Compositions and methods for cell reprogramming using circular RNA

Abstract

Improved compositions and methods for producing iPSCs are needed. [Solution] This specification provides a recombinant circular RNA comprising at least one protein-coding nucleic acid sequence, wherein the protein-coding nucleic acid sequence encodes a reprogramming factor (e.g., a transcription factor), and the reprogramming factor is Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc, or a fragment or variant thereof. This specification also provides a method for producing induced pluripotent stem cells (iPSCs), comprising contacting somatic cells with at least one of the recombinant circular RNAs described herein and maintaining the cells under conditions that will result in reprogrammed iPSCs.

Description

Technical Field

[0001] Cross - Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 046,976, filed Jul. 1, 2020, the content of which is hereby incorporated by reference in its entirety.

[0002] Description of Electronically Submitted Text File

[0002] The content of the text file submitted electronically with this specification is hereby incorporated by reference in its entirety. A computer - readable format copy of the sequence listing (file name: ELVT_011_01WO_SeqList_ST25.txt, recording date Jul. 1, 2021, file size ~89 kilobytes).

Background Art

[0003]

[0003] Induced pluripotent stem cells (iPSCs) have revolutionized drug discovery and healthcare. iPSCs are generated by reprogramming somatic cells into an embryoid - like pluripotent state that enables the development of various human cell types required for research and / or therapeutic purposes.

[0004]

[0004] iPSCs are typically induced by introducing one or more reprogramming factors (e.g., Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c - Myc, and / or L - Myc) into somatic cells. Reprogramming factors can be introduced into cells using standard approaches, but these approaches have various drawbacks. For example, self - replicating RNA systems use RNA replicons that are capable of self - replication. The nature of such replication vectors poses a risk of genomic integration. mRNA - based reprogramming is cumbersome and involves multiple transfections of mRNA due to the rapid turnover of mRNA molecules. Exogenous mRNA is also immunogenic and requires the use of immune - evasion factors (e.g., inhibitors of the interferon pathway) and / or modified nucleotides to minimize toxicity.

[0005]

[0005] Therefore, improved compositions and methods for producing iPSCs are needed in the art. [Overview of the project]

[0006]

[0006] This specification provides circular RNAs (circRNAs) encoding one or more reprogramming factors (e.g., transcription factors). The reprogramming factors may be, for example, Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and / or L-Myc. In some embodiments, the circular RNAs may be used to generate unintegrated iPSCs. The iPSCs may be used, for example, to generate disease-related cell types to lead to specific cell therapies or to advance drug discovery research.

[0007]

[0007] In some embodiments, the recombinant circular RNA comprises a protein-coding sequence which encodes at least one reprogramming factor, the at least one reprogramming factor being Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc, or a fragment or variant thereof.

[0008]

[0008] In some embodiments, the complex comprises recombinant cyclic RNA and lipid nanoparticles (LNPs) as described herein.

[0009]

[0009] In some embodiments, the vector comprises a nucleic acid encoding a recombinant circular RNA disclosed herein.

[0010]

[0010] In some embodiments, the composition comprises recombinant circular RNA, complex, or vector as described herein.

[0011]

[0011] In some embodiments, the composition comprises two or more recombinant circular RNAs, each recombinant circular RNA encoding a reprogramming factor selected from the reprogramming factors in Tables 1, 2, or 3.

[0012]

[0012] In some embodiments, the composition comprises two or more recombinant circular RNAs, the composition comprising a combination of recombinant circular RNAs encoding a reprogramming factor selected from (i) Oct3 / 4, Klf4, Sox2, and c-Myc, (ii) Oct3 / 4, Klf4, Sox2, and L-Myc, (iii) Oct3 / 4, Klf4, and Sox2, (iv) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc, or (iv) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc.

[0013]

[0013] In some embodiments, the cells include recombinant circular RNA, complex, vector, or composition described herein.

[0014]

[0014] In some embodiments, a method for expressing a protein in cells includes contacting cells with a circular RNA, complex, vector, or composition described herein, and maintaining the cells under conditions in which the protein is expressed.

[0015]

[0015] In some embodiments, a method for producing induced pluripotent stem cells (iPSCs) comprises contacting somatic cells with at least one recombinant circular RNA, complex, vector, and / or composition described herein, and maintaining the cells under conditions that result in reprogrammed iPSCs.

[0016]

[0016] In some embodiments, a method for producing induced pluripotent stem cells (iPSCs) comprises contacting CD34+ cells in suspension with at least one recombinant circular RNA, complex, vector, and / or composition described herein, and maintaining the cells under conditions that result in reprogrammed iPSCs.

[0017]

[0017] In some embodiments, a method for reprogramming cells includes contacting the cells with one or more of the following: (i) circular RNA encoding a reprogramming factor, (ii) circular RNA not encoding any protein or miRNA, (iii) circular or linear RNA encoding a miRNA, and / or (iv) circular or linear RNA encoding a viral protein.

[0018]

[0018] In some embodiments, a method for reprogramming cells includes contacting the cells with (i) circular RNA encoding a reprogramming factor, (ii) circular RNA not encoding any protein or miRNA, (iii) circular or linear RNA encoding a miRNA, and (iv) circular or linear RNA encoding a viral protein.

[0019]

[0019] In some embodiments, a method of reprogramming a cell involves (i) a circular RNA encoding a reprogramming factor, (ii) a circular or linear RNA encoding miRNA, and (iii) a circular or linear RNA encoding a viral protein. This includes making contact with each of them.

[0020]

[0020] In some embodiments, a method for reprogramming cells includes contacting the cells with (i) circular RNA encoding a reprogramming factor and (ii) circular or linear RNA encoding miRNA.

[0021]

[0021] In some embodiments, a method for increasing the duration of protein expression in cells comprises contacting cells with a circular RNA, complex, vector, or composition described herein, and maintaining the cells under conditions in which the protein is expressed, wherein the duration of protein expression is increased compared to transfection of cells with linear RNA encoding the same protein.

[0022]

[0022] In some embodiments, a method of improving cell reprogramming efficiency includes contacting a cell with a circular RNA, complex, vector, or composition described herein and maintaining the cell under conditions in which a protein is expressed, and the cell reprogramming efficacy is increased compared to a cell reprogramming method using linear RNA.

[0023]

[0023] In some embodiments, a method of increasing the number of colonies of reprogrammed cells formed after reprogramming includes contacting a cell with a circular RNA, complex, vector, or composition described herein and maintaining the cell under conditions in which a protein is expressed, and the number of colonies of reprogrammed cells formed after reprogramming is increased compared to a cell reprogramming method using linear RNA.

[0024]

[0024] In some embodiments, a method of reprogramming cells in suspension includes contacting the cells in suspension with a circular RNA, complex, vector, or composition described herein and maintaining the cells under conditions in which a protein is expressed.

[0025]

[0025] In some embodiments, a method of improving the morphological maturation of reprogrammed colonies includes contacting the cells in suspension with a circular RNA, complex, vector, or composition described herein and maintaining the cells under conditions in which a protein is expressed, and the morphological maturation is improved compared to a cell reprogramming method using linear RNA.

[0026]

[0026] In some embodiments, a method of reprogramming a cell that results in reduced cell death compared to a method using linear RNA includes contacting the cell with a circular RNA, complex, vector, or composition described herein and maintaining the cell under conditions in which a protein is expressed.

[0027]

[0027] In some embodiments, a method for shortening the time from reprogramming to picking (manual selection of iPSC colonies by mechanical dissociation) includes contacting the cells with the circular RNAs, complexes, vectors, or compositions described herein and maintaining the cells under conditions in which the protein is expressed, and the time is shortened compared to a reprogramming method using linear RNA.

[0028]

[0028] In some embodiments, a method for reducing the number of transfections that induce cell reprogramming compared to a method using linear RNA includes contacting the cells with the circular RNAs, complexes, vectors, or compositions described herein and maintaining the cells under conditions in which the protein is expressed.

[0029]

[0029] In some embodiments, the suspension culture contains one or more CD34-expressing cells, and the CD34-expressing cells contain one or more exogenous circRNAs encoding reprogramming factors.

[0030]

[0030] Circular RNAs encoding one or more transdifferentiation factors are also provided herein. The transdifferentiation factors can be, for example, one or more of the factors listed in Table 6. Circular RNAs encoding one or more transdifferentiation factors can be used to convert a first somatic cell type to a second somatic cell type.

[0031]

[0031] In some embodiments, a method for directly converting cells from a first cell type to a second cell type includes contacting the cells with the recombinant circular RNAs, complexes, vectors, and / or compositions described herein and maintaining the cells under conditions in which the cells are converted to the second cell type.

[0032]

[0032] In some embodiments, a method for reprogramming and editing the genome of a cell comprises contacting the cell with (i) a recombinant circular RNA comprising a protein-coding sequence encoding at least one reprogramming factor, and (ii) an enzyme capable of editing the cell's DNA or RNA, or a nucleic acid encoding such an enzyme.

[0033]

[0033] In some embodiments, a method for transdifferentiating and editing the genome of a cell comprises contacting the cell with (i) a recombinant circular RNA comprising a protein-coding sequence encoding at least one transdifferentiating factor, and (ii) an enzyme capable of editing the cell's DNA or RNA, or a nucleic acid encoding such an enzyme.

[0034]

[0034] In some embodiments, the composition comprises somatic cells containing one or more exogenous circular RNAs encoding reprogramming factors.

[0035]

[0035] In some embodiments, the composition comprises a converted cell containing one or more exogenous circular RNAs encoding a conversion factor.

[0036]

[0036] In some embodiments, a method for inducing somatic cell mesenchymal epithelial transition (MET) to iPSCs involves contacting somatic cells with one or more circular RNAs encoding reprogramming factors.

[0037]

[0037] In some embodiments, a method for transdifferentiating cells includes contacting the cells with recombinant circular RNA containing a protein-coding sequence encoding at least one transdifferentiating factor.

[0038]

[0038] In some embodiments, the kit comprises a recombinant circular RNA, complex, vector, or composition described herein.

[0039]

[0039] In some embodiments, the kit includes (i) a container containing circular RNA encoding OCT4 and a buffer, (ii) a container containing circular RNA encoding SOX2 and a buffer, (iii) a container containing cirRNA encoding KLF4 and a buffer, and (iv) a package and its instructions.

[0040]

[0040] Cells prepared using one or more of the methods disclosed herein are also provided herein.

[0041]

[0041] iPSCs prepared using one or more of the methods disclosed herein are also provided. To be served.

[0042]

[0042] Differentiated cells derived from iPSCs produced using one or more of the methods disclosed herein are also provided herein.

[0043]

[0043] Other objects, advantages and features of the present invention will become apparent from the following detailed description. [Brief explanation of the drawing]

[0044]

[0044] The patent or application file includes at least one drawing made in color. A copy of this patent or patent application publication including the color drawing will be provided by the Patent Office upon request and payment of the necessary fees.

[0045] [Figure 1]

[0045] Figure 1 is a schematic diagram illustrating an exemplary protocol for generating circular RNA by circularizing linear RNA produced using chemical synthesis or in vitro transcription (IVT). First, linear RNA is prepared. Next, the 5' end of the linear RNA is phosphorylated by amplification using a primer specific to the flanking sequence. Subsequently, the 5' and 3' ends are ligated using T4 RNA ligase. The circular RNA is purified, or the linear byproduct is enzymatically denatured. The circular RNA can then be brought into contact with cells (e.g., transfection) and / or conjugated to lipid nanoparticles. [Figure 2]

[0046] Figures 2A-2G are schematic diagrams illustrating exemplary methods for cyclizing linear RNA, including enzymatic linkage of the 5' phosphate to the 3'-OH end (Figure 2A), chemical linkage of the phosphate to the OH end (the 5' or 3' end may be phosphorylated) (Figure 2B), chemical linkage of the 3' thiophosphate to the tosylated 5' end (Figure 2C), chemical linkage of the 3'-thiophosphate to the iodized 5'- end (Figure 2D), chemical linkage of the 3'-aldehyde to a 50-oxoamine (oxime cyclization) (Figure 2E), chemical linkage of the 5'- or 3'-azide to a 3'- or 5'-alkyne (click cyclization) (Figure 2F), and cyclization by metal chelation (M=Zn2+ or Fe2+, (=terpyridine)) (Figure 2G). [Figure 3]

[0047] Figure 3 is a schematic diagram illustrating an exemplary method for circularizing linear RNA. In the shown intron-exon construct, the group I catalytic intron of the T4 phage Td gene is bifurcated in a manner that preserves structural elements crucial for ribozyme folding. Next, exon fragment 2 (E2) is ligated upstream of exon fragment 1 (E1), and a coding region of approximately 1.1 kb in length is inserted between the exon-exon junctions. During splicing, the 3' hydroxyl group of the guanosine nucleotide is involved in a transesterification reaction at the 5' splicing site, resulting in the circularization of the intervening region and the excision of the 3' intron. [Figure 4]

[0048] Figure 4 shows the design of a permutation-substitution intron-exon (PIE) based circRNA construct and the production of circRNA. [Figure 5]

[0049] Figures 5A and 5B show nicked circular RNA. Figure 5A shows a diagram of the circular RNA, and Figure 5B shows the expected nicked RNA resulting from nicking at each of the three nicking sites indicated by the white triangles labeled "A". Since nicking can occur anywhere along the length of the circRNA, the degradation products shown in B are illustrative. [Figure 6]

[0050] Figure 6 shows agarose gel electrophoresis of in vitro transcripts from DNA templates corresponding to permutation-substituted intron-exon (PIE) precursor RNA, either full-length (WT) or truncated (ΔSS). [Figure 7]

[0051] Figure 7 shows splicing junction-specific RT-PCR results to verify that the circRNA band contains circular RNA. [Figure 8A]

[0052] Figure 8A shows the distribution of RNA types remaining after each step for each of the six reprogramming factors. [Figure 8B]

[0052] Figure 8B shows the results of RNaseR digestion of the circRNA preparation. [Figure 9A]

[0053] Figures 9A–9F show the results from fibroblast reprogramming using linear and circular RNA. Figure 9A shows the timeline for reprogramming HDF using linear and circular RNA. [Figure 9B]

[0053] Figure 9B shows the expression levels of nuclear GFP (nGFP) protein encoded by linear or circular coding RNA of nGFP, spiked in a reprogramming cocktail as shown (Stemgent linear RNA or TriLink linear RNA or circRNA). The graph shows nGFP expression normalized as a percentage of peak expression. [Figure 9C]

[0053] Figure 9C shows a representative image illustrating the morphological transition from fibroblasts to iPSCs during RNA reprogramming. [Figure 9D]

[0053] Figure 9D shows an image of the entire well of a reprogrammed iPSC colony on day 18 expressing the pluripotency marker Tra-1-81. [Figure 9E]

[0053] Figure 9E shows a typical image of an iPSC that has been reprogrammed with circRNA. [Figure 9F]

[0053] Figure 9F shows the concentration density of iPSC colonies as a quantification of the iPSC reprogramming shown in Figure 9D. [Figure 10A]

[0054] Figures 10A–10C provide data illustrating the physical characteristics of iPSCs reprogrammed by the methods described herein. Figure 10A shows representative images of iPSCs derived from cultures between passages 3 and 5, using the Stemgent mRNA reprogramming kit (top), linear mRNA synthesized by Trilink (middle), and circRNA (bottom). [Figure 10B]

[0054] Figure 10B shows the population doubling time (PDT) of iPSCs induced by RNA reprogramming, including five clones induced from circRNA, two clones induced from the Stemgent kit, and three clones induced from Trilink linear mRNA. [Figure 10C]

[0054] Figure 10C shows SSEA expression in iPSC clones induced by RNA reprogramming, as measured by flow cytometry. S = Stemgent mRNA kit induction, L = Trilink linear mRNA induction, C = circRNA induction. [Figure 11]

[0055] Figure 11 shows the transfection schedule for the iPSC reprogramming experiment in Example 6. [Figure 12A]

[0056] Figures 12A-12D show the morphological progression during reprogramming. Figure 12A-4 shows the Tx+EKB group. Tx = transfection. [Figure 12B]

[0056] Figure 12B-4 Tx-EKB group. Tx = transfection. [Figure 12C]

[0056] Figure 12C-2 Tx group. Tx = transfection. [Figure 12D]

[0056] Figure 12D-1 Tx group. Tx = transfection. [Figure 13]

[0057] Figure 13 shows cell culture images on day 6 to evaluate the cytotoxicity resulting from the transfection conditions shown. [Figure 14A]

[0058] Figure 14A shows the co-staining of cell culture wells with Tra-1-81 and Oct4 to evaluate iPSC reprogramming. [Figure 14B]

[0058] Figure 14B shows the quantification of iPSC reprogramming shown in Figure 14A. [Figure 15A]

[0059] Figures 15A–15D show the results of myocyte differentiation from fibroblasts using linear (TriLink) or MyoD-encoding circRNAs. Figure 15A shows MyoD expression in cells transfected with mock, circRNA, or linear mRNA. [Figure 15B]

[0059] Figure 15B shows myotube formation in cells transfected with mock, circRNA, or linear mRNA. [Figure 15C]

[0059] Figure 15C shows the expression of muscle-specific markers (myogenin, desmin, and myosin heavy chain (MHC)) in fibroblasts transfected with circRNA encoding MyoD. [Figure 15D]

[0059] Figure 15D shows the expression of myogenin, desmin, and myosin heavy chain (MHC) in fibroblasts transfected with linear mRNA MyoD. [Figure 16A]

[0060] Figure 16A shows the verification of protein expression of target genes encoded by linear mRNA (TriLink) or circRNA. Images were acquired using a 20X objective lens. Scale bar = 100 μM [Figure 16B]

[0060] Figure 16B shows verification of protein expression of the target gene encoded by linear mRNA (TriLink) or circRNA. Images were acquired using a 20X objective lens. Scale bar = 100 μM [Figure 17A]

[0061] Figures 17A-17C show the quantification of myoplastic transformation and myotube formation in human dermal fibroblasts using linear mRNA versus circRNA. Figure 17A shows the fusion index, which is the ratio of nuclei in desmin-positive myotubes (DAPI-positive) to the total number of nuclei in the population. [Figure 17B]

[0061] Figure 17B shows the overlap percentage between MYOG-positive nuclei and desmin-positive myotubes. [Figure 17C]

[0061] Figure 17C shows the overlap percentage between myosin heavy chain (MHC), a muscle-specific marker, and desmin-positive myotubes. [Modes for carrying out the invention]

[0046]

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those with ordinary skill in the art to which this disclosure belongs. The terms used in the detailed description herein are for the purpose of describing specific embodiments only and are not intended to limit them.

[0047]

[0063] Unless otherwise indicated in the context, it is particularly intended that the various features described herein may be used in any combination. Furthermore, in some embodiments, any feature or combination of features described herein may be excluded or omitted. To elaborate further, for example, where the specification indicates that a particular amino acid may be A, G, I, L, and / or V, this wording indicates that the amino acid may be any subset of these amino acids, e.g., A, G, I, or L; A, G, I, or V; A or G; L only, as if each such subcombination were explicitly described herein. Furthermore, such wording also indicates that one or more of a particular amino acid may be abandoned. For example, in some embodiments, the amino acid may not be A, G, or I; not A; not G or V, as if each such possible abandonment were explicitly described herein.

[0048]

[0064] All publications, patent applications, patents, GenBank or other access numbers, and other references referred to herein are incorporated in whole by reference for all purposes.

[0049] General method

[0065] The implementation of this invention will employ conventional techniques of cell culture, molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, unless otherwise indicated, within the scope of those skilled in the art. Such techniques are referenced in Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook et al., 2001) Cold Spring Harbor Press; Oligonucleotide Synthesis (P. Herdewijn, ed., 2004); Animal Cell Culture (RIFreshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (M.D. Weir & C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller & M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Oligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Manual of Clinical Laboratory Immunology (B. Detrick, N.R. Ose, and J.D. Folds, eds., 2006); Immunochemical Protocols This topic is fully explained in the following literature: Protocols) (J. Pound, ed., 2003); Lab Manual in Biochemistry: Immunology and Biotechnology (A. Nigam and A. Ayyagari, eds., 2007); Immunology Methods Manual: The Comprehensive Sourcebook of Techniques (Ivan Lefkovits, ed., 1996); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, eds., 1988); and others.

[0050] definition

[0066] The following terms are used in this specification and in the appended claims.

[0051]

[0067] The singular forms "a," "an," and "the" are intended to include the plural form unless the context clearly indicates otherwise.

[0052]

[0068] When used herein to refer to measurable values ​​such as the length, dose, time, or temperature of a polynucleotide or polypeptide, the term "about" is meant to include variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of a particular quantity.

[0053]

[0069] As used herein, "and / or" means and encompasses any and all possible combinations of one or more of the enumerated items in question, as well as any omission of combinations when interpreted selectively as "or".

[0054]

[0070] As used herein, “circular RNA” or “circRNA” refers to a type of single-stranded RNA that forms a continuous loop closed by covalent bonds, unlike the more well-known linear RNA. In this specification, any protein name preceded by “circ” refers to the circular RNA that codes for that gene. RNA can be circularized within a cell by cell splicing mechanisms. For example, circular RNA can be produced when the mRNA precursor splicing mechanism “reverse splices,” binding a splicing donor to an upstream splicing receptor, thereby producing circular RNA with covalently linked ends. Alternatively, circular RNA can be produced in vitro, for example, by the circularization of linear RNA produced by in vitro transcription (IVT). There are three common strategies for in vitro RNA circularization: chemical methods using cyanide bromide or similar condensing agents; enzymatic methods using RNA or DNA ligases (such as T4 RNA ligase position I or II); and lysozyme methods using self-splicing introns. Lysozyme-like methods utilizing substitution group I catalytic introns are applicable to the cyclization of long RNAs, with GTP and Mg as cofactors. 2+Only the addition of is required. This permutation-substitution intron-exon (PIE) splicing strategy consists of fused partial exons adjacent by a half-intron sequence. In vitro, these constructs undergo the double transesterification reaction characteristic of group I catalytic introns, but since the exons are already fused, they are excised as a circle covalently linked from 5' to 3' (see Figure 3). Figure 1 provides an exemplary protocol for circularizing linear RNA, and Figures 2A to 2G provide a list of exemplary linear RNA circularization strategies.

[0055]

[0071] The terms “linear RNA” and “linear mRNA” are used interchangeably herein, as will be apparent to those skilled in the art from the context.

[0056]

[0072] As used herein, “pluripotency” refers to a cell having the ability to differentiate into two or more differentiated cell types under different conditions, and the ability to differentiate into cell types characteristic of all three germ cell layers. In some embodiments, pluripotency may be manifested by the expression of one or more pluripotent stem cell markers.

[0057]

[0073] As used herein, the terms “induced pluripotent stem cells” and “iPSCs” refer to pluripotent cells generated from various differentiated (i.e., multipotent or non-pluripotent) somatic cells. iPSCs are substantially genetically identical to each of the original differentiated somatic cells and exhibit characteristics similar to cells with higher potential, including the ability to self-regenerate indefinitely in culture and to differentiate into other cell types, such as embryonic stem (ES) cells. In some embodiments, iPSCs exhibit morphological characteristics (i.e., round shape, large nucleolus, and poor cytoplasm) and proliferative characteristics (i.e., doubling time) similar to ES cells. In some embodiments, iPSCs express pluripotency-specific markers (e.g., Oct-4, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, but not SSEA-1).

[0058]

[0074] As used herein, “differentiated cell” or “somatic cell” refers to any cell that is not pluripotent in its original form, as defined herein. The term “somatic cell” also includes progenitor cells that are multipotent (e.g., capable of producing two or more cell types) but not pluripotent (e.g., capable of producing cells from all three germ layers).

