WO2026006203A2 - Compositions et procédés de production d'arn circulaire - Google Patents

Compositions et procédés de production d'arn circulaire

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Publication number
WO2026006203A2
WO2026006203A2 PCT/US2025/034866 US2025034866W WO2026006203A2 WO 2026006203 A2 WO2026006203 A2 WO 2026006203A2 US 2025034866 W US2025034866 W US 2025034866W WO 2026006203 A2 WO2026006203 A2 WO 2026006203A2
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WO
WIPO (PCT)
Prior art keywords
t4td
self
group
nucleotides
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/034866
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English (en)
Other versions
WO2026006203A3 (fr
Inventor
Srivats VENKATARAMANAN
David Eric WEINBERG
Gilles BESIN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Orbital Therapeutics Inc
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Orbital Therapeutics Inc
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Publication date
Application filed by Orbital Therapeutics Inc filed Critical Orbital Therapeutics Inc
Publication of WO2026006203A2 publication Critical patent/WO2026006203A2/fr
Publication of WO2026006203A3 publication Critical patent/WO2026006203A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • RNAs form covalently closed, continuous stable loops. Therefore, circular RNAs are resistant to exonuclease digestion, making them more stable as compared to linear RNA.
  • the circular form makes the RNA molecule more stable and results in the circular RNA having increased protein production capabilities and increased efficacy as therapeutics.
  • the present invention provides nucleic acids and methods for making circular RNAs (circRNAs), and circular RNAs, compositions and methods of use thereof.
  • the present invention utilizes permuted intronic sequences derived from self-splicing Group I intron, td intron of T4 bacteriophage (T4td intron) to produce circular RNAs.
  • the permuted intron sequences described herein mediate efficient self-splicing to circularize an RNA sequence.
  • the present disclosure provides optimized T4 bacteriophage td gene (T4td) intron fragments for more efficient RNA circularization.
  • the precursor nucleic acid molecule comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment Attorney Docket No: ORB-014WO1 comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is between 220-230 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is between 170-190 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule is linear precursor RNA.
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 244 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is between 170-190 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 225 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is between 170-190 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is between 200-250 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 189 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is between 200-250 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 176 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 244 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment Attorney Docket No: ORB-014WO1 comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 189 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 226 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 189 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 227 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 176 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 225 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 176 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 244 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 176 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 225 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 189 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment Attorney Docket No: ORB-014WO1 comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 185 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 165 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 183 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 112 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 183 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 104 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 244 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 182 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 850 to +7 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -5 to 99 of SEQ ID NO: 1.
  • the precursor nucleic acid molecule comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 800 to +26 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -20 to 169 of SEQ ID NO: 1.
  • the precursor nucleic acid molecule comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 850 to +17 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron Attorney Docket No: ORB-014WO1 fragment comprising a 5’ splice site and the nucleotide positions -20 to 169 of SEQ ID NO: 1.
  • the precursor nucleic acid molecule comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 800 to +8 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -20 to 169 of SEQ ID NO: 1.
  • the precursor nucleic acid molecule comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 852 to +17 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -13 to 99 of SEQ ID NO: 1.
  • the precursor nucleic acid molecule comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 852 to +17 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -5 to 99 of SEQ ID NO: 1.
  • the precursor nucleic acid molecule comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 800 to +9 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -7 to 169 of SEQ ID NO: 1.
  • the precursor nucleic acid molecule comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 800 to +26 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -13 to 169 of SEQ ID NO: 1.
  • precursor nucleic acid molecule comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 800 to +7 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -7 to 169 of SEQ ID NO: 1.
  • precursor nucleic acid molecule comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 800 to +26 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -7 to 169 of SEQ ID NO: 1.
  • the precursor nucleic acid molecule comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide Attorney Docket No: ORB-014WO1 positions 800 to +7 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -20 to 169 of SEQ ID NO: 1.
  • the precursor nucleic acid molecule does not include 5’ or 3’ homology arms (i.e., outer homology arms).
  • the precursor nucleic acid molecule further comprises a 5’ spacer sequence between the upstream intron sequence and the sequence to be circularized (i.e., the sequence of interest).
  • the 5’ spacer sequence is close (i.e., directly downstream) to the upstream intron sequence.
  • the 5’ spacer sequence comprises a 5’ inner homology element.
  • the 5’ inner homology element in some cases, is located at the 5’ end of the 5’ spacer sequence.
  • the precursor nucleic acid molecule further comprises a 3’ inner homology element at the 5’ end of the downstream intron sequence.
  • the 5’ spacer sequence does not comprise a 5’ inner homology element.
  • the precursor nucleic acid molecule does not comprise a 3’ inner homology element at the 5’ end of the downstream intron sequence.
  • the precursor nucleic acid molecule does not include the 5’ spacer sequence.
  • the precursor nucleic acid molecule further comprises a 3’spacer sequence between the sequence of interest (i.e., to be circularized) and the downstream intron sequence.
  • the 3’ spacer sequence is a 3’ untranslated region (3’ UTR) between the sequence of interest and the downstream intron sequence.
  • the sequence of interest (i.e., to be circularized) is an RNA sequence encoding a polypeptide of interest.
  • the sequence of interest (i.e., to be circularized) is a non-coding sequence.
  • the sequence of interest encodes for a therapeutic protein (e.g., a chimeric antigen receptor, an enzyme replacement protein, a cytokine, a chemokine, an antibody), or an antigen (e.g, a tumor antigen and a pathogen antigen).
  • the precursor nucleic acid molecule further comprises an internal ribosome entry site (IRES).
  • the IRES may be operably linked to the RNA sequence of interest that encodes a polypeptide of interest.
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order, (A) an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment Attorney Docket No: ORB-014WO1 comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is between 180-260 nucleotides in length, (B) 5’ spacer, (C) optionally an IRES, (D) a sequence of interest, (E) 3’ UTR, and (F) a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is between 110-210 nucleotides in length.
  • the precursor nucleic acid molecule does not include a 5’ or 3’ homology arm.
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order, (A) an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is between 180-260 nucleotides in length, (B) 5’ spacer comprising a 5’ inner homology element, (C) optionally an IRES, (D) a sequence of interest, (E) 3’ UTR, (F) a 3’ inner homology element and (G) a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is between 110-210 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor RNA does not include 5’ or 3’ homology arm.
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order, (A) an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 244 nucleotides in length, (B) a 5’ spacer sequence, (C) optionally, an internal ribosome entry site (IRES), (D) a sequence of interest, (E) a 3’ spacer sequence, and (F) a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 189 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • Group I self-splicing intron fragment comprising a 3
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order, (A) an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 244 nucleotides in length, (B) a 5’ spacer sequence comprising a 5’ inner homology element, (C) optionally, an internal ribosome entry site (IRES), (D) a sequence of interest, (E) a 3’ spacer sequence, (F) a 3’ inner homology element, and (G) a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 189 nucleotides in length, wherein the precursor nucleic acid molecule does not include 5’ or 3’ homology arm.
  • T4td upstream
  • the precursor nucleic acid molecule comprises, in 5’ to 3’ order, (A) an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment Attorney Docket No: ORB-014WO1 is 226 nucleotides in length, (B) a 5’ spacer sequence, (C) optionally, an internal ribosome entry site (IRES), (D) a sequence of interest, (E) a 3’ spacer sequence, and (F) a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 189 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • T4td upstream T4 bacteriophage Td
  • the precursor nucleic acid molecule comprises in 5’ to 3’ order, (A) an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 226 nucleotides in length, (B) a 5’ spacer sequence comprising a 5’ inner homology element, (C) optionally, an internal ribosome entry site (IRES), (D) a sequence of interest, (E) a 3’ spacer sequence, (F) a 3’ inner homology element, and (G) a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 189 nucleotides in length, wherein the nucleic acid molecule does not include 5’ or 3’ homology arm.
  • T4td upstream T4
  • the sequence of interest (i.e., sequence to be circularized) is a coding sequence. In other embodiments, the sequence of interest (i.e., sequence to be circularized) is a non-coding sequence. In some embodiments, the sequence of interest comprises a protein coding sequence. In some embodiments, the sequence of interest encodes a therapeutic protein. In other embodiments, the sequence of interest encodes an antigen, e.g., a tumor antigen and an antigen derived from a pathogen (e.g., a microorganism such as a virus, a bacterium, a fungus and a protozoa). In some embodiments, the sequence of interest encodes a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the sequence of interest expresses a CAR in vivo.
  • the sequence of interest comprises one or more non-coding functional sequence, including but not limited to an aptamer, microRNA, miRNA sponge, an antisense RNA and a long non-coding RNA.
  • the precursor nucleic acid molecule i.e., the nucleic acid molecule for making a circular RNA
  • the DNA construct can be packaged into a viral vector.
  • the DNA construct for making a circular RNA is a non-viral vector, e.g., a plasmid.
  • the nucleic acid molecule for making a circular RNA is a linear RNA.
  • the linear RNA can be synthesized by in vitro transcription of a DNA construct of the present disclosure.
  • the present invention provides a circular RNA molecule that is transcribed from a nucleic acid described herein. Attorney Docket No: ORB-014WO1 [050]
  • the present invention provides a method for making a circular RNA using permuted self-splicing T4td intron fragments described herein.
  • the method comprises co-transcriptional circularization, in which transcription of a linear precursor RNA from the nucleic acid and circularization of the precursor RNA into a circular RNA occur in one reaction.
  • the method comprises transcribing the nucleic acid template in the presence of agents for the circularization reaction.
  • agents for such reaction include, for example, magnesium cation (Mg2+) and guanosine (GMP, GDP and/or GTP).
  • the present invention provides a method for making a circular RNA comprises (a) providing an RNA precursor polynucleotide, wherein the precursor polynucleotide comprises, in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment of greater than 180 nucleotides in length, i. an internal ribosome entry site (IRES), ii.
  • T4td upstream T4 bacteriophage Td
  • I self-splicing intron fragment of greater than 180 nucleotides in length, i. an internal ribosome entry site (IRES), ii.
  • the present invention provides a circular RNA.
  • the circular RNA is made from the precursor nucleic acid molecule, including linear RNA, or by the methods described herein.
  • the circular RNA comprises unmodified nucleotides.
  • the circular RNA comprises modified nucleotides.
  • the circular RNA comprises one or more modified nucleotides N1- methylpseudouridine. In other embodiments, the circular RNA comprises one or more modified nucleotides 5-methoxyuridine. In yet other embodiments, the circular RNA comprises one or more modified nucleotides m5C. In yet other embodiments, the circular RNA comprises one or more modified nucleotides m6A. [055] In some embodiments, the circular RNA encodes a chimeric antigen receptor (CAR), a therapeutic protein, a replacement enzyme, an antigen, an antibody and variants thereof. [056] In some embodiments, the present invention provides a composition comprising the circular RNA described herein.
  • CAR chimeric antigen receptor
  • the circular RNA is encapsulated Attorney Docket No: ORB-014WO1 in a lipid nanoparticle (LNP). In other embodiments, the circular RNA is encapsulated in a virus like particle (VLP).
  • the present invention provides a method for expressing a protein of interest in a cell comprising delivering to the cell the circular RNA described herein.
  • the present invention provides a method for treating a disease in a subject, the method comprising administering to the subject a linear precursor polynucleotide, including a linear precursor RNA, or the circular RNA described herein.
  • the sequence of interest in the RNA encodes a chimeric antigen receptor (CAR), a therapeutic protein, an enzyme replacement protein, an antigen, or an antibody.
  • CAR chimeric antigen receptor
  • the linear RNA is unmodified.
  • the linear RNA is modified.
  • the linear RNA comprises one or more modified nucleotides selected from N1-methylpseudouridine and/or 5-methoxyuridine.
  • the method makes intact circular RNA.
  • the circular RNA is formulated in a delivery vehicle.
  • the delivery vehicle is a lipid nanoparticle.
  • the lipid nanoparticle is conjugated to a targeting moiety.
  • a circular RNA produced from the precursor nucleic acid molecule of the present disclosure.
  • a circular RNA produced by the method of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS [069]
  • FIG. 1 is a diagram showing the structure and sequence of T4td intron.
  • FIG. 2 is a representative diagram of a nucleic acid construct for making a circular RNA.
  • the RNA transcript further comprises a 5’ spacer, an Internal Ribosome Entry Site (IRES), and a 3’ UTR. Splicing of the US and DS intron fragments generates a circular RNA sequence.
  • ORB-014WO1 ORB-014WO1
  • FIG. 3A shows an RNA transcript construct comprising inner homology elements (i.e., IHE) and 5’ and 3’ homology arms (i.e., outer homology arms)(OHE/IHE).
  • FIG. 3B shows an RNA transcript construct including inner homology elements (i.e., IHE) only (-/IHE), without outer homology arms.
  • FIG. 3C shows an RNA transcript construct that lacks both inner homology elements, and 5’ and 3’ homology arms (-/-).
  • FIG. 3D shows an RNA transcript construct including 5’ and 3’ homology arms only, without inner homology elements (OHE/-).
  • FIG. 4 shows diagrams of “DS” and “US” intron fragments split at different exemplary positions of the T4td intron.
  • the DS and US intron fragments are cut or split or permuted (the terms “cut”, “split”, and “permutated” are used interchangeably herein) at a site such that the catalytic core of the T4td intron is retained.
  • the DS and US intron fragments are cut such that an internal guide sequence of the T4td intron is retained.
  • the DS and US intron fragments are cut so that the self-splicing activity of the T4td intron is retained.
  • FIG. 5A shows an exemplary gel image of T4td-v2 , as compared to the legacy split (labeled as T4td).
  • FIG. 5B shows circularization efficiency.
  • T4td-v2 and T4td-v3 circularized RNA, and generated about over 50% circular RNA over total RNAs (including circular RNAs and linear RNAs) (FIG. 5B).
  • FIG. 6 is a representative gel image showing circularization of T4td-v1 and T4td- v2 at different temperatures. The results showed that T4td intron sequences were used to circularize an RNA sequence at temperatures ranging from 37°C to 61°C. Use of the T4td-v1 and T4td-v2 intron fragments at 56°C and 52°C, respectively, had moderately increased circularization efficiency. [075] FIGs.
  • FIG. 7A-7D show circularization using T4td-v1 intron sequences in combination with different parts in the RNA construct: FIG. 7A: T4td-v1and different 5’ spacer sequences; FIG. 7B: T4td-v1and different coding sequences (CDS); FIG. 7C: T4td-v1and different IRES; FIG. 7D: T4td-v1and different 3’UTR sequences.
