Synthetic internal ribosome entry sites and uses thereof

Abstract

The disclosure provides a polynucleotide construct (DNA or RNA) that includes a 5' IRES sequence and a 3' IRES sequence.

Description

Attorney Docket No. : 2019398-0005SYNTHETIC INTERNAL RIBOSOME ENTRY SITES AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application No.63 / 761,738, filed February 21, 2025, the entire contents of which are hereby incorporated by reference herein.TECHNICAL FIELD

[0002] The following generally relates to polynucleotide constructs and more particularly to circular RNA and constructs for generating circular RNA.BACKGROUND

[0003] circRNAs are closed single- stranded RNA that lack a 5' cap and poly- A tail. Tn nature, circular RNAs (circRNAs) have a wide range of origins and tissue specificity and play a variety of roles in the development and homeostasis of organisms, in aging, and have also been linked to the occurrence and development of various human diseases (e.g., cardiovascular diseases, diabetes mellitus, and cancer).SUMMARY

[0004] The present disclosure provides, among other things, polynucleotide constructs and circular RNAs (circRNA). The present disclosure recognizes that circRNA presents certain advantages as therapeutics. In particular, the present disclosure provides the recognition that circRNAs can be particularly useful for as therapeutic agents. circRNA has certain characteristics that can be beneficial for use in cells, tissues, and subjects. For example, circRNA can be more resistant to exonuclease degradation compares to linear RNA molecules. Nonetheless, methods for producing circRNAs suitable for delivery to cells, tissues, and subjects, which could leverage these benefits, has remained a challenge.

[0005] Prior research has shown that circRNA can be manufactured through the use of self-splicing introns. See, e.g., Wesselhoeft, Kowalski, & Anderson, Nat Comm, 2018, which is incorporated herein by reference in its entirety. However, there are disadvantages to using self-Page 1 of 14413314218vlAttorney Docket No. : 2019398-0005splicing introns for manufacturing. Use of therapeutic circRNAs has been limited in part, due to difficulty in purifying circRNAs, low fidelity and reproducibility of a circularization reaction, and / or need for sequence elements that can be used in various therapeutic circRNAs in a modular fashion. Current polynucleotide constructs that generate therapeutic circRNAs may include synthetic spacer sequences, which may increase immunogenicity of therapeutic circRNAs.

[0006] The present disclosure provides polynucleotide constructs and methods of circularizing the same to generate circRNAs that address the limitations of prior methods. The present disclosure also provides circRNAs that have improved characteristics and / or that address the limitations of circRNAs generated using prior methods. For example, polynucleotide constructs provided herein utilize a permuted IRES sequence which improves a circularization reaction and / or generates a scarless circRNA.

[0007] The present disclosure provides, among other things, polynucleotide constructs that can generate circRNAs with improved immunogenicity compared to circRNAs produced using prior methods. In some embodiments, the present disclosure provides polynucleotide constructs that can generate circRNAs without increased immunogenicity (e.g., immunogenicity of the circRNA itself and not the immunogenicity of, e.g., a payload). In some embodiments, the present disclosure provides polynucleotide constructs that produce therapeutic circRNAs, wherein the therapeutic circRNAs do not have increased immunogenicity (e.g., immunogenicity of the circRNA itself and not the immunogenicity of, e.g., a payload).

[0008] In some embodiments, the present disclosure provides polynucleotide constructs comprising a permuted IRES sequence that can produce a circRNA that does not comprise an excision scar. In some embodiments, the present disclosure provides a scarless circRNA. In some embodiments, a scarless circRNA produced by a polynucleotide construct of the present disclosure does not have increased immunogenicity (e.g., immunogenicity of the circRNA itself and not the immunogenicity of, e.g., a payload). In some embodiments, a circRNA described herein does not have increased immunogenicity (e.g., immunogenicity of the circRNA itself and not the immunogenicity of, e.g., a payload). In some embodiments, a circRNA described herein can be used as a therapeutic circRNA.

[0009] The present disclosure provides the insight that a polynucleotide construct comprising a permuted IRES sequence described herein can produce a scarless circRNA. ThePage 2 of 144133142i8vlAttorney Docket No. : 2019398-0005present disclosure also provides, a circRNA with an improved immunogenicity profde compared to the same circRNA comprising an excision scar.

[0010] Among other things, the present disclosure provides a polynucleotide construct comprising a permuted internal ribosomal entry signal (IRES) sequence. In some embodiments, a polynucleotide construct described herein comprises DNA, RNA, or both. In some embodiments, a polynucleotide construct described herein is a DNA nucleic acid molecule or a RNA nucleic acid molecule. In some embodiments, a polynucleotide construct is a DNA nucleic acid molecule. In some embodiments, a DNA polynucleotide construct comprises a promoter sequence. In some embodiments, a promoter sequence comprises a bacteriophage promoter sequence, a viral promoter sequence, a bacterial promoter sequence, an eukaryotic promoter sequence or an engineered promoter sequence. In some embodiments, a bacteriophage promoter sequence comprises a T7 promoter sequence or a functional variant or a functional fragment thereof, a T6 promoter sequence or a functional variant or a functional fragment thereof, a SP6 promoter sequence or a functional variant or a functional fragment thereof, a T3 promoter sequence or a functional variant or a functional fragment thereof, or a T4 promoter sequence or a functional variant or a functional fragment thereof. In some embodiments, a DNA polynucleotide construct is transcribed to a RNA nucleic acid molecule. In some embodiments, a polynucleotide construct is a RNA nucleic acid molecule.

[0011] The present disclosure provides, among other things, a polynucleotide construct comprising a first intronic sequence, a first portion of an IRES sequence, a coding sequence, a second portion of an IRES sequence, a second intronic sequence, a first internal guide sequence (IGS) and / or a second internal guide sequence (IGS'). Among other things, the present disclosure provides a polynucleotide construct comprising a first intronic sequence, a first portion of an IRES sequence, a coding sequence, a second portion of an IRES sequence, and a second intronic sequence.

[0012] In some embodiments, a polynucleotide construct comprises an IGS and an IGS'.

[0013] In some embodiments, an IGS' is capable of hybridizing to an IGS. In some embodiments, an IGS' hybridizes to an IGS. In some embodiments, an IGS' is capable of base pairing to an IGS. In some embodiments, an IGS' base pairs to an IGS. In some embodiments, an IGS’ is a reverse complement of an IGS. In some embodiments, an IGS' is capable of base pairing with at least 50% of an IGS. In some embodiments, an IGS' is capable of base pairing with at leastPage 3 of 14413314218vlAttorney Docket No. : 2019398-000560% of an IGS. Tn some embodiments, an IGS' is capable of base pairing with at least 3 nucleotides of an IGS. In some embodiments, an IGS' base pairs with at least 50% of an IGS. In some embodiments, an IGS' base pairs with at least 60% of an IGS. In some embodiments, an IGS' base pairs with at least 3 nucleotides of an IGS. In some embodiments, an IGS' comprises a nucleotide sequence that is complimentary to a nucleotide sequence comprised in an IGS. In some embodiments, a nucleotide sequence that is the reverse complement of an IGS' has at least 50% sequence identity to the nucleotide sequence of an IGS. In some embodiments, a nucleotide sequence that is the reverse complement of an IGS' has at least 60% sequence identity to the nucleotide sequence of an IGS. In some embodiments, an IGS comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 1-4. In some embodiments, an IGS comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 1. In some embodiments, an IGS comprises a nucleotide sequence of SEQ ID NO: 1. In some embodiments, an IGS comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 2. In some embodiments, an IGS comprises a nucleotide sequence of SEQ ID NO: 2. In some embodiments, an IGS comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 3. In some embodiments, an IGS comprises a nucleotide sequence of SEQ ID NO: 3. In some embodiments, an IGS comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 4. In some embodiments, an IGS comprises a nucleotide sequence of SEQ ID NO: 4. In some embodiments, an IGS’ comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 5-8. In some embodiments, an IGS’ comprises a sequence having at least 80%, at least 85%, at least 90%, atPage 4 of 14413314218vlAttorney Docket No. : 2019398-0005least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 5. In some embodiments, an IGS’ comprises a nucleotide sequence of SEQ ID NO: 5. In some embodiments, an IGS’ comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 6. In some embodiments, an IGS’ comprises a nucleotide sequence of SEQ ID NO: 6. In some embodiments, an IGS’ comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 7. In some embodiments, an IGS’ comprises a nucleotide sequence of SEQ ID NO: 7. In some embodiments, an IGS’ comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 8. In some embodiments, an IGS’ comprises a nucleotide sequence of SEQ ID NO: 8. In some embodiments, an IGS’ comprises single-stranded RNA. In some embodiments, an IGS comprises single-stranded RNA.

[0014] In some embodiments, an IRES sequence disclosed herein is a synthetic IRES sequence comprising an IGS' that is complementary to an IGS sequence. In some embodiments, an IRES sequence is split to form a first portion of the IRES sequence and a second portion of the IRES sequence. In some embodiments, a first portion of an IRES sequence and the second portion of an IRES sequence, together, form an IRES sequence. In some embodiments, an IRES sequence formed is a functional IRES sequence.

[0015] In some embodiments, an IRES sequence comprises a naturally occurring IRES sequence. In some embodiments, an IRES sequence comprises a synthetic IRES sequence. In some embodiments, a synthetic IRES sequence is derived from a naturally occurring IRES sequence. In some embodiments, a naturally occurring IRES sequence comprises a cellular IRES sequence or a viral IRES sequence. In some embodiments, an IRES sequence comprises a Type I IRES sequence, a Type II IRES sequence, a Type III IRES sequence, a Type IV IRES sequence, or a Type V IRES sequence. In some embodiments, a Type I IRES sequence comprises an Enterovirus IRES sequence or a Rhinovirus IRES sequence. In some embodiments, an Enterovirus IRES sequence comprises a Poliovirus (PV) IRES sequence, a Coxsackievirus B3 (CVB3) IRESPage 5 of 14413314218vlAttorney Docket No. : 2019398-0005sequence, or an Enterovirus 71 (EV71) IRES sequence. In some embodiments, an IRES sequence comprises a Coxsackievirus B3 (CVB3) IRES sequence. In some embodiments, a Type II IRES sequence comprises a Cardiovirus (EMCV) IRES sequence or a Aphthovirus (FMDV) IRES sequence.

[0016] In some embodiments, an IRES sequence comprises seven domains (domains I -VII), and wherein domain IV contains a proximal loop. In some embodiments, an IRES sequence is split within domain IV to form a first portion of the IRES sequence and a second portion of the IRES sequence. In some embodiments, a first portion of an IRES sequence comprises domains I -III and a 5' portion of domain IV of an IRES sequence. In some embodiments, a second portion of an IRES sequence comprises a 3' portion of domain IV and domains V - VII of an IRES sequence. In some embodiments, a first portion of an IRES sequence comprises domains I - III and a 5' portion of domain IV of an IRES sequence and a second portion of an IRES sequence comprises a 3' portion of domain IV and domains V - VII of an IRES sequence.

[0017] In some embodiments, an IRES sequence comprises one or more modifications to increase or reduce IRES activity. In some embodiments, one or more modifications comprise adding one or more accessory sequences to an IRES sequence, truncating the 5’ and / or 3’ ends of an IRES sequence, adding a spacer 5’ to an IRES sequence, modifying a Kozak sequence, modifying alternative translation initiation sites, creating a chimeric IRES sequence, creating a hybrid IRES sequence, or any combination thereof.

[0018] In some embodiments, an IRES sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 9-11. In some embodiments, an IRES sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 9. In some embodiments, an IRES sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 10. In some embodiments, an IRES sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 11. In some embodiments, a first portion of an IRESPage 6 of 14413314218vlAttorney Docket No. : 2019398-0005sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 12. In some embodiments, a second portion of an IRES sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 13.

[0019] In some embodiments, the present disclosure provides a polynucleotide construct comprising a first intronic sequence and / or a second intronic sequence.

[0020] In some embodiments, a polynucleotide construct described herein comprises a first intronic sequence. In some embodiments, a first intronic sequence comprises a naturally occurring intronic sequence. In some embodiments, a first intronic sequence is derived from a naturally occurring intronic sequence. In some embodiments, a first intronic sequence comprises a bacterial intronic sequence, a viral intronic sequence, or a eukaryotic intronic sequence. In some embodiments, an eukaryotic intronic sequence is a fungi intronic sequence, a plant intronic sequence, or an algae intronic sequence. In some embodiments, a first intronic sequence comprises a group I intronic sequence or a group II intronic sequence. In some embodiments, a first intronic sequence comprises an intronic sequence from a bacteriophage T4 genome, an Anabaena azollae genome, an Anabaena PCC7120 genome, an Aphanizomenon flos-aquae (e.g., strain NIVA-C YA 142) genome, a Synechococcus genome, an Azoarcus genome, a Tetrahymena thermophila genome, a trimorphomyces genome, or a Trimorphomyces papilionaceus genome, or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from a T4 phage genome, or a pre-tRNALEUfrom Anabaena azollae, Anabaena PCC7120, Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142), or Synechococcus, or a pre-tRNAILEfrom Azoarcus, or a 26S ribosomal RNA from Tetrahymena thermophila, or a cytochrome C oxidase subunit 2 (C0X2) from Trimorphomyces papilionaceus, or any combination thereof. In some embodiments, a first intronic sequence comprises an intronic sequence derived from a naturally occurring intronic sequence.

[0021] In some embodiments, a first intronic sequence comprises an IGS and / or a catalytic core. In some embodiments, a first intronic sequence comprises an IGS and a catalytic core. In some embodiments, a catalytic core is capable of mediating a self-splicing reaction. In some embodiments, a catalytic core mediates a self-splicing reaction. In some embodiments, a catalyticPage 7 of 14413314218vlAttorney Docket No. : 2019398-0005core comprises a nucleotide sequence of GAG and CC. In some embodiments, a catalytic core comprises a nucleotide sequence of GAG and CC separated by [N]n, wherein N is any one of A, C, G, T, or U, and n is an integer. In some embodiments, a catalytic core comprises a nucleotide sequence of ACG and CT(or U). In some embodiments, a catalytic core comprises a nucleotide sequence of ACG and CT(or U) separated by [N]n, wherein N is any one of A, C, G, T, or U, and n is an integer. In some embodiments, a catalytic core comprises a nucleotide sequence of any one of SEQ ID NOs: 33, 34, 50, or 51. In some embodiments, a catalytic core comprises a nucleotide sequence of AGAGA[N]6-4iA[N]43-4sGUCC (SEQ ID NO: 33), wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]6-4i, or [N]4 -45, can be independently any one of A, U, C, or G. In some embodiments, a catalytic core comprises a nucleotide sequence of AGAGA[N]6-4OA[N]42-44GUCC (SEQ ID NO: 34), wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]e-40, or [N]42-44, can be independently any one of A, U, C, or G.

[0022] In some embodiments, a polynucleotide construct described herein comprises a second intronic sequence. In some embodiments, a second intronic sequence comprises a naturally occurring intronic sequence. In some embodiments, a second intronic sequence is derived from a naturally occurring intronic sequence. In some embodiments, a second intronic sequence comprises an intronic sequence derived from a naturally occurring intronic sequence. In some embodiments, a second intronic sequence comprises a bacterial intronic sequence, a viral intronic sequence, or a eukaryotic intronic sequence. In some embodiments, an eukaryotic intronic sequence is a fungi intronic sequence, a plant intronic sequence, or an algae intronic sequence. In some embodiments, a second intronic sequence comprises a group I intronic sequence or a group II intronic sequence. In some embodiments, a second intronic sequence comprises an intronic sequence from a bacteriophage T4 genome, an Anabaena azollae genome, an Anabaena PCC7120 genome, an Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142) genome, a Synechococcus genome, an Azoarcus genome, a Tetrahymena thermophila genome, a trimorphomyces genome, or a Trimorphomyces papilionaceus genome, or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from a T4 phage genome, or a pre-tRNALEUfrom Anabaena azollae, Anabaena PCC7120, Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142), or Synechococcus, or a pre-tRNAILEfrom Azoarcus, or a 26S ribosomal RNA from Tetrahymena thermophila, or a cytochrome C oxidase subunit 2 (COY2)Page 8 of 14413314218vlAttorney Docket No. : 2019398-0005from Trimorphomyces papihonaceus. or any combination thereof In some embodiments, a second intronic sequence comprises an intronic sequence derived from a naturally occurring intronic sequence.

[0023] In some embodiments, a second intronic sequence comprises an IGS and / or a catalytic core. In some embodiments, a second intronic sequence comprises an IGS and a catalytic core. In some embodiments, a catalytic core is capable of mediating a self-splicing reaction. In some embodiments, a catalytic core mediates a self-splicing reaction. In some embodiments, a catalytic core comprises a nucleotide sequence of GAG and CC. In some embodiments, a catalytic core comprises a nucleotide sequence of GAG and CC separated by [N]n, wherein N is any one of A, C, G, T, or U, and n is an integer. In some embodiments, a catalytic core comprises a nucleotide sequence of ACG and CT(or U). In some embodiments, a catalytic core comprises a nucleotide sequence of ACG and CT(or U) separated by [N]n, wherein N is any one of A, C, G, T, or U, and n is an integer. In some embodiments, a catalytic core comprises a nucleotide sequence of any one of SEQ ID NOs: 33, 34, 50, or 51. In some embodiments, a catalytic core comprises a nucleotide sequence of AGAGA[N]6-4iA[N]43-4sGUCC (SEQ ID NO: 33), wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]6-4i, or [N]43-45, can be independently any one of A, U, C, or G. In some embodiments, a catalytic core comprises a nucleotide sequence of AGAGA[N]e-4oA[N]42-44GUCC (SEQ ID NO: 34), wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]e-4o, or [N]42-44, can be independently any one of A, U, C, or G.

[0024] In some embodiments, a first intronic sequence or a second intronic sequence comprise an IGS. In some embodiments, a first intronic sequence or a second intronic sequence comprise a catalytic core. In some embodiments, a first intronic sequence or a second intronic sequence comprise an IGS and a catalytic core. In some embodiments, a first intronic sequence comprises an IGS and a catalytic core. In some embodiments, a second intronic sequence comprises an IGS and a catalytic core. In some embodiments, a first intronic sequence comprises an IGS and a second intronic sequence comprises a catalytic core. In some embodiments, a first intronic sequence comprises a catalytic core and a second intronic sequence comprises an IGS.

[0025] In some embodiments, a polynucleotide construct comprises a first intronic sequence and a second intronic sequence. In some embodiments, a first intronic sequence and / or a second intronic sequence comprise a bacterial intronic sequence, a viral intronic sequence, or aPage 9 of 144133142i8vlAttorney Docket No. : 2019398-0005eukaryotic intronic sequence. In some embodiments, a first intronic sequence and / or a second intronic sequence comprise a group I intronic sequence or a group II intronic sequence. In some embodiments, a first intronic sequence and / or a second intronic sequence comprise an intronic sequence from a bacteriophage T4 genome, an Anabaena azollae genome, an Anabaena PCC7120 genome, an Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142) genome, a Synechococcus genome, an Azoarcus genome, a Tetrahymena thermophila genome, a trimorphomyces genome, or a Trimorphomyces papilionaceus genome, or an RNA transcribed therefrom. In some embodiments, a first intronic sequence and / or a second intronic sequence comprise an intronic sequence from a T4 phage genome, or a pre-tRNALEU from Anabaena azollae, Anabaena PCC7120, Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142), or Synechococcus, or a pre-tRNAILE from Azoarcus, or a 26S ribosomal RNA from Tetrahymena thermophila, or a cytochrome C oxidase subunit 2 (COX2) from Trimorphomyces papilionaceus, or any combination thereof.

[0026] In some embodiments, a first intronic sequence and / or a second intronic sequence comprise a nucleotide sequence derived from a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 35-42. In some embodiments, a first intronic sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 35-38. In some embodiments, a first intronic sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 29. In some embodiments, a second intronic sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 39-42. In some embodiments, a second intronic sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at leastPage 10 of 144133142i8vlAttorney Docket No. : 2019398-000595%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a nucleotide sequence of SEQ ID NO: 32.

[0027] In some embodiments, a polynucleotide construct described herein comprises a first intronic sequence that is directly j oined to a first portion of an IRES sequence at a first junction, and a second portion of an IRES sequence that is directly joined to a second intronic sequence at a second junction.

[0028] In some embodiments, a polynucleotide construct described herein comprises at least one splice site. In some embodiments, a polynucleotide construct comprises a first splice site. In some embodiments, a first splice site comprises a first junction. In some embodiments, a polynucleotide construct comprises a second splice site. In some embodiments, a second splice site comprises a second junction. In some embodiments, a polynucleotide construct comprises a first splice site and a second splice site. In some embodiments, a first splice site sequence comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with a nucleotide sequence of SEQ ID NO: 17 or 19. In some embodiments, a second splice site sequence comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with a nucleotide sequence of SEQ ID NO: 18 or 20. In some embodiments, a second splice site sequence comprises a nucleotide sequence of NAGUAAAA, wherein N is A, U, C, or G, and wherein “A” indicates the position of the phosphodiester bond that is cleaved during a splicing reaction.

[0029] In some embodiments, a polynucleotide construct described herein comprises a 5' homology arm and / or a 3' homology arm. In some embodiments, a 5' homology arm and / or a 3' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 21-30. In some embodiments, a polynucleotide construct comprises a 5' homology arm. In some embodiments, a 5' homology arm is about 5-50 nucleotides in length. In some embodiments, a 5' homology arm is capable of hybridizing to a 3' homology arm. In some embodiments, a 5' homology arm is capable of base pairing to a 3' homology arm. In some embodiments, a 5' homology arm hybridizes to a 3' homology arm. In some embodiments, a 5' homology arm base pairs to a 3' homology arm. In some embodiments, a 5' homology arm base pairs with at least 75% of the 3' homology arm. In some embodiments, a 5' homology arm basePage 11 of 14413314218vlAttorney Docket No. : 2019398-0005pairs with at least 95% of the 3' homology arm. In some embodiments, a 5' homology arm comprises a nucleotide sequence that is the reverse complement of a nucleotide sequence comprised in a 3' homology arm. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 21-30. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 21-25. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 21. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 22. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 23. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 24. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 25.

[0030] In some embodiments, a polynucleotide construct comprises a 3’ homology arm. In some embodiments, a 3’ homology arm is about 5-50 nucleotides in length. In some embodiments, a 3’ homology arm comprises a nucleotide sequence that is the reverse complement of a nucleotide sequence comprised in the 5’ homology arm. . In some embodiments, a 3' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%,Page 12 of 14413314218vlAttorney Docket No. : 2019398-0005at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 21-30. In some embodiments, a 3’ homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 26-30. In some embodiments, a 3’ homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID No: 26. In some embodiments, a 3’ homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID No: 27. In some embodiments, a 3’ homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID No: 28. In some embodiments, a 3’ homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID No: 29. In some embodiments, a 3’ homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID No: 30.

[0031] In some embodiments, a polynucleotide construct described herein comprises a coding sequence. In some embodiments, a coding sequence is about 50-50,000 nucleotides in length. In some embodiments, a coding sequence is or comprises a payload sequence. In some embodiments, a payload sequence is about 100-20,000 nucleotides in length. In some embodiments, a payload sequence comprises a sequence encoding at least one target gene, target protein, or target peptide.

[0032] In some embodiments, a payload sequence comprises a sequence encoding at least one target gene. In some embodiments, a target gene is a therapeutic RNA.Page 13 of 14413314218vlAttorney Docket No. : 2019398-0005

[0033] In some embodiments, a payload sequence comprises a sequence encoding at least one target protein. In some embodiments, a payload sequence comprises a sequence that encodes at least one target protein and a non-protein encoding sequence. In some embodiments, a target protein is a therapeutic protein. In some embodiments, a therapeutic protein comprises a chimeric protein. In some embodiments, a therapeutic protein comprises a fusion protein. In some embodiments, a therapeutic protein comprises an enzyme, a cytokine, an antibody, and / or a receptor. In some embodiments, a therapeutic protein comprises a structural protein. In some embodiments, a therapeutic protein comprises an immune modulatory ligand. In some embodiments, a therapeutic protein comprises an antigen-binding protein or a functional fragment thereof. In some embodiments, a therapeutic protein comprises a transcription factor.

[0034] In some embodiments, a polynucleotide construct described herein comprises one or more untranslated regions (UTRs). In some embodiments, a payload sequence described herein comprises one or more untranslated regions (UTRs). In some embodiments, one or more UTRs comprise a 5' UTR. In some embodiments, one or more UTRs comprise a 3' UTR. In some embodiments, a 5’ UTR comprises a Kozak sequence.

[0035] In some embodiments, a polynucleotide construct disclosed herein comprises a polyA sequence and / or a polyA-C sequence. In some embodiments, a polyA sequence or a polyA-C sequence is at least 10, at least 15, at least 30, or at least 60 nucleotides in length. In some embodiments, a polyA-C sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 43 or 44.

[0036] In some embodiments, a polynucleotide construct disclosed herein comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 45 or 46.

[0037] In some embodiments, a polynucleotide construct disclosed herein comprises one or more modified nucleotides. In some embodiments, polynucleotide constructs disclosed herein comprises a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.Page 14 of 14413314218vlAttorney Docket No. : 2019398-0005

[0038] In some embodiments, the present disclosure provides, RNA or DNA polynucleotide constructs capable of forming cirRNAs disclosed herein (e.g., a linear RNA polynucleotide construct or a linear DNA polynucleotide construct from which a linear RNA polynucleotide construct can be transcribed) comprising a first intronic sequence, a first portion of an IRES sequence, a coding sequence, a second portion of an IRES sequence, and a second intronic sequence, wherein a first intronic sequence comprises an IGS and a second portion of an IRES sequence comprises a IGS', and wherein an IGS' is capable of hybridizing to an IGS.

[0039] The present disclosure provides, among other things, a DNA polynucleotide construct comprising a promoter sequence, a 5' homology arm, a first intronic sequence comprising an IGS and a catalytic core, a first splice site sequence, an exonic sequence comprising a permuted IRES sequence and a coding sequence, a second splice site sequence, a second intronic sequence, and a 3' homology arm. In some embodiments, an exonic sequence comprises a first portion of an IRES sequence, a coding sequence, and a second portion of an IRES sequence comprising an IGS'.

[0040] The present disclosure also provides, among other things, a RNA polynucleotide construct comprising a 5' homology arm, a first intronic sequence comprising an IGS and a catalytic core, a first splice site sequence, an exonic sequence comprising a permuted IRES sequence and a coding sequence, a second splice site sequence, a second intronic sequence, and a 3' homology arm. In some embodiments, an exonic sequence comprises a first portion of an IRES sequence, a coding sequence, and a second portion of an IRES sequence comprising an IGS'.