[0059]

[0075] As used herein, the term “reprogramming” refers to the process of altering the differentiation state of cells, such as somatic cells, pluripotent cells, or progenitor cells. In some embodiments, reprogramming cells may include converting cells from a first cell type to a second cell type. In some embodiments, reprogramming may include changing the phenotype of differentiated cells to a pluripotent phenotype. In some embodiments, reprogramming may refer to the process of “induced differentiation” or “transcription factor-directed differentiation” in which iPSCs are converted into differentiated cells.

[0060]

[0076] As used herein, the term “reprogramming factor” means any factor or combination of factors that promote cell reprogramming. Reprogramming factors may be, for example, transcription factors. Exemplary reprogramming factors for generating iPSCs from differentiated cells include Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and L-Myc. Exemplary reprogramming factors and their combinations for generating differentiated cells are provided in Table 6.

[0061]

[0077] As used herein, “transdifferentiation” refers to a type of cellular reprogramming in which one somatic cell type is directly converted into a second somatic cell type. In some embodiments, transdifferentiation is performed without passing through an intermediate pluripotent state or progenitor cell type, resulting in the conversion of a somatic cell of a first cell type into a second somatic cell type. This can be described as "direct reprogramming" or "direct cell fate reversal," in which a cell is converted into a somatic cell, a second cell type.

[0062]

[0078] As used herein, “internal ribosome entry site” or “IRES” is an RNA element that enables translation initiation in a cap-independent manner. An IRES may be, for example, a viral IRES or a mammalian IRES (e.g., a human IRES).

[0063]

[0079] A "nucleotide triphosphate" or "NTP" is a molecule containing a nitrogenous base bonded to a five-carbon sugar (either ribose or deoxyribose), with three phosphate groups attached to the sugar.

[0064]

[0080] As used herein, “modified NTP” is NTP that has been chemically modified to confer desirable properties to a nucleic acid containing NTP. Such desirable properties may include, for example, reduced immunogenicity, improved stability, chemical functionality, or altered binding affinity.

[0065]

[0081] The term "modified RNA" (e.g., "modified linear RNA" or "modified circular RNA") is used to describe RNA molecules containing one or more modified NTPs.

[0066]

[0082] The term “vector” refers to a carrier for nucleic acids (i.e., DNA or RNA molecules) that can be used to introduce nucleic acids into cells. An “expression vector” is a vector containing a sequence encoding a protein or RNA (e.g., circular RNA) and essential regulatory regions necessary for the expression of that sequence in cells. In some embodiments, the protein or RNA encoding sequence is operably ligated to another sequence within the vector. The term “operably ligated” means that the regulatory sequences necessary for the expression of the protein or RNA encoding sequence are positioned on the nucleic acid molecule at the appropriate location relative to the sequence resulting in protein or RNA expression.

[0067]

[0083] As used herein, the terms “lipid nanoparticles” and “LNP” refer to lipid-based particles in the submicron range. LNPs may have liposome structural features and / or alternative non-bilayer type structures. LNPs may be conjugated to nucleic acids (e.g., DNA or RNA molecules) and used to deliver nucleic acids to cells.

[0068]

[0084] Methods for determining sequence similarity or identity between two or more nucleic acid sequences or amino acid sequences are known in the art. For example, sequence similarity or identity can be determined by the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48, 443 (1970), by the study of similarity methods of Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 85, 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA at Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), by the Best Fit sequence program described by Develeux et al., Nucl. Acid Res. 12, 387-395 (1984), or by examination.

[0069]

[0085] Another suitable algorithm is the BLAST algorithm described by Altschul et al., J.Mol.Biol.215, 403-410 (1990) and Karlin et al., Proc.Natl.Acad.Sci.USA, 90, 5873-5787 (1993). A particularly useful BLAST program is Altschul et al., Hand in Enzymology The WU-BLAST-2 program is derived from Methods in Enzymology, 266, 460-480 (1996); blast.wustl / edu / blast / README.html. WU-BLAST-2 uses several search parameters, which are optionally set to default values. These parameters are dynamic values, set by the program itself depending on the composition of a particular sequence and the configuration of the specific database against which the target sequence is being searched; however, the values ​​can be adjusted to increase sensitivity. Furthermore, an additional useful algorithm is gapped BLAST reported by Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. Unless otherwise indicated, identity % is determined herein using the algorithm available at internet address:blast.ncbi.nlm.nih.gov / Blast.cgi.

[0070] Recombinant circular RNA

[0086] Recombinant circular RNAs are provided herein. In certain embodiments, the recombinant circular RNA encodes a reprogramming factor that enables (alone or in combination with other reprogramming factors) to reprogram differentiated cells into iPSCs, enables iPSCs to differentiate into differentiated cells, and / or enables one differentiated cell type to differentiate into another differentiated cell type. For example, in some embodiments, the circular RNA encodes a reprogramming factor for induced differentiation or transcription factor-directed differentiation.

[0071]

[0087] In some embodiments, the recombinant circular RNA contains about 200 to about 5,000 nucleotides. In some embodiments, the recombinant circular RNA contains about 200 to about 1,000 nucleotides. In some embodiments, the recombinant circular RNA contains about 1,000 to about 2,500 nucleotides. In some embodiments, the circular RNA contains about 2,500 to about 5,000 nucleotides. In some embodiments, the circular RNA contains more than about 5,000 nucleotides.

[0072]

[0088] In some embodiments, the recombinant circular RNA includes one or more open reading frames. In some embodiments, the recombinant circular RNA includes one or more protein-coding sequences. In some embodiments, the recombinant circular RNA does not include open reading frames and / or protein-coding sequences.

[0073]

[0089] In some embodiments, the recombinant circular RNA contains a sequence encoding a reprogramming factor. In some embodiments, the reprogramming factor is a human or humanized reprogramming factor. In some embodiments, the reprogramming factor is a transcription factor.

[0074]

[0090] In some embodiments, the reprogramming factor may be, for example, one of the reprogramming factors listed in Table 1. In some embodiments, the reprogramming factor is a fragment or variant of one of the reprogramming factors listed in Table 1. In some embodiments, the reprogramming factor has at least 90%, at least 95%, or at least 99% sequence identity with respect to one of the reprogramming factors listed in Table 1.

[0075] [Table 1] TIFF2026091847000003.tif249127TIFF2026091847000004.tif48126

[0076]

[0091] In some embodiments, the reprogramming factor is RNA, such as microRNA (miRNA). miRs, such as the miRNA302(ad) cluster and miR367, have been shown to improve reprogramming efficiency when used in conjunction with other reprogramming factors (see U.S. Patents 8,791,248; 8,852,940; Poleganov et al., Human Gene Therapy, Nov, 2015, 751-766). For example, the miRNA may be one of the miRNA302 family (e.g., miR302d, miR302a, miR302c, and miR302b) or miR367, or a fragment or variant thereof. In some embodiments, the reprogramming factor is one of the following reprogramming factors, or a fragment or variant thereof: Oct4, Sox2, Klf4, c-Myc, Lin28, Nanog, Sall4, Utf1, p53, p21, p16 Ink4a GLIS1, L-Myc, TGF-beta, MDM2, REM2, cyclin D1, SV40 large T antigen, DOT1L, CX43, MBD3, SIRT6, TCL1a, RARy, SNAIL, Lrh-1, or RCOR2.

[0077]

[0092] In some embodiments, the recombinant circular RNA comprises a protein-coding sequence, which encodes a reprogramming factor (e.g., a transcription factor). In some embodiments, the reprogramming factor is Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and / or L-Myc, or a fragment or variant thereof. In some embodiments, the reprogramming factor is human or a humanized reprogramming factor.

[0078]

[0093] In some embodiments, the recombinant circular RNA encodes the reprogramming factor Oct3 / 4. In some embodiments, the encoded Oct3 / 4 has the sequence of SEQ ID NO: 1, or a sequence that is at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the circular RNA encodes the reprogramming factor Oct3 / 4 and contains or consists of the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the circular RNA encodes the reprogramming factor Oct3 / 4 and contains a nucleic acid sequence that is at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37.

[0079]

[0094] In some embodiments, the recombinant circular RNA encodes the reprogramming factor Klf4. In some embodiments, the encoded Klf4 has the sequence of SEQ ID NO: 2 or 3, or a sequence that is at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the circular RNA encodes the reprogramming factor Klf4 and contains the nucleic acid sequence of SEQ ID NO: 37. or consisting thereof. In some embodiments, the circular RNA comprises a nucleic acid sequence encoding the reprogramming factor Klf4 and being at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37.

[0080]

[0095] In some embodiments, the recombinant circular RNA encodes the reprogramming factor Sox2. In some embodiments, Sox2 has the sequence of SEQ ID NO: 4, or a sequence that is at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the circular RNA encodes the reprogramming factor Sox2 and comprises or consists of the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, the circular RNA encodes the reprogramming factor Sox2 and comprises a nucleic acid sequence that is at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34.

[0081]

[0096] In some embodiments, the recombinant circular RNA encodes the reprogramming factor Nanog. In some embodiments, Nanog has the sequence of SEQ ID NO: 5 or 6, or a sequence that is at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the circular RNA encodes the reprogramming factor Nanog and comprises or consists of the nucleic acid sequence of SEQ ID NO: 36. In some embodiments, the circular RNA encodes the reprogramming factor Nanog and comprises a nucleic acid sequence that is at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 36.

[0082]

[0097] In some embodiments, the recombinant circular RNA encodes the reprogramming factor Lin28. In some embodiments, Lin28 has the sequence of SEQ ID NO: 7, or a sequence that is at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the circular RNA encodes the reprogramming factor Lin28 and comprises or consists of the nucleic acid sequence of SEQ ID NO: 35. In some embodiments, the circular RNA encodes the reprogramming factor Lin28 and comprises a nucleic acid sequence that is at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 35.

[0083]

[0098] In some embodiments, the recombinant circular RNA encodes the reprogramming factor c-Myc. In some embodiments, c-Myc has the sequence of SEQ ID NO: 8 or 9, or a sequence that is at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the circular RNA encodes the reprogramming factor c-Myc and comprises or consists of the nucleic acid sequence of SEQ ID NO: 38. In some embodiments, the circular RNA encodes the reprogramming factor c-Myc and comprises a nucleic acid sequence that is at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38.

[0084]

[0099] In some embodiments, recombinant circular RNA encodes the reprogramming factor L-Myc. In some embodiments, L-Myc has at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical sequences of sequence numbers 10-12.

[0085]

[0100] In some embodiments, the circular RNA encodes the reprogramming factor MyoD and comprises or consists of the nucleic acid sequence of SEQ ID NO: 32. In some embodiments, the circular RNA encodes the reprogramming factor MyoD and comprises a nucleic acid sequence that is at least 90%, or at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32.

[0086]

[0101] In some embodiments, the recombinant circular RNA comprises two or more protein-coding nucleic acid sequences. For example, the recombinant circular RNA may contain three, four, five, or six protein-coding sequences. In some embodiments, at least one of the protein-coding sequences encodes a reprogramming factor (e.g., a transcription factor).

[0087]

[0102] In some embodiments, the recombinant circular RNA comprises two or more protein-coding sequences, at least one of which encodes a reprogramming factor. In some embodiments, the recombinant circular RNA comprises two or more protein-coding sequences, at least one of which encodes Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc, or a fragment or variant thereof. In some embodiments, the recombinant circular RNA comprises two or more protein-coding sequences, each of which is independently selected from Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and L-Myc, or a fragment or variant thereof.

[0088]

[0103] In some embodiments, the disclosure provides compositions of recombinant circular RNA encoding a reprogramming factor. In some embodiments, the composition further comprises a buffer, which may, for example, contain 1 to 10 mM sodium citrate. In some embodiments, the pH of the buffer is about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, or about 12. In some embodiments, the pH of the buffer is about 6.5.

[0089]

[0104] In some embodiments, the composition comprises two or more recombinant circular RNAs, each encoding a reprogramming factor selected from Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and L-Myc. In some embodiments, the composition comprises two or more recombinant circular RNAs, each encoding a reprogramming factor selected from combinations provided in Table 2.

[0090] [Table 2]

[0091]

[0105] In embodiments where the recombinant circular RNA comprises two or more protein-coding nucleic acid sequences, each sequence may be separated by a sequence encoding a self-cleaving peptide, such as a 2A peptide. Exemplary 2A peptides include, but are not limited to, EGRGSLLTCGDVEENPGP (SEQ ID NO: 17), ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 19), and VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 20). In some embodiments, each protein-coding nucleic acid sequence may be separated by IRES.

[0092]

[0106] In some embodiments, the recombinant circular RNA comprises a protein-coding sequence and a second sequence. In some embodiments, the protein-coding sequence encodes Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc, or fragments or variants thereof. In some embodiments, the second sequence is a sequence from one or more of circBIRC6, circCORO1C, or circMAN1A2. circBIRC6, circCORO1C, and circMAN1A2 are endogenously expressed circRNAs that have been shown to be rich in human ESCs and are thought to function as a "miR sponge." Therefore, they may have a regulatory role in promoting pluripotency by counteracting certain miRNAs (e.g., miR34a and / or miR145) that are known to repress the expression of pluripotency-related transcription factors NANOG, SOX2, and OCT4 (Yu et al., Nat Commun 8, 1149 (2017)).

[0093]

[0107] Circular RNA lacks the 5' 7-methylguanosine cap structure necessary for the efficient translation of linear mRNA. Therefore, circular RNA requires ribosome replenishment to be translated. Alternative mechanisms may be used. For example, an internal ribosome entry site (IRES) that directly binds to an initiation factor or the ribosome itself may be used. Thus, in some embodiments, recombinant circular RNA includes an internal ribosome entry site (IRES). In some embodiments, the IRES is involved with eukaryotic ribosomes. In some embodiments, the IRES is operably ligated to a protein-coding nucleic acid sequence.

[0094]

[0108] Examples of IRES sequences include, for example, picornavirus UTR (such as encephalomyocarditis virus (EMCV)) leader sequences, polio leader sequences, hepatitis A virus leader, hepatitis C virus IRES, human rhinovirus type 2 IRES, IRES elements from foot-and-mouth disease virus, and giardiavirus IRES, among others, and sequences derived from a wide variety of viruses. Various non-viral IRES sequences may also be used, including but not limited to IRES sequences from yeast, as well as human angiotensin II type 1 receptor IRES, fibroblast growth factor IRES, vascular endothelial growth factor IRES, and insulin-like growth factor 2 IRES. Additional IRES sequences suitable for use in recombinant circular RNA described herein include sequences listed in databases available at http: / / iresite.org / .

[0095]

[0109] In some embodiments, the circular RNA includes an intron element adjacent to the protein-coding sequence. The intron element can be reverse-spliced ​​by the cellular splicing mechanism to produce a covalently closed circular RNA. Thus, in some embodiments, the circular RNA includes a first intron element located at 5' of the protein-coding sequence and a second intron element located at 3' of the protein-coding sequence.

[0096]

[0110] In some embodiments, circular RNA is produced by circularizing linear RNA. In some embodiments, linear RNA can be self-circularized, for example, if it contains self-splicing introns. Because circular RNA lacks a 5' or 3' end, it may be resistant to exonuclease-mediated degradation and may be more stable in cells than most linear RNA.

[0097]

[0111] In some embodiments, intron elements are selected from any known intron elements in any combination and in any multiple and / or ratio. Examples of intron elements include those in the circBase circular RNA database (Glazar et al., RNA Examples include those described in 20:1666~1670 (2014); and www.circbase.org) and those described in Rybak-Wolf et al., Mol. Cell 58(5):870~885 (2015), each of which is incorporated herein by reference in whole. In some embodiments, the intron element is a mammalian intron or a fragment thereof. In some embodiments, the intron element is a non-mammalian intron (e.g., a self-splicing group I intron, a self-splicing group II intron, a splicesome intron, or a tRNA intron) or a fragment thereof.

[0098]

[0112] In some embodiments, the circular RNA includes one or more additional elements that improve the stability of the protein-coding sequence from the circular RNA and / or facilitate translation. For example, in some embodiments, the circular RNA may include a Kozak sequence. One example of a Kozak consensus sequence is RCC(AUG)G (SEQ ID NO: 21), which has a start codon in parentheses and an "R" at position -3 representing a purine (A or G). Another example of a Kozak consensus sequence is RXY(AUG) (SEQ ID NO: 22), where R is a purine (A or G), Y is either C or G, and X is any base.

[0099]

[0113] In some embodiments, circular RNA has a first intron element, protein It includes a protein-coding sequence and a second intron element. In some embodiments, the circular RNA includes an IRES and a protein-coding sequence. In some embodiments, the circular RNA includes a first intron sequence, an IRES, a protein-coding sequence, and a second intron sequence.

[0100]

[0114] In some embodiments, the circular RNA includes a sequence encoding a reprogramming factor (e.g., a transcription factor). In some embodiments, the circular RNA includes a first intron element, a sequence encoding a reprogramming factor, and a second intron element.

[0101]

[0115] In some embodiments, the circular RNA includes sequences encoding IRES and reprogramming factors. In some embodiments, the circular RNA includes a first intron sequence, an IRES, a sequence encoding a reprogramming factor, and a second intron sequence. In some embodiments, the circular RNA includes sequences encoding IRES and reprogramming factors. In some embodiments, the circular RNA includes a first intron element, an IRES, a sequence encoding a reprogramming factor, and a second intron element. An exemplary schematic diagram of the arrangement of elements in the circular RNA is provided in Figure 4. See also U.S. Patent Application Publication No. 2020 / 0080106, incorporated herein by reference.

[0102]

[0116] In some embodiments, the circular RNA includes a sequence encoding Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc. In some embodiments, the circular RNA includes a first intron element, a sequence encoding Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc, and a second intron element.

[0103]

[0117] In some embodiments, the circular RNA comprises an IRES and a sequence encoding Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc. In some embodiments, the circular RNA comprises a first intron sequence, an IRES and a sequence encoding Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc, and a second intron sequence. In some embodiments, the circular RNA comprises an IRES and a sequence encoding Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc. In some embodiments, the circular RNA comprises a first intron element, an IRES and a sequence encoding Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc, and a second intron element.

[0104]

[0118] Circular RNA may also contain modified bases and / or NTPs. In some embodiments, recombinant circular RNA contains modified NTPs. In some embodiments, recombinant circular RNA is modified circular RNA.

[0105]

[0119] Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, 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-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, 6-azocytosine and 6-azothimine, 5-uracil (pseuducracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted This includes synthetic and natural bases such as uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Furthermore, modified bases include G-clamps such as phenoxazinecytidine (1H-pyrimido[5,4-b][1,4]benzoxazine-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiadin-2(3H)-one), substituted phenoxazinecytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazine-2(3H)-one), and tricyclic pyrimidines such as carbazolecytidine (2H-pyrimido[4,5-b]indole-2-one) and pyridoindolecytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one). Modified bases also include those in which a purine or pyrimidine base is substituted with another heterocycle, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.

[0106]

[0120] In some embodiments, recombinant circular RNA includes a modified backbone. Examples of modified RNA backbones include methyl and other alkylphosphonates, phosphinates, phosphotriesters, aminoalkyl-phosphotriesters, 3-alkylenephosphonates, 5'-alkylenephosphonates, and chiral phosphonates, phosphoamides including 3'-aminophosphoramides and aminoalkyl-phosphoramides, thionophosphoramides, thionoalkylphosphonates, thionoalkyl-phosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, their 2'-5' linkage analogs, and those having inverted polarity with one or more internucleotide linkages being 3'-3', 5'-5', or 2'-2' links.

[0107]

[0121] In some embodiments, circular RNA can be modified by chemically linking one or more moieties or conjugates to the RNA that enhance activity, cell distribution, or cell uptake. For example, circular RNA can be linked to intercalators, reporter molecules, polyamines, polyamides, polyethylene glycol, polyethers, oligomer-enhancing groups, or oligomer-enhancing groups. In some embodiments, circular RNA can be linked to cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, or dyes. Groups that enhance pharmacodynamic properties include groups that improve RNA uptake, groups that enhance oligomer resistance to degradation, and / or groups that enhance sequence-specific hybridization with RNA. Groups that enhance pharmacokinetic properties include groups that improve oligomer uptake, distribution, metabolism, or excretion. Circular RNA can also be conjugated to, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansyl sarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepine, indomethicine, barbiturates, cephalosporins, sulfonamides, antidiabetic agents, antibacterial agents, or antibiotics. In some embodiments, recombinant circular RNA is conjugated to lipid nanoparticles (LNPs).

[0108]

[0122] In some embodiments, circular RNA is part of a complex. In some embodiments, the complex includes recombinant circular RNA and lipid nanoparticles (LNPs). In some embodiments, the recombinant circular RNA and LNPs are conjugated. In some embodiments, the recombinant circular RNA and LNPs are conjugated by covalent bonds. In some embodiments, the recombinant circular RNA and LNPs are conjugated by non-covalent bonds.

[0109]

[0123] LNPs are, for example, one or more cationic lipids, non-cationic lipids, and / or It may contain PEG-modified lipids. In some embodiments, LNP may contain at least one of the following cationic lipids: C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, or HGT5001. In some embodiments, LNP contains cholesterol and / or PEG-modified lipids. In some embodiments, LNP contains DMG-PEG2K. In some embodiments, LNP contains one of the following: C12-200, doped, cholesterol, DMG-PEG2K; DODAP, doped, cholesterol, DMG-PEG2K; HGT5000, doped, cholesterol, DMG-PEG2K; HGT5001, doped, or DMG-PEG2K. In some embodiments, LNP contains polyethyleneimine (PEI).

[0110]

[0124] In some embodiments, recombinant circular RNA is substantially non-immunogenic. In some embodiments, circular RNA is considered non-immunogenic if it does not induce the expression or activity of one or more interferon regulatory genes (e.g., one or more genes listed on www.interferome.org). In some embodiments, the interferon regulatory genes are selected from IFN-alpha, IFN-beta, and / or TNF-alpha. Various modifications can be made to the circular RNA to reduce its immunogenicity. For example, in some embodiments, the circular RNA is modified to include one or more M-6-methyladenosine (m 6 A) It may be modified to include a 5-methylcytosine (5mC) or pseudouridine residue.

[0111]

[0125] In some embodiments, the circular RNAs described herein are less immunogenic than linear RNAs. For example, in some embodiments, circular RNAs substantially do not induce the expression and / or activity of one or more interferon regulatory genes. In some embodiments, circular RNAs induce the expression and / or activity of one or more interferon regulatory genes about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% lower than linear RNAs.

[0112]

[0126] In some embodiments, the circular RNAs described herein have a longer cellular half-life than linear RNAs. For example, circular RNAs may have a half-life that is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer than linear RNAs. In some embodiments, circular RNAs may have a half-life that is about 4 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 10 days, or 10 days longer than linear RNAs.

[0113]

[0127] In some embodiments, recombinant circular RNA does not replicate within the cell. In some embodiments, recombinant circular RNA does not pose a risk of genomic integration.

[0114]

[0128] Circular RNA can be produced using in vitro transcription (IVT) according to standard protocols and / or by using commercially available kits (e.g., ThermoFisher®'s MAXIscript® or MEGAscript® kits). For example, in an exemplary IVT protocol, circular RNA is produced using a purified linear DNA template (i.e., a DNA molecule encoding the circular RNA described herein), a buffer system containing ribonucleotide triphosphates, DTT, and magnesium ions, and a suitable phage RNA polymerase. The DNA template contains a double-stranded promoter region to which the phage polymerase binds and initiates RNA synthesis. Reaction conditions (e.g., type of nucleotide salt, type and concentration of salts in the transcription buffer, enzyme concentration, and pH) are optimized for the specific polymerase and the entire set of components used to achieve optimal yield. Large-scale IVT reactions can produce up to 120–180 μg of RNA per microgram of template in 20 μl of reaction mixture. In some embodiments, circular RNA can be produced using RNA synthesis according to a standard protocol.

