribonucleotides described herein (e.g., polyribonucleotide cargoes of cyclic polyribonucleotides) include at least one coding sequence encoding a secreted polypeptide effector. Exemplary secreted polypeptide effectors or proteins that may be expressed include, for example, cytokines and cytokine receptors, polypeptide hormones and receptors, growth factors, coagulation factors, therapeutic replacement enzymes and therapeutic non-enzymatic effectors, regeneration, repair and fibrosis factors, transformation factors, and proteins that stimulate cell regeneration, and non-limiting examples thereof are listed herein, for example, in the table below.

[0266] Cytokines and cytokine receptors In some embodiments, the effectors described herein include a cytokine from Table 2, or a functional variant or fragment thereof, a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, or 99% identity with a protein sequence disclosed in Table 11 with reference to its UniProt ID. In some embodiments, the functional variant binds to the corresponding cytokine receptor at a Kd 10%, 20%, 30%, 40%, or 50% higher or lower than the Kd of the corresponding wild-type cytokine for the same receptor under the same conditions. In some embodiments, the effector includes a fusion protein comprising a first region (e.g., a cytokine polypeptide from Table 1 or a functional variant or fragment thereof) and a second heterogeneous region. In some embodiments, the first region is the first cytokine polypeptide from Table 11. In some embodiments, the second region is the second cytokine polypeptide from Table 11, and the first and second cytokine polypeptides form cytokine heterodimers with each other in wild-type cells. In some embodiments, the polypeptides or functional variants thereof listed in Table 11 include signal sequences, such as endogenous signal sequences for effectors or heterologous signal sequences.

[0267] In some embodiments, the effectors described herein include antibodies or fragments thereof that bind to cytokines listed in Table 11. In some embodiments, the antibody molecule includes a signal sequence.

[0268] [Table 11-1]

[0269] [Table 11-2]

[0270] [Table 11-3]

[0271] polypeptide hormones and receptors In some embodiments, the effectors described herein include a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, or 99% identity with the hormones or functional variants thereof in Table 12, for example, the protein sequences disclosed in Table 12 with reference to UniProt ID. In some embodiments, the functional variants bind to the corresponding receptor at a Kd 10%, 20%, 30%, 40%, or 50% higher than that of the corresponding wild-type hormone for the same receptor under the same conditions. In some embodiments, the polypeptides or functional variants thereof in Table 12 include a signal sequence, for example, an endogenous signal sequence or a heterologous signal sequence for the effector.

[0272] In some embodiments, the effector described herein includes an antibody molecule (e.g., scFv) that binds to a hormone in Table 12. In some embodiments, the effector described herein includes an antibody molecule (e.g., scFv) that binds to a hormone receptor in Table 12. In some embodiments, the antibody molecule includes a signal sequence.

[0273] [Table 12-1]

[0274] [Table 12-2]

[0275] growth factors In some embodiments, the effectors described herein include a growth factor in Table 13, or a functional variant thereof, for example, a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, or 99% identity to the protein sequences disclosed in Table 13 with reference to their UniProt ID. In some embodiments, the functional variant binds to the corresponding receptor at a Kd 10%, 20%, 30%, 40%, or 50% higher than that of the corresponding wild-type growth factor for the same receptor under the same conditions. In some embodiments, the polypeptide in Table 13 or its functional variant includes a signal sequence, for example, an endogenous signal sequence or a heterologous signal sequence for the effector.

[0276] In some embodiments, the effector described herein comprises an antibody or fragment thereof that binds to the growth factors in Table 13. In some embodiments, the effector described herein comprises an antibody molecule (e.g., scFv) that binds to the growth factor receptors in Table 13. In some embodiments, the antibody molecule comprises a signal sequence.

[0277] [Table 13-1]

[0278] [Table 13-2]

[0279] clotting factors In some embodiments, the effectors described herein include polypeptides from Table 14, or functional variants thereof, for example, proteins having at least 80%, 85%, 90%, 95%, 967%, 98%, or 99% identity with the protein sequences disclosed in Table 14 with reference to their UniProt IDs. In some embodiments, the functional variants catalyze the same reactions as the corresponding wild-type protein, but at a rate 10%, 20%, 30%, 40%, or 50% or more lower or higher than, for example, the wild-type protein. In some embodiments, the polypeptides from Table 14 or their functional variants include signal sequences, for example, endogenous signal sequences for the effector, or heterologous signal sequences.

[0280] [Table 14]

[0281] Therapeutic replacement enzymes In some embodiments, the effectors described herein include the enzymes in Table 15, or functional variants thereof, for example, proteins having at least 80%, 85%, 90%, 95%, 967%, 98%, or 99% identity with the protein sequences disclosed in Table 15 with reference to UniProt IDs. In some embodiments, the functional variants catalyze the same reaction as the corresponding wild-type protein, for example, at a rate at least 10%, 20%, 30%, 40%, or 50% lower than that of the wild-type protein.

[0282] [Table 15-1]

[0283] [Table 15-2]

[0284] [Table 15-3]

[0285] [Table 15-4]

[0286] [Table 15-5]

[0287] [Table 15-6]

[0288] Other non-enzymatic effects pedals In some embodiments, the therapeutic polypeptides described herein include polypeptides or functional variants thereof from Table 16, for example, proteins having at least 80%, 85%, 90%, 95%, 967%, 98%, and 99% identity with the protein sequences disclosed in Table 16 with reference to UniProt IDs.

[0289] [Table 16-1]

[0290] [Table 16-2]

[0291] [Table 16-3]

[0292] Regeneration, repair, and fibrosis factors The therapeutic polypeptides described herein also include, for example, growth factors disclosed in Table 17, or functional variants thereof, proteins having at least 80%, 85%, 90%, 95%, 967%, 98%, and 99% identity to the protein sequences disclosed in Table 17 with reference to their NCBI protein accession numbers. Antibodies or fragments thereof against such growth factors, or miRNAs that promote regeneration and repair, are also included.

[0293] [Table 17]

[0294] Transformation factor The therapeutic polypeptides described herein also include transforming factors, such as protein factors that transform fibroblasts into differentiated cells, such as the factors disclosed in Table 18 or their functional variants, such as proteins having at least 80%, 85%, 90%, 95%, 967%, 98%, and 99% identity to the protein sequences disclosed in Table 18 with reference to UniProt ID.

[0295] [Table 18]

[0296] Proteins that stimulate cell regeneration The therapeutic polypeptides described herein also include proteins that stimulate cell regeneration, such as the proteins disclosed in Table 19 or their functional variants, such as proteins having at least 80%, 85%, 90%, 95%, 967%, 98%, and 99% identity to the protein sequences disclosed in Table 19 with reference to their UniProt IDs.

[0297] [Table 19]

[0298] Cystic fibrosis membrane conductance regulator (CFTR) In some embodiments, the effectors described herein include the CFTR gene in Table 20, or functional variants or fragments thereof, for example, proteins having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the protein sequences disclosed in Table 20.

[0299] [Table 20-1]

[0300] [Table 20-2]

[0301] [Table 20-3]

[0302] [Table 20-4]

[0303] [Table 20-5]

[0304] In some embodiments, the cyclic polyribonucleotide comprises one or more expression sequences (coding sequences) and is configured for sustained expression in the target cell in vivo. In some embodiments, the cyclic polyribonucleotide is configured such that the expression of one or more expression sequences in the cell at a later time point is greater than or equal to that at a previous time point. In such embodiments, the expression of one or more expression sequences can be maintained at a relatively stable level or can increase over time. The expression of expression sequences can be relatively stable over long periods. For example, in some cases, the expression of one or more expression sequences in the cell over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 days or longer does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some cases, the expression of one or more expression sequences in a cell is maintained at a level that does not fluctuate by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% over a period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 days or longer.

[0305] Plant-modified polypeptides In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargoes of polyribonucleotides) include at least one expression sequence encoding a plant-modified polypeptide. A plant-modified polypeptide refers to a polypeptide that can alter a plant's genetic characteristics (e.g., increasing or decreasing gene expression, or otherwise altering the nucleotide sequence of DNA or RNA), epigenetic properties, or physiological or biochemical properties in a manner that results in a change in the plant's physiology or phenotype, e.g., an increase or decrease in the plant's fitness. In some embodiments, the polyribonucleotide encodes two, three, four, five, six, seven, eight, nine, ten or more different plant-modified polypeptides, or multiple copies of one or more plant-modified polypeptides. A plant-modified polypeptide may alter the physiology or phenotype of various plants, increase or decrease their fitness, or influence such changes in one or more specific plants (e.g., a specific species or genus of plants).

[0306] Examples of polypeptides that may be used herein include enzymes (e.g., metabolic recombinases, helicases, integrases, RNAse, DNAse, or ubiquitinated proteins), pore-forming proteins, signaling ligands, cell-permeable peptides, transcription factors, receptors, antibodies, nanobodies, gene-editing proteins (e.g., CRISPR-Cas endonucleases, TALENs, or zinc fingers), riboproteins, protein aptamers, or chaperones.

[0307] Agricultural polypeptides In some embodiments, the polyribonucleotides described herein (e.g., polyribonucleotide cargo of polyribonucleotides) include at least one expression sequence encoding an agricultural polypeptide. The agricultural polypeptide is a polypeptide suitable for agricultural use. In embodiments, the agricultural polypeptide is applied to plants or seeds (e.g., by foliar spray, spray, injection, or seed coating) or to the plant environment (e.g., by soil drenching or granular soil application) to bring about changes in the plant's physiology, phenotype, or fitness. Embodiments of agricultural polypeptides include polypeptides that alter the level, activity, or metabolism of one or more microorganisms commensal in or on a plant or non-human animal host, the alteration resulting in an increase in the host's fitness. In some embodiments, the agricultural polypeptide is a plant polypeptide. In some embodiments, the agricultural polypeptide is an insect polypeptide. In some embodiments, the agricultural polypeptide has a biological effect when in contact with non-human vertebrates, invertebrates, microorganisms, or plant cells.

[0308] In some embodiments, the polyribonucleotide encodes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more agricultural polypeptides, or multiple copies of one or more agricultural polypeptides.