[0041] Among other things, the present disclosure provides a circRNA. In some embodiments, a circRNA described herein is produced by a polynucleotide construct of the present disclosure. In some embodiments, a circRNA comprises a first portion of an IRES sequence, a second portion of an IRES sequence, and a coding sequence, wherein the first portion of an IRES sequence and the second portion of an IRES sequence, together, comprise an IRES sequence. In some embodiments, a circRNA comprises an IRES sequence and a coding sequence. In some embodiments, an IRES sequence is directly connected to coding sequence. In some embodiments, an IRES sequence comprises a second IGS'. In some embodiments, an IRES sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a nucleotide sequence of SEQ ID NO: 47. In some embodiments, a circRNA comprising a full and / or functional IRES sequence. In some embodiments, a circRNAPage 15 of 14413314218vlAttorney Docket No. : 2019398-0005described herein comprises a functional IRES sequence. Tn some embodiments, a circRNA comprises an IRES sequence capable of initiating translation. In some embodiments, a circRNA comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a nucleotide sequence of SEQ ID NO: 48 or 49. In some embodiments, a circRNA comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a nucleotide sequence of SEQ ID NO: 48. In some embodiments, a circRNA comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a nucleotide sequence of SEQ ID NO: 49. In some embodiments, a circRNA is at least about Ikb.

[0042] The present disclosure provides, among other things, a circRNA that does not comprise an excision scar. In some embodiments, a circRNA is a scarless circRNA. In some embodiments, a circRNA induces less immunogenicity compared to a same circRNA comprising an excision scar. In some embodiments, a circRNA induces less production of IFN-pi, RIG-I, IL-2, IL-6, IFN-y, and / or TNFa transcript when exposed to an immune system of an organism or a certain type of immune cell compared to a same circRNA comprising an excision scar. In some embodiments, a circRNA modulates production of cytokine when exposed to an immune system of an organism or a certain type of immune cell compared to a same circRNA comprising an excision scar. In some embodiments, a circRNA induces less immunogenicity compared to a same circRNA comprising an excision scar. In some embodiments, a circRNA has a half-life or functional half-life of at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 30 hours, at least 40 hours, at least 50 hours, at least 60 hours, at least 70 hours or at least 80 hours.

[0043] Among other things, the present disclosure provides compositions and methods for making circRNA comprising a polynucleotide construct described herein. In some embodiments, a linear polynucleotide construct described herein circularizes to generate a circRNA. In some embodiments, a linear polynucleotide construct comprises a permuted IRES sequence separated by a coding sequence. In some embodiments, a linear polynucleotide construct does not comprise a functional IRES sequence.Page 16 of 14413314218vlAttorney Docket No. : 2019398-0005

[0044] In some embodiments, a linear RNA polynucleotide construct circularizes to generate in a circRNA comprising a full and / or functional IRES sequence. In some embodiments, a linear RNA polynucleotide construct provided herein can be generated from a linear DNA polynucleotide construct provided herein. In some embodiments, a linear DNA polynucleotide construct (e.g., comprised in a vector) is incubated inside of a cell and transcribed by a bacteriophage RNA polymerase or by a RNA polymerase II. In some embodiments, a linear RNA polynucleotide construct is generated by performing in vitro transcription using a linear DNA polynucleotide construct (e g., comprised in a vector) provided herein. In some embodiments, a linear RNA polynucleotide construct provided herein can be used to generate a circRNA provided herein by incubating. In some embodiments, a method of circularizing a RNA polynucleotide construct comprises incubating a composition comprising a RNA polynucleotide construct. In some embodiments, a RNA polynucleotide construct circularizes when a composition is incubated for 1 hour to 3 hours at 20 °C to 60 °C. In some embodiments, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of a RNA polynucleotide construct circularizes when a composition is incubated for 1 hour to 3 hours at 20 °C to 60 °C.

[0045] Among other things, the present disclosure provides an expression vector or expression vectors comprising a polynucleotide construct and / or a circRNA of the present disclosure. In some embodiments, a vector is a DNA vector. In some embodiments, a vector is a RNA vector. In some embodiments, a vector is a DNA / RNA hybrid vector. In some embodiments, a vector is a plasmid.

[0046] Among other things, the present disclosure also provides a cell comprising a polynucleotide construct and / or a circRNA as described herein or a composition as described herein. In some embodiments, a host cell comprises a polynucleotide construct and / or a circRNA. In some embodiments, a host cell is a prokaryotic cell. In some embodiments, a host cell is a eukaryotic cell. In some embodiments, a eukaryotic cell is a human cell.

[0047] The present disclosure provides, among other things, a composition comprising a polynucleotide construct and / or a circRNA as described herein. In some embodiments, a composition comprises one or more polynucleotide constructs and / or one or more circRNAs. In some embodiments, a polynucleotide construct comprised in a composition is a linearPage 17 of 14413314218vlAttorney Docket No. : 2019398-0005polynucleotide construct. In some embodiments, a subset of one or more polynucleotide constructs are linear polynucleotide constructs. In some embodiments, a linear polynucleotide construct is a DNA polynucleotide construct. In some embodiments, a subset of linear polynucleotide constructs are DNA polynucleotide constructs. In some embodiments, a linear polynucleotide construct is a RNA polynucleotide construct. In some embodiments, a subset of linear polynucleotide constructs are RNA polynucleotide constructs. In some embodiments, a polynucleotide construct comprised in a composition is a circular polynucleotide construct. In some embodiments, a subset of one or more polynucleotide constructs are circular polynucleotide constructs. In some embodiments, a composition comprises one or more circRNAs. In some embodiments, a composition comprises one or more polynucleotide constructs and one or more circRNAs.

[0048] Among other things, the present disclosure provides, a pharmaceutical composition. In some embodiments, a pharmaceutical composition comprises a polynucleotide construct as described herein. In some embodiments, a pharmaceutical composition comprises a circRNA as described herein. In some embodiments, a pharmaceutical composition comprises one or more circRNAs. In some embodiments, a pharmaceutical composition as described herein further comprises one or more pharmaceutically acceptable carriers or excipients. In some embodiments, a pharmaceutical composition comprises a plurality of lipid nanoparticles or liposomes. In some embodiments, a circular RNA is partially or fully encapsulated by lipid nanoparticles of liposomes of a plurality.

[0049] The present disclosure provides, among other things, a method comprising administering a pharmaceutical composition as described herein to a subject. Also provided herein is a method of delivering a payload comprised in a circRNA of the present disclosure, comprising administering a pharmaceutical composition as described herein to a subject. In some embodiments, a method described herein comprises a method of treating a disease comprising administering the pharmaceutical composition.

[0050] Among other things, the present disclosure provides use of a polynucleotide construct, a circRNA, a composition or a pharmaceutical composition as described herein. In some embodiments, a polynucleotide construct as described herein is used in the manufacture of a medicament for treating a subject. In some embodiments, a circRNA as described herein is used in the manufacture of a medicament for treating a subject. In some embodiments, a composition as described herein is used in the manufacture of a medicament for treating a subject. In somePage 18 of 14413314218vlAttorney Docket No. : 2019398-0005embodiments, a pharmaceutical composition as described herein is used in the manufacture of a medicament for treating a subject.

[0051] These, and other features encompassed by the present disclosure, are described in more detail below and in the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawings.

[0053] Embodiments of the present disclosure may take form in various features and arrangements of features, and in various steps and arrangements of steps. It should be understood that the drawings described below are only for illustration purpose of select embodiments of the present disclosure and are not intended to limit the scope of the present teachings in any way. The foregoing and other objects, aspects, features, and advantages of the disclosure will become more apparent and may be better understood by referring to the following description taken in conjunction with the accompanying drawings, in which:

[0054] FIG. 1 is as schematic representation of an exemplary polynucleotide construct encoding (if DNA) or comprising (if RNA) a permuted IRES described herein. Such polynucleotide constructs can be configured according to a formula:5'-(P)-H-A-3S-E-5S-B-H'-3'.In the formula, “(P)” represents a promoter. The parentheses around “P” reflect that, when the polynucleotide construct described herein comprises DNA, the polynucleotide construct includes a promoter, whereas when the polynucleotide construct described herein comprises RNA, it does not. “H” represents a 5' homology arm, and “H'” represents a 3' homology arm. “A” represents a first intronic sequence (which can comprise e.g., a group I 3' intronic sequence) comprising a first internal guide sequence (IGS). “3S” represents a first splice site sequence (e.g. a 3' splice site sequence), and “5S” represents a second splice site sequence (e.g. a 5' splice site sequence). “E” represents an exonic sequence comprising a permuted IRES sequence as provided herein and a coding sequence - e.g., as shown, a first portion of an IRES sequence (e g. a 3' IRES sequence) (C), a second portion of an IRES sequence (e.g. a 5' IRES sequence) (D), and a coding sequencePage 19 of 14413314218vlAttorney Docket No. : 2019398-0005(CDS). A second portion of an IRES sequence comprises a second internal guide sequence (IGS1). “B” represents a second intronic sequence, which can comprise, e.g., a group 15' intronic sequence.

[0055] FIG. 2 is a plasmid map of a polynucleotide construct depicted in FIG. 1.

[0056] FIG. 3 is a schematic representation of a circularization process of a polyribonucleotide construct provided herein. As shown, a polyribonucleotide construct (A) according to FIG. 1 forms a circular RNA (circRNA) (B), e.g., via interactions between sequences on the 5' and 3' ends of the polyribonucleotide construct (e.g., H and H' and / or IGS and IGS') and a catalytic reaction driven by the first and second intronic sequences.

[0057] FIG. 4 is a schematic representation of a circularization process using a Permuted Intron-Exon (PIE) method.

[0058] FIG. 5 depicts the structure of a Type I IRES sequence (here, a wild-type CVB3 IRES), which can be used in a permuted IRES sequence as described herein. As shown, Domain IV includes a proximal loop. An IGS' as provided herein can be located at a proximal loop of a Type I IRES sequence.

[0059] FIG. 6 depicts the structure of a synthetic Type I IRES sequence (here, a synthetic CVB3 IRES), which can be used in a permuted IRES sequence as described herein. As shown, a synthetic IRES sequence can comprise a proximal loop that comprises an IGS' beginning at nucleotide position 386.

[0060] FIG. 7 depicts sequencing results for a reverse-transcribed circRNA as described herein. Following a circularization reaction, the splice junction in the circRNA was confirmed to match a sequence of the CVB3 IRES sequence comprising IGS'.

[0061] FIG. 8 shows results of an in vitro transcription (IVT) reaction of an exemplary polyribonucleotide construct described herein. The electropherogram depicted shows an in vitro transcribed polyribonucleotide construct. The peak annotated “1650” contains circRNA, whereas the peak annotated “1843” contains un-spliced linear precursor polyribonucleotide construct. Minimal splicing of the linear precursor into circRNA was observed following the IVT reaction.

[0062] FIG. 9 shows results of an in vitro transcription (IVT) reaction comprising an exemplary polyribonucleotide construct described herein followed by a circularization reaction. The electropherogram depicted shows the in vitro transcribed polyribonucleotide construct described herein, which was subjected to a standard PIE recircularization reaction. The peak annotated “1655” contains spliced circRNA, whereas the peak annotated “1851” contains un-Page 20 of 14413314218vlAttorney Docket No. : 2019398-0005spliced linear precursor polyribonucleotide construct. Under these conditions, polyribonucleotide constructs circularized with high efficiency.

[0063] FIG. 10 shows expression results obtained by comparing an exemplary circRNA described herein encoding the reporter protein Enhanced Green Fluorescent Protein (EGFP). A first sample comprised circRNA prepared using a standard PIE architecture (e.g. circRNA does not comprise a permuted IRES sequence disclosed herein), a second sample comprised circRNA prepared using an exemplary permuted IRES sequence disclosed herein, and a third sample comprised Jurkat cells electroporated with water (e.g., circRNA was not transfected; “Mock”). The median GFP expression was calculated after the transfected Jurkat cells were assayed via flow cytometry 24 hours post-transfection.

[0064] FIG. 11 shows circRNA half-life results calculated from qPCR data obtained by comparing an exemplary circRNA encoding the reporter protein Gaussia Luciferase (Glue) prepared using either the standard PIE architecture or a permuted IRES disclosed in the present disclosure. circRNA half-life was calculated after the transfected Jurkat cells were assayed via qPCR at four-hour and six-day post-transfection time points.

[0065] FIG. 12 shows normalized Glue expression results obtained by comparing an exemplary circRNA encoding the reporter protein Glue prepared using permuted IRES as described herein, and a circRNA encoding the reporter protein Glue prepared using the standard PIE architecture. Normalized Glue expression was calculated after the transfected Jurkat cells were assayed using Luminescence reader at one-day, three-day and ten-day post-transfection time points.

[0066] FIG. 13 shows Glue expression half-life (RLU per hour) results obtained by comparing an exemplary circRNA encoding the reporter protein Glue prepared using permuted IRES as described herein, and a circRNA encoding the reporter protein Glue prepared using the standard PIE architecture. Glue expression half-life was calculated after the transfected Jurkat cells were assayed using Luminescence reader at one-day and ten-day post-transfection time points.DEFINITIONS

[0067] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including the definitions, will control. UnlessPage 21 of 144133142i8vlAttorney Docket No. : 2019398-0005otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0068] As used herein, the term “a” or “an” refers to one or more of that entity. For example, “a polynucleotide,” is understood to represent one or more polynucleotides. As such, the terms “a” or “an” and “one or more” or “at least one” can be used interchangeably herein.

[0069] As used herein, the term “and / or” is to be taken as specific disclosure of each of the two specified features or features with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone) and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0070] As used herein, the term “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. As used herein, the term “consisting of’ excludes any element, step, or ingredient not specified in the claim element. As used herein, the term “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0071] As used herein, the terms “about” and “approximately,” when used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” or “approximately” in that context. For example, in some embodiments, the term “about” or “approximately” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0072] As used herein, the term “administering” or “administration” typically refers to administration of a composition to a subject to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, bronchialPage 22 of 144133142i8vlAttorney Docket No. : 2019398-0005(e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0073] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence,” “nucleic acid sequence,” “polynucleotide,” and grammatical variants thereof are used interchangeably and refer to a polymer of 3 nucleotides or more. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. In some embodiments, a nucleic acid comprises messenger RNA (mRNA). In some embodiments, a nucleic acid is single stranded. In some embodiments, a nucleic acid is double stranded. In some embodiments, a nucleic acid comprises both single and double stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphor othioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxy thy mi dine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C 5 -methyl cytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-Page 23 of 14413314218vlAttorney Docket No. : 2019398-0005oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13.500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19.500, or 20,000 or more residues or nucleotides long. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0074] As used herein, the symbol in the formula of any polynucleotide constructs disclosed herein can be a phosphodiester bond, or one or more nucleotides inserted between two nucleotides. For example, when a formula requires 5'-A-E-B-3', wherein A comprises a 3' intron icsequence, E comprises an exonic sequence (i.e., one or more exons or fragments thereof), and B comprises a 5' intronic sequence, and wherein between A and E and / or between E and B can be a phosphodiester bond or one or more nucleotides between A and E and / or E and B.

[0075] As used herein, the term “polypeptide” generally has its art-recognized meaning of a polymer of at least three amino acids or more. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional, biologically active, or characteristic fragments, portions or domains (e.g., fragments, portions, or domains retaining at least one activity) of such complete polypeptides. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g.,Page 24 of 14413314218vlAttorney Docket No. : 2019398-0005terminal acetylation, amidation, methylation, etc. In some embodiments, polypeptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

[0076] As used herein, the term the terms “polyribonucleotide” and “RNA” are used interchangeably to refer to a polymer of 3 ribonucleotides or more. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA comprises a backbone structure as described in the definition of “nucleic acid” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA) oligonucleotide. In some embodiments, where an RNA is a mRNA oligonucleotide, the RNA comprises at its 3’ end a poly(A) region, an art-recognized cap structure at its 5’ end (e.g, for recognizing and attachment of an mRNA to a ribosome to initiate translation), and a coding sequence. In some embodiments, a polyribonucleotide can be referred to as an RNA oligonucleotide. When a number of ribonucleotides is used as an indication of size, e.g., for an RNA, a certain number of nucleotides refers to the number of ribonucleotides on a single strand.

[0077] As used herein, the term “pre-circular RNA” refers to a RNA (e.g., mRNA) that is capable of directing its own circularization to form a circular RNA (also referred to herein as “pre-circRNA”).

[0078] As used herein, the term “circular RNA,” or “circRNA” refers to an RNA (e.g., mRNA) that forms a circular structure through covalent bonds. CircRNAs are single- stranded RNA molecules lacking free 5' and 3' ends, and therefore lacking a 5' cap and a 3' polyA tail.

[0079] As used interchangeably herein, the terms “delivery,” “delivering,” or “contacting” refers to introduction of a polynucleotide (e.g., as described herein) into a target cell. A target cell can be cultured in vitro or ex vivo or be present in a subject (in vivo). Methods of introducing a polynucleotide (e.g., as described herein) into a target cell can vary with in vitro, ex vivo, or in vivo applications. In some embodiments, a polynucleotide (e.g., as described herein) can be introduced into a target cell in a cell culture by in vitro transfection. In some embodiments, a polynucleotide (e.g., as described herein) can be introduced into a target cell via delivery vehicles (e.g., nanoparticles, liposomes, and / or complexation with a cell-penetrating agent). In some embodiments, a polynucleotide (e.g., as described herein) can be introduced into a target cell in a subject by administering a polynucleotide (e.g., as described herein) to a subject.Page 25 of 144133142I8vlAttorney Docket No. : 2019398-0005

[0080] As used herein, the term “functional” is used to refer to a form or fragment of an entity that exhibits a particular property and / or activity.

[0081] As used herein, the term “sequence identity” is used herein to mean a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In certain embodiments, sequence identity is calculated based on the full length of two given SEQ ID NO or on part thereof. Part thereof can mean at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of both SEQ ID NO, or any other specified percentage. The term “identity” can also mean the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case can be, as determined by the match between strings of such sequences. In some embodiments, methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.

[0082] As used herein, the terms “effective amount” or “therapeutically effective amount” of, e.g., a circRNA disclosed herein, refers to a quantity sufficient to, when administered to the subject, including a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends on the context in which it is being applied.

[0083] As used herein, the term “target cell” refers to a cell in which a payload (e.g., encoded by the payload sequence) is desired to be expressed. As used herein, the term “non-target cell” refers to a cell in which a payload is not intended to be expressed.

[0084] As used herein, the term “polynucleotide construct” refers to an artificially-designed nucleic acid molecule, which can be borne on a vector. A polynucleotide construct can be an RNA nucleic acid molecule (e.g., a pre-circRNA) or a DNA nucleic acid molecule (e.g., an expression cassette from which the pre-circRNA can be transcribed, or a vector comprising an expression cassette).

[0085] As used herein, the term “vector” refers to any nucleic acid molecule for the cloning of a nucleic acid, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc. A vector can be a replicon to which another nucleic acid segment can be attached so as to bring about the replication of the attached segment. A “replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as anPage 26 of 144133142i8vlAttorney Docket No. : 2019398-0005autonomous unit of replication in vivo, i.e., capable of replication under its own control. A vector can be a “delivery vector” includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A vector can be also used in vitro in the absence of cells to perform any of the procedures known in the art. For example, a vector can be used for in vitro transcription. A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. In some embodiments, insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini. Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), P-galactosidase (LacZ), P-glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters. In some embodiments, the delivery vector is selected from the group consisting of a viral vector (e.g., an AAV vector), a plasmid, a lipid, a protein particle, a bacterial vector, and a lysosome.

[0086] As used herein the term “splicing” refers to the process by which exons are joined while introns are removed from primary transcripts (pre-RNAs) to form the mature RNAs.

[0087] As used herein the term “exons” are the coding sections of a DNA molecule, or of an RNA molecule which is transcribed from a DNA molecule that are then translated into protein, or are part of a mature structural RNA (e.g., tRNA or rRNA). In most eukaryotic genes, exons are separated by “introns,” intervening non-coding sections of DNA. Therefore, introns are segments of nucleic acid that are transcribed into RNA, but are excised via splicing and therefore are not present in the mature RNA transcript, which comprises only exons. The new, immature strands of messenger or structural RNAs, before splicing occurs are called pre-RNAs, and may contain both introns and exons, the RNA molecules resulting from splicing are called RNAs, or mature RNAs. pre-RNA molecules go through splicing and only the exons remain in the “mature RNA,” whichPage 27 of 144133142i8vlAttorney Docket No. : 2019398-0005is then, in the case of mRNA, translated into a protein. “Structural RNAs” or “non-coding RNAs” are RNA molecules that are not translated into proteins and exert a function in the form of RNA molecules. Exemplary structural RNA molecules are rRNAs and tRNAs. During splicing, introns are removed from the pre-RNA by cleavage at conserved sequences called “splice sites,” which are located at the 5' and 3' ends of the exons.

[0088] As used herein, the term “exonic sequence” refers to a nucleotide sequence which is comprised in the mature RNA. As used herein, the term “intronic sequence” refers to a nucleotide sequence which is removed from the RNA molecule during splicing and is thus not comprised in the mature RNA. An exonic sequence can comprise one or more exons, or portions thereof, can comprise a nucleotide sequence derived from a naturally occurring nucleotide sequence, or can be an artificial sequence. An intronic sequence can comprise a complete naturally occurring intron, or portions thereof, can comprise a nucleotide sequence derived from a naturally occurring nucleotide sequence, or can be an artificial sequence.

[0089] As used herein, the term “5’ intronic sequence” and “3' intronic sequence” refer to intronic sequences that are located at the 5' and at the 3’, respectively, of an intronic sequence (e g., a naturally occurring intron). In some embodiments, the constructs disclosed herein comprise “51intronic sequence” and “31intronic sequence” that are in a reverse position. Thus, in some embodiments, a “31intronic sequence” can be located at the 5', and a “5' intronic sequence” can be located at the 3' of the constructs disclosed herein.

[0090] As used herein, the term “splice site” refers to a sequence at the border between an exon and an intron. A splice site comprises a pair of nucleotides linked by a phosphodiester bond that is cleaved during a splicing reaction. A 5' splice site is located at the border between the 3' end of an exon and the 5' end of an intron. For example, a 5' splice site comprises a pair of nucleotides between which cleavage of the phosphodiester bond occurs during a splicing reaction. The nucleotide at the 5' of the pair of nucleotides is comprised in the exon, the nucleotide at the 3' of the pair of nucleotides is comprised in the intron. For example, a 3' splice site is located at the border between the 5' end of an exon and the 3' end of an intron. A 3' splice site comprises a pair of nucleotides between which cleavage of the phosphodi ester bond occurs during a splicing reaction. The nucleotide at the 5' of the pair of nucleotides is comprised in the intron, the nucleotide at the 3' of the pair of nucleotides is comprised in the exon.Page 28 of 144133142I8vlAttorney Docket No. : 2019398-0005

[0091] As used herein, the term “catalytic nucleotide” refers to a nucleotide sequence that facilitates the circularization of pre-circRNA by interacting with a catalytic core of an intronic sequence.

[0092] As used herein, the terms “linear nucleic acid molecules” or “linear polynucleotide molecules” are said to have a “5'-terminus” (5' end) and a “3'- terminus” (3' end) because nucleic acid phosphodiester linkages occur at the 5' carbon and 3' carbon of the sugar moieties of the substituent mononucleotides. The end nucleotide of a polynucleotide at which a new linkage would be to a 5' carbon is its 5' terminal nucleotide. The end nucleotide of a polynucleotide at which a new linkage would be to a 3' carbon is its 3' terminal nucleotide. A terminal nucleotide, as used herein, is the nucleotide at the end position of the 3'- or 5 '-terminus.

[0093] As used herein, the term “transcription” means the formation or synthesis of an RNA molecule by an RNA polymerase using a DNA molecule as a template. The disclosure is not limited with respect to the RNA polymerase that is used for transcription. For example, in some embodiments, a T7- type RNA polymerase can be used.

[0094] As used herein, the term “translation” means the formation of a polypeptide molecule by a ribosome based upon an RNA template.

[0095] As used herein, the term “coding sequence “, “coding region,” or “expression sequence” refers to a nucleic acid sequence that encodes a product, e.g., a peptide or polypeptide (i.e., a protein coding sequence), or a regulatory or structural nucleic acid (i.e., a tRNA or a rRNA). In some embodiments, the nucleic acid sequence refers to a DNA or RNA sequence which “encodes” a gene product (e.g., an RNA), polypeptide, or protein, e.g., such as a payload.

[0096] As used herein, the term “spacer” refers to a region of a polynucleotide sequence ranging from 1 nucleotide to hundreds or thousands of nucleotides separating two other elements along a polynucleotide sequence. The sequences can be defined or can be random. A spacer is typically non-coding. In some embodiments, spacers include duplex forming regions.

[0097] As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or morePage 29 of 144133142t8vlAttorney Docket No. : 2019398-0005features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0098] As used herein, the term “internal ribosome entry site” or “IRES” refers to an RNA sequence or structural element capable of recruiting ribosomes to initiate translation of a polypeptide independent of a typical RNA cap structure. An IRES is typically about 500 nt to about 700 nt in length.

[0099] As used herein, the term “5* IRES sequence” and a “3' IRES sequence” refer to fragments of an IRES sequence that are located at the 5' and the 3' end, respectively, of a functional IRES sequence. These fragments individually are not capable of recruiting ribosomes to initiate translation but form a functioning ribosome when covalently joined into a continuous RNA transcript, for example, via splicing. In some embodiments, polynucleotide constructs disclosed herein comprise a 5' IRES sequence and a 3' IRES sequence that are in a reverse position. Thus, in some embodiments, a 3' IRES sequence can be located at the 5' and a 5' IRES sequence can be located at the 3' of the constructs disclosed herein.

[0100] As used herein, the phrase “therapeutic agent” refers to an agent that, when administered to a subject, has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0101] As used herein, the term “variant” refers to an entity that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moi eties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity. For example, a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities.Page 30 of 144133142I8vlAttorney Docket No. : 2019398-0005In some embodiments, a variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide shows a reduced level of one or more biological activities as compared with the reference polypeptide.

[0102] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, e.g., RNA synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated herein by reference for any purpose.DETAILED DESCRIPTION

[0103] The present disclosure provides, among other things, polynucleotide constructs and circular RNAs (circRNA). The present disclosure also provides improved methods for producing circRNAs. Methods provided herein utilize a Permuted IRES design which can yield higher circularization efficiency. Methods provided herein can generate a scarless circRNA. The present disclosure also provides recognition that circRNAs can be particularly useful as therapeutic agents. circRNAs as disclosed herein can have certain advantages as therapeutic agents. circRNAs have certain characteristics that can be beneficial for use in cells, tissues, and / or subjects. For example, a circRNA can be more resistant to exonuclease degradation than a linear RNA molecule. Nonetheless, methods for producing circRNAs suitable for delivery to cells, tissues, and / or subjects, which could leverage these benefits, remain a challenge.