[0115]

[0129] Various methods for circularizing RNA are known in the art. For example, Figure 1 provides an exemplary protocol for circularizing linear RNA, and Figures 2A to 2G provide a list of exemplary linear RNA circularization strategies. In some embodiments, RNA is self-circularized, for example, if the RNA contains self-splicing introns.

[0116]

[0130] Nucleic acids (i.e., DNA molecules) encoding circular RNA as described herein, and vectors containing the same, are also provided herein.

[0117] A method for expressing proteins (e.g., reprogramming factors) in cells using circular RNA.

[0131] This specification provides a method for expressing a protein in a cell, where the protein is encoded by circular RNA. In some embodiments, the protein is a reprogramming factor. In some embodiments, the reprogramming factor is a transcription factor. In some embodiments, the protein is Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and / or L-Myc. In some embodiments, the protein is Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and / or c-Myc.

[0118]

[0132] In some embodiments, a method for expressing a protein in a cell includes contacting the cell with at least one of the recombinant circular RNA, vector, complex, or composition described herein, and maintaining the cell under conditions in which the protein is expressed.

[0119]

[0133] In some embodiments, a method for expressing a protein in a cell includes contacting a cell with a first circular RNA and at least one additional circular RNA, and maintaining the cell under conditions in which the protein is expressed. In some embodiments, a method for expressing a protein in a cell includes contacting a cell with a first circular RNA and a second circular RNA, and maintaining the cell under conditions in which the protein is expressed. In some embodiments, a method for expressing a protein in a cell includes contacting a cell with first, second, and third circular RNAs, and maintaining the cell under conditions in which the protein is expressed. In some embodiments, a method for expressing a protein in a cell includes contacting a cell with at least four circular RNAs, at least five circular RNAs, at least six circular RNAs, at least seven circular RNAs, at least eight circular RNAs, at least nine circular RNAs, or at least ten circular RNAs, and maintaining the cell under conditions in which the protein is expressed.

[0120]

[0134] In some embodiments, a method for expressing a protein in a cell includes contacting a cell with a first circular RNA encoding Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc, and at least one additional circular RNA, and maintaining the cell under conditions in which the protein is expressed. In some embodiments, a method for expressing a protein in a cell includes contacting a cell with a first circular RNA encoding Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc, and at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten additional circular RNAs, and maintaining the cell under conditions in which the protein is expressed. In some embodiments, a method for expressing a protein in a cell includes contacting a cell with multiple circular RNAs (e.g., at least one) each encoding Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc. This also includes contacting the cells with two, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten circular RNAs, and maintaining the cells under conditions in which the protein is expressed.

[0121]

[0135] In some embodiments, a method for expressing a protein in a cell comprises contacting a cell with (i) a first circular RNA encoding Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc, and (ii) at least one additional circular RNA, which is circBIRC6, circCORO1C, or circMAN1A2, and maintaining the cell under conditions in which the protein is expressed. In some embodiments, the additional circular RNA is circBIRC6. In some embodiments, circBIRC6 has the sequence of SEQ ID NO: 13, or a sequence that is at least 90% or at least 95% identical thereto. In some embodiments, the additional circular RNA is circCORO1C. In some embodiments, circCORO1C has the sequence of SEQ ID NO: 14, or a sequence that is at least 90% or at least 95% identical thereto. In some embodiments, the additional circular RNA is circMAN1A2. In some embodiments, circMAN1A2 has the sequence of SEQ ID NO: 15, or a sequence that is at least 90% or at least 95% identical thereto.

[0122]

[0136] In some embodiments, a method for expressing a protein in a cell includes contacting the cell with circular RNAs, each encoding one of Oct4, Sox2, Klf4, and cMyc. In some embodiments, a method for expressing a protein in a cell includes contacting the cell with circular RNAs, each encoding one of Oct4, Sox2, Klf4, cMyc, and Lin28. In some embodiments, a method for expressing a protein in a cell includes contacting the cell with (i) circular RNAs, each encoding one of Oct4, Sox2, Klf4, cMyc, and Lin28, and (ii) circBIRC6, circCORO1C, and circMAN1A2.

[0123]

[0137] In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are animal cells. In some embodiments, the cells are mammalian cells (e.g., mouse, cow, monkey, pig, horse, sheep, or human cells). In some embodiments, the cells are human cells. In some embodiments, the cells are yeast, fungal, or plant cells.

[0124]

[0138] In some embodiments, the cells are somatic cells. In some embodiments, the cells are fibroblasts, peripheral blood-derived cells, endothelial progenitor cells, umbilical cord blood-derived cells, hepatocytes, keratinocytes, melanocytes, adipose tissue-derived cells, or urine-derived cells (e.g., renal epithelial progenitor cells). In some embodiments, the cells are epithelial cells, endothelial cells, nerve cells, adipocytes, cardiac cells, skeletal muscle cells, immune cells, hepatocytes, spleen cells, lung cells, circulating blood cells, gastrointestinal cells, renal cells, bone marrow cells, progenitor cells, or pancreatic cells. In some embodiments, the cells are isolated from any body tissue, including but not limited to the brain, liver, lungs, digestive tract, stomach, intestines, fat, muscle, uterus, skin, spleen, endocrine organs, bone, etc.

[0125]

[0139] In some embodiments, the cells are adherent cells. In some embodiments, the cells are non-adherent cells (e.g., suspension cells such as CD34+ cells).

[0126]

[0140] In some embodiments, the cell comes into contact with the circular RNA once. In some embodiments, the cell comes into contact with the circular RNA two or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In some embodiments, the contacts are made at effective intervals. Effective intervals may be, for example, once a day, every other day, once every three days, once a week, once every two weeks, or once a month.

[0127]

[0141] In some embodiments, contact involves transfecting cells with a vector containing circular RNA or a nucleic acid (i.e., a DNA molecule) encoding it. In some embodiments, circular RNA is transfected into cells using lipid-mediated transfection. Lipid-mediated transfection stimulates the active uptake of nucleic acids by endocytosis. An example of a lipid-mediated transfection reagent is lipofectamine® (e.g., lipofectamine® RNAiMAX® from ThermoFisher®). In some embodiments, a method for transfecting cells includes (i) diluting the RNA or DNA and the transfection reagent in separate tubes, (ii) forming a complex with the DNA or RNA and the transfection reagent, (iii) adding the complex to cells, and (iv) assaying the protein expression of the cells. Detection of intracellular protein expression can be achieved by several techniques, among others, Western blotting, immunocytochemistry, and fluorescence-mediated detection (e.g., FACS).

[0128]

[0142] In some embodiments, contact involves electroporating a vector containing circular RNA, or nucleic acid (i.e., a DNA molecule) encoding it, into a cell. Electroporation delivers the nucleic acid by creating a temporary pore in the cell membrane while the cell is in a solution containing a high concentration of the nucleic acid.

[0129]

[0143] In some embodiments, contact involves incubating cells with a circRNA-LNP complex.

[0130]

[0144] In some embodiments, the contact includes one or more techniques such as ballistic transfection (i.e., gene gun or particulate gun transfection), magnetofection, peptide-mediated transfection (either by non-covalent peptide / RNA nanoparticle-based transfection such as Sigma-Aldrich's N-TER® transfection system, or by covalent attachment of peptides to RNA), and / or microinjection. Combinations of these techniques used sequentially or simultaneously may also be used.

[0131]

[0145] As described above, methods for expressing proteins in cells may include maintaining the cells under conditions in which the protein is expressed. Such conditions are well known to those skilled in the art and may vary depending on the cell type. For example, in some embodiments, cells may be maintained in a normal culture medium (with or without serum) at about 37°C in an atmosphere containing about 5% CO2. Method for creating iPSCs

[0132]

[0146] Methods for reprogramming somatic cells and methods for producing iPSCs are also provided herein. In some embodiments, a method for producing iPSCs includes contacting somatic cells with at least one of the recombinant circular RNAs, complexes, vectors, or compositions described herein, and maintaining the cells under conditions that result in reprogrammed iPSCs.

[0133]

[0147] In some embodiments, a method for producing iPSCs includes contacting somatic cells with at least one circular RNA encoding a reprogramming factor (e.g., a transcription factor) and maintaining the cells under conditions that result in reprogrammed iPSCs. The reprogramming factor is, for example, any of the reprogramming factors shown in Table 1. It may be. In some embodiments, the reprogramming factor is Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc. In some embodiments, the reprogramming factor is Oct3 / 4. In some embodiments, the reprogramming factor is Klf4. In some embodiments, the reprogramming factor is Sox2. In some embodiments, the reprogramming factor is Nanog. In some embodiments, the reprogramming factor is Lin28. In some embodiments, the reprogramming factor is c-Myc. In some embodiments, the reprogramming factor is L-Myc.

[0134]

[0148] In some embodiments, a method for producing iPSCs includes contacting somatic cells with two or more circular RNAs, each of which encodes a reprogramming factor, and maintaining the cells under conditions that result in reprogrammed iPSCs. In some embodiments, cells are contacted with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more circular RNAs, each of which encodes a reprogramming factor. In some embodiments, a method for producing iPSCs includes contacting somatic cells with six circular RNAs encoding the reprogramming factors Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc. In some embodiments, a method for producing iPSCs includes contacting somatic cells with four circular RNAs encoding the reprogramming factors Oct3 / 4, Klf4, Sox2, and c-Myc. In some embodiments, a method for producing iPSCs includes contacting somatic cells with four circular RNAs encoding the reprogramming factors Oct3 / 4, Klf4, Sox2, and L-Myc. In some embodiments, a method for producing iPSCs includes contacting somatic cells with six circular RNAs encoding the reprogramming factors Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc. In some embodiments, a method for producing iPSCs includes contacting somatic cells with five circular RNAs encoding the reprogramming factors Oct3 / 4, Klf4, Sox2, Lin28, and c-Myc. In some embodiments, a method for producing iPSCs includes contacting somatic cells with five circular RNAs encoding the reprogramming factors Oct3 / 4, Klf4, Sox2, Lin28, and L-Myc.

[0135]

[0149] In some embodiments, a method for producing iPSCs includes contacting somatic cells with two circular RNAs and maintaining the cells under conditions that result in reprogrammed iPSCs. In some embodiments, the first and second circular RNAs each encode a reprogramming factor selected from Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and L-Myc, and the first and second circular RNAs do not encode the same reprogramming factor. In some embodiments, the first circular RNA encodes Oct3 / 4 and the second circular RNA encodes Sox2.

[0136]

[0150] In some embodiments, a method for producing iPSCs includes contacting somatic cells with three circular RNAs and maintaining the cells under conditions that result in reprogrammed iPSCs. In some embodiments, the first, second, and third circular RNAs each encode a reprogramming factor selected from Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and L-Myc, respectively, and none of the first, second, and third circular RNAs encode the same reprogramming factor.

[0137]

[0151] In some embodiments, a method for producing iPSCs includes contacting somatic cells with four circular RNAs and maintaining the cells under conditions that result in reprogrammed iPSCs. In some embodiments, the first, second, third, and fourth circular RNAs are Oct3 / 4, Klf4, Sox2, Nanog, and Lin2, respectively. 8. Encodes a reprogramming factor selected from c-Myc and L-Myc, and none of the first, second, third, and fourth circular RNAs encode the same reprogramming factor. In some embodiments, the first circular RNA encodes Oct3 / 4, the second circular RNA encodes Sox2, the third circular RNA encodes c-Myc, and the fourth circular RNA encodes Klf4.

[0138]

[0152] In some embodiments, a method for producing iPSCs includes contacting somatic cells with four circular RNAs and maintaining the cells under conditions that result in reprogrammed iPSCs. In some embodiments, the first, second, third, and fourth circular RNAs each encode a reprogramming factor selected from Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, respectively, while none of the first, second, third, fourth, and fifth circular RNAs encode the same reprogramming factor. In some embodiments, the first circular RNA encodes Oct3 / 4, the second circular RNA encodes Sox2, the third circular RNA encodes Klf4, and the fourth circular RNA encodes Lin28.

[0139]

[0153] In some embodiments, a method for producing iPSCs includes contacting somatic cells with five circular RNAs and maintaining the cells under conditions that result in reprogrammed iPSCs. In some embodiments, the first, second, third, fourth, and fifth circular RNAs each encode a reprogramming factor selected from Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and L-Myc, and no two of the first, second, third, fourth, and fifth circular RNAs encode the same reprogramming factor. In some embodiments, the first circular RNA encodes Oct3 / 4, the second circular RNA encodes Sox2, the third circular RNA encodes Klf4, the fourth circular RNA encodes cMyc, and the fifth circular RNA encodes Lin28.

[0140]

[0154] In some embodiments, a method for producing iPSCs includes contacting somatic cells with five circular RNAs and maintaining the cells under conditions that result in reprogrammed iPSCs. In some embodiments, the first, second, third, fourth, and fifth circular RNAs each encode a reprogramming factor selected from Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, and no two of the first, second, third, fourth, and fifth circular RNAs encode the same reprogramming factor. In some embodiments, the first circular RNA encodes Oct3 / 4, the second circular RNA encodes Sox2, the third circular RNA encodes Klf4, the fourth circular RNA encodes Lin28, and the fifth circular RNA encodes Nanog. In some embodiments, the first circular RNA encodes Oct3 / 4, the second circular RNA encodes Sox2, the third circular RNA encodes Klf4, the fourth circular RNA encodes Lin28, and the fifth circular RNA encodes c-Myc.

[0141]

[0155] In some embodiments, a method for producing iPSCs includes contacting somatic cells with six circular RNAs and maintaining the cells under conditions that result in reprogrammed iPSCs. In some embodiments, the first, second, third, fourth, fifth, and sixth circular RNAs contain reprogramming factors selected from Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and L-Myc, respectively. Furthermore, none of the first, second, third, fourth, fifth, and sixth circular RNAs encode the same reprogramming factor. In some embodiments, the first circular RNA encodes Oct3 / 4, the second circular RNA encodes Sox2, the third circular RNA encodes Klf4, the fourth circular RNA encodes cMyc, the fifth circular RNA encodes Lin28, and the sixth circular RNA encodes Nanog.

[0142]

[0156] In some embodiments, a method for producing iPSCs includes contacting somatic cells with six circular RNAs and maintaining the cells under conditions that result in reprogrammed iPSCs. In some embodiments, the first, second, third, fourth, fifth, and sixth circular RNAs each encode a reprogramming factor selected from Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and L-Myc, and no two of the first, second, third, fourth, fifth, and sixth circular RNAs encode the same reprogramming factor. In some embodiments, the first circular RNA encodes Oct3 / 4, the second circular RNA encodes Sox2, the third circular RNA encodes Klf4, the fourth circular RNA encodes cMyc, the fifth circular RNA encodes Lin28, and the sixth circular RNA encodes Nanog. In some embodiments, the first circular RNA encodes Oct3 / 4, the second circular RNA encodes Sox2, the third circular RNA encodes Klf4, the fourth circular RNA encodes cMyc, the fifth circular RNA encodes Lin28, and the sixth circular RNA encodes Nanog.

[0143]

[0157] In some embodiments, a method for producing iPSCs includes contacting somatic cells with seven circular RNAs and maintaining the cells under conditions that result in reprogrammed iPSCs.

[0144]

[0158] In some embodiments, cells are exposed to multiple circular RNAs, each encoding a reprogramming factor selected from the reprogramming factors shown in Table 1, and no two circular RNAs encode the same reprogramming factor.

[0145]

[0159] In some embodiments, cells are exposed to multiple circular RNAs as shown in Table 3. In Table 3, each row represents a different combination of circular RNAs that can be exposed to the cell, and "X" indicates that the circular RNA is exposed to the cell. For example, in combination number 1, the cell is exposed to the circular RNA encoding Oct3 / 4 and the circular RNA encoding Klf4. In combination number 104, the cell is exposed to the circular RNAs encoding Oct3 / 4, Klf4, Sox2, and Nanog, Lin28, and L-Myc. In each of the following combinations, the cell may optionally further expose to one or more acyclic RNA nucleic acids encoding one or more reprogramming factors (e.g., one or more plasmids or mRNAs).

[0146] [Table 3] TIFF2026091847000007.tif247168TIFF2026091847000008.tif248168TIFF2026091847000009.tif111168

[0147]

[0160] In some embodiments, a method for producing iPSCs involves contacting somatic cells with the circular RNA of combination number 100 in Table 3 above. In some embodiments, a method for producing iPSCs involves contacting somatic cells with a combination of circular RNAs that does not include any circular RNA expressing C-Myc or L-Myc. In some such embodiments, the combination is selected from the combinations listed in Table 3 above, which include C-Myc and / or L-Myc, but the combination is modified to omit C-Myc and / or L-Myc.

[0148]

[0161] In some embodiments, a method for producing iPSCs includes contacting somatic cells with a circular RNA encoding Oct4, and in addition, contacting somatic cells with one or more linear RNAs encoding differentiation factors, circular RNAs encoding differentiation factors, or viral vectors encoding differentiation factors. In some embodiments, the level of Oct4 expression is lower compared to similar methods in which linear RNA encoding Oct4 is contacted with cells. In some embodiments, Oct4 expression persists for a longer period compared to similar methods in which linear RNA encoding Oct4 is contacted with cells.

[0149]

[0162] In some embodiments, a method for producing iPSCs comprises contacting somatic cells with one or more circular RNAs encoding reprogramming factors as described above (e.g., Table 3), and further comprising contacting the cells with one or more additional circular RNAs. In some embodiments, the one or more additional circular RNAs are selected from circBIRC6, circCORO1C, and circMAN1A2. In some embodiments, the additional circular RNA is circBIRC6. In some embodiments, the additional circular RNA is circCORO1C, and in some embodiments, the additional circular RNA is circBIRC6. The RNA in question is circMAN1A2.

[0150]

[0163] In some embodiments, a method for producing iPSCs comprises contacting somatic cells with one or more circular RNAs encoding the reprogramming factors described above (e.g., Table 3), and further comprising contacting the cells with the B18R protein, or a circular RNA encoding the B18R protein. In some embodiments, a method for producing iPSCs comprises contacting somatic cells with one or more circular RNAs encoding the reprogramming factors described above (e.g., Table 3), one or more additional circular RNAs selected from circBIRC6, circCORO1C, and circMAN1A2, and the B18R protein, or a circular RNA encoding the B18R protein. The B18R protein, encoded by the open reading frame of B18R in the Western Reserve (WR) strain of vaccinia virus, is a type I interferon (IFN) binding protein known to inhibit the IFN response and protect cells from the effects of interferon. An exemplary B18R sequence is provided in SEQ ID NO: 16. In some embodiments, the B18R protein has a sequence that is at least 90% or at least 95% identical to SEQ ID NO: 16.

[0151]

[0164] In some embodiments, a method for producing iPSCs comprises contacting somatic cells with one or more circular RNAs encoding reprogramming factors listed above (e.g., Table 3), and further comprising contacting the cells with one or more additional reprogramming factors. The additional reprogramming factors may be, for example, non-coding RNAs (e.g., LINcRNA-ROR, miR302 (miR302d, miR302a, miR302c, or miR302b), miR367, miR766, miR200c, miR369, miR372, Let7, miR19a / b), vitamin C, valproic acid, CHIR99021, Parnate, SB431542, PD0325901, BIX-01294, lithium maxizadilan, 8-Br-cAMP, A-83-01, thiazovibin, Y-27632, EPZ004777, or DAPT.

[0152]

[0165] In some embodiments, a method for reprogramming cells may involve contacting cells in any combination with (i) at least one circular RNA encoding a reprogramming factor, (ii) at least one circular RNA not encoding either a protein or miRNA, (iii) at least one circular or linear RNA encoding a miRNA, and / or (iv) at least one circular or linear RNA encoding a viral protein. The at least one reprogramming factor may be, for example, one of the reprogramming factors listed in Table 1. The at least one circular RNA not encoding either a protein or miRNA may be, for example, circBIRC6 (SEQ ID NO: 13), circCORO1C (SEQ ID NO: 14), and / or circMAN1A2 (SEQ ID NO: 15). The miRNA may be, for example, a miRNA from the miRNA302 family (e.g., miR302d, miR302a, miR302c, and miR302b) or miR367. The viral protein may be, for example, B18R, E3, or K3.

[0153]

[0166] In some embodiments, a method for reprogramming cells may include treating cells to suppress or prevent an innate immune response. For example, a method for reprogramming cells may include contacting cells with one or more viral proteins that inhibit an innate immune response, or with circular RNA encoding a viral protein. The viral protein may be, for example, an inhibitor of the RIG-1 (retinoic acid-inducible gene I) or PKR (protein kinase R) pathway. Exemplary viral proteins suitable for use in the methods described herein include, but are not limited to, B18R, E3, or K3 from vaccinia virus. Additional viral proteins are listed in Table 4 below. .

[0154] [Table 4] TIFF2026091847000011.tif22127

[0155]

[0167] Another way to suppress or prevent innate immune responses is to treat cells with miRNAs (or circular RNAs encoding miRNAs) that target RIG-1 (retinoic acid-inducible gene I) or PKR (protein kinase R). Examples of miRNAs include miR146a, miR485, miR182, nc886, miR-155, miR526a, or miR132. In some embodiments, a method for reprogramming cells may involve treating cells with miRNAs or circular RNAs encoding RIG-1 or PKR.

[0156]

[0168] Table 5 below shows exemplary combinations of RNA for use in methods of reprogramming cells. In Table 5, each row represents a different combination that may come into contact with the cell. The symbol "X" indicates that RNA comes into contact with a cell. For example, in combination number 1, the cell comes into contact with circular RNA encoding a reprogramming factor. In combination number 15, the cell comes into contact with circular RNA encoding a reprogramming factor, circular RNA that does not encode a protein or miRNA, circular or linear RNA encoding miRNA, and circular or linear RNA encoding a viral protein.

[0157] [Table 5] TIFF2026091847000013.tif35161

[0158]

[0169] Contact can be carried out by any of the above methods, such as transfection, electroporation, and / or the use of a circRNA-LNP complex. In some embodiments, contact involves incubating cells with one or more circular RNAs, such as circular RNAs encoding reprogramming factors.

[0159]

[0170] In some embodiments, the circular RNA comes into contact with the cell once. In some embodiments, the circular RNA comes into contact with the cell two or more times, for example, two, three, four, five, six, seven, eight, nine, or ten times. In some embodiments, the contacts are made at effective intervals. Effective intervals may be, for example, once a day, every other day, once every three days, once a week, once every two weeks, or once a month. In some embodiments, the circular RNA comes into contact with the cell for the duration of the reprogramming process so that the contacts continue throughout the reprogramming process.

[0160]

[0171] As explained above, the method for creating iPSCs is to reprogram i This may include maintaining the cells under conditions that will result in PSCs. Such conditions are known to those skilled in the art and may vary depending on the cell type. As an example, somatic cells may first be placed in a flask containing a suitable medium at a concentration of about 75% to about 90% on the day of contact with circRNA (day 0). The cells may then be brought into contact with circRNA (e.g., by transfection). The transfected cells may be seeded onto a culture disk and incubated overnight. The medium may be changed as needed for the next 10 to 14 days. In some embodiments, the medium may be supplemented with one or more additional agents to enhance cell reprogramming. The cells may be monitored for the appearance of iPSC colonies, and the iPSC colonies may be harvested and transferred to separate dishes for growth.

[0161]

[0172] To confirm the pluripotency of iPSCs, isolated clones may be tested for the expression of one or more stem cell markers. Stem cell markers may be selected from, for example, Oct4, Lin28, SOX2, SSEA4, SSEA3, TRA-1-81, TRA-1-60, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Nat1. Methods for detecting the expression of such markers may include, for example, RT-PCR and immunological methods for detecting the presence of encoded polypeptides.

[0162]

[0173] In some embodiments, cellular pluripotency is confirmed by measuring the ability of a cell to differentiate into cells of each of the three germ layers. In some embodiments, teratoma formation in immunodeficient rodents may be used to assess the pluripotency of isolated clones.