[0309] Examples of polypeptide embodiments useful for agricultural applications include, for example, bacteriocins, lysines, antimicrobial peptides, nodule C-rich peptides, and bacterial cell regulatory peptides. Such polypeptides can be used to alter the levels, activity, or metabolism of target microorganisms to enhance the fitness of insects (e.g., honeycombs and silkworms). Examples of agriculturally useful polypeptide embodiments include, as known in the art, entomopathogenic bacteria (e.g., Bacillus thuringiensis, Photorhabdus luminescens, Serratia entomophila, or Xenorhabdus nematophylla). This includes peptide toxins such as peptide toxins naturally produced by Nematophila. Examples of agriculturally useful polypeptide embodiments include polypeptides for controlling agriculturally important pests or pathogens (e.g., including small peptides such as cyclodipeptides or diketopiperazines), such as antimicrobial polypeptides or antifungal polypeptides for controlling plant diseases, or pest-killing polypeptides for controlling invertebrate pests such as insects or nematodes (e.g., insecticidal polypeptides or nematode-killing polypeptides). Examples of agriculturally useful polypeptide embodiments include antibodies, nanobodies, and their fragments, such as antibody or nanobodyle fragments that retain at least a portion (e.g., at least 10%) of the specific binding activity of an intact antibody or nanobody. Examples of agriculturally useful polypeptide embodiments include transcription factors, such as plant transcription factors. For example, the model plant Arabidopsis thaliana. Please refer to the "AtTFDB" database, which lists the transcription factor families identified in thaliana, and is publicly available at agris-knowledgebase.org / AtTFDB / . Agriculturally useful polypeptide embodiments include nucleases, such as exonucleases or endonucleases (e.g., Cas nucleases such as Cas9 or Cas12a).Examples of agriculturally useful polypeptides further include cell-permeable peptides, enzymes (e.g., amylase, cellulase, peptidase, lipase, chitinase), and peptide pheromones (e.g., yeast conjugation pheromones, invertebrate reproductive and larval fish signaling pheromones; see Altstein (2004) Peptides, 25:1373-1376).

[0310] Termination element In some embodiments, the polyribonucleotides described herein include at least one termination element. In some embodiments, the polyribonucleotide includes a termination element operably linked to an expression sequence. In some embodiments, the polynucleotide lacks a termination element.

[0311] In some embodiments, the polyribonucleotide comprises one or more expression sequences, each expression sequence may or may not have a termination element. In some embodiments, the polyribonucleotide comprises one or more expression sequences, and the expression sequences lack a termination element so that the polyribonucleotide is translated sequentially. The exclusion of the termination element may result in rolling circle translation or sequential expression of the expression product.

[0312] In some embodiments, the cyclic polyribonucleotide comprises one or more expression sequences, each expression sequence may or may not have a termination element. In some embodiments, the cyclic polyribonucleotide comprises one or more expression sequences, and the expression sequences lack a termination element so that the cyclic polyribonucleotide is translated sequentially. The exclusion of the termination element can result in rolling circle translation or sequential expression of an expression product, such as a peptide or polypeptide, due to the absence of ribosome stalling or detachment. In such embodiments, rolling circle translation expresses a sequential expression product through each expression sequence. In some other embodiments, the termination element of the expression sequence may be part of a stagger element. In some embodiments, one or more expression sequences in the cyclic polyribonucleotide include a termination element. However, rolling circle translation or expression of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in the cyclic polyribonucleotide takes place. In such cases, the expression product may detach from the ribosome when the ribosome encounters a stop element, such as a stop codon, and terminates translation. In some embodiments, translation is completed while the ribosome, for example, at least one subunit of the ribosome, remains in contact with the cyclic polyribonucleotide.

[0313] In some embodiments, the cyclic polyribonucleotide includes termination elements at the ends of one or more expression sequences. In some embodiments, one or more expression sequences include two or more consecutive termination elements. In such embodiments, translation is completed, and circular translation is completed. In some embodiments, the ribosome completely disengages from the cyclic polyribonucleotide. In some such embodiments, the production of subsequent expression sequences (e.g., second, third, fourth, fifth, etc.) in the cyclic polyribonucleotide may require the ribosome to reengage from the cyclic polyribonucleotide before translation initiation. Generally, the termination elements include an in-frame nucleotide triplet, e.g., UAA, UGA, UAG, which signals the termination of translation. In some embodiments, one or more cyclic terminal elements of the polyribonucleotide are frameshifted termination elements, e.g., off-frame or -1 and +1 shifted leading frames (e.g., hidden stop elements), which can terminate translation. Frameshifted termination elements include nucleotide triples, TAA, TAG, and TGA appearing in the second and third reading frames of the expression sequence. Frameshifted termination elements can be important in preventing misreading of mRNA, which is often harmful to cells. In some embodiments, the termination element is a stop codon.

[0314] In some embodiments, the expression sequence includes a polyA sequence (e.g., the 3' end of the expression sequence, e.g., the termination element from 3'). In some embodiments, the length of the polyA sequence exceeds 10 nucleotides. In one embodiment, the polyA sequence is longer than 15 nucleotides (for example, at least or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500 and 3,000 nucleotides or more). In some embodiments, the polyA sequence is designed in accordance with the description of the polyA sequence in sections

[0202] to

[0204] of International Publication No. 2019 / 118919, which is incorporated herein by reference in its entirety. In some embodiments, the expression sequence lacks the polyA sequence (e.g., the 3' end of the expression sequence).

[0315] In some embodiments, the cyclic polyribonucleotide contains poly(A), lacks poly(A), or has modified poly(A) to modulate one or more features of the cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide lacking poly(A) or having modified poly(A) improves one or more functional features, such as immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response), half-life, and / or expression efficiency.

[0316] Further examples of terminal elements are described in paragraphs

[0169] to

[0170] of International Publication No. 2019 / 118919, which is incorporated herein by reference in its entirety.

[0317] qualification Polyribonucleotides may include one or more substitutions, insertions and / or additions, deletions and covalent modifications of a reference sequence, particularly a parent polyribonucleotide, and are included within the scope of this disclosure.

[0318] In some embodiments, the polyribonucleotide comprises one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation and nitrosylation of thiol and tyrosine residues, etc.). The one or more post-transcriptional modifications may be any post-transcriptional modification, e.g., any of the more than 100 different nucleoside modifications identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 updated Nucleoside Acids Res 27:196-197). In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the polyribonucleotide comprises at least one nucleoside selected from the group such as those described in

[0311] of International Publication No. 2019 / 118919, which is incorporated herein in its entirety by reference.

[0319] Polyribonucleotides may include any useful modifications to sugars, nucleic acid bases, or nucleoside bonds (e.g., to linked phosphates / phosphodiester bonds / phosphodiester skeletons). One or more atoms of the pyrimidine nucleic acid bases may be replaced or substituted with optionally substituted aminos, optionally substituted thiols, optionally substituted alkyls (e.g., methyl or ethyl) or halos (e.g., chloro or fluoro). In certain embodiments, the modifications (e.g., one or more modifications) are present in both the sugar and the nucleoside bonds. The modifications may be from ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof. Further modifications are described herein.

[0320] In some embodiments, the polyribonucleotide includes at least one N(6) methyladenosine (m6A) modification to enhance translation efficiency. In some embodiments, the m6A modification can reduce the immunogenicity of the polyribonucleotide (e.g., by reducing the level of one or more markers of an immune or inflammatory response).

[0321] In some embodiments, modifications may include chemical or cell-induced modifications. For example, some non-limiting examples of intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to guide RNA-protein interactions” in Nat Reviews Mol Cell Biol, 2017, 18:202-210.

[0322] In some embodiments, chemical modification of ribonucleotides in polyribonucleotides can enhance immune evasion. Polyribonucleotides can be synthesized and / or modified by methods well established in the art, such as those described in Nucleic Acid Chemistry, Beaucage, S. Let al., which are incorporated herein by reference. Modifications include, for example, terminal modifications, e.g., 5'-terminal modifications (phosphorylation (mono-, di-, and tri-), conjugation, reverse bond, etc.), 3'-terminal modifications (conjugation, DNA nucleotide, reverse bond, etc.), base modifications (e.g., stabilizing bases, destabilizing bases, or substitution with bases that base-pair with an extended repertoire of partners), base removal (baseless nucleotides), or conjugated bases. Modified ribonucleotide bases may also include 5-methylcytidine and pseudouridine. In some embodiments, base modifications can modulate polyribonucleotide expression, immune response, stability, and intracellular localization, to name a few functional effects. In some embodiments, the modifications include biorthogonal nucleotides, such as non-natural bases. See, for example, Kimoto et al., Chem Commun (Camb), 2017, 53:12309, DOI:10.1039 / c7cc06661a, incorporated herein by reference.

[0323] In some embodiments, sugar modification (e.g., at the 2' or 4' position) or sugar substitution of one or more ribonucleotides of a polyribonucleotide may include modification or substitution of phosphodiester bonds, as well as skeletal modifications. Specific examples of polyribonucleotides include, but are not limited to, polyribonucleotides containing a modified skeleton, or polyribonucleotides that do not contain natural internucleoside bonds, such as internucleoside modifications including modification or substitution of phosphodiester bonds. Polyribonucleotides having a modified skeleton include, in particular, those that do not have a phosphorus atom in the skeleton. For the purposes of this application and as is sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in the internucleoside skeleton can also be considered oligonucleosides. In certain embodiments, polyribonucleotides will include ribonucleotides that have a phosphorus atom in their internucleoside skeleton.

[0324] Modified polyribonucleotide skeletons may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, methyl and other alkylphosphonates, such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotryesters, and boranophosphorates having the usual 3'-5' bond, their 2'-5' bond analogues, and those having reverse polarity in which adjacent pairs of nucleoside units are bonded from 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments, the polyribonucleotides may be negatively or positively charged.

[0325] Modified nucleotides that can be incorporated into polyribonucleotides may be modified by internucleoside bonds (e.g., phosphate backbone). In this specification, the terms “phosphate” and “phosphodiester” are used interchangeably in the context of the polynucleotide backbone. The phosphate group of the backbone can be modified by substituting one or more oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides may include massive substitutions of the unmodified phosphate moiety by other internucleoside bonds as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotryesters. In phosphorodithioates, both unbonded oxygen atoms are substituted with sulfur. Phosphate linkers can also be modified by substituting linked oxygen with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate), and carbon (bridged methylene phosphonate).

[0326] The α-thio-substituted phosphate moiety is provided to confer stability to RNA and DNA polymers via non-natural phosphorothioate backbone binding. Phosphorothioate DNA and RNA exhibit increased nuclease resistance, followed by a longer half-life in the cellular environment. Phosphorothioate bound to polyribonucleotides is expected to reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.

[0327] In certain embodiments, the modified nucleoside includes alpha-thionucleosides (e.g., 5'-O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine (α-thiocytidine), 5'-O-(l-thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine).

[0328] Other nucleoside bonds that may be used pursuant to this disclosure, including nucleoside bonds that do not contain a phosphorus atom, are described herein.