[0104] Among other things, the present disclosure provides polynucleotide constructs comprising a permuted IRES sequence. Provided herein are also linear RNA polynucleotide constructs, linear DNA polynucleotide constructs from which linear RNA polynucleotide constructs can be transcribed, and / or circRNAs. The present disclosure also provides methods of synthesizing linear polynucleotide constructs and / or circRNAs, compositions comprising linearPage 31 of 14413314218vlAttorney Docket No. : 2019398-0005polynucleotide constructs and / or circRNAs described herein, pharmaceutical compositions comprising linear polynucleotide constructs and / or circRNAs described herein, and / or methods of use comprising linear polynucleotide constructs and / or circRNAs described herein. Accordingly, in some embodiments, the present disclosure provides for administration of compositions comprising linear polynucleotide constructs and / or circRNAs described herein. In some embodiments, the present disclosure provides technologies for delivering one or more linear polynucleotide constructs and / or circRNAs described herein to cells, tissues, and / or subjects. In some embodiments, the present disclosure provides related technologies, including but not limited to, cells comprising linear polynucleotide constructs and / or circRNAs described herein, and related methods.

[0105] Embodiments of the present disclosure include synthesizing a circRNA from a polynucleotide construct comprising a permuted internal ribosomal entry signal (IRES) sequence. In some embodiments, a polynucleotide construct provided herein can have the following benefits over a polynucleotide construct comprising a standard Permuted Intron-Exon (PIE) architecture:a) A Permuted IRES design can eliminate a structured RNA motif at a splice junction included in a circRNA synthetized using standard PIE method. A structured RNA motif at a splice junction can increase the immunogenicity of a circRNA.b) circRNA is shorter in terms of nucleotide length. A shorter circRNA in a therapeutic context can require a lower dose to achieve a desired expression level.c) A circRNA synthetized using a Permuted IRES design as described herein can possess a longer half-life, and / or can be active longer in cells compared to an equivalent circRNA synthetized using standard PIE method.d) Circularization primarily occurs during the recircularization reaction rather than during the in vitro transcription reaction, which can increase ease of analyzing a composition comprising a circRNA designed using a Permuted IRES design as described herein. In some embodiments, this property can aid in fine-tuning a circularization reaction and / or maximize circularization efficiency. In some embodiments, a production process of a composition comprising circRNAs designed using a Permuted IRES design as described herein can be scaled up.Page 32 of 14413314218vlAttorney Docket No. : 2019398-0005e) A circRNA synthetized using a Permuted IRES design as described herein can yield higher circularization efficiency following a recircularization reaction when compared to an equivalent circRNA synthetized using standard PIE method.f) A circRNA synthetized using a Permuted IRES design as described herein can show lower rates of concatemer and / or multimer formation. In some embodiments, multimeric circRNAs (e.g splice product of two or more linear precursors into a single circular RNA molecule) can be considered an impurity. In some embodiments, multimeric circRNAs can have potential effects on formulation and immunogenicity of a composition comprising circRNAs described herein.

[0106] Provided herein are (e.g. RNA or DNA) polynucleotide constructs. In some embodiments, a polynucleotide construct provided herein is a linear polynucleotide construct. In some embodiments, a polynucleotide construct provided herein can be used for synthesis of a circRNA. The present disclosure also provides, among other things, for circRNAs produced from such polynucleotide constructs. In some embodiments, the present disclosure provides circRNAs. In some embodiments, a polynucleotide construct is a DNA polynucleotide construct or a RNA polynucleotide construct. In some embodiments, a polynucleotide construct is a linear polynucleotide construct or a circular polynucleotide construct. In some embodiments, a polynucleotide construct comprises a permuted IRES sequence.I. Features of Polynucleotide constructs

[0107] The present disclosure provides, among other things, a polynucleotide construct comprising (i) a first intronic sequence, (ii) a first portion of an internal ribosomal entry signal (IRES) sequence, (iii) a coding sequence, (iv) a second portion of an IRES sequence, and / or (v) a second intronic sequence. In some embodiments, a polynucleotide construct comprises a first intronic sequence. In some embodiments, a polynucleotide construct comprises a first portion of an internal ribosomal entry signal (IRES) sequence. In some embodiments, a polynucleotide construct comprises a coding sequence. In some embodiments, a polynucleotide construct comprises a second portion of an IRES sequence. In some embodiments, a polynucleotide construct comprises a second intronic sequence. In some embodiments, a polynucleotide construct comprising (i) a first intronic sequence, (ii) a first portion of an internal ribosomal entry signal (IRES) sequence, (iii) a coding sequence, (iv) a second portion of an IRES sequence, and (v) a second intronic sequence. In some embodiments, a first intronic sequence comprises a first internalPage 33 of 144133142i8vlAttorney Docket No. : 2019398-0005guide sequence (IGS). In some embodiments, a second portion of an IRES sequence comprises a second internal guide sequence (IGS'). In some embodiments, a IGS' is capable of hybridizing to an IGS. In some embodiments, a first intronic sequence comprises an IGS, and a second portion of an IRES sequence comprises an IGS', wherein an IGS' is capable of hybridizing to an IGS.

[0108] In some embodiments, a polynucleotide construct described herein comprises (i) a first intronic sequence, (ii) a first portion of an internal ribosomal entry signal (IRES) sequence, (iii) a coding sequence, (iv) a second portion of an IRES sequence, and (v) a second intronic sequence, wherein the first intronic sequence comprises an IGS and the second portion of an IRES sequence comprises an IGS', and wherein an IGS' is capable of hybridizing to an IGS. In some embodiments, a polynucleotide construct described herein is linear. In some embodiments, a polynucleotide construct is a DNA nucleic acid molecule. In some embodiments, a polynucleotide construct is a RNA nucleic acid molecule.First Internal Guide Sequence (IGS) / Second Internal Guide Sequence (IGS’)

[0109] In some embodiments, a polynucleotide construct as described herein (e.g. a linear RNA polynucleotide construct or linear DNA polynucleotide construct) comprises an IGS and / or an IGS'. In some embodiments, a polynucleotide construct comprises an IGS and an IGS'.IGS

[0110] In some embodiments, a polynucleotide construct comprises an IGS. In some embodiments, a polynucleotide construct comprises an IGS, wherein an IGS is comprised in a first intronic sequence. In some embodiments, an IGS comprises single-stranded DNA. In some embodiments, an IGS comprises single- stranded RNA. In some embodiments, an IGS comprises a nucleotide sequence that is or comprises a nucleotide sequence that is the reverse complement of an IGS'. In some embodiments, an IGS is capable of hybridizing with an IGS’. In some embodiments, an IGS hybridizes with an IGS’. In some embodiments, an IGS hybridizes with an IGS’ to facilitate a splicing reaction. In some embodiments, an IGS is excised after a circularization reaction. In some embodiments, an IGS comprised in a first intronic sequence is excised after a circularization reaction. In some embodiments, a circRNA described herein does not comprise an IGS.

[0111] In some embodiments, an IGS is about 2 to about 20 nucleotides in length. In some embodiments, an IGS is about 2 to about 10 nucleotides in length. In some embodiments, an IGS is about 3 to about 15 nucleotides in length. In some embodiments, an IGS is about 10 to about 20Page 34 of 144133142t8vlAttorney Docket No. : 2019398-0005nucleotides in length. In some embodiments, an IGS is at least about 3 nucleotides in length. In some embodiments, an IGS is at least about 6 nucleotides in length. In some embodiments, an IGS is at least about 10 nucleotides in length. In some embodiments, an IGS is at most about 20 nucleotides in length. In some embodiments, an IGS is at least about 2, at least about 3, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or at least about 15 nucleotides in length. In some embodiments, an IGS is about 2, about 3, about 5, about 6, about 7, about 8, about 9, about 10, or about 15 nucleotides in length. In some embodiments, an IGS is at most about 20, at most about 15, at most about 10, at most about 5, or at most about 3 nucleotides in length.

[0112] In some embodiments, an IGS comprises a nucleotide sequence that may contain sequence identity (i.e., similarity) to a sequence as described herein. For example, in some embodiments, an IGS comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence as described herein.

[0113] In some embodiments, an IGS comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical with a nucleotide sequence of any one of SEQ ID NOs: 1-4. In some embodiments, an IGS comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 1-4. In some embodiments, an IGS comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 1. In some embodiments, an IGS comprises a nucleotide sequence of SEQ ID NO: 1. In some embodiments, an IGS comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 2. In some embodiments, an IGS comprises aPage 35 of 144133142I8vlAttorney Docket No. : 2019398-0005nucleotide sequence of SEQ ID NO: 2. In some embodiments, an IGS comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 3. In some embodiments, an IGS comprises a nucleotide sequence of SEQ ID NO: 3. In some embodiments, an IGS comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 4. In some embodiments, an IGS comprises a nucleotide sequence of SEQ ID NO: 4. In some embodiments, an IGS is or comprises a nucleotide sequence of any one of SEQ ID NOs: 1-4. In some embodiments, an IGS is or comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 1-4. In some embodiments, an IGS disclosed herein consists of a nucleotide sequence of any one of SEQ IDS NOs: 1-4.

[0114] In some embodiments, an IGS comprises a nucleotide sequence that is or comprises a nucleotide sequence described in Table 1. In some embodiments, an IGS comprises a nucleotide sequence that is or comprises a nucleotide sequence comprising a complement of a nucleotide sequence described in Table 1. In some embodiments, an IGS comprises a nucleotide sequence that is or comprises a nucleotide sequence comprising a reverse complement of a nucleotide sequence described in Table 1.

[0115] Non-limiting examples of internal guide sequences that can be used according to the present disclosure are shown in Table 1.Table 1. Exemplary Internal Guide Sequences.Page 36 of 14413314218vlAttorney Docket No. : 2019398-0005IGS'

[0116] In some embodiments, a polynucleotide construct comprises an IGS’. In some embodiments, a polynucleotide construct comprises an IGS', wherein an IGS' is comprised in a second portion of an IRES sequence. In some embodiments, an IGS’ comprises single-stranded DNA. In some embodiments, an IGS’ comprises single-stranded RNA. In some embodiments, an IGS’ comprises a nucleotide sequence that is or comprises a nucleotide sequence that is a reverse complement of an IGS.

[0117] In some embodiments, an IGS’ is capable of hybridizing and / or base pairing with an IGS. In some embodiments, an IGS’ hybridizes and / or base pairs with an IGS. In some embodiments, an IGS’ hybridizes and / or base pairs with an IGS to facilitate a splicing reaction. In some embodiments, an IGS’ is not excised after a circularization reaction. In some embodiments, a circRNA as described herein comprises an IGS'. In some embodiments, a circRNA described herein comprises an IGS’ within an IRES sequence.

[0118] In some embodiments, an IGS’ is about 2 to about 20 nucleotides in length. In some embodiments, an IGS’ is about 2 to about 10 nucleotides in length. In some embodiments, an IGS’ is about 3 to about 15 nucleotides in length. In some embodiments, an IGS’ is about 10 to about 20 nucleotides in length. In some embodiments, an IGS’ is at least about 3 nucleotides in length. In some embodiments, an IGS’ is at least about 6 nucleotides in length. In some embodiments, an IGS’ is at least about 10 nucleotides in length. In some embodiments, an IGS’ is at most about 20 nucleotides in length. In some embodiments, an IGS’ is at least about 2, at least about 3, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or at least about 15 nucleotides in length. In some embodiments, an IGS’ is at most about 20, at most about 15, at most about 10, at most about 5, or at most about 3 nucleotides in length. In some embodiments, an IGS’ is about 2, about 3, about 5, about 6, about 7, about 8, about 9, about 10, or about 15 nucleotides in length. In some embodiments, an IGS’ is about 3 nucleotides in length.

[0119] In some embodiments, an IGS’ comprises a nucleotide sequence that may contain sequence identity (i.e., similarity) to a sequence as described herein. For example, in some embodiments, an IGS’ comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least aboutPage 37 of 144133142t8vlAttorney Docket No. : 2019398-0005^ / o, at least about 98%, at least about 99%, or about 100% identical to a sequence as described herein.

[0120] In some embodiments, an IGS’ comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of SEQ ID NOs: 5-8. In some embodiments, an IGS’ comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 5-8. In some embodiments, an IGS’ comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 5. In some embodiments, an IGS’ comprises a nucleotide sequence of SEQ ID NO: 5. In some embodiments, an IGS’ comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 6. In some embodiments, an IGS’ comprises a nucleotide sequence of SEQ ID NO: 6. In some embodiments, an IGS’ comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 7. In some embodiments, an IGS’ comprises a nucleotide sequence of SEQ ID NO: 7. In some embodiments, an IGS’ comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 8. In some embodiments, an IGS’ comprises a nucleotide sequence of SEQ ID NO: 8. In some embodiments, an IGS’ is or comprises a nucleotide sequence of any one of SEQ ID NOs: 5-8. In some embodiments, an IGS’ is or comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 5-8. In some embodiments, an IGS' disclosed herein consists of a nucleotide sequence of any one of SEQ IDS NOs: 5-8.

[0121] In some embodiments, an IGS' is capable of base pairing with at least about 70%, at least about 80%, at least about 90%, or about 100% of an IGS. In some embodiments, an IGS'Page 38 of 14413314218vlAttorney Docket No. : 2019398-0005is capable of base pairing with at least 50% of an TGS. Tn some embodiments, an IGS' is capable of base pairing with at least 60% of an IGS. In some embodiments, an IGS’ is capable of base pairing with at least about 2, at least about 3, at least about 5, at least about 10, or at least about 15 nucleotides of an IGS. In some embodiments, an IGS’ is capable of base pairing with at least about 2 nucleotides of an IGS. In some embodiments, an IGS’ is capable of base pairing with at least about 3 nucleotides of an IGS. In some embodiments, an IGS' base pairs with at least about 70%, at least about 80%, at least about 90%, or about 100% of an IGS. In some embodiments, an IGS' base pairs with at least 50% of an IGS. In some embodiments, an IGS' base pairs with at least 60% of an IGS. In some embodiments, an IGS’ base pairs with at least about 2, at least about 3, at least about 5, at least about 10, or at least about 15 nucleotides of an IGS. In some embodiments, an IGS’ base pairs with at least about 2 nucleotides of an IGS. In some embodiments, an IGS’ base pairs with at least about 3 nucleotides of an IGS.

[0122] In some embodiments, an IGS' comprises a nucleotide sequence that is complimentary to a nucleotide sequence comprised in an IGS. In some embodiments, an IGS' comprises a nucleotide sequence that is complimentary to an IGS. In some embodiments, an IGS' is complimentary to an IGS.

[0123] In some embodiments, a nucleotide sequence that is the reverse complement of an IGS' has at least about 70%, at least about 80%, at least about 90%, or about 100% sequence identity to a nucleotide sequence of an IGS. In some embodiments, a nucleotide sequence that is the reverse complement of an IGS' has at least 50% sequence identity to a nucleotide sequence of an IGS. In some embodiments, a nucleotide sequence that is the reverse complement of an IGS' has at least 60% sequence identity to a nucleotide sequence of an IGS.

[0124] In some embodiments, an IGS’ inserted into an IRES sequence determines an IGS. In some embodiments, an IGS determines an IGS’ sequence inserted into an IRES sequence. For example, aRNA polynucleotide construct described herein comprises an IGS sequence comprising a 5'-UUGAG-3' sequence. In some embodiments, an IGS' is inserted in an IRES sequence comprising nucleotides that do not pair with an IGS. In some embodiments, an IRES sequence comprises nucleotides that do not pair with an IGS comprising an AA sequence and / or other nucleotide sequences. In some embodiments, an IGS' comprises a CUU sequence. In some embodiments, an IGS' is a CUU sequence. In some embodiments, an IGS' comprising a CUU sequence can be inserted upstream of an AA sequence comprised in an IRES sequence. In somePage 39 of 144133142t8vlAttorney Docket No. : 2019398-0005embodiments, an IRES sequence comprises a 5-CUUAA-3' sequence. In some embodiments, an IRES sequence comprising a 5'-CUUAA-3' sequence is capable of pairing with an IGS sequence. In some embodiments, an IRES sequence comprising a 5'-CUUAA-3' sequence pairs with an IGS sequence. In some embodiments, an IRES sequence comprising a 5'-CUUAA-3' sequence is capable of hybridizing with an IGS sequence. In some embodiments, an IRES sequence comprising a 5'-CUUAA-3' sequence hybridizes with an IGS sequence.

[0125] In some embodiments, a circularization reaction occurs via an autocatalytic reaction and / or is facilitated by a base-pairing interaction between sequences of an IGS and an IGS'. In some embodiments, a circularization reaction can be facilitated by a base-pairing interaction between sequences of an IGS and an IGS'. In some embodiments, an IGS' base pairs or hybridizes with an IGS facilitating a circularization reaction.Internal ribosome entry site (IRES) Sequence

[0126] In some embodiments, a polynucleotide construct as described herein comprises an internal ribosomal entry signal IRES sequence, a first portion of an internal ribosomal entry signal (IRES) sequence, and / or a second portion of an internal ribosomal entry signal (IRES) sequence. In some embodiments, a polynucleotide construct described herein comprises an IRES sequence. In some embodiments, a polynucleotide construct comprises a naturally occurring IRES sequence. In some embodiments, a polynucleotide construct comprises a synthetic IRES sequence. In some embodiments, a polynucleotide construct described herein comprises a synthetic IRES sequence comprising an IGS' that is complementary to an IGS sequence. In some embodiments, an IRES sequence comprises an IGS' that is complementary to an IGS sequence. In some embodiments, an IRES sequence disclosed herein is a synthetic IRES sequence comprising an IGS' that is complementary to an IGS sequence.

[0127] First identified as a feature Picorna virus RNA, an internal ribosome entry site (IRES) can play an important role in initiating protein synthesis in absence of a 5' cap structure. In some embodiments, an IRES can act as sole ribosome binding site on an RNA polynucleotide construct. In some embodiments, an IRES can be one of multiple ribosome binding sites on an RNA polynucleotide construct. In some embodiments, a polynucleotide construct comprises more than one functional ribosome binding sites encoding one or more peptides or polypeptides. In some embodiments, a polynucleotide construct comprising more than one functional ribosome binding sites and encoding more than one peptides or polypeptides, can translate the more than one peptidesPage 40 of 14413314218vlAttorney Docket No. : 2019398-0005or polypeptides independently. Tn some embodiments, a polynucleotide construct comprising an IRES can provide a second translatable region.

[0128] Naturally occurring IRESs may lack a sequence capable of base-pairing interactions. Accordingly, in some embodiments, an IRES sequence disclosed herein may include a modification. In some embodiments, an IRES sequence comprises a naturally occurring IRES sequence. In some embodiments, an IRES sequence disclosed herein comprises a synthetic IRES sequence. In some embodiments, an IRES sequence is a synthetic IRES sequence. In some embodiments, a synthetic IRES sequence is derived from a naturally occurring IRES sequence. In some embodiments, an IRES sequence comprises an IRES sequence derived from a naturally occurring IRES sequence. In some embodiments, an IRES sequence comprises or is derived from a cellular IRES sequence or a viral IRES sequence. In some embodiments, an IRES sequence comprises or is derived from a Type I IRES sequence, a Type II IRES sequence, a Type III IRES sequence, a Type IV IRES sequence, or a Type V IRES sequence. In some embodiments, an IRES sequence comprises or is derived from a Type II IRES sequence comprising a Cardiovirus (EMCV) IRES sequence or a Aphthovirus (FMDV) IRES sequence. In some embodiments, an IRES sequence comprises or is derived from a Type I IRES sequence comprising an Enterovirus IRES sequence or a Rhinovirus IRES sequence. In some embodiments, an IRES sequence comprises or is derived from an Enterovirus IRES sequence comprising a Poliovirus (PV) IRES sequence, a Coxsackievirus B3 (CVB3) IRES sequence, or an Enterovirus 71 (EV71) IRES sequence. In some embodiments, a Type I IRES described herein comprises a structure as shown in FIG. 5 or FIG. 6

[0129] In some embodiments, an IRES sequence comprises or is derived from a naturally occurring IRES sequence from Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute beePage 41 of 144133142t8vlAttorney Docket No. : 2019398-0005paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA 1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human ATIR, 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-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SHI, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or an aptamer to eIF4G. In some embodiments, an IRES sequence comprises oris derived from a Coxsackievirus B3 (CVB3) IRES sequence. In some embodiments, an IRES sequence comprises a Coxsackievirus B3 (CVB3) IRES sequence. In some embodiments, an IRES sequence comprises a natural occurring Coxsackievirus B3 (CVB3) IRES sequence. In some embodiments, an IRES sequence comprises a synthetic Coxsackievirus B3 (CVB3) IRES sequence.

[0130] In some embodiments, a polynucleotide construct described herein comprises an IRES sequence comprising one or more domains. In some embodiments, a polynucleotide construct described herein comprises an IRES sequence comprising seven domains (domains I -VII). In some embodiments, an IRES sequence comprises one or more domains. In some embodiments, an IRES sequence comprises seven domains (domains I - VII). In some embodiments, an IRES sequence comprising one or more domains comprises at least one domain containing a proximal loop. In some embodiments, an IRES sequence comprising seven domainsPage 42 of 14413314218vlAttorney Docket No. : 2019398-0005(domains I - VTI) comprising at least one domain containing a proximal loop. In some embodiments, an IRES sequence comprises seven domains (domains I - VII), and domain IV contains a proximal loop.

[0131] In some embodiments, an IRES sequence comprises one or more modifications. In some embodiments, an IRES sequence comprises one or more modifications that do not affect IRES activity. In some embodiments, an IRES sequence comprises one or more modifications to increase or reduce IRES activity. In some embodiments, an IRES sequence comprises one or more modifications to increase IRES activity. In some embodiments, an IRES sequence comprises one or more modifications to reduce IRES activity. In some embodiments, an IRES sequence comprises one or more modifications such as inserting one or more nucleotides. In some embodiments, an IRES sequence comprises one or more modifications such as adding one or more accessory sequences to an IRES sequence. In some embodiments, an IRES sequence comprises one or more modifications such as truncating 5' and / or 3' ends of an IRES sequence. In some embodiments, an IRES sequence comprises one or more modifications such as adding a spacer 5' to an IRES sequence. In some embodiments, an IRES sequence comprises one or more modifications such as modifying a Kozak sequence. In some embodiments, an IRES sequence comprises one or more modifications such as modifying alternative translation initiation sites. In some embodiments, an IRES sequence comprises one or more modifications such as creating a chimeric IRES sequence. In some embodiments, an IRES sequence comprises one or more modifications such as creating a hybrid IRES sequence. In some embodiments, an IRES sequence comprises one or more modifications comprising adding one or more accessory sequences to an IRES sequence, truncating the 5' and / or 3' ends of an IRES sequence, adding a spacer 5' to an IRES sequence, modifying a Kozak sequence, modifying alternative translation initiation sites, creating a chimeric IRES sequence, creating a hybrid IRES sequence, or any combination thereof.

[0132] In some embodiments, an IRES sequence comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence of any one of SEQ ID NOs: 9-11. In some embodiments, an IRES sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atPage 43 of 14413314218vlAttorney Docket No. : 2019398-0005least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 9-11. In some embodiments, an IRES sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 9-11. In some embodiments, an IRES sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 9. In some embodiments, an IRES sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 10. In some embodiments, an IRES sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 11. In some embodiments, an IRES sequence is or comprises a nucleotide sequence of any one of SEQ ID NOs: 9-11. In some embodiments, an IRES sequence is or comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 9-11. In some embodiments, an IRES sequence disclosed herein consists of a nucleotide sequence of any one of SEQ IDS NOs: 9-11.

[0133] Non-limiting examples of IRES sequences that can be used according to the present disclosure are shown in Table 2.Page 44 of 14413314218vlAttorney Docket No.: 2019398-0005Table 2. Exemplary IRES Sequences.Page 45 of 14413314218vlAttorney Docket No. : 2019398-0005

[0134] In some embodiments, an IRES sequence comprises a first portion of the IRES sequence and a second portion of the IRES sequence. For example, in some embodiments, an IRES sequence is split to form a first portion of the IRES sequence and a second portion of the IRES sequence. In some embodiments, an IRES sequence is split within domain IV to form a first portion of the IRES sequence and a second portion of the IRES sequence. In some embodiments, domain IV comprised in an IRES sequence comprises a homologous nucleotide sequence that can be split and / or exploited to circularize a polynucleotide construct disclosed herein. In some embodiments, an IRES sequence is split within a proximal loop comprised in domain IV to form a first portion of the IRES sequence and a second portion of the IRES sequence. In some embodiments, a first portion of an IRES sequence and a second portion of an IRES sequence, together, form an IRES sequence. In some embodiments, a first portion of an IRES sequence comprises domains I - III and a 5' portion of domain IV of an IRES sequence. In some embodiments, a second portion of an IRES sequence comprises a 3' portion of domain IV and domains V - VII of an IRES sequence.First Portion of IRES Sequence

[0135] In some embodiments, a first portion of an IRES sequence comprises a naturally occurring IRES sequence. In some embodiments, a first portion of an IRES sequence disclosed herein comprises a synthetic IRES sequence. In some embodiments, a first portion of an IRES sequence is a synthetic IRES sequence. In some embodiments, a first portion of a synthetic IRES sequence is derived from a naturally occurring IRES sequence. In some embodiments, a first portion of an IRES sequence comprises an IRES sequence derived from a naturally occurring IRES sequence. In some embodiments, a first portion of an IRES sequence comprises or is derived from a naturally occurring IRES sequence comprising a cellular IRES sequence or a viral IRES sequence. In some embodiments, a first portion of an IRES sequence comprises or is derived from a Type I IRES sequence, a Type II IRES sequence, a Type III IRES sequence, a Type IV IRES sequence, or a Type V IRES sequence. In some embodiments, a first portion of an IRES sequence comprises or is derived from a Type II IRES sequence comprising a Cardiovirus (EMCV) IRES sequence or a Aphthovirus (FMDV) IRES sequence. In some embodiments, a first portion of an IRES sequence comprises or is derived from a Type I IRES sequence comprising an Enterovirus IRES sequence or a Rhinovirus IRES sequence. In some embodiments, a first portion of an IRES sequence comprises or is derived from an Enterovirus IRES sequence comprising a PoliovirusPage 46 of 144133142t8vlAttorney Docket No. : 2019398-0005(PV) IRES sequence, a Coxsackievirus B3 (CVB3) IRES sequence, or an Enterovirus 71 (EV71) IRES sequence.

[0136] In some embodiments, a first portion of an IRES sequence comprises or is derived from a naturally occurring IRES sequence from Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA 1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human ATIR, 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-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SHI, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or an aptamer to eIF4G. In some embodiments, a first portion ofPage 47 of 14413314218vlAttorney Docket No. : 2019398-0005an IRES sequence comprises or is derived from a Coxsackievirus B3 (CVB3) IRES sequence. In some embodiments, a first portion of an IRES sequence comprises a Coxsackievirus B3 (CVB3) IRES sequence. In some embodiments, a first portion of an IRES sequence comprises or is derived from a natural occurring Coxsackievirus B3 (CVB3) IRES sequence. In some embodiments, a first portion of an IRES sequence comprises a synthetic Coxsackievirus B3 (CVB3) IRES sequence.