[0163]

[0174] In some embodiments, circRNA reprogramming requires fewer transfections and / or fewer transfections (compared to linear RNA-based approaches) to achieve iPSC reprogramming. For example, circRNA reprogramming may require about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% fewer transfections to achieve reprogramming compared to linear RNA-based approaches.

[0164]

[0175] In some embodiments, circRNA reprogramming results in improved reprogramming efficiency compared to linear RNA-based approaches. “Reprogramming efficiency” refers to a quantitative or qualitative measure of iPSC generation from an initial cell population. Reading out reprogramming efficiency includes quantifying the number of iPSC colonies present at a specific point in time during the reprogramming protocol (as an assessment of colony formation rate) or at the completion of the reprogramming protocol (as an assessment of the total number of iPSC colonies generated during a particular protocol). See, for example, Example 6 and Figure 12. iPSC colonies can be identified quantitatively (e.g., by staining with a pluripotent cell surface marker and counting the number of stained cells – see Figure 14) or qualitatively by assessment of morphological features (e.g., densely packed cells in colonies where each cell has more or less uniform shape and diameter, colonies with clearly defined boundaries, and cells within iPSC colonies with a high nucleus-to-cytoplasmic ratio and prominent nucleoli). Reprogramming efficiency may also include an assessment of the relative maturity of iPSC colonies across different reprogramming protocols. The maturation of iPSC colonies can be determined by the morphological characteristics described above.

[0165]

[0176] An increase in reprogramming efficiency means an increase in the readout of one or more reprogramming efficiencies when comparing two or more reprogramming protocols. For example, as detailed in the examples, reprogramming with circRNA-encoded reprogramming factors is compared to reprogramming with linear RNA-encoded reprogramming factors. Compared to reprogramming, this results in increased reprogramming efficiency.

[0166]

[0177] In some embodiments, the increase in reprogramming efficiency includes an increase in the total number of iPSC colonies present at the end of the first reprogramming protocol compared to the total number of iPSC colonies present at the end of the second and / or third reprogramming protocol. In some embodiments, the increase in reprogramming efficiency includes an increase in the total number of iPSC colonies present at a specific point in time in the first reprogramming protocol compared to the total number of iPSC colonies present at the same point in time in the second and / or third reprogramming protocol (i.e., an increase in the iPSC colony formation rate).

[0167]

[0178] In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells (e.g., mouse, cattle, monkey, pig, horse, sheep, or human cells). In some embodiments, the cells are human cells. In some embodiments, the cells are yeast, fungal, or plant cells.

[0168]

[0179] In some embodiments, the cells are somatic cells. In some embodiments, the cells are fibroblasts, peripheral blood-derived cells, endothelial progenitor cells, umbilical cord blood-derived cells, hepatocytes, keratinocytes, melanocytes, adipose tissue-derived cells, or urine-derived cells (e.g., renal epithelial progenitor cells). In some embodiments, the cells are epithelial cells, endothelial cells, nerve cells, adipocytes, cardiac cells, skeletal muscle cells, immune cells, hepatocytes, spleen cells, lung cells, circulating blood cells, gastrointestinal cells, renal cells, bone marrow cells, progenitor cells, or pancreatic cells. In some embodiments, the cells are isolated from body tissues including, but not limited to, the brain, liver, lungs, digestive tract, stomach, intestines, fat, muscle, uterus, skin, spleen, endocrine organs, bone, etc. In some embodiments, the cells are amniotic fluid cells, adipose stem cells, dental pulp cells, or pancreatic islet beta cells.

[0169]

[0180] In some embodiments, the cells are adherent cells. In some embodiments, the cells are non-adherent cells (i.e., suspension cells such as CD34+ cells).

[0170] Methods for cell differentiation

[0181] In addition, methods for transdifferentiating cells using circular RNA are provided herein. In some embodiments, a method for directly transdifferentiating cells from a first cell type to a second cell type includes contacting the cells with recombinant circular RNA or composition described herein and maintaining the cells under conditions in which they are transdifferentiated to the second cell type. In some embodiments, the cells do not enter an intermediate pluripotent state. In some embodiments, the cells are transdifferentiated directly from the first cell type to the second cell type without becoming progenitor cells.

[0171]

[0182] In some embodiments, the circular RNA encodes one or more reprogramming factors that enable cells to differentiate from a first cell type to a second cell type. In some embodiments, the circular RNA encodes MyoD, C / EBPα, C / EBPβ, Pdx1, Ngn3, Mafa, Pdx1, Hnf4α, Foxa1, Foxa2, Foxa3, Ascl1 (also known as Mash1), Brn2, Myt1l, miR-124, Brn2, Myt1l, Ascl1, Nurr1, Lmx1a, Ascl1, Brn2, Myt1l, Lmx1a, FoxA2, ​​Oct4, Sox2, Klf4, and c-Myc, Tbx5, Mef2c, Gata-4, and / or Mesp1. In some embodiments, the circular RNA encodes one or more reprogramming factors listed in Table 1.

[0172]

[0183] In some embodiments, the first cell type is an iPSC. In some embodiments, the first cell type is a differentiated fibroblast.

[0173]

[0184] In some embodiments, the second cell type is muscle cells, neurons, cardiomyocytes, hepatocytes, pancreatic islets, keratinocytes, T cells, or NK cells.

[0174]

[0185] In some embodiments, a method for directly converting cells from a first cell type to a second cell type involves exposing the cells to a plurality of circular RNAs, each circular RNA encoding a conversion factor according to one of the combinations listed in Table 6.

[0175]

[0186] In some embodiments, a method for directly converting cells from a first cell type to a second cell type involves contacting the cells with a plurality of circular RNAs, each of which encodes a conversion factor listed in Table 6. In some embodiments, the cells come into contact with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more circular RNAs.

[0176]

[0187] In some embodiments, a method for directly converting cells from a first cell type to a second cell type includes contacting the cells with two circular RNAs and maintaining the cells under conditions in which they are converted to the second cell type. In some embodiments, the first and second circular RNAs each encode a conversion factor listed in Table 6, and the first and second circular RNAs do not encode the same conversion factor.

[0177]

[0188] In some embodiments, a method for directly converting cells from a first cell type to a second cell type includes contacting the cells with three circular RNAs and maintaining the cells under conditions in which they are converted to the second cell type. In some embodiments, the first, second, and third circular RNAs each encode a conversion factor listed in Table 6, and the first, second, and third circular RNAs do not encode the same conversion factor.

[0178]

[0189] In some embodiments, a method for directly converting cells from a first cell type to a second cell type includes contacting the cells with four circular RNAs and maintaining the cells under conditions in which they are converted to the second cell type. In some embodiments, the first, second, third, and fourth circular RNAs each encode a conversion factor listed in Table 6, and the first, second, third, and fourth circular RNAs do not encode the same conversion factor.

[0179]

[0190] In some embodiments, a method for directly converting cells from a first cell type to a second cell type includes contacting the cells with five circular RNAs and maintaining the cells under conditions in which they are converted to the second cell type. In some embodiments, the first, second, third, fourth, and fifth circular RNAs each encode a conversion factor listed in Table 6, while the first, second, third, fourth, and fifth circular RNAs do not encode the same conversion factor.

[0180]

[0191] In some embodiments, a method for directly converting cells from a first cell type to a second cell type includes contacting the cells with six circular RNAs and maintaining the cells under conditions in which they are converted to the second cell type. In some embodiments, the first, second, third, fourth, fifth, and sixth circular RNAs each encode a conversion factor listed in Table 6, while the first, second, third, fourth, fifth, and sixth circular RNAs do not encode the same conversion factor.

[0181]

[0192] In some embodiments, cells are directly converted from a first cell type to a second cell type. The method involves exposing cells to seven circular RNAs and maintaining the cells under conditions in which they are converted to a second cell type. In some embodiments, the first, second, third, fourth, fifth, and sixth circular RNAs each encode a conversion factor listed in Table 6, while the first, second, third, fourth, fifth, and sixth circular RNAs do not encode the same conversion factor.

[0182]

[0193] In some embodiments, a method for directly converting cells from a first cell type to a second cell type includes contacting the cells with a plurality of circular RNAs and maintaining the cells under conditions in which they are converted to the second cell type. In some embodiments, each of the circular RNAs codes for one of the conversion factors listed in Table 6, and no two circular RNAs code for the same conversion factor.

[0183]

[0194] In some embodiments, cells are exposed to circular RNA encoding one or more reprogramming factors listed in Table 6. In some embodiments, a method for directly converting cells from a first cell type shown in Table 6 to a second cell type shown in Table 6 comprises exposing cells to recombinant circular RNA encoding one or more reprogramming factors listed in Table 6 and maintaining the cells under conditions in which they are converted to the second cell type. The first cell type may be, for example, any of the cell types listed in Table 6. The second cell type may be, for example, any of the cell types listed in Table 6.

[0184]

[0195] In some embodiments, the Disclosure provides compositions comprising one or more circular RNAs, each of which encodes one or more of the conversion factors listed in Table 6. In some embodiments, the Disclosure provides compositions comprising multiple circular RNAs, each of which encodes at least one of the conversion factors listed in Table 6.

[0185]

[0196] In some embodiments, a method for transdifferentiating cells comprises contacting the cells with one or more circular RNAs, each of which encodes a transdifferentiating factor listed in Table 6.

[0186]

[0197] In some embodiments, a method for transdifferentiating cells involves contacting a first cell type listed in column A for any combination number shown in Table 6 with the corresponding transdifferentiating factor listed in column B for the same transdifferentiating combination to produce a second cell type shown in column C for the same transdifferentiating combination, where at least one transdifferentiating factor listed in column B is encoded by circular RNA. In some embodiments, all of the transdifferentiating factors listed in column B for a given transdifferentiating combination are encoded by one or more circularized RNAs. In some embodiments, the first cell type is transdifferentiated to the second cell type using the transdifferentiating factors listed in column B for any one of combination numbers 1 to 151. In some embodiments, the first cell type is one of the cell types listed in column A for any one of combination numbers 1 to 151. In some embodiments, the second cell type is one of the second cell types listed in column C for any one of combination numbers 1 to 151.

[0187] [Table 6] TIFF2026091847000015.tif247162TIFF2026091847000016.tif242162TIFF2026091847000017.tif245162 TIFF2026091847000018.tif249162TIFF2026091847000019.tif242162TIFF2026091847000020.tif222162

[0188]

[0198] Contact can be carried out by any of the methods described above (e.g., by transfection, electroporation, and / or by the use of circRNA-LNP complexes).

[0189]

[0199] In some embodiments, the cell comes into contact with the circular RNA once. In some embodiments, the cell comes into contact with the circular RNA two or more times, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the contacts are made at effective intervals. Effective intervals may be, for example, once a day, every other day, once every three days, once a week, once every two weeks, or once a month.

[0190]

[0200] As described above, a method for directly converting cells from a first cell type to a second cell type may involve maintaining the cells under conditions under which they will be converted to the second cell type. Such conditions are known to those skilled in the art and may vary depending on the cell type. As an example, cells may be exposed to one or more circular RNAs and then cultured in standard media supplemented with various reprogramming factors of choice. The cells are monitored to observe their morphology and the presence of markers characteristic of the second cell type.

[0191]

[0201] Differentiated cells prepared using the methods described herein are also provided herein.

[0192]

[0202] Compositions comprising transdifferentiated cells are also provided herein, the transdifferentiated cells comprising one or more exogenous circular RNAs encoding transdifferentiating factors. In some embodiments, the transdifferentiating factors are any one of the transdifferentiating factors or combinations of transdifferentiating factors listed in Table 6. In some embodiments, the transdifferentiated cells are any one of the second cell types listed in Table 6. In some embodiments, the transdifferentiated cells are derived from a first cell type which is any one of the first cell types listed in Table 6.

[0193] iPSC differentiation using circular RNA

[0203] iPSCs prepared using the methods described herein are also provided. In some embodiments, the iPSCs express one or more of the following: Oct4, SOX2, Lin28, SSEA4, SSEA3, TRA-1-81, TRA-1-60, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Nat1.

[0194]

[0204] Differentiated cells derived from iPSCs produced using the methods described herein are also provided herein. Methods for differentiating iPSCs are known to those skilled in the art. In some embodiments, the differentiated cells are muscle cells, neurons, cardiomyocytes, hepatocytes, islet cells, keratinocytes, T cells, or NK cells.

[0195]

[0205] In some embodiments, iPSCs described herein (or iPSCs prepared using methods not described herein) can be differentiated by contacting the iPSC with one or more circular RNAs encoding differentiation factors. For example, in some embodiments, the iPSC is contacted with a circular RNA, or a DNA molecule encoding such a differentiation factor, which can differentiate the iPSC into a target cell type such as a T cell. In some embodiments, the differentiation factor is selected from RORA, HLF, MYB, KLF4, ERG, SOX4, LUC, HOXA9, HOXA10, and HOXA5. In some embodiments, the iPSC is contacted with at least one, at least two, or at least three circular RNAs, each encoding a differentiation factor selected from RORA, HLF, MYB, KLF4, ERG, SOX4, LUC, HOXA9, HOXA10, and HOXA5. The iPSC comes into contact with at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or at least eleven circular RNAs. In some embodiments, the iPSC comes into contact with at least one, at least two, at least three, at least four, or at least five circular RNAs, each encoding a differentiation factor selected from HOXA9, ERG, RORA, SOX4, or MYB. In some embodiments, the iPSC comes into contact with multiple circular RNAs, each encoding at least one of HOXA9, ERG, RORA, SOX4, or MYB. In some embodiments, the iPSC comes into contact with at least one circular RNA, the circRNA encoding one or more differentiation factors listed in Table 6. In some embodiments, the iPSC comes into contact with EZH1 shRNA. EXH1 shRNA expression may facilitate the transition from lineage-restricted hematopoietic progenitor cells to progenitor cells with polylymphocytic potential.

[0196]

[0206] In some embodiments, iPSCs differentiate into CD34+CD38- cells. In some embodiments, iPSCs differentiate into CD34+CD38- cells by contacting them with one or more circular RNAs encoding one or more of the following differentiation factors: RORA, HLF, MYB, KLF4, ERG, SOX4, LUC, HOXA9, HOXA10, or HOXA5.

[0197]

[0207] In some embodiments, CD34+CD45+ myeloid progenitor cells are contacted with a circular RNA encoding one or more of RORA, HLF, MYB, KLF4, ERG, SOX4, LUC, HOXA9, HOXA10, or HOXA5, or a DNA molecule encoding one or more of these. In some embodiments, CD34+CD45+ cells are transdifferentiated into CD34+CD38- cells by contacting iPSCs with one or more circular RNAs as described above. In some embodiments, the resulting cells after contact are self-replicating HSPCs (hematopoietic stem cells and progenitor cells) with the potential to be erythrocytes and lymphocytes.

[0198]

[0208] In some embodiments, iPSCs produced using the methods described herein are younger than iPSCs produced using conventional methods such as the use of a viral vector encoding a reprogramming factor or transfection with linear RNA encoding a reprogramming factor. Where herein, “younger” means that the cells are reprogrammed more quickly (i.e., within about 5, 6, 7, or 8 days after transfection) compared to conventional methods (i.e., about 9 days or more).

[0199]

[0209] In some embodiments, iPSCs express one or more biomarkers at different levels compared to iPSCs produced using conventional methods. For example, in some embodiments, iPSCs express markers related to cell stress and / or cell death (apoptosis) at lower levels compared to iPSCs produced using conventional methods. For example, in some embodiments, iPSCs express one or more heat shock proteins or caspases at lower levels.

[0200]

[0210] In some embodiments, the genome of an iPSC has different epigenetic modifications compared to an iPSC produced using conventional methods. For example, in some embodiments, the iPSC may have altered levels of DNA methylation and / or histone modifications.

[0201]

[0211] In some embodiments, T cells can co-opt factors that can improve T cell efficacy. The T cell comes into contact with one or more circular RNAs (or DNA molecules encoding them). In this context, improving potency means promoting T cell survival and / or its antitumor activity when used in an immuno-oncology setting. For example, a T cell may come into contact with one or more circular RNAs encoding IL-12, IL-18, IL-15, or IL-7.

[0202]

[0212] In some embodiments, T cells come into contact with one or more circular RNAs (or DNA molecules encoding them) that enhance the T cells' ability to home to tumor tissue. For example, T cells may come into contact with one or more circular RNAs encoding CXCR2, CCR2B, or heparanase.

[0203]

[0213] In some embodiments, T cells come into contact with one or more circular RNAs (or DNA molecules encoding them) that help improve survival and / or facilitate switching to a central memory phenotype. For example, T cells may come into contact with one or more circular RNAs encoding Suv39h1.

[0204] A combination of methods for reprogramming and editing the genome of cells.

[0214] Combining methods for generating iPSCs with methods for editing their genomes enhances the diagnostic and therapeutic capabilities of iPSCs. As used herein, the terms “genome editing” and “editing a genome” refer to the modification of a specific gene locus in the nucleic acid (e.g., DNA or RNA) of a cell. Genome editing can correct pathological gene mutations originating from diseased patients and can also be used to induce specific mutations in disease-free wild-type cells (such as iPSCs). Therefore, this disclosure provides combined methods for reprogramming and editing the genome of a cell. In some embodiments, the circular RNAs described herein may be used in methods for reprogramming and editing the genome of a cell.

[0205]

[0215] Genome editing may include, for example, inducing double-strand DNA breaks in a region of genetic modification. In some embodiments, the DNA locus is replaced with an exogenous sequence by supplementation with a target vector. Any of the following enzymes may be used to edit cellular DNA: zinc fingernuclease, homing endonuclease, TALEN (transcription activator-like effectanucleases), NgAgo (argonaut endonuclease), SGN (structure-inducing endonuclease), RGN (RNA-inducing nuclease), or modified or cleavage variants thereof. In some embodiments, the RNA-inducing nuclease is described in International Publication No. 2019 / 236566 (e.g., RNA-inducing nucleases of APG05083.1, APG07433.1, APG07513.1, APG08290.1, APG05459.1, APG04583.1, and APG1688.1), International Publication No. 2021 / 0 30344 (e.g., APG05733.1, APG06207.1, APG01647.1, APG08032.1, APG05712.1, APG01658.1, APG06498.1, APG09106.1, APG09882.1, APG02675.1, APG01405.1, APG06250.1, APG0687) RNA-inducing nucleases disclosed in any one of the following: 7.1, APG09053.1, APG04293.1, APG01308.1, APG06646.1, APG09748, and APG07433.1 (RNA-inducing nucleases), and International Publication No. 2020 / 139783 (RNA-inducing nucleases of APG00969, APG03128, APG09748, APG00771, APG02789, APG09106, APG02312, APG07386, APG09980, APG05840, APG05241, APG07280, APG09866, and APG00868), each of which is incorporated herein by reference in whole. In some embodiments, RNA-inducing nucleases include Cas9 nuclease, Cas12(a) nuclease (Cpf1), Cas12b nuclease, Cas12c nuclease, TrpB-like nuclease, and Cas13a nuclease. These are crease (C2c2), Cas13b nuclease, Cas14 nuclease, or modified or cleavage mutants thereof.

[0206]

[0216] In some embodiments, the Cas9 nuclease is used to edit the genome of a cell. Cas9 is a large, multifunctional protein with two putative nuclease domains, HNH and RuvC-like. The HNH and RuvC-like domains cleave a complementary 20-nucleotide sequence on the crRNA and the DNA strand opposite the complementary strand, respectively. Several variants of the CRISPR-Cas9 system exist, and any one of these variants may be used in the methods disclosed herein. (1) The original CRISPR-Cas9 system functions by inducing DNA double-strand breaks caused by the wild-type Cas9 nuclease directed by a single RNA. (2) A Cas9 nickase variant (D10A variant) produced by a mutation in either the HNH or RuvC-like domain of Cas9 is directed by a paired guide RNA. (3) An engineered nuclease variant of Cas9 with enhanced specificity (eSpCas9). (4) Catalytically inactive Cas9 (dCas9) mutants are generated by mutating both domains (HNH and RuvC-like). dCas9 can be used to modify the transcription of endogenous genes (CRISPRa or CRISPRi) when combined with a transcription repressor or activator, or to image genomic loci when fused with a fluorescent protein. (5) CRISPR-Cas9 fused with cytidine deaminase avoids DNA double-strand breaks by resulting in mutants that induce direct conversion from cytidine to uridine. In some embodiments, Cas9 nucleases are isolated or derived from Streptococcus pyogenes or Staphylococcus aureus.

[0207]

[0217] Cas9 requires an RNA guide sequence ("guide RNA" or "gRNA") to target a specific gene locus. In some embodiments, the gRNA is a single guide ("sgRNA"). The sgRNA may include a spacer sequence and a scaffold sequence. The spacer sequence is complementary to the target cleavage sequence and directs the enzyme there. The scaffold region binds to the Cas9 enzyme.

[0208]

[0218] Exemplary enzymes that may be used to edit cellular RNA include, but are not limited to, enzymes of the ADAR (adenosine deaminase acting on RNA) family. For example, the enzyme may be human ADAR1, ADAR2, or ADAR3, or modified or cleavage mutants thereof. In some embodiments, the enzyme may be ADAR from squid (e.g., Loligo pealeii), such as sqADAR2, or modified or cleavage mutants thereof. In some embodiments, the enzyme may be ADAR from Caenorhabditis elegans (e.g., ceADAR1 or ceADAR2) or ADAR from Drosophila melanogaster (e.g., dADAR), or modified or cleavage mutants thereof.

[0209]

[0219] In some embodiments, a method for reprogramming and editing a cell's genome comprises contacting the cell with (i) a recombinant circular RNA containing a protein-coding sequence encoding at least one reprogramming factor, and (ii) an enzyme capable of editing the cell's DNA or RNA.

[0210]

[0220] In some embodiments, a method for reprogramming and editing a cell's genome comprises contacting the cell with (i) a recombinant circular RNA comprising a protein-coding sequence encoding at least one reprogramming factor, and (ii) a nucleic acid encoding an enzyme capable of editing the cell's DNA or RNA.

[0211]

[0221] In some embodiments, cells come into contact with recombinant circular RNA before coming into contact with the enzyme or the nucleic acid encoding it. In some embodiments, cells come into contact with recombinant circular RNA after coming into contact with the enzyme or the nucleic acid encoding it. In some embodiments, cells come into contact with recombinant circular RNA almost simultaneously with coming into contact with the enzyme or the nucleic acid encoding it.

[0212]

[0222] In some embodiments, a method for reprogramming and editing the genome of a cell further includes contacting the cell with a nucleic acid or guide RNA encoding a guide RNA.

[0213]

[0223] A composition for reprogramming and editing the genome of a cell may include, for example, recombinant circular RNA (or a DNA molecule encoding it) and an enzyme (or a DNA or RNA molecule encoding it) capable of editing DNA or RNA. In some embodiments, the recombinant circular RNA contains a protein-coding sequence. In some embodiments, the circular RNA does not encode a protein. In some embodiments, the circular RNA is circBIRC6 (SEQ ID NO: 13), circCORO1C (SEQ ID NO: 14), or circMAN1A2 (SEQ ID NO: 15).

[0214] Combination methods for differentiating and editing the genome of cells

[0224] The circular RNAs described herein may also be used in methods for transdifferentiating and editing the genomes of cells. Accordingly, compositions and methods for transdifferentiating and editing the genomes of cells are provided herein.

[0215]

[0225] In some embodiments, a method for transdifferentiating and editing the genome of a cell comprises contacting the cell with (i) a recombinant circular RNA containing a protein-coding sequence encoding at least one transdifferentiating factor, and (ii) an enzyme capable of editing the cell's DNA or RNA. In some embodiments, the transdifferentiating factor is selected from any of those listed in Table 6.

