Ribozyme-mediated RNA assembly and expression

Abstract

The present invention provides compositions, systems and methods for expressing a protein of interest or a fusion protein using ribozyme-mediated cis-cleavage and trans-splicing of RNA molecules.

Description

[0001] Citations of Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 971,356, filed on February 7, 2020, the contents of which are incorporated herein by reference in their entirety. Background Art

[0003] In some cases, the expression of full-length proteins is limited due to size constraints of plasmids and vectors. For example, in therapeutic settings, some nucleic acids encoding full-length proteins exceed the packaging size of AAV, thus limiting their applicability in gene therapy settings. In addition, certain biologically and industrially relevant proteins contain large repeats, which can make expression difficult.

[0004] Therefore, there is a need in the art for improved compositions and methods for efficient protein expression. The present invention satisfies this unmet need. Summary of the Invention

[0005] In one embodiment, the present invention includes a system for producing RNA molecules encoding a protein of interest, the system comprising: a nucleic acid molecule encoding a first RNA molecule comprising a coding region encoding a first portion of the protein of interest and a 3' ribozyme; and a nucleic acid molecule encoding a second RNA molecule comprising a coding region encoding a second portion of the protein of interest and a 5' ribozyme.

[0006] In one embodiment, the 3' ribozyme catalyzes its own detachment from a first RNA molecule, thereby generating a 3'P or 2'3'cP terminus. In one embodiment, the 5' ribozyme catalyzes its own detachment from a second RNA molecule, thereby generating a 5'OH terminus. In one embodiment, the 3'P or 2'3'cP terminus is linked to the 5'OH terminus, thereby forming an RNA molecule comprising the coding region of the first RNA molecule and the coding region of the second RNA molecule. In one embodiment, the 3' ribozyme is a member of the HDV ribozyme family. In one embodiment, the 5' ribozyme is a member of the HH ribozyme family.

[0007] In one embodiment, the system further comprises one or more additional nucleic acid molecules encoding one or more additional RNA molecules, each additional RNA molecule comprising a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme.

[0008] In one embodiment, the system further comprises one or more additional nucleic acid molecules encoding one or more additional RNA molecules, each of which comprises a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme recognition sequence. In one embodiment, the system further comprises a ribozyme that interacts with the 3' ribozyme recognition sequence, wherein the ribozyme induces removal of the 3' recognition sequence. In one embodiment, the 3' ribozyme recognition sequence comprises VS-S and wherein the ribozyme is VS-Rz.

[0009] In one embodiment, the present invention relates to a method for producing an RNA molecule encoding a protein of interest, the method comprising: administering to a cell or tissue a nucleic acid molecule encoding a first RNA molecule, the first RNA molecule comprising a coding region encoding a first portion of the protein of interest and a 3' ribozyme; and administering to the cell or tissue a nucleic acid molecule encoding a second RNA molecule, the second RNA molecule comprising a coding region encoding a second portion of the protein of interest and a 5' ribozyme.

[0010] In one embodiment, the 3' ribozyme catalyzes its own detachment from a first RNA molecule, thereby generating a 3'P or 2'3'cP terminus. In one embodiment, the 5' ribozyme catalyzes its own detachment from a second RNA molecule, thereby generating a 5'OH terminus. In one embodiment, the 3'P or 2'3'cP terminus is linked to the 5'OH terminus, thereby forming an RNA molecule comprising the coding region of the first RNA molecule and the coding region of the second RNA molecule. In one embodiment, the 3' ribozyme is a member of the HDV ribozyme family. In one embodiment, the 5' ribozyme is a member of the HH ribozyme family.

[0011] In one embodiment, the method further comprises administering to the cell or tissue one or more additional nucleic acid molecules encoding one or more additional RNA molecules, each additional RNA molecule comprising a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme.

[0012] In one embodiment, the method further comprises administering to the cell or tissue one or more additional nucleic acid molecules encoding one or more additional RNA molecules, each additional RNA molecule comprising a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme recognition sequence. In one embodiment, the method further comprises administering to the cell or tissue a ribozyme that interacts with the 3' ribozyme recognition sequence, which induces the removal of the 3' recognition sequence. In one embodiment, the 3' ribozyme recognition sequence comprises VS-S and wherein the ribozyme is VS-Rz. In one embodiment, the method further comprises administering to the cell or tissue a ligase to induce the assembly of RNA molecules. In one embodiment, the ligase is an RNA 2', 3'-cyclic phosphate and 5'-OH (RtcB) ligase.

[0013] In one embodiment, the present invention includes an in vitro method for producing an RNA molecule encoding a protein of interest, the method comprising: providing a first RNA molecule comprising a coding region encoding a first portion of the protein of interest and a 3' ribozyme; providing a second RNA molecule comprising a coding region encoding a second portion of the protein of interest and a 5' ribozyme; and providing a ligase to induce assembly of the RNA molecule from the coding region of the first RNA molecule and the coding region of the second RNA molecule.

[0014] In one embodiment, the present invention includes an in vitro method for producing an RNA molecule encoding a repeat domain protein of interest, the method comprising the following steps: a) providing a first RNA molecule comprising a coding region encoding a first portion of the protein of interest and a 3' ribozyme; b) providing one or more additional RNA molecules comprising a coding region encoding a domain of the protein of interest, a 5' ribozyme, and a 3' ribozyme recognition sequence; c) providing a ligase to join the coding region of the first RNA molecule to the coding region of the one or more additional RNA molecules; d) providing a ribozyme that recognizes the 3' ribozyme recognition sequence and catalyzes the removal of the 3' ribozyme recognition sequence; e) repeating steps b)-d) one or more times to produce RNA molecules encoding multiple repeat domains; f) providing a last RNA molecule comprising a coding region encoding the last portion of the protein of interest and a 5' ribozyme; and g) providing a ligase to join the coding region of the one or more additional RNA molecules to the coding region of the last RNA molecule, thereby producing a complete RNA molecule encoding the repeat domain protein.

[0015] In one embodiment, the invention includes a method of treating a disease or condition caused by a mutation in a large protein of interest in a subject, the method comprising: administering to the subject a first nucleic acid molecule comprising a coding region encoding a first portion of the protein of interest and a 3' ribozyme; and administering to the subject a second nucleic acid molecule comprising a coding region encoding a second portion of the protein of interest and a 5' ribozyme.

[0016] In one embodiment, the disease or condition is one or more selected from the group consisting of: Duchenne muscular dystrophy; autosomal recessive polycystic kidney disease; hemophilia A; Stargardt macular degeneration; limb-girdle muscular dystrophy; DFNB9; neurosensory nonsyndromic recessive deafness; cystic fibrosis; Wilson disease; autosomal recessive Miyoshi muscular dystrophy and deafness type 9; autosomal recessive 9); Usher syndrome type 1 and autosomal recessive hearing loss type 2; autosomal recessive hearing loss type 3 and nonsyndromic hearing loss; Usher syndrome type 1; autosomal recessive hearing loss 16 (DFNB16); Meniere's disease (MD); autosomal dominant hearing loss type 12 and autosomal recessive hearing loss type 21; Usher syndrome type 1F (USH1F) and DFNB23; autosomal recessive hearing loss type 28 and nonsyndromic hearing loss; autosomal recessive hearing loss type 30 and nonsyndromic hearing loss; autosomal recessive aurispondylosis and autosomal dominant aurispondylosis; autosomal recessive hearing loss type 77 and autosomal recessive nonsyndromic sensorineural hearing loss Dfnb type; autosomal recessive nonsyndromic hearing impairment DFN B84; autosomal recessive deafness type 84B and rare hereditary forms of deafness; autosomal dominant peripheral neuropathy, myopathy, hoarseness, and hearing loss, and deafness type 4A; congenital thrombocytopenia; sensorineural hearing loss; DFNA56; HXB; autosomal dominant deafness 56; hexabrachion; epileptic encephalopathy; Timothy syndrome and long Qt syndrome type 8; X-linked retinal disorders; hyperaldosteronism; spinocerebellar ataxia type 42; primary aldosteronism; seizures and neurologic abnormalities and sinus node dysfunction and deafness; neurodevelopmental disorders; hypokalemic periodic paralysis; epilepsy; developmental and epileptic encephalopathy; Brodymyopathy; Darier disease / heart disease; von Willebrand disease and Zellweger syndrome.

[0017] In one embodiment, the invention includes a system for producing RNA molecules encoding a protein of interest and circular RNA molecules comprising a nucleic acid encoding: a first portion of the protein of interest; a synthetic intron comprising a 5' ribozyme, a cargo sequence, and a 3' ribozyme; and a second portion of the protein of interest.

[0018] In one embodiment, the protein of interest is one or more selected from the group consisting of: a therapeutic protein, a reporter protein, and a Cas9 protein.

[0019] In one embodiment, the cargo sequence is one or more selected from the group consisting of: a sequence encoding a therapeutic protein of interest, a CRISPR guide RNA sequence, a small RNA sequence, and a trans-cleaving ribozyme sequence. In one embodiment, the small RNA sequence comprises one or more selected from the group consisting of: microRNA (miRNA), Piwi interacting RNA (piRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small tRNA-derived RNA (tsRNA), small rDNA-derived RNA (srRNA), and small nuclear RNA (snRNA).

[0020] In one embodiment, the 3' ribozyme of the synthetic intron is a member of the HH ribozyme family. In one embodiment, the 5' ribozyme of the synthetic intron is one or more selected from the group consisting of: a member of the HDV ribozyme family, a member of the HDV ribozyme family, and a VS-S ribozyme recognition sequence. In one embodiment, the system further comprises one or more selected from the group consisting of: an RtcB ligase and a nucleic acid encoding an RtcB ligase.

[0021] In one embodiment, the invention includes a method for delivering RNA molecules and circular RNA molecules encoding a protein of interest, the method comprising administering to a cell or tissue a nucleic acid encoding: a first portion of the protein of interest; a synthetic intron comprising a cis-cutting 5' ribozyme, a cargo sequence, and a cis-cutting 3' ribozyme; and a second portion of the protein of interest.

