Patent Application
20240035025
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 61057-718.601.xml, created on or about Sep. 6, 2023, which is 66,455 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.
Linear RNA molecules have many applications in biological research and therapeutic development. However, the relatively short half-life of long linear RNA molecules can limit their use in applications where long-term protein translation is desired. Circular RNAs (circRNAs) have been reported to exist in various organisms, including C. elegans, Drosophila melanogaster, mice, and humans and have been explored for a applications including microRNA sponges and protein expression. Methods for synthesizing circRNAs include the use of a splint molecule to bring the 5′ and 3′ ends of linear RNA in close proximity for ligation and the use of ribozymatic methods in conjunction with self-splicing introns to covalently link the 5′ and 3′ ends of an in vitro-transcribed (iVT) RNA molecule. However, these methods suffer from low efficiency or result in cytotoxic byproducts that must be removed using high-performance liquid chromatography (HPLC). What is needed is improved circRNA compositions, and methods of making circRNAs.
Accordingly, the present disclosure relates to, in aspects, RNA molecules which are engineered to promote formation of Circular RNAs (circRNAs), methods for making circRNAs, and the circRNAs themselves.
In aspects, the present disclosure provides methods of making circRNA and the circRNA that result therefrom.
In aspects, the present disclosure provides methods of making circRNA that are substantially free of cytotoxic byproducts. In embodiments, the present disclosure provides methods of making circRNA that do not require a linear oligonucleotide (splint) to pre-orient the two reacting ends of a linear RNA to assist in ligation to yield a circRNA. In embodiments, the present disclosure provides methods of making circRNA that do not require ribozymes to yield a circRNA. In embodiments, the present disclosure provides methods of making circRNA that do not require HPLC-based purification, e.g. post-ligation.
An aspect of the present disclosure is a composition comprising a nucleic acid of the structure: 5′-X—Y-A-IRES-B—CDS—C—Y′—Z-3′. In this aspect, Y and Y′ each independently comprise one or more nucleotides and Y and Y′ are substantially complementary; X and Z each independently comprise one or more nucleotides and X and Z are not substantially complementary; IRES comprises an internal ribosome entry site; CDS comprises a coding sequence; and A, B, and C are each independently a spacer comprising one or more nucleotides or null.
In embodiments, the protein of interest is selected from Table 1, Table 2, or Table 3, inclusive of the protein product of any gene of Table 1, Table 2, or Table 3.
In some embodiments, the protein of interest is one or more gene-editing proteins comprising (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG or LTPvQVVAIAwxyzGTHG and is between 36 and 39 amino acids long, wherein: “v” is Q, D or E, “w” is S or N, “x” is H, N, or I, “y” is D, A, I, N, G, H, K, S, or null, and “z” is GGKQALETVQRLLPVLCQD or GGKQALETVQRLLPVLCQA and (ii) a nuclease domain comprising a catalytic domain of a nuclease.
In various embodiments, the protein of interest is one or more gene-editing proteins comprising (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα and is between 36 and 39 amino acids long, wherein: v is Q, D or E, w is S or N, x is I, H, N, or I, y is D, A, I, N, H, K, S, G or null, z is GGRPALE, GGKQALE, GKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG, GKQALETVQRLLPVLCQAHG, GGKQALETVQRLLPVLCQD or GGKQALETVQRLLPVLCQA, a is four consecutive amino acids; and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In some cases, α is selected from GHGG, HGSG, HGGG, GGHD, GAHD, AHDG, PHDG, GPHD, GHGP, PHGG, PHGP, AHGA, LHGA, VHGA, IVHG, IHGM, RHGD, RDHG, RHGE, HRGE, RHGD, HRGD, GPYE, NHGG, THGG, GTHG, GSGS, GSGG, GGGG, Another aspect of the present disclosure is a pharmaceutical composition comprising any herein-disclosed nucleic acid composition or comprising the any herein-disclosed circRNA composition, and a pharmaceutically acceptable carrier, vehicle or excipient.
Yes another aspect of the present disclosure is a host cell comprising any herein-disclosed composition.
In an aspect, the present disclosure provides a method of making a circRNA. The method comprising a step of contacting any herein-disclosed nucleic acid composition with one or more RNA ligases to result in circularization of the nucleic acid and formation of the circRNA.
In another aspect, the present disclosure provides a method of expressing a protein of interest in a cell. The method comprising a step of contacting the cell with any herein-disclosed nucleic acid or any herein-disclosed circRNA composition.