[0329] In some embodiments, the polyribonucleotide may contain one or more cytotoxic nucleosides. For example, the cytotoxic nucleosides may be incorporated into the polyribonucleotide through difunctional modifications or other means. Examples of cytotoxic nucleosides include, but are not limited to, adenosine arabinoside, 5-azacitidine, 4'-thio-aracitidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, phloxuridine, gemcitabine, combinations of tegafur and uracil, tegafur ((RS)-5-fluoro-l-(tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione), troxacitabine, tezacitabine, 2'-deoxy-2'-methylidencytidine (DMDC), and 6-mercaptopurine. Further examples include fludarabine phosphate, N4-behenoyl-l-beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4-palmitoyl-l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5'-elaidic acid ester).

[0330] Polyribonucleotides may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally occurring nucleotides, purines or pyrimidines, or one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the polyribonucleotide or in a given predetermined sequence region thereof. In some embodiments, the polyribonucleotide contains pseudouridine. In some embodiments, the polyribonucleotide contains inosine, which may support the immune system, characterizing the polyribonucleotide as endogenous RNA versus viral RNA. Inosine incorporation may also mediate improved RNA stability / reduced degradation. See, for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res. 25, 1283-1284, each of which is incorporated in whole by reference.

[0331] In some embodiments, all nucleotides in a polyribonucleotide (or a given sequence region thereof) are modified. In some embodiments, the modifications may include m6A, which may enhance expression; inosine, which may attenuate the immune response; pseudouridine, which may increase RNA stability; or m5C, which may increase stability; which is a translational readthrough (stagger element); and 2,2,7-trimethylguanosine, which may aid in intracellular translocation (e.g., nuclear localization).

[0332] Different sugar modifications, nucleotide modifications, and / or internucleoside bonds (e.g., in the main chain structure) can be located at various positions on polyribonucleotides. Those skilled in the art will understand that nucleotide analogs or other modifications can be located at any position on a polyribonucleotide so as not to substantially impair the function of the polyribonucleotide. Modifications can also be non-coding region modifications. Polyribonucleotides are composed of approximately 1% to approximately 100% modified nucleotides (either in relation to the overall nucleotide content or in relation to one or more types of nucleotides, i.e., A, G, U, or C) or any intervening percentage (e.g., 1% to less than 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10%). This may include percentages of 90%, 10%~95%, 10%~100%, 20%~25%, 20%~50%, 20%~60%, 20%~70%, 20%~80%, 20%~90%, 20%~95%, 20%~100%, 50%~60%, 50%~70%, 50%~80%, 50%~90%, 50%~95%, 50%~100%, 70%~80%, 70%~90%, 70%~95%, 70%~100%, 80%~90%, 80%~95%, 80%~100%, 90%~95%, 90%~100%, and 95%~100%).

[0333] Generation method This disclosure provides methods for generating cyclic polyribonucleotides, including, for example, recombinant techniques or chemical synthesis. For example, the DNA molecules used to generate the RNA ring may include DNA sequences of naturally occurring nucleic acid sequences, modified versions thereof, or DNA sequences encoding synthetic polypeptides not typically found in nature (e.g., chimeric molecules or fusion proteins). DNA and RNA molecules can be modified using a variety of techniques, but are not limited to classical mutagenesis and recombinant techniques, including, for example, site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification or mutagenesis of selected regions of nucleic acid sequences, synthesis of oligonucleotide mixtures and ligation "construction" of mixtures into mixtures of nucleic acid molecules and combinations thereof.

[0334] Cyclic polyribonucleotides can be prepared according to any available techniques, including but not limited to chemical synthesis and enzymatic synthesis. In some embodiments, linear primary constructs or linear polyribonucleotides for cyclization can be cyclized or ligated to produce the circRNAs described herein. In some embodiments, linear polyribonucleotides for cyclization can be cyclized in vitro before formulation and / or delivery. In some embodiments, cyclic polyribonucleotides may be a mixture with linear polyribonucleotides. In some embodiments, the linear polyribonucleotides have the same nucleic acid sequence as the cyclic polyribonucleotides.

[0335] The cyclization or ligation mechanism can occur by methods such as chemical, enzymatic, sprint ligation, or ribozyme-catalyzed methods. The newly formed 5'-3' bond can be intramolecular or intermolecular. For example, sprint ligases such as Sprint® ligase can be used for sprint ligation. By this method, a single-stranded polynucleotide (sprint), such as single-stranded DNA or RNA, can be designed to hybridize with both ends of a linear polyribonucleotide, so that the two ends can be juxtaposed during hybridization with the single-stranded sprint. Thus, a sprint ligase can catalyze the ligation of the two juxtaposed ends of a linear polyribonucleotide to produce circRNA. In some embodiments, DNA ligases or RNA ligases may be used in the synthesis of cyclic polyribonucleotides. In non-limiting examples, the ligase may be a circuligase or a cyclic ligase.

[0336] In some embodiments, either the 5' or 3' end of the linear polyribonucleotide can encode a ligase ribozyme sequence, and as a result, the linear polyribonucleotide for cyclization can contain an active ribozyme sequence that can ligate the 5' end of the linear polyribonucleotide for cyclization to the 3' end of the linear polyribonucleotide for cyclization during in vitro transcription. The ligase ribozyme may be derived from a group I intron, hepatitis delta virus, hairpin ribozyme, or may be selected by SELEX (phylogenetic evolution of ligands by exponential enrichment).

[0337] In another example, a linear polyribonucleotide may be cyclized or linked by using at least one non-nucleic acid moiety. In one embodiment, at least one non-nucleic acid moiety may react with a region or feature near the 5' or 3' end of the linear polyribonucleotide for cyclization in order to cyclize or link the linear polyribonucleotide. In another embodiment, at least one non-nucleic acid moiety may be located within the 5' or 3' end of the linear polyribonucleotide for cyclization, or may be linked near the 5' and / or 3' end. The non-nucleic acid moiety may be homologous or heterogeneous. In an unrestricted example, the non-nucleic acid moiety may be a hydrophobic bond, an ionic bond, a biodegradable bond, or a cleavable bond. In another unrestricted example, the non-nucleic acid moiety may be a ligation moiety. In yet another unrestricted example, the non-nucleic acid moiety may be an oligonucleotide or a peptide moiety, such as the aptamer or non-nucleic acid linker described herein.

[0338] In some embodiments, the linear polyribonucleotide for cyclization may include the 5' triphosphate of a nucleic acid that has been converted to 5' monophosphate, for example, by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or ATP diphosphohydrolase (apirase). In some embodiments, at least a portion of the 5' end of the linear polyribonucleotide includes a monophosphate moiety. In some embodiments, a population of polyribonucleotides, including cyclic and linear polyribonucleotides, is contacted with RppH before at least a portion of the linear polyribonucleotides are digested with a 5' exonuclease and / or a 3' exonuclease. Alternatively, the conversion of the 5' triphosphate of a linear polyribonucleotide for cyclization to a 5' monophosphate may occur by a two-step reaction comprising: (a) contacting the 5' nucleotide of the linear polyribonucleotide for cyclization with a phosphatase (e.g., Antarctic phosphatase, shrimp alkaline phosphatase, or calf intestinal phosphatase) to remove all three phosphates; and (b) contacting the 5' nucleotide after step (a) with a single-phosphate-adding kinase (e.g., polynucleotide kinase). In another embodiment, the linear polyribonucleotide for cyclization may be cyclized or ligated by self-splicing. In some embodiments, the linear polyribonucleotide may include a sequence that mediates self-ligation. In some embodiments, the linear polyribonucleotide may include a loop E sequence (e.g., PSTVd) for self-ligation. In some embodiments, the linear polyribonucleotide may include an HDV sequence, e.g., an HDV replication domain conservation sequence, for self-ligation. [ka] This includes. In another embodiment, the linear polyribonucleotide may include autocyclizing introns, e.g., 5' and 3' slice junctions, or autocyclizing catalytic introns, e.g., group I, group II, or group III introns. Non-limiting examples of group I intron autosplifying sequences include autosplifying substitution intron-exon sequences derived from the T4 bacteriophage gene td, and Tetrahymena intervening sequence (IVS) rRNA, the cyanobacterial Anabaena pre-tRNA-Leu gene, or Tetrahymena pre-rRNA.

[0339] In some embodiments, the polyribonucleotide comprises catalytic intron fragments such as the 3' half and 5' half of the Group I catalytic intron fragment. The first and second annealing regions may be located within the catalytic intron fragment. The catalytic introns of Group I are self-splicing ribozymes that catalyze their own excision from mRNA, tRNA, and rRNA precursors via a two-metal ion phoryl transfer mechanism. Importantly, the RNA itself autocatalyzes intron removal without the need for exogenous enzymes such as ligases.

[0340] In some embodiments, the 3' half and 5' half of the group I catalytic intron fragment are derived from the cyanobacterial Anabaena pre-tRNA-Leu gene or Tetrahymena pre-rRNA.

[0341] In some embodiments, the 3' half of the catalytic intron fragment of group I and the 5' half of the catalytic intron fragment of group I are derived from the cyanobacterial Anabaena pre-tRNA-Leu gene, where the 3' exon fragment comprises a first annealing region and the 5' exon fragment comprises a second annealing region. The first annealing region may comprise, for example, 5 to 50, for example, 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides, and the second annealing region may comprise, for example, 5 to 50, for example, 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides.

[0342] In some embodiments, the 3' half and 5' half of the group I catalytic intron fragment are derived from Tetrahymena pre-rRNA, the 3' half of the group I catalytic intron fragment comprises a first annealing region, and the 5' exon fragment comprises a second annealing region. In some embodiments, the 3' exon comprises a first annealing region, and the 5' half of the group I catalytic intron fragment comprises a second annealing region. The first annealing region may comprise, for example, 6 to 50, for example, 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the second annealing region may comprise, for example, 6 to 50, for example, 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

[0343] In some embodiments, the 3' half and 5' half of the group I catalytic intron fragment are derived from the cyanobacterial Anabaena pre-tRNA-Leu gene, Tetrahymena pre-rRNA, or T4 phage td gene.

[0344] In some embodiments, the 3' half and 5' half of the catalytic intron fragment of group I are derived from the T4 phage td gene. The 3' exon fragment may comprise a first annealing region, and the 5' half of the catalytic intron fragment of group I may comprise a second annealing region. The first annealing region may comprise, for example, 2-16, or for example, 10-16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the second annealing region may comprise, for example, 2-16, or for example, 10-16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

[0345] In some embodiments, the 3' half of the group I catalytic intron fragment is the 5' end of a linear polyribonucleotide.

[0346] In some embodiments, the 5' half of the group I catalytic intron fragment is the 3' end of a linear polyribonucleotide.

[0347] In some embodiments, the 3' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0348] In some embodiments, the 5' half of the catalytic intron fragment of group I is 5'- [ka] and have at least 80% (for example, at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity.