[0022] In one embodiment, the protein of interest is one or more selected from the group consisting of: a therapeutic protein, a reporter protein, and a Cas9 protein.

[0023] In one embodiment, the cargo sequence is one or more selected from the group consisting of: a sequence encoding a therapeutic protein of interest, a CRISPR guide RNA sequence, a small RNA sequence, and a trans-cleaving ribozyme sequence. In one embodiment, the small RNA sequence comprises one or more selected from the group consisting of: microRNA (miRNA), Piwi interacting RNA (piRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small tRNA-derived RNA (tsRNA), small rDNA-derived RNA (srRNA), and small nuclear RNA (snRNA).

[0024] In one embodiment, the method further comprises administering to the cell or tissue one or more selected from the group consisting of: an RtcB ligase and a nucleic acid encoding an RtcB ligase. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the accompanying drawings.It should be understood that this invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0026] Figure 1 include Figure 1 A to Figure 1 E, Depicts ribozyme-mediated trans-splicing and expression in mammalian cells. Figure 1 A shows a diagram depicting vectors encoding the N-terminal (Nt) half of GFP with the HDV ribozyme at 3' and the C-terminal (Ct) half of GFP with the hammerhead (HH) ribozyme at 5'. Figure 1 B depicts exemplary results demonstrating that co-expression of Nt-GFP-HDV and HH-Ct-GFP in COS7 and HEK293T cells resulted in detectable GFP fluorescence, but not when transfected individually. Figure 1 C-1D depicts RT-PCR amplification using primers specific for each individual RNA (G1 and G2) ( Figure 1 C) and Sanger sequence analysis ( Figure 1 D) Exemplary results showing removal of the ribozyme and scarless trans-splicing and restoration of the GFP coding sequence. Figure 1 E depicts exemplary Western blot results using an antibody specific for GFP, showing the predicted full-length protein size for GFP.

[0027] Figure 2 include Figure 2 A to Figure 2 E, Depicts the development of a luciferase-based reporter gene for quantifying the effects of ribozyme sequences on trans-splicing in mammalian cells. Figure 2 A shows a diagram depicting vectors encoding the N-terminal (Nt) half of luciferase with a 3' HDV ribozyme and the C-terminal (Ct) half of luciferase with a 5' hammerhead (HH) ribozyme. Figure 2 B-2C depicts RT-PCR amplification using primers specific for each individual Luc RNA (L1 and L2) ( Figure 2 B) and Sanger sequence analysis ( Figure 2 C) Exemplary results showing removal of the ribozyme and scarless trans-splicing of the luciferase open reading frame. Figure 2D-2E shows different HDV ( Figure 2 D) and HH( Figure 2 E) Effect of ribozyme sequence on trans-splicing in mammalian cells. In addition, mutation of the catalytic nucleotide of the ribozyme results in loss of luciferase activity ( Figure 2 D, the last column and Figure 2 E, last column).

[0028] Figure 3 include Figure 3 A to Figure 3 D, shows the regulation of protein expression by Nt and Ct vectors. Figure 3 A shows a diagram depicting the placement of a C-terminal protein degradation sequence that prevents expression of Nt vector-encoded proteins. Figure 3 B depicts exemplary results demonstrating the efficiency of different protein degradation sequences in preventing the expression of GFP-HDV from Nt vectors encoding full-length GFP. Figure 3 C shows a diagram depicting the placement of N-terminal translation control sequences used to prevent translation of protein sequences in the Ct vector. Figure 3 D depicts exemplary results demonstrating the efficiency of different GFP sequence modifications or translation control sequences in preventing GFP fluorescence in mammalian cells.

[0029] Figure 4 include Figure 4 A to Figure 4 D, Demonstration of single and multiple ribozyme-mediated trans-splicing in mammalian cells. Figure 4 A shows a diagram depicting a vector encoding 4xMTS and full-length GFP (without the initiating ATG codon), where ribozymes mediate trans-splicing and expression of mitochondrial-targeted GFP protein. Figure 4 B depicts exemplary results demonstrating that co-expression of these vectors resulted in overlapping green fluorescence localized to mitochondria and red fluorescence of mitotracker CMXRos. Figure 4 C shows a diagram depicting the vectors used for multiple trans-splicing and expression of mitochondrial-targeted GFP protein in reading frame 1 (4xMTS-GFP) and myristoylated membrane-targeted red fluorescent protein in reading frame 2 (F2-Myr-RFP). Figure 4 D depicts exemplary results demonstrating that co-expression of all four vectors in mammalian Cos7 cells results in specific green fluorescence in mitochondria and red fluorescence in membranes.

[0030] Figure 5 include Figure 5 A and Figure 5 B, Demonstrates enhanced ribozyme-mediated trans-splicing using optimized ribozyme sequences and cis-splicing splice acceptor and splice donor sequences. Figure 5A shows a diagram depicting the arrangement of chimeric splice donor (SD) and splice acceptor (SA) sequences in the universal Nt-GFP-3'Rz and 5'Rz-Ct-GFP trans-splicing GFP reporter genes (where Rz represents the cis-cleaving ribozyme). Figure 5 B depicts exemplary results of GFP fluorescence in Cos7 cells after single vector transfection (first two columns) or co-transfection (last two columns) 18 hours after transfection (first three columns) or 36 hours after transfection (last column). The first row depicts the use of non-optimized HH and HDV ribozymes, the second row depicts the use of optimized Twister ribozymes and RzB ribozymes, and the last row depicts the combination of Twister ribozymes and RzB ribozymes with SD and SA sequences.

[0031] Figure 6 include Figure 6 A to Figure 6 D, Demonstrates ribozyme-mediated trans-splicing of a large protein-coding gene. Figure 6 A shows a diagram depicting a vector encoding a split μ Dystrophin-GFP fusion protein for delivery using an AAV vector. Figure 6 B-6C depicts RT-PCR performed on cells transfected with Nt-Dys and Ct-Dys vectors ( Figure 6 B) and Sanger sequencing ( Figure 6 C) Exemplary results of an analysis showing specific trans-splicing. Figure 6 D depicts exemplary results of GFP fluorescence of cells transfected with Nt and Ct dystrophin vectors imaged using confocal microscopy, showing predicted membrane localization of dystrophin.

[0032] Figure 7 include Figure 7 A to Figure 7 C, shows lentiviral delivery of ribozyme-containing RNA for trans-splicing in target cells. Figure 7 A shows a diagram depicting the negative orientation of the Nt and Ct split GFP expression cassette in the lentiviral gene transfer vector. Figure 7 B depicts exemplary results demonstrating that only cells co-transduced with lentivirus encoding Nt-GFP and Ct-GFP genes exhibited GFP fluorescence. Figure 7 C shows a diagram depicting the negative orientation of the Nt and Ct split Dys expression cassette in the lentiviral gene transfer vector.

[0033] Figure 8 include Figure 8 A and Figure 8 B, shows ribozyme-mediated trans-splicing and expression of the toxic DTA gene. Figure 8A shows a diagram depicting the vector encoding the split Nt and Ct DTA genes. Figure 8 B depicts exemplary results demonstrating that co-transfection of cells with Nt-DTA and Ct-DTA resulted in reduced expression of the co-transfected GFP reporter gene, consistent with the translational repression function of DTA in mammalian cells.

[0034] Figure 9 Depicted are exemplary results demonstrating that co-expression of exogenous RNA regulatory enzymes can enhance or inhibit ribozyme-mediated trans-splicing in mammalian cells.

[0035] Figure 10 include Figure 10 A to Figure 10 D, Demonstrates that RtcB is sufficient to catalyze ribozyme-mediated trans-splicing in vitro. Figure 10 A shows a diagram depicting a split luciferase trans-splicing reporter gene containing an upstream T7 RNA promoter to allow in vitro RNA transcription. Figure 10 B shows exemplary RT-PCR results demonstrating in vitro trans-splicing of luciferase RNA dependent on the addition of RtcB protein (NEB) using the manufacturer's recommended reaction conditions. Figure 10 C shows a diagram depicting the trans-splicing vectors of the conserved N-terminal (N1L) and C-terminal (N3R) domains of spidroin. Figure 10 D depicts exemplary Sanger sequencing results demonstrating that RtcB ligase from E. coli is sufficient to catalyze trans-ligation of ribozyme-cleaved N1L and N3R encoding RNAs.

[0036] Figure 11 Depicted are the in vitro directed ligation of RNA catalyzed by ribozymes using RtcB, VS-S, and VS-Rz.

[0037] Figure 12 include Figure 12 A to 12D, depict trans-splicing of RNA using a trans-cleaving ribozyme. Figure 12 A depicts the secondary structure of a ribozyme cleaving in cis. Figure 12 B depicts an engineered ribozyme capable of cleaving in trans. Figure 12 C and Figure 12 D depicts a diagram demonstrating the potential use of trans-cleaving ribozymes to delete disease-causing mutations (such as frameshifts or premature stop codons) to restore protein expression and function.

[0038] Figure 13 include Figure 13 A and Figure 13 B, Depicted are the secondary structures of representative ribozymes that can be used for scarless trans-splicing of RNA. Figure 13A depicts a representative ribozyme that can be used for scarless 5' cleavage. Figure 13 B depicts a representative ribozyme capable of scarless 3' cleavage. N = any nucleotide. Red scissors delineate the cleavage site. Red nucleotides indicate catalytic mutations. Orange nucleotides represent the RNA sequence to be trans-spliced. Dark blue nucleotides indicate the ribozyme sequence required for stem formation. Light blue indicates the tertiary stabilizing motif (TSM) in stem 1, which interacts with the stem 2 loop. HH – hammerhead, HDV – hepatitis delta virus, Rz – ribozyme.