  • FIG. 7A T4td-v1and different 5’ spacer sequences
  • FIG. 7B T4td-v1and different coding sequences (CDS)
  • FIG. 7C T4td-v1and different IRES
  • FIG. 7D T4td-v1and different 3’UTR sequences.
  • ORB-014WO1 DETAILED DESCRIPTION [076]
  • the present disclosure provides nucleic acid molecules useful in the generation of circular RNAs containing a sequence of interest, as well as methods of generating circular RNA
  • the present disclosure provides optimized intron fragments derived from T4td intron (a self-splicing Group I intron). Accordingly, in certain embodiments, the present disclosure is directed to nucleic acid molecules comprising permuted T4td intron sequences that facilitate the efficient production of circularized RNA containing a sequence of interest.
  • T4td intron a self-splicing Group I intron
  • the present disclosure is directed to nucleic acid molecules comprising permuted T4td intron sequences that facilitate the efficient production of circularized RNA containing a sequence of interest.
  • the terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions.
  • Circular RNA refers to an RNA that forms a circular structure through covalent or non-covalent bonds.
  • the terms “circRNA” or “circular polyribonucleotide” or “circular RNA” are used interchangeably.
  • circRNAs are covalently closed, single stranded RNA molecules.
  • a circular RNA can be produced by back-splicing of a linear precursor RNA, by chemical ligation and/or enzymatic ligation.
  • Circular RNAs can be endogenous or synthetic. Synthetically created and exogenously delivered circRNAs can be synthesized in vitro using self-splicing permuted introns (e.g., self-splicing Group I or Group II intron) from in vitro transcribed constructs. Unlike linear RNAs, circular RNAs are more resistant to the degradation by exonuclease and have a longer half-life than their corresponding linear Attorney Docket No: ORB-014WO1 counterparts.
  • a circular RNA can be a circular mRNA that encodes a polypeptide of interest (e.g., an immunogen and a therapeutic polypeptide).
  • Circularization efficiency refers to a measurement of resultant circular RNA versus its non-circular starting material (e.g., a linear precursor RNA).
  • corresponding to refers to a nucleic acid sequence or an amino acid sequence at particular positions of an intron, or the corresponding positions in another intron.
  • a sequence corresponding to the sequence at particular positions of an intron may comprise a corresponding substitution or a variant, e.g., the substituted nucleotides or amino acids do not naturally occur at the corresponding positions.
  • the substituted nucleotides or amino acids may be the corresponding residues in another intron (e.g., Group I or II intron).
  • Delivery refers to the act or manner of delivering a circular RNA, a construct, a linear RNA precursor, a cell comprising a circular RNA, a construct or a linear RNA precursor, or a composition comprising a circular RNA, a construct or a linear RNA precursor, a protein, cargo and/or payload.
  • Downstream As used herein, the term “downstream” refers to sequence that is 3’ to a particular sequence.
  • Encapsulate As used herein, the term “encapsulate” means to enclose, surround, or encase. As it relates to the formulation of the compositions of the disclosure, encapsulation may be substantial, complete or partial. The term “substantially encapsulated” means that at least greater than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.9% or greater than 99.999% of the pharmaceutical composition of the disclosure may be enclosed, surrounded or encased within the delivery agent.
  • Partially encapsulated means that less than 10%, 20%, 30%, 40%, 50%, or less of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent. In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or greater than 99.99% of the pharmaceutical composition of the present disclosure are encapsulated in the delivery vehicle (e.g., a lipid nanoparticle (LNP)).
  • the delivery vehicle e.g., a lipid nanoparticle (LNP)
  • Encode As used herein, the term “encode” or “encoding” refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule.
  • Enhance As used herein, the terms “enhance” and “enhancement” refers to an increase of at least about 5%, 10%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more of a reference; the reference may be a biological function of a nucleic acid or protein and a gene expression level, etc.
  • expression refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.
  • Feature refers to a characteristic, a property, or a distinctive element.
  • a “formulation” includes at least one compound, substance, entity, moiety, cargo or payload, and a delivery agent.
  • Fragment refers to a portion.
  • an intron fragment may comprise a portion of the full intron sequence.
  • Fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells.
  • homology refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar.
  • the term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).
  • two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids.
  • homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4–5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in Attorney Docket No: ORB-014WO1 length, homology is determined by the ability to encode a stretch of at least 4–5 uniquely specified amino acids.
  • two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.
  • Identity refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • Calculation of the percent identity of two polynucleotide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • a sequence can be at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a reference sequence.
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M.
  • the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, Attorney Docket No: ORB-014WO1 alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • Introns are non-coding sequences of DNA or RNA.
  • pre-mRNA messenger RNA
  • exons are any sequence of DNA or RNA that encode for proteins.
  • the pre-mRNA molecule goes through a modification process called splicing during which the noncoding introns are cut out, and only the coding exons remain. Splicing produces a mature messenger RNA molecule that is then translated into a protein.
  • the Group I or Group II introns are self-splicing introns.
  • the term “self-splicing intron” refers to introns that can act as ribozymes to autocatalytically splice them out from the parent RNA in the absence of any added protein or RNA.
  • An autocatalytic intron can be a Group I intron or a Group II intron.
  • Ionizable Lipid As used herein, “ionizable lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH.
  • Lipid Nanoparticle As used herein, “lipid nanoparticle” or “LNP” refers to a delivery vehicle comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, PEG- modified lipids).
  • liposome As used herein, “liposome” generally refers to a vesicle composed of lipids (e.g., amphiphilic lipids) arranged in one or more spherical bilayers or bilayers.
  • Modified As used herein, “modified” or, as appropriate “modification” refers to a changed state or structure of a molecule.
  • Molecules may be modified in many ways including chemically, structurally, and/or functionally.
  • nucleic acid molecules e.g., DNA and RNA
  • A, G, C, T (if DNA), or U (if RNA) nucleotides are modified.
  • polypeptides the term “modification” refers to a modification as compared to the canonical set of 20 amino acids.
  • mRNA As used herein, the term “messenger RNA” (mRNA) means a polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.
  • mRNA messenger RNA
  • Non-Cationic Lipid As used herein, “non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid.
  • nucleic acid molecule refers to a polymeric form of nucleotides, either deoxyribonucleotides (DNAs) or ribonucleotides (RNAs), or analogs thereof.
  • the terms include single stranded or double-stranded molecules comprised of nucleic acid bases.
  • the term includes, and may be used interchangeably with “plasmids”, “constructs”, or “vectors.”
  • the terms “polynucleotide” and “nucleic acid” are used interchangeably.
  • composition refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.
  • PEG As used herein “PEG” means any polyethylene glycol or other polyalkylene ether polymer.
  • Spacer refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. The spacer can be a 5’ spacer or 3’ spacer.
  • the nucleic acid for making a circular RNA of the present invention comprises a 5’ spacer that is located between the upstream intron fragment and a sequence of interest (i.e., to be circularized). In some embodiments, the 5’ spacer can be inserted between the upstream intron fragment and the IRES. In some embodiments, the nucleic acid for making a circular RNA of the present invention comprises a 3’ spacer that is located between a sequence of interest (to be circularized) and the downstream intron fragment.
  • the 5’ and 3’ spacer sequences may be 10 nucleotides to 100 nucleotides in length, or 20 nucleotides to 50 nucleotides in length.
  • the 5’ spacer is at least 10 nucleotides in length. In some embodiments, the 5’ spacer sequence is at least 15 nucleotides in length. In some embodiments, the 5’ spacer is at least 20 nucleotides in length. In some embodiments, the 5’ spacer sequence is at least 30 nucleotides in length. In some embodiments, a 3’ spacer is located between a sequence to be circularized and the downstream intron sequence.
  • Sterol is a subgroup of steroids consisting of steroid alcohols.
  • Structural lipid refers to sterols and lipids containing sterol moieties.
  • Transcription refers to the formation or synthesis of an RNA molecule by an RNA polymerase using a DNA molecule as a template.
  • Translation refers to the formation of a polypeptide molecule by a ribosome based upon an RNA template.
  • Treat refers to a prophylactic or therapeutic treatment of a disease or disorder (e.g., an infectious disease, a cancer, an autoimmune disorder, a genetic disease) in a subject, including a human subject.
  • a disease or disorder e.g., an infectious disease, a cancer, an autoimmune disorder, a genetic disease
  • the effect of treatment can include reversing, alleviating, reducing the severity of, curing, inhibiting the progression of, reducing the likelihood of recurrence of the disease or one or more symptoms or manifestations of the disease or disorder, stabilizing (i.e., not worsening) the state of the disease or disorder, or preventing the spread of the disease or disorder as compared to the state or the condition of the disease or disorder in the absence of the therapeutic treatment.
  • Unmodified refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequent modification.
  • Upstream As used herein, the term “upstream” refers to sequence that is 5’ to a particular sequence.
  • Vector As used herein, a “vector” is any molecule or moiety which transports, transduces or otherwise acts as a carrier of a heterologous molecule.
  • Vectors of the present disclosure may be produced recombinantly and may be based on and/or may comprise viral parent or reference sequences.
  • Such parent or reference viral sequences may serve as an original, second, third, or subsequent sequence for engineering vectors.
  • such parent or reference viral sequences may comprise any one or more of the following sequences: a polynucleotide sequence encoding a polypeptide or multi-polypeptide, which sequence may be wild-type or modified from wild-type and which sequence may encode full-length or partial sequence of a protein, protein domain, or one or more subunits of a protein; a polynucleotide comprising a modulatory or regulatory nucleic acid which sequence may be wild-type or modified from wild-type; and a transgene that may or may not be modified from wild-type sequence.
  • the present invention provides nucleic acid molecules and methods for synthesizing circular RNAs.
  • the circular RNA comprises a sequence of interest.
  • the nucleic acid molecules described herein are synthetic and/or recombinant. Synthetic and/or recombinant nucleic acid molecules are made by any known method in the art. For example, recombinant nucleic acid molecules, such as constructs described herein, are generated using standard molecular biology techniques.
  • a nucleic acid molecule for generating a circular RNA described herein comprises an upstream intron sequence corresponding to the 3’ splicing fragment of the T4td intron and a downstream intron sequence corresponding to the 5’ splicing fragment of the T4td intron.
  • the nucleic acid construct comprising intron sequences of the T4td intron is optimized to increase circularization efficiency.
  • a nucleic acid molecule for generating a circular RNA described herein comprises, from 5’ to 3’ end, an upstream intron sequence corresponding to the 3’ splicing fragment of the T4td intron, a sequence of interest, and a downstream intron sequence corresponding to the 5’ splicing fragment of the T4td intron.
  • nucleic acid molecule or “construct” are used interchangeably.
  • a “precursor RNA” is a linear precursor RNA that may be circularized to make circular RNA.
  • the present invention also provides circular RNAs synthesized using the nucleic acid molecules and methods disclosed herein and methods of use of circular RNAs.
  • the circular RNAs are distinguished from linear polynucleotides (e.g., mRNA) in their functional and/or structural design features which serve to, as evidenced herein, overcome existing problems of effective polypeptide production using nucleic acid-based methodologies.
  • the circular RNAs described herein comprise additional features to improve one or more of the stability and/or clearance in tissues, receptor uptake and/or kinetics, cellular access by the compositions, engagement with translational machinery, half-life, translation efficiency, immune evasion, and other functions and/or activities.
  • the present invention provides a precursor nucleic acid molecule for making a circular RNA.
  • the nucleic acid molecule is a DNA construct (e.g., a vector) that is transcribed into precursor RNA, and the precursor RNA circularizes into a circular RNA.
  • the nucleic acid molecule is a linear precursor Attorney Docket No: ORB-014WO1 RNA (e.g., a linear mRNA) that circularizes into a circular RNA.
  • the precursor nucleic acid molecule described herein comprises different elements essential for circular RNA synthesis and function.
  • the nucleic acid molecule for making a circular RNA comprises self-splicing intron sequences derived from T4td intron and a sequence of interest (i.e., to be circularized).
  • the sequence of interest encodes a chimeric antigen receptor (CAR).
  • a rational design of a synthetic circular RNA polynucleotide cassette includes at least two self-splicing intron sequences flanking the sequence to be circularized.
  • the upstream and downstream intron sequences are self-spliced to generate a circular RNA comprising the RNA sequence of interest.
  • the nucleic acid molecule comprises one or more additional sequences that facilitate circularization and functions of circular RNA.
  • a 5’ spacer and/or 3’ spacer e.g., 3’ untranslated region (UTR) sequences are included between the intron sequences and the sequence of interest (i.e., to be circularized).
  • one or more homology elements are included.
  • a 5’ inner homology element and a 3’ homology element are included between the intron sequences and the sequence of interest (i.e., to be circularized).
  • at least one internal ribosome entry site is included within the construct and linked to the sequence of interest, for example, a coding sequence.
  • the present invention provides a circular RNA that is made from a precursor nucleic acid molecule or by a method described herein.
  • T4td Intron is a self-splicing Group I intron.
  • Bacteriophage T4 contains a self- splicing Group I intron in the gene (Td) coding for thymidylate synthase.
  • the T4td intron also contains an open reading frame encoding a homing endonuclease. It has been known that Group I introns have autocatalytic activity (acting as ribozymes), i.e., are self-splicing. Group I introns splice themselves out without assistance from the spliceosome or other proteins, and the splicing results in joining of the flanking exons and circularization of the intervening intron to produce an intronic circRNA.
  • Group I introns can be found naturally within the rRNA, tRNA, and mRNA genes of bacteria and non-metazoan eukaryotes. A general discussion of the catalytic activity of Group I introns can be found in the review article by Hausner et al., (Mobile DNA, vol 5(8)(2014)). Attorney Docket No: ORB-014WO1 [0121] Similar to other Group I introns, naturally, the T4td intron is autocatalytically excised from linear RNA precursors and the flanking exons ligated in tandem transesterification reactions. [0122] Permuting the T4td intron to generate circular exon has been used.
  • the intron is genetically permuted (i.e., split) into 5’ and 3’ portion fragments and assembled with the DNA equivalent of the RNA of interest (i.e., to be circularized) in a circularization construct (the nucleic acid molecule described herein), in an order that promotes backsplicing and circularization.
  • the order within the nucleic acid construct is, from the 5’ to 3’ end, an upstream intron sequence corresponding to a 3’ splicing fragment of T4td intron, a sequence of interest, and a downstream intron sequence corresponding to a 5’ splicing fragment of T4td intron.