In yet another aspect, the present disclosure provides a method of gene editing a target nucleic acid in a cell. The method comprising a step of contacting the cell with any herein-disclosed composition or any herein-disclosed pharmaceutical composition. In this aspect, the composition comprises a circRNA in which the CDS encodes one or more proteins of interest, the proteins of interest being one or more gene-editing proteins, optionally selected from a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, CRISPR/Cas9, Cas9, xCas9, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, and a gene-editing protein comprising a repeat sequence comprising LTPvQVVAIAwxyzα, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof. In this aspect, the gene-editing protein is directed to the target nucleic acid.
In embodiments, the target nucleic acid is a gene selected from Table 2 or encodes a peptide or protein selected from Table 1 or Table 3.
In some embodiments, the method further comprises reprogramming the cell, e.g., comprising contacting the cell with a herein-disclosed composition comprising a circRNA comprising a CDS encoding a reprogramming factor.
An aspect of the present disclosure is a method of reprogramming a cell. The method comprising a step of contacting the cell with any herein-disclosed composition or any herein-disclosed pharmaceutical composition. In this aspect, the composition comprises a circRNA and the CDS encodes one or more proteins of interest, the protein of interest being one or more reprogramming factors, optionally selected from Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, Lin28, Glis1, Utf1, Aicda, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof. In some cases, the CDS encodes two, three, four, five, six, seven, eight, nine, ten, eleven, or more reprogramming factor(s).
In various embodiments, the method comprises (a) providing a differentiated or a non-pluripotent cell; (b) culturing the differentiated or a non-pluripotent cell; (c) transfecting the differentiated or a non-pluripotent cell with the circRNA.
In embodiments, the method further comprises gene-editing the cell, e.g., comprising contacting the cell with any herein composition comprising a circRNA in which the CDS encodes one or more proteins of interest, the proteins of interest being one or more gene-editing proteins, optionally selected from a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, CRISPR/Cas9, Cas9, xCas9, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, and a gene-editing protein comprising a repeat sequence comprising LTPvQVVAIAwxyzα, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.
In embodiments, the gene-editing targets a nucleic acid which is a gene selected from Table 2 or which encodes a peptide or protein selected from Table 1 or Table 3.
Another aspect of the present disclosure is a method of treating a disease, disorder, or condition. The method comprising steps of (1) contacting a cell with any herein-disclosed composition or any herein-disclosed pharmaceutical composition and administering the cell to a patient in need thereof or (2) administering any herein-disclosed composition or any herein-disclosed pharmaceutical composition of to a patient in need thereof. In embodiments, the disease, disorder, or condition is selected from Table 1 or Table 3.
Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.
The present disclosure is based, in part, on the discovery of methods to yield circRNA in an efficient manner.
In aspects, the present disclosure provides RNA that is engineered to yield circRNA, e.g. via non-complementary and complementary sequence elements.
In aspects, the present disclosure provides methods of making circRNA and the circRNA that result therefrom.
circRNAs and Compositions Comprising circRNAs
circRNAs comprise single-stranded RNAs that are joined head to tail. circRNAs were initially discovered in pathogenic genomes such as hepatitis D virus (HDV) and plant viroids. circRNAs may function as potential molecular markers of disease and may play an important role in the initiation and progression of human diseases, including in tumors.