[0137] In some embodiments, a first portion of an IRES sequence comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence of SEQ ID NO: 12. In some embodiments, a first portion of an IRES sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 12. In some embodiments, a first portion of an IRES sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 12. In some embodiments, a first portion of an IRES sequence is or comprises a nucleotide sequence of SEQ ID NO: 12. In some embodiments, a first portion of an IRES sequence is or comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 12. In some embodiments, a first portion of an IRES sequence disclosed herein consists of a nucleotide sequence of SEQ ID NO: 12.

[0138] Non-limiting examples of first portion of IRES sequences that can be used according to the present disclosure are shown in Table 3.Table 3. Exemplary First Portion of IRES Sequences.Page 48 of 14413314218vlAttorney Docket No. : 2019398-0005Second Portion of IRES Sequence

[0139] In some embodiments, a second portion of an IRES sequence comprises a naturally occurring IRES sequence. In some embodiments, a second portion of an IRES sequence disclosed herein comprises a synthetic IRES sequence. In some embodiments, a second portion of an IRES sequence is a synthetic IRES sequence. In some embodiments, a second portion of a synthetic IRES sequence is derived from a naturally occurring IRES sequence. In some embodiments, a second portion of an IRES sequence comprises an IRES sequence derived from a naturally occurring IRES sequence. In some embodiments, a second portion of an IRES sequence comprises or is derived from a naturally occurring IRES sequence comprising a cellular IRES sequence or a viral IRES sequence. In some embodiments, a second portion of an IRES sequence comprises or is derived from a Type I IRES sequence, a Type II IRES sequence, a Type III IRES sequence, a Type IV IRES sequence, or a Type V IRES sequence. In some embodiments, a second portion of an IRES sequence comprises or is derived from a Type II IRES sequence comprising a Cardiovirus (EMCV) IRES sequence or a Aphthovirus (FMDV) IRES sequence. In some embodiments, a second portion of an IRES sequence comprises or is derived from a Type I IRES sequence comprising an Enterovirus IRES sequence or a Rhinovirus IRES sequence. In some embodiments, a second portion of an IRES sequence comprises or is derived from an Enterovirus IRES sequence comprising a Poliovirus (PV) IRES sequence, a Coxsackievirus B3 (CVB3) IRES sequence, or an Enterovirus 71 (EV71) IRES sequence.

[0140] In some embodiments, a second portion of an IRES sequence comprises or is derived from a naturally occurring IRES sequence from Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA 1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, HumanPage 49 of 14413314218vlAttorney Docket No. : 2019398-0005ATIR, Human BAG-1 , Human BCL2, Human BiP, Human c-TAPl , 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-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SHI, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or an aptamer to eIF4G. In some embodiments, a second portion of an IRES sequence comprises or is derived from a Coxsackievirus B3 (CVB3) IRES sequence. In some embodiments, a second portion of an IRES sequence comprises a Coxsackievirus B3 (CVB3) IRES sequence. In some embodiments, a second portion of an IRES sequence comprises or is derived from a natural occurring Coxsackievirus B3 (CVB3) IRES sequence. In some embodiments, a second portion of an IRES sequence comprises a synthetic Coxsackievirus B3 (CVB3) IRES sequence.

[0141] In some embodiments, a second portion of an IRES sequence comprises an IGS'. In some embodiments, a second portion of an IRES sequence comprises an IGS', wherein the second portion of an IRES sequence comprises or is a synthetic CVB3 IRES sequence. In some embodiments, a second portion of an IRES sequence comprises an IGS' within domain IV of the IRES sequence. In some embodiments, a second portion of an IRES sequence comprises an IGS' within a proximal loop in domain IV of the IRES sequence. In some embodiments, a second portion of an IRES sequence comprises an IGS', wherein the IGS' defines a junction between aPage 50 of 144133142t8vlAttorney Docket No. : 2019398-0005first portion of an IRES sequence and a second portion of an IRES sequence. In some embodiments, a second portion of an IRES sequence comprises an IGS' at 3' end.

[0142] In some embodiments, a second portion of an IRES sequence comprises an IGS', wherein an IGS' is complementary to an IGS sequence. In some embodiments, a second portion of an IRES sequence comprises an IGS', wherein an IGS' is capable of hybridizing and / or base pairing to an IGS. In some embodiments, a second portion of an IRES sequence comprises an IGS', wherein an IGS' is capable of hybridizing to an IGS. In some embodiments, a second portion of an IRES sequence comprises an IGS', wherein an IGS' hybridizes and / or base pairs to an IGS. In some embodiments, a second portion of an IRES sequence comprises an IGS’, wherein an IGS' hybridizes and / or base pairs with an IGS to facilitate a splicing reaction and / or a circularization reaction.

[0143] In some embodiments, a second portion of an IRES sequence comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence of SEQ ID NO: 13. In some embodiments, a second portion of an IRES sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 13. In some embodiments, a second portion of an IRES sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 13. In some embodiments, a second portion of an IRES sequence is or comprises a nucleotide sequence of SEQ ID NO: 13. In some embodiments, a second portion of an IRES sequence is or comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 13. In some embodiments, a second portion of an IRES sequence disclosed herein consists of a nucleotide sequence of SEQ ID NO: 13.

[0144] Non-limiting examples of second portion of IRES sequences that can be used according to the present disclosure are shown in Table 4. IGS' appear as underlined in Table 4.Page 51 of 14413314218vlAttorney Docket No. : 2019398-0005Table 4. Exemplary Second Portion of IRES Sequences.

[0145] In some embodiments, a polynucleotide construct described herein comprises a first portion of an IRES sequence and / or a second portion of an IRES sequence. In some embodiments, a polynucleotide construct described herein comprises a first portion of an IRES sequence comprising domains I - III and a 5' portion of domain IV of an IRES sequence. In some embodiments, a polynucleotide construct comprises a first portion of an IRES sequence comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 12. In some embodiments, a polynucleotide construct comprises a first portion of an IRES sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 12. In some embodiments, a polynucleotide construct described herein comprises a second portion of an IRES sequence comprising a 3' portion of domain IV and domains V - VII of an IRES sequence. In some embodiments, a polynucleotide construct comprises a second portion of an IRES sequence comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 13. In some embodiments, a polynucleotide construct comprises a second portion of an IRES sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 13. In some embodiments, a polynucleotide construct described herein comprises a first portion of an IRES sequence comprising domains I - III and a Page 52 of 14413314218vlAttorney Docket No. : 2019398-00055' portion of domain IV of an IRES sequence; and a second portion of an IRES sequence comprising a 3' portion of domain IV and domains V - VII of an IRES sequence. In some embodiments, a polynucleotide construct comprises an IRES sequence comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 9-11. In some embodiments, a polynucleotide construct comprises an IRES sequence comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 9. In some embodiments, a polynucleotide construct comprises an IRES sequence comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 10. In some embodiments, a polynucleotide construct comprises an IRES sequence comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 11. In some embodiments, a polynucleotide construct comprises an IRES sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 9-11. In some embodiments, a polynucleotide construct comprises an IRES sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 9. In some embodiments, a polynucleotide construct comprises an IRES sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 10. In some embodiments, a polynucleotide construct comprises an IRES sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 11. In some embodiments, a polynucleotide construct of the present disclosure comprises permuted IRES sequences. For example, in somePage 53 of 144133142I8vlAttorney Docket No. : 2019398-0005embodiments, a polynucleotide construct disclosed herein comprises a first portion of an IRES sequence comprising a 3' portion of domain IV of the IRES sequence located at 5' end of a linear polynucleotide construct. In some embodiments, a polynucleotide construct disclosed herein comprises a second portion of an IRES sequence comprising a 5' portion of domain IV of the IRES sequence located at 3' end of a linear polynucleotide construct. In some embodiments, a linear polynucleotide construct disclosed herein comprises a first portion of an IRES sequence and a second portion of an IRES sequence that are permuted. In some embodiments, a linear polynucleotide construct disclosed herein comprising a first portion of an IRES sequence and a second portion of an IRES sequence that are permuted, cannot form a functional IRES sequence. In some embodiments, a linear polynucleotide construct disclosed herein comprises a first portion of an IRES sequence and a second portion of an IRES sequence that are permuted, wherein the first portion of an IRES sequence and the second portion of an IRES sequence cannot form a functioning IRES sequence and / or the linear polynucleotide construct is not capable of being translated. In some embodiments, a linear polynucleotide construct comprising a first portion of an IRES sequence and a second portion of an IRES sequence that are permuted can circularize to produce a circRNA of the present disclosure. In some embodiments, a linear polynucleotide construct comprising a first portion of an IRES sequence and a second portion of an IRES sequence that are permuted, circularizes to produce a circRNA comprising a functional IRES sequence. In some embodiments, a circRNA described herein comprises a functional IRES sequence and / or is capable of being translated. In some embodiments, a circRNA described herein is produced from a linear polynucleotide construct comprising a first portion of an IRES sequence and a second portion of an IRES sequence that are permuted, wherein circularization of the linear polynucleotide construct produces a circRNA comprising a first portion of an IRES sequence and a second portion of an IRES sequence that forms a functional IRES sequence. In some embodiments, circularization of a linear polynucleotide construct described herein produces a circRNA comprising a functional IRES sequence that is capable of being translated.Promoter

[0146] In some embodiments, a polynucleotide construct described in the present disclosure comprises a promoter sequence. In some embodiments, a polynucleotide construct described herein is a DNA polynucleotide construct comprising a promoter sequence. In some embodiments, a polynucleotide construct encoding a permuted IRES sequence described hereinPage 54 of 144133142I8vlAttorney Docket No. : 2019398-0005comprises a promoter sequence. In some embodiments, a promoter sequence comprises an RNA polymerase promoter. In some embodiments, a promoter sequence comprises a bacteriophage promoter sequence, a viral promoter sequence, a bacterial promoter sequence, an eukaryotic promoter sequence or an engineered promoter sequence. In some embodiments, a promoter sequence comprises a bacteriophage promoter sequence. In some embodiments, a bacteriophage promoter sequence comprises a T7 promoter sequence or a variant or a fragment thereof, a T6 promoter sequence or a variant or a fragment thereof, a SP6 promoter sequence or a variant or a fragment thereof, a T3 promoter sequence or a variant or a fragment thereof, or a T4 promoter sequence or a variant or a fragment thereof. In some embodiments, a bacteriophage promoter sequence comprises a T7 promoter sequence or a variant or a fragment thereof, a SP6 promoter sequence or a variant or a fragment thereof, or a T3 promoter sequence or a variant or a fragment thereof. In some embodiments, a promoter sequence is a T7 promoter sequence. In some embodiments, a promoter sequence is a SP6 promoter sequence. In some embodiments, a promoter sequence is a T3 promoter sequence.

[0147] In some embodiments, a promoter sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 14-16. In some embodiments, a promoter sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 14. In some embodiments, a promoter sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 14-16. In some embodiments, a promoter sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID No: 14. In some embodiments, a promoter sequence is or comprises a nucleotide sequence of any one of SEQ ID NOs: 14-16. In some embodiments, a promoter sequence is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of any one of SEQ ID NOs: 14-16. In some embodiments, aPage 55 of 144133142i8vlAttorney Docket No. : 2019398-0005promoter sequence disclosed herein consists of a nucleotide sequence of any one of SEQ ID NOs: 14-16.

[0148] Non-limiting examples of promoter sequences that can be used according to the present disclosure are shown in Table 5.Table 5. Exemplary Promoter Sequences.Splice site sequence

[0149] In some embodiments, a polynucleotide construct described in the present disclosure comprises at least one splice site. In some embodiments, a polynucleotide construct described herein is an RNA polynucleotide construct comprising at least one splice site. A splice site is a nucleotide sequence comprising a pair of nucleotides linked by a phosphodiester bond that can cleaved during a splicing reaction.

[0150] In some embodiments, a polynucleotide construct capable of forming a circRNA disclosed herein comprises a first splice site and a second splice site. In some embodiments, a first splice site and a second splice site can be transposed with respect to relative position of same splice sites in a naturally occurring configuration. In some embodiments, a polynucleotide construct comprises a first splice site and a second splice site. In some embodiments, a polynucleotide construct comprises a first intronic sequence, a first portion of an IRES sequence, a second portion of an IRES sequence, and a second intronic sequence. In some embodiments, a polynucleotide construct comprises a first intronic sequence directly j oined to a first portion of an IRES sequence at a first junction. In some embodiments, a polynucleotide construct comprises a second portion of an IRES sequence directly joined to a second intronic sequence at a second junction. In some embodiments, a polynucleotide construct comprises a first intronic sequence directly joined to a first portion of an IRES sequence at a first junction, and a second portion of an IRES sequence directly joined to a second intronic sequence at a second junction. In some embodiments, a first junction is comprised in a first splice site. In some embodiments, a first splice site comprises a first junction. In some embodiments, a first splice site is a first junction.Page 56 of 144133142I8vlAttorney Docket No. : 2019398-0005

[0151] In some embodiments, a first splice site comprises a sequence of formula S'-CfN-3', wherein N is any one of A, C, G, T, or U, and wherein “A” indicates the position of the phosphodiester bond that is cleaved during a splicing reaction. In some embodiments, a first splice site comprises a sequence of formula 5'-GAA-3'. In some embodiments, a first splice site comprises a sequence of formula 5'-GAC-3'. In some embodiments, a first splice site comprises a sequence of formula 5'-GAG-3'. In some embodiments, a first splice site comprises a sequence of formula 5'-GAT-3'. In some embodiments, a first splice site comprises a sequence of formula 5'-GAU-3'. In some embodiments, a first splice site comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 17 or 19. In some embodiments, a first splice site comprises a nucleotide sequence having at least 85%, at least 90%, at least 95% or 100% identity with a nucleotide sequence of SEQ ID NO: 17 or 19. In some embodiments, a first splice site is or comprises a nucleotide sequence of SEQ ID NO: 17 or 19. In some embodiments, a first splice site is or comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 17 or 19. In some embodiments, a first splice site disclosed herein consists of a nucleotide sequence of SEQ ID NO: 17 or 19.

[0152] In some embodiments, a second junction is comprised in a second splice site. In some embodiments, a second splice site comprises a second junction. In some embodiments, a second splice site is a second junction. In some embodiments, a second splice site comprises a nucleotide sequence of 5'-UAN-3', wherein N is A, U, C, T, or G, and wherein “A” indicates the position of the phosphodiester bond that is cleaved during a splicing reaction. In some embodiments, a second splice site comprises a nucleotide sequence of 5'-UAA-3', wherein “A” indicates the position of the phosphodiester bond that is cleaved during a splicing reaction. In some embodiments, a second splice site comprises a nucleotide sequence of NAGUAAAA, wherein N is A, U, C, or G, and wherein “A” indicates the position of the phosphodiester bond that is cleaved during a splicing reaction.

[0153] In some embodiments, a second splice site comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 18 or 20. In some embodiments, a second splice site comprises a nucleotide sequence having at least 85%, at least 90%, at least 95% or 100% identity with aPage 57 of 144133142i8vlAttorney Docket No. : 2019398-0005nucleotide sequence of SEQ ID NO: 18 or 20. Tn some embodiments, a second splice site is or comprises a nucleotide sequence of SEQ ID NO: 18 or 20. In some embodiments, a second splice site is or comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 18 or 20. In some embodiments, a second splice site disclosed herein consists of a nucleotide sequence of SEQ ID NO: 18 or 20.

[0154] Non-limiting examples of splice sites sequences that can be used according to the present disclosure are shown in Table 6.Table 6. Exemplary Splice Sites Sequences (“A” indicates the position of the phosphodiester bond that is cleaved during a splicing reaction).Homology Arms

[0155] In some embodiments, a polynucleotide construct described in the present disclosure comprises at least one homology arm. Tn some embodiments, a polynucleotide construct described herein comprises a 5' homology arm and / or a 3' homology arm. A homology arm can refer to a nucleotide sequence that is capable of hybridizing and / or base paring with another sequence (e.g., another homology arm). In some embodiments, a polynucleotide construct capable of forming a circRNA disclosed herein comprises a 5' homology arm and a 3' homology arm. In some embodiments, a linear polynucleotide construct described in the present disclosure comprises a 5' homology arm at 5' end of a linear polynucleotide construct. In some embodiments, a linear polynucleotide construct described in the present disclosure comprises a 3' homology arm at 3' end of a linear polynucleotide construct. In some embodiments, a polynucleotide construct comprises a 5' homology arm at 5' end of a polynucleotide construct and a 3' homology arm at 3' end of a polynucleotide construct.

[0156] In some embodiments, a polynucleotide construct described herein comprises a 5’ homology arm capable of hybridizing and / or base pairing with a 3’ homology arm. In some embodiments, a 5’ homology arm is capable of base pairing with a 3’ homology arm. In somePage 58 of 14413314218vlAttorney Docket No. : 2019398-0005embodiments, a 5’ homology arm is capable of hybridizing with a 3’ homology arm. In some embodiments, a polynucleotide construct described herein comprises a 5’ homology arm that hybridizes and / or base pairs with a 3’ homology arm. In some embodiments, a 5’ homology arm hybridizes with a 3’ homology arm. In some embodiments, a 5’ homology arm base pairs with a 3’ homology arm. In some embodiments, a 5’ homology arm base pairs with a 3’ homology arm and produces a change in a spatial conformation of a polynucleotide construct. In some embodiments, a 5’ homology arm base pairs with a 3’ homology arm and produces a change in a spatial conformation of a polynucleotide construct, wherein the change in spatial conformation of a polynucleotide construct brings a 3' splice site and a 5' splice site into proximity. In some embodiments, a 5’ homology arm hybridizes and / or base pairs with a 3’ homology arm to facilitate a splicing reaction and / or a circularization reaction. In some embodiments, a 5’ homology arm base pairs with a 3’ homology arm. In some embodiments, a 5’ homology arm base pairs with a 3’ homology arm to facilitate a splicing reaction and / or a circularization reaction.5' Homology Arm

[0157] In some embodiments, a 5’ homology arm comprises a nucleotide sequence that is complimentary to a nucleotide sequence comprised in a 3’ homology arm. In some embodiments, a 5’ homology arm comprises a nucleotide sequence that is the reverse complement of a nucleotide sequence comprised in a 3’ homology arm. In some embodiments, a nucleotide sequence that is the reverse complement of a 5’ homology arm has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to a nucleotide sequence of a 3’ homology arm. In some embodiments, a nucleotide sequence that is the reverse complement of a 5’ homology arm has at least 75% sequence identity to a nucleotide sequence of a 3’ homology arm. In some embodiments, a nucleotide sequence that is the reverse complement of a 5’ homology arm has at least 95% sequence identity to a nucleotide sequence of a 3’ homology arm.

[0158] In some embodiments, a 5’ homology arm is capable of base pairing with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of a 3’ homology arm. In some embodiments, a 5’ homology arm is capable of base pairing with at least 75% of a 3’ homology arm. In some embodiments, a 5’ homology arm is capable of base pairing with at least 95% of a 3’ homology arm. In somePage 59 of 14413314218vlAttorney Docket No. : 2019398-0005embodiments, a 5’ homology arm base pairs with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of a 3’ homology arm. In some embodiments, a 5’ homology arm base pairs with at least 75% of a 3’ homology arm. In some embodiments, a 5’ homology arm base pairs with at least 95% of a 3’ homology arm.

[0159] In some embodiments, a 5’ homology arm is capable of hybridizing with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of a 3’ homology arm. In some embodiments, a 5’ homology arm is capable of hybridizing with at least 75% of a 3’ homology arm. In some embodiments, a 5’ homology arm is capable of hybridizing with at least 95% of a 3’ homology arm. In some embodiments, a 5’ homology arm hybridizes with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of a 3’ homology arm. In some embodiments, a 5’ homology arm hybridizes with at least 75% of a 3’ homology arm. In some embodiments, a 5’ homology arm hybridizes with at least 95% of a 3’ homology arm.

[0160] In some embodiments, a 5’ homology arm is about 5-50 nucleotides in length. In some embodiments, a 5’ homology arm is about 9-19 nucleotides in length. In some embodiments, a 5’ homology arm is 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 16, at least 17, at least 18 or at least 19 nucleotides in length. In some embodiments, a 5’ homology arm is at most 20, at most 30, at most 40 or at most 50 nucleotides in length.

[0161] . In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 21-30. In some embodiments, a 5' homology arm comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence of any one of SEQ IDNOs: 21-25. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at leastPage 60 of 14413314218vlAttorney Docket No. : 2019398-000593%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 21-25.. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 21. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 22. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 23. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 24. In some embodiments, a 5' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 25. In some embodiments, a 5' homology arm has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 21-25. In some embodiments, a 5' homology arm has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 21. In some embodiments, a 5' homology arm has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 22. In some embodiments, a 5' homology arm has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 23. In some embodiments, a 5' homology arm has at least 75%, at least 80%, at least 85%,Page 61 of 14413314218vlAttorney Docket No. : 2019398-0005at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 24. In some embodiments, a 5' homology arm has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 25. In some embodiments, a 5' homology arm is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of any one of SEQ ID NOs: 21-25. In some embodiments, a 5' homology arm is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 21. In some embodiments, a 5' homology arm is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 22. In some embodiments, a 5' homology arm is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 23. In some embodiments, a 5' homology arm is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 24. In some embodiments, a 5' homology arm is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 25. In some embodiments, a 5' homology arm is or comprises a nucleotide sequence of any one of SEQ ID NOs: 21-25. In some embodiments, a 5' homology arm is or comprises a nucleotide sequence of SEQ ID NO: 21. In some embodiments, a 5' homology arm is or comprises a nucleotide sequence of SEQ ID NO: 22. In some embodiments, a 5' homology arm is or comprises a nucleotide sequence of SEQ ID NO: 23. In some embodiments, a 5' homology arm is or comprises a nucleotide sequence of SEQ ID NO: 24. In some embodiments, a 5' homology arm is or comprises a nucleotide sequence of SEQ ID NO: 25. In some embodiments, a 5' homology arm disclosed herein consists of a nucleotide sequence of any one of SEQ ID NOs: 21-25. In some embodiments, a 5' homology arm disclosed herein consists of a nucleotide sequence of SEQ ID NO: 21. In some embodiments, a 5' homology arm disclosed herein consists of a nucleotide sequence of SEQ ID NO: 22. In some embodiments, a 5' homology arm disclosed herein consists of a nucleotide sequence of SEQ ID NO: 23. In some embodiments, a 5' homology arm disclosed herein consists of a nucleotide sequence of SEQ ID NO: 24. In some embodiments, a 5' homology arm disclosed herein consists of a nucleotide sequence of SEQ ID NO: 25.Page 62 of 14413314218vlAttorney Docket No. : 2019398-00053' Homology Arm

[0162] In some embodiments, a 3’ homology arm comprises a nucleotide sequence that is complimentary to a nucleotide sequence comprised in a 5’ homology arm. In some embodiments, a 3’ homology arm comprises a nucleotide sequence that is the reverse complement of a nucleotide sequence comprised in a 5’ homology arm. In some embodiments, a nucleotide sequence that is the reverse complement of a 3’ homology arm has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to a nucleotide sequence of a 5’ homology arm. In some embodiments, a nucleotide sequence that is the reverse complement of a 3’ homology arm has at least 75% sequence identity to a nucleotide sequence of a 5’ homology arm. In some embodiments, a nucleotide sequence that is the reverse complement of a 3’ homology arm has at least 95% sequence identity to a nucleotide sequence of a 5’ homology arm.

[0163] In some embodiments, a 3’ homology arm is capable of base pairing with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of a 5’ homology arm. In some embodiments, a 3’ homology arm is capable of base pairing with at least 75% of a 5’ homology arm. In some embodiments, a 3’ homology arm is capable of base pairing with at least 95% of a 5’ homology arm. In some embodiments, a 3’ homology arm base pairs with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of a 5’ homology arm. In some embodiments, a 3’ homology arm base pairs with at least 75% of a 5’ homology arm. In some embodiments, a 3’ homology arm base pairs with at least 95% of a 5’ homology arm.

[0164] In some embodiments, a 3’ homology arm is capable of hybridizing with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of a 5’ homology arm. In some embodiments, a 3’ homology arm is capable of hybridizing with at least 75% of a 5’ homology arm. In some embodiments, a 3’ homology arm is capable of hybridizing with at least 95% of a 5’ homology arm. In some embodiments, a 3’ homology arm hybridizes with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of a 5’ homology arm. In some embodiments, a 3’ homology arm hybridizes with at least 75% of a 5’Page 63 of 14413314218vlAttorney Docket No. : 2019398-0005homology arm. In some embodiments, a 3’ homology arm hybridizes with at least 95% of a 5’ homology arm.

[0165] In some embodiments, a 3’ homology arm is about 5-50 nucleotides in length. In some embodiments, a 3’ homology arm is about 9-19 nucleotides in length. In some embodiments, a 3’ homology arm is 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 16, at least 17, at least 18 or at least 19 nucleotides in length. In some embodiments, a 3’ homology arm is at most 20, at most 30, at most 40 or at most 50 nucleotides in length.

[0166] In some embodiments, a 3' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 21-30. In some embodiments, a 3' homology arm comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence of any one of SEQ ID NOs: 26-30. In some embodiments, a 3' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 26-30. In some embodiments, a 3' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 26. In some embodiments, a 3' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 27. In some embodiments, a 3' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 28. In some embodiments, a 3' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%,Page 64 of 14413314218vlAttorney Docket No. : 2019398-0005at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 29. In some embodiments, a 3' homology arm comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 30. In some embodiments, a 3' homology arm has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 26-30. In some embodiments, a 3' homology arm has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 26. In some embodiments, a 3' homology arm has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 27. In some embodiments, a 3' homology arm has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 28. In some embodiments, a 3' homology arm has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 29. In some embodiments, a 3' homology arm has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 30. In some embodiments, a 3' homology arm is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of any one of SEQ ID NOs: 26-30. In some embodiments, a 3' homology arm is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 26. In some embodiments, a 3' homology arm is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 27. In some embodiments, a 3' homology arm is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 28. In some embodiments, a 3' homology arm is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence ofPage 65 of 144133142I8vlAttorney Docket No. : 2019398-0005SEQ ID NO: 29. Tn some embodiments, a 3' homology arm is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 30. In some embodiments, a 3' homology arm is or comprises a nucleotide sequence of any one of SEQ ID NOs: 26-30. In some embodiments, a 3' homology arm is or comprises a nucleotide sequence of SEQ ID NO: 26. In some embodiments, a 3' homology arm is or comprises a nucleotide sequence of SEQ ID NO: 27. In some embodiments, a 3' homology arm is or comprises a nucleotide sequence of SEQ ID NO: 28. In some embodiments, a 3' homology arm is or comprises a nucleotide sequence of SEQ ID NO: 29. In some embodiments, a 3' homology arm is or comprises a nucleotide sequence of SEQ ID NO: 30. In some embodiments, a 3' homology arm disclosed herein consists of a nucleotide sequence of any one of SEQ ID NOs: 26-30. In some embodiments, a 3' homology arm disclosed herein consists of a nucleotide sequence of SEQ ID NO: 26. In some embodiments, a 3' homology arm disclosed herein consists of a nucleotide sequence of SEQ ID NO: 27. In some embodiments, a 3' homology arm disclosed herein consists of a nucleotide sequence of SEQ ID NO: 28. In some embodiments, a 3' homology arm disclosed herein consists of a nucleotide sequence of SEQ ID NO: 29. In some embodiments, a 3' homology arm disclosed herein consists of a nucleotide sequence of SEQ ID NO: 30.