  • the “upstream intron fragment sequence” or “US” and “downstream intron fragment sequence” or “DS” derived from the T4td intron optionally comprise exon fragment and in this context, are also referred to as “upstream intron-exon fragment sequence” and “downstream intron-exon fragment sequence,” respectively.
  • the nucleic acid molecule for making a circular RNA comprises an upstream intron sequence and a downstream intron sequence generated from a cut site or splitting position (the terms “cut site”, “permutation site”, and “splitting position” are used interchangeably herein), wherein the cut site retains the structural integrity of the intron.
  • the term “catalytic core” refers to an intron region (e.g., the internal stem structure, or internal guide sequence) needed for the intron to self-splice, which is well- understood in the art.
  • the catalytic core of an intron particularly, a Group I intron, including for example, T4td intron, is a highly conserved small region of about 70 nucleotides composed of paired regions (e.g., P1-P6) that form elongated domains (e.g., helical domains). More detailed description of the catalytic core of an intron can be found in, e.g., Michel and Westhof, J. Mol.
  • the cut site is designed to retain the catalytic core of the intron, which is a region of nucleotides containing sequences and structures needed for the intron to self-splice.
  • a suitable cut site is chosen outside the catalytic core of the intron such that the structural integrity of the intron is retained, and the resulting upper intron fragments and lower intron fragments allows for RNA circularization.
  • a cut site of the T4Td intron is chosen such that an “internal guide” sequence is retained that comprises base pairing association of the distal exons to the P1-P10 helix
  • the T4td intron comprises a sequence in SEQ ID NO: 1.
  • nucleic acid molecules described herein comprises permuted intron sequences derived from the T4td intron.
  • the nucleic acid molecule for making a circular RNA comprises upstream and downstream intron sequences corresponding to a 3’ splicing fragment and a 5’ splicing fragment of the T4td intron, respectively.
  • the precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is between 180-260 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is between 110-210 nucleotides in length.
  • the upstream T4td self-splicing intron fragment is between 180-240 nucleotides, 200-260 nucleotides, 220-240 nucleotides, or 230-250 nucleotides in length.
  • the upstream T4td self-splicing intron fragment is 180 nucleotides, 181 nucleotides, 182 nucleotides, 183 nucleotides, 184 nucleotides, 185 nucleotides, 186 nucleotides, 187 nucleotides, 188 nucleotides, 189 nucleotides, 190 nucleotides, 191 nucleotides, 192 nucleotides, 193 nucleotides, 194 nucleotides, 195 nucleotides, 196 nucleotides, 197 nucleotides, 198 nucleotides, 199 nucleotides, 200 nucleotides, 201 nucleotides, 202 nucleotides, 203 nucleotides, 204 nucleotides, 205 nucleotides, 206 nucleotides, 207 nucleotides, 208 nucleotides, 209 nucle
  • the downstream T4td self-splicing intron fragment is between 110-200 nucleotides, 110-160 nucleotides, 120-180 nucleotides, 140-210 nucleotides, 150-200 nucleotides, or 160-210 nucleotides in length.
  • the downstream T4td self-splicing intron fragment is 110 nucleotides, 111 nucleotides, 112 nucleotides, 113 nucleotides, 114 nucleotides, 115 nucleotides, 116 nucleotides, 117 nucleotides, 118 nucleotides, 119 nucleotides, 120 nucleotides, 121 nucleotides, 122 nucleotides, 123 nucleotides, 124 nucleotides, 125 nucleotides, 126 nucleotides, 127 nucleotides, 128 nucleotides, 129 nucleotides, 130 nucleotides, 131 nucleotides, 132 Attorney Docket No: ORB-014WO1 nucleotides, 133 nucleotides, 134 nucleotides, 135 nucleotides, 136 nucleotides, 137 nucleotides, 138
  • the precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is between 220-230 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is between 170-190 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment 244 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is between 170-190 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 225 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is between 170-190 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is between 200-250 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 176 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 244 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 189 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 226 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 189 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 227 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 176 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 225 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 176 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 244 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, Attorney Docket No: ORB-014WO1 wherein the downstream T4td Group I self-splicing intron fragment is 176 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 225 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 189 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 183 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 104 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor RNA comprises in 5’ to 3’ order: an upstream T4 bacteriophage Td (T4td) Group I self-splicing intron fragment comprising a 3’ splice site, wherein the upstream T4td Group I self-splicing intron fragment is 244 nucleotides in length, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site, wherein the downstream T4td Group I self-splicing intron fragment is 182 nucleotides in length.
  • T4td upstream T4 bacteriophage Td
  • the precursor RNA comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 850 to Attorney Docket No: ORB-014WO1 +7 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -5 to 99 of SEQ ID NO: 1.
  • the precursor RNA comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 800 to +26 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -20 to 169 of SEQ ID NO: 1.
  • the precursor RNA comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 850 to +17 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -20 to 169 of SEQ ID NO: 1.
  • the precursor RNA comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 852 to +17 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -13 to 99 of SEQ ID NO: 1.
  • the precursor RNA comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 800 to +26 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -13 to 169 of SEQ ID NO: 1.
  • the precursor RNA comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 800 to +7 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -7 to 169 of SEQ ID NO: 1.
  • the precursor RNA comprises an upstream T4td Group I self-splicing intron fragment comprising a 3’ splice site and the nucleotide positions 800 to +26 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -7 to 169 of SEQ ID NO: 1.
  • precursor RNA comprises an upstream T4td Group I self- splicing intron fragment comprising a 3’ splice site and the nucleotide positions 800 to +7 of SEQ ID NO: 1, and a downstream T4td Group I self-splicing intron fragment comprising a 5’ splice site and the nucleotide positions -20 to 169 of SEQ ID NO: 1.
  • 5’ Spacer [0154]
  • the nucleic acid molecule for making a circular RNA comprises a 5’ spacer between the upstream intron fragment and the sequence of interest (i.e., to be circularized), or the IRES.
  • the 5’spacer sequence comprises a random sequence that increases circularization efficiency.
  • the 5’ spacer sequence may be of any length (e.g., 10 to 100 nucleotides, 10 to 90 nucleotides, 10 to 80 nucleotides, 10 to 70 nucleotides, 10 to 60 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides, 10 to 20 nucleotides, 20 to 100 nucleotides, 20 to 90 nucleotides, 20 to 80 nucleotides, 20 to 70 nucleotides, 20 to 60 nucleotides, 20 to 50 nucleotides, 20 to 40 nucleotides, 20 to 30 nucleotides, 30 to 100 nucleotides, 30 to 90 nucleotides, 30 to 80 nucleotides, 30 to 70 nucleotides, 30 to 60 nucleotides, 30 to 50 nucleotides, 30 to 40 nucleo
  • the length of the 5’ spacer is selected to optimize translation of the protein-coding nucleic acid sequence.
  • the 5’ spacer sequence is between 20 and 50 nucleotides in length. In some embodiments, the 5’ spacer sequence is between 30 and 100 nucleotides in length. In certain embodiments, the 5’ spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length.
  • the 5’ spacer sequence includes a 5’ inner homology element.
  • the terms “internal homology region” and “inner homology element (IHE)” are used interchangeably.
  • the internal homology element is about 5-50 nucleotides in length. In some embodiments, the internal homology element is about 5-30 nucleotides in length. In some embodiments, the internal homology region is about 10-25 nucleotides in length. In some embodiments, the internal homology element is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.
  • the 5’ internal homology element is located at the 5’ end of the 5’ spacer sequence.
  • the internal homology element forms base-pairing with an internal homology region, e.g., at the 3’ end (3’ inner homology element).
  • the nucleic acid described herein comprising 5’ and 3’ inner homology elements that are 75%, 80%, 85%, 90%, 95%, or 100% complementary to each other.
  • the 5’ spacer sequence comprises a polyA sequence.
  • the polyA sequence may comprise 15-30 As.
  • the polyA sequence comprises 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 As.
  • the 5’ spacer sequence comprises a poly A-C sequence.
  • the nucleic acid molecule described herein comprises an internal ribosome entry site (IRES) sequence.
  • IRES internal ribosome entry site
  • the term “internal ribosome entry site” or “IRES” refers to an RNA sequence or structural element ranging in size from 10 nucleotides to 1,000 nucleotides or more which is capable of initiating translation of a polypeptide in the absence of a normal RNA cap structure.
  • An IRES element may engage a eukaryotic ribosome for translation, or initiate cap-independent translation and protein synthesis.
  • the IRES has a sequence of an IRES, or is a functional fragment or variant thereof.
  • the IRES sequence may be derived from a viral genome or is a cellular IRES.
  • the nucleic acid molecule described herein comprises a viral IRES.
  • the IRES may include but is not limited to, the encephalomyocarditis virus (EMCV) IRES, polio virus IRES, Kaposi sarcoma-associated herpesvirus (KSHV) vFLIP IRES, or hepatitis C virus (HCV) IRES.
  • EMCV encephalomyocarditis virus
  • polio virus IRES polio virus IRES
  • KSHV Kaposi sarcoma-associated herpesvirus
  • HCV hepatitis C virus
  • the IRES that can be included in a circular RNA is a Type 1 IRES.
  • the IRES that can be included in a circular RNA is a viral IRES sequence.
  • Attorney Docket No: ORB-014WO1 [0166]
  • the IRES is an enterovirus IRES.
  • the IRES is a human rhinovirus (HRV) IRES.
  • the IRES that can be included in a circular RNA is a non- viral IRES sequence, including but not limited to IRES sequences from yeast, the human angiotensin II type 1 receptor IRES, fibroblast growth factor IRESs (e.g., FGF-1 IRES and FGF-2 IRES), vascular endothelial growth factor IRES, and insulin-like growth factor 2 IRES.
  • the IRES that can be included in a circular RNA is a synthetic IRES sequence.
  • a “synthetic IRES” is an IRES that is modified relative to a wild- type IRES in order to modulate its structure and/or activity.
  • an IRES that is modified to incorporate an aptamer sequence is a synthetic IRES.
  • the IRES sequence in the circular RNA comprises at least one RNA secondary structure element or feature.
  • the IRES may be of any length or size.
  • the IRES may be about 200 to about 400, about 400 to about 600, about 600 to about 700, or about 600 to about 800 nucleotides in length. In some embodiments, the IRES is about 210 nucleotides in length. In some embodiments, the IRES may be about 100 to about 3,000 nucleotides in length. [0171] In some embodiments, the IRES sequence may be operably linked to a protein- coding sequence. Different IRES elements (and exonic elements in general) affect the strength of protein expression as well as the cell/tissue specificity. Selection of an IRES element depends on the purpose of protein expression.
  • the IRES is “in-frame” with respect to the protein-coding nucleic acid sequence, that is, the IRES is positioned in the circular RNA molecule in the correct reading frame for the encoded protein.
  • the IRES may be “out of frame” with respect to the protein-coding nucleic acid sequence, such that the position of the IRES disrupts the open reading frame (ORF) of the protein-coding nucleic acid sequence.
  • the IRES may overlap with one or more ORFs of the protein-coding nucleic acid sequence.
  • 3’ UTR [0173]
  • the nucleic acid molecule provide herein comprises 3’ UTR.
  • the 3’ UTR is also a 3’ spacer sequence.
  • the regulatory features of a UTR can be incorporated into the nucleic acid molecules and circular RNAs made from the nucleic acid constructs. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organ sites.
  • the 3’ UTR may be derived from human beta-globin, human alpha-globin, xenopus beta-globin, xenopus alpha-globin.
  • the nucleic acid molecule comprises an additional 3’ spacer sequence at 3’ end of 3’ UTR.
  • the 3’ UTR sequence comprises a 3’ inner homology element at the 3’ end of the 3’ UTR.
  • a 3’ inner homology element is added downstream of the 3’ UTR.
  • the 3’ inner homology element pairs with the inner homology element in the 5’ spacer.
  • the 3’ internal homology element is about 5-50 nucleotides in length.
  • the 3’ inner homology element is 5-30 nucleotides in length.
  • the 3’ internal homology region is about 10-25 nucleotides in length.
  • the 3’ inner homology element has the same length as the inner homology element in the 5’ spacer. In some embodiments, the inner homology element is 10, 15, 20, or 25 nucleotides in length. In some embodiments, the 3’ inner homology element is at least 75%, 80%, 85%, 90%, 95%, or 100% complementary to the inner homology element in the 5’ spacer. [0177] In some embodiments, the nucleic acid molecule does not include inner homology elements. Attorney Docket No: ORB-014WO1 Homology arms [0178] As discussed herein, the inventors generated four different embodiments of RNA constructs (as shown in FIG. 3A- 3D).
  • the inventors of the present disclosure found that, in some embodiments, the nucleic acid molecule that does not include 5’ or 3’ homology arm (HA) had higher circularization efficiency.
  • the terms “homology arm” and “outer homology element (OHE)” are used interchangeably.
  • a 5’ homology arm (HA) is also referred to a 5’ outer homology element (OHE).
  • a 3’ homology arm (HA) is also referred to a 3’ outer homology element (OHE).
  • the results indicated that 5’ and 3’ homology arms (i.e., outer homology elements (OHEs) are not necessarily required for RNA circularization.
  • Homology arms are complementary sequences (e.g., 10-50 nucleotides) that pair with each other at the ends of a linear nucleic acid (e.g., a linear precursor mRNA).
  • the 5’ homology arm is located at the 5’ end of the upstream intron sequence (i.e., before and adjacent to or within the upstream intron sequence).
  • the 3’ homology arm is located at the 3’ end of the downstream intron sequence (i.e., after and adjacent to or within the downstream intron sequence).
  • the nucleic acid molecule comprises, from the 5’ to 3’ end, an upstream intron sequence corresponding to a 3’ splicing fragment of the T4td intron of SEQ ID NO: 1, a 5’ spacer comprising a 5’ inner homology element, optionally an IRES, a RNA sequence of interest, a 3’UTR, a 3’ inner homology element, and a downstream intron sequence corresponding to the 5’ splicing fragment of T4td intron of SEQ ID NO: 1, wherein the nucleic acid molecule does not comprise 5’ or 3’ homology arm.
  • a polypeptide of interest may include, but is not limited to, whole polypeptides, a plurality of polypeptides or fragments of polypeptides, which independently may be encoded by one or more nucleic acids, a plurality of nucleic acids, fragments of nucleic acids or variants of any of the aforementioned.