[0039] Figure 14 include Figure 14 A to Figure 14 C, Depicts scarless cleavage and inducible RNA trans-splicing and expression using transactivating ribozymes. Figure 14 A depicts a diagram showing that the VS ribozyme can be split into two components: a small VS-S stem-loop that lacks autocatalytic activity and a larger VS-Rz that induces VS-S cleavage when delivered in trans. This VS-S / VS-Rz ribozyme pair can be used to generate inducible scarless trans-splicing. Figure 14 B shows a diagram depicting a method for generating an inducible RNA trans-splicing system using the VS-S / VS-Rz transactivating ribozyme pair. Only after delivery or expression of VS-Rz does the Nt-GFP-VS-S RNA generate appropriate RNA ends that can participate in trans-splicing using the co-expressed Ct-GFP RNA. Figure 14 C shows a diagram depicting a method for generating RNAs having an N-terminal sequence, a variable or non-variable repeat region, and a C-terminal sequence. The "repeat" RNA contains a 5' autocatalytic ribozyme and a 3' transactivating ribozyme (such as VS-S) that allows for controlled repeat addition based on the selective addition of the transactivating VS-Rz and a ligase (such as RtcB).

[0040] Figure 15 include Figure 15 A to Figure 15 E, Depicts ribozyme-mediated trans-splicing and the generation of stable intronic RNA sequences. Figure 15 A shows a diagram depicting the use of a cis-cutting ribozyme to mediate trans-splicing of two independent RNAs. Figure 15 B shows a diagram depicting the use of internal cis-cutting ribozymes to generate synthetic introns. Figure 15 C depicts exemplary results demonstrating efficient cis-cleavage of a synthetic intron and trans-splicing of an independent RNA to produce a functional protein (GFP). Figure 15 D and Figure 15E shows a diagram depicting the use of an internal cis-cutting ribozyme to generate a trans-spliced ​​and translated reporter gene and intron sequence "cargo" (which can be any useful RNA sequence or gene expression cassette).

[0041] Figure 16 include Figure 16 A to Figure 16 C, depicts exemplary results of optimized ribozyme sequences for in vivo ribozyme-mediated trans-splicing. Figure 16 A depicts a comparison of relative ribozyme activities using a luciferase trans-splicing reporter gene. RzB hammerhead ribozyme variants containing a tertiary stabilization motif and active at low magnesium concentrations exhibited maximal luciferase activity in mammalian cells. Figure 16 B depicts a comparison of HDV ribozymes (HDV68 and genomic HDV) and the Twister ribozyme (Twst). The Twister ribozyme located at the 3' end of Nt-Luc provided maximal luciferase activity, which was abolished by a catalytically inactive mutation (Twst mut). Figure 16 C depicts a comparison of Twister ribozyme sequence modifications. Shortening of the P1 stem reduces reporter gene activity. Modification of the first residue reveals that the Twister ribozyme can tolerate an A nucleotide at position 1 (U1A). DETAILED DESCRIPTION

[0042] definition

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0044] Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, nucleic acid chemistry and hybridization are those well known and commonly used in the art.

[0045] Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to the conventional methods in the art and throughout the various general references provided herein (e.g., Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, and Ausubel et al., 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

[0046] The nomenclature used herein and the laboratory procedures in analytical chemistry and organic syntheses described below are those well known and commonly employed in the art. Standard techniques or their modifications were used for chemical syntheses and chemical analyses.

[0047] The terms "a," "an," "the," and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0048] As used herein, "about" when referring to a measurable value such as an amount, duration, etc., is intended to encompass variations of ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% of the specified value, as such variations are applicable to performing the disclosed methods.

[0049] "Antisense" refers specifically to a nucleic acid sequence of the non-coding strand of a double-stranded DNA molecule encoding a protein, or a sequence that is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double-stranded DNA molecule encoding a protein. An antisense sequence need not be complementary only to the coding portion of the coding strand of a DNA molecule. An antisense sequence can be complementary to a specified regulatory sequence on the coding strand of a DNA molecule encoding a protein, which regulatory sequence controls the expression of the coding sequence.

[0050] As used herein, the term "attached" when referring to the immobilization of a molecule (eg, a nucleic acid molecule) to a solid support is intended to encompass direct or indirect, covalent or non-covalent attachment, unless expressly or context indicates otherwise.

[0051] As used interchangeably herein, "microspheres," "beads," or grammatical equivalents thereof, describe small, discrete particles capable of serving as solid supports for the attachment of biomolecules (eg, nucleic acid molecules).

[0052] "Disease" is a health condition in an animal in which the animal cannot maintain homeostasis and in which, if the disease does not improve, the animal's health continues to deteriorate.

[0053] In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is less than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily result in a further decline in the animal's health.

[0054] A disease or condition is "relieved" if the severity of the signs or symptoms of the disease or condition, the frequency with which the patient experiences such signs or symptoms, or both, is reduced.

[0055] "Coding" refers to the inherent properties of a specific nucleotide sequence (such as a gene, cDNA or mRNA) in a polynucleotide, which is used to serve as a template for synthesizing other polymers and macromolecules with a determined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a determined amino acid sequence and the resulting biological properties in a biological process. Therefore, if the transcription and translation of the mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene encodes a protein. Coding strand (its nucleotide sequence is identical to the mRNA sequence, and is generally provided in a sequence table) and non-coding strand (as a template for gene or cDNA transcription) can be referred to as encoding a protein or other product of the gene or cDNA.

[0056] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cell suitable for the methods described herein, whether in vitro or in vivo. In one embodiment, the subject includes vertebrates and invertebrates. Invertebrates include, but are not limited to, Drosophila melanogaster and Caenorhabditis elegans. Vertebrates include, but are not limited to, primates, rodents, domestic animals, or game animals. Primates include, but are not limited to, chimpanzees, crab-eating macaques, spider monkeys, and macaques (e.g., rhesus macaques). Rodents include, but are not limited to, mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic animals and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffaloes, feline species (e.g., house cats), canine species (e.g., dogs, foxes, wolves), bird species (e.g., chickens, emu, ostriches), and fish (e.g., zebrafish, trout, catfish, and salmon). In some embodiments, the subject is a mammal, such as a primate, such as a human. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0057] As used herein, the term "specifically binds" with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, this cross-species reactivity itself does not change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, this cross-reactivity itself does not change the specific classification of the antibody.

[0058] In some cases, the term "specific binding" or "specifically binds" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical substance to indicate that the interaction is dependent on a specific structure (e.g., an antigenic determinant or epitope) present on the chemical substance; for example, the antibody recognizes and binds to a specific protein structure, rather than proteins in general. If the antibody is specific for epitope "A," then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0059] The "coding region" of a gene is composed of nucleotide residues of the coding strand of the gene and nucleotides of the non-coding strand of the gene that are homologous or complementary to the coding region of the mRNA molecule transcribed from the gene.

[0060] The "coding region" of an mRNA molecule also consists of the nucleotide residues of the mRNA molecule that match the anticodon region of the transfer RNA molecule or encode a stop codon during translation of the mRNA molecule. Thus, the coding region can include nucleotide residues that contain codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0061] As used herein to refer to "complementarity" of nucleic acids, it refers to the broad concept of sequence complementarity between the regions of two nucleic acid chains or between two regions of the same nucleic acid chain. It is known that if the residue is thymine or uracil, the adenine residues of the first nucleic acid region can form specific hydrogen bonds ("base pairing") with the residues of the second nucleic acid region that is antiparallel to the first region. Similarly, it is known that the cytosine residues of the first nucleic acid chain can pair with the residues of the second nucleic acid chain, and if the residue is guanine, the second nucleic acid chain is antiparallel to the first chain. The first region of a nucleic acid is complementary to the second region of the same or different nucleic acids under the following circumstances: if when the two regions are arranged in an antiparallel manner, at least one nucleotide residue in the first region can pair with the residues in the second region. In one embodiment, the first region includes a first portion and the second region includes a second portion, whereby, when the first portion and the second portion are arranged in an antiparallel manner, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues in the first portion can base pair with the nucleotide residues in the second portion. In one embodiment, all nucleotide residues in the first portion are capable of base pairing with nucleotide residues in the second portion.

[0062] The term "DNA" as used herein is defined as deoxyribonucleic acid.

[0063] The term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0064] As used herein, the term "expression vector" refers to a vector containing a nucleic acid sequence that encodes at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, siRNA, ribozymes, etc. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possible translation of an operably linked coding sequence in a specific host organism. In addition to control sequences for regulating transcription and translation, vectors and expression vectors can also contain nucleic acid sequences with other functions.

[0065] As used herein, the term "wild type" is a term understood by those skilled in the art and means the typical form of an organism, strain, gene or trait as found in nature, as distinguished from mutant or variant forms.

[0066] The term "homology" refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). Homology is often measured using sequence analysis software (e.g., sequence analysis software packages from Genetics Computer Group. (University of Wisconsin Biotechnology Center. 1710 University Avenue. Madison, Wis. 53705)). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, insertions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0067] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in the normal environment of a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural environment is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-natural environment, such as a host cell.

[0068] The term "isolated" when used in relation to nucleic acids, such as "isolated oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid sequence that has been identified and separated from at least one contaminant normally associated with its source. Thus, an isolated nucleic acid exists in a form or environment different from that in which it is found in nature. In contrast, non-isolated nucleic acids (e.g., DNA and RNA) are found in the state in which they exist in nature. For example, a given DNA sequence (e.g., a gene) is found adjacent to adjacent genes on a host cell chromosome; an RNA sequence (e.g., a specific mRNA sequence encoding a specific protein) is found in a cell as a mixture with many other mRNAs encoding numerous proteins. However, an isolated nucleic acid includes, for example, a nucleic acid in a cell that normally expresses the nucleic acid, that is located at a chromosomal location different from that of the natural cell, or that is otherwise flanked by a nucleic acid sequence different from that found in nature. An isolated nucleic acid or oligonucleotide can exist in single-stranded or double-stranded form. When isolated nucleic acids or oligonucleotides are used to express a protein, the oligonucleotide comprises at least the sense or coding strand (ie, the oligonucleotide can be single-stranded), but can comprise both sense and antisense strands (ie, the oligonucleotide can be double-stranded).