CircRNAs of the present disclosure may be about 100 nucleotides in length, about 200 nucleotides in length, about 300 nucleotides in length, about 400 nucleotides in length, about 500 nucleotides in length, about 600 nucleotides in length, about 700 nucleotides in length, about 800 nucleotides in length, about 900 nucleotides in length, about 1000 nucleotides in length, about 1100 nucleotides in length, about 1200 nucleotides in length, about 1300 nucleotides in length, about 1400 nucleotides in length, about 1500 nucleotides in length, about 2000 nucleotides in length, about 2500 nucleotides in length, about 3000 nucleotides in length, about 3500 nucleotides in length, about 4000 nucleotides in length, about 4500 nucleotides in length, about 5000 nucleotides in length, about 5500 nucleotides in length, about 6000 nucleotides in length, about 6500 nucleotides in length, about 7000 nucleotides in length, about 7500 nucleotides in length, about 8000 nucleotides in length, about 8500 nucleotides in length, about 9000 nucleotides in length, about 9500 nucleotides in length, or about 10000 nucleotides in length. CircRNAs of the present disclosure may be at least about 100 nucleotides in length, at least about 200 nucleotides in length, at least about 300 nucleotides in length, at least about 400 nucleotides in length, at least about 500 nucleotides in length, at least about 600 nucleotides in length, at least about 700 nucleotides in length, at least about 800 nucleotides in length, at least about 900 nucleotides in length, at least about 1000 nucleotides in length, at least about 1100 nucleotides in length, at least about 1200 nucleotides in length, at least about 1300 nucleotides in length, at least about 1400 nucleotides in length, at least about 1500 nucleotides in length, at least about 1600 nucleotides in length, at least about 1700 nucleotides in length, at least about 1800 nucleotides in length, at least about 1900 nucleotides in length, at least about 2000 nucleotides in length, at least about 2500 nucleotides in length, at least about 3000 nucleotides in length, at least about 3500 nucleotides in length, at least about 4000 nucleotides in length, at least about 4500 nucleotides in length, at least about 5000 nucleotides in length, at least about 5500 nucleotides in length, at least about 6000 nucleotides in length, at least about 6500 nucleotides in length, at least about 7000 nucleotides in length, at least about 7500 nucleotides in length, at least about 8000 nucleotides in length, at least about 8500 nucleotides in length, at least about 9000 nucleotides in length, at least about 9500 nucleotides in length, or at least about 10000 nucleotides in length. CircRNAs of the present disclosure may be from about 100 to about 200 nucleotides in length, from about 200 to about 300 nucleotides in length, from about 300 to about 400 nucleotides in length, from about 400 to about 500 nucleotides in length, from about 500 to about 600 nucleotides in length, from about 600 to about 700 nucleotides in length, from about 700 to about 800 nucleotides in length, from about 800 to about 900 nucleotides in length, from about 900 to about 1000 nucleotides in length, from about 1000 to about 1100 nucleotides in length, from about 1100 to about 1200 nucleotides in length, from about 1200 to about 1300 nucleotides in length, from about 1300 to about 1400 nucleotides in length, from about 1400 to about 1500 nucleotides in length, from about 1500 to about 2000 nucleotides in length, from about 2000 to about 2500 nucleotides in length, from about 2500 to about 3000 nucleotides in length, from about 3000 to about 3500 nucleotides in length, from about 3500 to about 4000 nucleotides in length, from about 4000 to about 4500 nucleotides in length, from about 4500 to about 5000 nucleotides in length, from about 5000 to about 5500 nucleotides in length, from about 5500 to about 6000 nucleotides in length, from about 6000 to about 6500 nucleotides in length, from about 6500 to about 7000 nucleotides in length, from about 7000 to about 7500 nucleotides in length, from about 7500 to about 8000 nucleotides in length, from about 8000 to about 8500 nucleotides in length, from about 8500 to about 9000 nucleotides in length, from about 9000 to about 9500 nucleotides in length, or from about 9500 to about 10000 nucleotides in length, and any length therebetween.
Preparation of circRNAs from linear precursors can pose serious challenges, due to, inter alia, a negative entropy associated with the circularization step. The most significant side reaction can be intramolecular bond forming leading to oligomerization of the linear precursor instead of circularization.
In aspects, the present disclosure relates to engineered nucleic acids, e.g. RNAs, that are suitable for efficient circularization to yield circRNAs. In aspects, the present disclosure relates to RNA sequences that form a hairpin loop in close proximity to the 5′ and 3′ ends and better support intramolecular ligation than RNA sequences without such a hairpin loop.
In aspects, the present disclosure relates to a composition comprising a nucleic acid of the structure:
5′-X—Y-A-IRES-B—CDS—C—Y′—Z-3′,
where Y and Y′ each independently comprise one or more nucleotides and Y and Y′ are substantially complementary; X and Z each independently comprise one or more nucleotides and X and Z are not substantially complementary; IRES comprises an internal ribosome entry site; CDS comprises a coding sequence; and A, B, and C are each independently a spacer comprising one or more nucleotides or null.
In embodiments, the nucleic acid is RNA. In embodiments, the RNA is synthetic RNA.
In embodiments, the nucleic acid forms a circular RNA (circRNA), e.g., when contacted with an RNA ligase.
In embodiments, the CDS comprises one more exons. In embodiments, the CDS encodes one or more proteins of interest. In embodiments, the protein of interest is a soluble protein. In embodiments, the protein of interest is selected from Table 1, Table 2, or Table 3, inclusive of the protein product of any gene of Table 1, Table 2, or Table 3.
In embodiments, the protein of interest is one or more reprogramming factors, optionally selected from Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, Lin28, Glis1, Utf1, Aicda, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof.
In embodiments, the protein of interest is one or more gene-editing proteins, optionally selected from a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, CRISPR/Cas9, Cas9, xCas9, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, and MAD7, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.