  • polypeptides of interest refer to any polypeptide which is selected to be encoded in the primary construct of the present disclosure.
  • polypeptide means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds.
  • polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • a polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides.
  • the term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • polypeptide variant refers to molecules which differ in their amino acid sequence from a native or reference sequence.
  • the amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, Attorney Docket No: ORB-014WO1 as compared to a native or reference sequence.
  • variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a native or reference sequence.
  • the coding nucleic acid sequence may be sufficient to encode a polypeptide of at least 10 amino acids in length, e.g., 10 to 5000 amino acids in length, or 10- 1000 amino acids in length, or 50-2000 amino acids in length, or 30-3000 amino acids in length, or 100-1000 amino acids in length, or 100-3000 amino acids in length, or 200-1000 amino acids in length, or 200-500 amino acids in length, or 500-5000 amino acids in length, or 500-4000 amino acids in length, or 500-1500 amino acids in length, or 1000-5000 amino acids in length.
  • the coding nucleic acid sequence element may be greater than 30 nucleotides in length, e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1, 100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2250, 2,500, and 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides.
  • the sequence of interest may encode a therapeutic protein or polypeptide.
  • the therapeutic protein may be an enzyme, a replacement therapy protein, etc.
  • one or more therapeutic proteins or peptides currently being marketed or in development may be encoded by the circular RNAs of the present disclosure.
  • the therapeutic polypeptide is a recombinant or chimeric polypeptide.
  • the recombinant polypeptide is a chimeric antigen receptor (CAR).
  • the therapeutic polypeptide is a cytokine (e.g., IFNg, IL2, IL7, IL12, IL15, IL21, IL27 etc.) [0190] In some embodiments, the therapeutic polypeptide is a transcription factor. [0191] In some embodiments, the sequence of interest may encode an antigen of interest.
  • the antigen may be an antigen that causes infection such as a viral antigen, a fungal antigen and a bacterial antigen.
  • the antigen may also be a cancer antigen such as a neoantigen and an antigen that is specific or associated with a cancer (e.g., a tumor associated antigen (TAA)).
  • TAA tumor associated antigen
  • a “neoantigen” refers to a class of tumor antigens which arises from tumor-specific mutations in an expressed protein.
  • the encoded polypeptide may be an antibody, a heavy chain of an antibody, a light chain of an antibody, a variable region of a heavy chain of an antibody, a variable region of a light chain of an antibody, a Fab fragment, and the like.
  • antibody is referred to in the broadest sense and specifically covers various embodiments including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., diabodies) so long as they exhibit a desired biological activity (e.g., “functional”).
  • Antibodies are primarily amino acid-based molecules but may also comprise one or more modifications (including, but not limited to the addition of sugar moieties, fluorescent moieties, chemical tags, etc.).
  • Non-limiting examples of antibodies or fragments thereof include VH and VL domains, scFvs, Fab, Fab’, F(ab’)2, Fv fragment, diabodies, linear antibodies, single chain antibody molecules, multispecific antibodies, bispecific antibodies, intrabodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, codon-optimized antibodies, tandem scFv antibodies, bispecific T-cell engagers, mAb2 antibodies, chimeric antigen receptors (CAR), tetravalent bispecific antibodies, biosynthetic antibodies, native antibodies, miniaturized antibodies, unibodies, maxibodies, antibodies to senescent cells, antibodies to conformers, antibodies to disease specific epitopes, or antibodies to innate defense molecules.
  • VH and VL domains scFvs, Fab, Fab’, F(ab’)2, Fv fragment, diabodies, linear antibodies, single chain antibody molecules, multispecific antibodies, bispecific antibodies, intrabodies, monoclonal antibodies,
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to Attorney Docket No: ORB-014WO1 corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.
  • antibody fragment refers to any portion of an intact antibody.
  • antibody fragments comprise antigen binding regions from intact antibodies.
  • Examples of antibody fragments may include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab” fragments, each with a single antigen-binding site. Also produced is a residual "Fc" fragment, whose name reflects its ability to crystallize readily.
  • an “antibody” may comprise a heavy and light variable domain as well as an Fc region.
  • antibody variant refers to a biomolecule resembling an antibody in structure and/or function comprising some differences in their amino acid sequence, composition or structure as compared to a native antibody.
  • the recombinant protein is a chimeric antigen receptor (CAR).
  • the CAR comprises an antigen binding domain, a hinge, a transmembrane domain and at least one intracellular signaling domain.
  • the CAR comprises an antigen binding domain, a hinge, a transmembrane domain, at least one co-stimulating signaling domain and an activation signaling domain.
  • the antigen binding domain of the CAR recognizes cellular antigen. Numerous antigen binding domains are known in the art, including those based on the antigen binding Attorney Docket No: ORB-014WO1 site of an antibody, antibody mimetics and T cell receptors.
  • the hinge domain may comprise about 20- 100 amino acids in length. In some embodiments, the hinge domain comprises at least 25, 30, 35, 40, 45, 50, or 60 amino acids. In some embodiments, the hinge domain comprises a sequence derived from an IgG Fc region (e.g., CH1, CH2 and/or CH3 regions), an IgG hinge or CD8 stalk region.
  • the transmembrane domain of the CAR may be any protein structure which is thermodynamically stable in a membrane. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the CAR described herein.
  • the CAR comprises a transmembrane domain derived from CD28, CD3 ⁇ , CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD2, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137/4-1BB, CD154, ICOS/CD278, GITR/CD357, or NKG2D.
  • the intracellular portion of the CAR comprises at least one co-stimulatory domain and an activation signaling domain.
  • the intracellular activation signaling domain is derived from CD3 zeta.
  • one or more costimulatory signaling domains are inserted between the transmembrane domain and the CD3zeta intracellular domain.
  • the costimulatory domain may be derived from, but not limited to, CD28, OX40, CD27, 4-1BB/CD137, CD2, CD7, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), CD8 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF1.4), NKG2C, Ig alpha (CD79a), Fc gamma receptor, MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell Attorney Docket No: ORB-014WO1 receptors, BTLA
  • miRNA sponge refers to a circular polynucleotide comprising a single-stranded non-coding polynucleotide with repeat copies of at least one specific microRNA binding site to hold microRNA molecules of interest.
  • the miRNA sponge acts as an artificial microRNA inhibitor, when expressed in a cell, would decrease the cellular level of the microRNA of interest.
  • the miRNA sponge sequence may comprise at least one miRNA response element (MRE) that binds to a miRNA and negatively regulates its activity.
  • miRNA response element (MRE) refers to a target site (i.e., a short nucleic acid fragment) that binds to a miRNA.
  • the circular polynucleotide may comprise two or more MREs.
  • the number of MREs in the circular polynucleotide is variable and relates to the length of the circular polynucleotide.
  • the circular polynucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more MREs.
  • the multiple MREs may have the same nucleic acid sequences and bind to the same miRNA; or alternatively, the MREs have different nucleic acid sequences and bind to different miRNAs, such as 2, 3, 4, 5, or more different miRNAs.
  • the miRNA sponge sequence may comprise 1-150 conserved miRNA target sites to bind to a miRNA to sponge the miRNA, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, Attorney Docket No: ORB-014WO1 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95, 100, or 150 miRNA target sites to sponge the miRNA.
  • the circular RNA described herein may regulate more than one miRNA, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more miRNAs.
  • the circular RNA described herein may be used to affect target miRNA activities, i.e., by acting as miRNAs sponge, circular RNAs may down-regulate miRNA activity and/or up-regulate the expression of miRNA target genes.
  • the circular RNA comprises a nucleic acid sequence that binds to one or more RNA binding proteins (RBPs) acting as a protein sponge.
  • RBPs RNA binding proteins
  • the circular RNA comprises a nucleic acid sequence that interacts with one or more protein to enhance protein function.
  • the circular RNA comprises a nucleic acid sequence that acts as scaffold to mediate complex formation between specific enzymes and substrates.
  • the circular RNA comprises a nucleic acid sequence that binds to one or more protein to recruit proteins to specific locations.
  • the circular RNA may comprise one or more long noncoding RNA (lncRNA, or lincRNA), a small nucleolar RNA (sno-RNA), microRNA (miRNA), small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA) and/or a portion thereof.
  • lncRNA long noncoding RNA
  • siRNA microRNA
  • siRNA small interfering RNA
  • piRNA Piwi-interacting RNA
  • the circular RNAs may comprise regions that partially or substantially not translatable, e.g., having a noncoding region. Such noncoding regions are different from the non-coding functional sequences and may located in any region of the circular RNA. Those non-coding regions include but are not limited to the linker, the spacer and/or the flanking regions. The noncoding regions may locate in more than one region of the circular RNA.
  • the nucleic acid molecule described herein is a DNA construct.
  • the construct comprises an upstream intron sequence and a downstream intron sequence which flanks exonic sequence (e.g., a protein coding RNA sequence).
  • viral vectors may be used to package the constructs for making circular RNAs.
  • the viral vectors may be AAV vectors.
  • the construct is non-viral vector. Exemplary non-viral vectors may include plasmids, cosmids and artificial chromosomes.
  • the construct includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000
  • the constructs and vectors provided herein can be made using standard techniques of molecular biology.
  • the various elements of the vectors provided herein can be obtained using recombinant methods, or by deriving the polynucleotides from a vector known to include the same.
  • the various elements of the vectors provided herein can also be produced synthetically, rather than cloned, based on the known sequences.
  • the complete sequence can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into the complete sequence.
  • Linear RNA [0226]
  • the nucleic acid molecules described herein are linear RNA (e.g., a linear mRNA).
  • the vector is incubated inside of a cell by a bacteriophage RNA polymerase or in the nucleus of a cell by host RNA polymerase II.
  • the resulting linear RNA can be used to generate circular RNA by incubating it in the presence of magnesium ions and guanosine nucleotide or nucleoside at a temperature at which RNA circularization occurs (e.g., between 30 °C and 60 °C).
  • Circular RNAs [0229] In accordance with the present disclosure, provided herein include circular RNAs.
  • the circular RNA can be made from a nucleic acid molecule described herein.
  • the circular RNA is synthesized by the Group I or II intron sequences mediated self-splicing of the nucleic acid molecule described herein.
  • an IVT linear RNA polynucleotide is circularized to produce the circular RNA.
  • the circular RNA comprises a portion of upstream intron fragment and a portion of downstream Attorney Docket No: ORB-014WO1 intron fragment.
  • the portion of upstream intron fragment and/or the portion of downstream intron fragment optionally comprises an exon fragment.
  • the circular RNA of the present disclosure has increased stability in vivo.
  • the circular RNA of the present disclosure has reduced immunogenicity.
  • the modifications may also increase the biological functions of nucleic acid molecules as compared to unmodified polynucleotides, such as binding to an RBP or another polynucleotide.
  • the modifications may be structural and/or chemical modifications.
  • the chemical modification may be a nucleotide and/or nucleoside modification including a nucleobase modification and/or a sugar modification, and a backbone linkage modification (i.e., the internucleoside linkage, e.g., a linking phosphate, a phosphodiester linkage, and a Attorney Docket No: ORB-014WO1 phosphodiester backbone).
  • the structural modification may include a secondary structural modification, and a tertiary structural modification.
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • GNAs glycol nucleic acids
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • nucleoside is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or a pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • a sugar molecule e.g., a pentose or ribose
  • an organic base e.g., a purine or a pyrimidine
  • nucleobase also referred to herein as “nucleobase”.
  • nucleobases Five primary/canonical nucleobases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) are the fundamental units of nucleic acid molecules, in which adenine and guanine, referred to purine bases, have a fused-ring skeletal structure derived of purine while uracil, and thymine, derived of pyrimidine, are referred to pyrimidine bases.
  • nucleotide is defined as a nucleoside including a phosphate group or other backbone linkage (internucleoside linkage).
  • the circular RNA comprises at least one modification described herein.
  • the circular RNA comprises two, three, four, or more (optionally different) chemical modifications described herein.
  • the modifications may be combinations of nucleobase (purine and/or pyrimidine), sugar and backbone (internucleoside) linkage modifications.
  • the modifications may be located on one or more nucleotides of the circular RNA.
  • all the nucleotides of the circular RNA are chemically modified.
  • all the nucleotides of the nucleic acid sequence with a biological function are chemically modified.
  • the circular RNA described herein may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, T/U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from Attorney Docket No: ORB-014WO1 50%
  • the polynucleotides are at least 50% modified, e.g., at least 50% of the nucleotides are modified. In some embodiments, the polynucleotides are at least 75% modified, e.g., at least 75% of the nucleotides are modified. It is to be understood that since a nucleotide (sugar, base and phosphate moiety, e.g., linkage) may each be modified, any modification to any portion of a nucleotide, or nucleoside, will constitute a modification.
  • the polynucleotides are at least 10% modified in only one component of the nucleotide, with such component being the nucleobase, sugar, or linkage between nucleosides. For example, modifications may be made to at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the nucleobases, sugars, or linkages of a polynucleotide described herein.
  • the circular RNA described herein can be designed with a patterned array of sugar, nucleobase or linkage modifications.
  • the polynucleotides can comprise modifications to maximize stability.
  • the modified nucleosides and nucleotides can include a modified nucleobase.
  • nucleobases in RNA include, but are not limited to, adenine(A), guanine(G), cytosine(C), and uracil(U).
  • nucleobases in DNA include, but are not limited to, adenine(A), guanine(G), cytosine(C), and thymine(T).
  • the modified nucleobase is a modified uracil(U).
  • nucleobases and nucleosides having a modified uracil include pseudouridine ( ⁇ ), pyridin-4- one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4- thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine (I 5 U) or 5-bromo-uridine (br 5 U)), 3- methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl- pseudouridine, 5-
  • the modified nucleobase is a modified cytosine(C).
  • exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl-cytidine (ac 4 C), 5- formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2 C), 2-thio-5-methyl-cytidine, 4-thio-pse
  • the modified nucleobase is a modified adenine(A).
  • exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7- deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m 1 A), 2-methyl- adenosine (m 1 A), 2-methyl- adenosine (
  • the modified nucleobase is a modified guanine(G).
  • exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1- methyl-inosine (m 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7- deaza-guanosine (preQ0), 7-aminomethyl-7-d
  • the nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog.
  • the nucleobase and/or analog may be each be independently selected from adenine, cytosine, guanine, uracil, naturally-occurring and synthetic derivatives of a base, including but not limited to pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2- thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine,
  • the circular polynucleotide of the present disclosure may comprise a nucleoside modification.