[0216]

[0226] In some embodiments, a method for transdifferentiating and editing the genome of a cell comprises contacting the cell with (i) a recombinant circular RNA comprising a protein-coding sequence encoding at least one transdifferentiating factor, and (ii) a nucleic acid encoding an enzyme capable of editing the cell's DNA or RNA.

[0217]

[0227] The enzymes used to edit DNA or RNA in methods for transdifferentiating and editing the genome of cells may be any of the enzymes listed above.

[0218]

[0228] In some embodiments, cells come into contact with recombinant circular RNA before coming into contact with the enzyme or the nucleic acid encoding it. In some embodiments, cells come into contact with recombinant circular RNA after coming into contact with the enzyme or the nucleic acid encoding it. In some embodiments, cells come into contact with recombinant circular RNA almost simultaneously with coming into contact with the enzyme or the nucleic acid encoding it.

[0219]

[0229] In some embodiments, a method for transdifferentiating and editing the genome of cells further includes contacting the cells with a nucleic acid or guide RNA encoding a guide RNA.

[0220]

[0230] Compositions for transdifferentiating and editing the genome of cells may include, for example, recombinant circular RNA (or a DNA molecule encoding it) and an enzyme (or a DNA or RNA molecule encoding it) capable of editing DNA or RNA. In some embodiments, Recombinant circular RNA contains a protein-coding sequence. In some embodiments, the circular RNA does not encode a protein. In some embodiments, the circular RNA is circBIRC6 (SEQ ID NO: 13), circCORO1C (SEQ ID NO: 14), or circMAN1A2 (SEQ ID NO: 15). In some embodiments, the circular RNA encodes a reprogramming factor disclosed herein. In some embodiments, the circular RNA encodes one or more of Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and L-Myc. In some embodiments, the circular RNA encodes one or more of the conversion factors listed in Table 6.

[0221] Additional methods

[0231] As will be understood by those skilled in the art, the circular RNAs and related compositions described herein may be useful in one or more of the following methods.

[0222]

[0232] In some embodiments, methods for reprogramming cells that result in reduced cell death compared to methods using linear RNA are provided herein, the methods comprising contacting cells with circular RNA, complexes, vectors, or compositions described herein, and maintaining the cells under conditions in which a protein is expressed. In some embodiments, reprogramming-induced cell death is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more, compared to methods using linear RNA. In some embodiments, cells come into contact with a combination of circular RNAs, the combination of circular RNAs being selected from (i) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circ c-Myc, (ii) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (iii) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circL-Myc, (iv) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (v) circOct3 / 4, circKlf4, circSox2, and circC-Myc, (vi) circOct3 / 4, circKlf4, circSox2, and circL-Myc, or (vii) circOct3 / 4, circKlf4, and circSox2. In some embodiments, the cells come into contact with circMyoD.

[0223]

[0233] Methods for shortening the time from reprogramming to picking are also provided herein, comprising contacting cells with a circular RNA, complex, vector, or composition described herein and maintaining the cells under conditions in which a protein is expressed, thereby shortening the time from reprogramming to picking compared to reprogramming methods using linear RNA. As used herein, the term “picking” means manual selection of iPSC colonies by mechanical dissociation. In some embodiments, the time is shortened by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more compared to reprogramming methods using linear RNA. In some embodiments, cells come into contact with a combination of circular RNAs, the combination of circular RNAs being (i) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circ c-Myc, (ii) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (iii) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circL-Myc, (iv) circOct3 / 4, Selected from circKlf4, circSox2, circNanog, and circLin28, (v)circOct3 / 4, circKlf4, circSox2, and circC-Myc, (vi)circOct3 / 4, circKlf4, circSox2, and circL-Myc, or (vii)circOct3 / 4, circKlf4, and circSox2. In some embodiments, the cells come into contact with circMyoD.

[0224]

[0234] Methods for reducing the number of transfections that induce cellular reprogramming are also provided herein, which include contacting cells with a circular RNA, complex, vector, or composition described herein and maintaining the cells under conditions in which a protein is expressed. In some embodiments, the number of transfections is reduced compared to methods using linear RNA. In some embodiments, the number of transfections that induce cellular reprogramming is 1, 2, 3, 4, 5, 6, or 7. In some embodiments, the number of transfections is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more compared to methods using linear RNA. In some embodiments, cells come into contact with a combination of circular RNAs, the combination of circular RNAs being selected from (i) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circ c-Myc, (ii) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (iii) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circL-Myc, (iv) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (v) circOct3 / 4, circKlf4, circSox2, and circC-Myc, (vi) circOct3 / 4, circKlf4, circSox2, and circL-Myc, or (vii) circOct3 / 4, circKlf4, and circSox2. In some embodiments, the cells come into contact with circMyoD.

[0225]

[0235] Methods for increasing the duration of protein expression in cells are also provided herein, which include contacting cells with a circular RNA, complex, vector, or composition described herein, and maintaining the cells under conditions in which the protein is expressed. In some embodiments, the duration of protein expression is increased compared to methods comprising transfection of cells with linear RNA encoding the same protein. In some embodiments, the duration of protein expression is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more compared to methods comprising transfection of cells with linear RNA encoding the same protein. In some embodiments, the duration of protein expression is increased by at least 1 hour, at least 4 hours, at least 8 hours, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, or more, compared to a method involving transfection of cells with linear RNA encoding the same protein. In some embodiments, cells are exposed to a combination of circular RNAs, the combination of circular RNAs being (i) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circ c-Myc, (ii) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (iii) circOct3 / 4, circKlf4, circSox2, circNanog, circL (iv)circOct3 / 4,circKlf4,circSox2,circNanog, and circLin28, (v)circOct3 / 4,circKlf4,circSox2, and circC-Myc, (vi)circOct3 / 4,circKlf4,circSox2, and circL-Myc, or (vii)circOct3 / 4,circKlf4, and circSox2. In some embodiments, the cells come into contact with circMyoD.

[0226]

[0236] Methods for improving the efficiency of cell reprogramming are also provided herein, comprising contacting cells with a circular RNA, complex, vector, or composition described herein and maintaining the cells under conditions in which a protein is expressed, thereby increasing the efficacy of cell reprogramming compared to cell reprogramming methods using linear RNA. In some embodiments, the cell reprogramming efficiency increases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more compared to methods using linear RNA. The improvement in the efficiency of cell reprogramming may be observed based on qualitative and / or qualitative assessments, including but not limited to a reduction in cell death, a reduction in stress induced by immune responses measured by IFN-gamma secretion, and a reduction in the induction of stress response genes. In some embodiments, cells come into contact with a combination of circular RNAs, the combination of circular RNAs being selected from (i) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circ c-Myc, (ii) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (iii) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circL-Myc, (iv) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (v) circOct3 / 4, circKlf4, circSox2, and circC-Myc, (vi) circOct3 / 4, circKlf4, circSox2, and circL-Myc, or (vii) circOct3 / 4, circKlf4, and circSox2. In some embodiments, the cells come into contact with circMyoD.

[0227]

[0237] Methods for increasing the number of reprogrammed cell colonies formed after reprogramming are also provided herein, which comprises contacting cells with circular RNA, complexes, vectors, or compositions and maintaining the cells under conditions in which a protein is expressed, thereby increasing the number of reprogrammed cell colonies formed after reprogramming compared to cell reprogramming methods using linear RNA. In some embodiments, the number of reprogrammed cell colonies increases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more compared to methods using linear RNA. In some embodiments, the increase in colony number may be observed for about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 days after transfection with one or more circRNAs encoding transcription factors. In some embodiments, cells come into contact with a combination of circular RNAs, the combination of circular RNAs being (i) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circ c-Myc, (ii) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (iii) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circL-Myc, (iv) circOct3 / 4, circKlf4, circSox2, c ircNanog and selected from circLin28, (v)circOct3 / 4, circKlf4, circSox2, and circC-Myc, (vi)circOct3 / 4, circKlf4, circSox2, and circL-Myc, or (vii)circOct3 / 4, circKlf4, and circSox2. In some embodiments, the cells come into contact with circMyoD.

[0228]

[0238] A method for reprogramming cells in suspension is also provided herein, which comprises contacting cells in suspension with a circular RNA, complex, vector, or composition described herein, and maintaining the cells under conditions in which a protein is expressed. In some embodiments, the cells express CD34 (i.e., they are CD34+). In some embodiments, cells come into contact with a combination of circular RNAs, the combination of circular RNAs being selected from (i) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circ c-Myc, (ii) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (iii) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circL-Myc, (iv) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (v) circOct3 / 4, circKlf4, circSox2, and circC-Myc, (vi) circOct3 / 4, circKlf4, circSox2, and circL-Myc, or (vii) circOct3 / 4, circKlf4, and circSox2. In some embodiments, the cells come into contact with circMyoD.

[0229]

[0239] Methods for improving the morphological maturation of reprogrammed colonies are also provided herein, comprising contacting cells in suspension with a circular RNA, complex, vector, or composition described herein, and maintaining the cells under conditions in which a protein is expressed, wherein morphological maturation is improved compared to cell reprogramming methods using linear RNA. The improvement in morphological maturation may include, for example, more densely packed colonies, colonies in which more cells have a uniform shape and diameter, colonies with clearly defined boundaries, and cells in iPSC colonies with a higher nucleo-to-cytoplasmic ratio and / or prominent nucleoli. In some embodiments, the morphological maturation of reprogrammed colonies is improved by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more compared to methods using linear RNA. In some embodiments, cells come into contact with a combination of circular RNAs, the combination of circular RNAs being selected from (i) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circ c-Myc, (ii) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (iii) circOct3 / 4, circKlf4, circSox2, circNanog, circLin28, and circL-Myc, (iv) circOct3 / 4, circKlf4, circSox2, circNanog, and circLin28, (v) circOct3 / 4, circKlf4, circSox2, and circC-Myc, (vi) circOct3 / 4, circKlf4, circSox2, and circL-Myc, or (vii) circOct3 / 4, circKlf4, and circSox2. In some embodiments, the cells come into contact with circMyoD.

[0230]

[0240] Suspension cultures containing one or more CD34-expressing cells are also provided herein, and the CD34-expressing cells contain one or more exogenous circRNAs encoding reprogramming factors. In some embodiments, the reprogramming factors are Oct3 / 4, Klf4, The schema is selected from Sox2, Nanog, Lin28, c-Myc, and L-Myc.

[0231]

[0241] A method for inducing mesenchymal epithelial transition (MET) of somatic cells to iPSCs is also provided herein, comprising contacting somatic cells with one or more circular RNAs encoding reprogramming factors.

[0232]

[0242] A method for inducing mesenchymal epithelial transition (MET) of somatic cells to iPSCs is also provided herein, comprising contacting somatic cells with one or more circular RNAs encoding reprogramming factors.

[0233] Vectors, compositions, and cells

[0243] This disclosure also provides vectors comprising nucleic acids (i.e., DNA molecules) encoding circular RNA as described herein. In some embodiments, the vector is a non-viral vector, such as a plasmid. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, retroviral vectors, herpesvirus vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, baculovirus vectors, alphavirus vectors, picornavirus vectors, vacciniavirus vectors, and lentivirus vectors. In some embodiments, the viral vector is a replication-deficient viral vector. A replication-deficient viral vector retains its infectivity and enters cells in a similar manner to a replicating vector, but once inside a cell, it does not replicate or proliferate.

[0234]

[0244] Figure 4 provides a schematic diagram of an exemplary vector construct that may be used to produce the circular RNA described herein. In some embodiments, the nucleic acid encoding the circular RNA includes a sequence encoding a reprogramming factor operably ligated to the IRES. In some embodiments, the nucleic acid encoding the circular RNA includes a sequence encoding a reprogramming factor operably ligated to the IRES, flanked by a permutation-substitution type I intron. In some embodiments, the nucleic acid encoding the circular RNA includes a promoter and a sequence encoding a reprogramming factor operably ligated to the IRES. In some embodiments, the nucleic acid encoding the circular RNA includes a promoter and a sequence encoding a reprogramming factor operably ligated to the IRES, flanked by a permutation-substitution type I intron. In some embodiments, the nucleic acid further includes an exon or a portion thereof.

[0235]

[0245] Exemplary vector sequences that can be used to produce circular RNA are shown in SEQ ID NOs. 23–30. These vectors encode linear RNA that, upon transcription, becomes circularized to form circular RNA (i.e., the circular RNAs of SEQ ID NOs. 30–38), and are therefore referred to herein as circular RNA "precursors."

[0236]

[0246] In some embodiments, the circular RNA precursor encodes an nGFP reprogramming factor. In some embodiments, the circular RNA precursor contains the sequence of SEQ ID NO: 23, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the circular RNA encodes an nGFP reprogramming factor. In some embodiments, the circular RNA contains the sequence of SEQ ID NO: 31, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0237]

[0247] In some embodiments, the circular RNA precursor encodes a MyoD reprogramming factor. In some embodiments, the circular RNA precursor encodes the MyoD reprogramming factor of SEQ ID NO: 24 The sequence, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the circular RNA encodes a MyoD reprogramming factor. In some embodiments, the circular RNA contains the sequence of SEQ ID NO: 32, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0238]

[0248] In some embodiments, the circular RNA precursor encodes an OCT4 reprogramming factor. In some embodiments, the circular RNA precursor contains the sequence of SEQ ID NO: 25, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the circular RNA encodes an OCT4 reprogramming factor. In some embodiments, the circular RNA contains the sequence of SEQ ID NO: 33, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0239]

[0249] In some embodiments, the circular RNA precursor encodes a SOX2 reprogramming factor. In some embodiments, the circular RNA precursor contains the sequence of SEQ ID NO: 26, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the circular RNA encodes a SOX2 reprogramming factor. In some embodiments, the circular RNA contains the sequence of SEQ ID NO: 34, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0240]

[0250] In some embodiments, the circular RNA precursor encodes the LIN28 reprogramming factor. In some embodiments, the circular RNA precursor comprises the sequence of SEQ ID NO: 27, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the circular RNA encodes the LIN28 reprogramming factor. In some embodiments, the circular RNA comprises the sequence of SEQ ID NO: 35, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0241]

[0251] In some embodiments, the circular RNA precursor encodes the NANOG reprogramming factor. In some embodiments, the circular RNA precursor comprises the sequence of SEQ ID NO: 28, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the circular RNA encodes the NANOG reprogramming factor. In some embodiments, the circular RNA comprises the sequence of SEQ ID NO: 36, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0242]

[0252] In some embodiments, the circular RNA precursor encodes the KLF4 reprogramming factor. In some embodiments, the circular RNA precursor comprises the sequence of SEQ ID NO: 29, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the circular RNA encodes the KLF4 reprogramming factor. In some embodiments, the circular RNA comprises the sequence of SEQ ID NO: 37, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. comprises a sequence that is at least 99% identical.

[0243]

[0253] In some embodiments, the circular RNA precursor encodes a cMYC reprogramming factor. In some embodiments, the circular RNA precursor comprises the sequence of SEQ ID NO: 30, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the circular RNA encodes a cMYC reprogramming factor. In some embodiments, the circular RNA comprises the sequence of SEQ ID NO: 38, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0244]

[0254] Compositions comprising circular RNA or vectors as described herein are also provided herein. In some embodiments, the composition comprises (i) circular RNA and (ii) a carrier or medium. In some embodiments, the composition comprises (i) a vector and (ii) a carrier or medium. Suitable carriers or mediums include, for example, sterile water, sterile buffer solutions (e.g., solutions buffered with phosphates, citrates, or acetates), sterile culture media, polyalkylene glycols, hydrogenated naphthalenes (e.g., biocompatible lactide polymers), lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In some embodiments, the carrier or medium may include substances for covalent attachment of polymers such as lactose, mannitol, polyethylene glycol, complex formation with metal ions, or in or on specific preparations of polymer compounds such as polylactic acid, polyglycolic acid, hydrogels, or on liposomes, microemulsions, micelles, monolayers or multilayer vesicles, erythrocyte fragments, or spheroplasts. In some embodiments, the pH of the carrier or medium is in the range of 5.0 to 8.0, such as in the range of about 6.0 to about 7.0. In some embodiments, the carrier or medium comprises a salt component (e.g., sodium chloride, potassium chloride) or other components that make the solution isotonic, for example. Furthermore, the carrier or medium may contain additional components such as fetal bovine serum, growth factors, human serum albumin (HSA), polysorbate 80, sugars, or amino acids.

[0245]

[0255] Cells comprising recombinant circular RNA, vectors, or compositions described herein are also provided herein. In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells (e.g., mouse, cattle, monkey, pig, horse, sheep, or human cells). In some embodiments, the cells are human cells.

[0246] kit

[0256] Kits for expressing proteins in cells are also provided. In some embodiments, the kit includes a vector containing at least one circular RNA, or nucleic acid (i.e., a DNA molecule) encoding it, as described herein. In some embodiments, the kit includes a container containing the circular RNA or the DNA molecule encoding it. In some embodiments, the kit includes a plurality of containers, each containing the circular RNA or the DNA molecule encoding it. In some embodiments, the kit includes a container containing a plurality of circular RNA molecules, each containing a protein-coding sequence. In some embodiments, the kit includes a container containing a plurality of DNA molecules, each encoding a circular RNA molecule that can be used to express a protein in cells. In some embodiments, the kit also includes a set of instructions for using at least one circular RNA (or the DNA molecule encoding it) to express a protein in cells.

[0247]

[0257] In some embodiments, the kit contains one or more circular RNAs, or it contains one or more circular RNAs. The kit includes a DNA molecule encoding a circular RNA, and each circular RNA, or DNA molecule encoding a circular RNA, includes a sequence encoding at least one protein. In some embodiments, the kit may further include a circular RNA that does not encode a protein or miRNA, or a DNA molecule encoding one such RNA. In some embodiments, the kit may further include a circular RNA that encodes a miRNA, or a DNA molecule encoding one such miRNA. In some embodiments, the kit may include a single container containing each of (i) one or more circular RNAs, or DNA molecules encoding one such miRNA, each of which is a circular RNA (or DNA sequence) encoding a protein, (ii) optionally a circular RNA, or DNA molecule encoding one such miRNA, and (iii) optionally a circular RNA, or DNA molecule encoding one such miRNA. In some embodiments, the kit may include multiple containers, each containing one of the following: (i) at least one circular RNA, or DNA molecule encoding one such miRNA, (ii) optionally a circular RNA, or DNA molecule encoding one such miRNA, and (iii) optionally a circular RNA, or DNA molecule encoding one such miRNA. In some embodiments, the kit also includes a set of instructions for using at least one circular RNA (or a DNA sequence encoding it) to express a protein in a cell.

[0248]

[0258] In some embodiments, kits are provided for reprogramming somatic cells and / or generating iPSCs. In some embodiments, the kit includes a vector containing at least one circular RNA encoding a reprogramming factor (e.g., a transcription factor), or a nucleic acid (i.e., a DNA molecule) encoding it. In some embodiments, the kit includes a container containing the circular RNA or the DNA molecule encoding it. In some embodiments, the kit includes a plurality of containers, each containing the circular RNA or the DNA molecule encoding it. In some embodiments, the kit includes a container containing a plurality of circular RNA molecules, each containing a sequence encoding a transcription factor. In some embodiments, the kit includes a container containing a plurality of DNA molecules, each containing a circular RNA molecule that can be used to express a transcription factor in a cell. In some embodiments, the kit also includes a set of instructions for using at least one circular RNA for reprogramming somatic cells and / or generating iPSCs.

[0249]

[0259] In some embodiments, the kit comprises one or more circular RNAs or DNA molecules encoding them, each circular RNA or DNA molecule encoding a circular RNA comprising a sequence encoding at least one reprogramming factor. The reprogramming factor may be, for example, one of the reprogramming factors listed in Table 1. In some embodiments, the kit may further comprise circular RNAs or DNA molecules encoding them that do not encode a protein or miRNA. In some embodiments, the kit may further comprise circular RNAs or DNA molecules encoding miRNA. In some embodiments, the kit may comprise a single container comprising each of (i) one or more circular RNAs or DNA molecules encoding them, each circular RNA (or DNA sequence) encoding a reprogramming factor, (ii) optionally, circular RNAs or DNA molecules encoding them that do not encode either a protein or miRNA, and (iii) optionally, circular RNAs or DNA molecules encoding miRNA. In some embodiments, the kit may comprise a plurality of containers, each containing one of the following: (i) at least one circular RNA or DNA molecule encoding a reprogramming factor; (ii) optionally a circular RNA or DNA molecule encoding neither a protein nor a miRNA; or (iii) optionally a circular RNA or DNA molecule encoding a miRNA. In some embodiments, the kit comprises a set of instructions for using at least one circular RNA (or DNA sequence encoding it) to express a reprogramming factor in a cell. Includes manuals.

[0250]

[0260] In some embodiments, kits for transdifferentiating cells are provided. In some embodiments, the kit includes a vector containing at least one circular RNA encoding a reprogramming factor (e.g., a transcription factor), or a nucleic acid (i.e., a DNA molecule) encoding it. In some embodiments, the kit includes a container containing the circular RNA or the DNA molecule encoding it. In some embodiments, the kit includes a plurality of containers, each containing the circular RNA or the DNA molecule encoding it. In some embodiments, the kit includes a container containing a plurality of circular RNA molecules, each containing a sequence encoding a transdifferentiating factor. In some embodiments, the kit includes a container containing a plurality of DNA molecules, each containing a circular RNA molecule that can be used to express the transdifferentiating factor in cells. In some embodiments, the kit also includes a set of instructions for using at least one circular RNA for transdifferentiating cells.

[0251]

[0261] In some embodiments, the kit comprises one or more circular RNAs or DNA molecules encoding them, each circular RNA or DNA molecule encoding a circular RNA comprising a sequence encoding at least one conversion factor. The conversion factor may be, for example, one of the conversion factors listed in Table 6. In some embodiments, the kit may further comprise circular RNAs or DNA molecules encoding them that do not encode a protein or miRNA. In some embodiments, the kit may further comprise circular RNAs or DNA molecules encoding miRNA. In some embodiments, the kit may comprise a single container comprising each of (i) one or more circular RNAs or DNA molecules encoding them, each circular RNA (or DNA sequence) encoding a conversion factor, (ii) optionally, circular RNAs or DNA molecules encoding them that do not encode either a protein or miRNA, and (iii) optionally, circular RNAs or DNA molecules encoding miRNA. In some embodiments, the kit may comprise a plurality of containers, each containing one of the following: (i) at least one circular RNA or DNA molecule encoding a conversion factor; (ii) optionally a circular RNA or DNA molecule encoding neither a protein nor a miRNA; or (iii) optionally a circular RNA or DNA molecule encoding a miRNA. In some embodiments, the kit also comprises a set of instructions for using at least one circular RNA (or DNA sequence encoding it) to express a conversion factor in a cell.

[0252]

[0262] In some embodiments, the kit comprises several circular RNAs (or DNA molecules encoding them), each circular RNA encoding a reprogramming factor selected from Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and L-Myc. Each of the circular RNAs (or DNA molecules encoding them) may be provided in a separate container or in a single container.

[0253]

[0263] In some embodiments, the kit comprises several circular RNAs (or DNA molecules encoding them), each of which encodes a reprogramming factor selected from Oct3 / 4, Sox2, and Klf4. Each of the circular RNAs (or DNA molecules encoding them) may be provided in a separate container or in a single container.

[0254]

[0264] In some embodiments, the kit comprises multiple circular RNAs (or DNA molecules encoding them), each of which encodes a reprogramming factor selected from Oct3 / 4, Sox2, c-Myc, and Klf4. Each of the DNA molecules encoding it may be provided in a separate container or in a single container.

[0255]

[0265] In some embodiments, the kit comprises several circular RNAs (or DNA molecules encoding them), each of which encodes a reprogramming factor selected from Oct3 / 4, Sox2, L-Myc, and Klf4. Each of the circular RNAs (or DNA molecules encoding them) may be provided in a separate container or in a single container.