[0349] In some embodiments, the 3' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 215, and the 5' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 216.

[0350] In some embodiments, the 3' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0351] In some embodiments, the 5' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0352] In some embodiments, the 3' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 217, and the 5' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 218.

[0353] In some embodiments, the 3' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0354] In some embodiments, the 5' half of the catalytic intron fragment of group I has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence 5'-TAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACT-3' (SEQ ID NO: 220).

[0355] In some embodiments, the 3' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 219, and the 5' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 220.

[0356] In some embodiments, the 3' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0357] In some embodiments, the 5' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0358] In some embodiments, the 3' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 221, and the 5' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 222.

[0359] In some embodiments, the 3' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0360] In some embodiments, the 5' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0361] In some embodiments, the 3' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 223, and the 5' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 224.

[0362] In some embodiments, the 3' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0363] In some embodiments, the 5' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0364] In some embodiments, the 3' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 225, and the 5' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 226.

[0365] In some embodiments, the 3' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0366] In some embodiments, the 5' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0367] In some embodiments, the 3' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 227, and the 5' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 228.

[0368] In some embodiments, the 3' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0369] In some embodiments, the 5' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0370] In some embodiments, the 3' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 229, and the 5' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 230.

[0371] In some embodiments, the 3' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0372] In some embodiments, the 5' half of the catalytic intron fragment of group I is 5'- [ka] It has sequence identity with at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) of the sequence.

[0373] In some embodiments, the 3' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 231, and the 5' half of the catalytic intron fragment of group I has the sequence of SEQ ID NO: 232.

[0374] In some embodiments, the catalytic intron fragment of group I originates from the nrdB or nrdD gene of the T4 phage. In some embodiments, the 3' half of the group I catalytic intron is 5'- [ka] It contains sequences that have at least 80% sequence identity with respect to [the given sequence].

[0375] In some embodiments, the 3' half of the catalytic intron fragment of group I of (A) is 5'- [ka] and include sequences having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity.

[0376] In some embodiments, the 5' half of a group I catalytic intron fragment derived from the T4 phage nrdB gene.

[0377] In some embodiments, the 3' half of the catalytic intron fragment of group I originates from the T4 phage nrdB gene, and the 5' half of the catalytic intron fragment of group I originates from the T4 phage nrdB gene.

[0378] In some embodiments, the 5' half of the catalytic intron of group I contains a sequence having at least 80% sequence identity with 5'-AAAATGCGCCTTTAAACGGTAACGTTTATCGAAAACTCCTTTAATTGCTGGAAAGTCCTTTATGGAAAACTAGCAGCCAAGGTTTTGCTT-3' (SEQ ID NO: 235).

[0379] In some embodiments, the 5' half of the catalytic intron of group I contains a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity with 5'-AAAATGCGCCTTTAAACGGTAACGTTTATCGAAAACTCCTTTAATTGCTGGAAAGTCCTTTATGGAAAACTAGCAGCCAAGGTTTTGCTT-3' (SEQ ID NO: 235).

[0380] In some embodiments, the 3' half of the catalytic intron fragment of group I originates from the T4 phage nrdD gene.

[0381] In some embodiments, the 3' half of the catalytic intron fragment of group I is 5'- [ka] It contains sequences that have at least 80% sequence identity with respect to [the given sequence].

[0382] In some embodiments, the 3' half of the catalytic intron fragment of group I is 5'- [ka] and include sequences having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity.

[0383] In some embodiments, the 5' half of the catalytic intron fragment of group I originates from the T4 phage nrdD gene.

[0384] In some embodiments, the 3' half of the catalytic intron fragment of group I originates from the T4 phage nrdD gene, and the 5' half of the catalytic intron fragment of group I originates from the T4 phage nrdD gene.

[0385] In some embodiments, the 5' half of the group I catalytic intron contains a sequence having at least 80% sequence identity with 5'-TAACGTAAGTCAAGCTCATGTAAAATCTGCCTAAAACGGGAAACTCTCACTGAGACAATCCGTTGCTAAATCAG-3' (SEQ ID NO: 239).

[0386] In some embodiments, the 5' half of the catalytic intron of group I contains a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity with 5'-TAACGTAAGTCAAGCTCATGTAAAATCTGCCTAAAACGGGAAACTCTCACTGAGACAATCCGTTGCTAAATCAG-3' (SEQ ID NO: 239).

[0387] In some embodiments, the 3' exon fragment contains a sequence having at least 80% sequence identity with 5'-GTACCTTTAACTTCCATAAGAACATGGAAATCATGGAAGGTAATGCCAAG-3' (SEQ ID NO: 241).

[0388] In some embodiments, the 3' exon fragment contains a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity with 5'-GTACCTTTAACTTCCATAAGAACATGGAAATCATGGAAGGTAATGCCAAG-3' (SEQ ID NO: 241).

[0389] In some embodiments, the 3' exon fragment contains a sequence having at least 80% sequence identity with 5'-GTACCTTTAACTTCCAAAAGATACATAAAAATCATGGAAGGTAATGCCAAG-3' (sequence number 243).

[0390] In some embodiments, the 3' exon fragment contains a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity with 5'-GTACCTTTAACTTCCAAAAGATACATAAAAATCATGGAAGGTAATGCCAAG-3' (SEQ ID NO: 243).

[0391] In some embodiments, the 5' exon fragment includes a sequence having at least 80% sequence identity with 5'-TTTTTATGTATCTTTTGCGT-3' (SEQ ID NO: 245).

[0392] In some embodiments, the 5' exon fragment comprises a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity with 5'-TTTTTATGTATCTTTTGCGT-3' (SEQ ID NO: 245).

[0393] In some embodiments, the 3' exon fragment contains a sequence having at least 80% sequence identity with 5'-ATGAAGTGAACACGTTATTCAGTTCAAACGGACAGACTCCTTTTGTAACA-3' (SEQ ID NO: 247).

[0394] In some embodiments, the 3' exon fragment contains a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity with 5'-ATGAAGTGAACACGTTATTCAGTTCAAACGGACAGACTCCTTTTGTAACA-3' (SEQ ID NO: 247).

[0395] In some embodiments, the 3' exon fragment contains a sequence having at least 80% sequence identity with 5'-ATGAAGTGAACACGTTACATAAGCTTGGAATGCAGACTCCTTTTGTAACA-3' (SEQ ID NO: 249).

[0396] In some embodiments, the 3' exon fragment contains a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity with 5'-ATGAAGTGAACACGTTACATAAGCTTGGAATGCAGACTCCTTTTGTAACA-3' (SEQ ID NO: 249).

[0397] In some embodiments, the 5' exon fragment contains a sequence having at least 80% sequence identity with 5'-TGCATTCCAAGCTTATGAGT-3' (SEQ ID NO: 251).

[0398] In some embodiments, the 5' exon fragment comprises a sequence having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity with 5'-TGCATTCCAAGCTTATGAGT-3' (SEQ ID NO: 251).

[0399] In another embodiment, linear polyribonucleotides for cyclization may be cyclized or linked at or near the 5' and 3' ends of the linear polyribonucleotides for cyclization, or by atoms linked thereto, non-nucleic acid moieties that cause attractive forces between molecular surfaces. In one embodiment, one or more linear polyribonucleotides are cyclized or linked by intermolecular or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonance bonds, invisible bonds, bipolar bonds, conjugation, hyperconjugation, and antibonding.

[0400] In some embodiments, the linear polyribonucleotide for cyclization may include ribozyme RNA sequences near the 5' and 3' ends. The ribozyme RNA sequences may covalently bind to the peptide when the sequence is exposed to the remainder of the ribozyme. In one embodiment, peptides covalently bound to the ribozyme RNA sequences near the 5' and 3' ends may associate with each other to cyclize or ligate the linear polyribonucleotide. In another example, peptides covalently bound to the ribozyme RNA near the 5' and 3' ends may cyclize or ligate the linear primary construct or linear mRNA after being subjected to ligation using various methods known in the art, such as protein ligation, but not limited to these. A non-exclusive list of ribozymes for use in the linear primary construct or linear polyribonucleotide of the present invention, or of methods for incorporating or covalently ligating peptides, is described in U.S. Patent Application No. US20030082768, the contents of which are incorporated herein by reference in their entirety.

[0401] In another embodiment, chemical methods of cyclization may be used to generate cyclic polyribonucleotides. Such methods may include, but are not limited to, click chemistry (e.g., methods based on alkynes and azides, or clickable bases), olefin metathesis, phosphoramidal ligation, hemiaminalhymine crosslinking, base modification, and any combination thereof. In some chemical methods, the 5' and 3' ends of the linear polyribonucleotide for cyclization contain chemically reactive groups that, when in close proximity to each other, can form new covalent bonds between the 5' and 3' ends of the molecule. The 5' end may contain an NHS-ester reactive group, and the 3' end may contain a 3'-amino-terminal nucleotide, thereby undergoing nucleophilic attack on the 5'-NHS-ester moiety of the 3' end of the linear RNA molecule in an organic solvent to form a new 5' / 3' amide bond.

[0402] In another embodiment, cyclic polyribonucleotides may be generated using a deoxyribonucleotide template that is transcribed in a cell-free system (e.g., by in vitro transcription) to produce linear RNA. The linear polyribonucleotides generate splicing-compatible polyribonucleotides that can self-splice to produce cyclic polyribonucleotides.

[0403] In some embodiments, the disclosure provides a method for producing cyclic polyribonucleotides (e.g., in a cell-free system) by providing linear polyribonucleotides. The linear polyribonucleotides are self-spliced ​​under conditions suitable for splicing the 3' and 5' splice sites of the linear polyribonucleotides; thereby producing cyclic polyribonucleotides.

[0404] In some embodiments, the disclosure provides a method for producing a cyclic polyribonucleotide by providing a deoxyribonucleotide encoding a linear polyribonucleotide. This method includes the steps of: transcribing the deoxyribonucleotide in a cell-free system to produce a linear polyribonucleotide; optionally purifying the splicing-compatible linear polyribonucleotide; and self-splicing the linear polyribonucleotide under conditions suitable for splicing the 3' and 5' splice sites of the linear polyribonucleotide, thereby producing a cyclic polyribonucleotide.

[0405] In some embodiments, the disclosure provides a method for producing a cyclic polyribonucleotide by providing a deoxyribonucleotide encoding a linear polyribonucleotide, the deoxyribonucleotide being transcribed in a cell-free system to produce a linear polyribonucleotide, the transcription being carried out in solution under conditions suitable for splicing the 3' and 5' splice sites of the linear polyribonucleotide, thereby producing a cyclic polyribonucleotide. In some embodiments, the linear polyribonucleotide includes a 5' split intron and a 3' split intron (e.g., a self-splicing construct for producing a cyclic polyribonucleotide). In some embodiments, the linear polyribonucleotide includes a 5' annealing region and a 3' annealing region.