  • One or more atoms of a pyrimidine nucleobase may be replaced or substituted, for example, with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), optionally substituted or halo (e.g., chloro or fluoro) atoms or groups.
  • the uracil nucleosides of the circular RNA described herein are all modified. The modifications may be the same or different.
  • the guanine nucleosides of the circular polynucleotide of the present disclosure are all modified.
  • the modifications may be the same or different.
  • the guanine nucleosides of the circular polynucleotide of the present disclosure are all modified.
  • the modifications may be the same or different.
  • the cytosine nucleosides of the circular polynucleotide of the present disclosure are all modified.
  • the modifications may be the same or different.
  • the adenine nucleosides of the circular polynucleotide of the present disclosure are all modified. The modifications may be the same or different.
  • the circular RNA described herein is modified to comprise N6-methyladenosine (m6A) nucleotides.
  • m6A N6-methyladenosine
  • the circular RNA described herein comprises at least one sugar modification.
  • RNA includes the sugar subunit: ribose, which is a 5- membered ring having an oxygen atom.
  • the 2’ hydroxyl group (OH) can be modified or replaced with a number of different substituents.
  • substitutions at the 2′OH-position include, but are not limited to, H, halo, optionally substituted C1-6 alkyl; optionally substituted C1-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C6-10 aryloxy; optionally substituted C6-10 aryl-C1-6 alkoxy, optionally substituted C1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG)- O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from
  • Other exemplary sugar modifications include replacement of the oxygen atom (O) in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (GNA)
  • the sugar subunit can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • polynucleotides as described herein, including circRNAs can include nucleotides containing, e.g., arabinose, as the sugar.
  • Attorney Docket No: ORB-014WO1 [0256]
  • Nonlimiting examples of the sugar modification may include the modifications provided in Table 1.
  • the polynucleotides of the present disclosure can have one or more nucleotides carrying a modification as provided in Table 1.
  • each of the nucleotides of a polynucleotide described herein carries any one of the modifications as provided in Table 1, or none of the modifications as provided in Table 1.
  • nucleotide Sugar Modifications Attorney Docket No: ORB-014WO1 Attorney Docket No: ORB-014WO1 [0257]
  • at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the polynucleotides is substituted with -O- Methoxyethyl, referred to as 2’-OMe.
  • at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the polynucleotides is substituted with -F, referred to as 2’-F.
  • the sugar modification can be one or more locked nucleic acids (LNAs).
  • the polynucleotides can be fully 2’-MOE-sugar modified.
  • Attorney Docket No: ORB-014WO1 [0258]
  • modifications e.g., one or more modifications
  • the internucleoside linkage the linking phosphate or the phosphodiester linkage or the phosphodiester backbone.
  • the phrases “phosphate” and “phosphodiester” are used interchangeably.
  • Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein.
  • modified phosphate groups include, but are not limited to, phosphorothioate, methylphosphonates phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).
  • the ⁇ -thio substituted phosphate moiety is provided to confer stability to RNA and DNA polynucleotides through the unnatural phosphorothioate backbone linkages.
  • Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
  • Phosphorothioate linked polynucleotide molecules are expected to also reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
  • the circular RNA described herein comprise at least one phosphorothioate linkage, methylphosphonate linkage between nucleotides, 5’-(E)- vinylphosphonate (5’-E-VP), a phosphate mimic, as a modification.
  • the internucleoside linkages of the polynucleotides may be partially or fully modified.
  • Modified nucleotides incorporated the circular polynucleotides of the present disclosure may include for example, 2’-O-Methyl-modified or 2’-O-Methoxyethyl-modified nucleotides (2’-OMe and 2’-MOE modifications, respectively), an alpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine ( ⁇ -thio-cytidine), 5′- O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)- pseudouridine.
  • an alpha-thio-nucleoside e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cyt
  • Additional modifications to the circular RNA described herein include, but are not limited to, any modifications as described in PCT Publication WO2017070626, including, for example, modification or deletion of nucleotides (or codons) encoding one or more N-linked Attorney Docket No: ORB-014WO1 glycosylation site in a translated polypeptide. Modifications may also comprise any modifications as described in PCT Publication WO2018200892.
  • the circular polynucleotides of the present disclosure may further comprise features or modifications as described in PCT patent application publications WO2020255063, WO2020182869, WO2016011222, WO2016011226, WO2016005004, WO2016000792, WO2015176737, WO2015085318, WO2015048744, and WO2015034925, and United States patent application publications US20200254086, US20200206362, US20180311336 and US20180303929; the contents of each of which are incorporated herein by reference in their entireties.
  • nucleoside linkages e.g., backbone structures
  • nucleotide analogs or other modification(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased.
  • Codon optimization [0266] The circular RNAs and nucleic acid molecules for making circular RNAs, their regions or parts or subregions may be codon optimized. In some embodiments, the coding sequences of the circular RNAs are codon optimized.
  • Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include, but are not limited to, match codon frequencies in target and host organisms to ensure proper folding, alter GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove/add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide.
  • a codon optimized sequence may be one in which codons in a polynucleotide encoding a polypeptide have been substituted in order to increase the expression, stability and/or activity of the polypeptide.
  • Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) Attorney Docket No: ORB-014WO1 systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence,
  • a codon optimized polynucleotide may minimize ribozyme collisions and/or limit structural interference between the expression sequence and the IRES.
  • Codon optimization tools, algorithms and services are known in the art, non- limiting examples include, but are not limited to, services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods.
  • the coding sequence is optimized using optimization algorithms.
  • Pharmaceutical compositions Provided by the present disclosure include compositions such as pharmaceutical compositions comprising at least one circular RNA or nucleic acid molecule for making a circular RNA as described herein.
  • compositions described herein may be formulated for administration to a particular target cell, a target tissue, or a target organ and/or a subject.
  • Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • Such preparatory methods include the step of bringing the active ingredient (e.g., circular RNAs) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition in accordance with the disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1 and 30%, between 5 and 80%, between 10 and 50%, between 20 and 90%, at least 70% (w/w), or at least 80% (w/w) active ingredient.
  • the formulations described herein may contain at least one circular RNA molecule.
  • the formulations may contain one, two, three, four or five circular RNAs with different sequences.
  • the formulation contains at least two circular RNAs.
  • the formulation contains at least three circular RNAs.
  • the formulation contains at least four circular RNAs.
  • the formulation contains at least five circular RNAs.
  • compositions and formulations of the present disclosure can be formulated with one or more excipients to increase the stability of circular RNA; increase cell penetration; permit the sustained, controlled or delayed release; alter the biodistribution (e.g., target the nucleic acid vaccine composition to specific tissues or cell types); increase the translation of encoded protein in vivo; and/or alter the release of encoded protein in vivo.
  • excipients of the present disclosure can include, without limitation, lipids, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, nucleic acid molecules, cells, organelles, explants, nanoparticle mimics and combinations thereof.
  • nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle, degree of loading, polynucleotide to lipid/lipidoid ratio, nature of polynucleotides such as sequence contents, single-stranded or double-stranded, linear or circular, length and modifications, particle sizes and charges, and administration routes, etc.
  • the present disclosure contemplates the formulation and use in delivering at least one circular RNA compositions and at least one pharmaceutically acceptable carrier, such as circular RNAs encoding proteins including antigen proteins for nucleic acid vaccines.
  • Complexes, micelles, liposomes or particles can be prepared containing any suitable lipids and lipidoids and therefore, can result in an effective delivery of the circular polynucleotide compositions following the injection of a formulation via localized and/or systemic routes of administration, e.g., by various means including, but not limited to, intravenous (IV), intramuscular (IM), subcutaneous (SC), intraparenchymal (IPa), intrathecal (IT), sub-retinal, intranasal, or intracerebroventricular (ICV) administration.
  • IV intravenous
  • IM intramuscular
  • SC subcutaneous
  • IPa intraparenchymal
  • IMV intrathecal
  • IMV intracerebroventricular
  • LNPs Lipid nanoparticles
  • the circular RNAs, nucleic acid molecules and compositions thereof described herein may be formulated in a delivery vehicle, e.g., a lipid nanoparticle (LNP).
  • delivery vehicle lipid nanoparticle
  • nanoparticle nanoparticle
  • grammatical equivalent are used interchangeably.
  • LNPs can be characterized as small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-LNP environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space.
  • LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers.
  • the LNPs for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may have a diameter from 10-1000 nm.
  • the nanoparticle may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500
  • the lipid nanoparticles for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may have a diameter from about 1 to about 100 nm, such as but not limited to, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about
  • the lipid nanoparticles for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may have a diameter from about 10 to about 100 nm, such as, but not limited to, about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 70 nm, about 10 nm to about 80 nm, about 10 nm to about 90 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 30 nm, about 10 nm
  • the lipid nanoparticles for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may have a diameter from about 50 nm to about 100 nm.
  • LNPs useful herein are known in the art and generally comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more polyethylene glycol (PEG) modified lipids.
  • PEG polyethylene glycol
  • a LNP comprises no more than three distinct lipid components. The components of the LNP may be selected based on the desired target, tropism, cargo (e.g., a circular RNA), size, or other desired feature or property.
  • the relative amounts (ratio) of ionizable lipid, helper lipid, cholesterol and PEG-modified lipids substantially affect the efficacy of lipid nanoparticles and may be optimized for a given application and administration route.
  • the circular RNAs, nucleic acid molecules and compositions thereof described herein may be formulated using LNPs into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof.
  • Ionizable lipids [0287]
  • the LNP for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein comprises at least one ionizable (i.e. cationic) lipid.
  • the LNPs may contain one or more cationic lipids selected from C12-200, DLin-KC2-DMA, HGT4003, HGT5000, HGT5001, MC3,DLinDMA, DLinkC2DMA, cKK-El2, ICE, , DODAC, DDAB, DMRIE DOSPA, DOGS, DODAP, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, and combinations thereof.
  • Suitable cationic lipids include (20Z,23Z)-N,N-dimethylnonacosa-20,23- dien-10-amine, (17Z,20Z)-N,N-dimemylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N- dimethylpentacosa-l 6, 19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5- amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N- dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, (15 ⁇ ,18 ⁇
  • Suitable cationic lipids which may be used in the compositions and methods of the present disclosure include ionizable cationic lipids described in PCT Patent Application Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865 and WO2008103276, US Patent Nos. 7,893,302, 7,404,969 and 8,283,333 and US Patent Publication No. US20100036115 and US20120202871; the contents of each of which are herein incorporated by reference in their entirety.
  • the cationic lipid may be synthesized by methods known in the art and/or as described in PCT Patent Application Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 and WO201021865; the contents of each of which are herein incorporated by reference in their entirety.
  • the LNP for formulating the circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise a plurality of cationic lipids, such as a first and a second cationic lipid.
  • the first cationic lipid can be selected on the basis of a first property and the second cationic lipid can be selected on the basis of a second property.
  • the first and second properties may be complementary.
  • the nanoparticles described herein may comprise at least one cationic polymer described herein and/or known in the art.
  • the compositions of the present disclosure include one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, Attorney Docket No: ORB-014WO1 55%, 60%, 65%, or 70%, measured by weight, of the total lipid content in the lipid nanoparticle.
  • the compositions of the present disclosure include one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured as a mol %, of the total lipid content in the lipid nanoparticle.
  • compositions of the present disclosure include one or more cationic lipids that constitute about 30-70 % (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured by weight, of the total lipid content in the lipid nanoparticle.
  • compositions of the present disclosure include one or more cationic lipids that constitute about 30-70 % (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured as mol %, of the total lipid content in the lipid nanoparticle.
  • the ionizable lipid comprises a compound disclosed in WO 2021/141969 A1 (Hamilton et al.), the entirety of which is incorporated by reference herein.
  • the ionizable lipid comprises a compound of Formula (I) of WO 2021/141969 A1 (Hamilton et al.).
  • R 1 in Formula (I) comprises C9-C20 alkyl or C9-C20 alkenyl with 1-3 units of unsaturation.
  • R 1 comprises a C 9 -C 20 alkenyl with 2 units of unsaturation, such as, without limitation, a C 17 alkenyl with 2 units of unsaturation.
  • X 3 , X 5 , and X 6 in Formula (I) are independently absent.
  • X 1 is -O-. In some embodiments, X 1 is absent. -(CH) a - [0299] In some embodiments, X 2 is X7 . In some embodiments, X 2 is -(CH2)a- or - CH(OH)-. In some embodiments, a is an integer between 0 and 6. In some embodiments, a is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, a is 0 and X 2 is absent. In some embodiments, a is 1. Attorney Docket No: ORB-014WO1 [0300] In some embodiments, X 7 is independently hydrogen or hydroxyl. In some embodiments, X 7 is hydroxyl.
  • X 7 is hydrogen.
  • X 4 is a 6-membered heterocyclyl optionally substituted with 1 or 2 C 1 -C 6 alkyl groups.
  • the heterocyclyl comprises at least one nitrogen.
  • X 4 is piperidinyl.
  • X 4 is ethylpiperidinyl.
  • a 1 and A 2 are independently C 5 -C 12 alkyl or C 5 -C 12 alkenyl with 1-3 units of unsaturation.
  • a 1 and A 2 are independently C5-C12 alkenyl with 1 unit of unsaturation.
  • a 1 is C8 alkenyl with 1 unit of unsaturation.
  • a 2 is C 8 alkenyl with 1 unit of unsaturation.
  • n1 is an integer between 1 and 6. In some embodiments, n1 is 1, 2, 3, 4, 5, or 6. In some embodiments, n1 is 2.
  • the ionizable lipid comprises a compound disclosed in WO 2022/140252 A1 (Patwardhan et al.), the entirety of which is incorporated by reference herein.
  • the ionizable lipid comprises a compound of Formula (III-a-i) of WO 2022/140252 A1 (Patwardhan et al.), or its N-oxide: (Formula (III-a-i))
  • R 1 is hydrogen.
  • L 1 is C2-C6 heteroalkylenyl comprising at least 1 heteroatom.
  • the heteroatom is oxygen.
  • L 1 is a C4 heteroalkylenyl comprising 1 oxygen atom, such as, for example and without limitation, - OCH2CH2CH2-.
  • L 1 is a C3 heteroalkylenyl comprising 1 oxygen atom, such as, for example and without limitation, - OCH 2 CH 2 -.
  • each R is independently C 6 -C 12 alkyl or C 6 -C 12 alkenyl with 1-3 units of unsaturation.
  • each L is independently C1-C5 alkylenyl.