[0256]

[0266] In some embodiments, the kit may include linear RNA that can be circularized, or a DNA sequence encoding it. In some embodiments, the kit may further include RNA or DNA ligase, or one or more reagents for circularizing the linear RNA, such as Mg2+ and guanosine 5' triphosphate (GTP).

[0257]

[0267] In some embodiments, the kit includes (i) a container containing circular RNA encoding OCT4 and a buffer (e.g., 1–10 mM sodium citrate, pH 6.5), (ii) a container containing circular RNA encoding SOX2 and a buffer (e.g., 1–10 mM sodium citrate, pH 6.5), (iii) a container containing cirRNA encoding KLF4 and a buffer (e.g., 1–10 mM sodium citrate, pH 6.5), and (iv) a package and its instructions. The kit may further include a container containing circular RNA encoding c-MYC or L-MYC and a buffer (e.g., 1–10 mM sodium citrate, pH 6.5), a container containing cirRNA encoding LIN28 and a buffer (e.g., 1–10 mM sodium citrate, pH 6.5), a container containing cirRNA encoding NANOG and a buffer (e.g., 1–10 mM sodium citrate, pH 6.5), or a combination thereof.

[0258]

[0268] In some embodiments, the kit includes (i) (a) reprogramming factors of any one or more circularized RNAs of the combination of circularized reprogramming factors listed in Table 2, each factor individually contained in a separate container, or two or more such factors combined together in one or more containers, and / or (b) circular RNAs of any one or more combinations of circular RNAs for generating iPSCs listed in Table 3, each such circular RNA individually contained in a separate container, or two or more such circular RNAs combined together in one or more containers, and (ii) a package and its instructions.

[0259]

[0269] In some embodiments, the kit comprises: (i) (a) one or more reprogramming factors as circular RNAs of any combination of the circularized reprogramming factors listed in Table 2, wherein each factor is individually contained in a separate container, or two or more of such factors are combined together in a single or multiple containers; and / or (b) circular RNAs of any one or more combinations of circular RNAs for generating iPSCs listed in Table 3, wherein each such circular RNA is individually contained in a separate container, or two or more of such circular RNAs are combined together in a single or multiple containers (for either (i)(a) or (i)(b), the circularized reprogramming factors and / or circular RNAs in Table 2 and Table 3 are each suspended in a buffer), and (iii) a package and its instructions.

[0260]

[0270] In any of the above kits, the circular RNA, or the DNA molecule encoding it, may be provided in a composition further comprising a buffer. The buffer may contain, for example, 1 to 10 mM sodium citrate. In some embodiments, the pH of the buffer ranges from about 2 to about 12, such as about 6.5.

Example

[0261]

[0271] The following examples are included herein for the purpose of illustration only and are not intended to be limiting.

[0262] Example 1: Generation of Circular RNA and Linear mRNA

[0272] Circular RNA expression vectors containing RNA sequences encoding circOct3 / 4 (SEQ ID NO: 1), circKlf4 (SEQ ID NOs: 2, 3), circSox2 (SEQ ID NO: 4), circNanog (SEQ ID NOs: 5, 6), circLin28 (SEQ ID NO: 7), circC-Myc (SEQ ID NOs: 8, 9), or circL-Myc (SEQ ID NOs: 10-12) were generated. Additional expression vectors encoding circBIRC6 (SEQ ID NO: 13), circCORO1C (SEQ ID NO: 14), or circMAN1A2 (SEQ ID NO: 15) were generated. Circular RNA expression vectors encoding circnGFP or circmCherry were prepared for use as reporters.

[0263]

[0273] A general protocol for circular RNA production is shown in Figure 4. A permutation-substitution intron-exon (PIE) circRNA construct includes a 3' intron and exon fragment of a group I ribozyme, followed by a target sequence (e.g., an internal ribosome entry site (IRES) and a coding sequence (CDS) of the desired protein product), followed by a 5' exon fragment and a 5' intron. The PIE construct was cloned into a suitable plasmid to enable amplification and purification of plasmid DNA. Plasmid DNA was linearized with restriction enzymes and used as a template for in vitro transcription of the precursor RNA. The 5' and 3' ends of the precursor RNA fold via long-range base pairing and tertiary structure interactions to form a ribozyme. In the presence of Mg2+ and free guanosine (e.g., guanosine 5' triphosphate (GTP)), the ribozyme spontaneously splices the exon fragment via a series of transesterification reactions that form circular RNA and release the intron. Further heating or other manipulation may cause half of the introns to dissociate. Nicking of circular RNA can lead to relinearization and the formation of nicked circRNA degradation products, as shown in Figure 5.

[0264]

[0274] Construct design and synthesis: Plasmids containing the desired circularized construct were purchased from a gene synthesis vendor. The circularized construct includes a sequence corresponding to the 3' half of the T7 promoter and the subsequent permutational ribozyme (consisting of a 3' intron, 3' exon fragment, and flanking sequence), the subsequent sequence of interest (IRES and the target gene), the subsequent 5' half of the permutational ribozyme (flanking sequence, 5' exon fragment, and 5' intron), and a restriction site for plasmid linearization.

[0265]

[0275] Plasmid linearization: Plasmid (usually 20 μg) is added to the reaction mixture prepared according to the product instructions using the appropriate restriction enzyme (Thermo Scientific: Fast The reaction was linearized by incubation with Digest Eco32I or MssI for 1 hour, and the resulting reaction was cleaned up using a silica-based spin column (Thermo Scientific: GeneJET PCR Purification Kit) according to the product's instructions.

[0266]

[0276] In vitro transcription: A linearized plasmid was used as a template for in vitro transcription of the precursor RNA for circularization. Exemplary precursor RNA sequences are provided in SEQ ID NOs. 23-30, listed below in Table 7. The in vitro transcription reaction was prepared as shown in the product insert (Invitrogen MEGAscript T7 Transcription Kit), and DNase (Invitrogen Turbo DNAse) was added to the reaction (4 units of DNase per 1 μg of template DNA). After adding the ingredients in the specified ratio, incubate at 37°C for 2-3 hours, mix, and incubate again at 37°C for a further 30 minutes.

[0267] [Table 7]

[0268]

[0277] Post-transcription RNA cleanup and cyclization: 200 μg of IVT precursor RNA product was prepared with 2 mM GTP (guanosine triphosphate) and 10 mM Mg2+ in a total volume of 100 μL. The reaction mixture was incubated at 55°C for 15 minutes and immediately cleaned up with the MEGAClear Transcription cleanup kit. Eluted RNA was collected and quantified using Nanodrop One operating in RNA mode.

[0269]

[0278] Size exclusion chromatography: Circular RNA was purified from other cyclization by-products via size exclusion chromatography. 50–500 μg of post-transcriptional cyclized RNA products were injected into an FPLC system equipped with a suitable SEC column, and fractions corresponding to the peak concentration of circular RNA were collected and pooled. The mobile phase was TE pH 6. The pooled fractions were concentrated and filtered through a centrifugal MWCO filter (Amicon Ultra 0.5 mL). The buffer was replaced with 10 mM Tris pH 7.4 using 100K MWCO (Millipore-Sigma). The resulting RNA was quantified using Nanodrop One operating in RNA mode.

[0270]

[0279] The peak fraction of circular RNA was identified by visualizing the relative intensity of bands associated with circular RNA or other cyclization byproducts, after electrophoresis of 50–600 ng of RNA from a given fraction on a 2% agarose gel (Thermo Scientific 2% EX Gel). Peak fractions were identified via visual inspection or quantification of band intensity using the ImageLab software package (Bio-Rad).

[0271]

[0280] Phosphatase treatment: RNA purified by SEC is prepared in the reaction mixture at a ratio of 1 U of alkaline phosphatase per μg of RNA according to the product's package insert (Thermo The reaction mixture was incubated at 37°C for 1 hour using a silica column-based kit (GeneJET RNA Cleanup). The RNA was cleaned up using the Thermo Scientific Concentration Micro Kit. The RNA was eluted at TE pH 6 and stored at -20°C until use.

[0272]

[0281] Exemplary sequences of circularized RNA are provided in SEQ ID NOs. 31–38 and are detailed in Table 8 below.

[0273] [Table 8]

[0274]

[0282] Linear RNA vectors for producing linear RNA encoding a reporter gene (nGFP or mCherry) were also generated by Trilink. Linear RNA is produced by IVT using either modified or unmodified nucleotide triphosphates (NTPs). A 5' cap and poly(A) tail may be added before or during IVT. Linear RNA produced using modified NTPs is referred to herein as “modified linear RNA,” and linear RNA produced using unmodified NTPs is referred to herein as “unmodified linear RNA.”

[0275] Example 2: Characterization of circular RNA

[0283] Experiments were conducted to further characterize the circular RNA generated in Example 1.

[0276] PIE-based circular RNA production depends on the autocatalytic splicing activity of permutation group I introns.

[0284] Figure 6 shows agarose gel electrophoresis of in vitro transcripts (100 ng) from DNA templates corresponding to either full-length (WT) or truncated (ΔSS) permutation-substituted intron-exon (PIE) precursor RNA. Full-length precursor RNA is cotranscribed into a circular RNA, resulting in the formation of a circular RNA, a nicked circular RNA, and half of an excised intron. 3'-truncate precursor RNA (ΔSS) lacks permutation-substituted 5' introns and splicing sites, making circularization impossible, thus resulting in a single RNA product.

[0277]

[0285] Precursor RNA bands were identified by comparing their known lengths in the ssRNA ladder (not shown) with the known lengths of the cleaved precursor RNA products. Similarly, bands of nicked circular RNA and introns were identified by comparing their known lengths in the ladder with their relative positions on the gel.

[0278]

[0286] Circular RNA is known to move more slowly (with a higher apparent molecular weight) than linear RNA of the same size when separated on a 2% agarose gel (see Wesselhoeft et al., Nat Commun 9, 2629 (2018) https: / / doi.org / 10.1038 / s41467-018-05096-6), allowing for the identification of the remaining bands as circular RNA.

[0279] Verification of RNA circularization

[0287] Figure 7 shows splicing junction-specific RT-PCR analysis to verify that circular RNA is present in the circRNA band. IVT product and gel purification. Both circRNA bands were used as templates for first-strand cDNA synthesis using random hexama (Hex) or splicing junction (SJ) specific primers. The resulting cDNA was then used as a template for PCR amplification using forward and reverse primer pairs spanning the splicing junction expected to be formed during circularization.

[0280]

[0288] RT-PCR using all combinations of RNA template and first-strand cDNA primer generated PCR products of the expected 507 nucleotides (lanes 3-6). Splicing junction formation was confirmed by Sanger sequencing of the PCR products (not shown).

[0281]

[0289] As a control, the same PCR primer was used to amplify DNA from a plasmid containing a circRNA PIE construct. Because the plasmid does not contain a splicing junction, the primer faces "outside" from either end of the PIE construct, and DNA polymerase must traverse the plasmid backbone to generate the amplicon. The resulting amplification product corresponded to the expected product of 3,594 base pairs (lane 2).

[0282] Purification and characterization of circular RNA

[0290] During circRNA generation, the initial in vitro transcription (IVT) and co-transcription cyclization products were subjected to an additional post-transcriptional cyclization step (Circ) and separated by size exclusion chromatography (SEC). The selected SEC fractions were then pooled and treated with phosphatase prior to transfection. Figure 8A shows the distribution of RNA types remaining after each step for each of the six reprogramming factors. In particular, most of the precursor RNA remaining after the in vitro transcription reaction is consumed by the post-transcriptional cyclization step, leading to both additional circRNA formation and circRNA nicking. While the SEC step is effective in removing high and low molecular weight byproducts, its ability to purify circRNA from nicked linearized circRNA is less pronounced.

[0283]

[0291] RNaseR is a 3'→5' progressive exonuclease that digests linear RNA. Circular RNA lacks a 3' end and is therefore expected to be protected from RNase degradation. SEC fractions containing both putative circular and putative nicked circular RNA were selected and incubated with and without RNaseR to confirm the identity of circRNA and linear contaminants. The resulting products were then separated by agarose gel electrophoresis. As shown in Figure 8B, slower-moving bands in each lane (A, circular RNA) were observed to be resistant to RNaseR digestion, while faster-moving bands (B, linear RNA) were highly susceptible.

[0284] Example 3: Use of circular RNA for protein expression

[0292] The circular RNA from Example 1 was used to express proteins in fibroblasts. The stability of protein expression from circular RNA, modified linear mRNA, and unmodified linear mRNA was compared.

[0285]

[0293] Human dermal fibroblasts (HDFs) were seeded at a density of 50 K / well in 24-well plates and grown for approximately 24 hours in Cascade 106 medium containing a low serum growth supplement. Following manufacturer instructions, 30 ng of RNA (linear mRNA or circRNA from TriLink) and circular RNA encoding Oct4, Klf4, Sox2, cMyc, Nanong, and Lin28 were transfected using RNAiMax reagent. The cultures were fixed after 24 hours and treated for immunofluorescence chemistry (IFC) using antibodies specific to the target proteins. Images were taken using a Nikon Ti2 inverted microscope. The samples were acquired and captured using a high-resolution PCO sCMOS camera. Additional experiments are performed in which circular RNA is conjugated to lipid nanoparticles ("LNPs") to form a circRNA-LNP complex. The circRNA-LNP complex can then be used to directly introduce circular RNA into cells without the need for any transfection reagents.

[0286]

[0294] The results are provided in Figures 16A and 16B. All reprogramming factors used were transcription factors and showed almost exclusively nuclear localization (stained using DAPI). Lin28A is primarily an RNA-binding protein of the cell matrix. As shown, the circRNA construct led to protein expression in transduced fibroblasts. Note that protein expression levels were generally lower for circRNA than for linear mRNA. Interestingly, as shown in the fibroblast reprogramming experiments, the circRNA cocktail of reprogramming factors produced more iPSC colonies compared to the linear mRNA cocktail. While not bound by any theory, it is thought that lower but more persistent expression of reprogramming factors promotes reprogramming more than high, short-term expression.

[0287] Example 4: Testing of immunogenicity of circRNA and circRNA-LNP complexes

[0295] The immunogenicity of circular RNA and circRNA-LNP complexes is compared to that of modified and unmodified linear mRNA.

[0288]

[0296] Simply put, circular RNA, circRNA-LNP complexes, modified linear mRNA, or unmodified linear mRNA are introduced into cells. At various time points, the expression levels of interferon regulatory genes (e.g., one or more of the genes listed on www.interferome.org) are investigated using qPCR and / or ELISA according to standard protocols. In some experiments, circRNA or linear mRNA is introduced into cells in combination with B18R, and optionally with additional immune evasion factors such as E3 and K3. B18R and additional immune evasion factors are supplied in the form of linear mRNA, circular RNA, or added directly to the culture medium as proteins.

[0289]

[0297] To determine whether circRNA and / or linear mRNA affect cell viability, cell viability is monitored after RNA is introduced into cells. Specifically, the dynamics of cell proliferation / viability are tracked from 24 hours to 10 days after RNA introduction into cells. Cell viability is also measured after single or multiple transfection.

[0290] Example 5: Generation of iPSCs using circRNA reprogramming of adherent cells

[0298] Experiments were conducted to compare fibroblast reprogramming with that of iPSCs using unmodified linear mRNA and circular RNAs encoding various reprogramming factors.

[0291]

[0299] The experimental groups were as follows: (a) Group 1-mock-without RNA (b) Group 2-ReproCELL Stemgent, StemRNA 3 for human fibroblasts rd Gen reprogramming kit (unmodified linear mRNA) (c) Unmodified linear mRNA synthesized by group 3-Trilink (d) Group 4-unmodified circular RNA

[0292]

[0300] For each group, a cocktail of three RNAs encoding reprogramming factors, vaccinia virus immunosuppressive proteins, and miRNA mimics was combined and dispensed into the desired number of transfections (Human Gene Therapy, 26(11), DOI:10.1089 / hum.2015.045). The RNA cocktails are as follows: (a) A reprogramming factor mRNA cocktail containing mRNAs encoding Oct4, Sox2, Klf4, Lin28, cMyc, and Nanog (OSKLMN) is present in a molar ratio of 3:1:1:1:1:1. (b) A vaccinia immune-evading mRNA cocktail containing mRNA encoding E3, K3, and B18R(EKB). (c) A microRNA mimic cocktail containing mimics of miR302a, miR302b, miR302c, miR302d, and miR367.

[0293]

[0301] In group 4 (circular RNA group), linear mRNA was used in the vaccinia EKB gene cocktail. A small amount of RNA encoding nGFP was spiked into each group to aid in the visualization of RNA delivery to cells. Linear nGFP mRNA was used in groups 2 and 3, and circular nGFP mRNA was used. RNA was used in group 4. MicroRNA mimics were purchased from Dharmacon. ReproCELL's Stemgent kit was used as the overall reprogramming control. The RNA constructs in groups 3 and 4 have the same ORF sequence for each reprogramming factor. Therefore, the linear mRNA in group 3 is a direct control for the circular RNA in group 4.

[0294]

[0302] Human dermal fibroblasts (HDFs) were seeded in 6-well plates at three different densities (25,000 cells / well, 50,000 cells / well, and 75,000 cells / well). On day 1, the fibroblast medium was replaced with Nutristom-hPSC-XF medium. Transfection was performed using RNAiMax lipofectamine reagent as directed by the manufacturer. Cells were grown under hypoxic conditions (37°C, 5% O2, 5% CO2) until the end of the experiment. Three additional transfections were performed on days 2, 3, and 4 (see schematic diagram in Figure 9A). Fibroblast reprogramming was performed under hypoxic conditions from day 1, when iPSC colonies were manually harvested, until day 16 / 18, in 6-well plates coated with Nutristom medium and iMatrix-511. On day 16 or 18, selected colonies were collected in 24-well plates coated with vitronectin, and the iPSC culture medium was changed to E8. Individual iPSC clones continued to grow in E8 medium and were subculturised using Barzen's solution.

[0295]

[0303] Cells were imaged and phenotypic changes were investigated (e.g., acquisition of pluripotent stem cell (PSC)-like features such as a high nucleus-to-cytoplasmic ratio and colony formation, as well as mesenchymal epithelial transition (MET) in the early stages of survival and reprogramming).

[0296]

[0304] By day 16, when the PSC-like colonies had grown sufficiently large (containing several thousand cells per colony), 3–10 colonies were manually sampled under a dissecting microscope and harvested for further growth and characterization. Reprogramming plates were fixed on day 18 and prepared for IFC. Co-staining with anti-OCT4 and anti-TRA1-81 was performed along with DAPI and imaged using a Nikon Ti2 microscope to obtain high-resolution images. Plates were also imaged with Incucyte to capture images of the entire well.

[0297]

[0305] Figure 9A provides a timeline for reprogramming HDF using linear and circular RNA. HDF was seeded on day 0 at three densities (25K, 50K, and 75K per well in a 6-well plate), and transfection was performed four times daily thereafter. iPSC colonies formed and appeared around days 8–10.

[0298]

[0306] To monitor RNA delivery to fibroblasts, a small amount of nGFP RNA is included in the daily transfection cocktail. Trilink mRNA encoding nGFP is used in the Stemgent mRNA cocktail and the Trilink mRNA cocktail. CircRNAs encoding nGFP, which were present in both, were included in the circRNA cocktail. IncuCyte was used to image the reprogramming of the culture and measure nGFP protein expression daily. Figure 9B shows nGFP expression normalized as the percentage of peak expression. CircRNA-encoded nGFP protein showed extended expression (slower turnover) compared to linear mRNA-encoded nGFP protein.

[0299]

[0307] Characteristic morphological changes during mesenchymal epithelial transition (MET) and iPSC reprogramming were observed in all three experimental groups (Stemgent RNA, Trilink mRNA, and circRNA). However, cultures transfected with circRNA showed accelerated morphological transition from fibroblast-like cells to polygonal cell clusters (arrows), followed by a transition to densely packed cell clusters resembling early iPSC colonies (asterisks), compared to the linear mRNA group (Figure 9C).

[0300]

[0308] Figure 9D shows a full-well image of reprogrammed cultures stained with the pluripotency marker Tra-1-81 at day 18. Green indicates regions containing Tra-1-81+ cells, which are presumed to represent iPSCs. Cultures transfected with circRNA produced significantly more Tra-1-81 positive regions than wells transfected with either Stemgent mRNA or Trilink mRNA, suggesting that circRNA provided improved reprogramming efficiency (i.e., resulted in more pluripotent cells at day 18 of reprogramming compared to the linear mRNA method). mRNA reprogramming using the Stemgent kit is more efficient than Trilink. This resulted in higher reprogramming efficiency than mRNA reprogramming using mRNA. iPSCs induced by Trilink mRNA appeared only at the edges of the wells. Figure 9E provides a representative image of iPSCs from circRNA reprogramming at day 18 of the culture, stained for Tra-1-81 and Oct4 expression. The results are quantified in Figure 9F. In short, reprogramming was quantified for each reprogramming condition at day 18 using IncuCyte. Each well was analyzed for the area covered by iPSC colonies as well density % based on the morphology of the phase image. At all seeding densities (25K, 50K, and 75K), wells reprogrammed with circRNA produced the largest area covered by iPSC colonies compared to wells reprogrammed with Stemgent mRNA or Trilink mRNA, suggesting the highest reprogramming efficiency with circRNA.

[0301]

[0309] Additional readouts were performed to further characterize iPSCs induced by circRNA reprogramming. Figure 10A shows representative images of iPSCs from cultures between passages 3–5, induced from the Stemgent mRNA reprogramming kit (top), mRNA synthesized from Trilink (center), and circRNA (bottom). Each of these iPSC clones exhibited characteristic iPSC morphology. Figure 10B shows the population doubling time (PDT) of iPSCs induced by RNA reprogramming. The growth rate of early passage iPSC clones (i.e., before passage 6) is dynamic and is often reflected in the variability of the population doubling time. After passage 6, the doubling time of most clones stabilized, remaining within the typical iPSC doubling time range of approximately 30 hours. Figure 10C shows the expression of the pluripotency marker, SSEA4, in iPSC clones (early passages - P6–P9) induced from different RNA reprogramming cocktails. Clones S1 and S2 were induced from the Stemgent kit. Clones L1, L2, and L3 were derived from Trilink linear mRNA. Clones C2, C3, C8, C9, and C10 were derived from circRNA. All clones exhibited over 90% SSEA4+ cell population. Epi-iPSC lines were used as a positive control, while HEK293 cells were used as a negative control. OCT4 expression was evaluated. Additional experiments were conducted. These assays confirmed that iPSCs reprogrammed with circRNA exhibited similar morphological, growth, and expression characteristics to iPSCs reprogrammed with linear mRNA.

[0302]

[0310] The above experiments demonstrate that protein expression during reprogramming is prolonged by circRNA (based on nGFP expression, see Figure 9B), and MET dynamics are accelerated by circRNA (Figure 9C). Overall, more iPSC colonies were generated using circRNA, meaning that reprogramming with circRNA showed higher reprogramming efficiency compared to the method using linear mRNA (Figure 9D). Furthermore, iPSCs induced from circRNA showed consistent proliferation and expressed pluripotency markers (Figure 10).

[0303]

[0311] Additional experiments will be conducted to evaluate gene expression patterns, epigenetics, and triphyletic differentiation. The target clones will be grown, frozen, and stored in liquid nitrogen for later use.