[0406] Suitable conditions for in vitro transcription and / or self-splicing may include any conditions that mimic physiological conditions in one or more respects (e.g., solutions or buffers, e.g., aqueous buffers or aqueous solutions). In some embodiments, suitable conditions include 0.1–100 mM Mg²⁺ ions or salts thereof (e.g., 1–100 mM, 1–50 mM, 1–20 mM, 5–50 mM, 5–20 mM, or 5–15 mM). In some embodiments, suitable conditions include 1–1000 mM K⁺ ions or salts thereof, e.g., KCl (e.g., 1–1000 mM, 1–500 mM, 1–200 mM, 50–500 mM, 100–500 mM, or 100–300 mM). In some embodiments, suitable conditions include 1 to 1000 mM Cl- ions or salts thereof, e.g., KCl (e.g., 1 to 1000 mM, 1 to 500 mM, 1 to 200 mM, 50 to 500 mM, 100 to 500 mM, or 100 to 300 mM). In some embodiments, suitable conditions include 0.1 to 100 mM Mn²⁺ ions or salts thereof, e.g., MnCl₂ (e.g., 0.1 to 100 mM, 0.1 to 50 mM, 0.1 to 20 mM, 0.1 to 10 mM, 0.1 to 5 mM, 0.1 to 2 mM, 0.5 to 50 mM, 0.5 to 20 mM, 0.5 to 15 mM, 0.5 to 5 mM, 0.5 to 2 mM, or 0.1 to 10 mM). In some embodiments, preferred conditions include dithiothreitol (DTT) (e.g., 1-1000 μM, 1-500 μM, 1-200 μM, 50-500 μM, 100-500 μM, 100-300 μM, 0.1-100 mM, 0.1-50 mM, 0.1-20 mM, 0.1-10 mM, 0.1-5 mM, 0.1-2 mM, 0.5-50 mM, 0.5-20 mM, 0.5-15 mM, 0.5-5 mM, 0.5-2 mM, or 0.1-10 mM). In some embodiments, preferred conditions include a ribonucleoside triphosphate (NTP) concentration of 0.1 mM to 100 mM (e.g., 0.1 to 100 mM, 0.1 to 50 mM, 0.1 to 10 mM, 1 to 100 mM, 1 to 50 mM, or 1 to 10 mM). In some embodiments, preferred conditions include a pH of 4 to 10 (e.g., pH 5 to 9, pH 6 to 9, or pH 6.5 to 8.5).In some embodiments, suitable conditions include temperatures ranging from 4°C to 50°C (e.g., 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, or 30°C to 40°C).

[0407] In some embodiments, linear polyribonucleotides are produced from deoxyribonucleic acid, such as the deoxyribonucleic acid described herein, such as a DNA vector, a linearized DNA vector, or cDNA. In some embodiments, linear polyribonucleotides are transcribed from deoxyribonucleic acid by transcription in a cell-free system (e.g., in vitro transcription).

[0408] In another embodiment, cyclic polyribonucleotides may be produced in cells, such as prokaryotic or eukaryotic cells. In some embodiments, exogenous polyribonucleotides are provided to cells (e.g., a linear polyribonucleotide as described herein or a DNA molecule encoding the transcription of a linear polyribonucleotide as described herein). Linear polyribonucleotides can be transcribed intracellularly from the exogenous DNA molecule provided to the cell. Linear polyribonucleotides can be transcribed intracellularly from an exogenous recombinant DNA molecule transiently provided to the cell. In some embodiments, the exogenous DNA molecule is not integrated into the cell's genome. In some embodiments, linear polyribonucleotides are transcribed intracellularly from a recombinant DNA molecule integrated into the cell's genome.

[0409] In some embodiments, the cells are prokaryotic cells. In some embodiments, the prokaryotic cells containing the polyribonucleotides described herein are bacterial cells or archaeal cells. For example, prokaryotic cells containing polyribonucleotides as described herein include Escherichia coli, halophilic archaea (e.g., Haloferax volcaniii, Sphingomonas), cyanobacteria (e.g., Synechococcus elongatus, Spirulina (Arthrospira) spp., and Synechocystis spp.), Streptomyces, actinomycetes (e.g., Nonomuraea, Kitasatospora, or Thermobifida), Bacillus species (e.g., Bacillus subtilis, Bacillus anthracis, Bacillus cereus) These may include prokaryotic cells (e.g., Cereus), beta-proteobacteria (e.g., Burkholderia), alpha-proteobacteria (e.g., Agrobacterium), Pseudomonas (e.g., Pseudomonas putida), and enterobacteria. Prokaryotic cells can be grown in culture medium. Prokaryotic cells may be contained in a bioreactor.

[0410] In some embodiments, the cells are eukaryotic cells. In some embodiments, the eukaryotic cells are unicellular eukaryotic cells. In some embodiments, the unicellular eukaryotes are yeast cells (e.g., Saccharomyces cerevisiae and other Saccharomyces spp.), Brettanomyces spp., Schizosaccharomyces spp., Torulaspora spp. and Pichia These are single-celled fungal cells such as spp. In some embodiments, single-celled eukaryotic cells are single-celled animal cells. Single-celled animal cells may be cells isolated from multicellular animals and grown in culture, or their daughter cells. In some embodiments, single-celled animal cells are dedifferentiated. In some embodiments, single-celled eukaryotic cells are single-celled plant cells. Single-celled plant cells may be cells isolated from multicellular plants and grown in culture, or their daughter cells. In some embodiments, single-celled plant cells are dedifferentiated. In some embodiments, single-celled plant cells are derived from plant callus. In some embodiments, single-celled cells are plant cell protoplasts. In some embodiments, single-celled eukaryotic cells are single-celled eukaryotic algal cells such as single-celled green algae, diatoms, euglenoids, or dinoflagellates. Non-limiting examples of single-celled eukaryotic algae of interest include Dunaliella salina, Chlorella vulgaris, Chlorella zophingiensis Examples include *Haematococcus zofingiensis*, *Haematococcus pluvialis*, *Neochloris oleoabundance* and other *Neochloris* species, *Protosiphon botryoides*, *Botryococcus braunii*, *Cryptococcus* spp., *Chlamydomonas reinhardtii* and other *Chlamydomonas* spp.In some embodiments, the single-celled eukaryotic cell is a protist cell. In some embodiments, the single-celled eukaryotic cell is a protozoan cell.

[0411] In some embodiments, eukaryotic cells are cells of multicellular eukaryotes. For example, multicellular eukaryotes may be selected from the group consisting of vertebrates, invertebrates, multicellular fungi, multicellular algae, and multicellular plants. In some embodiments, eukaryotes are humans. In some embodiments, eukaryotes are non-human vertebrates. In some embodiments, eukaryotes are invertebrates. In some embodiments, eukaryotes are multicellular fungi. In some embodiments, eukaryotes are multicellular plants. In embodiments, eukaryotic cells are human cells or cells of non-human mammals, such as non-human primates (e.g., monkeys, apes), ungulates (e.g., Bovidae including cattle, buffalo, bison, sheep, goats, and musk cows; pigs; camels including camels, llamas, and alpacas; deer, antelopes; horses and donkeys), carnivores (e.g., dogs, cats), rodents (e.g., rats, mice, guinea pigs, hamsters, squirrels), or lagomorphs (e.g., rabbits). In embodiments, the eukaryotic cells are avian cells, for example, members of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., domestic pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the eukaryotic cells are arthropod cells (e.g., insects, spiders, crustaceans), nematodes, annelids, helminths, or mollusks. In embodiments, the eukaryotic cells are cells of multicellular plants, for example, angiosperms (which may be dicotyledonous or monocotyledonous) or gymnosperms (e.g., conifers, buckwheat, ginkgo, ginkgo), ferns, cotton, hemlock, or mosses. In a preferred configuration, the eukaryotic cells are cells of eukaryotic multicellular algae.

[0412] Eukaryotic cells can be grown in culture medium. Eukaryotic cells may also be contained in a bioreactor.

[0413] In some embodiments, any method for producing the cyclic polyribonucleotides described herein may be carried out in a bioreactor. A bioreactor refers to any container in which a chemical or biological process involving a living organism or a biochemically active substance derived from such an organism is carried out. The bioreactor may be compatible with the cell-free method for producing the cyclic RNA described herein. Containers for the bioreactor may include culture flasks, dishes, or bags that are single-use (disposable), autoclavable, or sterilizable. The bioreactor may be made of glass, polymer-based, or other materials.

[0414] Examples of bioreactors include, but are not limited to, agitated tank (e.g., well-mixed) bioreactors and tubular (e.g., plug-flow) bioreactors, air-lift bioreactors, membrane agitated tanks, spin-filter agitated tanks, vibromixers, fluidized bed reactors, and membrane bioreactors. The mode of operation for the bioreactor may be a batch process or a continuous process. A bioreactor is continuous when reagent and product streams are continuously supplied and withdrawn from the system. A batch bioreactor may have a continuous recirculating stream but does not provide continuous supply of reagents or product recovery. Some methods of this disclosure relate to the large-scale production of cyclic polyribonucleotides. For large-scale production methods, the method may be carried out in volumes of 1 liter (L) to 50 L or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L or more). In some embodiments, the method can be carried out in volumes of 5L-10L, 5L-15L, 5L-20L, 5L-25L, 5L-30L, 5L-35L, 5L-40L, 5L-45L, 10L-15L, 10L-20L, 10L-25L, 20L-30L, 10L-35L, 10L-40L, 10L-45L, 10L-50L, 15L-20L, 15L-25L, 15L-30L, 15L-35L, 15L-40L, 15L-45L, or 15-50L. In some embodiments, the bioreactor can produce at least 1g of circular RNA. In some embodiments, the bioreactor may produce 1 to 200 g of circular RNA (e.g., 1 to 10 g, 1 to 20 g, 1 to 50 g, 10 to 50 g, 10 to 100 g, 50 to 100 g, or 50 to 200 g of circular RNA). In some embodiments, the amount produced is measured per liter (e.g., 1 to 200 g / liter), per batch or reaction (e.g., 1 to 200 g per batch or reaction), or per unit time (e.g., 1 to 200 g per hour or per day). In some embodiments, two or more bioreactors can be used in series to increase production capacity (e.g., one, two, three, four, five, six, seven, eight, or nine bioreactors can be used in series).