[0069] The term "isolated," when used in relation to a polypeptide, as in "isolated protein" or "isolated polypeptide," refers to a polypeptide that has been identified and separated from at least one contaminant normally associated with its source. Thus, an isolated polypeptide exists in a form or environment different from that in which it is found in nature. In contrast, non-isolated polypeptides (e.g., proteins and enzymes) are found in the state in which they occur in nature.

[0070] By "nucleic acid" is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, imidoacetate, carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate, or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil). The term "nucleic acid" generally refers to large polynucleotides.

[0071] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction.

[0072] The direction in which nucleotides are added to the nascent RNA transcript, from 5' to 3', is called the direction of transcription. The DNA strand with the same sequence as the mRNA is called the "coding strand." The sequence on the DNA strand 5' from a reference point on the DNA is called the "upstream sequence," and the sequence on the DNA strand 3' from the reference point on the DNA is called the "downstream sequence."

[0073] "Expression cassette" means a nucleic acid molecule comprising a coding sequence operably linked to promoter / regulatory sequences required for the transcription and, optionally, translation of the coding sequence.

[0074] As used herein, the term "operably linked" refers to the linkage of nucleic acid sequences that results in a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule. The term also refers to the linkage of amino acid encoding sequences that results in a functional (e.g., enzymatically active, capable of binding to a binding partner, capable of inhibition, etc.) protein or polypeptide.

[0075] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, the sequence may be a core promoter sequence, while in other cases, the sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be a sequence that expresses a gene product in an inducible manner.

[0076] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity with a target sequence primarily hybridizes to the target sequence and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and depend on many factors. Generally speaking, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology - Hybridization With Nucleic Acid Probes Part 1, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY.

[0077] "Hybridization" refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonding between the bases of the nucleotide residues. This hydrogen bonding can occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may include two chains that form a duplex structure, three or more chains that form a multi-chain complex, a single self-hybridizing chain, or any combination of these. A hybridization reaction can constitute a step in a broader process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence that is capable of hybridizing to a given sequence is referred to as the "complementary sequence" of that given sequence.

[0078] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced essentially only in the presence of an inducer corresponding to the promoter.

[0079] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions in that cell.

[0080] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. It is common knowledge among those skilled in the art that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant methods, i.e., cloning nucleic acid sequences from a recombinant library or a cell genome using conventional cloning techniques and PCR, as well as synthetic methods.

[0081] In the context of the present invention, the following abbreviations for commonly used nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0082] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids and there is no limit on the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) and longer chains (which are generally referred to in the art as proteins), of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0083] The term "RNA" as used herein is defined as ribonucleic acid.

[0084] As used herein, the term "ribozyme" refers to an RNA molecule that can act as an enzyme. For example, some ribozymes can cut RNA molecules. RNA-cleaving ribozymes generally consist of at least a catalytic domain and a recognition sequence recognized by the catalytic domain. The catalytic domain can be a portion of an RNA molecule identical to the recognition sequence, thereby mediating cis-cleavage. Alternatively, the catalytic domain can be an RNA molecule separate from the RNA molecule comprising the recognition sequence, thereby mediating trans-cleavage.

[0085] "Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally linked together. The amplified or assembled recombinant polynucleotide can be contained in a suitable vector, and the vector can be used to transform a suitable host cell.

[0086] A recombinant polynucleotide may also have non-coding functions (eg, promoter, origin of replication, ribosome binding site, etc.).

[0087] The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by the use of recombinant DNA methodologies.

[0088] As used herein, the terms "solid surface," "solid support," and other grammatical equivalents thereof refer to any material that is or can be modified to be suitable for the attachment of biomolecules (eg, nucleic acid molecules).

[0089] As used herein, the term "tag" refers to any chemical modification of a biomolecule (eg, a nucleic acid molecule) that provides additional functionality (eg, attachment to a solid support, fluorescent visualization, etc.).

[0090] As used herein, the term "variant" is a nucleic acid sequence or peptide sequence that is different in sequence from a reference nucleic acid sequence or peptide sequence but retains the basic biological properties of the reference molecule. Changes in the nucleic acid variant sequence may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Changes in peptide variant sequences are generally limited or conservative, so the sequences of the reference peptide and variant are very similar overall and are identical in many regions. The amino acid sequences of variants and reference peptides may differ by one or more substitutions, additions, deletions in any combination. The variant of a nucleic acid or peptide may be naturally occurring (such as an allelic variant), or may be a variant for which it is not yet known whether it occurs naturally. Non-naturally occurring variants of nucleic acids and peptides can be prepared by mutagenesis techniques or by direct synthesis.

[0091] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as including non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0092] Range: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that descriptions in range format are for convenience and brevity only and should not be construed as rigid limitations on the scope of the invention. Thus, descriptions of ranges should be considered to have specifically disclosed all possible subranges and individual numbers within that range. For example, descriptions of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies to any breadth of range.

[0093] Details

[0094] The present invention provides compositions and methods for effectively and reliably linking two or more independent RNA molecules to produce larger single RNA molecules of coded proteins and fusion proteins. The present invention utilizes the trans-splicing of ribozyme-mediated multiple RNA molecules to assemble single RNA molecules encoding target proteins or fusion proteins. The present invention can be used for effectively producing fusion proteins, chimeric proteins, etc. Further, the present invention can be used for producing large full-length proteins, and its coding sequence may be too large to be packaged into a single vector. Further, the technology of the present invention also allows fast and easy combination of two different sequences, which can have a multiplier effect (multiplier effect) for producing new protein combinations or library sequences. This may, for example, be particularly useful for producing synthetic antibodies (such as nano antibodies) or for the functional selection of enzymes.

[0095] The present invention also provides compositions and methods for effectively delivering one or more RNA molecules having synthetic introns flanked by ribozymes. The synthetic intron flanked by ribozymes can be placed between a first RNA portion encoding the N-terminal portion of a target protein and a second RNA portion encoding the C-terminal portion of the target protein. The synthetic intron flanked by ribozymes can contain a cargo sequence, for example, a sequence encoding a therapeutic protein or comprising a functional RNA. The use of two ribozymes allows cis-splicing to produce the following three RNA fragments: 1) a first RNA portion encoding the N-terminal portion of the target protein, 2) a synthetic intron flanked by ribozymes, and 3) a second RNA portion encoding the C-terminal portion of the target protein. The cis-splicing produces compatible ends for ligation. The ligation of the compatible ends of the cis-spliced ​​synthetic introns produces a circular RNA molecule, which is more resistant to degradation than linear RNA molecules. The ligation of the compatible ends of the first RNA portion encoding the N-terminal portion of the target protein and the second RNA portion encoding the C-terminal portion of the target protein produces an RNA molecule encoding the full-length target protein. The full-length protein of interest can be, for example, a therapeutic protein, a CRISPR-Cas protein, or a reporter protein used to provide a proxyindicator for the delivery and expression of a cargo sequence in a circular RNA molecule comprising a synthetic intron flanked by a ribozyme.

[0096] On the one hand, the invention provides one or more nucleic acid molecules encoding two or more RNA molecules.In certain embodiments, one or more in this RNA molecule comprise ribozyme.In one embodiment, one or more in this RNA molecule comprise coding region and ribozyme.In certain embodiments, ribozyme self-cutting breaks away from RNA molecule, thereby leaves coding region.The exemplary ribozyme that can use in the context of the present invention includes but is not limited to the member of hammerhead (HH) ribozyme, hepatitis D virus (HDV) ribozyme, Varkud satellite (VS) ribozyme, Sister ribozyme, Twister-sister ribozyme, hairpin ribozyme, Hatchet ribozyme and Pistol ribozyme family.

[0097] For example, in one embodiment, the composition comprises a nucleic acid molecule encoding a first RNA molecule, wherein the first RNA molecule comprises a coding region and a 3' ribozyme, wherein the 3' ribozyme is capable of catalyzing its own detachment from the RNA molecule, thereby leaving a coding region having a 3'P or 2'3' cyclic phosphate (cP) end. In one embodiment, the 3' ribozyme comprises an HDV ribozyme. Further, in one embodiment, the composition comprises a nucleic acid molecule encoding a second RNA molecule, wherein the second RNA molecule comprises a coding region and a 5' ribozyme, wherein the 5' ribozyme is capable of catalyzing its own detachment from the RNA molecule, thereby leaving a coding region having a 5'OH end. In one embodiment, the 5' ribozyme comprises an HH ribozyme. In some cases, a ligase connects the coding region of the first RNA molecule to the coding region of the second RNA molecule, thereby forming a longer RNA molecule encoding a protein of interest.

[0098] For example, in one embodiment, the composition comprises a first RNA molecule, wherein the first RNA molecule comprises a coding region and a 3' ribozyme, wherein the 3' ribozyme is capable of catalyzing its own detachment from the RNA molecule, thereby leaving a coding region having a 3'P or 2'3' cyclic phosphate (cP) end. In one embodiment, the 3' ribozyme comprises an HDV ribozyme. Further, in one embodiment, the composition comprises a second RNA molecule, wherein the second RNA molecule comprises a coding region and a 5' ribozyme, wherein the 5' ribozyme is capable of catalyzing its own detachment from the RNA molecule, thereby leaving a coding region having a 5'OH end. In one embodiment, the 5' ribozyme comprises an HH ribozyme. In some cases, a ligase connects the coding region of the first RNA molecule to the coding region of the second RNA molecule, thereby forming a longer RNA molecule encoding a protein of interest.