In embodiments, the protein of interest is one or more gene-editing proteins comprising (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG or LTPvQVVAIAwxyzGTHG and is between 36 and 39 amino acids long, wherein: “v” is Q, D or E, “w” is S or N, “x” is H, N, or I, “y” is D, A, I, N, G, H, K, S, or null, and “z” is GGKQALETVQRLLPVLCQD or GGKQALETVQRLLPVLCQA and (ii) a nuclease domain comprising a catalytic domain of a nuclease.
In embodiments, the protein of interest is one or more gene-editing proteins comprising (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα and is between 36 and 39 amino acids long, wherein: v is Q, D or E, w is S or N, x is I, H, N, or I, y is D, A, I, N, H, K, S, G or null, z is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG, GKQALETVQRLLPVLCQAHG, GGKQALETVQRLLPVLCQD or GGKQALETVQRLLPVLCQA, a is four consecutive amino acids; and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In embodiments, a is selected from
In embodiments, the IRES is selected from cMyc, CVB3 (coxsackievirus B3), EMCV (encephalomyocarditis virus), HCV (hepatitis C virus) HRV (human rhinovirus), or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof. In embodiments, the IRES is selected from poliovirus (PV), encephalomyelocarditis virus (EMCV), classical swine-fever virus (CSFV), foot-and-mouth disease virus (FMDV), human immunodeficiency virus (HIV), bovine viral diarrhoea virus (BVDV) and cricket paralysis virus (CrPV), or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.
In embodiments, the nucleic acid further comprises a Type IIS restriction enzyme site at or near the 3′ end.
In embodiments, A comprises one or more nucleotides, e.g. about 5-25, or about 5-20, or about 5-15, or about 5-15 nucleotides, e.g. about 5, or about 10, or about 15, or about 20, or about 25 nucleotides. In embodiments, A is null.
In embodiments, B comprises one or more nucleotides, e.g. about 5-25, or about 5-20, or about 5-15, or about 5-15 nucleotides, e.g. about 5, or about 10, or about 15, or about 20, or about 25 nucleotides. In embodiments, B is null.
In embodiments, C comprises one or more nucleotides, e.g. about 5-25, or about 5-20, or about 5-15, or about 5-15 nucleotides, e.g. about 5, or about 10, or about 15, or about 20, or about 25 nucleotides. In embodiments, C is null.
In embodiments, A comprises one or more nucleotides, e.g. about 1-200, or about 50-200, or about 100-200, or about 150-200 nucleotides, e.g. about 50, or about 75, or about 100, or about 150, or about 200 nucleotides.
In embodiments, B comprises one or more nucleotides, e.g. about 1-200, or about 50-200, or about 100-200, or about 150-200 nucleotides, e.g. about 50, or about 75, or about 100, or about 150, or about 200 nucleotides.
In embodiments, C comprises one or more nucleotides, e.g. about 1-200, or about 50-200, or about 100-200, or about 150-200 nucleotides, e.g. about 50, or about 75, or about 100, or about 150, or about 200 nucleotides.
In embodiments, A, B, and C are identical. In embodiments, two of A, B, and C are identical.
In embodiments, A, B, and C are non-identical.
In embodiments, Y and Y′ each independently comprise one or more nucleotides, e.g. about 1-25, or about 1-20, or about 1-15, or about 1-15 nucleotides, or about 5-10 nucleotides e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 10, or about 15, or about 20, or about 25 nucleotides.
In embodiments, X and Z each independently comprise one or more nucleotides, e.g. about 1-25, or about 1-20, or about 1-15, or about 1-15 nucleotides, or about 5-10 nucleotides e.g. about 1, or about 2, or about 3, or about 4, or about 5, or about 10, or about 15, or about 20, or about 25 nucleotides.
In embodiments, Y and Y′ are fully complementary. In embodiments, Y and Y′ are partially complementary.
In embodiments, X, Z, Y and Y′ form a hairpin structure. In embodiments, the hairpin structure is a loose hairpin of 24 or fewer nucleotides being preferentially bound within the hairpin. In embodiments, the hairpin structure is a tight hairpin with more than 24 nucleotides being preferentially bound within the hairpin. In embodiments, X, Z, Y and Y′ do not form a hairpin structure. In embodiments, X, Z, Y and Y′ form one of the structures illustrated in of
In embodiments, X and Z are suitable for interaction with an RNA ligase. In embodiments, the RNA ligase is a single strand RNA ligase. In embodiments, the single strand RNA ligase is T4 RNA Ligase 1, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.