[0415] In some embodiments, the cyclization efficiency is 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 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, the cyclization efficiency is at least about 40%. In some embodiments, the cyclization efficiency is about 10% to about 100%. For example, the cyclization efficiency is 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%, about 95%, and about 99%. In some embodiments, the cyclization efficiency is approximately 20% to 80%. In some embodiments, the cyclization efficiency is approximately 30% to 60%. In some embodiments, the cyclization efficiency is approximately 40%. Further methods for producing the cyclic polyribonucleotides described herein are, for example, described in Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press (2002); in Zhao, Synthetic Biology: Tools and Applications, (First Edition), Academic Press (2013); Muller and Appel (from RNA Biol, 2017, 14(8):1018-1027); and Egli & Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, (First Edition), Wiley-VCH (2012). Other methods for producing cyclic polyribonucleotides are described, for example, in International Publication No. 2022 / 247943, U.S. Patent No. 11000547, International Publication No. 2018 / 191722, International Publication No. 2019 / 236673, International Publication No. 2020 / 023595, International Publication No. 2022 / 204460, International Publication No. 2022 / 204464, International Publication No. 2022 / 204466, and International Publication No. 2022 / 261490.

[0416] Various methods for synthesizing cyclic polyribonucleotides are also described elsewhere (see, for example, U.S. Patent No. 6,210,931, U.S. Patent No. 5,773,244, U.S. Patent No. 5,766,903, U.S. Patent No. 5,712,128, U.S. Patent No. 5,426,180, U.S. Patent Publication No. 20100137407, International Publication No. 1992001813, International Publication No. 2010084371, and Petkovic et al., Nucleic Acids Res. 43:2454-65 (2015), the contents of which are incorporated herein by reference in their entirety).

[0417] Purification method One or more purification steps may be included in the methods described herein. For example, in some embodiments, the linear polyribonucleotides are substantially concentrated or pure (e.g., purified) before self-splicing. In other embodiments, the linear polyribonucleotides are not purified before self-splicing. In some embodiments, the resulting circular RNA is purified.

[0418] Purification may involve separating or concentrating a desired reaction product from one or more undesirable components, such as any unreacted starting materials, by-products, enzymes, or other reaction components. For example, the purification of post-transcriptional linear polyribonucleotides in a cell-free system (e.g., in vitro transcription) may involve separating or concentrating the linear polyribonucleotides from the DNA template prior to self-splicing. The purification of post-splicing cyclic RNA products may be used to separate or concentrate cyclic polyribonucleotides from their corresponding linear polyribonucleotides. Methods for purifying polyribonucleotides are known to those skilled in the art and include enzymatic or chromatographic purification.

[0419] In some embodiments, the purification method yields cyclic polyribonucleotides having less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%) of linear polyribonucleotides.

[0420] In some embodiments, the amount of linear polyribonucleotide molecules present in the preparation (e.g., a pharmaceutical or agricultural preparation) is specified as 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng The levels present are 1 / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 1 μg / ml, 10 μg / ml, 50 μg / ml, 100 μg / ml, 200 g / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1 mg / ml, 1.5 mg / ml, or 2 mg / ml or less.

[0421] In some embodiments, the reference standard for the amount of cyclic polyribonucleotide molecules present in a preparation (e.g., a pharmaceutical or agricultural preparation) is at least 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), or 92% (w / w) of the total ribonucleotide molecules in the pharmaceutical preparation. These are molecules with 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w), 99.1% (w / w), 99.2% (w / w), 99.3% (w / w), 99.4% (w / w), 99.5% (w / w), 99.6% (w / w), 99.7% (w), 99.8% (w), 99.9% (w / w), or 100% (w / w) content.

[0422] In some embodiments, the reference standard for the amount of linear polyribonucleotide molecules present in the preparation (e.g., a pharmaceutical or agricultural preparation) is 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 40% (w / w), and 50% (w / w) or less of the total ribonucleotide molecules in the preparation.

[0423] In some embodiments, the reference standard for the amount of nicked polyribonucleotide molecules present in the preparation (e.g., a pharmaceutical or agricultural preparation) is 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), or 15% (w / w) or less of nicked polyribonucleotide molecules of the total ribonucleotide molecules in the preparation.

[0424] In some embodiments, the reference standard for the amount of nicked polyribonucleotide molecules and linear polyribonucleotide molecules present in the preparation (e.g., pharmaceutical or agricultural formulation) is the amount of nicked polyribonucleotide molecules and linear polyribonucleotide molecules less than or equal to 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 40% (w / w), and 50% (w / w) of the total ribonucleotide molecules in the preparation. In some embodiments, the preparation (e.g., pharmaceutical or agricultural formulation) is an intermediate preparation of the final cyclic polyribonucleotide drug product. In some embodiments, the preparation (e.g., pharmaceutical or agricultural formulation) is a drug substance or active pharmaceutical ingredient (API). In some embodiments, the formulation (e.g., pharmaceutical or agricultural formulation) is a formulation for administration to a target.

[0425] In some embodiments, preparations of cyclic polyribonucleotides (e.g., pharmaceutical or agricultural formulations) are further processed to substantially remove DNA, protein contamination (e.g., cellular proteins such as host cell proteins or protein process impurities), endotoxins, mononucleotide molecules, and / or process-related impurities (before, during, or after reduction of linear polyribonucleotides).

[0426] Linear polyribonucleotide element Linear polyribonucleotides include elements such as those described below, as described herein.

[0427] The linear polyribonucleotides described herein are polyribonucleotide molecules having 5' and 3' ends. In some embodiments, the linear RNA has a free 5' or 3' end. In some embodiments, the linear RNA has a modified or degradation-protected 5' or 3' end. In some embodiments, the linear RNA has a non-covalently bonded 5' or 3' end. In some embodiments, the linear RNA is mRNA.

[0428] In some embodiments, the linear polyribonucleotides are at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

[0429] The linear polyribonucleotides of this disclosure may include any elements or combinations of elements described herein, for example, any elements or combinations of elements described herein with respect to cyclic polyribonucleotides. A linear polyribonucleotide may include any one or more IRESs, signal sequences, regulatory elements, cleavage domains, translation initiation sequences, untranslated regions, termination elements, or modifications as described herein (for example, with respect to the cyclic polyribonucleotides described herein). A linear polyribonucleotide may include any number or configuration of such elements as described herein (for example, with respect to the cyclic polyribonucleotides described herein).

[0430] How to use The polyribonucleotides described herein may be administered to a subject (e.g., in a pharmaceutical or agricultural composition). For example, the cyclic or linear polyribonucleotides described herein may be administered to a subject (e.g., in a pharmaceutical or agricultural composition). In some embodiments, the cyclic or linear polyribonucleotides described herein are delivered to cells.

[0431] In some embodiments, the polyribonucleotides described herein (e.g., cyclic polyribonucleotides, linear polyribonucleotides) (e.g., in pharmaceutical or agricultural compositions) are used for the treatment, improvement and / or prevention of syndromes, conditions, diseases and / or disorders.

[0432] Method of administration The polyribonucleotides described herein (e.g., cyclic polyribonucleotides, linear polyribonucleotides) (e.g., in pharmaceutical or agricultural compositions) may be administered to cells, tissues, or subjects in single or multiple doses.

[0433] A method for administering multiple doses of a polyribonucleotide composition (e.g., cyclic polyribonucleotide, linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition) described herein comprises providing two or more compositions to cells, tissues or subjects (e.g., mammals) over a period of time. According to certain embodiments, multiple doses of a polyribonucleotide composition (e.g., cyclic polyribonucleotide, linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition) described herein may be administered to a subject over a defined period of time. A method according to this aspect of the present invention comprises the step of administering multiple doses of a polyribonucleotide composition (e.g., cyclic polyribonucleotide, linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition) to a subject. As used herein, “administer sequentially” means that each dose of a polyribonucleotide composition described herein (e.g., cyclic polyribonucleotide, linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition) is administered to a subject at different times, for example, on different days separated by a predetermined interval (e.g., several hours, several days, several weeks, or several months). In some embodiments, the present invention provides a method comprising the steps of sequentially administering a single initial dose of a polyribonucleotide composition described herein (e.g., cyclic polyribonucleotide, linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition) to a subject, followed by the administration of one or more secondary doses of the composition, and optionally, followed by the administration of one or more tertiary doses of the composition.

[0434] The terms “initial dose,” “secondary dose,” and “tertiary dose” refer to the chronological order of administration of the polyribonucleotide compositions described herein (e.g., cyclic polyribonucleotides, linear polyribonucleotides) (e.g., in pharmaceutical or agricultural compositions). Therefore, the “initial dose” is the dose administered at the start of a treatment regimen (also called the “baseline dose”); the “secondary dose” is the dose administered after the initial dose; and the “tertiary dose” is the dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of the polyribonucleotide compositions described herein (e.g., cyclic polyribonucleotides, linear polyribonucleotides) (e.g., in pharmaceutical or agricultural compositions), and in certain embodiments, they may differ from each other in terms of administration frequency. In certain embodiments, the amounts of the polyribonucleotide compositions described herein (e.g., cyclic polyribonucleotides, linear polyribonucleotides) (e.g., in pharmaceutical or agricultural compositions) included in the initial dose, secondary dose, and / or tertiary dose vary from one another during the course of treatment (e.g., adjusted up or down as needed). In certain embodiments, one or more (e.g., 2, 3, 4, or 5) doses are administered as a “loading dose” at the start of the treatment regimen, followed by subsequent doses (e.g., “maintenance doses”) administered on a lower frequency basis.

[0435] In certain embodiments, each secondary and / or tertiary dose is administered after the preceding dose. As used herein, the term “preceding dose” means a dose of the polyribonucleotide composition described herein (e.g., cyclic polyribonucleotide, linear polyribonucleotide) (e.g., in a pharmaceutical or agricultural composition) administered to the subject in a series of multiple doses without an intervening dose, immediately before the next dose in that order. In certain embodiments, each secondary and / or tertiary dose is administered daily, every two days, every three days, every four days, every five days, every six days, or every seven days after the preceding dose. In certain embodiments, each secondary and / or tertiary dose is administered every 0.5 weeks, every week, every two weeks, every three weeks, or every four weeks of the preceding dose.

[0436] Methods according to this aspect of the present invention may involve administering any number of secondary and / or tertiary doses of the polyribonucleotide compositions described herein (e.g., cyclic polyribonucleotides, linear polyribonucleotides) (e.g., in pharmaceutical or agricultural compositions) to a subject. For example, in certain embodiments, only a single secondary dose is administered to the subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the subject. Similarly, in certain embodiments, only a single tertiary dose is administered to the subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the subject.

[0437] In certain embodiments, the frequency with which secondary and / or tertiary doses are administered to the subject may vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment.