[0304] Example 6: Optimized reprogramming protocol using circular RNA

[0312] Experiments were conducted to determine the optimal reprogramming protocol for adherent cells. Experimental groups were set up with reduced transfection numbers and with or without the use of the vaccinia EKB immune evasion cocktail. The RNA encoding the reprogramming factor was the same as that described in Example 5. (a) Mock RNA-free (b) Reprocell's Stemgent, StemRNA 3 for human fibroblasts rd Gen reprogramming kit (unmodified mRNA) (c) Unmodified mRNA synthesized by Trilink (d) Unmodified circRNA

[0305]

[0313] Four transfection conditions were tested for each RNA group. (a) Four transfections (4Tx, +EKB cocktail) (standard) - Days 1, 2, 3, and 4 after sowing (b) Two transfections (2Tx, +EKB cocktail) - Day 1 and Day 3 after sowing (c) One transfection (1Tx, +EKB cocktail) - Day 1 after sowing (d) Four transfections without EKB cocktail (4Tx, -EKB cocktail) - Days 1, 2, 3, and 4 after sowing

[0306]

[0314] Each transfection includes three cocktails (only (a) and (b) - excluding EKB conditions). (a) Reprogramming factor mRNA cocktail OSKLMN (Oct4 / Sox2 / Klf4 / Lin28 / cMyc / Nanog) (b) MicroRNA Mimic Cocktail (c) Vaccinia immune-evading mRNA cocktail EKB (E3 / K3 / B18R)

[0307]

[0315] Transfection was carried out according to the method outlined in Example 4. A schematic diagram of the transfection schedule is provided in Figure 11.

[0308]

[0316] Figure 12 shows the morphological progression of cultures in each experimental group. The subgroup transfected with circRNA in the 4Tx+EKB group (Figure 12A) showed iPSC colony-like morphology as early as day 5 and produced several hundred colonies by day 9. In contrast, the Stemgent and Trilink linear RNA conditions did not produce iPSC-like morphology until day 7. The colonies did not show iPSC-colony-like morphology, and by day 9 there were only a few dozen colonies. Figure 12B shows the morphological progress during reprogramming of the 4Tx-EKB group. Figure 12C shows the morphological progress during reprogramming of the 2Tx group. The inset under conditions transfected with circRNA showed iPSC-colony-like morphology by day 5 and hundreds of colonies by day 9. Figure 12D shows the morphological progress during reprogramming of the 1Tx group. Images were acquired with a 4X objective lens to capture the largest possible field of view. No iPSC colonies were observed in any of the groups with a single transfection.

[0309]

[0317] Further analysis was performed on two 4x transfection groups (4Tx with and without the EKB cocktail). Culture images were taken on day 6 (two days after the fourth and final transfection), and cytotoxicity was assayed based on the number of round dead cells in the culture (Figure 13). CircRNA cultures, with or without EKB, showed little round light-reflecting cells, indicating low cytotoxicity. In contrast, both the Trilink mRNA and Stemgent kits produced a large number of round or suspension cells in the culture, suggesting toxicity. Furthermore, morphological mesenchymal epithelial transition (MET) was far more pronounced in circRNA cultures at this early stage than in Trilink and Stemgent cultures (Trilink showed the lowest amount of MET and still exhibited spindle-shaped fibroblast morphology). Based on these data, transfection and reprogramming with circRNA resulted in less cell death and lower toxicity during early reprogramming (i.e., during the days of active transfection) than mRNA transfection / reprogramming. See Figure 13.

[0310]

[0318] Reprogramming efficiency was determined by semi-quantitative analysis of Tra-1-81 / Oct4 staining of cultures on day 16. On day 16 of reprogramming, cultures were fixed, stained with Tra-1-81 and Oct4, and images of the entire well were scanned using IncuCyte (Figure 14). Double-positive regions for Tra-1-81 and Oct4 are presumed to be iPSC colonies. No RNA type successfully formed iPSC colonies with only one transfection (left panel). Under 2Tx+EKB, 4Tx+EKB, and 4Tx-EKB transfection conditions, cultures transfected with circRNA showed the largest number of Tra-1-81 / Oct4 double-positive regions compared to stemgent or trilink mRNA. This was true regardless of seeding density (25K, 50K, and 75K), suggesting that reprogramming efficiency from circRNA is highest.

[0311]

[0319] The reprogramming efficiency for each experimental group is summarized in Table 9 below.

[0312] [Table 9] TIFF2026091847000024.tif59162

[0313]

[0320] As shown in Table 9, fibroblast reprogramming using circRNA resulted in improved reprogramming efficiency, independent of the initial fibroblast seeding density, regardless of the experimental transfection protocol used. The results are further quantified in Figure 14B. Reprogramming was quantified for each reprogramming condition on day 16 using IncuCyte. Each well was analyzed for the area covered by iPSC colonies as well concentration % based on the morphology of the phase image. For all transfection conditions except 1Tx+EKB, circRNA produced the largest area covered by iPSC colonies (i.e., the most iPSC colonies) compared to Stemgent mRNA or Trilink mRNA. No RNA type successfully produced iPSC colonies in a single transfection. Among the different transfection conditions, the greatest difference between circRNA and mRNA was observed with 2Tx+EKB (two transfections of the circRNA cocktail produced a large number of iPSC colonies, while two transfections of Stemgent or Trilink mRNA did not).

[0314]

[0321] In short, circRNA-based reprogramming is more efficient in fibroblast reprogramming compared to linear mRNA methods. CircRNA reprogramming showed lower toxicity during the initial reprogramming period (active transfection period, Figure 13), resulted in the generation of more iPSC-like colonies at an early stage (Figure 12), produced a similar or greater number of iPSC-like colonies with fewer cells at the start compared to linear mRNA (Table 9 and Figure 14), resulted in a faster reprogramming rate (colonies formed as early as day 5 or 6, Figure 12), and resulted in faster colony maturation (based on morphology, Figure 13).

[0315] Example 7: Delivery of circRNA to CD34+ cells

[0322] CD34+ suspension culture cells were unsuccessful to reprogram using conventional methods because they were inefficient and required repeated transfection, which is toxic to the cells. Results shown in previous examples indicate that circular RNA reprogramming is more efficient and results in significantly less cell death than conventional methods. Therefore, it was hypothesized that CD34+ cell reprogramming might be possible with circRNA. Experiments were conducted to determine the optimal method for delivering linear and circular RNA to CD34+ hematopoietic stem cells. Using a Neon electroporation system and liposome-based reagents, the transfection efficiency of nGFP RNA (linear and circular) was investigated. The rate was evaluated.

[0316]

[0323] Purified CD34+ cells were transfected with linear or circular RNA (nGFP) using one of the following three transfection methods, and nGFP protein expression was evaluated after RNA transfection. (a) Neon nucleofection (using ThermoFisher's Neon transfection system) (b) Lipofectamine RNAiMAX reagent (a reagent used for transfection of fibroblasts) (c) DOTAP liposome transfection reagent (MilliporeSigma)

[0317]

[0324] Nucleofection yielded a transfection efficiency of 80-100% (most cells accepted nGFP regardless of whether mRNA or circRNA was used). RNAiMAX transfection resulted in very low transfection efficiency. DOTAP transfection caused cell aggregation, and no transfection occurred.

[0318] Example 8: Generation of iPSCs using circRNA reprogramming of suspended cells

[0325] Suspended cells (such as CD34+ cells) are reprogrammed using circRNA to generate iPSCs. Briefly, purified CD34+ cells (Hemacare) are grown for 3 days ("-3 to 0 days") in hematopoietic stem cell (HSC) medium containing a cocktail of five cytokines (SCF, TPO, FLT3-L, IL3, and IL6, each at 100 ng / mL).

[0319]

[0326] On day 0 (3 days post-growth), 100K cells are combined with an RNA cocktail for reprogramming and electroporated using a Neon electroporator. The electroporated cells are transferred to 0.5 mL of SCGM medium containing cytokines (SCF, TPO, FLT3-L, IL3, and IL6, each at 100 ng / mL) in non-adherent wells of a 24-well plate. The cells are allowed to recover for approximately 48 hours before being transferred to a 6-well plate coated with VTN on day 3, or before a second transfection.

[0320]

[0327] Transfection cells cultured in VTN-coated wells are gradually transferred to pluripotent stem cell (PSC) medium as follows: (a) On days 4 and 6, replace 1 ml of used medium with 1 ml of "minus" medium (HSC medium without cytokines). (b) On day 7, replace 1 mL of used medium from each with 1 mL of PSC medium. (c) On days 8-18, replace the used culture medium in the wells with 100% PSC culture medium. (d) The estimated iPSC clone is expected to appear between 12 and 18 days.

[0321]

[0328] In some experiments, cells also come into contact with circB18R, optionally combined with additional immune evasion factors such as E3 and K3. In some experiments, cells also come into contact with circBIRC6, circCORO1C, or circMAN1A2.

[0322]

[0329] The morphological progression of cells toward pluripotency is tracked, and reprogramming efficiency is quantified. iPSC clones are selected and characterized. Specifically, gene expression patterns are analyzed. The expression of pluripotency markers is analyzed along with turnover, epigenetics, and triphyletic differentiation (using human embryonic stem cells (hES) or iPSCs as controls). The target clones are grown, frozen, and stored in liquid nitrogen for later use.

[0323] Example 9: Use of circular RNA encoding MyoD to induce muscle cell differentiation

[0330] Transduction of MyoD into fibroblasts (non-muscle cells) has been shown to be sufficient to convert them into myoblasts (muscle cells). In this example, the circRNA encoding MyoD was used to generate muscle cells.

[0324]

[0331] In short, human dermal fibroblasts (HDFs) were seeded on day 0 in 10% fibroblast growth medium (FEM) in 6-well plates at three different densities: 25K, 50K, or 75K per well. On day 1, 200 ng / ml of recombinant B18R protein was added to the 10% FEM. Cells were grown under normal oxygen conditions (and 5% CO2 at 37°C) until the end of the experiment. Cells were transfected daily for 6 days with 50 ng of circRNA or linear RNA (Trilink) encoding MyoD using RNAiMAX. Approximately 16 hours after transfection, the medium was replaced with 10% FEM containing 200 ng / ml of B18R protein. The 10% FEM medium was changed daily, cells were imaged, and phenotypic changes (e.g., survival, multinucleated myotubation) were investigated. Following the final transfection on day 6, the culture medium was changed to 2% FEM containing 200 ng / ml of B18R protein from day 7 onwards.

[0325]

[0332] Reprogramming plates were fixed on day 12 and processed for iFC. Co-staining with antibodies specific to desmin, myosin heavy chain (MHC), and myogenin (MYOG) was performed along with DAPI and imaged using a Nikon Ti2 microscope.

[0326]

[0333] The results are shown in Figures 15A to 15C. Figure 15A shows MyoD expression in transfected cells. Cultures transfected with both circRNA and linear mRNA stained positively for MyoD protein, but cultures transfected with mock were not stained, thus verifying protein expression by both types of RNA. 24 hours after transfection, the amount of protein expressed by circRNA was lower than that expressed by linear mRNA.

[0327]

[0334] Six days after the final transfection, the culture medium was changed to reduced serum (from 10% serum to 2% serum) to induce myoblast fusion and multinucleated myotube formation. The phase-contrast images shown in Figure 15B show examples of myotubes (arrows) observed in both cultures transfected with circRNA MyoD and cultures transfected with linear mRNA MyoD.

[0328]

[0335] Figures 15C and 15D show the expression of muscle-specific markers in cultures transfected with MyoD. Myotubes induced from cultures transfected with circRNA MyoD (Figure 15C) expressed the muscle-specific markers myogenin, desmin, and myosin heavy chain (MHC). The arrows in the merged image of myogenin and desmin indicate multinuclear fusion cells. However, myotubes induced from cultures transfected with linear mRNA MyoD expressed desmin but not myogenin or MHC (Figure 15D). The data from this experiment are quantified in Figures 17A to 17C.

[0329]

[0336] Desmin, myogenin, and myosin heavy chain (MHC) are generally considered to be markers for early, intermediate, and late muscle differentiation, respectively. CircRNA MyoD-inducible myotubes express all three markers, while linear mRNA MyoD-inducible myotubes express only desmin. The observation that ogenin and MHC were not expressed indicated that circRNA MyoD leads to final muscle differentiation more effectively than linear mRNA within the same timeframe (12 days).

[0330]

[0337] In summary, this data shows the overall viability of cells in cultures during the initial stages of reprogramming (day 6, see Figure 13) and after complete reprogramming (see, for example, Figures 9D and 9F (compared to wells at 25K for stemgent, linear, and circRNA), and also Figure 14A (compared to wells at 25K for stemgent, linear, and circRNA after 4 transfections (+ or -EKB)) and Figure 14B).

[0331] Example 10: Use of circular RNA in a combined method for reprogramming and editing the genome of cells

[0338] A composition is prepared comprising (i) recombinant circular RNAs, each containing a sequence encoding at least one reprogramming factor; (ii) a nucleic acid encoding a Cas9 nuclease; and (iii) a nucleic acid encoding a gRNA targeting the sequence of interest. The composition is brought into contact with cells. Cas9 edits the cells' DNA with the sequence of interest. The reprogramming factors reprogram the cells into a pluripotent state. Thus, the cells' genotype and phenotype are altered.

[0332] Example 11: Use of circular RNA in a combination method for transdifferentiation and editing of the cell genome

[0339] A composition is prepared comprising (i) recombinant circular RNAs, each containing a sequence encoding at least one conversion factor; (ii) a nucleic acid encoding a Cas9 nuclease; and (iii) a nucleic acid encoding a gRNA targeting the sequence of interest. The composition is brought into contact with differentiated cells. Cas9 edits the cells' DNA with the sequence of interest. The conversion factors reprogram the differentiated cells into a different differentiated cell type. Thus, the genotype and phenotype of the cells are altered.

[0333]

[0340] The foregoing is illustrative of the present invention and should not be construed as limiting the present invention. The present invention is defined by the following claims, which include equivalents thereof.

[0334] References 1. Cell Stem Cell (2010) 7: 618 2. SCIENTIFIC REPORTS (2012) 2: 657 3. Nature Review Genetics (2019) 20:675 4. NATURE COMMUNICATIONS (2017) 8: 1149

Claims

1. Recombinant circular RNA containing a protein-coding sequence, The protein coding sequence is a recombinant circular RNA that codes for at least one reprogramming factor, wherein the at least one reprogramming factor is Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, or L-Myc, or a fragment or variant thereof.

2. The recombinant circular RNA according to claim 1, wherein the at least one reprogramming factor is human or a humanized reprogramming factor.

3. The recombinant circular RNA according to claim 1 or 2, wherein the at least one reprogramming factor is Oct3 / 4, and the Oct3 / 4 has the sequence of SEQ ID NO: 1, or a sequence that is at least 90% or at least 95% identical thereto.

4. The recombinant circular RNA according to claim 3, wherein the recombinant circular RNA comprises the nucleic acid sequence of Sequence ID No. 33, or a sequence that is at least 90% or at least 95% identical thereto.

5. The recombinant circular RNA according to claim 1 or 2, wherein the at least one reprogramming factor is Klf4, and the Klf4 has the sequence of SEQ ID NO: 2 or 3, or a sequence that is at least 90% or at least 95% identical thereto.

6. The recombinant circular RNA according to claim 4, wherein the recombinant circular RNA comprises the nucleic acid sequence of Sequence ID No. 37, or a sequence that is at least 90% or at least 95% identical thereto.

7. The recombinant circular RNA according to claim 1 or 2, wherein the at least one reprogramming factor is Sox2, and the Sox2 has the sequence of SEQ ID NO: 4, or a sequence that is at least 90% or at least 95% identical thereto.

8. The recombinant circular RNA according to claim 7, wherein the recombinant circular RNA comprises the nucleic acid sequence of Sequence ID No. 34, or a sequence that is at least 90% or at least 95% identical thereto.

9. The recombinant circular RNA according to claim 1 or 2, wherein the at least one reprogramming factor is Nanog, and the Nanog has the sequence of SEQ ID NO: 5 or 6, or a sequence that is at least 90% or at least 95% identical thereto.

10. The recombinant circular RNA according to claim 9, wherein the recombinant circular RNA comprises the nucleic acid sequence of Sequence ID No. 36, or a sequence that is at least 90% or at least 95% identical thereto.

11. The recombinant circular RNA according to claim 1 or 2, wherein the at least one reprogramming factor is Lin28, and the Lin28 has the sequence of SEQ ID NO: 7, or a sequence that is at least 90% or at least 95% identical thereto.

12. The recombinant circular RNA according to claim 11, wherein the recombinant circular RNA comprises the nucleic acid sequence of Sequence ID No. 35, or a sequence that is at least 90% or at least 95% identical thereto.

13. The recombinant circular RNA according to claim 1 or 2, wherein the at least one reprogramming factor is c-Myc, and the c-Myc has the sequence of SEQ ID NO: 8 or 9, or a sequence that is at least 90% or at least 95% identical thereto.

14. The recombinant circular RNA according to claim 13, wherein the recombinant circular RNA comprises the nucleic acid sequence of Sequence ID No. 38, or a sequence that is at least 90% or at least 95% identical thereto.

15. The recombinant circular RNA according to claim 1 or 2, wherein the at least one reprogramming factor is L-Myc, and the L-Myc has any one sequence of sequence numbers 10 to 12, or a sequence that is at least 90% or at least 95% identical thereto.

16. The recombinant circular RNA according to any one of claims 1 to 15, wherein the circular RNA is substantially non-immunogenic.

17. The aforementioned circular RNA is one or more M-6-methyladenosine (m ６ A) Recombinant circular RNA according to claim 16, comprising a residue.

18. The recombinant circular RNA according to any one of claims 1 to 17, wherein the circular RNA comprises approximately 200 nucleotides to approximately 5,000 nucleotides.

19. The recombinant circular RNA according to any one of claims 1 to 18, wherein the circular RNA includes an internal ribosome entry site (IRES) operably ligated to the protein-coding sequence.

20. A complex comprising recombinant cyclic RNA according to any one of claims 1 to 19 and lipid nanoparticles (LNPs).

21. The composite according to claim 20, wherein the LNP comprises a cationic lipid.

22. The complex according to claim 20 or 21, wherein the recombinant circular RNA and the LNP are conjugated.

23. The complex according to claim 22, wherein the recombinant circular RNA and the LNP are covalently joined.

24. The complex according to claim 22, wherein the recombinant circular RNA and the LNP are joined non-covalently.

25. A vector comprising a nucleic acid encoding a recombinant circular RNA according to any one of claims 1 to 19.

26. The vector according to claim 25, wherein the vector is a non-viral vector.

27. The vector according to claim 26, wherein the non-viral vector is a plasmid.

28. The vector according to claim 25, wherein the vector is a viral vector.

29. The vector according to claim 28, wherein the viral vector is a retroviral vector, a herpesvirus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, a baculovirus vector, an alphavirus vector, a picornavirus vector, a vacciniavirus vector, or a lentivirus vector.

30. A recombinant circular RNA according to any one of claims 1 to 19, a complex according to any one of claims 20 to 24, or a vector according to any one of claims 25 to 29 Composition containing.

31. The composition according to claim 30, comprising a carrier and / or a medium.

32. A composition comprising two or more recombinant circular RNAs according to any one of claims 1 to 19, wherein the composition comprises a combination of recombinant circular RNAs encoding the reprogramming factors selected from the reprogramming factors of Table 2.

33. A composition comprising two or more recombinant circular RNAs, The aforementioned composition, (i) Oct3 / 4, Klf4, Sox2, and c-Myc (ii) Oct3 / 4, Klf4, Sox2, and L-Myc (iii) Oct3 / 4, Klf4, and Sox2 (iv) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc, or A composition comprising a combination of recombinant circular RNAs encoding the reprogramming factor selected from (iv)Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc.

34. A kit comprising a recombinant circular RNA according to any one of claims 1 to 19, a complex according to any one of claims 20 to 24, a vector according to any one of claims 25 to 29, or a composition according to any one of claims 30 to 33.

35. A cell comprising a recombinant circular RNA according to any one of claims 1 to 19, a complex according to any one of claims 20 to 24, a vector according to any one of claims 25 to 29, or a composition according to any one of claims 30 to 33.

36. The cell according to claim 35, wherein the cell is a eukaryotic cell.

37. The cell according to claim 36, wherein the cell is a mammalian cell.

38. The cell according to claim 37, wherein the cell is a human cell.

39. The cell according to any one of claims 35 to 38, wherein the cell is a CD34+ cell.

40. A method for expressing a protein in a cell, comprising contacting the cell with a circular RNA according to any one of claims 1 to 19, a complex according to any one of claims 20 to 24, a vector according to any one of claims 25 to 29, or a composition according to any one of claims 30 to 33, and maintaining the cell under conditions in which the protein is expressed.

41. The method according to claim 40, wherein the method comprises contacting the cells with an additional circular RNA, the additional circular RNA being circBIRC6, circCORO1C, or circMAN1A2.

42. The method according to claim 41, wherein the additional circular RNA is circBIRC6, and circBIRC6 has the sequence of SEQ ID NO: 13, or a sequence that is at least 90% or at least 95% identical thereto.

43. The aforementioned additional circular RNA is circCORO1C, and circCORO1C is, The method according to claim 41, having the sequence of sequence number 14, or a sequence that is at least 90% or at least 95% identical thereto.

44. The method according to claim 41, wherein the additional circular RNA is circMAN1A2, and circMAN1A2 has the sequence of Sequence ID No. 15, or a sequence that is at least 90% or at least 95% identical thereto.

45. The method according to any one of claims 40 to 44, comprising contacting the cells with circular RNA encoding B18R.

46. The method according to claim 45, wherein B18R has the sequence of sequence number 16, or a sequence that is at least 90% or at least 95% identical thereto.

47. A method for producing induced pluripotent stem cells (iPSCs), Contacting somatic cells with at least one of the recombinant circular RNA according to any one of claims 1 to 19, the complex according to any one of claims 20 to 24, the vector according to any one of claims 25 to 29, and / or the composition according to any one of claims 30 to 33, A method comprising maintaining the cells under conditions that yield reprogrammed iPSCs.

48. The method according to claim 47, comprising contacting the cells with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine circular RNAs.

49. The method according to claim 47 or 48, comprising contacting the cells with a first circular RNA encoding Oct4, a second circular RNA encoding Sox2, a third circular RNA encoding Klf4, a fourth circular RNA encoding C-Myc or L-Myc, and a fifth circular RNA encoding Lin28.

50. The method according to claim 47 or 48, wherein the method comprises contacting the cells with a first circular RNA encoding Oct4, a second circular RNA encoding Sox2, a third circular RNA encoding Klf4, a fourth circular RNA encoding C-Myc or L-Myc, a fifth circular RNA encoding Lin28, and a sixth circular RNA encoding Nanog.

51. The method according to claim 47, comprising contacting the somatic cells with at least one acyclic RNA nucleic acid encoding one or more reprogramming factors.

52. The method according to claim 51, wherein the one acyclic RNA nucleic acid is selected from mRNA or plasmid.

53. The method includes contacting the cells with at least one additional circular RNA, The method according to any one of claims 47 to 52, wherein the at least one additional circular RNA is circBIRC6, circCORO1C, or circMAN1A2.

54. The at least one additional circular RNA is BIRC6, which has the sequence of SEQ ID NO: 13, or a sequence that is at least 90% or at least 95% identical thereto. The method according to claim 53.

55. The method according to claim 53, wherein the at least one additional circular RNA is CORO1C, and CORO1C has the sequence of SEQ ID NO: 14, or a sequence that is at least 90% or at least 95% identical thereto.

56. The method according to claim 53, wherein the at least one additional circular RNA is MAN1A2, and MAN1A2 has the sequence of SEQ ID NO: 15, or a sequence that is at least 90% or at least 95% identical thereto.

57. The method according to any one of claims 47 to 56, wherein the method comprises contacting cells with circular RNA encoding B18R.

58. The method according to claim 57, wherein B18R has the sequence of sequence number 16, or a sequence that is at least 90% or at least 95% identical thereto.