[0167] Non-limiting examples of 5' homology arm and 3' homology arm sequences that can be used according to the present disclosure are shown in Table 7.Table 7. Exemplary 5' Homology Arm and 3* Homology Arm Sequences.Page 66 of 14413314218vlAttorney Docket No. : 2019398-0005Intronic Sequence

[0168] In some embodiments, a polynucleotide construct described in the present disclosure comprises at least one intronic sequence. In some embodiments, a polynucleotide construct described herein comprises a first intronic sequence and / or a second intronic sequence. In some embodiments, a polynucleotide construct described herein comprises a first intronic sequence comprising an IGS. In some embodiments, a polynucleotide construct capable of forming a circRNA disclosed herein comprises a first intronic sequence and a second intronic sequence, wherein the first intronic sequence comprises an IGS. In some embodiments, an IGS comprised in a first intronic sequence can facilitate a splicing reaction. A splicing reaction comprises removal of intronic sequences in a precursor RNA transcript (i.e., primary transcript) to form a mature RNA molecule (e.g., a mature mRNA, tRNA, or rRNA). In some embodiments, at least one intronic sequence is excised from a polynucleotide construct described herein after a circularization reaction. In some embodiments, a first intronic sequence and / or a second intronic sequence are excised from a polynucleotide construct described herein after a circularization reaction. In some embodiments, a first intronic sequence and / or a second intronic sequence comprises a self-splicing intronic sequence. In some embodiments, a self-splicing intronic sequence comprises a group I intronic sequence. In some embodiments, a first intronic sequence and / or a second intronic sequence can catalyze a splicing reaction. In some embodiments, a polynucleotide construct comprising a first intronic sequence and a second intronic sequence can undergo an autocatalytic reaction. In some embodiments, a circRNA described herein does not comprise a first intronic sequence and / or a second intronic sequence.

[0169] In some embodiments, a polynucleotide construct described in the present disclosure comprises a first intronic sequence at 5' end of a polynucleotide construct. In some embodiments, a first intronic sequence comprises a 3' intronic sequence. In some embodiments, a polynucleotide construct described in the present disclosure comprises a second intronic sequence at 3' end of a polynucleotide construct. In some embodiments, a second intronic sequence comprises a 5' intronic sequence. In some embodiments, a polynucleotide construct comprises a first intronic sequence at 5' end of a polynucleotide construct and a second intronic sequence at 3' end of a polynucleotide construct, wherein a first intronic sequence comprises a 3' intronic sequence and a second intronic sequence comprises a 5' intronic sequence. In some embodiments,Page 67 of 14413314218vlAttorney Docket No. : 2019398-0005a 5' intronic sequence and a 3' intronic sequence refer to a relative position between each intronic sequence within a full length intronic sequence.

[0170] In some embodiments, a full length intronic sequence can be spliced to form a first intronic sequence and a second intronic sequence. In some embodiments, a full length intronic sequence can be spliced to form a first intronic sequence and a second intronic sequence, wherein the first intronic and the second intronic sequence comprise a nucleotide sequence comprising nucleotides that are not contiguous in the full length intronic sequence.

[0171] Non-limiting examples of full length intronic sequences from which an intronic sequence can be derived (e.g., a first intronic sequence or a second intronic sequence) that can be used according to the present disclosure are shown in Table 8.Table 8. Exemplary Full Length Intronic Sequences.

[0172] In some embodiments, a full length intronic sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 31 or 32. In some embodiments, an intronic sequence comprises a nucleotide sequence derived from a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 31 or 32. In some embodiments, an intronic sequencePage 68 of 14413314218vlAttorney Docket No. : 2019398-0005comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 31 or 32. In some embodiments, an intronic sequence comprises a nucleotide sequence derived from a nucleotide sequence consisting of a nucleotide sequence of SEQ ID NO: 31 or 32. In some embodiments, an intronic sequence comprises a nucleotide sequence consisting of a nucleotide sequence of SEQ ID NO: 31 or 32. In some embodiments, an intronic sequence comprises a nucleotide sequence derived from a nucleotide sequence consisting of a nucleotide sequence of SEQ ID NO: 31. In some embodiments, an intronic sequence comprises a nucleotide sequence consisting of a nucleotide sequence of SEQ ID NO: 31. In some embodiments, an intronic sequence comprises a nucleotide sequence derived from a nucleotide sequence consisting of a nucleotide sequence of SEQ ID NO: 32. In some embodiments, an intronic sequence comprises a nucleotide sequence consisting of a nucleotide sequence of SEQ ID NO: 32.

[0173] In some embodiments, a first intronic sequence and / or a second intronic sequence comprise any portion of a full length intronic sequence. In some embodiments, a first intronic sequence and / or a second intronic sequence comprise a portion of a full length intronic sequence, wherein the portion of a full length intronic sequence comprises intronic sequences required for a circularization reaction. In some embodiments, a first intronic sequence and a second intronic sequence, together, comprise a full length intronic sequence or a fragment thereof. In some embodiments, a first intronic sequence and a second intronic sequence, together, comprise a full length intronic sequence. In some embodiments, a first intronic sequence and a second intronic sequence, together, comprise a fragment of a full length intronic sequence.

[0174] In some embodiments, a first intronic sequence and a second intronic sequence comprise a nucleotide sequence from a same full length intronic sequence. In some embodiments, a first intronic sequence and a second intronic sequence comprise a nucleotide sequence from different full length intronic sequences. In some embodiments, a first intronic sequence and / or a second intronic sequence comprise a nucleotide sequence from two or more full length intronic sequences. In some embodiments, a first intronic sequence comprises a nucleotide sequence from two or more full length intronic sequences. In some embodiments, a first intronic sequence comprises or is a chimeric intronic sequence. In some embodiments, a second intronic sequence comprises a nucleotide sequence from two or more full length intronic sequences. In somePage 69 of 144133142t8vlAttorney Docket No. : 2019398-0005embodiments, a second intronic sequence comprises or is a chimeric intronic sequence. In some embodiments, a first intronic sequence and / or a second intronic sequence comprise or is derived from a naturally occurring intronic sequence.First Intronic Sequence

[0175] In some embodiments, a first intronic sequence comprises a naturally occurring intronic sequence. In some embodiments, a first intronic sequence is derived from a naturally occurring intronic sequence. In some embodiments, a first intronic sequence comprises an intronic sequence derived from a naturally occurring intronic sequence.

[0176] In some embodiments, a first intronic sequence comprises or is derived from a group 1 intronic sequence or a non-group 1 intronic sequence. In some embodiments, a first intronic sequence comprises a group I intronic sequence or a non-group I intronic sequence. In some embodiments, a first intronic sequence comprises or is derived from a group I intronic sequence or a group II intronic sequence. In some embodiments, a first intronic sequence comprises a group I intronic sequence or a group II intronic sequence. In some embodiments, a first intronic sequence comprises a group I intronic sequence. In some embodiments, a first intronic sequence comprises a group II intronic sequence.

[0177] In some embodiments, a first intronic sequence comprises or is derived from a bacterial intronic sequence, a viral intronic sequence, or a eukaryotic intronic sequence. In some embodiments, a first intronic sequence comprises a bacterial intronic sequence, a viral intronic sequence, or a eukaryotic intronic sequence. In some embodiments, a first intronic sequence is derived from a bacterial intronic sequence, a viral intronic sequence, or a eukaryotic intronic sequence. In some embodiments, a first intronic sequence comprises or is derived from a bacterial intronic sequence. In some embodiments, a first intronic sequence comprises or is derived from a viral intronic sequence. In some embodiments, a first intronic sequence comprises or is derived from a eukaryotic intronic sequence. In some embodiments, a first intronic sequence comprises a eukaryotic intronic sequence. In some embodiments, a eukaryotic intronic sequence is a fungi intronic sequence, a plant intronic sequence, or an algae intronic sequence. In some embodiments, a eukaryotic intronic sequence is a fungi intronic sequence. In some embodiments, a eukaryotic intronic sequence is a plant intronic sequence. In some embodiments, a eukaryotic intronic sequence is an algae intronic sequence.Page 70 of 144133142t8vlAttorney Docket No. : 2019398-0005

[0178] In some embodiments, a first intronic sequence comprises or is derived from an intronic sequence from a phage genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from a phage genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises or is derived from an intronic sequence from a cyanobacterium genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from a cyanobacterium genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises or is derived from an intronic sequence from a nitrogen-fixing bacterium genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from a nitrogen-fixing bacterium genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises or is derived from an intronic sequence from a protozoan genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from a protozoan genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises or is derived from an intronic sequence from a ciliophoran genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from a ciliophoran genome or an RNA transcribed therefrom.

[0179] In some embodiments, a first intronic sequence comprises or is derived from an intronic sequence from a bacteriophage T4 genome, an Anabaena azollae genome, an Anabaena PCC7120 genome, an Aphanizomenon flos-aquae (e g., strain NIVA-CYA 142) genome, a Synechococcus genome, an Azoarcus genome, a Tetrahymena thermophila genome, a trimorphomyces genome, or a Trimorphomyces papilionaceus genome, or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from a bacteriophage T4 genome, an Anabaena azollae genome, an Anabaena PCC7120 genome, an Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142) genome, a Synechococcus genome, an Azoarcus genome, a Tetrahymena thermophila genome, a trimorphomyces genome, or a Trimorphomyces papilionaceus genome, or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from a bacteriophage T4 genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from an Anabaena azollae genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from an AnabaenaPage 71 of 144133142t8vlAttorney Docket No. : 2019398-0005PCC7120 genome or an RNA transcribed therefrom. Tn some embodiments, a first intronic sequence comprises an intronic sequence from an Aphanizomenon flos-aquae (e.g., strain NIVA- CYA 142) genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from a Synechococcus genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from an Azoarcus genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from a Tetrahymena thermophila genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from a trimorphomyces genome or an RNA transcribed therefrom. In some embodiments, a first intronic sequence comprises an intronic sequence from a Trimorphomyces papilionaceus genome or an RNA transcribed therefrom.

[0180] In some embodiments, a first intronic sequence comprises or is derived from an intronic sequence from a T4 phage genome, or a pre-tRNALEUfrom Anabaena azollae, Anabaena PCC7120, Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142), or Synechococcus, or a pre-tRNAILEfrom Azoarcus, or a 26S ribosomal RNA from Tetrahymena thermophila, or a cytochrome C oxidase subunit 2 (COX2) from Trimorphomyces papilionaceus, or any combination thereof. In some embodiments, a first intronic sequence comprises an intronic sequence from a T4 phage genome, or a pre-tRNALEUfrom Anabaena azollae, Anabaena PCC7120, Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142), or Synechococcus, or a pre-tRNAILEfrom Azoarcus, or a 26S ribosomal RNA from Tetrahymena thermophila, or a cytochrome C oxidase subunit 2 (COX2) from Trimorphomyces papilionaceus, or any combination thereof. In some embodiments, a first intronic sequence comprises an intronic sequence from a T4 phage genome. In some embodiments, a first intronic sequence comprises an intronic sequence from a pre-tRNALEUfrom Anabaena azollae, Anabaena PCC7120, Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142), or Synechococcus. In some embodiments, a first intronic sequence comprises an intronic sequence from a pre-tRNALEUfrom Anabaena azollae. In some embodiments, a first intronic sequence comprises an intronic sequence from a pre-tRNALEUfrom Anabaena PCC7120. In some embodiments, a first intronic sequence comprises an intronic sequence from a pre-tRNALEUfrom Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142). In some embodiments, a first intronic sequence comprises an intronic sequence from a pre-tRNALEUfrom Synechococcus. In some embodiments, a first intronic sequencePage 72 of 14413314218vlAttorney Docket No. : 2019398-0005comprises an intronic sequence from a pre-tRNAILEfrom Azoarcus. Tn some embodiments, a first intronic sequence comprises an intronic sequence from a 26S ribosomal RNA from Tetrahymena thermophila. In some embodiments, a first intronic sequence comprises an intronic sequence from a cytochrome C oxidase subunit 2 (COX2) from Trimorphomyces papilionaceus . In some embodiments, a first intronic sequence comprises an intronic sequence derived from a naturally occurring intronic sequence.

[0181] In some embodiments, a first intronic sequence or a second intronic sequence comprise an IGS. In some embodiments, a first intronic sequence or a second intronic sequence comprise a catalytic core. In some embodiments, a first intronic sequence or a second intronic sequence comprise an IGS and a catalytic core. In some embodiments, a first intronic sequence comprises an IGS and a catalytic core. In some embodiments, a second intronic sequence comprises an IGS and a catalytic core. In some embodiments, a first intronic sequence comprises an IGS and a second intronic sequence comprises a catalytic core. In some embodiments, a first intronic sequence comprises a catalytic core and a second intronic sequence comprises an IGS.Catalytic Core

[0182] In some embodiments, a polynucleotide construct described herein comprises a first intronic sequence comprising a catalytic core. In some embodiments, a polynucleotide construct capable of forming a circRNA disclosed herein comprises a first intronic sequence and a second intronic sequence, wherein the first intronic sequence comprises a catalytic core. In some embodiments, a polynucleotide construct described herein comprises a first intronic sequence comprising an IGS and a catalytic core. In some embodiments, a polynucleotide construct capable of forming a circRNA disclosed herein comprises a first intronic sequence and a second intronic sequence, wherein the first intronic sequence comprises an IGS and a catalytic core.

[0183] In some embodiments, a catalytic core comprises one or more nucleotide sequences and / or structures that can facilitate a catalytic function. In some embodiments, a catalytic core is capable of facilitating a splicing reaction and / or a circularization reaction. In some embodiments, a catalytic core is capable of facilitating a circularization reaction. In some embodiments, a catalytic core facilitates a circularization reaction. In some embodiments, a catalytic core is capable of facilitating a splicing reaction. In some embodiments, a catalytic core is capable of mediating a self-splicing reaction. In some embodiments, a catalytic core mediates a self-splicing reaction.Page 73 of 144133142I8vlAttorney Docket No. : 2019398-0005

[0184] In some embodiments, a catalytic core comprises a nucleotide sequence of GAG and CC. In some embodiments, a catalytic core comprises a nucleotide sequence of GAG and CC separated by [N]n, wherein N is any one of A, C, G, T, or U, and n is an integer. In some embodiments, a catalytic core comprises a nucleotide sequence of ACG and CT(or U). In some embodiments, a catalytic core comprises a nucleotide sequence of ACG and CT(or U) separated by [N]n, wherein N is any one of A, C, G, T, or U, and n is an integer.

[0185] In some embodiments, a catalytic core sequence comprises a nucleotide sequence that is or comprises any one of nucleotide sequences described in Table 9. In some embodiments, a catalytic core sequence comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence of SEQ ID NO: 33 or 34. In some embodiments, a catalytic core comprises a nucleotide sequence of AGAGA[N]6-4iA[N]43-45GUCC (SEQ ID NO: 33), wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]e-4i, or [N]43-45, can be independently any one of A, U, C, or G. In some embodiments, a catalytic core comprises a nucleotide sequence of AGAGA[N]6-4OA[N]42-44GUCC (SEQ ID NO: 34), wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]e-4o, or [N]42-44, can be independently any one of A, U, C, or G. In some embodiments, a catalytic core comprises a nucleotide sequence of any one of AGAGA[N]6-4iA[N]43-45GUCC (SEQ ID NO: 33), ACAGA[N]6-4iA[N]43-4sGUCG (SEQ ID NO: 50), or ACGACU[N]7-6sUAGUCU (SEQ ID NO: 51), wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]e-4i, [N]43-45, or [N]7-68, can be independently any one of A, U, C, or G. In some embodiments, a catalytic core consists of a nucleotide sequence consisting of AGAGA[N]6-4iA[N]43-45GUCC (SEQ ID NO: 33), ACAGA[N]6-4iA[N]43-45GUCG (SEQ ID NO: 50), or ACGACU[N]7-68UAGUCU (SEQ ID NO: 51), wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]e-4i, [N]43-45, or [N]7-68, can be independently any one of A, U, C, or G.

[0186] Non-limiting examples of catalytic core sequences that can be used according to the present disclosure are shown in Table 9.Page 74 of 14413314218vlAttorney Docket No. : 2019398-0005Table 9. Exemplary Catalytic Core Sequences.

[0187] In some embodiments, a catalytic core consists of a nucleotide sequence of AGAGA[N]6-4iA[N]43-45GUCC (SEQ ID NO: 33), wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]e-4i, or [N]43-45, can be independently any one of A, U, C, or G. Tn some embodiments, a catalytic core consists of a nucleotide sequence of AGAGA[N]6-4OA[N]42-44GUCC (SEQ ID NO: 34), wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]e-40, or [N]42-44, can be independently any one of A, U, C, or G.

[0188] In some embodiments, a first intronic sequence comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence of any one of SEQ ID NOs: 35-38. In some embodiments, a first intronic sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 35-38. In some embodiments, a first intronic sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 35-38. In some embodiments, a first intronic sequence comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of any one of SEQ ID NOs: 35-38. In some embodiments, a first intronic sequence is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 35. In some embodiments, a first intronic sequence is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 36. In some embodiments, a first intronic sequence is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 37. In some embodiments, a first intronic sequence is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 38. In some embodiments, a first Page 75 of 144133142I8vlAttorney Docket No. : 2019398-0005intronic sequence is or comprises a nucleotide sequence of any one of SEQ ID NOs: 35-38. In some embodiments, a first intronic sequence is or comprises a nucleotide sequence of SEQ ID NO: 35. In some embodiments, a first intronic sequence is or comprises a nucleotide sequence of SEQ ID NO: 36. In some embodiments, a first intronic sequence is or comprises a nucleotide sequence of SEQ ID NO: 37. In some embodiments, a first intronic sequence is or comprises a nucleotide sequence of SEQ ID NO: 38. In some embodiments, a first intronic sequence disclosed herein consists of a nucleotide sequence of any one of SEQ ID NOs: 35-38.

[0189] Non-limiting examples of examples of first intronic sequences that can be used according to the present disclosure are shown in Table 10.Table 10. Exemplary First Intronic Sequences.Second Intronic Sequence

[0190] In some embodiments, a second intronic sequence comprises a naturally occurring intronic sequence. In some embodiments, a second intronic sequence is derived from a naturally occurring intronic sequence. In some embodiments, a second intronic sequence comprises an intronic sequence derived from a naturally occurring intronic sequence.

[0191] In some embodiments, a second intronic sequence comprises or is derived from a group I intronic sequence or a non-group I intronic sequence. In some embodiments, a second intronic sequence comprises a group I intronic sequence or a non-group I intronic sequence. InPage 76 of 144133142t8vlAttorney Docket No. : 2019398-0005some embodiments, a second intronic sequence comprises or is derived from a group I intronic sequence or a group II intronic sequence. In some embodiments, a second intronic sequence comprises a group I intronic sequence or a group II intronic sequence. In some embodiments, a second intronic sequence comprises a group I intronic sequence. In some embodiments, a second intronic sequence comprises a group II intronic sequence.

[0192] In some embodiments, a second intronic sequence comprises or is derived from a bacterial intronic sequence, a viral intronic sequence, or a eukaryotic intronic sequence. In some embodiments, a second intronic sequence comprises a bacterial intronic sequence, a viral intronic sequence, or a eukaryotic intronic sequence. In some embodiments, a second intronic sequence is derived from a bacterial intronic sequence, a viral intronic sequence, or a eukaryotic intronic sequence. In some embodiments, a second intronic sequence comprises or is derived from a bacterial intronic sequence. In some embodiments, a second intronic sequence comprises or is derived from a viral intronic sequence. In some embodiments, a second intronic sequence comprises or is derived from a eukaryotic intronic sequence. In some embodiments, a second intronic sequence comprises a eukaryotic intronic sequence. In some embodiments, a eukaryotic intronic sequence is a fungi intronic sequence, a plant intronic sequence, or an algae intronic sequence. In some embodiments, a eukaryotic intronic sequence is a fungi intronic sequence. In some embodiments, a eukaryotic intronic sequence is a plant intronic sequence. In some embodiments, a eukaryotic intronic sequence is an algae intronic sequence.

[0193] In some embodiments, a second intronic sequence comprises or is derived from an intronic sequence from a phage genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from a phage genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises or is derived from an intronic sequence from a cyanobacterium genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from a cyanobacterium genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises or is derived from an intronic sequence from a nitrogen-fixing bacterium genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from a nitrogen-fixing bacterium genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises or is derived from an intronic sequence from a protozoan genome or an RNA transcribed therefrom. In somePage 77 of 144133142t8vlAttorney Docket No. : 2019398-0005embodiments, a second intronic sequence comprises an intronic sequence from a protozoan genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises or is derived from an intronic sequence from a ciliophoran genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from a ciliophoran genome or an RNA transcribed therefrom.

[0194] In some embodiments, a second intronic sequence comprises or is derived from an intronic sequence from a bacteriophage T4 genome, an Anabaena azollae genome, an Anabaena PCC7120 genome, an Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142) genome, a Synechococcus genome, an Azoarcus genome, a Tetrahymena thermophila genome, a trimorphomyces genome, or a Trimorphomyces papilionaceus genome, or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from a bacteriophage T4 genome, an Anabaena azollae genome, an Anabaena PCC7120 genome, an Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142) genome, a Synechococcus genome, an Azoarcus genome, a Tetrahymena thermophila genome, a trimorphomyces genome, or a Trimorphomyces papilionaceus genome, or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from a bacteriophage T4 genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from an Anabaena azollae genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from an Anabaena PCC7120 genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from an Aphanizomenon flos-aquae (e.g., strain NIVA- CYA 142) genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from a Synechococcus genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from an Azoarcus genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from a Tetrahymena thermophila genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from a trimorphomyces genome or an RNA transcribed therefrom. In some embodiments, a second intronic sequence comprises an intronic sequence from a Trimorphomyces papilionaceus genome or an RNA transcribed therefrom.Page 78 of 14413314218vlAttorney Docket No. : 2019398-0005

[0195] In some embodiments, a second intronic sequence comprises or is derived from an intronic sequence from a T4 phage genome, or a pre-tRNALEUfrom Anabaena azollae, Anabaena PCC7120, Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142), or Synechococcus, or a pre-tRNAILEfrom Azoarcus, or a 26S ribosomal RNA from Tetrahymena thermophila, or a cytochrome C oxidase subunit 2 (COX2) from Trimorphomyces papilionaceus, or any combination thereof. In some embodiments, a second intronic sequence comprises an intronic sequence from a T4 phage genome, or a pre-tRNALEUfrom Anabaena azollae, Anabaena PCC7120, Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142), or Synechococcus, or a pre-tRNAILEfrom Azoarcus, or a 26S ribosomal RNA from Tetrahymena thermophila, or a cytochrome C oxidase subunit 2 (COX2) from Trimorphomyces papilionaceus, or any combination thereof. In some embodiments, a second intronic sequence comprises an intronic sequence from a T4 phage genome. In some embodiments, a second intronic sequence comprises an intronic sequence from a pre-tRNALEUfrom Anabaena azollae, Anabaena PCC7120, Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142), or Synechococcus. In some embodiments, a second intronic sequence comprises an intronic sequence from a pre-tRNALEUfrom Anabaena azollae. In some embodiments, a second intronic sequence comprises an intronic sequence from a pre-tRNALEUfrom Anabaena PCC7120. In some embodiments, a second intronic sequence comprises an intronic sequence from a pre-tRNALEUfrom Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142). In some embodiments, a second intronic sequence comprises an intronic sequence from a pre-tRNALEUfrom Synechococcus. In some embodiments, a second intronic sequence comprises an intronic sequence from a pre-tRNAILEfrom Azoarcus. In some embodiments, a second intronic sequence comprises an intronic sequence from a 26S ribosomal RNA from Tetrahymena thermophila. In some embodiments, a second intronic sequence comprises an intronic sequence from a cytochrome C oxidase subunit 2 (COX2) from Trimorphomyces papilionaceus. In some embodiments, a second intronic sequence comprises an intronic sequence derived from a naturally occurring intronic sequence.

[0196] In some embodiments, a second intronic sequence comprises an IGS and / or a catalytic core. In some embodiments, a second intronic sequence comprises an IGS and a catalytic core. In some embodiments, a catalytic core is capable of mediating a self-splicing reaction. In some embodiments, a catalytic core mediates a self-splicing reaction. In some embodiments, a catalytic core comprises a nucleotide sequence of GAG and CC. In some embodiments, a catalyticPage 79 of 144133142t8vlAttorney Docket No. : 2019398-0005core comprises a nucleotide sequence of GAG and CC separated by [N]n, wherein N is any one of A, C, G, T, or U, and n is an integer. In some embodiments, a catalytic core comprises a nucleotide sequence of ACG and CT(or U). In some embodiments, a catalytic core comprises a nucleotide sequence of ACG and CT(or U) separated by [N]n, wherein N is any one of A, C, G, T, or U, and n is an integer. In some embodiments, a catalytic core comprises a nucleotide sequence of any one of SEQ ID NOs: 33, 34, 50, or 51. In some embodiments, a catalytic core comprises a nucleotide sequence of AGAGA[N]6-4iA[N]43-4sGUCC (SEQ ID NO: 33), wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]6-4i, or [N]43-45, can be independently any one of A, U, C, or G. In some embodiments, a catalytic core comprises a nucleotide sequence of AGAGA[N]6-4OA[N]42-44GUCC (SEQ ID NO: 34), wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]6-40, or [N]42-44, can be independently any one of A, U, C, or G.