In embodiments, the circRNA is substantially stable from an exonuclease.
In embodiments, the nucleic acid is suitable for synthesis by in vitro transcription.
In embodiments, the circRNA molecule has better pharmacokinetic behavior, such as absorption, efficacy, bioavailability and/or half-life, than a linear RNA molecule comprising the same sequence, or comparable sequence, as the circRNA molecule.
In embodiments, the circRNA molecule has better stability than a linear RNA molecule comprising the same sequence, or comparable sequence, as the circRNA molecule. In embodiments, the circRNA molecule has better intracellular stability than a linear RNA molecule comprising the same sequence, or comparable sequence, as the circRNA molecule.
In embodiments, the circRNA molecule has a longer intracellular half-life than a linear RNA molecule comprising the same sequence, or comparable sequence, as the circRNA molecule.
In embodiments, the intracellular half-life of the circRNA molecule is at least about 3 hours longer, or at least about 6 hours longer, or at least about 12 hours longer than a linear RNA molecule comprising the same sequence, or comparable sequence, as the circRNA molecule
In embodiments, the intracellular half-life of the circRNA molecule is at least about 1.5 times as long, or at least about 3 times as long, or at least about 5 times as long, or at least about 10 times as long, or at least about 30 times as long, or at least about 100 times as long as a linear RNA molecule comprising the same sequence, or comparable sequence, as the circRNA molecule.
In embodiments, there is provided a pharmaceutical composition comprising a composition comprising a herein-disclosed nucleic acid molecule (e.g., comprising the linear nucleic acid of the structure: 5′-X—Y-A-IRES-B—CDS—C—Y′—Z-3′), and a pharmaceutically acceptable carrier, vehicle or excipient.
In embodiments, there is provided a pharmaceutical composition comprising a composition comprising a herein-disclosed circRNA molecule, and a pharmaceutically acceptable carrier, vehicle or excipient.
In embodiments, there is provided a host cell comprising a herein-disclosed nucleic acid molecule (e.g., comprising the linear nucleic acid of the structure: 5′-X—Y-A-IRES-B—CDS—C—Y′—Z-3′) or a composition comprising the herein-disclosed nucleic acid.
In embodiments, there is provided a host cell comprising a herein-disclosed circRNA molecule or a composition comprising the herein-disclosed circRNA molecule.
Methods of Making
In embodiments, the present disclosure provides methods of making circRNA that are substantially free of cytotoxic byproducts.
In embodiments, the present disclosure provides methods of making circRNA that do not require a linear oligonucleotide (splint) to pre-orient the two reacting ends of a linear RNA to assist in ligation to yield a circRNA. In embodiments, the present disclosure provides methods of making circRNA that do not require ribozymes to yield a circRNA. In embodiments, the present disclosure provides methods of making circRNA that do not require a linear oligonucleotide (splint) to pre-orient the two reacting ends of a linear RNA to assist in ligation to yield a circRNA and do not require ribozymes to yield a circRNA. In embodiments, the present disclosure provides methods of making circRNA that have better ligation efficiency than alternative methods, e.g., those involving splints or ribozymes.
In embodiments, the present disclosure provides methods of making circRNA that do not require HPLC-based purification, e.g. post-ligation.
In embodiments, the present disclosure provides nucleic acids (e.g., a linear RNA molecules of the structure: 5′-X—Y-A-IRES-B—CDS—C—Y′—Z-3′) that form a hairpin loop in close proximity to the 5′ and 3′ ends and support intramolecular ligation, such intramolecular ligation being improved, e.g. relative to RNA sequences without such a hairpin loop.
In embodiments, the present disclosure provides a method of making a circRNA comprising contacting a herein-disclosed composition comprising a nucleic acid (e.g., a linear RNA molecule of the structure: 5′-X—Y-A-IRES-B—CDS—C—Y′—Z-3′) with one or more RNA ligases to result in circularization of the nucleic acid. In embodiments, the RNA ligase is a single strand RNA ligase. In embodiments, the single strand RNA ligase is T4 RNA Ligase 1, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.
In embodiments, the nucleic acid (e.g., a linear RNA molecule of the structure: 5′-X—Y-A-IRES-B—CDS—C—Y′—Z-3′) is synthesized in vitro transcription (iVT). In embodiments, the nucleic acid is synthesized using both guanosine monophosphate and guanosine triphosphate in the iVT reaction. In embodiments guanosine monophosphate and guanosine triphosphate are present at a ratio e.g. about 1:1, or about 2:1, or about 5:1, or about 10:1 or about 20:1.