[0099] In certain embodiments, the first RNA comprises a coding region encoding a first portion of a protein of interest, and the second RNA comprises a coding region encoding a second portion of a protein of interest, such that ribozyme-mediated RNA molecule cleavage and ligase-mediated RNA molecule assembly result in the production of RNA molecules encoding proteins having the first and second portions. The present invention can be used to produce full-length proteins from a variety of RNAs, each of which comprises a coding region encoding a portion of a full-length protein. Further, the present invention can be used to produce fusion proteins comprising multiple domains, wherein each of which comprises a coding region encoding a domain of a fusion protein. For example, the present invention can be used to produce RNA molecules encoding proteins having a leader sequence, an N-terminal tag, a C-terminal tag, etc. by assembling RNA from a first RNA comprising a coding sequence encoding a leader sequence, an N-terminal tag, or a C-terminal tag and a second RNA molecule comprising a coding sequence encoding a protein.

[0100] In certain embodiments, the present invention relates to forming a single RNA molecule by three or more separate RNA molecules. For example, in some aspects, the composition comprises a nucleic acid molecule encoding a first RNA molecule, wherein the first RNA molecule comprises a coding region for an N-terminal region encoding a protein; a nucleic acid molecule encoding a second RNA molecule, wherein the second RNA molecule comprises a coding region for a C-terminal region encoding a protein; and one or more nucleic acid molecules encoding one or more additional RNA molecules, each of which comprises a coding region for a domain (e.g., a repeat domain) encoding a protein. In one embodiment, the first RNA molecule comprises a coding region encoding an N-terminal region and a 3' ribozyme, wherein the 3' ribozyme is capable of catalyzing itself from the RNA molecule, thereby leaving a coding region with a 3'P or 2'3' cyclic phosphate (cP) end. In one embodiment, the 3' ribozyme comprises an HDV ribozyme. In one embodiment, the second RNA molecule comprises a coding region encoding a C-terminal region and a 5' ribozyme, wherein the 5' ribozyme is capable of catalyzing itself from the RNA molecule, thereby leaving a coding region with a 5'OH end. In one embodiment, the 5' ribozyme comprises an HH ribozyme. In one embodiment, the additional RNA molecules each comprise a coding region encoding a protein domain, a 3' ribozyme, and a 5' ribozyme. In one embodiment, the 3' ribozyme is an HDV ribozyme. In one embodiment, the 5' ribozyme is an HH ribozyme. In some aspects, the 3' ribozyme is capable of catalyzing its own detachment from the RNA molecule and the 5' ribozyme is capable of catalyzing its own detachment from the RNA molecule, thereby leaving a coding region having a 5'OH and 3'P or 2'3'cP terminus. In one embodiment, the additional RNA molecules each comprise a coding region encoding a protein domain, a 5' ribozyme, and a 3' ribozyme recognition sequence. In some aspects, the 5' ribozyme is capable of catalyzing its own detachment from the RNA molecule, thereby leaving a coding region having a 5'OH terminus; and the 3' ribozyme recognition sequence interacts with the ribozyme to induce the 3' ribozyme recognition sequence to be spliced ​​out of the RNA molecule, thereby leaving a coding region having a 3'P or 2'3'cP terminus. In one embodiment, the 3' ribozyme recognition sequence comprises a Vsv1 sequence that interacts with the VS ribozyme. This technology can be used to generate RNA molecules encoding proteins with multiple repeat domains by sequentially adding coding regions encoding repeat domains by sequentially providing ribozymes (e.g., VS ribozymes) to interact with the 3' ribozyme recognition sequence to generate a 3'P or 2'3'cP terminus, and then ligating the coding regions to the 5'OH terminus of another coding region encoding a repeat domain. In certain aspects, the sequential addition of repeat domains can be performed on a solid substrate or support, wherein the first RNA molecule encoding the N-terminal region is bound to the substrate or support.

[0101] In some aspects, multiple RNA molecules are linked together after ribozyme-mediated generation of 5'OH and 3'P or 2'3'cP termini. In some cases, the RNA molecules are linked together by endogenous ligases present in the natural cells or tissues where RNA assembly occurs. In some cases, the methods of the present invention include the step of adding an exogenous ligase to induce the processed RNA molecules to link together. In one embodiment, the ligase is RNA 2', 3'-cyclic phosphate and 5'-OH (RtcB) ligase.

[0102] Composition

[0103] In one embodiment, the present invention relates to a composition comprising one or more nucleic acid molecules encoding one or more ribozymes. In one embodiment, the present invention comprises one or more RNA molecules comprising one or more ribozymes. In some embodiments, the one or more RNA molecules comprise at least a first RNA molecule and a second RNA molecule.

[0104] In some embodiments, the one or more ribozymes of the composition are capable of spontaneously cis-cleaving from the one or more RNA molecules. In some embodiments, the one or more ribozymes are 3' ribozymes. In some embodiments, the 3' ribozymes produce 3'P or 2'3'cP termini on the remaining one or more RNA molecules after spontaneous cis-cleavage. In some embodiments, the one or more ribozymes are 5' ribozymes. In some embodiments, the 5' ribozymes produce 5'OH termini on the remaining one or more RNA molecules after spontaneous cis-cleavage. In some embodiments, the 3'P or 2'3'cP termini and the 5'OH termini can be linked together.

[0105] In some embodiments, the first RNA molecule comprises a 3' ribozyme. In some embodiments, the 3' ribozyme is from one or more families selected from the group consisting of: hammerhead (HH) ribozyme, hepatitis D virus (HDV) ribozyme, Varkud satellite (VS) ribozyme, Twister (Twst) ribozyme, Sister ribozyme, Twister-sister (TS) ribozyme, hairpin ribozyme, Hatchet ribozyme and Pistol ribozyme, or variants or fragments thereof that have retained cis-cleavage function. In some embodiments, the 3' ribozyme comprises an overhang of one or more nucleotides. In one embodiment, the overhang comprises a nucleotide sequence that hybridizes with a sequence upstream of the 3' ribozyme in the first RNA molecule. In some embodiments, the overhang improves the efficiency of spontaneous cis-cleavage.

[0106] In some embodiments, the second RNA molecule comprises a 5' ribozyme. In some embodiments, the 5' ribozyme is from one or more families selected from the group consisting of: hammerhead (HH) ribozyme, hepatitis D virus (HDV) ribozyme, Varkud satellite (VS) ribozyme, Twister (Twst) ribozyme, Sister ribozyme, Twister-sister (TS) ribozyme, hairpin ribozyme, Hatchet ribozyme and Pistol ribozyme, or variants or fragments thereof that retain cis-cleavage function. In some embodiments, the 5' ribozyme comprises an overhang of one or more nucleotides. In one embodiment, the overhang comprises a nucleotide sequence that hybridizes with a sequence downstream of the 5' ribozyme in the second RNA molecule. In some embodiments, the overhang improves the efficiency of spontaneous cis-cleavage.

[0107] In one embodiment, the HDV ribozyme of the composition comprises one or more selected from the group consisting of HDV, HDV68, HDV67, HDV56, genHDV, and anti-HDV, or variants or fragments thereof. In one embodiment, HDV68 comprises the nucleic acid sequence of SEQ ID NO:9. In one embodiment, HDV67 comprises the nucleic acid sequence of SEQ ID NO:10. In one embodiment, HDV56 comprises the nucleic acid sequence of SEQ ID NO:11. In one embodiment, genHDV comprises the nucleic acid sequence of SEQ ID NO:12. In one embodiment, anti-HDV comprises the nucleic acid sequence of SEQ ID NO:13.

[0108] In one embodiment, the HH ribozyme comprises one or more nucleotides in the stem 1 overhang that hybridize to nucleotides of the upstream or downstream sequence of the HH ribozyme. In one embodiment, the number of nucleotides in the stem 1 overhang can be 1 or more nucleotides, 2 or more nucleotides, 4 or more nucleotides, 6 or more nucleotides, 8 or more nucleotides, 10 or more nucleotides, 12 or more nucleotides, 14 or more nucleotides, 16 or more nucleotides, 18 or more nucleotides, or 20 or more nucleotides. In one embodiment, the HH ribozyme comprising one or more nucleotide stem 1 overhangs comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118, wherein the nucleotide designated as N corresponds to a nucleotide that hybridizes to a nucleotide of the downstream sequence of the HH ribozyme. In one embodiment, the HH ribozyme has one or more nucleotides in the stem 3 overhang. In one embodiment, the HH ribozyme has a 5-nucleotide stem 3 overhang. In one embodiment, the HH ribozyme comprises the nucleic acid sequence of SEQ ID NO: 105, wherein the nucleotide designated as N corresponds to a nucleotide that hybridizes to a nucleotide of the upstream sequence of the HH ribozyme. In one embodiment, the HH ribozyme is modified in the stem 2 loop. In one embodiment, the HH ribozyme having a modified stem 2 loop comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 124, wherein the nucleotide designated as N corresponds to a nucleotide that hybridizes to a nucleotide of the downstream sequence of the HH ribozyme. In one embodiment, the HH ribozyme is modified in stem 1 to include a tertiary stabilization motif (TSM). In one embodiment, the HH ribozyme is modified in the stem 2 loop and in stem 1 to include a tertiary stabilization motif (TSM). In one embodiment, the modified HH ribozyme cleaves more efficiently in cis than the HH ribozyme. In one embodiment, the modified HH ribozyme is RzB. In one embodiment, RzB comprises the nucleic acid sequence of SEQ ID NO: 125, wherein the nucleotide designated as N corresponds to a nucleotide that hybridizes to a nucleotide of the downstream sequence of the HH ribozyme.

[0109] In one embodiment, the Twister ribozyme comprises the nucleic acid sequence of SEQ ID NO: 32. In one embodiment, the Twister ribozyme comprises one or more nucleotides in the P1 stem overhang. In one embodiment, the number of nucleotides in the P1 stem overhang can be 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more.

[0110] In one embodiment, the Twister ribozyme comprising one or more nucleotide P1 stem overhangs comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110, wherein the nucleotide designated as N corresponds to a nucleotide that hybridizes to a nucleotide of the downstream sequence of the Twister ribozyme.