[0197] In some embodiments, a second intronic sequence comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence of any one of SEQ ID NOs: 39-42. In some embodiments, a second intronic sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 39-42. In some embodiments, a second intronic sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of any one of SEQ ID NOs: 39-42. In some embodiments, a second intronic sequence comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of any one of SEQ ID NOs: 39-42. In some embodiments, a second intronic sequence is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 39. In some embodiments, a second intronic sequence is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 40. In some embodiments, a second intronic sequence is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 41. In some embodiments, a second intronic sequence is or comprises aPage 80 of 14413314218vlAttorney Docket No. : 2019398-0005nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 42. In some embodiments, a second intronic sequence is or comprises a nucleotide sequence of any one of SEQ ID NOs: 39-42. In some embodiments, a second intronic sequence is or comprises a nucleotide sequence of SEQ ID NO: 39. In some embodiments, a second intronic sequence is or comprises a nucleotide sequence of SEQ ID NO: 40. In some embodiments, a second intronic sequence is or comprises a nucleotide sequence of SEQ ID NO: 41. In some embodiments, a second intronic sequence is or comprises a nucleotide sequence of SEQ ID NO: 42. In some embodiments, a second intronic sequence disclosed herein consists of a nucleotide sequence of any one of SEQ ID NOs: 39-42.

[0198] Non-limiting examples of examples of second intronic sequences that can be used according to the present disclosure are shown in Table 11.Table 11. Exemplary Second Intronic Sequences.Coding Sequence

[0199] Among other things, a polynucleotide construct described in the present disclosure comprises a coding sequence. In some embodiments, a polynucleotide construct described herein comprises a coding sequence and / or a non-coding sequence. In some embodiments, a polynucleotide construct described herein encodes a RNA coding sequence. In some embodiments, a polynucleotide construct described herein comprises a RNA coding sequence. In some embodiments, a polynucleotide construct capable of forming a circRNA disclosed herein comprises a coding sequence, wherein the coding sequence comprises a payload sequence. In some Page 81 of 14413314218vlAttorney Docket No. : 2019398-0005embodiments, a payload sequence encodes a target payload (target-encoding region), such as an encoded RNA (e.g., miRNA, siRNA), a target gene, protein, or peptide. In some embodiments, a payload sequence comprises a sequence encoding at least one target gene, target protein, or target peptide.

[0200] In some embodiments, a payload sequence comprises at least one target gene. In some embodiments, a payload sequence comprises a sequence encoding at least one target gene. In some embodiments, a payload sequence encodes a RNA. In some embodiments, a target gene comprises a synthetic nucleotide sequence. In some embodiments, a target gene comprises a single-stranded RNA (ssRNA) oligonucleotide, a double-stranded RNA (dsRNA) oligonucleotide, a single-stranded DNA (ssDNA) oligonucleotide, or a double- stranded DNA (dsDNA) oligonucleotide. In some embodiments, a target gene is a DNA sequence. In some embodiments, a target gene is a RNA sequence. In some embodiments, a target gene is a viral RNA. In some embodiments, a target gene is a non-viral RNA. In some embodiments, a target gene is a synthetic RNA. In some embodiments, a target gene is a therapeutic RNA.

[0201] In some embodiments, a payload sequence comprises a sequence encoding at least one target peptide. In some embodiments, a payload sequence comprises a sequence encoding two or more polypeptides in a same oligonucleotide. In some embodiments, a payload sequence comprises a sequence encoding at least one target polypeptide. In some embodiments, a payload sequence comprises a sequence encoding at least one target fusion polypeptide and / or chimeric polypeptide. In some embodiments, a payload sequence comprises a sequence encoding at least one target fusion polypeptide. In some embodiments, a payload sequence comprises a sequence encoding at least one target chimeric polypeptide.

[0202] In some embodiments, a payload sequence comprises a sequence encoding at least one target protein. In some embodiments, a payload sequence comprises a sequence encoding one target protein. In some embodiments, a payload sequence comprises a sequence encoding two or more target proteins. In some embodiments, a payload sequence comprises a sequence that encodes at least one target protein and a non-protein encoding sequence. In some embodiments, a nonprotein encoding sequence comprises a ribosomal skipping element and / or a nucleotide sequence encoding a protease cleavage site. In some embodiments, a nucleotide sequence encoding a protease cleavage site comprises one or more self-cleaving 2A peptides. In some embodiments, a self-cleaving 2A peptide comprises or is a thosea-asigna virus 2 A peptide (T2A), porcinePage 82 of 144133142i8vlAttorney Docket No. : 2019398-0005teschovirus-1 2 A peptide (P2A), foot-and-mouth disease virus 2 A peptide (F2A), equine rhinitis A vims 2A peptide (E2A), cytoplasmic polyhedrosis vims 2A peptide (BmCPV 2A), or flacherie vims of B. mori 2A peptide (BmIFV 2A).

[0203] In some embodiments, a target protein is a protein of eukaryotic or prokaryotic origin. In some embodiments, a target protein is a human protein or a non-human protein.

[0204] In some embodiments, a target protein is a protein for diagnostic use. In some embodiments, a target protein comprises a fluorescent protein or a luminescent protein. In some embodiments, a target protein comprises a Gaussia luciferase (Glue), Firefly luciferase (Flu), or enhanced green fluorescent protein (EGFP). In some embodiments, a target protein comprises a erythropoietin EPO. In some embodiments, a target protein comprises a human erythropoietin (hEPO). In some embodiments, a target protein comprises a Cas endonuclease. In some embodiments, a target protein comprises a Cas9 endonuclease or a Cas 12 endonuclease.

[0205] In some embodiments, a target protein is a therapeutic protein. In some embodiments, a therapeutic protein comprises an enzyme, a cytokine, an antibody, and / or a receptor. In some embodiments, a therapeutic protein comprises a viral antigenic polypeptide or an immunogenic fragment thereof. In some embodiments, a therapeutic protein comprises a adjuvant or adjuvant-like polypeptide or an immunogenic fragment thereof. In some embodiments, a therapeutic protein comprises an immunotherapy protein, a replacement therapy protein, an immune response inducing protein, and / or a gene editing protein.

[0206] In some embodiments, a therapeutic protein comprises a chimeric antigen receptor (CAR), T-cell receptor (TCR), B-cell receptor (BCR), immune cell activation or inhibitory receptor, Cas9 protein, bispecific T cell engager, recombinant fusion protein, chimeric mutant protein, fusion protein, or any combination thereof.

[0207] In some embodiments, a therapeutic protein comprises an antibody, nanobody, nonantibody protein, antigen binding protein or a functional fragment thereof, immune modulatory ligand, receptor, structural protein, growth factor ligand or receptor, hormone or hormone receptor, transcription factor, checkpoint inhibitor or agonist, Fc fusion protein, anticoagulant, blood clotting factor, chaperone protein, antimicrobial protein, structural protein, biochemical enzyme, tight junction protein, mitochondrial stress response protein, cytoskeletal protein, metal-binding protein, small molecule-binding protein, or any combination thereof.Page 83 of 14413314218vlAttorney Docket No. : 2019398-0005

[0208] In some embodiments, a therapeutic protein comprises an immune modulatory ligand. In some embodiments, an immune modulatory ligand comprises an interferon, cytokine, chemokine, and / or interleukin.

[0209] In some embodiments, a therapeutic protein comprises a structural protein. In some embodiments, a structural protein comprises a channel protein or nuclear pore protein, a cytokine, an immune checkpoint inhibitor, an agonist, an antagonist, a chimeric antigen receptor, one or more TCR chains, a secreted T cell or immune cell engager, a transcription factor, and / or an immunosuppressive enzyme.

[0210] In some embodiments, a therapeutic protein comprises a cytokine. In some embodiments, non-limiting examples of cytokines comprise, but are not limited to, IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-35, IL-12p70, IL-27beta, IFN-a, IFN-0, IFN-y, TGF beta, or a functional fragment thereof.

[0211] In some embodiments, a therapeutic protein comprises an antigen-binding protein or a functional fragment thereof. In some embodiments, an antigen binding protein or a functional fragment thereof, comprises a monopecific antigen-binding protein, a bispecific antigen-binding protein, or trispecific antigen-binding protein. In some embodiments, non-limiting examples of antigens comprise, but are not limited to, Ebola virus glycoprotein Ebola virus glycoprotein, a4 integrin, Amyloid beta, Amyloid beta protofibrils, Amyloid beta, N3pG (N-terminal truncated), Angiopoietin-like 3, B. anthrasis PA, B7-H3, B-cell maturation antigen, BLyS, Cis, C5, CCR4, CDlla, CD19, CD20, CD22, CD3, CD30, CD33, CD38, CD4, CD52, CD62 (aka P-selectin), CD79b, CGRP, CGRP receptor, Clostridium difficile enterotoxin B, Complement 5, CTLA-4, Dabigatran, Ebola virus, EGFR, EGFR, cMET, Endotoxin, EpCAM, EPCAM / CD3, Factor Ixa, X, FcRn, FGF23, Folate receptor alpha, G protein-coupled receptor 5D, CD3, GD2, gplOO, CD3, GPIIb / IIIa, HER2, IFNAR1, IFNg, IgE, IGF-1R, IL-12 / 23, IL-13, IL-17a, IL-17A, F, IL-17R, IL-ip, IL-23 p!9, IL-23pl9, IL-2R, IL-36 receptor, IL-4R a, IL-5, IL-5R a, IL-6, IL-6R, LAG-3, MASP-2, Nectin-4, PCSK9, PD-1, PDGFRa, PD-L1, Plasma kallikrelin, RANK-L, RSV, SARS-CoV-2, Sclerostin, SLAMF7, Thymic stromal lymphopoietin, Tissue factor, Tissue factor pathway inhibitor, TNF, TROP-2, EPO, VEGF, VEGF-A, VEGF-A, Ang-2, VEGFR2, von Willebrand factor, and / or a4p7 integrin.

[0212] In some embodiments, an antigen binding protein or a functional fragment thereof comprises or is an antibody or a functional fragment thereof. In some embodiments, non-limitingPage 84 of 14413314218vlAttorney Docket No. : 2019398-0005examples of antibodies or functional fragments thereof comprise, but are not limited to, Murom onab-CD3, Efalizumab, Tositumomab-1131, Nebacumab, Edrecolomab, Catumaxomab, Daclizumab, Olaratumab, Abciximab, Rituximab, Basiliximab, Palivizumab, Infliximab, Trastuzumab, Adalimumab, Ibritumomab tiuxetan, Omalizumab, Cetuximab, Bevacizumab, Natalizumab, Panitumumab, Ranibizumab, Eculizumab, Certolizumab pegol, Ustekinumab, Canakinumab, Golimumab, Ofatumumab, Tocilizumab, Denosumab, Belimumab, Ipilimumab, Brentuximab vedotin, Pertuzumab, Ado-trastuzumab emtansine, Raxibacumab, Obinutuzumab, Siltuximab ,Ramucirumab, Vedolizumab, Nivolumab, Pembrolizumab, Blinatumomab, Alemtuzumab, Evolocumab, Idarucizumab, Necitumumab, Dinutuximab, Secukinumab, Mepolizumab, Alirocumab, Daratumumab, Elotuzumab, Ixekizumab, Reslizumab, Bezlotoxumab, Atezolizumab, Obiltoxaximab, Brodalumab, Dupilumab, Inotuzumab ozogamicin, Guselkumab, Sarilumab, Avelumab, Emicizumab, Ocrelizumab, Benralizumab, Durvalumab, Gemtuzumab ozogamicin, Erenumab, erenumab-aooe, Galcanezumab, galcanezumab-gnlm, Burosumab, burosumab-twza, Lanadelumab, lanadelumab-flyo, Mogamulizumab, mogamulizumab-kpkc, Tildrakizumab, tildrakizumab-asmn, Fremanezumab, fremanezumab-vfrm, Ravulizumab, ravulizumab-cwvz, Cemiplimab, cemiplimab-rwlc, Ibalizumab, ibalizumab-uiyk, Emapalumab, emapalumab-lzsg, Moxetumomab pasudotox, moxetumomab pasudotox-tdfk, Caplacizumab, caplacizumab-yhdp, Risankizumab, risankizumab-rzaa, Polatuzumab vedotin, polatuzumab vedotin-piiq, Romosozumab, romosozumab-aqqg, Brolucizumab, brolucizumab-dbll, Crizanlizumab, crizanlizumab-tmca, Enfortumab vedotin, enfortumab vedotin-ejfv, [fam-]trastuzumab deruxtecan, fam-trastuzumab deruxtecan-nxki, Isatuximab, isatuximab-irfc, Belantamab mafodotin, belantamab mafodotin-blmf, Sacituzumab govitecan, sacituzumab govitecan-hziy, Tafasitamab, tafasitamab-cxix ,Satralizumab, satralizumab-mwge, Eptinezumab, eptinezumab-jjmr, Inebilizumab, inebilizumab-cdon, Teprotumumab, teprotumumab-trbw, Evinacumab, Dostarlimab, dostarlimab-gxly, Amivantamab, amivantamab-vmjw, Tralokinumab, tralokinumab-ldrm, Anifrolumab, anifrolumab-fnia, Loncastuximab tesirine, loncastuximab tesirine-lpyl, Atoltivimab, maftivimab, odesivimab-ebgn, Naxitamab-gqgk, Margetuximab-cmkb, Ansuvimab-zykl, Aducanumab, aducanumab-avwa, Regdanvimab, Sotrovimab, Tisotumab vedotin, tisotumab vedotin-tftv, Bimekizumab, Casirivimab + imdevimab, Tezepelumab, tezepelumab-ekko, Faricimab, faricimab-svoa, Sutimlimab, sutimlimab-jome, Tixagevimab, cilgavimab, Spesolimab, Nirsevimab, Teplizumab, teplizumab-mzwv, Ublituximab, Tebentafusp,Page 85 of 144133142I8vlAttorney Docket No. : 2019398-0005tebentafusp-tebn, Relatlimab, Mosunetuzumab, Teclistamab, Tremelimumab, Mirvetuximab soravtansine, mirvetuximab soravtansine-gynx, Lecanemab, Toripalimab, Trastuzumab duocarmazine, Epcoritamab, Mirikizumab, Glofitamab, Pozelimab, Lebrikizumab, Talquetamab, Rozanolixizumab, Cosibelimab, Concizumab, Elranatamab, Sugemalimab, Penpulimab, Donanemab, Sintilimab, Tislelizumab, Retifanlimab, Narsoplimab, and / or Omburtamab.

[0213] In some embodiments, a therapeutic protein comprises a chimeric antigen receptor (CARs or CAR-Ts). In some embodiments, non-limiting examples of CARs comprise, but are not limited to, CAR comprising an antigen binding domain specific for an antigen such as CD 19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule- 1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)), prostatespecific membrane antigen (PSMA), Receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-Like Tyrosine Kinase 3 (FLT3), Tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, Carcinoembryonic antigen (CEA), Epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), Interleukin- 13 receptor subunit alpha-2, mesothelin, Interleukin 11 receptor alpha (IL-llRa), prostate stem cell antigen (PSCA), Protease Serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis(Y) antigen, CD24, Platelet-derived growth factor receptor beta (PDGFR-beta), Stage-specific embryonic antigen-4 (SSEA-4), CD20, Folate receptor alpha, HER2, HER3, Mucin 1, cell surface associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), Prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), Ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF -I receptor), carbonic anhydrase IX (CAIX), Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2), glycoprotein 100 (gplOO), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type-A receptor 2 (EphA2), Fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight-melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), Folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), PolysialicPage 86 of 144133142I8vlAttorney Docket No. : 2019398-0005acid, placenta-specific 1 (PLAC1), hexasaccharide portion of globoH glycoceramide (GloboH), mammary gland differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), Hepatitis A virus cellular receptor 1 (HAVCR1), adrenoceptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K 9 (LY6K), Olfactory receptor 51E2 (OR51E2), TCR Gamma Alternate Reading Frame Protein (TARP), Wilms tumor protein (WT1), Cancer / testis antigen 1 (NY-ESO-1), Cancer / testis antigen 2 (LAGE-la), MAGE family members (including MAGE-A1, MAGE- A3 and MAGE-A4), ETS translocation- variant gene 6, located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X Antigen Family, Member 1 A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD- CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, surviving, telomerase, prostate carcinoma tumor antigen-1, melanoma antigen recognized by T cells 1, Rat sarcoma (Ras) mutant, human Telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoints, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-Acetyl glucosaminyl-transf erase V (NA17), paired box protein Pax-3 (PAX3), Androgen receptor, Cyclin Bl, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), Ras Homolog Family Member C (RhoC), Tyrosinase-related protein 2 (TRP-2), Cytochrome P450 1B1 (CYPIBI), CCCTC-Binding Factor (Zinc Finger Protein)-Like, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3), Paired box protein Pax-5 (PAX5), proacrosin binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchor protein 4 (AKAP-4), synovial sarcoma, X breakpoint 2 (SSX2), Receptor for Advanced Gly cation Endproducts (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papilloma virus E6 (HPV E6), human papilloma virus E7 (HPV E7), intestinal carboxyl esterase, heat shock protein 70-2 mutated (mut hsp70-2), CD79a, CD79b, CD72, Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF- like module-containing mucinlike hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), Glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), MUC16, 5T4, 8H9, u. P? integrin, avpe integrin, alpha fetoprotein (AFP), B7-H6, ca-125, CA9, CD44, CD44v7 / 8, CD52, E- cadherin, EMA (epithelial membrane antigen),Page 87 of 14413314218vlAttorney Docket No. : 2019398-0005epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB4, epithelial tumor antigen (ETA), folate binding protein (FBP), kinase insert domain receptor (KDR), k-light chain, LI cell adhesion molecule, MFJC18, NKG2D, oncofetal antigen (h5T4), tumor / testis-antigen IB, GAGE, GAGE-1, BAGE, SCP-1, CTZ9, SAGE, CAGE, CT 10, MART-1, immunoglobulin lambda-like polypeptide 1 (IGLL1), Hepatitis B Surface Antigen Binding Protein (HBsAg), viral capsid antigen (VC A), early antigen (EA), EBV nuclear antigen (EBNA), HHV-6 p41 early antigen, HHV-6B U94 latent antigen, HHV-6B p98 late antigen , cytomegalovirus (CMV) antigen, large T antigen, small T antigen, adenovirus antigen, respiratory syncytial virus (RSV) antigen, haemagglutinin (HA), neuraminidase (NA), parainfluenza type 1 antigen, parainfluenza type 2 antigen, parainfluenza type 3 antigen, parainfluenza type 4 antigen, Human Metapneumovirus (HMPV) antigen, hepatitis C virus (HCV) core antigen, HIV p24 antigen, human T-cell lympotrophic virus (HTLV-1) antigen, Merkel cell polyoma virus small T antigen, Merkel cell polyoma virus large T antigen, Kaposi sarcoma-associated herpesvirus (KSHV) lytic nuclear antigen and / or KSHV latent nuclear antigen.

[0214] In some embodiments, a therapeutic protein comprises a transcription factor. In some embodiments, non-limiting examples of transcription factors comprise, but are not limited to, Forkhead box P3 (Foxp3), and / or signal transducer and activator of transcription (STAT, e.g., STAT1, STAT2, STAT3, STAT4, STAT 5 (including STAT5 A and STAT5B), and STATE).

[0215] In some embodiments, a coding sequence comprises a payload sequence and / or one or more additional elements. In some embodiments, a coding sequence comprises one or more untranslated regions (UTRs), spacers, binding motifs, gene regulatory elements, and / or transcription terminators. In some embodiments, non-limiting examples of gene regulatory elements comprise, but are not limited to, promoters, transcriptional activators, enhancers, and / or polyadenylation signals.

[0216] In some embodiments, a payload sequence comprises a sequence of interest and / or one or more additional elements. In some embodiments, a payload sequence comprises one or more untranslated regions (UTRs). In some embodiments, a payload sequence comprises a 5' UTR and / or a 3' UTR. In some embodiments, a payload sequence comprises one or more UTRs, wherein one or more UTRs comprise a 5' UTR. In some embodiments, a 5' UTR comprises a Kozak sequence. In some embodiments, a payload sequence comprises one or more UTRs, wherein one or more UTRs comprise a 3' UTR.Page 88 of 14413314218vlAttorney Docket No. : 2019398-0005

[0217] In some embodiments, a coding sequence can be of any length, for example, between about 2 and about 100,000,000 nucleotides in length (or any integer value therebetween). In some embodiments, a coding sequence is about 50-50,000 nucleotides in length. In some embodiments, a coding sequence is about 50-25,000 nucleotides in length. In some embodiments, a coding sequence is about 100-20,000 nucleotides in length. In some embodiments, a coding sequence is about 500-10,000 nucleotides in length. In some embodiments, a coding sequence is about 1,000-8,000 nucleotides in length. In some embodiments, a coding sequence is about 2,000-5,000 nucleotides in length. In some embodiments, a coding sequence is at least 50, at least 100, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 8,000, at least 20,000, at least 25,000, at least 35,000, or at least 45,000 nucleotides in length. In some embodiments, a coding sequence comprises a payload sequence that is about 50-25,000 nucleotides in length.

[0218] In some embodiments, a payload sequence is about 100-20,000 nucleotides in length. In some embodiments, a payload sequence is about 500-10,000 nucleotides in length. In some embodiments, a payload sequence is about 1,000-8,000 nucleotides in length. In some embodiments, a payload sequence is about 2,000-5,000 nucleotides in length. In some embodiments, a payload sequence comprises at least 20 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, at least 550 nucleotides, at least 600 nucleotides, at least 650 nucleotides, at least 700 nucleotides, at least 750 nucleotides, at least 800 nucleotides, at least 850 nucleotides, at least 900 nucleotides, at least 950 nucleotides, at least 1000 nucleotides, at least 1100 nucleotides, at least 1200 nucleotides, at least 1300 nucleotides, at least 1400 nucleotides, at least 1500 nucleotides, at least 1600 nucleotides, at least 1700 nucleotides, at least 1800 nucleotides, at least 2000 nucleotides, at least 2500 nucleotides, at least 3000 nucleotides, at least 3000 nucleotides, at least 4000 nucleotides, at least 5000 nucleotides, at least 6000 nucleotides, at least 7000 nucleotides, at least 8000 nucleotides, at least 9000 nucleotides, at least 10,000 nucleotides, at least 11,000 nucleotides, at least 12,000 nucleotides, at least 13,000 nucleotides, at least 14,000 nucleotides, at least 15,000 nucleotides, at least 16,000 nucleotides, at least 17,000 nucleotides, at least 18,000 nucleotides, at least 19,000 nucleotides, at least 20,000 nucleotides, at least 21,000 nucleotides, at least 22,000 nucleotides, at least 23,000 nucleotides, at least 24,000 nucleotides, or at least 25,000 nucleotides.Page 89 of 144133142t8vlAttorney Docket No. : 2019398-0005Other FeaturesUTRs and Kozak Sequence

[0219] In some embodiments, polynucleotide constructs disclosed herein comprise one or more untranslated regions (UTRs) and / or one or more Kozak sequences. In some embodiments, a polynucleotide construct described in the present disclosure comprises a payload sequence comprising one or more untranslated regions (UTRs) and / or one or more Kozak sequences. In some embodiments, a polynucleotide construct comprises a 5' UTR and / or a 3' UTR. In some embodiments, UTRs of a gene comprise nucleotide sequences that can be transcribed but not translated. In some embodiments, a 5'-UTR comprises a nucleotide sequence comprising initial sequence of a transcription start site. In some embodiments, a start codon is not comprised in a nucleotide sequence following a transcription start site. In some embodiments, a 3'-UTR comprises a nucleotide sequence that starts after a stop codon and / or continues until a transcriptional termination signal. In some embodiments, UTRs can contribute to stability of a polynucleotide construct and / or a translation reaction comprising a polynucleotide construct.

[0220] In some embodiments, a polynucleotide construct disclosed herein comprises one or more UTRs, wherein the polynucleotide construct has an increased stability. In some embodiments, a payload sequence disclosed herein comprises one or more UTRs, wherein the payload sequence has an increased stability compared to a payload sequence without an UTR. In some embodiments, an increased stability of a payload sequence can result in an increased expression of an encoded protein. In some embodiments, natural occurring 5'-UTRs comprise nucleotide sequences that can mediate translation initiation. In some embodiments, natural occurring 5'-UTRs comprise Kozak sequences. In some embodiments, Kozak sequences can be involved in a process by which a ribosome initiates translation of a gene. In some embodiments, Kozak sequences comprise a nucleotide sequence of CCR(A / G)CCAUGG, wherein R is a purine (adenine or guanine) three bases upstream of a start codon (AUG), and wherein the start codon if followed by G.

[0221] In some embodiments, natural occurring 5'-UTRs comprise nucleotide sequences that can form secondary structures and / or mediate elongation factor binding. In some embodiments, 5'-UTR secondary structures that mediate elongation factor binding can interact with other RNA binding molecules in a 5'-UTR or 3'-UTR and / or regulate gene expression. In some embodiments, microRNA mediated repression comprise an elongation factor EIF4A2Page 90 of 14413314218vlAttorney Docket No. : 2019398-0005binding and / or a secondary structure element in a 5 -UTR Tn some embodiments, a 5'-UTR comprises secondary structures in a flanking region to stabilize and / or destabilize a mRNA.

[0222] In some embodiments, a 5'-UTR of liver-expressed mRNA can be used to enhance expression of a nucleotide sequence in hepatic cell lines or liver. In some embodiments, nonlimiting examples of a 5'-UTR of liver-expressed mRNA comprises, but not limited to, albumin, serum amyloid A, Apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, and / or Factor VIII. In some embodiments, a 5'-UTR derived from a tissue-specific mRNA can improve expression in that tissue. In some embodiments, non-limiting examples of a 5'-UTR of muscle -expressed mRNA comprises, but not limited to, MyoD, Myosin, Myoglobin, Myogenin, and / or Herculin. In some embodiments, non-limiting examples of a 5'-UTR of endothelial cells-expressed mRNA comprises, but not limited to, Tie-1 and / or CD36). In some embodiments, nonlimiting examples of a 5'-UTR of myeloid cells-expressed mRNA comprises, but not limited to, C / EBP, AML1, G-CSF, GM-CSF, CD1 lb, MSR, Fr-1, and / or i-NOS. In some embodiments, nonlimiting examples of a 5’-UTR of leukocytes-expressed mRNA comprises, but not limited to, CD45 and / or CD18. In some embodiments, non-limiting examples of a 5'-UTR of adipose tissue-expressed mRNA comprises, but not limited to, CD36, GLUT4, ACRP30, and / or adiponectin. In some embodiments, non-limiting examples of a 5'-UTR of lung epithelial cells-expressed mRNA comprises, but not limited to, SP-A, SP-B, SP-C, and / or SP-D.

[0223] In some embodiments, a 5' UTR comprises a nucleotide sequence from a human beta globin, Xenopus laevis beta globin, human alpha globin, Xenopus laevis alpha globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70kDa protein 1 A, tobacco alcohol dehydrogenase, tobacco etch virus, turnip crinkle virus, or the adenovirus tripartite leader.