59. The method according to any one of claims 47 to 58, wherein the cells are fibroblasts, peripheral blood-derived cells, endothelial progenitor cells, umbilical cord blood-derived cells, keratinocytes, melanocytes, adipose tissue-derived cells, or urine-derived cells.

60. The method according to any one of claims 47 to 58, wherein the cells are CD34+ cells.

61. The method according to any one of claims 47 to 60, wherein the cells are adherent cells.

62. The method according to any one of claims 47 to 60, wherein the cells are in a suspension.

63. A method for producing induced pluripotent stem cells (iPSCs), comprising contacting CD34+ in a suspension of cells with at least one of the recombinant circular RNA described in any one of claims 1 to 19, the complex described in any one of claims 20 to 24, the vector described in any one of claims 25 to 29, and / or the composition described in any one of claims 30 to 33, and maintaining the cells under conditions that will result in reprogrammed iPSCs.

64. The above method yields results, (i) Compared to methods using one or more linear RNAs to produce iPSCs, the number of reprogrammed iPSCs present at the end of culture is increased. (ii) Compared to methods of producing iPSCs using one or more linear RNAs, an increase in the maturation rate of reprogrammed iPSCs, and / or (iii) Compared to methods for producing iPSCs using one or more linear RNAs, reduction in cytotoxicity at one or more time points during reprogramming. The method according to any one of claims 47 to 63, wherein one or more of the above are met.

65. The above method yields results, (i) Compared to methods using one or more linear RNAs to produce iPSCs, the number of reprogrammed iPSCs present at the end of culture is increased. (ii) Compared to methods of producing iPSCs using one or more linear RNAs, the rate of maturation of reprogrammed iPSCs is increased, and (iii) Compared to methods for producing iPSCs using one or more linear RNAs, reduction in cytotoxicity at one or more time points during reprogramming. The method according to any one of claims 47 to 63, wherein each of the above is true.

66. The method according to any one of claims 47 to 65, comprising contacting the cells once or more with at least one of the recombinant circular RNA according to any one of claims 1 to 19, the complex according to any one of claims 20 to 24, the vector according to any one of claims 25 to 29, and / or the composition according to any one of claims 30 to 33.

67. The method according to claim 66, comprising bringing the cells into contact two, three, four, or more times.

68. The method according to claim 66, comprising bringing the cells into contact less than four times.

69. The method according to claim 66, comprising bringing the cells into contact two to four times.

70. iPSC prepared using the method described in any one of claims 47 to 69.

71. Differentiated cells derived from iPSCs according to claim 70.

72. The differentiated cells according to claim 71, wherein the differentiated cells are muscle cells, neurons, cardiomyocytes, hepatocytes, islet cells, keratinocytes, T cells, or NK cells.

73. A method for directly converting cells from a first cell type to a second cell type, comprising contacting the cells with a recombinant circular RNA according to any one of claims 1 to 19, a complex according to any one of claims 20 to 24, a vector according to any one of claims 25 to 29, and / or a composition according to any one of claims 30 to 33, and maintaining the cells under conditions that allow them to be converted to the second cell type.

74. The method according to claim 73, wherein the first cell type is a somatic cell and the second cell type is a somatic cell.

75. The method according to claim 73, wherein the second cell type is a muscle cell, neuron, cardiomyocyte, hepatocyte, pancreatic islet, keratinocyte, T cell, or NK cell.

76. The method according to claim 74 or 75, wherein the first cell type is a fibroblast.

77. The method according to claim 76, wherein the cells are contacted with a plurality of recombinant circular RNAs, the plurality of circular RNAs comprising a circular RNA encoding any one of the combinations of differentiation factors listed in column B of Table 6.

78. The method according to any one of claims 73 to 77, wherein the cells do not enter an intermediate pluripotent state.

79. The method according to any one of claims 73 to 78, wherein the cells are directly converted from the first cell type to the second cell type without becoming progenitor cells.

80. The method according to any one of claims 78 to 79, wherein the first cell type is a fibroblast, the second cell type is a muscle cell, and the recombinant circular RNA encodes myoD.

81. Cells prepared by the method described in any one of claims 73 to 80.

82. A method for reprogramming and editing the genome of a cell, wherein the cell is (i) Recombinant circular RNA containing a protein-coding sequence encoding at least one reprogramming factor, (ii) A method comprising contacting the DNA or RNA of the cell with an enzyme capable of editing it, or a nucleic acid encoding it.

83. The method according to claim 82, wherein the recombinant circular RNA is the recombinant circular RNA described in any one of claims 1 to 19.

84. The method according to claim 82 or 83, wherein the enzyme is TALEN, NgAgo, SGN, or RGN, or a modified or cleavage variant thereof.

85. The method according to any one of claims 82 to 84, wherein the enzyme is Cas9 nuclease, Cas12(a) nuclease (Cpf1), Cas12b nuclease, Cas12c nuclease, TrpB-like nuclease, Cas13a nuclease (C2c2), Cas13b nuclease, Cas14 nuclease, or a modified or cleavage variant thereof.

86. The method according to claim 85, wherein the enzyme is a Cas9 nuclease, and the Cas9 nuclease is isolated or derived from Streptococcus pyogenes or Staphylococcus aureus.

87. The method according to claim 82 or 83, wherein the enzyme is ADAR.

88. The method according to any one of claims 82 to 84, wherein the enzyme is an RNA-inducing nuclease.

89. The RNA-inducing nucleases are APG05083.1, APG07433.1, APG07513.1, APG08290.1, APG05459.1, APG04583.1, APG1688.1, APG05733.1, APG06207.1, APG01647.1, APG08032.1, APG05712.1, APG01658.1, APG06498.1, APG09106.1, APG09882.1, APG02675.1, APG01405.1, APG06250.1, APG The method according to claim 88, selected from any one of 06877.1, APG09053.1, APG04293.1, APG01308.1, APG06646.1, APG09748, APG07433.1, APG00969, APG03128, APG09748, APG00771, APG02789, APG09106, APG02312, APG07386, APG09980, APG05840, APG05241, APG07280, APG09866, and APG00868.

90. The method according to any one of claims 82 to 89, further comprising contacting the cells with guide RNA or nucleic acid encoding it.

91. The method according to any one of claims 82 to 90, wherein the cells contact the recombinant circular RNA before contacting the enzyme or the nucleic acid encoding it.

92. The method according to any one of claims 82 to 90, wherein the cells are contacted with the enzyme or the nucleic acid encoding it, and then contacted with the recombinant circular RNA.

93. The method according to any one of claims 82 to 90, wherein the cell comes into contact with the recombinant circular RNA at approximately the same time as it comes into contact with the enzyme or the nucleic acid encoding it.

94. Cells produced by the method according to any one of claims 82 to 93.

95. A method for transdifferentiating and editing the genome of a cell, wherein the cell (i) Recombinant circular RNA containing a protein-coding sequence encoding at least one differentiation factor, (ii) A method comprising contacting the DNA or RNA of the cell with an enzyme capable of editing it, or a nucleic acid encoding it.

96. The method according to claim 95, wherein the at least one differentiation factor is one of MyoD, C / EBPα, C / EBPβ, Pdx1, Ngn3, Mafa, Pdx1, Hnf4α, Foxa1, Foxa2, Foxa3, Ascl1 (also known as Mash1), Brn2, Myt1l, miR-124, Brn2, Myt1l, Ascl1, Nurr1, Lmx1a, Ascl1, Brn2, Myt1l, Lmx1a, FoxA2, ​​Oct4, Sox2, Klf4 and c-Myc, Tbx5, Mef2c, Gata-4, or Mesp1.

97. The method according to claim 95, wherein the at least one differentiation factor is one of the differentiation factors listed in Table 6.

98. The method according to claim 95, comprising two or more differentiation factors selected from those listed in Table 6.

99. The method according to any one of claims 95 to 98, wherein the enzyme is TALEN, NgAgo, SGN, or RGN, or a modified or cleavage variant thereof.

100. The method according to any one of claims 95 to 98, wherein the enzyme is Cas9 nuclease, Cas12(a) nuclease (Cpf1), Cas12b nuclease, Cas12c nuclease, TrpB-like nuclease, Cas13a nuclease (C2c2), Cas13b nuclease, Cas14 nuclease, or a modified or cleavage variant thereof.

101. The method according to claim 100, wherein the nuclease is a Cas9 nuclease, and the Cas9 nuclease is isolated or derived from Streptococcus pyogenes or Staphylococcus aureus.

102. The method according to any one of claims 95 to 98, wherein the enzyme is ADAR.

103. The method according to any one of claims 95 to 98, wherein the enzyme is an RNA-inducing nuclease.

104. The RNA-inducing nucleases are APG05083.1, APG07433.1, APG07513.1, APG08290.1, APG05459.1, APG04583.1, APG1688.1, APG05733.1, APG06207.1, APG01647.1, APG08032.1, APG05712.1, APG01658.1, APG06498.1, APG09106.1, APG09882.1, APG02675.1, APG01405.1, APG06250.1, APG06877.1, APG09053.1, APG04293.1, APG01308.1, AP G06646.1, APG09748, APG07433.1, APG00969, APG03128, APG09748, APG00771, APG02789, APG09106, APG0231 2. The method according to claim 103, selected from any one of APG07386, APG09980, APG05840, APG05241, APG07280, APG09866, and APG00868.

105. The method according to any one of claims 95 to 104, further comprising contacting the cells with a guide RNA or a nucleic acid encoding it.

106. The method according to any one of claims 95 to 105, wherein the cells contact the recombinant circular RNA before contacting the enzyme or the nucleic acid encoding it.

107. The method according to any one of claims 95 to 105, wherein the cells are contacted with the enzyme or the nucleic acid encoding it, and then contacted with the recombinant circular RNA.

108. The method according to any one of claims 95 to 105, wherein the cell comes into contact with the recombinant circular RNA at approximately the same time as it comes into contact with the enzyme or the nucleic acid encoding it.

109. Cells produced by the method according to any one of claims 95 to 108.

110. A method for reprogramming cells, wherein cells (i) circular RNA encoding a reprogramming factor, (ii) Circular RNA that does not encode either a protein or a miRNA, (iii) circular or linear RNA encoding miRNA, and / or (iv) Circular or linear RNA encoding a viral protein, A method that includes bringing one or more of them into contact.

111. A method for reprogramming cells, wherein cells (i) circular RNA encoding a reprogramming factor, (ii) Circular RNA that does not encode either a protein or a miRNA, (iii) circular or linear RNA encoding miRNA, and (iv) Circular or linear RNA encoding a viral protein, A method that includes making contact with each of them.

112. A method for reprogramming cells, wherein cells (i) circular RNA encoding a reprogramming factor, (ii) circular or linear RNA encoding miRNA, and (iii) Circular or linear RNA encoding a viral protein, A method that includes making contact with each of them.

113. A method for reprogramming cells, wherein cells (i) circular RNA encoding a reprogramming factor, and (ii) Circular or linear RNA encoding miRNA, A method that includes making contact with each of them.

114. The method according to any one of claims 110 to 113, wherein either the circular RNA or the linear RNA is attached to a lipid nanoparticle.

115. The method according to any one of claims 110 to 113, wherein the reprogramming factor is one of the reprogramming factors listed in Table 1, Table 2, or Table 3.

116. The circular RNA is the recombinant circular RNA described in any one of claims 1 to 19. The method according to any one of claims 110 to 115.

117. The method according to claim 111, wherein the circular RNA that does not encode either a protein or a miRNA is circBIRC6, circCORO1c, or circMAN1A2.

118. The method according to any one of claims 111 to 113, wherein the miRNA is miR302d, miR302a, miR302c, miR302b, or miR367.

119. The method according to any one of claims 111 to 113, wherein the miRNA is miR146a, miR485, miR182, nc886, miR-155, miR526a, or miR132.

120. The method according to any one of claims 111 to 112, wherein the viral protein is B18R, E3, or K3.

121. The method according to any one of claims 111 to 112, wherein the viral protein is one of the viral proteins listed in Table 4.

122. The method according to any one of claims 111 to 112, wherein the viral proteins are B18R, E3, and K3.

123. Cells produced by the method according to any one of claims 111 to 122.

124. A composition comprising isolated somatic cells containing one or more exogenous circular RNAs encoding reprogramming factors.

125. The composition according to claim 124, wherein the somatic cells comprise one or more exogenous circular RNAs encoding reprogramming factors selected from the reprogramming factors listed in Table 1, Table 2, or Table 3.

126. The composition according to claim 124, wherein the somatic cells comprise one or more exogenous circular RNAs, each encoding a reprogramming factor selected from Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc.

127. The composition according to claim 124, wherein the somatic cells comprise six exogenous circular RNAs, each circular RNA encoding one of Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc.

128. The composition according to claim 124, wherein the somatic cell comprises one or more exogenous circular RNAs, and each of the one or more endogenous circular RNAs encodes a reprogramming factor selected from Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc.

129. The composition according to claim 124, wherein the somatic cells comprise six exogenous circular RNAs, each circular RNA encoding one of Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc.

130. The composition according to claim 124, wherein the somatic cell comprises four exogenous circular RNAs, each circular RNA encoding one of Oct3 / 4, Klf4, Sox2, and c-Myc.

131. The composition according to claim 124, wherein the somatic cells comprise four exogenous circular RNAs, each circular RNA encoding one of Oct3 / 4, Klf4, Sox2, and L-Myc.

132. The composition according to claim 124, wherein the somatic cells comprise four exogenous circular RNAs, each circular RNA encoding one of Oct3 / 4, Klf4, and Sox2.

133. The composition according to any one of claims 124 to 132, wherein the cells comprise at least one, at least two, or all three exogenous viral proteins selected from B18R, E3, and K3.

134. The cell comprises exogenous miRNA, according to any one of claims 124 to 133.

135. The composition according to any one of claims 124 to 133, wherein the cell comprises a circular RNA encoding an exogenous miRNA.

136. The composition according to claim 134 or 135, wherein the miRNA is selected from miR302a, miR302b, miR302c, miR302d, and miR367.

137. A composition comprising a converted cell, wherein the converted cell comprises one or more exogenous circular RNAs encoding a conversion factor.

138. The composition according to claim 137, wherein the differentiation factor is one of the differentiation factors listed in Table 6 or a combination of the differentiation factors.

139. The composition according to claim 137 or 138, wherein the differentiated cells are any one of the second cell types listed in Table 6.

140. The composition according to claim 137 or 138, wherein the differentiated cells are derived from a first cell type which is one of the first cell types listed in Table 6.

141. A method for reprogramming cells to produce reduced cell death compared to a method using linear RNA, comprising contacting the cells with the circular RNA described in any one of claims 1 to 19, the complex described in any one of claims 20 to 24, the vector described in any one of claims 25 to 29, or the composition described in any one of claims 30 to 33, and maintaining the cells under conditions in which the protein is expressed.

142. The method described above includes contacting the cells with a plurality of circular RNAs, each circular RNA being (i) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc, (ii) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (iii) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc, (iv) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (v) Oct3 / 4, Klf4, Sox2, and c-Myc, (vi) Oct3 / 4, Klf4, Sox2, and L-Myc, or (vii) Oct3 / 4, Klf4, and Sox2, The method according to claim 141, wherein one of the reprogramming factors is coded.

143. A method for shortening the time from reprogramming to picking, comprising contacting cells with a circular RNA according to any one of claims 1 to 19, a complex according to any one of claims 20 to 24, a vector according to any one of claims 25 to 29, or a composition according to any one of claims 30 to 33, and maintaining the cells under conditions in which the protein is expressed, wherein the time is shortened compared to a reprogramming method using linear RNA.

144. The method described above includes contacting the cells with a plurality of circular RNAs, each circular RNA being (i) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc, (ii) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (iii) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc, (iv) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (v) Oct3 / 4, Klf4, Sox2, and c-Myc, (vi) Oct3 / 4, Klf4, Sox2, and L-Myc, or (vii) Oct3 / 4, Klf4, and Sox2, The method according to claim 143, wherein one of the reprogramming factors is coded.

145. A method for reducing the number of transfections required to induce cell reprogramming compared to a method using linear RNA, comprising contacting cells with a circular RNA according to any one of claims 1 to 19, a complex according to any one of claims 20 to 24, a vector according to any one of claims 25 to 29, or a composition according to any one of claims 30 to 33, and maintaining the cells under conditions in which the protein is expressed.

146. The method described above includes contacting the cells with a plurality of circular RNAs, each circular RNA being (i) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc, (ii) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (iii) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc, (iv) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (v) Oct3 / 4, Klf4, Sox2, and c-Myc, (vi) Oct3 / 4, Klf4, Sox2, and L-Myc, or (vii) Oct3 / 4, Klf4, and Sox2, The method according to claim 145, wherein one of the reprogramming factors is coded.

147. A method for increasing the duration of protein expression in cells, comprising: contacting cells with a circular RNA according to any one of claims 1 to 19, a complex according to any one of claims 20 to 24, a vector according to any one of claims 25 to 29, or a composition according to any one of claims 30 to 33; and maintaining the cells under conditions in which the protein is expressed, wherein the duration of protein expression is increased compared to transfection of the cells with linear RNA encoding the same protein.

148. The method includes contacting the cells with a plurality of circular RNAs, each circular RNA is 、 (i) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc, (ii) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (iii) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc, (iv) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (v) Oct3 / 4, Klf4, Sox2, and c-Myc, (vi) Oct3 / 4, Klf4, Sox2, and L-Myc, or (vii) Oct3 / 4, Klf4, and Sox2, The method according to claim 147, wherein one of the reprogramming factors is coded.

149. A method for improving the efficiency of cell reprogramming, comprising contacting cells with a circular RNA according to any one of claims 1 to 19, a complex according to any one of claims 20 to 24, a vector according to any one of claims 25 to 29, or a composition according to any one of claims 30 to 33, and maintaining the cells under conditions in which the protein is expressed, wherein the efficacy of cell reprogramming is increased compared to cell reprogramming methods using linear RNA.

150. The method described above includes contacting the cells with a plurality of circular RNAs, each circular RNA being (i) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc, (ii) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (iii) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc, (iv) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (v) Oct3 / 4, Klf4, Sox2, and c-Myc, (vi) Oct3 / 4, Klf4, Sox2, and L-Myc, or (vii) Oct3 / 4, Klf4, and Sox2, The method according to claim 149, wherein one of the reprogramming factors is coded.

151. A method for increasing the number of reprogrammed cell colonies formed after reprogramming, comprising contacting cells with a circular RNA according to any one of claims 1 to 19, a complex according to any one of claims 20 to 24, a vector according to any one of claims 25 to 29, or a composition according to any one of claims 30 to 33, and maintaining the cells under conditions in which the protein is expressed, wherein the number of reprogrammed cell colonies formed after reprogramming is increased compared to a cell reprogramming method using linear RNA.

152. The method described above includes contacting the cells with a plurality of circular RNAs, each circular RNA being (i) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc, (ii) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (iii) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc, (iv) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (v) Oct3 / 4, Klf4, Sox2, and c-Myc, (vi) Oct3 / 4, Klf4, Sox2, and L-Myc, or (vii) Oct3 / 4, Klf4, and Sox2, The method according to claim 151, wherein one of the reprogramming factors is coded.

153. A method for reprogramming cells in a suspension, comprising contacting the cells in the suspension with a circular RNA according to any one of claims 1 to 19, a complex according to any one of claims 20 to 24, a vector according to any one of claims 25 to 29, or a composition according to any one of claims 30 to 33, and maintaining the cells under conditions in which the protein is expressed.

154. The method described above includes contacting the cells in a suspension with a plurality of circular RNAs, each circular RNA being (i) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc, (ii) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (iii) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc, (iv) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (v) Oct3 / 4, Klf4, Sox2, and c-Myc, (vi) Oct3 / 4, Klf4, Sox2, and L-Myc, or (vii) Oct3 / 4, Klf4, and Sox2, The method according to claim 153, wherein one of the reprogramming factors is coded.

155. The method according to claim 153 or 154, wherein the cells express CD34.

156. A method for improving the morphological maturation of a reprogrammed colony, comprising contacting cells in a suspension with a circular RNA according to any one of claims 1 to 19, a complex according to any one of claims 20 to 24, a vector according to any one of claims 25 to 29, or a composition according to any one of claims 30 to 33, and maintaining the cells under conditions in which the protein is expressed, wherein the morphological maturation is improved compared to cell reprogramming methods using linear RNA.

157. The method described above includes contacting the cells in a suspension with a plurality of circular RNAs, each circular RNA being (i) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc, (ii) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (iii) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc, (iv) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (v) Oct3 / 4, Klf4, Sox2, and c-Myc, (vi) Oct3 / 4, Klf4, Sox2, and L-Myc, or (vii) Oct3 / 4, Klf4, and Sox2, The method according to claim 156, wherein one of the reprogramming factors is coded.

158. A suspension culture comprising one or more CD34-expressing cells, wherein the CD34-expressing cells contain one or more exogenous circRNAs encoding reprogramming factors.

159. The suspension culture according to claim 158, wherein the reprogramming factor is selected from Oct3 / 4, Klf4, Sox2, Nanog, Lin28, c-Myc, and L-Myc.

160. CD34-expressing cells are, (i) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and c-Myc, (ii) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (iii) Oct3 / 4, Klf4, Sox2, Nanog, Lin28, and L-Myc, (iv) Oct3 / 4, Klf4, Sox2, Nanog, and Lin28, (v) Oct3 / 4, Klf4, Sox2, and c-Myc, (vi) Oct3 / 4, Klf4, Sox2, and L-Myc, or (vii) Oct3 / 4, Klf4, and Sox2, The suspension culture according to claim 158, comprising multiple circRNAs encoding one of the combinations of reprogramming factors.

161. (i) A container containing circular RNA encoding OCT4 and a buffer, (ii) A container containing circular RNA encoding SOX2 and a buffer, (iii) A container containing cirRNA encoding KLF4 and a buffer, and (iv) A kit including the package and its instructions.

162. A container containing circular RNA encoding c-MYC or L-MYC and a buffer solution. A container containing cirRNA encoding LIN28 and a buffer, A container containing cirRNA encoding NANOG and a buffer, or The kit according to claim 161, including a combination thereof.

163. A method for inducing mesenchymal epithelial migration (MET) of somatic cells to iPSCs, comprising contacting the somatic cells with one or more circular RNAs encoding reprogramming factors.

164. A method for inducing mesenchymal epithelial migration (MET) of somatic cells to iPSCs, comprising contacting the somatic cells with one or more circular RNAs encoding reprogramming factors.

165. A method for transdifferentiating cells, comprising contacting the cells with recombinant circular RNA containing a protein-coding sequence, wherein the protein-coding sequence encodes at least one transdifferentiating factor.

166. The method according to claim 165, wherein the at least one differentiation factor is one of MyoD, C / EBPα, C / EBPβ, Pdx1, Ngn3, Mafa, Pdx1, Hnf4α, Foxa1, Foxa2, Foxa3, Ascl1 (also known as Mash1), Brn2, Myt1l, miR-124, Brn2, Myt1l, Ascl1, Nurr1, Lmx1a, Ascl1, Brn2, Myt1l, Lmx1a, FoxA2, ​​Oct4, Sox2, Klf4 and c-Myc, Tbx5, Mef2c, Gata-4, or Mesp1.

167. The method according to claim 165, wherein the at least one differentiation factor is one of the differentiation factors listed in Table 6.

168. The method according to claim 167, wherein the cells come into contact with a circular RNA encoding a combination of differentiation factors listed in any one of the combinations shown in column B of Table 6.

169. A method for differentiating iPSCs, comprising contacting the iPSCs with a circular RNA encoding one or more differentiation factors of RORA, HLF, MYB, KLF4, ERG, SOX4, LUC, HOXA9, HOXA10, or HOXA5.

170. The method according to claim 169, wherein iPSCs differentiate into T cells.

171. Cells produced by the method according to any one of claims 165 to 170.

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