  • each L 2 is independently C 4 -C 8 alkylenyl.
  • the ionizable lipid comprises a compound of Formula (I’’- a) of WO 2022/140252 A1 (Patwardhan et al.).
  • R 1 is hydrogen.
  • L 1 is C 2 -C 6 heteroalkylenyl comprising at least 1 heteroatom.
  • the heteroatom is oxygen.
  • L 1 is a C3 heteroalkylenyl comprising 1 oxygen atom, such as, for example and without limitation, - OCH2CH2-.
  • each R is independently C 6 -C 12 alkyl or C 6 -C 12 alkenyl with 1-3 units of unsaturation.
  • R’’ is C6-C12 alkyl.
  • each L is independently C 1 -C 5 alkylenyl.
  • each L 2 is independently C4-C8 alkylenyl.
  • Exemplary ionizable lipids include 3-((((1-ethylpiperidin-3- yl)methoxy)carbonyl)oxy)-2-(((4-(((Z)-oct-5-en-1-yl)oxy)-4-(((Z)-oct-5-en-1- yl)oxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate ( Compound 1).
  • Non-cationic lipids/helper lipids may comprise one or more non-cationic lipids (helper lipids).
  • the helper lipids in LNPs may contribute to their stability and delivery efficiency, and/or mitigate the toxicity owing to the cationic lipids.
  • a “non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid.
  • Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-
  • the non-cationic lipid is a phospholipid such as a synthetic phospholipid, including but not limited to, DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC and DEPC; DMPG, DPPG, DSPG and POPG; DMPA, DPPA and DSPA; DMPE, DPPE, DSPE and DOPE; DOPS; and polyglycerin attached phospholipids (PG phospholipid).
  • the phospholipid may be selected based on administration routes, e.g., DPPC, POPC and POPG used in LNPs for injection and DOPC, POPC and DDPC used in LNPs for pulmonary delivery.
  • the phospholipid may be a purified lipid from a natural source.
  • the LNP for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise one or more neutral helper lipids such as dioleoyl phosphoethanolamine (DOPE), prostaglandins, eicosanoids, glycerides, glycosylated diacyl glycerols, oxygenated fatty acids, NAGly and PAHSA.
  • DOPE dioleoyl phosphoethanolamine
  • prostaglandins prostaglandins
  • eicosanoids eicosanoids
  • glycerides glycosylated diacyl glycerols
  • oxygenated fatty acids NAGly and PAHSA.
  • ORB-014WO1 neutral lipid is a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered.
  • the non-cationic lipid may comprise a molar ratio of about 5% to about 90%, or about 10 % to about 70% of the total lipid present in an LNP. In some embodiments, the percentage of non-cationic lipid in a LNP may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.
  • Cholesterol-derived lipids [0323]
  • the LNP for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein comprises one or more cholesterol derived lipids.
  • the cholesterol derived lipids can be a cholesterol, a naturally occurring cholesterol analogue, or a synthetic cholesterol like compound and the cholesterol derivatives.
  • a naturally occurring cholesterol analog may be selected from those by Patel et al., (Nature Communications, 2020; 983); the contents of which are incorporated herein by reference in their entirety.
  • the LNPs comprise one or more cholesterol derivatives, e.g., PtdChol.
  • the cholesterol-based lipid may comprise a molar ration of about 2% to about 30%, or about 5% to about 20% of the total lipid present in an LNP.
  • the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.
  • PEG-modified lipids [0325]
  • the LNP described herein comprises one or more PEG modified lipids, such as PEG polymers and PEGylated lipids.
  • Suitable PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C 6 - C20 length. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid- nucleic acid composition to the target tissues, or they may be selected to rapidly exchange out of the formulation in vivo.
  • Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18).
  • the PEG-modified may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the LNP.
  • LNPs for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may include at least one of the Attorney Docket No: ORB-014WO1 PEGylated lipids described in PCT Patent Application Publication No. WO2012099755, the contents of which are herein incorporated by reference in their entirety.
  • the ratio of PEG in the lipid nanoparticle (LNP) formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the LNP formulations.
  • the LNP comprises PEG-c-DOMG.
  • the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DPG (1,2-Dipalmitoyl-sn- glycerol, methoxypolyethylene glycol), or PEG-DMG 2000 (1,2-dimyristoyl-sn-glycero-3- phophoethanolamine-N-[methoxy(polyethylene glycol)-2000).
  • the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol.
  • the LNP formulation may contain PEG-DMG 2000, DLin- DMA, DSPC and cholesterol in a molar ratio of 2:40:10:48 (see e.g., Geall et al., PNAS, 2012, 109(36): 14604-14609; herein incorporated by reference in its entirety).
  • Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP).
  • Ionizable cationic lipids such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity.
  • the rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles.
  • Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation.
  • the ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.
  • the LNP for delivering circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise a cleavable lipid such as those described in PCT Patent Application Publication No. WO2012170889, the contents of which are herein incorporated by reference in their entirety.
  • the LNP for delivering circular RNAs, nucleic acid molecules and compositions thereof described herein may comprises a conjugated lipid.
  • the conjugated lipid may have a formula such as described in US Pub. No. US 20120264810 to Lin et al., the contents of which are incorporated herein by reference Attorney Docket No: ORB-014WO1 in their entirety.
  • the conjugate lipid may form a lipid particle which further comprises a cationic lipid, a neutral lipid, and a lipid capable of reducing aggregation.
  • the LNP for formulating the circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise a mixture of cationic compounds and neutral lipids.
  • the cationic compounds may be formula (I) disclosed in PCT Patent Application Publication No.: WO 1999010390 to Ansell et al., the contents of which are described herein by reference in their entirety, and the neutral lipid may be selected from the group consisting of diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide and sphingomyelin.
  • the LNP formulations described herein may additionally comprise a permeability enhancer molecule.
  • Non-limiting permeability enhancer molecules are described in US Patent Publication No. US20050222064; the contents of which are herein incorporated by reference in their entirety.
  • the LNP for formulating the circular RNAs, nucleic acid molecules and compositions thereof described herein may be encapsulated into any polymer known in the art which may form a gel when injected into a subject.
  • the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.
  • the LNP for formulating the circular RNAs, nucleic acid molecules and compositions thereof described herein may be encapsulated in the lipid formulation to form a stable nucleic acid-lipid particle (SNALP) such as described in US Pat. No.
  • SNALP stable nucleic acid-lipid particle
  • the SNALP includes 40% 2,2- Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Lipid A), 10% dioleoylphosphatidylcholine (DSPC), 40% cholesterol, 10% polyethylene glycol (PEG)-C- DOMG (mole percent) with a particle size of 63.0 ⁇ 20 nm and a 0.027 nucleic acid/lipid ratio.
  • Lipid A 2,2- Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane
  • DSPC dioleoylphosphatidylcholine
  • cholesterol 10% polyethylene glycol (PEG)-C- DOMG (mole percent) with a particle size of 63.0 ⁇ 20 nm and a 0.027 nucleic acid/lipid ratio.
  • the LNPs for formulating the circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise an endosomal membrane destabilizer as disclosed in US Pat. No. US 7,189,705 to Lam et al., the contents of which are incorporated herein by reference in their entirety.
  • the endosomal membrane destabilizer may be a Ca 2+ ion.
  • the LNPs for formulating the circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise a charged lipid or an amino lipid.
  • charged lipid is meant to include those lipids having Attorney Docket No: ORB-014WO1 one or two fatty acyl or fatty alkyl chains and a quaternary amino head group.
  • the quaternary amine carries a permanent positive charge.
  • the head group can optionally include an ionizable group, such as a primary, secondary, or tertiary amine that may be protonated at physiological pH.
  • the presence of the quaternary amine can alter the pKa of the ionizable group relative to the pKa of the Group In a structurally similar compound that lacks the quaternary amine (e.g., the quaternary amine is replaced by a tertiary amine).
  • the charged lipid used in any of the formulations described herein may be any charged lipid described in EP2509636 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety.
  • a charged lipid is referred to as an “amino lipid.”
  • the amino lipid may be any amino lipid described in US Pub. No.
  • the amino lipids may have the structure disclosed in Tables 3-7 of Hope, such as structure (II), DLin-K-C2-DMA, DLin-K2- DMA, DLin-K6-DMA, etc.
  • the amino lipids may be any amino lipid described in US 20110117125 to Hope et al., the contents of which are incorporated herein by reference in their entirety, such as a lipid of structure (I), DLin-K- DMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLin-S-DMA, etc.
  • the amino lipid may have the structure (I), (II), (III), or (IV), or 4-(R)-DLin-K- DMA (VI), 4-(S)-DLin-K-DMA (V) as described in PCT Patent Application Publication No. WO2009132131 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety.
  • the LNPs for formulating the circular polynucleotide compositions of the present disclosure may comprise reverse head group lipids, e.g., formulated with a zwitterionic lipid comprising a headgroup wherein the positive charge is located near the acyl chain region and the negative charge is located at the distal end of the head group, such as a lipid having structure (A) or structure (I) described in PCT Patent Application Publication No. WO2011056682 to Leung et al., the contents of which are incorporated herein by reference in their entirety.
  • a lipid having structure (A) or structure (I) described in PCT Patent Application Publication No. WO2011056682 to Leung et al. the contents of which are incorporated herein by reference in their entirety.
  • the lipid components of the LNP to nucleic acid ratio may be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1, or 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1. 9:1, 10:1, 11:1, 12:1, 13:1.
  • the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or WO2008103276.
  • the circular polynucleotide compositions of the present disclosure may be encapsulated in any of the lipid nanoparticle (LNP) formulations described in WO2011127255 and/or WO2008103276; the contents of each of which are herein incorporated by reference in their entirety.
  • LNP lipid nanoparticle
  • Other nanoparticles and delivery agents are used to deliver circular RNAs, nucleic acid molecules and compositions thereof described herein.
  • the vehicles may include other lipid-based particles such as lipidoids, liposomes, lipoplexes, micelles, multilamellar vesicle (MLV), unicellular vesicle (SUV), polymer-based nanoparticles and exosomes.
  • compositions described herein may also be constructed or altered such that their properties are suitable for different administration routes, such as parenteral (intravenously, intramuscularly, intradermally, intraperitoneally or subcutaneously), oral, rectal, ophthalmic and/or topical administration.
  • compositions of the present disclosure can be formulated for controlled release and/or targeted delivery.
  • controlled release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to affect a therapeutic outcome.
  • sustained release refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time.
  • the period of time may include, but is not limited to, hours, days, weeks, months and years.
  • the compositions may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery.
  • Engineered exosomes [0345] Exosomes are tiny vesicles smaller than 50 nm secreted by mature reticulocytes, which are associated with transferrin receptors and function in antigen presentation during the regulation of immune cells. In some embodiments, engineered exosomes act as cargo carriers and deliver small hydrophilic or lipophilic molecules, including some therapeutic drugs to cells, participating in the regulation of many major diseases.
  • RNA therapeutics discussed herein are delivered using virus-like particles.
  • Viral particles include recombinant viruses and virus-like particles (VLPs).
  • VLPs virus-like particles
  • the term “Virus-like particles (VLPs)” are molecules that closely resemble viruses, but are non-infectious. VLPs can be naturally occurring or synthesized through the individual expression of viral structural proteins, which can then self-assemble into the virus-like structure. Combinations of structural capsid proteins from different viruses can be used to create recombinant VLPs.
  • VLPs can be produced from different viruses, such as adeno-associated viruses, retroviruses, lentiviruses and vesiculoviruses.
  • VLPs can be produced in multiple cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast and plant cells. VLPs possess diverse applications in therapeutics, immunization, and diagnostics. VLPs have been synthesized in a wide range of expression systems (ESs), including prokaryotic (bacteria) and eukaryotic (insect cells, mammalian cell lines, plant cells, or yeast).
  • ESs expression systems
  • VLPs can be increased through modifying their exterior or interior surface by displaying the heterologous epitopes of interest using different methods like peptide conjugation, genetic fusion, and chemical crosslinking.
  • the VLP is derived from a Vesiculovirus.
  • the VLP is derived from VSV (Indiana vesiculovirus, formerly Vesicular stomatitis Indiana virus (VSIV or VSV).
  • the virus like particle comprises a mutated VSV-G protein.
  • VSV-G protein is a single transmembrane glycoprotein (G) which plays a critical role during the initial steps of virus infection. it is responsible for virus attachment to specific receptor, LDL-R.
  • VSV-G protein triggers the fusion between the viral and endosomal membranes, which releases the viral genome in the cytosol for the subsequent steps of infection.
  • VSV-G protein is mutated to abolish its binding to LDL-R receptor.
  • a VSV-G envelope protein may be a mutated at one or more of any one of H8, K47, Y209, and/or R354.
  • a VLP may comprise a mutated VSV-G protein described in the PCT patent application Publication No. WO2019057974; the contents of which are incorporated herein by reference in their entireties.
  • the VLP for delivery RNA therapeutics is a viral particle disclosed in the PCT Publication Nos.
  • the virus like particle is pseudotyped.
  • the virus like particle is VSV-G-pseudotyped lentiviruses (VSV-G-LVs).
  • the viral particle for delivering RNA therapeutics is a retrovirus, a recombinant AAV, or an adenovirus.
  • the present disclosure encompasses the delivery of circular RNAs, nucleic acid molecules and compositions thereof described herein for any therapeutic, prophylactic, pharmaceutical, diagnostic or research use.
  • the circular RNAs and compositions thereof described herein may be loaded to delivery vehicles such as those formulation components discussed herein in order to be administered to target cells, tissues, organs and/or subjects.
  • the formulated circular RNA compositions may be delivered to the cell using routes of administration known in the art and described herein. [0351] Delivery may also be naked.
  • the circular RNAs and compositions may be delivered to a cell naked. As used herein in, “naked” refers to delivering the compositions described herein free from agents which promote transfection.
  • the naked circular RNA compositions may be delivered to the cell using routes of administration known in the art and described herein.
  • the circular RNA compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like.
  • the compositions of the present disclosure may be administered by any route which results in a therapeutically effective outcome.
  • compositions may be administered in a way which allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.
  • Routes of administration disclosed in International Publication WO 2013/090648 filed December 14, 2012, the contents of which are incorporated herein by reference in their entirety, may be used to administer the circular polynucleotide-based compositions of the present disclosure.