[0224] In some embodiments, natural occurring 3'-UTRs comprise nucleotide sequences comprising a sequence of adenosines and / or uridines. In some embodiments, natural occurring 3'-UTRs comprise nucleotide sequences comprising one or more sequences of AU. In some embodiments, natural occurring 3'-UTRs comprise AU rich elements (AREs). In some embodiments, AU rich elements (AREs) comprise one or more nucleotides that are mutated, replaced and / or removed. In some embodiments, modified AU rich elements (AREs) can modulate stability of a nucleotide sequence.Page 91 of 144133142I8vlAttorney Docket No. : 2019398-0005

[0225] In some embodiments, a 3' UTR comprises a nucleotide sequence from a human beta globin, human alpha globin Xenopus beta globin, Xenopus alpha globin, human prolactin, human GAP -43, human eEFlal, human Tau, human TNFa, dengue virus, hantavirus small mRNA, bunyavirus small mRNA, turnip yellow mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human tubulin, hibiscus chlorotic ringspot virus, woodchuck hepatitis virus post translationally regulated element, sindbis virus, turnip crinkle virus, tobacco etch virus, or Venezuelan equine encephalitis virus.

[0226] In some embodiments, UTRs comprise non-UTR sequences. In some embodiments, UTRs comprise one ore more intronic sequences or a fragment thereof in flanking regions of a nucleotide sequence.

[0227] In some embodiments, a UTR comprises one or more nucleotides that are mutated, replaced and / or removed. In some embodiments, a UTR comprises one or more nucleotides upstream of a start codon that are replaced with another nucleotide. In some embodiments, a UTR comprises one or more nucleotides upstream of a start codon that are removed.Poly(A)

[0228] In some embodiments, polynucleotide constructs disclosed herein comprise a polyA sequence and / or a polyA-C sequence. In some embodiments, a polynucleotide construct comprises a polyA sequence. In some embodiments, a polynucleotide construct comprises a polyA-C sequence. In some embodiments, a polyA sequence is removed upon a circularization reaction comprising a polynucleotide construct described herein. In some embodiments, a polyA sequence is not removed upon a circularization reaction comprising a polynucleotide construct described herein. In some embodiments, a polyA-C sequence is removed upon a circularization reaction comprising a polynucleotide construct described herein. In some embodiments, a polyA-C sequence is not removed upon a circularization reaction comprising a polynucleotide construct described herein. In some embodiments, a polyA sequence or a polyA-C sequence is at least 10, at least 15, at least 30, or at least 60 nucleotides in length. In some embodiments, a polyA sequence or a polyA-C sequence can hybridize with an oligonucleotide to separate a circRNA from a precursor linear RNA polynucleotide construct comprising a polyA sequence. In some embodiments, a polyA sequence or a polyA-C sequence comprised in a polynucleotide construct disclosed herein hybridizes with a deoxythymine oligonucleotide (oligo(dT)) conjugated to a solid surface (e.g., a resin). In some embodiments, a polyA sequence or a polyA-C sequence comprisedPage 92 of 144133142i8vlAttorney Docket No. : 2019398-0005in linear polynucleotide constructs can hybridize with an oligonucleotide during circRNA purification.

[0229] In some embodiments, a polyA-C sequence comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence of SEQ ID NO: 43 or 44. In some embodiments, a polyA-C sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 43 or 44. In some embodiments, a polyA-C sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 43 or 44. In some embodiments, a polyA-C sequence is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 43. In some embodiments, a polyA-C sequence is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 44. In some embodiments, a polyA-C sequence is or comprises a nucleotide sequence of SEQ ID NO: 43. In some embodiments, a polyA-C sequence is or comprises a nucleotide sequence of SEQ ID NO: 44. In some embodiments, a polyA-C sequence disclosed herein consists of a nucleotide sequence of any one of SEQ ID NO: 43 or 44.

[0230] Non-limiting examples of second intronic sequences that can be used according to the present disclosure are shown in Table 12.Table 12. Exemplary polyA Sequences.Modified Nucleotides

[0231] Over one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-Page 93 of 14413314218vlAttorney Docket No. : 2019398-0005197, which is incorporated herein by reference in its entirety). In some embodiments, a polyribonucleotide described herein may have modified nucleosides. In some embodiments, a polyribonucleotide comprises a modified nucleoside in place of at least one (e.g., every) uridine.

[0232] In some embodiments, polynucleotide constructs disclosed herein comprise one or more modified nucleotides. In some embodiments, polynucleotide constructs disclosed herein comprise a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0233] In some embodiments, a polynucleotide construct comprises one or more modified nucleotides comprising: N4-acetylcytidine (ac4C), 5-hydroxymethyluridine (5hmu), Nl-methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5-methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1 -methylinosine (ml I), wyosine (imG), methylwyosine (mimG), 5-hydroxy cytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5 -hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N 1 -hydroxypseudouridine, 2'-O-methyl-Nl -methylpseudouridine, Nl-ethylpseudouridine, Nl-hydroxymethylpseudouridine, and arauridine, N 6-methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 7-deazaadenosine, 8-oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 6-O-methylguanine, or any combination thereof.

[0234] In some embodiments, a polynucleotide construct comprises one or more uridines replaced by a modified nucleoside. In some embodiments, one or more uridines are replaced with a modified nucleoside comprising pseudouridine (y), Nl-methyl-pseudouridine (mly) or 5-methyl-uridine (m5U).

[0235] In some embodiments, a polynucleotide construct comprises one or more modified nucleosides comprising: 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, pseudo-iso-cytidine, 5-Page 94 of 14413314218vlAttorney Docket No. : 2019398-0005ami noallyl -uridine, 5-iodo-uridine, N1 -methyl -pseudouridine, 5,6-dihydrouridine, a-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudo-uridine, inosine, a-thio-guanosine, 8-oxo-guanosine, O6-methyl-guanosine, 7-deaza-guanosine, Nl-methyl adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-purine, N6-methyl-adenosine, a-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrolo-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5 -iodo-cytidine, or any combination thereof.

[0236] In some embodiments, polynucleotide constructs disclosed herein comprise one or more uridines replaced by a modified nucleoside. In some embodiments, one or more uridines are replaced with a modified nucleoside comprising pseudouridine (y), Nl-methyl-pseudouridine (ml\| / ) or 5-methyl-uridine (m5U).II. Linear polynucleotide construct

[0237] The present disclosure provides, among other things, linear polynucleotide constructs (e.g. RNA or DNA). In some embodiments, a polynucleotide construct is a RNA polynucleotide construct. In some embodiments, a polynucleotide construct is a linear RNA polynucleotide construct. In some embodiments, a polynucleotide construct is a linear RNA polynucleotide construct comprising a permuted IRES sequence. In some embodiments, a linear RNA polynucleotide construct comprising a permuted IRES sequence can be used for synthesis of a circRNA.

[0238] In some embodiments, a polynucleotide construct is a DNA polynucleotide construct. In some embodiments, a polynucleotide construct is a linear DNA polynucleotide construct. In some embodiments, a polynucleotide construct is a linear DNA polynucleotide construct encoding a permuted IRES sequence. In some embodiments, a linear DNA polynucleotide construct encoding a permuted IRES sequence can be transcribed into a linear RNA polynucleotide construct. In some embodiments, a linear RNA polynucleotide construct comprising a permuted IRES sequence transcribed from a linear DNA polynucleotide construct encoding a permuted IRES sequence can be used for synthesis of a circRNA.

[0239] In some embodiments, a polynucleotide construct disclosed herein is a RNA polynucleotide construct produced by transcription of a DNA polynucleotide construct. In some embodiments, a polynucleotide construct is a RNA polynucleotide construct produced by in vivo transcription of a DNA polynucleotide construct. In some embodiments, a polynucleotide construct is a linear RNA polynucleotide construct produced by in vivo transcription of a DNAPage 95 of 144133142i8vlAttorney Docket No. : 2019398-0005polynucleotide construct. Tn some embodiments, a polynucleotide construct is a RNA polynucleotide construct produced by in vitro transcription of a DNA polynucleotide construct. In some embodiments, a polynucleotide construct is a linear RNA polynucleotide construct produced by in vitro transcription of a DNA polynucleotide construct. In some embodiments, a polynucleotide construct disclosed herein is a RNA polynucleotide construct produced by chemical synthesis. In some embodiments, a polynucleotide construct disclosed herein is a linear RNA polynucleotide construct produced by chemical synthesis. Polynucleotide constructs (e.g., linear polynucleotide construct) disclosed herein can be unmodified, partially modified or completely modified.

[0240] In some embodiments, a polynucleotide construct disclosed herein can undergo a splicing reaction. In some embodiments, a RNA polynucleotide construct disclosed herein can undergo a splicing reaction. In some embodiments, a RNA polynucleotide construct transcribed from a DNA polynucleotide construct disclosed herein, can undergo a splicing reaction. In some embodiments, a splicing reaction is an auto-catalytic splicing reaction (i.e., self-splicing reaction). In some embodiments, a splicing reaction removes one or more intronic sequences from a RNA polynucleotide construct. In some embodiments, a splicing reaction circularizes one or more coding sequences comprised in a RNA polynucleotide construct.

[0241] Among other things, the present disclosure provides a polynucleotide construct comprising a first intronic sequence, a first portion of an IRES sequence, a coding sequence, a second portion of an IRES sequence, and / or a second intronic sequence. In some embodiments, a polynucleotide construct comprises a first intronic sequence, a first portion of an IRES sequence, a coding sequence, a second portion of an IRES sequence, a second intronic sequence, first internal guide sequence (IGS) and / or a second internal guide sequence (IGS'). In some embodiments, an IGS' is capable of hybridizing to an IGS. In some embodiments, a polynucleotide construct disclosed herein comprises a permuted IRES sequence. In some embodiments, a polynucleotide construct disclosed herein can produce circRNAs that do not comprise an excision scar. In some embodiments, a scarless circRNA produced by a polynucleotide construct of the present disclosure do not have increased immunogenicity.Exemplary Linear Polynucleotide constructs

[0242] The present disclosure provides RNA or DNA polynucleotide constructs capable of forming circular RNAs disclosed herein (e g., a linear RNA polynucleotide construct or a linearPage 96 of 14413314218vlAttorney Docket No. : 2019398-0005DNA polynucleotide construct from which a linear RNA polynucleotide construct can be transcribed) comprising a first intronic sequence, a first portion of an IRES sequence, a coding sequence, a second portion of an IRES sequence, and a second intronic sequence.

[0243] In some embodiments, a polynucleotide construct described in the present disclosure can be a polynucleotide construct encoding a permuted IRES sequence described herein. In some embodiments, a polynucleotide construct encoding a permuted IRES sequence is a DNA polynucleotide construct. In some embodiments, a polynucleotide construct encoding a permuted IRES sequence is a linear DNA polynucleotide construct.

[0244] In some embodiments, a DNA polynucleotide construct can be configured according to formula I: 5'-P-H-A-3S-E-5S-B-H'-3'. In some embodiments, a DNA polynucleotide construct configured according to formula I comprises a promoter sequence represented as “P”. In some embodiments, a DNA polynucleotide construct configured according to formula I comprises one or more homology arms. In some embodiments, a DNA polynucleotide construct configured according to formula I comprises a 5' homology arm represented as “H”. In some embodiments, a DNA polynucleotide construct configured according to formula I comprises a 3' homology arm represented as “H1”. In some embodiments, a DNA polynucleotide construct configured according to formula I comprises a first intronic sequence (which can comprise e.g., a group I 3' intronic sequence) represented as “A”. In some embodiments, a DNA polynucleotide construct configured according to formula I comprises a first intronic sequence comprising a first internal guide sequence (IGS). In some embodiments, a DNA polynucleotide construct configured according to formula I comprises a first intronic sequence comprising a catalytic core. In some embodiments, a DNA polynucleotide construct configured according to formula I comprises a first splice site sequence (e.g. a 3' splice site sequence) represented as “3S”. In some embodiments, a DNA polynucleotide construct configured according to formula I comprises a second splice site sequence (e.g. a 5' splice site sequence) represented as “5S”. In some embodiments, a DNA polynucleotide construct configured according to formula I comprises an exonic sequence, represented as “E”. In some embodiments, a DNA polynucleotide construct configured according to formula I comprises an exonic sequence comprising a permuted IRES sequence as provided herein and a coding sequence. In some embodiments, a DNA polynucleotide construct configured according to formula I comprises a second intronic sequence (which can comprise e.g., a group I 5' intronic sequence) represented as “B”.Page 97 of 144133142I8vlAttorney Docket No. : 2019398-0005

[0245] In some embodiments, a DNA polynucleotide construct can be configured according to formula la: 5'-P-H-A-3S-C-CDS-D-5S-B-H'-3'. In some embodiments, a DNA polynucleotide construct configured according to formula la comprises an exonic sequence comprising a first portion of an IRES sequence (e.g. a 3' IRES sequence) represented as “C”. In some embodiments, a DNA polynucleotide construct configured according to formula la comprises an exonic sequence comprising a second portion of an IRES sequence (e.g. a 5' IRES sequence) represented as “D”. In some embodiments, a DNA polynucleotide construct configured according to formula la comprises an exonic sequence comprising a coding sequence represented as “CDS”. In some embodiments, a DNA polynucleotide construct configured according to formula la comprises an exonic sequence comprising a first portion of an IRES sequence (e.g. a 3' IRES sequence) (C), a second portion of an IRES sequence (e.g. a 5' IRES sequence) (D), and a coding sequence (CDS). In some embodiments, a DNA polynucleotide construct configured according to formula la comprises a second portion of an IRES sequence comprising a second internal guide sequence (IGS').

[0246] In some embodiments, a polynucleotide construct described in the present disclosure can be a polynucleotide construct comprising a permuted IRES sequence described herein. In some embodiments, a polynucleotide construct comprising a permuted IRES sequence is a RNA polynucleotide construct. In some embodiments, a polynucleotide construct comprising a permuted IRES sequence is a linear RNA polynucleotide construct.

[0247] In some embodiments, a RNA polynucleotide construct can be configured according to formula II: 5'-H-A-3S-E-5S-B-H'-3'. In some embodiments, a RNA polynucleotide construct configured according to formula II comprises one or more homology arms. In some embodiments, a RNA polynucleotide construct configured according to formula II comprises a 5' homology arm represented as “H”. In some embodiments, a RNA polynucleotide construct configured according to formula II comprises a 3' homology arm represented as “H1”. In some embodiments, a RNA polynucleotide construct configured according to formula II comprises a first intronic sequence (which can comprise e.g., a group I 3' intronic sequence) represented as “A”. In some embodiments, a RNA polynucleotide construct configured according to formula II comprises a first intronic sequence comprising a first internal guide sequence (IGS). In some embodiments, a RNA polynucleotide construct configured according to formula II comprises a first intronic sequence comprising a catalytic core. In some embodiments, a RNA polynucleotidePage 98 of 144133142i8vlAttorney Docket No. : 2019398-0005construct configured according to formula TI comprises a first splice site sequence (e.g. a 3' splice site sequence) represented as “3S”. In some embodiments, a RNA polynucleotide construct configured according to formula II comprises a second splice site sequence (e.g. a 5' splice site sequence) represented as “5S”. In some embodiments, a RNA polynucleotide construct configured according to formula II comprises an exonic sequence, represented as “E”. In some embodiments, a RNA polynucleotide construct configured according to formula II comprises an exonic sequence comprising a permuted IRES sequence as provided herein and a coding sequence. In some embodiments, a RNA polynucleotide construct configured according to formula II comprises a second intronic sequence (which can comprise e.g., a group I 5' intronic sequence) represented as “B”

[0248] In some embodiments, a RNA polynucleotide construct can be configured according to formula Ila: 5'-H-A-3S-C-CDS-D-5S-B-H'-3'. In some embodiments, a RNA polynucleotide construct configured according to formula Ila comprises an exonic sequence comprising a first portion of an IRES sequence (e.g. a 3' IRES sequence) represented as “C”. In some embodiments, a RNA polynucleotide construct configured according to formula Ila comprises an exonic sequence comprising a second portion of an IRES sequence (e.g. a 5' IRES sequence) represented as “D”. In some embodiments, a RNA polynucleotide construct configured according to formula Ila comprises an exonic sequence comprising a coding sequence represented as “CDS”. In some embodiments, a RNA polynucleotide construct configured according to formula Ila comprises an exonic sequence comprising a first portion of an IRES sequence (e.g. a 3' IRES sequence) (C), a second portion of an IRES sequence (e.g. a 5' IRES sequence) (D), and a coding sequence (CDS). In some embodiments, a RNA polynucleotide construct configured according to formula Ila comprises a second portion of an IRES sequence comprising a second internal guide sequence (IGS1).

[0249] In some embodiments, the disclosure comprises a RNA polynucleotide construct produced by a chemical synthesis. In some embodiments, a RNA polynucleotide construct is transcribed from a linear DNA polynucleotide construct or method(s) described herein.

[0250] Exemplary embodiments of polynucleotide constructs disclosed herein are described in detail below. For simplicity, exemplary polynucleotide constructs are described with reference to a RNA polynucleotide construct. It is to be understood that a DNA polynucleotide construct, from which such RNA polynucleotide construct can be transcribed, is also to bePage 99 of 144133142i8vlAttorney Docket No. : 2019398-0005considered as encompassed by the following description. As such, for example, any RNA nucleotide sequence reciting uracil (U) is to be understood as encompassing a corresponding DNA nucleotide sequence reciting thymine (T). In some embodiments, a RNA polynucleotide construct is capable of producing a circRNA. In some embodiments, a RNA polynucleotide construct is capable of producing a circRNA in the presence of an exogenous G.

[0251] In some embodiments, a polynucleotide construct is comprised in a vector. In some embodiments, a vector is a DNA vector. In some embodiments, a vector is a RNA vector. In some embodiments, a vector is a DNA / RNA hybrid vector. In some embodiments, a vector is a plasmid.

[0252] Non-limiting examples of polynucleotide constructs of the present disclosure are shown in Table 13.Table 13. Exemplary polynucleotide constructs.Page 100 of 14413314218vlAttorney Docket No. : 2019398-0005Page 101 of 14413314218vlAttorney Docket No. : 2019398-0005

[0253] In some embodiments, a polynucleotide construct comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a nucleotide sequence of SEQ ID NO: 45 or 46. In some embodiments, a polynucleotide construct comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a nucleotide sequence of SEQ ID NO: 45. In some embodiments, a polynucleotide construct comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of SEQ ID NO: 45. In some embodiments, a polynucleotide construct comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a nucleotide sequence of SEQ ID NO: 46. In some embodiments, a polynucleotide construct comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of SEQ ID NO: 46.III. Circular polynucleotide construct

[0254] The present disclosure provides, among other things, a circRNA produced by a polynucleotide construct or method(s) described herein. In some embodiments, a circRNA can be produced by a linear polynucleotide construct described herein. In some embodiments, a circRNA described in the present disclosure is produced by a linear RNA polynucleotide construct. In some embodiments, a circRNA is produced by a linear RNA polynucleotide construct, wherein the linear RNA polynucleotide construct is produced by chemical synthesis. In some embodiments, circRNAs as described herein can be candidates as therapeutic agents. In some embodiments, a therapeutic circRNA can be formed by covalently closing a linear RNA polynucleotide construct.

[0255] In some embodiments, circRNAs are closed single-stranded RNA nucleotide sequences that lack a 5' cap structure and a poly-A tail. In some embodiments, translation initiation in a circRNA depends on recognition of an internal ribosome entry site (IRES). In some embodiments, IRES sequences can enable protein translation independent of a 5' cap structure. In some embodiments, a circRNA is more stable than a linear RNA polynucleotide construct. In some embodiments, a circRNA does not require a 5' cap structure or a 3' poly (A) tail to function and / or are more resistant to degradation by RNA exonucleases.Page 102 of 14413314218vlAttorney Docket No. : 2019398-0005

[0256] Use of therapeutic circRNAs has been limited in part, due to difficulty in purifying circRNAs, low fidelity and reproducibility of a circularization reaction, and / or need for sequence elements that can be used in various therapeutic circRNAs in a modular fashion. Current polynucleotide constructs that generate therapeutic circRNAs may include synthetic spacer sequences, which may increase immunogenicity of therapeutic circRNAs.

[0257] The present disclosure also provides, polynucleotide constructs that can generate circRNAs without increased immunogenicity. In some embodiments, the present disclosure provides polynucleotide constructs that produce therapeutic circRNAs, wherein the therapeutic circRNAs do not have increased immunogenicity.

[0258] In some embodiments, the present disclosure provides polynucleotide constructs comprising a permuted IRES sequence that can produce circRNAs that do not comprise an excision scar. In some embodiments, the present disclosure provides a scarless circRNA. In some embodiments, a scarless circRNA produced by a polynucleotide construct of the present disclosure do not have increased immunogenicity. In some embodiments, a circRNA described herein does not have increased immunogenicity and / or can be a therapeutic circRNA.

[0259] Among other things, the present disclosure provides a circRNA produced by a polynucleotide construct described herein. In some embodiments, a circRNA does not comprise a sequence derived from an organism from which a first intronic sequence and / or a second intronic sequence are derived from (“excision scar”). In some embodiments, a circRNA produced as described herein is a scarless circRNA. In some embodiments, a circRNA does not comprise an excision scar.

[0260] The present disclosure provides a polynucleotide construct capable of producing a scarless circRNA. In some embodiments, a polynucleotide construct disclosed herein can circularize to produce a circRNA. In some embodiments, a polynucleotide construct circularizes to produce a circRNA.

[0261] In some embodiments, a circRNA described in the present disclosure comprises an IRES sequence and / or a coding sequence. In some embodiments, a circRNA comprises an IRES sequence. In some embodiments, a circRNA comprises a coding sequence. In some embodiments, a circRNA comprises an IRES sequence and / or a coding sequence. In some embodiments, a circRNA comprises an IRES sequence that is directly connected to a coding sequence. In some embodiments, an IRES sequence comprises a second internal guide sequence (IGS1). In somePage 103 of 14413314218vlAttorney Docket No. : 2019398-0005embodiments, an IRES sequence comprises a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence of SEQ ID NO: 47. In some embodiments, an IRES sequence comprises a nucleotide sequence having at least about 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 47. In some embodiments, an IRES sequence has at least about 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with a nucleotide sequence of SEQ ID NO: 47. In some embodiments, an IRES sequence is or comprises a nucleotide sequence of SEQ ID NO: 47. In some embodiments, an IRES sequence is or comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence of SEQ ID NO: 47. In some embodiments, an IRES sequence disclosed herein consists of a nucleotide sequence of SEQ ID NO: 47.

[0262] Non-limiting examples of IRES sequences that can be used according to the present disclosure are shown in Table 14. IGS' appear as underlined in Table 14.Table 14. Exemplary IRES Sequences.Page 104 of 14413314218vlAttorney Docket No. : 2019398-0005

[0263] In some embodiments, a circRNA is at least about 1000, at least about 1500, at least about 2000, at least about 2500, at least about 3000, at least about 3500, at least about 4000 or at least about 4500 nucleotides in size. In some embodiments, a circRNA is at least about Ikb.

[0264] In some embodiments, a circRNA described in the present disclosure has a half-life or functional half-life of at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 30 hours, at least 40 hours, at least 50 hours, at least 60 hours, at least 70 hours or at least 80 hours. In some embodiments, a circRNA provided herein has a half-life of about 5-80, about 10-70, about 15-60, and / or about 20-50 hours. In some embodiments, a circRNA has a half-life or functional half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days.

[0265] In some embodiments, a half-life or functional half-life is determined by a nucleic acid assay. In some embodiments, a functional half-life is determined by a functional protein assay. In some embodiments, a circRNA comprises a nucleotide sequence encoding a reporter gene. In some embodiments, a functional half-life is determined by an in vitro luciferase assay measuring expression of a reporter gene. In some embodiments, a functional half-life is determined by an in vitro luciferase assay, wherein activity of Gaussia luciferase (Glue) is measured in cells (e g. HepG2) expressing a circRNA. In some embodiments, activity of Glue is measured every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In some embodiments, a functional half-life is determined by an in vitro fluorescence assay, wherein fluorescence (e.g., from a GFP protein) is measured in cells (e.g. HepG2) expressing circRNA every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In other embodiments, a functional half-life is determined by an in vivo assay, wherein levels of a protein encoded by a circRNA are measured in patient serum or tissue samples every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days.

[0266] In some embodiments, a circRNA described herein a) induces less immunogenicity, b) induces less production of IFN-pi, RIG-I, IL-2, IL-6, IFN-y, and / or TNFa transcript when exposed to an immune system of an organism or a certain type of immune cell, and / or c) modulates production of cytokine when exposed to an immune system of an organism or a certain type of immune cell, compared to a same circRNA comprising an excision scar.

[0267] In some embodiments, a circRNA described herein does not comprise an excision scar and has less immunogenicity than a same circRNA comprising an excision scar and comprising a same sequence. In some embodiments, a circRNA provided herein has anPage 105 of 144133142i8vlAttorney Docket No. : 2019398-0005immunogenicity at least 0.5, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 folds lower than that of a same circRNA comprising an excision scar and comprising a same sequence. In some embodiments, a circRNA provided herein has less immunogenicity than a same circRNA comprising an excision scar and comprising a same sequence when administered to a cell. In some embodiments, a cell is comprised in an organism. In some embodiments, an organism is a human. In some embodiments, a circRNA provided herein has less immunogenicity than a same circRNA comprising an excision scar and comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload. In some embodiments, a circRNA provided herein has an immunogenicity at least 0.5, at least 1, 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 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 folds lower than that of a same circRNA comprising an excision scar and comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload. In some embodiments, a circRNA provided herein has less immunogenicity than a same circRNA comprising an excision scar and comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload when administered to a cell. In some embodiments, a cell is comprised in an organism. In some embodiments, an organism is a human. In some embodiments, a circRNA has an immunogenicity lower than or equal to that of a pre-determined threshold value. In some embodiments, the pre-determined threshold value is an immunogenicity of a reference circRNA comprising an excision scar and comprising a same sequence as a circRNA. In some embodiments, the pre-determined threshold value is an immunogenicity of a reference circRNA comprising an excision scar and comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload. In some embodiments, a circRNA has an immunogenicity lower than or equal to that of a pre-determined threshold value when administered to a cell. In some embodiments, a cell is comprised in an organism. In some embodiments, an organism is a human.Page 106 of 144133142t8vlAttorney Docket No. : 2019398-0005

[0268] In some embodiments, a circRNA described herein is less immunogenic than an equivalent circRNA comprising an excision scar (i.e., a circRNA comprising an excision scar and comprising a same sequence, same modifications, an same optimized UTR, and encoding a same payload) when exposed to an immune system of an organism or a certain type of immune cell.

[0269] In some embodiments, a circRNA provided herein does not comprise an excision scar. In some embodiments, a circRNA provided herein are associated with modulated production of cytokines when exposed to an immune system of an organism or a certain type of immune cell. In some embodiments, a circRNA provided herein are associated with reduced production of IFN-P 1 , RIG-I, IL-2, IL-6, IFN-y, and / or TNFa when exposed to an immune system of an organism or a certain type of immune cell as compared to an equivalent circRNA comprising an excision scar (i.e., a circRNA comprising a same sequence as the circRNA, and further comprising an excision scar; or a circRNA comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload as circRNA (e.g., mRNA), and further comprising an excision scar).