[0111] In some embodiments, the one or more ribozymes of the composition are composed of a first part and a second part. In some embodiments, the first part is incorporated into the one or more RNA molecules. In some embodiments, the first part is a ribozyme recognition sequence. In some embodiments, the second part is introduced separately. In some embodiments, the cis-cleavage of the first part from the one or more RNA molecules occurs only when the first part and the second part are in contact with each other. In some embodiments, the one or more ribozymes are VS ribozymes. In one embodiment, the VS ribozyme comprises the nucleic acid sequence of SEQ ID NO: 14. In one embodiment, the first part is a VS ribozyme stem-loop (VS-S). In one embodiment, VS-S comprises the nucleic acid sequence of SEQ ID NO: 15. In one embodiment, the second part is the remainder of VS without stem-loop (VS-Rz). In one embodiment, VS-Rz comprises the nucleic acid sequence of SEQ ID NO: 16.

[0112] Ribozymes are autocatalytic RNAs that cut in cis to produce unique RNA 3' and 5' ends as described herein. However, cis-cutting ribozymes can be engineered to cut in trans so that the target RNA can be cut in a nucleotide-specific manner, thereby producing similar RNA ends. In some embodiments, the present invention includes a composition comprising a single nucleic acid molecule encoding a single RNA molecule, which comprises a trans-cutting engineered ribozyme. In one embodiment, the trans-cutting engineered ribozyme is capable of trans-cutting an independent RNA molecule. In one embodiment, the trans-cutting engineered ribozyme recognizes a specific nucleic acid sequence in an independent RNA molecule. In some embodiments, the trans-cutting engineered ribozyme targets a pathogenic mutation for deletion. In some embodiments, the pathogenic mutation is located in an exon. In some embodiments, the pathogenic mutation is located in an intron. In some embodiments, the composition comprises two trans-cutting engineered ribozymes, which target the upstream and downstream of the pathogenic mutation. In some embodiments, trans-cutting upstream and downstream of the pathogenic mutation results in the removal of the pathogenic mutation. In some embodiments, after trans-cleavage of the pathogenic mutation, the remaining portions of the gene are trans-spliced ​​together. In some embodiments, the trans-spliced ​​gene is expressed as a functional protein.

[0113] As described herein, the 3'P or 2'3'cP end and the 5'OH end of the RNA molecule that has been subjected to ribozyme-mediated cleavage can be linked together. Thus, the isolated RNA sequences encoding the separate parts of the larger full-length protein can be trans-spliced ​​together in a scarless manner to achieve expression of the full-length protein. In one embodiment, the present invention relates to a composition comprising one or more nucleic acid molecules encoding two or more parts of a protein of interest and encoding one or more ribozymes. In one embodiment, the present invention relates to a composition comprising one or more RNA molecules encoding two or more parts of a protein of interest and comprising one or more ribozymes.

[0114] In one embodiment, the one or more nucleic acid molecules encoding two or more parts of the target protein include a first nucleic acid molecule encoding the first part of the target protein and a second nucleic acid molecule encoding the second part of the target protein. In one embodiment, the first nucleic acid comprises a first RNA molecule. In one embodiment, the second nucleic acid comprises a second RNA molecule. In one embodiment, the first RNA molecule is connected to a 3' ribozyme at the 3' end. In one embodiment, the second RNA molecule is connected to a 5' ribozyme at the 5' end. In one embodiment, after cis-cleavage of the 3' and 5' ribozyme sequences, the 3'P or 2'3'cP end of the first RNA molecule is linked to the 5'OH end of the second RNA molecule, thereby generating a single RNA molecule encoding the full-length target protein. In one embodiment, the function of the full-length target protein is identical to that of the endogenously expressed full-length protein of the same sequence.

[0115] In one embodiment, the full-length protein of interest comprises a therapeutic protein. In one embodiment, the therapeutic protein comprises one or more selected from the group consisting of, but not limited to, Utrophin, Dystrophin, Dysferlin, Myoferlin, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), Coagulation Factor VIII, Fibrocystin, Retinal-Specific Phospholipid-Transporting ATPase (ABCA4), Otoferlin, Copper-Transporting ATPase 2, MYO7A, MYO15A, CDH 23, STRC, OTOG, TECTA, PCDH15, TRIOBP, MYO3A, COL11A2, LOXHD1, PTPRQ, OTOGL, MYH14, MYH9, TNC, CACNA1A, CACNA1C, CACNA1F, CACNA1H, CACNA1G, CACNA1D, CACNA1B, CACNA1S, CACNA1I, CACNA1E, ATP2A1, ATP2A2, Adcy6, FKBP12-rapamycin binding domain and Cas9. In one embodiment, the full-length protein of interest is a recombinase. In one embodiment, the recombinase is one or more selected from the group consisting of (but not limited to) the following: CRE recombinase, FLP recombinase. In one embodiment, the full-length protein of interest is a eukaryotic / prokaryotic antibiotic resistance gene product. In one embodiment, the eukaryotic / prokaryotic antibiotic resistance gene product is one or more selected from the group consisting of (but not limited to) ampicillin, kanamycin, blasticidin, puromycin, neomycin and hygromycin. In certain embodiments, the full-length protein of interest is an antibody. In one embodiment, the antibody is capable of binding to the target protein of interest. In some embodiments, the antibody is an antibody fragment, a synthetic antibody, a nanobody or a fragment or variant thereof that retains the ability to bind to the target protein. In one embodiment, the full-length protein of interest includes synthetic repeat proteins, including but not limited to those that constitute hydrogels, synthetic spider silk and collagen. In one embodiment, the synthetic repeat protein comprises one or more selected from the group consisting of, but not limited to, Spidroin, Silk, Keratin, Collagen, Elastin, Resilin, Squid Ring Teeth, beta-solenoid protein, Zinc Finger Nuclease (ZFN) and Tal Effector Nuclease (TALEN).In one embodiment, the full-length protein of interest comprises a toxic protein or antiviral protein that can inhibit the production of lentiviral particles in mammalian packaging cells. In one embodiment, the toxic protein is a cell suicide gene. In one embodiment, the cell suicide gene comprises one or more selected from the group consisting of (but not limited to) diphtheria toxin A (DTA), HSV-tk, ricin, cholera toxin, major prion protein, pertussis toxin, ectatomin, conopeptide, abrin, verotoxin, tetanospasmin, botulinum toxin, Pseudomonas exotoxin A, anthrax, saporin, and pokeweed antiviral protein (PAP). In one embodiment, the antiviral protein comprises one or more selected from the group consisting of (but not limited to) interferon-induced GTP-binding protein (MxA), myeloperoxidase (MPO), and interferon.

[0116] The N-terminal or C-terminal RNA molecule encoding a portion of the target protein may undergo translation prior to ribozyme-mediated cleavage, or when expressed alone, may result in unwanted or truncated protein expression. However, the translational control of the protein degradation sequence can be utilized to limit this unwanted expression. In one embodiment, the one or more RNA molecules of the composition comprise the nucleic acid sequence of a translational control encoding a protein degradation sequence. In one embodiment, the first RNA molecule comprises the nucleic acid sequence of a translational control encoding a protein degradation sequence. In one embodiment, the second RNA molecule comprises the nucleic acid sequence of a translational control encoding a protein degradation sequence. In some embodiments, the translational control of the protein degradation sequence prevents partial expression of the protein prior to cleavage and splicing of the ribozyme sequence. In some embodiments, the translational controller of the protein degradation sequence comprises one or more selected from the group consisting of: an hCL1-PEST sequence, an E1A-PEST sequence, removal of the poly(A) sequence of the nucleic acid, mimicking translation by a poly A tail to generate a poly K tail, deletion of the ATG stop codon, a silent mutation within the N-terminal NTG codon, a 5′UTR of the yeast GCN4 sequence encoding four small upstream ORFs that function as translation inhibitors, or a small internal fragment of the 5′UTR of the yeast GCN4 sequence. In some embodiments, the translational controller of the protein degradation sequence comprises one or more nucleic acid sequences selected from the group consisting of: SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:77, SEQ ID NO:79, and SEQ ID NO:104.In some embodiments, the translational controller of the protein degradation sequence comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, and SEQ ID NO:80.

[0117] In certain aspects, in order to further prevent unwanted or truncated protein expression, RNA nuclear localization signal can be used to prevent the cytoplasmic export and translation of unspliced ​​RNA molecules. In one embodiment, the one or more RNA molecules of the composition include a nucleic acid sequence encoding an RNA nuclear localization sequence. In one embodiment, the first RNA molecule includes a nucleic acid sequence encoding an RNA nuclear localization sequence. In one embodiment, the second RNA molecule includes a nucleic acid sequence encoding an RNA nuclear localization sequence. In one embodiment, the RNA nuclear localization sequence prevents the cytoplasmic RNA export and translation of an incomplete protein (partial protein) before the cutting and splicing of the ribozyme sequence. In one embodiment, the RNA nuclear localization sequence includes one or more nucleic acid sequences selected from the group consisting of: SEQ ID NO: 50 and SEQ ID NO: 51.

[0118] In some embodiments, the composition further comprises one or more additional RNA molecules, each comprising a coding region encoding a domain of a protein of interest; a 5' ribozyme; and a 3' ribozyme. In some embodiments, the system further comprises one or more additional nucleic acid molecules encoding one or more additional RNA molecules, each comprising a coding region encoding a domain of a protein of interest; a 5' ribozyme; and a 3' ribozyme.

[0119] In some embodiments, the composition further comprises one or more additional RNA molecules, each comprising a coding region encoding a domain of a protein of interest; a 5' ribozyme; and a 3' ribozyme recognition sequence. In some embodiments, the system further comprises one or more additional nucleic acid molecules encoding one or more additional RNA molecules, each comprising a coding region encoding a domain of a protein of interest; a 5' ribozyme; and a 3' ribozyme recognition sequence.