  • the compositions described herein can be formulated into a dosage form and for a route of administration as described herein, such as liquid dosage forms, injectable preparations, pulmonary forms, and solid dosage. Methods of use thereof [0355]
  • the circular RNAs, nucleic acid molecules and compositions thereof are used for therapy.
  • the circular RNAs, nucleic acid molecules and compositions thereof are used for treating a disease, e.g., an autoimmune disease, an infectious disease, a genetic disorder and a cancer, in a subject, including a human subject.
  • a disease e.g., an autoimmune disease, an infectious disease, a genetic disorder and a cancer
  • the present disclosure provides a method for treating or preventing a disease using circular RNAs, nucleic acid molecules and compositions thereof described herein.
  • the sequence of interest encodes a chimeric antigen receptor (CAR), a therapeutic protein, an enzyme replacement protein, an antigen, or an antibody.
  • the linear RNA is unmodified.
  • the linear RNA is modified.
  • the linear RNA comprises one or more modified nucleotides selected from N1-methylpseudouridine and/or 5-methoxyuridine.
  • the method makes intact circular RNA.
  • the circular RNA is formulated in a delivery vehicle.
  • the delivery vehicle is a lipid nanoparticle.
  • the lipid nanoparticle is conjugated to a targeting moiety.
  • provided herein is a circular RNA produced from the precursor nucleic acid molecule of the present disclosure.
  • provided herein is a circular RNA produced by the method of the present disclosure.
  • the circular RNAs, nucleic acid molecules and compositions thereof are used for treating or preventing cancer.
  • Cancer includes a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. Examples of cancers that may be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system including lymphoma, leukemia, myeloma, and other leukocyte malignancies.
  • the methods disclosed herein may be used to reduce the tumor size of a tumor derived from, for example , bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, multiple myeloma, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, cancer of the urethra, cancer of the penis,
  • the methods disclosed herein may be used to reduce the tumor size of a tumor derived from, for example, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, Kaposi's sarcoma, sarcoma of soft tissue, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, lung cancer, colorectal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (for example adenocarcinoma of the pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, or esophagus), sweat gland carcinoma, sebaceous
  • the particular cancer may be responsive to chemo- or radiation therapy or the cancer may be refractory.
  • a refractory cancer refers to a cancer that is not amenable to surgical intervention and the cancer is either initially unresponsive to chemo- or radiation therapy or the cancer becomes unresponsive over time.
  • the circular RNAs, nucleic acid molecules and compositions thereof are used for treating or preventing an infectious disease, such as a viral infection.
  • the circular RNAs, nucleic acid molecules and compositions thereof may be used as vaccines.
  • the circular RNAs, nucleic acid molecules and compositions thereof are used for treating or preventing a genetic disorder.
  • the circular RNAs, nucleic acid molecules and compositions thereof are used for inducing an immune response in a subject.
  • the immune response includes the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast, cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • a cell of the immune system for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast, cells, dendritic cells and neutrophils
  • compositions of the present disclosure are used to treat an autoimmune disease, including systemic lupus erythematosus (SLE)/lupus nephritis.
  • SLE systemic lupus erythematosus
  • the compositions of the present disclosure are used to induce an immune response for therapeutic or prophylactic purposes.
  • the compositions of the present disclosure are used as vaccines to prevent infection, e.g., viral infections.
  • the compositions of the present disclosure may be used as cancer vaccines.
  • the circular RNA comprises an antigen coding nucleic acid sequence.
  • the antigen is a tumor associated antigen (TAA) or a fragment thereof.
  • compositions described herein are co-administered with one or more additional therapeutic agents (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions).
  • the compositions described herein can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa.
  • the therapeutic compositions described herein, and the one or more additional therapeutic agents can be administered simultaneously.
  • the subject is a mammal.
  • the mammal referred to herein can be any mammal, including, but not limited to, mice and hamsters, rabbits, cats, dogs, pigs and primates.
  • the mammal is a human (e.g., a patient).
  • PIE Permuted Intron-Exon constructs that allow RNA circularization
  • plasmid parts Part 0 for the upstream intron sequence (US intron); Part 1 for 5’ spacer; Part 345 for gene coding sequence, which is also referred to herein as “the sequence of interest”; Part 6 for 3’ UTR; and Part 7 for the downstream intron sequence (DS intron)
  • PCR polymerase chain reaction
  • the nucleic acid constructs for making circular RNA were assembled by cloning parts 1–7 into an entry vector including a T7 promotor and a T7 terminator via a Golden Gate reaction according to the method described in Chen et al (Nature Biotechnology, 2023; 41(2): 262–272).
  • FIG. 3A shows an RNA transcript construct comprising 5’ and 3’ inner homology elements (i.e., IHEs) and 5’ and 3’ homology arms (i.e., outer homology arms (OHEs) )(OHE/IHE).
  • FIG. 3B shows an RNA transcript construct including 5’ and 3’ inner homology elements (i.e., IHE) only (-/IHE), without outer homology arms.
  • FIG. 3C shows an RNA transcript construct that lacks both inner homology elements, and 5’ and 3’ homology arms (-/-).
  • FIG. 3A shows an RNA transcript construct comprising 5’ and 3’ inner homology elements (i.e., IHEs) and 5’ and 3’ homology arms (OHEs) )(OHE/IHE).
  • FIG. 3B shows an RNA transcript construct including 5’ and 3’ inner homology elements (i.e., IHE) only (-/IHE), without outer homology arms.
  • FIG. 3C shows an RNA transcript
  • 3D shows an RNA transcript construct including 5’ and 3’ homology arms only (OHE/-), without inner homology elements.
  • Attorney Docket No: ORB-014WO1 [0379] The nucleic acid constructs were transformed into E. coli. Colonies were picked, mini-prepped and sequenced to confirm the assembled constructs. [0380] The sequence encoding a GFP protein was cloned into the constructs (i.e., Part 345) and the IRES sequence from iHRVB3 was used.
  • Example 2 Co-transcriptional circularization [0381] The plasmids were linearized and used as linearized transcription templates for in vitro transcription (IVT) to generate linear RNA precursors.
  • IVTT in vitro transcription
  • IVT In vitro transcription
  • Mg+2 magnesium
  • Mg+2 Mg+2
  • GTP nucleoside triphosphate
  • ATP ATP
  • CTP CTP
  • GTP nucleoside triphosphate
  • UTP UTP
  • Mg+2 was also a component of the IVT reaction buffer.
  • Mg+2 and guanine nucleotide (GMP or GTP) were cofactors for the autocatalytic reaction of self-splicing Group I intron. IVT synthesis was initiated with the addition of T7 RNA polymerase.
  • the self-splicing reaction circularized the linear RNA transcript during the IVT reaction.
  • the reaction mixtures were conditioned for refolding and ribozyme activation to increase the yield of circular RNA products.
  • the splicing reactions, IVT, and refolding were analyzed by gel electrophoresis to measure circularization of the RNA precursors.
  • Example 3 Testing different Group I T4td intron sequences for RNA circularization
  • the T4 bacteriophage td gene (T4td) intron sequence was split at different positions illustrated in FIG.
  • FIG. 5A shows an Attorney Docket No: ORB-014WO1 exemplary gel image of T4td-v2, as compared to the legacy split (labeled as T4_td). The results also showed that the T4td-v2 introns resulted in less accumulation of nicked circRNAs during in vitro transcription and post refolding and ribozyme activation.
  • FIG. 5A shows the bands on top of the labeled “circle” bands in FIG. 5A.
  • the nicked circRNA band represents single nicks that occur at random positions in an intact circRNA.
  • FIG. 5B – FIG. 5C show results demonstrating circularization efficiency of the tested introns.
  • T4td-v2 and T4td-v3 introns circularized RNA, and generated about over 50% circular RNA over total RNAs (including circular RNAs and linear RNAs) (FIG. 5B).
  • FIG. 5C are representative gel images showing RNA circularization of T4td-v1 and T4td-v2 at different temperatures (post- refolding and ribozyme activation).
  • the “% spliced” circRNAs over total RNAs post refolding ribozyme activation were measured and the “% nicked RNA (post-refolding ribozyme activation) were also measured and subtracted from the “% spliced” RNA.
  • the overall % intact circRNA was measured for each intron sequence pair at each temperature (as shown in Table 2 below). The results indicated that at temperatures ranges of 37°C to 60°C, each pair of T4td intron sequences resulted in circularization of a precursor RNA sequence. .
  • Example 5 Outer and inner homology elements and RNA circularization [0389] This study tested RNA circularization of the constructs that removed the 5’ and 3’ homology arms
  • the four construct designs described in Example 1 (as illustrated in FIGs. 3A-3D): (1) both OHE/IHE (“OHE/IHE”, FIG. 3A), (2) IHE only, no OHE (“-/IHE”, FIG. 3B), (3) no OHE or IHE ( “-/-”, FIG. 3C) and (4) OHE only, no IHE (“OHE/-”, FIG.
  • RNA sequences were circularized.
  • Each construct included “US” and “DS” sequences from T4td-v1 and T4td-v2, respectively.
  • the results showed that both 5’ and 3’ outer homology arm (OHE) and inner homology elements (IHEs) are not necessary for RNA circularization, in the context of the T4td intron fragments from T4td-v1 and T4td-v2 splits.
  • OOE outer homology arm
  • IHEs inner homology elements
  • Circularization of T4td intron fragments with or without homology arms Attorney Docket No: ORB-014WO1 Table 3b. Circularization of T4td intron fragments without homology arms Table 4: Exemplary upstream and downstream sequences used in circular RNA constructs Attorney Docket No: ORB-014WO1 Attorney Docket No: ORB-014WO1 Attorney Docket No: ORB-014WO1 Attorney Docket No: ORB-014WO1 Attorney Docket No: ORB-014WO1 EQUIVALENTS AND SCOPE [0390] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the following claims:

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Abstract

La présente invention concerne des acides nucléiques et des procédés de production d'ARN circulaires (ARNcirc), des compositions comprenant des ARN circulaires, et leurs procédés d'utilisation. La présente invention utilise des séquences d'introns du gène td du bactériophage T4 à auto-épissage optimisé (T4td) pour produire des ARN circulaires. Les séquences d'introns du T4td optimisées décrites ici assurent une circularisation efficace d'une séquence d'ARN linéaire.