In embodiments, the nucleic acid and/or circRNA comprises at least one non-canonical nucleotide. In embodiments, the non-canonical nucleotide is selected from 5-methylcytidine, 5-hydroxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxy cytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-methoxyuridine, 5-formyluridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-methoxypseudouridine, and 5-formylpseudouridine
In embodiments, the nucleic acid and/or circRNA lacks any non-canonical nucleotides.
Methods of Expressing a Protein of Interest/Reprogramming/Gene-Editing/Disease Treatment
In embodiments, the present disclosure provides a method of expressing a protein of interest in a cell, comprising contacting the cell with a herein-disclosed composition comprising a circRNA.
In embodiments, the cell is contacted with mild hypothermic conditions, e.g., about 30° C. to about 36° C.
In embodiments, the present disclosure provides a method of gene editing a target nucleic acid in a cell, comprising contacting the cell with the composition or pharmaceutical composition described herein where the composition comprises a circRNA and the CDS encodes one or more proteins of interest, the proteins of interest being one or more gene-editing proteins, optionally selected from a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, CRISPR/Cas9, Cas9, xCas9, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, and a gene-editing protein comprising a repeat sequence comprising LTPvQVVAIAwxyzα as defined herein, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof, where the gene-editing protein is directed to the target nucleic acid. In some cases, the target nucleic acid is a gene selected from Table 2 or encodes a peptide or protein selected from Table 1 or Table 3. In embodiments, the present disclosure provides a method of increasing a replicative potential of a cell, comprising contacting the cell with the composition or pharmaceutical composition described herein where the composition comprises a circRNA and the CDS encodes one or more proteins of interest. In embodiments, the circRNA encodes a protein that increases the replicative potential of a cell. In embodiments, the encoded protein is TERT. In embodiments, the cell is selected from: a hematopoietic cell, a hematopoietic stem cell, a T cell, an NK cell, a myeloid cell, a macrophage, a tumor-infiltrating lymphocyte, a marrow-infiltrating lymphocytes, a peripheral blood lymphocyte, and a hair follicle stem cell.
In embodiments, the circRNA encodes a protein that increases the interaction between a cell and a protein. In embodiments, the encoded protein is chimeric antigen receptor. In embodiments, the cell is selected from: a hematopoietic cell, a hematopoietic stem cell, a T cell, an NK cell, a myeloid cell, a macrophage, a tumor-infiltrating lymphocyte, a marrow-infiltrating lymphocytes, a peripheral blood lymphocyte, and a hair follicle stem cell.
In embodiments, the cell is contacted with mild hypothermic conditions, e.g., about 30° C. to about 36° C.
In embodiments, the method further comprises reprogramming the cell. In embodiments, the present disclosure provides a method of reprogramming a cell, comprising contacting the cell with the composition described herein, where the composition is a circRNA and the CDS encodes one or more proteins of interest, the protein of interest being one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or more) reprogramming factors, optionally selected from Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, Lin28, Glis1, Utf1, Aicda, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.
In embodiments, method comprises (a) providing a differentiated or a non-pluripotent cell; (b) culturing the differentiated or a non-pluripotent cell; (c) transfecting the differentiated or a non-pluripotent cell with the circRNA. In embodiments, the transfecting is accomplished via electroporation. In embodiments, step (c) occurs in the presence of a medium containing ingredients that support reprogramming of the differentiated or a non-pluripotent to a less differentiated state. In embodiments, the method further comprises repeating step (c) at least twice during 5 consecutive days. In embodiments, the amount of one or more circRNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections. In embodiments, steps (a)-(c) are performed without using feeder cells and occur in the presence of a feeder cell conditioned medium. In embodiments, step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a feeder cell conditioned medium. In embodiments, the circRNA molecule encodes one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or more) reprogramming factor(s) selected from Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, Lin28, Glis1, Utf1, Aicda, miR200 micro-RNA, MiR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, MiR369 micro-RNA and biologically active fragments, analogues, variants and family members thereof. In embodiments, the differentiated or a non-pluripotent cell is derived from a biopsy. In embodiments, the differentiated or a non-pluripotent cell is from a human subject. In embodiments, the differentiated or a non-pluripotent cell is derived from a dermal punch biopsy sample. In embodiments, the differentiated or a non-pluripotent cell is a skin cell (e.g., a fibroblast or a keratinocyte). In embodiments, the method comprises or further comprises contacting the cell with at least one member of the group: poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, and laminin. In embodiments, the method further comprises gene-editing the cell.