[0120] Pre-mRNA splicing by the spliceosome has been shown to enhance mRNA translation by depositing factors that promote the first round of translation or by promoting RNA processing and export to the cytoplasm. The addition of chimeric cis-splicing introns within the transgene has also been shown to promote transgenic protein expression. Therefore, in certain embodiments, the addition of splice donor and splice acceptor sites that are recognized and cis-spliced ​​by the spliceosome can enhance protein expression from the split precursor RNA molecule. In one embodiment, the composition comprises one or more RNA molecules that comprise a splice donor or splice acceptor sequence. In one embodiment, the first RNA molecule of the composition comprises a splice donor sequence. In one embodiment, the splice donor sequence is linked to the 3' end of the first RNA molecule after the ribozyme sequence. In one embodiment, the second RNA molecule of the composition comprises a splice acceptor sequence. In one embodiment, the splice acceptor sequence is linked to the 5' end of the second RNA molecule before the ribozyme sequence. In one embodiment, the inclusion of splice donor and splice acceptor sequences into the nucleus enhances protein expression after enzyme-mediated trans-splicing.

[0121] In one embodiment, the composition of the present invention comprises at least four nucleic acid molecules, and these nucleic acid molecules comprise at least two pairs of nucleic acid molecules. In one embodiment, at least two parts of every pair of nucleic acid molecules encoding target protein and encoding at least two ribozymes. In one embodiment, the composition comprises at least four RNA molecules, and these at least four RNA molecules comprise at least two pairs of RNA molecules. In one embodiment, at least two parts of every pair of RNA molecules encoding target protein and comprising at least two ribozymes.

[0122] In one embodiment, the at least two pairs of RNA molecules include a first pair of RNA molecules and a second pair of RNA molecules. In one embodiment, the first pair of RNA molecules includes a first RNA molecule and a second RNA molecule. In one embodiment, the second pair of RNA molecules includes a third RNA molecule and a fourth RNA molecule. In some embodiments, the third RNA molecule and the fourth RNA molecule have different open reading frames from the first RNA molecule and the second RNA molecule, such that after spontaneous cis-cleavage, the first RNA molecule or the second RNA molecule is unable to translate a full-length functional protein product from the connection with the third RNA molecule or the fourth RNA molecule.

[0123] In one embodiment, the at least two pairs of RNA molecules further include a third pair of RNA molecules. In one embodiment, the third pair of RNA molecules includes a fifth RNA molecule and a sixth RNA molecule. In some embodiments, the fifth RNA molecule and the sixth RNA molecule have different open reading frames from the first pair of RNA molecules and the second pair of RNA molecules, such that after spontaneous cis-cleavage, only the junction of the first, second, or third pair of RNA molecules can translate a full-length functional protein product.

[0124] As described herein, when one RNA contains a 3' ribozyme and the other contains a 5' ribozyme, ribozyme-mediated trans-splicing can occur between the two independent RNAs. However, when transcribed in cis within the same RNA molecule, the two ribozymes can mediate their own scarless removal. This approach similarly produces two independent RNAs with 3'-P and 5'OH ends that can undergo trans-splicing and translation in cells. Including a cargo sequence between the 3' and 5' ribozymes also creates the possibility of generating a circularized RNA molecule upon ligation.

[0125] In one embodiment, the present invention relates to a composition comprising a single nucleic acid molecule encoding two or more portions of a protein of interest and encoding one or more ribozymes. In one embodiment, the present invention relates to a composition comprising a single RNA molecule encoding two or more portions of a protein of interest and comprising one or more ribozymes.

[0126] In one embodiment, the single nucleic acid molecule encodes a first portion of an RNA, a synthetic intron, and a second portion of the RNA. In one embodiment, the synthetic intron comprises a 5' ribozyme and a 3' ribozyme. In one embodiment, the first portion of the RNA encodes the first portion of a protein of interest. In one embodiment, the second portion of the RNA encodes the second portion of the protein of interest. In one embodiment, the single nucleic acid comprises a sequence linked in the following order: (first portion of the RNA encoding the first portion of the protein of interest)-(5' ribozyme of the synthetic intron)-(3' ribozyme of the synthetic intron)-(second portion of the RNA encoding the second portion of the protein of interest). In one embodiment, the first portion of the protein of interest is the N-terminal portion of GFP. In one embodiment, the 5' ribozyme of the synthetic intron comprises HDV. In one embodiment, the first portion of the RNA and the 5' ribozyme of the synthetic intron comprise the nucleic acid sequence of SEQ ID NO: 127, wherein lowercase letters represent the 5' ribozyme sequence and uppercase letters represent the sequence encoding the N-terminal portion of GFP (see Example 4, "GFP Containing an Internal Synthetic Ribozyme Intron with and Without Cargo"). In one embodiment, the second portion of the protein of interest is the C-terminal portion of GFP. In one embodiment, the 3' ribozyme of the synthetic intron comprises HH. In one embodiment, the second portion of the RNA and the 3' ribozyme of the synthetic intron comprise the nucleic acid sequence of SEQ ID NO: 128, wherein lowercase letters represent the 3' ribozyme sequence and uppercase letters represent the sequence encoding the C-terminal portion of GFP. (See Example 4, "GFP containing an internal synthetic ribozyme intron with and without cargo").

[0127] In one embodiment, the synthetic intron comprises a cargo sequence positioned between the 5' ribozyme and the 3' ribozyme. In one embodiment, the single nucleic acid comprises sequences linked in the following order: (first portion of RNA encoding the first portion of the protein of interest)-(5' ribozyme of the synthetic intron)-(cargo sequence)-(3' ribozyme of the synthetic intron)-(second portion of RNA encoding the second portion of the protein of interest).

[0128] In one embodiment, the 5' ribozyme sequence of the synthetic intron does not require bilateral flanking sequences for activity. In one embodiment, the circular RNA generated by ligating the ends of a synthetic intron comprising a 5' ribozyme sequence that does not require bilateral flanking sequences for activity can exist in a circular form and in a recleaved linear form. In one embodiment, the ribozyme sequence is an HDV ribozyme.

[0129] In one embodiment, the 5' ribozyme sequence of the synthetic intron does require bilateral flanking sequences for activity. In one embodiment, the circular RNA generated by ligating the ends of a synthetic intron comprising a 5' ribozyme sequence that does require bilateral flanking sequences for activity can exist only in a circular form. In one embodiment, the ribozyme sequence is an HH ribozyme.

[0130] In one embodiment, the 5' ribozyme sequence of the synthetic intron is a ribozyme recognition sequence. In one embodiment, the ribozyme recognition sequence requires the addition of a trans-cleaving ribozyme for inducible cleavage. In one embodiment, the ribozyme recognition sequence comprises VS-S. In some embodiments, VS-S is encoded by a nucleic acid sequence comprising SEQ ID NO: 15. In one embodiment, the trans-cleaving ribozyme comprises VS-Rz. In some embodiments, VS-S is encoded by a nucleic acid sequence comprising SEQ ID NO: 16.

[0131] In one embodiment, self-cleavage of the 5' and 3' ribozyme sequences produces three separate RNA molecules: 1) a first segment comprising a first portion of the RNA encoding the first portion of the protein of interest; 2) a second segment comprising a synthetic intron; and 3) a third segment comprising a second portion of the RNA encoding the second portion of the protein of interest. In one embodiment, the compatible ends of the second segment are ligated to produce a circular RNA molecule comprising the synthetic intron containing the cargo sequence. In another embodiment, the first and third segments are ligated together to produce a single, full-length linear RNA molecule.

[0132] In one embodiment, the cargo sequence of the synthetic intron is one or more selected from the group consisting of: a sequence encoding a therapeutic protein of interest, a CRISPR guide RNA sequence, a small RNA sequence, and a trans-cleaving ribozyme sequence. In one embodiment, the small RNA sequence comprises one or more selected from the group consisting of: microRNA (miRNA), Piwi interacting RNA (piRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small tRNA-derived RNA (tsRNA), small rDNA-derived RNA (srRNA), and small nuclear RNA (snRNA).

[0133] In one embodiment, the single full-length linear RNA molecule encodes a full-length protein of interest. In one embodiment, the full-length protein of interest is a therapeutic protein. In one embodiment, the therapeutic protein can be, but is not limited to, one or more selected from the group consisting of: Utrophin, Dystrophin, Dysferlin, Myoferlin, Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), Coagulation Factor VIII, Fibrocystin, Retina-specific Phospholipid-Transporting ATPase (ABCA4), Otoferlin, Copper-Transporting ATPase 2, MYO7A, MYO15A, CDH23, STRC, OTOG, TECTA, PCDH15, TRIOBP, MYO3A, COL11A2, LOXHD1, PTPRQ, OTOGL, MYH14, MYH9, TNC, CACNA1A, CACNA1C, CACNA1F, CACNA1H, CACNA1G, CACNA1D, CACNA1B, CACNA1S, CACNA1I, CACNA1E, ATP2A1, ATP2A2, Adcy6, FKBP12-rapamycin binding domain, and Cas9. In one embodiment, the full-length protein of interest is a recombinase. In one embodiment, the recombinase is one or more selected from the group consisting of (but not limited to) the following: CRE recombinase, FLP recombinase. In one embodiment, the full-length protein of interest is a eukaryotic / prokaryotic antibiotic resistance gene product. In one embodiment, the eukaryotic / prokaryotic antibiotic resistance gene product is one or more selected from the group consisting of (but not limited to) the following: ampicillin, kanamycin, blasticidin, puromycin, neomycin and hygromycin. In one embodiment, the full-length protein of interest is a reporter protein. In one embodiment, the reporter protein is one or more selected from the group consisting of: green fluorescent protein (GFP), red fluorescent protein (RFP) and luciferase (Luc). In one embodiment, the reporter protein is used as a representative indicator for evaluating the delivery and expression of cargo sequences. In certain embodiments, the full-length protein of interest is an antibody. In one embodiment, the antibody is capable of binding to the target protein of interest. In some embodiments, the antibody is an antibody fragment, a synthetic antibody, a nanobody or a fragment or variant thereof that retains the ability to bind to the target protein.