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Citations (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010390A1 (fr) 1997-08-22 1999-03-04 Idemitsu Petrochemical Co., Ltd. Constituants de catalyseurs solides destines a la polymerisation d'olefines, catalyseurs destines a la polymerisation d'olefines et procede de production de polymeres olefiniques
US20050222064A1 (en) 2002-02-20 2005-10-06 Sirna Therapeutics, Inc. Polycationic compositions for cellular delivery of polynucleotides
US7189705B2 (en) 2000-04-20 2007-03-13 The University Of British Columbia Methods of enhancing SPLP-mediated transfection using endosomal membrane destabilizers
US7404969B2 (en) 2005-02-14 2008-07-29 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
WO2008103276A2 (fr) 2007-02-16 2008-08-28 Merck & Co., Inc. Compositions et méthodes de potentialisation de l'activité de molécules biologiquement actives
WO2009132131A1 (fr) 2008-04-22 2009-10-29 Alnylam Pharmaceuticals, Inc. Formulation lipidique améliorée à base d'amino lipide
US20100036115A1 (en) 1997-07-23 2010-02-11 Sirna Therapeutics, Inc. Novel Compositions for the Delivery of Negatively Charged Molecules
WO2010021865A1 (fr) 2008-08-18 2010-02-25 Merck Sharp & Dohme Corp. Nouvelles nanoparticules lipidiques et nouveaux composants pour l'administration d'acides nucléiques
WO2010080724A1 (fr) 2009-01-12 2010-07-15 Merck Sharp & Dohme Corp. Nanoparticules lipidiques inédites et composants inédits utilisables pour l'administration d'acides nucléiques
US7893302B2 (en) 2005-02-14 2011-02-22 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
WO2011022460A1 (fr) 2009-08-20 2011-02-24 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques avec différents groupes de tête pour délivrance d’oligonucléotide
WO2011043913A2 (fr) 2009-10-08 2011-04-14 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques à chaînes lipidiques courtes pour une administration d'oligonucléotides
WO2011056682A1 (fr) 2009-10-27 2011-05-12 The University Of British Columbia Lipides à têtes polaires inversées, compositions particulaires lipidiques comprenant les lipides à têtes polaires inversées, et procédés d'administration d'acides nucléiques
US20110117125A1 (en) 2008-01-02 2011-05-19 Tekmira Pharmaceuticals Corporation Compositions and methods for the delivery of nucleic acids
WO2011090965A1 (fr) 2010-01-22 2011-07-28 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques pour transfert d'oligonucléotide
WO2011127255A1 (fr) 2010-04-08 2011-10-13 Merck Sharp & Dohme Corp. Préparation de nanoparticules de lipide
US20110256175A1 (en) 2008-10-09 2011-10-20 The University Of British Columbia Amino lipids and methods for the delivery of nucleic acids
WO2011149733A2 (fr) 2010-05-24 2011-12-01 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques alcools aminés pour l'administration d'oligonucléotides
WO2011153120A1 (fr) 2010-06-04 2011-12-08 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques de faible poids moléculaire pour l'administration d'oligonucléotides
WO2012040184A2 (fr) 2010-09-20 2012-03-29 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques de faible poids moléculaire pour l'administration d'oligonucléotides
WO2012044638A1 (fr) 2010-09-30 2012-04-05 Merck Sharp & Dohme Corp. Lipides cationiques de faible masse moléculaire utilisables en vue de l'administration d'oligonucléotides
WO2012054365A2 (fr) 2010-10-21 2012-04-26 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques à faible poids moléculaire destinés à une administration d'oligonucléotides
WO2012061259A2 (fr) 2010-11-05 2012-05-10 Merck Sharp & Dohme Corp. Amine cyclique inédite de faible masse moléculaire contenant des lipides cationiques en vue de l'administration d'oligonucléotides
WO2012099755A1 (fr) 2011-01-11 2012-07-26 Alnylam Pharmaceuticals, Inc. Lipides pégylés et leur utilisation pour une administration de médicament
US20120202871A1 (en) 2009-07-01 2012-08-09 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US8283333B2 (en) 2009-07-01 2012-10-09 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
EP2509636A2 (fr) 2009-12-07 2012-10-17 Alnylam Pharmaceuticals, Inc. Compositions utilisées pour l'administration d'acides nucléiques
US20120264810A1 (en) 2009-09-22 2012-10-18 The University Of British Columbia Compositions and methods for enhancing cellular uptake and intracellular delivery of lipid particles
WO2012170889A1 (fr) 2011-06-08 2012-12-13 Shire Human Genetic Therapies, Inc. Lipides clivables
WO2013090648A1 (fr) 2011-12-16 2013-06-20 modeRNA Therapeutics Nucléoside, nucléotide, et compositions d'acide nucléique modifiés
US8546554B2 (en) 2008-09-25 2013-10-01 Alnylam Pharmaceuticals, Inc. Lipid formulated compositions and methods for inhibiting expression of Serum Amyloid A gene
WO2015034925A1 (fr) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Polynucléotides circulaires
WO2015048744A2 (fr) 2013-09-30 2015-04-02 Moderna Therapeutics, Inc. Polynucléotides codant des polypeptides de modulation immunitaire
WO2015085318A2 (fr) 2013-12-06 2015-06-11 Moderna Therapeutics, Inc. Vaccins adaptatifs ciblés
WO2015176737A1 (fr) 2014-05-19 2015-11-26 Biontech Ag Particules comprenant une protamine et un arn combinés à des agents de déstabilisation d'endosome
WO2016000792A1 (fr) 2014-07-04 2016-01-07 Biontech Ag Préparations d'arn stabilisées
WO2016005004A1 (fr) 2014-07-11 2016-01-14 Biontech Rna Pharmaceuticals Gmbh Stabilisation de séquences d'adn codant pour une séquence poly (a)
WO2016011222A2 (fr) 2014-07-16 2016-01-21 Moderna Therapeutics, Inc. Polynucléotides circulaires
WO2016011226A1 (fr) 2014-07-16 2016-01-21 Moderna Therapeutics, Inc. Polynucléotides chimériques
WO2017070626A2 (fr) 2015-10-22 2017-04-27 Modernatx, Inc. Vaccins contre les virus respiratoires
US20180303929A1 (en) 2015-10-22 2018-10-25 Moderna TX, Inc. Herpes simplex virus vaccine
WO2018200892A1 (fr) 2017-04-27 2018-11-01 The Trustees Of The University Of Pennsylvania Vaccin à lignée de nanoparticules d'arnm-lipide modifié par nucléoside pour le virus de l'hépatite c
US20180311336A1 (en) 2015-10-22 2018-11-01 Moderna TX, Inc. Broad spectrum influenza virus vaccine
WO2019057974A1 (fr) 2017-09-22 2019-03-28 Centre National De La Recherche Scientifique (Cnrs) Glycoprotéine mutée du virus de la stomatite vésiculaire
US20200206362A1 (en) 2015-12-10 2020-07-02 Moderna TX, Inc. Compositions and methods for delivery of agents
US20200254086A1 (en) 2017-08-18 2020-08-13 Moderna TX, Inc. Efficacious mrna vaccines
WO2020182869A1 (fr) 2019-03-12 2020-09-17 Biontech Rna Pharmaceuticals Gmbh Arn thérapeutique contre le cancer de la prostate
WO2020236263A1 (fr) 2019-05-23 2020-11-26 Massachusetts Institute Of Technology Découverte de ligands et apport de gènes par l'intermédiaire d'une présentation à la surface de rétrovirus
WO2020255063A1 (fr) 2019-06-20 2020-12-24 Janssen Sciences Ireland Unlimited Company Administration par nanoporteurs glucidiques, de vaccins contre le virus de l'hépatite b (vhb)
WO2021141969A1 (fr) 2020-01-09 2021-07-15 Guide Therapeutics, Inc. Nanomatériaux
WO2022140252A1 (fr) 2020-12-21 2022-06-30 Beam Therapeutics Inc. Nanomatériaux comprenant des acétals à liaison ester
WO2023107886A1 (fr) 2021-12-06 2023-06-15 The Board Of Trustees Of The Leland Stanford Junior University Méthodes et compositions pour la découverte d'une spécificité de ligand de récepteur par une entrée de cellule modifiée

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7686574B2 (ja) * 2019-05-22 2025-06-02 マサチューセッツ インスティテュート オブ テクノロジー 環状ｒｎａ組成物及び方法
AU2022350873A1 (en) * 2021-09-26 2024-04-18 Center For Excellence In Molecular Cell Science, Chinese Academy Of Sciences Circular rna and preparation method thereof
CN119013404A (zh) * 2022-04-06 2024-11-22 新加坡科技研究局 用于产生环状rna的载体
US12297285B2 (en) * 2022-06-24 2025-05-13 Orna Therapeutics, Inc. Circular RNA encoding chimeric antigen receptors targeting BCMA
CN118028340B (zh) * 2024-01-31 2025-04-29 北京悦康科创医药科技股份有限公司 一种用于环状rna表达的核酸构建体及其在蛋白表达中的应用

Patent Citations (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100036115A1 (en) 1997-07-23 2010-02-11 Sirna Therapeutics, Inc. Novel Compositions for the Delivery of Negatively Charged Molecules
WO1999010390A1 (fr) 1997-08-22 1999-03-04 Idemitsu Petrochemical Co., Ltd. Constituants de catalyseurs solides destines a la polymerisation d'olefines, catalyseurs destines a la polymerisation d'olefines et procede de production de polymeres olefiniques
US7189705B2 (en) 2000-04-20 2007-03-13 The University Of British Columbia Methods of enhancing SPLP-mediated transfection using endosomal membrane destabilizers
US20050222064A1 (en) 2002-02-20 2005-10-06 Sirna Therapeutics, Inc. Polycationic compositions for cellular delivery of polynucleotides
US7404969B2 (en) 2005-02-14 2008-07-29 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
US7893302B2 (en) 2005-02-14 2011-02-22 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
WO2008103276A2 (fr) 2007-02-16 2008-08-28 Merck & Co., Inc. Compositions et méthodes de potentialisation de l'activité de molécules biologiquement actives
US20110117125A1 (en) 2008-01-02 2011-05-19 Tekmira Pharmaceuticals Corporation Compositions and methods for the delivery of nucleic acids
WO2009132131A1 (fr) 2008-04-22 2009-10-29 Alnylam Pharmaceuticals, Inc. Formulation lipidique améliorée à base d'amino lipide
WO2010021865A1 (fr) 2008-08-18 2010-02-25 Merck Sharp & Dohme Corp. Nouvelles nanoparticules lipidiques et nouveaux composants pour l'administration d'acides nucléiques
US8546554B2 (en) 2008-09-25 2013-10-01 Alnylam Pharmaceuticals, Inc. Lipid formulated compositions and methods for inhibiting expression of Serum Amyloid A gene
US20110256175A1 (en) 2008-10-09 2011-10-20 The University Of British Columbia Amino lipids and methods for the delivery of nucleic acids
WO2010080724A1 (fr) 2009-01-12 2010-07-15 Merck Sharp & Dohme Corp. Nanoparticules lipidiques inédites et composants inédits utilisables pour l'administration d'acides nucléiques
US20120202871A1 (en) 2009-07-01 2012-08-09 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US8283333B2 (en) 2009-07-01 2012-10-09 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
WO2011022460A1 (fr) 2009-08-20 2011-02-24 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques avec différents groupes de tête pour délivrance d’oligonucléotide
US20120264810A1 (en) 2009-09-22 2012-10-18 The University Of British Columbia Compositions and methods for enhancing cellular uptake and intracellular delivery of lipid particles
WO2011043913A2 (fr) 2009-10-08 2011-04-14 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques à chaînes lipidiques courtes pour une administration d'oligonucléotides
WO2011056682A1 (fr) 2009-10-27 2011-05-12 The University Of British Columbia Lipides à têtes polaires inversées, compositions particulaires lipidiques comprenant les lipides à têtes polaires inversées, et procédés d'administration d'acides nucléiques
EP2509636A2 (fr) 2009-12-07 2012-10-17 Alnylam Pharmaceuticals, Inc. Compositions utilisées pour l'administration d'acides nucléiques
WO2011090965A1 (fr) 2010-01-22 2011-07-28 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques pour transfert d'oligonucléotide
WO2011127255A1 (fr) 2010-04-08 2011-10-13 Merck Sharp & Dohme Corp. Préparation de nanoparticules de lipide
WO2011149733A2 (fr) 2010-05-24 2011-12-01 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques alcools aminés pour l'administration d'oligonucléotides
WO2011153120A1 (fr) 2010-06-04 2011-12-08 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques de faible poids moléculaire pour l'administration d'oligonucléotides
WO2012040184A2 (fr) 2010-09-20 2012-03-29 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques de faible poids moléculaire pour l'administration d'oligonucléotides
WO2012044638A1 (fr) 2010-09-30 2012-04-05 Merck Sharp & Dohme Corp. Lipides cationiques de faible masse moléculaire utilisables en vue de l'administration d'oligonucléotides
WO2012054365A2 (fr) 2010-10-21 2012-04-26 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques à faible poids moléculaire destinés à une administration d'oligonucléotides
WO2012061259A2 (fr) 2010-11-05 2012-05-10 Merck Sharp & Dohme Corp. Amine cyclique inédite de faible masse moléculaire contenant des lipides cationiques en vue de l'administration d'oligonucléotides
WO2012099755A1 (fr) 2011-01-11 2012-07-26 Alnylam Pharmaceuticals, Inc. Lipides pégylés et leur utilisation pour une administration de médicament
WO2012170889A1 (fr) 2011-06-08 2012-12-13 Shire Human Genetic Therapies, Inc. Lipides clivables
WO2013090648A1 (fr) 2011-12-16 2013-06-20 modeRNA Therapeutics Nucléoside, nucléotide, et compositions d'acide nucléique modifiés
WO2015034925A1 (fr) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Polynucléotides circulaires
WO2015048744A2 (fr) 2013-09-30 2015-04-02 Moderna Therapeutics, Inc. Polynucléotides codant des polypeptides de modulation immunitaire
WO2015085318A2 (fr) 2013-12-06 2015-06-11 Moderna Therapeutics, Inc. Vaccins adaptatifs ciblés
WO2015176737A1 (fr) 2014-05-19 2015-11-26 Biontech Ag Particules comprenant une protamine et un arn combinés à des agents de déstabilisation d'endosome
WO2016000792A1 (fr) 2014-07-04 2016-01-07 Biontech Ag Préparations d'arn stabilisées
WO2016005004A1 (fr) 2014-07-11 2016-01-14 Biontech Rna Pharmaceuticals Gmbh Stabilisation de séquences d'adn codant pour une séquence poly (a)
WO2016011222A2 (fr) 2014-07-16 2016-01-21 Moderna Therapeutics, Inc. Polynucléotides circulaires
WO2016011226A1 (fr) 2014-07-16 2016-01-21 Moderna Therapeutics, Inc. Polynucléotides chimériques
US20180311336A1 (en) 2015-10-22 2018-11-01 Moderna TX, Inc. Broad spectrum influenza virus vaccine
WO2017070626A2 (fr) 2015-10-22 2017-04-27 Modernatx, Inc. Vaccins contre les virus respiratoires
US20180303929A1 (en) 2015-10-22 2018-10-25 Moderna TX, Inc. Herpes simplex virus vaccine
US20200206362A1 (en) 2015-12-10 2020-07-02 Moderna TX, Inc. Compositions and methods for delivery of agents
WO2018200892A1 (fr) 2017-04-27 2018-11-01 The Trustees Of The University Of Pennsylvania Vaccin à lignée de nanoparticules d'arnm-lipide modifié par nucléoside pour le virus de l'hépatite c
US20200254086A1 (en) 2017-08-18 2020-08-13 Moderna TX, Inc. Efficacious mrna vaccines
WO2019057974A1 (fr) 2017-09-22 2019-03-28 Centre National De La Recherche Scientifique (Cnrs) Glycoprotéine mutée du virus de la stomatite vésiculaire
WO2020182869A1 (fr) 2019-03-12 2020-09-17 Biontech Rna Pharmaceuticals Gmbh Arn thérapeutique contre le cancer de la prostate
WO2020236263A1 (fr) 2019-05-23 2020-11-26 Massachusetts Institute Of Technology Découverte de ligands et apport de gènes par l'intermédiaire d'une présentation à la surface de rétrovirus
WO2020255063A1 (fr) 2019-06-20 2020-12-24 Janssen Sciences Ireland Unlimited Company Administration par nanoporteurs glucidiques, de vaccins contre le virus de l'hépatite b (vhb)
WO2021141969A1 (fr) 2020-01-09 2021-07-15 Guide Therapeutics, Inc. Nanomatériaux
WO2022140252A1 (fr) 2020-12-21 2022-06-30 Beam Therapeutics Inc. Nanomatériaux comprenant des acétals à liaison ester
WO2023107886A1 (fr) 2021-12-06 2023-06-15 The Board Of Trustees Of The Leland Stanford Junior University Méthodes et compositions pour la découverte d'une spécificité de ligand de récepteur par une entrée de cellule modifiée

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
"Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC PRESS
"Computer Analysis of Sequence Data", 1994, HUMANA PRESS
"Remington: The Science and Practice of Pharmacy", 2006, LIPPINCOTT, WILLIAMS & WILKINS
"Sequence Analysis Primer", 1991, M STOCKTON PRESS
ALTSCHUL, S. F. ET AL., J. MOLEC. BIOL., vol. 215, 1990, pages 403
CARILLO, H.LIPMAN, D., SIAM J APPLIED MATH., vol. 48, 1988, pages 1073
CHEN ET AL., NATURE BIOTECHNOLOGY, vol. 41, no. 2, 2023, pages 262 - 272
CHU, F.K. ET AL., CELL, vol. 45, no. 2, 25 April 1986 (1986-04-25), pages 157 - 166
DEVEREUX, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, no. 1, 1984, pages 387
GEALL ET AL., PNAS, vol. 109, no. 36, 2012, pages 14604 - 14609
HAUSNER ET AL., MOBILE DNA, vol. 5, no. 8, 2014
LUPTAKDOUDNA, NUCLEIC ACIDS RESEARCH, vol. 32, no. 7, 2004, pages 2272 - 80
MEYERSMILLER, CABIOS, vol. 4, 1989, pages 11 - 17
MICHELWESTHOF, J. MOL. BIO., vol. 216, 1990, pages 585 - 610
PATEL ET AL., NATURE COMMUNICATIONS, vol. 983, 2020
RAUSCH, J.W. ET AL., NUCLEIC ACIDS RESEARCH, vol. 49, no. 6, 6 January 2021 (2021-01-06), pages e35
VON HEINJE, G.: "Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS

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