[0438] In some embodiments, multiple doses are provided to produce a certain level of composition or to express a certain level of encoded polypeptide in cells, tissues, or subjects. In some embodiments, multiple doses are provided in cells, tissues, or subjects for a period of time, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 21, or 24 months, or at least 1, 2, 3, 4, or 5 years, to produce or maintain a certain level of composition or to produce or maintain a certain level of encoded polypeptide in cells, tissues, or subjects. In some embodiments, the method includes providing (e.g., administering) at least a first and second composition to cells, tissues, or subjects (e.g., mammals, e.g., humans). In some embodiments, the method further includes providing (e.g., administering) a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more compositions. In some embodiments, further compositions are provided over the lifespan of the cells. In some embodiments, additional compositions are provided (e.g., administered) while the cells, tissues, or subjects benefit from the compositions.

[0439] In some embodiments, the first composition in a multi-dose regimen comprises a first amount of the polyribonucleotide disclosed herein (e.g., cyclic polyribonucleotide, linear polyribonucleotide). In some embodiments, the second composition in a multi-dose regimen comprises a second amount of the polyribonucleotide disclosed herein (e.g., cyclic polyribonucleotide, linear polyribonucleotide). In some embodiments, the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more compositions in a multi-dose regimen comprises the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more amounts of the polyribonucleotide disclosed herein (e.g., cyclic polyribonucleotide, linear polyribonucleotide). In some embodiments, a second amount of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) is the same as a first amount of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide). In some embodiments, a third amount of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) is the same as a first amount of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide). In some embodiments, a fourth, fifth, sixth, seventh, eighth, ninth, tenth or more amount of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) is the same as a first amount of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide). In some embodiments, a second amount of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) is less than a first amount of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide). In some embodiments, the third amount of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) is less than the first amount of polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide).In some embodiments, the fourth, fifth, sixth, seventh, eighth, ninth, tenth or more amounts of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) are less than the first amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides). In some embodiments, the second amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) is greater than the first amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides). In some embodiments, the third amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) is greater than the first amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides). In some embodiments, the amount of the fourth, fifth, sixth, seventh, eighth, ninth, tenth or more polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) is greater than the amount of the first polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides). In some embodiments, the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the second composition varies by 1%, 5%, 10%, 15%, 20%, or 25% or less of the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the first composition. In some embodiments, the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the second composition is 1%, 5%, 10%, 15%, 20%, or 25% or less of the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the first composition. In some embodiments, the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the second composition is 0.1 to 1000 times greater than the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the first composition.In some embodiments, the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the second composition is 0.1 times, 1 time, 5 times, 10 times, 100 times, or 1000 times greater than the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the first composition. In some embodiments, the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in a subsequent composition (e.g., a composition administered after the first composition) is 0.1 times, 1 time, 5 times, 10 times, 100 times, or 1000 times greater than the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the first composition. In some embodiments, the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the second composition is 0.1 to 1000 times lower than the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the first composition. In some embodiments, the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the second composition is 0.1, 1, 5, 10, 100, or 1000 times lower than the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the first composition. In some embodiments, the amount of nucleic acid molecules (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in a subsequent composition (e.g., a composition administered after the first composition) is 0.1 times, 1 time, 5 times, 10 times, 100 times, or 1000 times lower than the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the first composition. In some embodiments, the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in a subsequent composition (e.g., after the first composition, a certain amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) is 0.1 to 1000 times the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the first composition.In some embodiments, the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in a subsequent composition (e.g., after a first composition, a certain amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) is 0.1 times, 1 time, 5 times, 10 times, 100 times, or 1000 times or less the amount of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) in the first composition. For example, the first composition contains 1 time the amount of polyribonucleotide molecules (e.g., cyclic polyribonucleotides), the second composition contains 5 times the amount of polyribonucleotides (e.g., cyclic polyribonucleotides) compared to the first composition, and the third composition contains 0.2 times the amount of polyribonucleotides (e.g., cyclic polyribonucleotides) compared to the first composition. In some embodiments, the second composition contains at least five times the amount of polyribonucleotides (e.g., cyclic polyribonucleotides) compared to the amount of polyribonucleotides (e.g., cyclic polyribonucleotides) in the first composition.

[0440] In some embodiments, the first composition contains a larger amount of polyribonucleotides (e.g., cyclic polyribonucleotides) than the second composition. In some embodiments, the first composition contains a larger amount of polyribonucleotides (e.g., cyclic polyribonucleotides) than the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth composition.

[0441] In some embodiments, multiple (e.g., two or more) compositions of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) administered in a multi-dose regimen as described herein are the same composition. In some embodiments, multiple (e.g., two or more) compositions of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) administered in a multi-dose regimen as described herein are different compositions. In some embodiments, the same composition comprises polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides). In some embodiments, different compositions comprise polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) or a combination thereof.

[0442] Compositions of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) disclosed herein can induce a response in a subject.

[0443] In some embodiments, a multi-dose regimen involves administering polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) provided herein to a subject requiring them in multiple doses (multiple administrations), for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 100, 150, 200, or 500 times over a period of 1 to 56 days, for example, about 49, 42, 35, 28, 21, 14, or 7 days. In some embodiments, a multi-dose regimen involves administering polyribonucleotides to a subject requiring them in at least 3 doses, at intervals of about 7 days. In some embodiments, in subjects receiving multiple doses of the polyribonucleotides provided herein (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses), the level of the encoded polypeptide is maintained at a level with variation of less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% over a period longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, or 20 weeks after the last dose. In some embodiments, in subjects receiving multiple doses of the polyribonucleotide provided herein (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses), the level of the encoded polypeptide is maintained at a first level for a period longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, or 20 weeks after the second, third, fourth, fifth, sixth, seventh, eighth, or last dose, where the first level is higher than the level of the encoded polypeptide measured immediately after the first dose (e.g., measured approximately 12, 24, 36, or 48 hours after the first dose).In some embodiments, in subjects receiving multiple doses of the polyribonucleotide provided herein (e.g., at least three doses) at intervals of about 7 days, the level of the encoded polypeptide is maintained at a first level for a period longer than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks after the second, third, fourth, fifth, sixth, seventh, eighth, or final dose, where the first level is higher than the level of the encoded polypeptide measured immediately after the first dose (e.g., measured about 12, 24, 36, or 48 hours after the first dose).

[0444] Delivery method The cyclic polyribonucleotides described herein may be included in compositions (e.g., pharmaceutical compositions or agricultural compositions) that include or do not include a carrier. The linear polyribonucleotides described herein may be included in compositions (e.g., pharmaceutical compositions or agricultural compositions) that include or do not include a carrier.

[0445] The pharmaceutical compositions described herein may be formulated to include, for example, a carrier, such as a pharmaceutical carrier and / or a polymeric carrier, such as a liposome, and may be delivered by known methods to an object requiring it (e.g., human or non-human agricultural or livestock animals, such as cattle, dogs, cats, horses, poultry). Such methods include, but are not limited to, transfection (e.g., lipid-mediated cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection); viral delivery (e.g., lentiviruses, retroviruses, adenoviruses, AAVs); microinjection; microprojectile impulsions ("gene guns"); fugene; direct sonic loading; cell squeezing; phototransfection; protoplast fusion; impulfection; magnetofection; exosome-mediated transfer; lipid nanoparticle-mediated transfer; and any combination thereof. Delivery methods include, for example, Gori et al., Delivery and Specificity of CRISPR / Cas9 Genome Editing Technologies for Human Gene Therapy. Human Gene Therapy. July 2015, 26(7):443-451. doi:10.1089 / hum.2015.074; and Zuris et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2014 Oct 30;33(1):73-80.

[0446] Pharmaceutical compositions can be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Examples of suitable aqueous and non-aqueous compositions that can be used in pharmaceutical compositions include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0447] Sterile injection solutions can be prepared by incorporating the required amount of the active compound (e.g., polyribonucleotides as described herein (e.g., cyclic polyribonucleotides or linear polyribonucleotides)) into a suitable solvent containing, for example, one or a combination of components as described herein, as necessary, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components, for example, those described herein. In the case of sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying (lyophilization), which yield powders of the active component and any further desired components from the pre-sterile filtered solution.

[0448] The polyribonucleotides of this disclosure (e.g., cyclic polyribonucleotides, linear polyribonucleotides) can be prepared in controlled-release formulations, including compositions that protect them from rapid release, such as implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are generally known to those skilled in the art. The compositions of this disclosure may be, for example, immediate-release forms or controlled-relea...

Claims

1. In the following order from 5' to 3', (a) The first cyclonized element; (b) A first spacer element having a length of at least 100 ribonucleotides; (c) Polyribonucleotide cargo; (d) Second spacer element; and (e) The second cyclonized element; A cyclic polyribonucleotide comprising, A cyclic polyribonucleotide in which the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

2. In the following order from 5' to 3', (a) The first cyclonized element; (b) First spacer element; (c) Polyribonucleotide cargo; (d) A second spacer element having a length of at least 100 ribonucleotides; and (e) The second cyclonized element; A cyclic polyribonucleotide comprising, A cyclic polyribonucleotide in which the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

3. The cyclic polyribonucleotide according to claim 1 or 2, wherein the first spacer element has a ribonucleotide length of 100 to 500.

4. The cyclic polyribonucleotide according to claim 3, wherein the second spacer element has a ribonucleotide length of 100 to 500.

5. The cyclic polyribonucleotide according to claim 4, wherein the first spacer element and the second spacer element each contain 110 to 500 ribonucleotides.

6. The cyclic polyribonucleotide according to claim 5, wherein the first spacer element and the second spacer element each contain 120 to 500 ribonucleotides.

7. The cyclic polyribonucleotide according to claim 6, wherein the first spacer element and the second spacer element each have a length of 150 to 500 ribonucleotides.

8. The cyclic polyribonucleotide according to claim 7, wherein the first spacer element and the second spacer element each have a length of 200 to 500 ribonucleotides.

9. The cyclic polyribonucleotide according to claim 4, wherein the first spacer element and the second spacer element each have a length of 100 to 300 ribonucleotides.

10. The cyclic polyribonucleotide according to claim 9, wherein the first spacer element and the second spacer element each have a length of 100 to 200 ribonucleotides.

11. The cyclic polyribonucleotide according to claim 9, wherein the first spacer element and the second spacer element each have a length of 110 to 300 ribonucleotides.

12. The cyclic polyribonucleotide according to claim 11, wherein the first spacer element and the second spacer element each have a length of 120 to 300 ribonucleotides.

13. The cyclic polyribonucleotide according to claim 12, wherein the first spacer element and the second spacer element each have a length of 150 to 300 ribonucleotides.

14. The cyclic polyribonucleotide according to any one of claims 1 to 13, wherein the difference between the length of the first spacer element and the length of the second spacer element is 0 to 500 nucleotides.