[0270] In some embodiments, a circRNA provided herein is associated with less IFN-pi, RIG-I, IL-2, IL-6, IFN-y, and / or TNFa transcript induction when exposed to an immune system of an organism or a certain type of immune cell as compared to an equivalent circRNA comprising an excision scar (i.e., a circRNA comprising a same sequence as the circRNA, and further comprising an excision scar; or a circRNA comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload as circRNA (e.g., mRNA), and further comprising an excision scar).

[0271] In some embodiments, a circRNA described herein does not comprise a spacer and has less immunogenicity than a same circRNA comprising a spacer and comprising a same sequence. In some embodiments, a circRNA provided herein has an immunogenicity at least 0.5, at least 1, 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 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 folds lower than that of a same circRNA comprising a spacer and comprising a same sequence. In some embodiments, a circRNA provided herein has less immunogenicity than a same circRNA comprising a spacer and comprising a same sequence when administered to a cell. In some embodiments, a cell is comprised in an organism. In somePage 107 of 144133142i8vlAttorney Docket No. : 2019398-0005embodiments, a organism is a human. In some embodiments, a circRNA provided herein has less immunogenicity than a same circRNA comprising a spacer and comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload. In some embodiments, a circRNA provided herein has an immunogenicity at least 0.5, at least 1, 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 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 folds lower than that of a same circRNA comprising a spacer and comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload. In some embodiments, a circRNA provided herein has less immunogenicity than a same circRNA comprising a spacer and comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload when administered to a cell. In some embodiments, a cell is comprised in an organism. In some embodiments, a organism is a human. In some embodiments, a circRNA has an immunogenicity lower than or equal to that of a pre-determined threshold value. In some embodiments, a pre-determined threshold value is an immunogenicity of a reference circRNA comprising a spacer and comprising a same sequence as a circRNA. In some embodiments, a pre-determined threshold value is an immunogenicity of a reference circRNA comprising a spacer and comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload. In some embodiments, a circRNA has an immunogenicity lower than or equal to that of a pre-determined threshold value when administered to a cell. In some embodiments, a cell is comprised in an organism. In some embodiments, an organism is a human.

[0272] In some embodiments, a circRNA described herein is less immunogenic than an equivalent circRNA comprising a spacer (i.e., a circRNA comprising a spacer and comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload) when exposed to an immune system of an organism or a certain type of immune cell.

[0273] In some embodiments, a circRNA provided herein does not comprise a spacer. In some embodiments, a circRNA provided herein are associated with modulated production of cytokines when exposed to an immune system of an organism or a certain type of immune cell. In some embodiments, a circRNA provided herein is associated with reduced production of IFN-[31,Page 108 of 144133142I8vlAttorney Docket No. : 2019398-0005RIG-I, IL-2, IL-6, IFN-y, and / or TNFa when exposed to an immune system of an organism or a certain type of immune cell as compared to an equivalent circRNA comprising a spacer (i.e., a circRNA comprising a same sequence as the circRNA, and further comprising an a spacer; or a circRNA comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload as circRNA (e.g., mRNA), and further comprising a spacer).

[0274] In some embodiments, a circRNA provided herein is associated with less IFN-pi, RIG-I, IL-2, IL-6, IFN-y, and / or TNFa transcript induction when exposed to an immune system of an organism or a certain type of immune cell as compared to an equivalent circRNA comprising a spacer (i.e., a circRNA comprising a same sequence as the circRNA, and further comprising a spacer; or a circRNA comprising a same sequence, a same modification, a same optimized UTR, and encoding a same payload as circRNA (e.g., mRNA), and further comprising a spacer).

[0275] In some embodiments, transcription of a DNA polynucleotide construct (e.g., comprised in a vector), is in vitro. In some embodiments, in vitro transcription of a DNA polynucleotide construct (e.g., comprised in a vector), is in a cell. In some embodiments, a cell is a prokaryotic cell. In some embodiments, a cell is a eukaryotic cell. In vitro transcription of a precursor RNA polynucleotide construct from a DNA polynucleotide construct (e.g., comprised in a vector) in a cell can be via added polymerases or polymerases encoded by nucleic acids transfected into a cell, or via endogenous (e.g., RNA polymerase II). In some embodiments, a polynucleotide construct is transcribed in cytoplasm of a cell. In some embodiments, a polynucleotide construct is transcribed in the nucleus of a cell. In some embodiments, in vitro transcription of a DNA polynucleotide construct (e.g., comprised in a vector), is in a cell-free reaction. In vitro transcription of a precursor RNA polynucleotide construct from a DNA polynucleotide construct (e.g., comprised in a vector) in a cell free reaction can be by any RNA polymerase. In some embodiments, a RNA polymerase is a bacteriophage RNA polymerase (e.g., T7, T3, or Sp6).

[0276] In some embodiments, circularization of a RNA polynucleotide construct to produce a circRNA, is in vitro. In some embodiments, in vitro circularization of a RNA polynucleotide construct to produce a circRNA, is in a cell. In some embodiments, a cell is a prokaryotic cell. In some embodiments, a cell is a eukaryotic cell. In some embodiments, a circRNA are produced in the cytoplasm of a cell. In some embodiments, a circRNA are producedPage 109 of 144133142I8vlAttorney Docket No. : 2019398-0005in the nucleus of a cell. In some embodiments, circularization of a RNA polynucleotide construct to produce a circRNA is in a cell-free reaction.

[0277] In some embodiments, transcription of a DNA polynucleotide construct (e.g., comprised in a vector), is in vivo. In some embodiments, DNA polynucleotide constructs (e.g., comprised in a vector) is administered to a cell comprised in a multicellular organism and a polynucleotide construct is transcribed from a DNA polynucleotide construct (e.g., comprised in a vector) in a cell comprised in the multicellular organism.

[0278] In some embodiments, circularization of a RNA polynucleotide construct to produce a circRNA is in vivo. In some embodiments, DNA polynucleotide constructs (e.g., comprised in a vector) are administered to a cell comprised in a multicellular organism, a polynucleotide construct is transcribed from a DNA polynucleotide construct (e.g., comprised in a vector) and a RNA polynucleotide construct circularizes in a cell comprised in a multicellular organism. In some embodiments, a RNA polynucleotide construct is administered to a cell comprised in a multicellular organism and a RNA polynucleotide construct circularizes in a cell comprised in a multicellular organism.

[0279] In certain embodiments, a circRNA provided herein is administered to a cell. In some embodiments, the sequence encoding the payload comprised in circRNAs described herein is expressed in a cell. In some embodiments, a cell in comprised in an organism. In some embodiments, an organism is an animal. In some embodiments, an animal is a human.Exemplary Circular Polynucleotide constructs

[0280] The present disclosure provides circRNA polynucleotide constructs. In some embodiments, the present disclosure provides a scarless circRNA. In some embodiments, a circRNA described herein does not have increased immunogenicity. In some embodiments, a circRNA described herein can be used as a therapeutic circRNA.

[0281] In some embodiments, a circRNA described in the present disclosure comprises an exonic sequence comprised in a linear RNA polynucleotide construct, represented as “E” in a RNA polynucleotide construct configured according to formula II.

[0282] In some embodiments, a circRNA can be configured according to formula III: C-CDS-D. In some embodiments, a circRNA configured according to formula III comprises a first portion of an IRES sequence (e.g. a 3' IRES sequence) represented as “C”. In some embodiments, a circRNA configured according to formula III comprises a second portion of an IRES sequencePage 110 of 14413314218vlAttorney Docket No. : 2019398-0005(e g. a 5' IRES sequence) represented as “D”. In some embodiments, a circRNA configured according to formula III comprises a coding sequence represented as “CDS”. In some embodiments, a circRNA configured according to formula III comprises a first portion of an IRES sequence (e.g. a 3' IRES sequence) (C), a second portion of an IRES sequence (e.g. a 5' IRES sequence) (D), and a coding sequence (CDS). In some embodiments, a circRNA configured according to formula III comprises a second portion of an IRES sequence comprising a second internal guide sequence (IGS1). In some embodiments, a circRNA configured according to formula III comprises a first portion of an IRES sequence (e.g. a 3' IRES sequence) (C), a second portion of an IRES sequence (e.g. a 5' IRES sequence) (D), wherein the first portion of an IRES sequence and the second portion of an IRES sequence, together, comprise an IRES sequence. In some embodiments, a circRNA configured according to formula III comprises a functional IRES sequence as provided herein and a coding sequence.

[0283] In some embodiments, a circRNA configured according to formula III does not comprise an excision scar. In some embodiments, a circRNA configured according to formula III is a scarless circRNA.

[0284] In some embodiments, a circRNA is comprised in a vector. In some embodiments, a vector is a DNA vector. In some embodiments, a vector is a RNA vector. In some embodiments, a vector is a DNA / RNA hybrid vector. In some embodiments, a vector is a plasmid.

[0285] Non-limiting examples of circRNAs of the present disclosure are shown in Table 15Page 111 of 14413314218vlAttorney Docket No. : 2019398-0005Table 15. Exemplary circRNAs constructs.Page 112 of 14413314218vlAttorney Docket No. : 2019398-0005

[0286] In some embodiments, a circRNA comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a nucleotide sequence of SEQ ID NO: 48 or 49. In some embodiments, a circRNA comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a nucleotide sequence of SEQ ID NO: 48. In some embodiments, a circRNA comprises a nucleotide sequence having at least 90% identity with a nucleotide sequencePage 113 of 14413314218vlAttorney Docket No. : 2019398-0005of SEQ ID NO: 48. In some embodiments, a circRNA comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a nucleotide sequence of SEQ ID NO: 49. In some embodiments, a circRNA comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of SEQ ID NO: 49.IV. Methods of Making circRNA

[0287] Among other things, the present disclosure provides methods of making circRNA. In some embodiments, a method of making a circRNA comprises a step of transcribing a RNA polynucleotide construct described herein. For example, in some embodiments, a RNA polynucleotide construct as described herein is transcribed from a DNA polynucleotide construct template as described herein. In some embodiments, a RNA polynucleotide construct is transcribed from a double-stranded DNA (dsDNA) template. In some embodiments, a dsDNA template may be linear. In some embodiments, a dsDNA template may be circular.

[0288] In some embodiments, transcription of a RNA polynucleotide construct from a template DNA polynucleotide construct may be performed using a polymerase. In some embodiments, a polymerase is an RNA polymerase. In some embodiments, a polymerase is a DNA dependent RNA-polymerase. In some embodiments, a polymerase is an RNA dependent RNA-polymerase. In some embodiments, an RNA polymerase is RNA polymerase I, RNA polymerase II, RNA polymerase III, T7 RNA polymerase, POLRMT, Primase, PrimPol, or a combination thereof. In some embodiments, an RNA polymerase is T7 RNA polymerase.

[0289] In some embodiments, a method of making a circRNA comprises a step of structure formation of a RNA polynucleotide construct. In some embodiments, structure formation will occur spontaneously. For example, in some embodiments, a RNA polynucleotide construct transcribed from a template DNA polynucleotide construct as described herein comprises sequences capable of hybridizing and / or base pairing that may form a structure (e.g., spontaneously). In some embodiments, a structure may be formed spontaneously by hydrogen bonds. For example, in some embodiments, a transcribed RNA polynucleotide construct comprises a first intronic sequence, a first portion of an internal ribosomal entry signal (IRES) sequence, a coding sequence, a second portion of an IRES sequence, and a second intronic sequence, wherein the first intronic sequence comprises a first internal guide sequence (IGS) and the second portionPage 114 of 144133142I8vlAttorney Docket No. : 2019398-0005of an IRES sequence comprises a second internal guide sequence (IGS'), and wherein an IGS' is capable of hybridizing to an IGS.

[0290] In some embodiments, a polynucleotide construct described herein comprises a first intronic sequence and / or a second intronic sequence derived from a group I intronic sequence. Group I introns are self-splicing introns, which can catalyze their own excision from primary transcripts (e.g., pre-mRNA, pre-tRNA and pre-rRNA) in a wide range of organisms (e.g., spontaneously). In some embodiments, a first intronic sequence and a second intronic sequence are removed from a RNA polynucleotide construct by an autocatalytic RNA splicing reaction. In some embodiments, an autocatalytic RNA splicing reaction is mediated by hybridizing and / or base-pairing between an IGS comprised in a first intronic sequence and an IGS' comprised in a second portion of an IRES sequence.

[0291] In some embodiments, a circularization of a RNA polynucleotide construct comprises a two-step transesterification reaction with an exogenous GTP (aG), wherein 3 -OH acts as an initiating nucleophile. In some embodiments, a aG in a G-binding site in P7 position binds, and a 3-OH of GTP attacks a 5' splice site. In some embodiments, a first transesterification step comprises attachment of a aG to a 5'-end of an intron RNA by a 3’-5’ phosphodiester bond. In some embodiments, a first transesterification step is followed by conformational changes allowing upstream terminal 3' guanosine (coG) to trade position with aG and occupy a G-binding site to initiate a second transesterification reaction. In some embodiments, 3 '-OH of an upstream exon attacks a 3' splice site.

[0292] In some embodiments, a RNA polynucleotide construct comprises a permuted IRES sequence separated by a coding sequence. That is, in some embodiments, a RNA polynucleotide construct comprises a first portion of an IRES sequence and a second portion of an IRES sequence that are in reverse position (e.g., the 3' IRES sequence is located at the 5' and the 5' IRES sequence is located at the 3' of a RNA polynucleotide construct). In some embodiments, a RNA polynucleotide construct does not comprise a functional IRES sequence. Circularizing a RNA polynucleotide construct results in a circRNA comprising a full and / or functional IRES sequence. In some embodiments, a circRNA comprises an IRES sequence capable of initiating translation.

[0293] A RNA polynucleotide construct provided herein can be generated by incubating a vector provided herein under conditions permissive of transcription of a precursor RNAPage 115 of 14413314218vlAttorney Docket No. : 2019398-0005polynucleotide construct encoded by a vector. In some embodiments, a precursor RNA polynucleotide construct is synthesized by incubating a DNA polynucleotide construct (e.g., comprised in a vector) provided herein that comprises an RNA polymerase promoter with a compatible RNA polymerase enzyme (e.g., a T7, T3, or Sp6 RNA polymerase) under conditions permissive of in vitro transcription. In some embodiments, a DNA polynucleotide construct (e.g., comprised in a vector) is incubated inside of a cell and transcribed by a bacteriophage RNA polymerase or by a RNA polymerase II.

[0294] In some embodiments, provided herein is a method of generating a RNA polynucleotide construct by performing in vitro transcription using a DNA polynucleotide construct (e.g., comprised in a vector) provided herein as a template (e.g., a vector provided herein comprising a RNA polymerase promoter). In some embodiments, a resulting RNA polynucleotide construct can be used to generate circRNAs provided herein by incubating it in the presence of magnesium and ammonium ions at a temperature at which RNA circularization occurs (e.g., between 20 °C and 60 °C). In some embodiments, a method of circularizing a RNA polynucleotide construct comprises incubating a composition. In some embodiments, a RNA polynucleotide construct circularizes when a composition is incubated. In some embodiments, a RNA polynucleotide construct circularizes when a composition is incubated for 1 hour to 3 hours at 20 °C to 60 °C. In some embodiments, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of a RNA polynucleotide construct circularizes when a composition is incubated for 1 hour to 3 hours at 20 °C to 60 °C.

[0295] In some embodiments, a composition comprising circRNA is purified. circRNA may be purified by any known method known in the art, such as column chromatography, gel filtration chromatography, and size exclusion chromatography. In some embodiments, purification comprises one or more of the following steps: phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some embodiments, purification comprises the following steps in order: RNase R digestion, phosphatase treatment, and HPLC size exclusion purification. In some embodiments, purification comprises reverse phase HPLC. In some embodiments, a purified composition contains less double stranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins, protein ligases, capping enzymes and / or nicked RNA than unpurified RNA.Page 116 of 14413314218vlAttorney Docket No. : 2019398-0005In some embodiments, a purified composition is less immunogenic than an unpurified composition. In some embodiments, immune cells exposed to a purified composition produce less IFN-pi, RIG-I, IL-2, IL-6, IFN-y, and / or TNFa than immune cells exposed to an unpurified composition.V. Vectors and Cells

[0296] Vectors and cells comprising each and every one of polynucleotide constructs described herein are also provided. In some embodiments, an expression vector comprises a polynucleotide construct and / or a circRNA described herein. In some embodiments, polynucleotide constructs can be comprised in a vector. In some embodiments, a DNA polynucleotide construct is comprised in a vector. In some embodiments, a RNA polynucleotide construct is comprised in a vector. In some embodiments, a circRNA is comprised in a vector. In some embodiments, a vector is a DNA vector. In some embodiments, a vector is a RNA vector. In some embodiments, a vector is a DNA / RNA hybrid vector. In some embodiments, a vector is a plasmid.

[0297] In some embodiments, a host cell comprises a polynucleotide construct described herein. In some embodiments, a host cell comprises a DNA polynucleotide construct described herein. In some embodiments, a host cell comprises a RNA polynucleotide construct described herein. In some embodiments, a host cell comprises a circRNA described herein. In some embodiments, a host cell comprises an expression vector comprising a polynucleotide construct described herein. In some embodiments, a host cell comprises an expression vector comprising a DNA polynucleotide construct described herein. In some embodiments, a host cell comprises an expression vector comprising a RNA polynucleotide construct described herein. In some embodiments, a host cell comprises an expression vector comprising a circRNA described herein. In some embodiments, a host cell is a prokaryotic cell. In some embodiments, a host cell is a eukaryotic cell. In some embodiments, a eukaryotic cell is a human cell.VI. Compositions

[0298] Among other things, the present disclosure provides compositions. Compositions disclosed herein comprise one or more polynucleotide constructs and / or one or more circRNAs described herein. In some embodiments, a composition comprises one or more polynucleotide constructs and one or more circRNAs. In some embodiments, a composition comprises one or more DNA polynucleotide constructs, RNA polynucleotide constructs, circRNAs, or any Page 117 of 144133142i8vlAttorney Docket No. : 2019398-0005combination thereof In some embodiments, a composition comprises one or more DNA polynucleotide constructs described herein. In some embodiments, a composition comprises one or more RNA polynucleotide constructs described herein. In some embodiments, a composition comprises one or more circRNAs described herein.

[0299] In some embodiments, compositions disclosed herein comprise one or more polynucleotide constructs described herein and one or more additional components. In some embodiments, one or more additional components comprise a salt, a buffer, or any additional feature that may facilitate the transcription of the DNA polynucleotide constructs disclosed herein into a RNA polynucleotide construct disclosed herein, and / or any additional feature that may facilitate circularization of a RNA polynucleotide construct disclosed herein to produce a circRNA disclosed herein.

[0300] In some embodiments, a composition comprising one or more polynucleotide constructs described herein is a pharmaceutical composition. In some embodiments, a composition comprising one or more circRNAs described herein is a pharmaceutical composition.VII. Pharmaceutical Compositions

[0301] In some embodiments, provided herein are pharmaceutical compositions comprising a DNA polynucleotide construct (e.g., comprised in a vector), a RNA polynucleoti...

Claims

Attorney Docket No. : 2019398-0005CLAIMSWhat is claimed is:

1. A polynucleotide construct comprising:a first intronic sequence,a first portion of an internal ribosomal entry signal (IRES) sequence,a coding sequence,a second portion of an IRES sequence, anda second intronic sequence,wherein the first intronic sequence comprises a first internal guide sequence (IGS) and the second portion of an IRES sequence comprises a second internal guide sequence (IGS'), and wherein an IGS' is capable of hybridizing to an IGS.

2. The polynucleotide construct of claim 1, wherein an IGS' is capable of base pairing with at least 50% of an IGS.

3. The polynucleotide construct of claim 1 or 2, wherein an IGS comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of any one of SEQ ID NOs: 1- 4.

4. The polynucleotide construct of claim 1 or 2, wherein an IGS’ comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of any one of SEQ ID NOs: 5-8.

5. The polynucleotide construct of any one of claims 1 to 4, wherein the first portion of an IRES sequence has at least 90% identity with a nucleotide sequence of SEQ ID NO: 12.

6. The polynucleotide construct of any one of claims 1 to 5, wherein the second portion of an IRES sequence has at least 90% identity with a nucleotide sequence of SEQ ID NO: 13.

7. The polynucleotide construct of any one of claims 1 to 6, wherein the first portion of an IRES sequence and the second portion of an IRES sequence, together, form an IRES sequence.Page 139 of 144133142I8vlAttorney Docket No. : 2019398-00058. The polynucleotide construct of any one of claims 1 to 7, wherein an IRES sequence comprises a Type I IRES sequence, a Type II IRES sequence, a Type III IRES sequence, a Type IV IRES sequence, or a Type V IRES sequence.

9. The polynucleotide construct of claim 7 or 8, wherein an IRES sequence comprises one or more modifications to increase or reduce IRES activity.

10. The polynucleotide construct of claim 9, wherein the one or more modifications comprise adding one or more accessory sequences to an IRES sequence, truncating the 5' and / or 3' ends of an IRES sequence, adding a spacer 5' to an IRES sequence, modifying a Kozak sequence, modifying alternative translation initiation sites, creating a chimeric IRES sequence, creating a hybrid IRES sequence, or any combination thereof.

11. The polynucleotide construct of any one of claims 1 to 10, wherein an IRES sequence has at least 90% identity with a nucleotide sequence of any one of SEQ ID NOs: 9-11.

12. The polynucleotide construct of claim 1, further comprising a promoter sequence.

13. The polynucleotide construct of any one of claims 1 to 12, wherein the polynucleotide construct comprises at least one splice site.

14. The polynucleotide construct of claim 13, wherein the polynucleotide construct comprises a first splice site and a second splice site.

15. The polynucleotide construct of any one of claims 1 to 14, wherein the first intronic sequence is directly joined to the first portion of an IRES sequence at a first junction, and the second portion of an IRES sequence is directly j oined to the second intronic sequence at a second junction.

16. The polynucleotide construct of claim 14 or 15, wherein(i) the first splice site comprises the first junction;(ii) the second splice site comprises the second junction;(iii) the first splice site sequence comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of SEQ ID NO: 17 or 19; and / orPage 140 of 144133142I8vlAttorney Docket No. : 2019398-0005(iv) the second splice site sequence comprises a) a nucleotide sequence having at least 90% identity with a nucleotide sequence of SEQ ID NO: 18 or 20; or b) a nucleotide sequence of NAGU AAA, wherein N is A, U, C, or G, and wherein “A” indicates the position of the phosphodiester bond that is cleaved during a splicing reaction.

17. The polynucleotide construct of any one of claims 1 to 16, wherein the polynucleotide construct further comprises a 5' homology arm and / or a 3' homology arm.

18. The polynucleotide construct of claim 17, wherein the 5' homology arm is capable of hybridizing to a 3' homology arm.

19. The polynucleotide construct of claim 17 or 18, wherein the 5' homology arm comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of any one of SEQ ID NOs: 21-25.

20. The polynucleotide construct of any one of claims 17 to 19, wherein the 3' homology arm comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of any one of SEQ ID NOs: 26-30.

21. The polynucleotide construct of any one of claims 1 to 20, wherein the first intronic sequence and / or the second intronic sequence comprise:(i) a bacterial intronic sequence, a viral intronic sequence, or a eukaryotic intronic sequence;(ii) a fungi intronic sequence, a plant intronic sequence, or an algae intronic sequence; (iii) a group I intronic sequence or a group II intronic sequence;(iv) an intronic sequence from a bacteriophage T4 genome, an Anabaena azollcie genome, an Anabaena PCC7120 genome, an Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142) genome, a Synechococcus genome, an Azoarcus genome, a Tetrahymena thermophila genome, a trimorphomyces genome, or a Trimorphomyces papilionaceus genome, or an RNA transcribed therefrom;(v) an intronic sequence from a T4 phage genome, or a pre-tRNALEUfrom Anabaena azollae, Anabaena PCC7120, Aphanizomenon flos-aquae (e.g., strain NIVA-CYA 142), or Synechococcus, or a pre-tRNAILEfrom Azoarcus, or a 26 S ribosomal RNA from TetrahymenaPage 141 of 14413314218vlAttorney Docket No. : 2019398-0005thermophila, or a cytochrome C oxidase subunit 2 (C0X2) from Trimorphomyces papilionaceus, or any combination thereof; and / or(vi) a nucleotide sequence derived from a nucleotide sequence having at least 90% identity with a nucleotide sequence of any one of SEQ ID NO: 31 or 32.

22. The polynucleotide construct of any one of claims 1 to 21, wherein the first intronic sequence comprises a catalytic core.

23. The polynucleotide construct of claim 22, wherein the catalytic core comprises a nucleotide sequence of AGAGA[N]e-4iA[N]43-45GUCC, wherein N is any one of A, U, C, or G, and wherein each N in each one of the chains of [N]6-4i, or [N]43-45, can be independently any one of A, U, C, or G.

24. The polynucleotide construct of any one of claims 1 to 23, wherein:(i) the first intronic sequence comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of any one of SEQ ID NOs: 35-38; and / or(ii) the second intronic sequence comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of any one of SEQ ID NOs: 39-42.

25. The polynucleotide construct of any one of claims 1 to 24, wherein the coding sequence comprises a payload sequence.

26. The polynucleotide construct of any one of claims 1 to 25, wherein the polynucleotide construct comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of SEQ ID NO: 45 or 46.

27. A circRNA produced by the polynucleotide construct of any one of claims 1 to 26.

28. A circRNA comprising an IRES sequence and a coding sequence.

29. The circRNA of claim 28, wherein an IRES sequence comprises a second internal guide sequence (IGS1).

30. The circRNA of claim 28 or 29, wherein an IRES sequence comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of SEQ ID NO: 47.Page 142 of 144133142I8vlAttorney Docket No. : 2019398-000531. The circRNA of any one of claims 28 to 30, wherein the circRNA comprises a nucleotide sequence having at least 90% identity with a nucleotide sequence of SEQ ID NO: 48 or 49.

32. A host cell comprising the polynucleotide construct of any one of claims 1 to 26, and / or the circRNA of any one of claims 27 to 29.

33. A composition comprising one or more of the polynucleotide constructs of any one of claims 1 to 26, one or more of the circRNA of any one of claims 27 to 29, or any combination thereof.

34. A pharmaceutical composition comprising one or more of the circRNA of any one of claims 27 to 29.

35. A method comprising administering the pharmaceutical composition of claim 34, to a subject.

36. A method of treating a disease comprising administering the pharmaceutical composition of claim 34, to a subject.

37. Use of the composition of claim 33 in the manufacture of a medicament for treating a subject.

38. Use of the pharmaceutical composition of claim 34 in the manufacture of a medicament for treating a subject.Page 143 of 144133142I8vl

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