In embodiments, the circRNA molecule comprises at least one non-canonical nucleotide, optionally selected from 5-methylcytidine, 5-hydroxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-methoxyuridine, 5-formyluridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-methoxypseudouridine, and 5-formylpseudouridine.
In embodiments, the circRNA molecule lacks any non-canonical nucleotides.
In embodiments, the medium is substantially free of immunosuppressants.
In embodiments, the cell is contacted with mild hypothermic conditions, e.g., about 30° C. to about 36° C.
In embodiments, the method further comprises gene-editing the cell.
In embodiments, the present disclosure provides a method of treating a disease, disorder, or condition comprising: (a) contacting a cell with the composition described herein (e.g., comprising a circRNA) and administering the cell to a patient in need thereof or (b) administering the composition (e.g., comprising a circRNA) to a patient in need thereof. In embodiments, the disease, disorder, or condition is selected from Table 1 or Table 3.
In embodiments, the circRNA molecule encodes one or more of: a T-cell receptor, a chimeric antigen receptor, a bispecific T-cell engagers (BiTE), a checkpoint inhibitor, an antibody, a nanobody, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.
In some cases, a cell transfected with a circRNA is a T-cell and the circRNA encodes a chimeric antigen receptor such that the transfected cell becomes a CAR-T. In other cases, a cell transfected with a circRNA is an NK cell and the circRNA encodes a chimeric antigen receptor such that the transfected cell becomes a CAR-NK.
In various cases, a reprogrammed cell (with or without gene editing) is provided to a subject in need thereof in the context of a stem cell therapy.
In embodiments, the circRNA molecule encodes on or more of Interleukin 7, Interleukin 12, Interleukin 15, Interleukin 18, dominant-negative TGF-β receptor II (dnTGF-βRII), constitutively active Akt (caAkt), or CD40 Ligand (CD40L), or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.
In embodiments, the disease/indication is associated with one or more cancers. The one or more cancers may comprise: adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann Syndrome, bile duct caner (Cholangiocarcinoma), Birt-Hogg Dube Syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, breast cancer (inflammatory), breast cancer (metastatic), breast cancer in men, camey complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden Syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma tumor, ependymoma, esophageal cancer, Ewing Sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor GIST, germ cell tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, HIV/AIDS related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, leukemia—acute lymphoblastic—ALL, leukemia, acute lymphocytic—ALL, leukemia—acute myeloid—ALL, leukemia—acute myeloid—AML, leukemia—B-cell prolymphocytic leukemia and hairy cell leukemia, leukemia—chronic lymphocytic—CLL, leukemia—chronic myeloid—CML, leukemia—chronic t-cell lymphocytic, leukemia—eosinophilic, Li-Fraumeni Syndrome, liver cancer, lung cancer—non-small cell, lung cancer—small cell, lymphoma—Hodgkin, lymphoma—Non-Hodgkin, Lynch Syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH/MYH—associated polyposis, myelodysplastic syndromes—MDS, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, fallopian tube cancer, peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers Syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma—Kaposi, sarcomas of specific organs, sarcomas—soft tissue, skin cancer (non-melanoma), skin cancer (melanoma), small bowel cancer, stomach cancer, testicular cancer, thyoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, unknown primary, uterine cancer, vaginal cancer, Von Hippel-Lindau Syndrome, vulvar cancer, Waldenstrom macroglobulinemia (lymphoplasmacytic lymphoma), Werner Syndrome, Wilms tumor, and xeroderma pigmentosum.
In embodiments, gene editing further comprises transfecting a cell with a nucleic acid that acts as a repair template by either causing the insertion of a DNA sequence in the region of a gene edit, e.g., a single-strand or double-strand break, or by causing the DNA sequence in the region of the gene edit to otherwise change.
In some cases, the gene edit targets a genomic safe harbor locus, e.g., TRAC and AAVS1.
In various embodiments, a circRNA encodes a protein of interest that serves as a vaccine when introduced into a subject in need of vaccination.
The circRNA and nucleic acids of the present disclosure may be adapted to and use in the methods or compositions described in WO/2013/086008, WO/2014/071219, WO/2015/117021, WO/2016/131052, WO/2018/035377, WO/2019/191341, WO/2021/003462, WO2021/231549, or WO2021/222389. The entire contents of which are incorporated by reference in their entirety.
The Entrez entries listed in the table above are hereby incorporated by reference in their entireties.