15. The cyclic polyribonucleotide according to claim 14, wherein the difference between the length of the first spacer element and the length of the second spacer element is 0 to 100 nucleotides.

16. The cyclic polyribonucleotide according to claim 15, wherein the difference between the length of the first spacer element and the length of the second spacer element is 0 to 50 nucleotides.

17. The first spacer element or the second spacer element (i) Poly-A regions containing 80% to 100% adenosine residues; (ii) Poly-AC regions containing 80% to 100% adenosine or cytosine residues; (iii) A poly-AU region containing 80% to 100% adenosine or uridine residues; or (iii) Poly-AG region containing 80% to 100% adenosine or guanosine residues A cyclic polyribonucleotide according to any one of claims 1 to 16, comprising:

18. The cyclic polyribonucleotide according to any one of claims 1 to 17, wherein the cyclic polyribonucleotide exhibits at least twice the stability of a polyribonucleotide that does not contain the first spacer element or the second spacer element.

19. The cyclic polyribonucleotide according to any one of claims 1 to 18, wherein the cyclic polyribonucleotide can be detected at least twice as long after the cyclic polyribonucleotide has been administered to a subject, compared to a polyribonucleotide that does not contain the first spacer element or the second spacer element.

20. The cyclic polyribonucleotide according to any one of claims 1 to 19, wherein the polynucleotide cargo encodes a polypeptide, and the polypeptide has at least four times greater expression compared to a polyribonucleotide that does not contain the first spacer element or the second spacer element.

21. The cyclic polyribonucleotide according to any one of claims 1 to 20, wherein the polyribonucleotide cargo comprises an expression sequence.

22. The cyclic polyribonucleotide according to claim 21, wherein the polyribonucleotide cargo comprises an IRES operably linked to an expression sequence.

23. The cyclic polyribonucleotide according to claim 21 or 22, wherein the expression sequence further comprises a 3' untranslated region or a 5' untranslated region.

24. The cyclic polyribonucleotide according to any one of claims 21 to 23, wherein the expression sequence encodes a polypeptide.

25. The cyclic polyribonucleotide according to any one of claims 1 to 24, wherein the polyribonucleotide comprises 500 to 20,000 ribonucleotides.

26. The cyclic polyribonucleotide according to claim 25, wherein the polyribonucleotide comprises 2,000 to 20,000 ribonucleotides.

27. The cyclic polyribonucleotide according to any one of claims 1 to 26, wherein the cyclization junction includes a splice junction.

28. The cyclic polyribonucleotide according to claim 27, wherein the first post-cyclization element comprises a first exon fragment, the second post-cyclization element comprises a second exon fragment, and the first exon fragment and the second exon fragment are linked by the splice junction.

29. The cyclic polyribonucleotide according to any one of claims 1 to 28, wherein the cyclization junction includes an oligonucleotide sprint that hybridizes to the first post-cyclization element and the second post-cyclization element.

30. The cyclic polyribonucleotide according to claim 29, wherein the oligonucleotide sprint is a DNA sprint or an RNA sprint.

31. The cyclic polyribonucleotide according to claim 29 or 30, wherein the first post-cyclization element includes a region that can hybridize to a first region of the oligonucleotide sprint, and the second post-cyclization element includes a region that can hybridize to a second region of the oligonucleotide sprint.

32. In the following order from 5' to 3', (a) The first cyclonized element; (b) A first spacer element having a length of at least 100 ribonucleotides; (c) Polyribonucleotide cargo; (d) Second spacer element; and (e) The second cyclonized element; A linear polyribonucleotide comprising, A linear polyribonucleotide in which the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

33. In the following order from 5' to 3', (a) The first cyclonized element; (b) First spacer element; (c) Polyribonucleotide cargo; (d) A second spacer element having a length of at least 100 ribonucleotides; and (e) The second cyclonized element; A linear polyribonucleotide comprising, A linear polyribonucleotide in which the first post-cyclization element and the second post-cyclization element together form a cyclization junction.

34. The linear polyribonucleotide according to claim 32 or 33, wherein the first spacer element has a ribonucleotide length of 100 to 500.

35. The linear polyribonucleotide according to claim 34, wherein the second spacer element has a ribonucleotide length of 100 to 500.

36. The linear polyribonucleotide according to claim 35, wherein the first spacer element and the second spacer element each contain 110 to 500 ribonucleotides.

37. The linear polyribonucleotide according to claim 36, wherein the first spacer element and the second spacer element each contain 120 to 500 ribonucleotides.

38. The linear polyribonucleotide according to claim 37, wherein the first spacer element and the second spacer element each have a length of 150 to 500 ribonucleotides.

39. The linear polyribonucleotide according to claim 38, wherein the first spacer element and the second spacer element each have a length of 200 to 500 ribonucleotides.

40. The linear polyribonucleotide according to claim 35, wherein the first spacer element and the second spacer element each have a length of 100 to 300 ribonucleotides.

41. The linear polyribonucleotide according to claim 40, wherein the first spacer element and the second spacer element each have a length of 100 to 200 ribonucleotides.

42. The linear polyribonucleotide according to claim 40, wherein the first spacer element and the second spacer element each have a length of 110 to 300 ribonucleotides.

43. The linear polyribonucleotide according to claim 42, wherein the first spacer element and the second spacer element each have a length of 120 to 300 ribonucleotides.

44. The linear polyribonucleotide according to claim 43, wherein the first spacer element and the second spacer element each have a length of 150 to 300 ribonucleotides.

45. The linear polyribonucleotide according to any one of claims 32 to 44, wherein the difference between the length of the first spacer element and the length of the second spacer element is 0 to 500 nucleotides.

46. The linear polyribonucleotide according to claim 45, wherein the difference between the length of the first spacer element and the length of the second spacer element is 0 to 100 nucleotides.

47. The linear polyribonucleotide according to claim 46, wherein the difference between the length of the first spacer element and the length of the second spacer element is 0 to 50 nucleotides.

48. The first spacer element or the second spacer element (i) Poly-A regions containing 80% to 100% adenosine residues; (ii) Poly-AC regions containing 80% to 100% adenosine or cytosine residues; (iii) A poly-AU region containing 80% to 100% adenosine or uridine residues; or (iv) Poly-AG region containing 80% to 100% adenosine or guanosine residues A linear polyribonucleotide according to any one of claims 32 to 47, comprising the above.

49. The linear polyribonucleotide according to any one of claims 32 to 48, wherein the polyribonucleotide exhibits at least twice the stability of a polyribonucleotide that does not contain the first spacer element or the second spacer element.

50. The linear polyribonucleotide according to any one of claims 32 to 49, wherein the polyribonucleotide can be detected at least twice as long after the polyribonucleotide has been administered to a subject compared to a polyribonucleotide that does not contain the first spacer element or the second spacer element.

51. The linear polyribonucleotide according to any one of claims 32 to 50, wherein the polynucleotide cargo encodes a polypeptide, and the polypeptide has at least four times greater expression compared to a polyribonucleotide that does not contain the first spacer element or the second spacer element.

52. The linear polyribonucleotide according to any one of claims 32 to 51, wherein the polyribonucleotide cargo includes an expression sequence.

53. The linear polyribonucleotide according to claim 52, wherein the polyribonucleotide cargo comprises an IRES operably linked to an expression sequence.

54. The linear polyribonucleotide according to claim 52 or 53, wherein the expression sequence further comprises a 3' untranslated region or a 5' untranslated region.

55. The linear polyribonucleotide according to any one of claims 52 to 54, wherein the expression sequence encodes a polypeptide.

56. The linear polyribonucleotide according to any one of claims 32 to 55, wherein the polyribonucleotide comprises 500 to 20,000 ribonucleotides.

57. The linear polyribonucleotide according to claim 56, wherein the polyribonucleotide comprises 2,000 to 20,000 ribonucleotides.

58. The linear polyribonucleotide according to any one of claims 32 to 57, wherein the cyclization junction includes a splice junction.

59. The linear polyribonucleotide according to claim 58, wherein the first post-cyclization element comprises a first exon fragment, the second post-cyclization element comprises a second exon fragment, and the first exon fragment and the second exon fragment are linked by the splice junction.

60. The linear polyribonucleotide according to any one of claims 32 to 59, wherein the cyclization junction includes an oligonucleotide sprint that hybridizes to the first post-cyclization element and the second post-cyclization element.

61. The linear polyribonucleotide according to claim 60, wherein the oligonucleotide sprint is a DNA sprint or an RNA sprint.

62. The linear polyribonucleotide according to claim 60 or 61, wherein the first post-cyclization element includes a region that can hybridize to a first region of the oligonucleotide sprint, and the second post-cyclization element includes a region that can hybridize to a second region of the oligonucleotide sprint.

63. The first cyclization element comprises a first catalytic intron fragment, a first splice site dinucleotide, and a first exon fragment. The second cyclization element comprises a second catalytic intron fragment, a second splice site dinucleotide, and a second exon fragment. A linear polyribonucleotide according to any one of claims 32 to 62.

64. The linear polyribonucleotide according to claim 63, wherein the first catalytic intron fragment and the second catalytic intron fragment can self-splice together, thereby covalently bonding the first exon region and the second exon region to produce a cyclic polyribonucleotide.

65. The linear polyribonucleotide according to claim 63 or 64, wherein the first catalytic intron fragment and the second catalytic intron fragment are derived from the cyanobacterium Anabaena pre-tRNA-Leu gene or Tetrahymena pre-rRNA.

66. The linear polyribonucleotide according to any one of claims 32 to 65, wherein the first cyclization element includes a region that can hybridize to a first region of the oligonucleotide sprint, and the second cyclization element includes a region that can hybridize to a second region of the oligonucleotide sprint.

67. A DNA vector comprising an RNA polymerase promoter operably ligated to a sequence encoding a linear polyribonucleotide according to any one of claims 32 to 66.

68. A cyclic polyribonucleotide produced from a linear polyribonucleotide according to any one of claims 32 to 66 or from a DNA vector according to claim 67.

69. A pharmaceutical composition comprising a linear polyribonucleotide, a cyclic polyribonucleotide, or a DNA vector according to any one of claims 1 to 66, and a pharmaceutically acceptable excipient.

70. A method for expressing a polypeptide in cells or a subject, comprising providing the cells or the subject with a linear polyribonucleotide, a cyclic polyribonucleotide, a DNA vector, or a pharmaceutical composition according to any one of claims 1 to 69.

71. A method for producing a cyclic polyribonucleotide from a linear polyribonucleotide according to any one of claims 32 to 66, comprising providing the linear polyribonucleotide under conditions suitable for self-splicing of the linear polyribonucleotide to produce the cyclic polyribonucleotide.

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