For each of the illustrative proteins, peptides, or genes identified in Table 1, Table 2, or Table 3, the present disclosure considers and covers natural or engineered variants, family members, orthologues, fragments or fusions construct thereof of the illustrative proteins, peptides, or genes.
The following definitions are used in connection with the invention disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this invention belongs.
The term “in vivo” refers to an event that takes place in a subject's body.
The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and/or organ which has been removed from a subject's body. Aptly, the cell, tissue and/or organ may be returned to the subject's body in a method of treatment or surgery.
As used herein, the term “variant” encompasses but is not limited to nucleic acids or proteins which comprise a nucleic acid or amino acid sequence which differs from the nucleic acid or amino acid sequence of a reference by way of one or more substitutions, deletions and/or additions at certain positions. The variant may comprise one or more conservative substitutions. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.
“Carrier” or “vehicle” as used herein refer to carrier materials suitable for drug administration. Carriers and vehicles useful herein include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, surfactant, lipid or the like, which is nontoxic and which does not interact with other components of the composition in a deleterious manner.
The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.
As used herein, “a,” “an,” or “the” can mean one or more than one.
Herein the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
The terms “comprise”, “comprising”, “contain,” “containing,” “including”, “includes”, “having”, “has”, “with”, or variants thereof as used in either the present disclosure and/or in the claims, are intended to be inclusive in a manner similar to the term “comprising.” Although the open-ended term “comprising,” is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.”
As used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.
By treating is meant, at least, ameliorating or avoiding the effects of a disease, including reducing a sign or symptom of the disease.
Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein set forth and as follows in the scope of the appended claims.
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.
All patents and publications referenced herein are hereby incorporated by reference in their entireties.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.
In embodiments, the present disclosure provides for any of the sequences below, or a sequence having at least about 95%, or at least about 97%, or at least about 98% identity thereto, e.g. as compositions and/or for use in the methods described herein.
In embodiments, the GFP part of a sequence (see the bold and underlined text) may be swapped with a different coding sequence to yield a circRNA.
ACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGG
CGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGC
CCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGAC
CACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT
CTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACC
GCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAAC
TACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGAT
CCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG
ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAAC
GAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA
GCTGTACAAGTACTCAGATCTCGAGCTCAAGTAG
ataaataaaaacccaaaccctgacgggtttaaaa
GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAA
CGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGT
TCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTG
CAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG
CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGT
TCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATC
CTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAA
CGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACT
ACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAG
TCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGC
CGGGATCACTCTCGGCATGGACGAGCTGTACAAGTACTCAGATCTCGAGCTCAAGTAG
CAAGTACTCA
GGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCA
GCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACC
GGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCG
CTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC
GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACC
CTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCT
GGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGA
ACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACC
CCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAA
AGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG
GCATGGACGAGCTGTACAAGTACTCAGATCTCGAGCTCAAGTAG
ACGCTTCGCTCAAATTAAAAGAAG
TGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTG
TCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAA
GCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC
CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACC
ATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGT
GAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGT
ACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTC
AAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCAT
CGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACC
CCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG
GACGAGCTGTACAAGTACTCAGATCTCGAGCTCAAGTAG
AAAGAAAAGAAAAAGAAAAAAGACGCTTC
GGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGA
GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCG
TGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCAC
ATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT
CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA
TCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTAC
AACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCG
CCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACG
GCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAG
AAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCT
GTACAAGTACTCAGATCTCGAGCTCAAGTAG
AAAGAAAAGAAAAAGAAAAAAGACGCTTCGCTCAAAT
GGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGA
GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCG
TGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCAC
ATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT
CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA
TCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTAC
AACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCG
CCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACG
GCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAG
AAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCT
GTACAAGTACTCAGATCTCGAGCTCAAGTAG
ACGCTTCGCTCAAATTaaaagaagagcgaattc
CGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGT
TCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC
ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAG
CCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGG
AGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGAC
ACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAA
GCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGG
TGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAAC
ACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAG
CAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTC
TCGGCATGGACGAGCTGTACAAGTACTCAGATCTCGAGCTCAAGTAG
AAAGAAAAGAAAAAGAAAAAA
circRNAs as described in
circRNA was tested for its efficiency in transfection of cells.
This application is a continuation of PCT Application No. PCT/US2022/026564, filed Apr. 27, 2022, which claims the benefit of US Provisional Patent Application. No. 63/180,387, filed Apr. 27, 2021. The entire contents of the aforementioned patent applications are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63180387 | Apr 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2022/026564 | Apr 2022 | US |
| Child | 18480405 | US |