[0134] In certain aspects, the technology of the present invention can be used to assemble full-length RNA viral genomes. In one embodiment, the one or more nucleic acid molecules encoding the one or more ribozymes of the present invention encode one or more portions of an RNA viral genome. In one embodiment, the one or more RNA molecules comprising the one or more ribozymes of the present invention comprise one or more portions of an RNA viral genome.

[0135] In one embodiment, the one or more nucleic acid molecules comprise a first nucleic acid molecule that encodes the first portion of an RNA viral genome and encodes a 3' ribozyme. In one embodiment, the one or more nucleic acid molecules comprise a second nucleic acid that encodes the second portion of an RNA viral genome and encodes a 5' ribozyme. In one embodiment, the one or more RNA molecules comprise a first RNA molecule that comprises the first portion of an RNA viral genome and a 3' ribozyme. In one embodiment, the one or more RNA molecules comprise a second RNA molecule that comprises the second portion of an RNA viral genome and a 5' ribozyme. In one embodiment, the composition comprises a nucleic acid or ligase that encodes a ligase. In one embodiment, after cis-cleavage by the 3' and 5' ribozymes, the first portion of the RNA viral genome and the second portion of the RNA viral genome are ligated together, thereby producing a full-length RNA viral genome. Exemplary RNA viruses include, but are not limited to, coronaviruses, paramyxoviruses, orthomyxoviruses, retroviruses, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles viruses, Newcastle disease viruses, and picornaviruses.

[0136] In some embodiments, the present invention includes a composition comprising a nucleic acid encoding a ligase. In some embodiments, the ligase mediates the joining of a 3'P or 2'3'cP end and a 5'OH end. In some embodiments, the ligase is an RNA 2', 3'-cyclic phosphate and 5'-OH (RtcB) ligase. In some embodiments, the RtcB ligase is from one or more domains of an organism selected from the group consisting of: eukaryotes, bacteria, and archaea. In some embodiments, the organism is selected from the group consisting of: humans, Escherichia coli, Deinococcus radiodurans, Pyrococcus horikoshii, Pyrococcus sp. ST04, and Pyrococcus sp. EP. In some embodiments, the nucleic acid sequence encoding the ligase is one or more selected from the group consisting of: SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92. In some embodiments, the nucleic acid sequence encoding the ligase encodes one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91.

[0137] Nucleic Acids

[0138] In some embodiments, one or more nucleic acids of the present invention comprise a nucleic acid sequence that is substantially homologous to a nucleic acid sequence as described herein. For example, in some embodiments, the nucleic acid has a degree of identity to the original nucleic acid sequence of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%.

[0139] In some embodiments, one or more nucleic acids of the invention comprise a nucleic acid sequence that is a portion of a nucleic acid sequence described herein. For example, in some embodiments, the nucleic acid has a length of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the original nucleic acid sequence.

[0140] In some embodiments, one or more nucleic acids of the invention comprise a nucleic acid sequence that is a portion of a nucleic acid sequence described herein and is substantially homologous to a nucleic acid sequence described herein. For example, in some embodiments, the nucleic acid is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99. 5%, and / or the nucleic acid has a length of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% relative to the original nucleic acid sequence.

[0141] Nucleic acids of the present invention can include any type of nucleic acid, including but not limited to DNA and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule encoding a fusion protein of the present invention, including, for example, an isolated cDNA molecule. In one embodiment, the composition comprises an isolated RNA molecule encoding a fusion protein of the present invention or a functional fragment thereof.

[0142] Nucleic acid molecules of the present invention can be modified to improve stability in serum or in the growth medium for cell culture. Modification can be added to enhance stability, functionality and / or specificity and minimize the immunostimulatory properties of nucleic acid molecules of the present invention. For example, in order to enhance stability, 3'-residues can be stabilized to prevent degradation, for example, they can be selected so that they are composed of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, pyrimidine nucleotides can be tolerated by the replacement of modified analogs (for example uridine is replaced by 2'-deoxythymidine) and do not affect the function of this molecule.

[0143] In one embodiment of the present invention, the nucleic acid molecule may contain at least one modified nucleotide analog. For example, the termini may be stabilized by incorporating modified nucleotide analogs.

[0144] Non-limiting examples of nucleotide analogs include ribonucleotides with modified sugars and / or backbones (i.e., including modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA can be modified to incorporate at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphate groups connected to adjacent ribonucleotides are replaced by modified groups (e.g., phosphorothioate groups). In exemplary sugar-modified ribonucleotides, the 2'OH- group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, wherein R is a C1-C6 alkyl, alkenyl, or alkynyl group, and the halo is F, Cl, Br, or I.

[0145] Other examples of modifications are ribonucleotides with modified nucleobases, i.e., ribonucleotides containing at least one non-natural nucleobase instead of a naturally occurring nucleobase. The base can be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8-position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O-alkylated nucleotides and N-alkylated nucleotides, such as N6-methyladenosine are suitable. It should be noted that the above modifications can be combined.

[0146] In some cases, the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-O-methyl or 2'-OH modifications of one or more nucleotides. In certain embodiments, the nucleic acid molecules of the present invention can have enhanced resistance to nucleases. In order to increase nuclease resistance, the nucleic acid molecule can comprise, for example, 2'-modified ribose units and / or thiophosphate linkages. For example, the 2'hydroxyl (OH) can be modified or replaced by many different "oxy" or "deoxy" substituents. In order to increase nuclease resistance, the nucleic acid molecules of the present invention can comprise 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino and / or thiophosphate linkages. The inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA) (e.g., 2'-4'-ethylene bridged nucleic acids), and certain core base modifications (such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications) can also increase binding affinity to the target.

[0147] In one embodiment, the nucleic acid molecule includes 2'-modified nucleotides, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA). In one embodiment, the nucleic acid molecule includes at least one 2'-O-methyl modified nucleotide, and in some embodiments, all nucleotides of the nucleic acid molecule include a 2'-O-methyl modification.

[0148] In certain embodiments, nucleic acid molecules of the invention have one or more of the following properties:

[0149] Nucleic acid agents discussed herein include otherwise unmodified RNA and DNA and RNA and DNA that have been modified, for example, to improve efficacy, and polymers of nucleoside substitutes. Unmodified RNA refers to molecules in which the components of nucleic acid (i.e., sugar, base, and phosphate moieties) are identical or substantially identical to naturally occurring components in nature or in the human body. Rare or uncommon but naturally occurring RNA is referred to in the art as modified RNA, see, for example, Limbach et al. (Nucleic Acids Res., 1994, 22: 2183-2196). Such rare or uncommon RNAs often referred to as modified RNAs are typically the result of post-transcriptional modifications and are within the scope of unmodified RNA as used herein. Modified RNA as used herein refers to molecules in which one or more components of nucleic acid components (i.e., sugar, base, and phosphate moieties) are different from one or more components present in nature or from one or more components present in the human body. Although they are referred to as "modified RNAs," they certainly include molecules that are not strictly speaking RNAs because of modification. Nucleoside surrogates are molecules in which the ribose phosphate backbone is replaced with a non-ribose phosphate construct that allows the bases to be presented in the correct spatial relationship, such that hybridization is substantially similar to that seen with the ribose phosphate backbone (e.g., an uncharged mimetic of the ribose phosphate backbone).

[0150] Modifications of the nucleic acids of the invention may occur at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobases.

[0151] carrier

[0152] The present invention also includes compositions comprising one or more vectors inserted with one or more nucleic acid molecules of the present invention. In one embodiment, the vector encodes at least two RNA molecules. In one embodiment, the system comprises at least two RNA molecules. In some embodiments, the at least two RNA molecules are encoded by the same vector. In some embodiments, the at least two RNA molecules are contained in the same vector. In one embodiment, the at least two RNA molecules comprise a first RNA molecule and a second RNA molecule.

[0153] In some embodiments, the present invention includes at least two vectors encoding at least two RNA molecules. In some embodiments, the at least two vectors comprise at least two RNA molecules. In some embodiments, the at least two vectors encode separate RNA molecules. In one embodiment, the at least two vectors comprise separate RNA molecules. In some embodiments, the at least two separate RNA molecules comprise a first RNA molecule and a second RNA molecule. In some embodiments, the first RNA molecule is encoded by a first vector and the second RNA molecule is encoded by a second vector. In some embodiments, the first RNA molecule comprises the first vector and the second RNA molecule comprises the second vector.

[0154] In some embodiments, the present invention further comprises a vector encoding one or more additional RNA molecules. In some embodiments, the present invention further comprises one or more vectors comprising one or more additional RNA molecules. In some embodiments, each additional RNA molecule comprises a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme. In some embodiments, each additional RNA molecule comprises a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme recognition sequence.

[0155] There are many suitable vectors that can be used for the present invention in this area.In short, the expression of the natural or synthetic nucleic acid encoding the fusion protein of the present invention is usually achieved by operably linking the nucleic acid encoding the fusion protein of the present invention or its part to a promoter, and incorporating the construct into the expression vector. The carrier to be used is suitable for replication and optionally integration in eukaryotic cells. Typical carriers contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0156] The vectors of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0157] The isolated nucleic acid of the present invention can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including but not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0158] In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.In some embodiments, the present invention provides the vector of the present invention.

[0159] Furthermore, many other viral-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art.

[0160] In one embodiment, the composition comprises a vector derived from an adeno-associated virus (AAV). The term "AAV vector" means a vector derived from an adeno-associated virus serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8 and AAV-9. AAV vectors have become powerful gene delivery tools for treating various diseases. AAV vectors have many features that make them very suitable for gene therapy, including lack of pathogenicity, minimal immunogenicity and the ability to transduce post-mitotic cells in a stable and effective manner. By selecting an appropriate combination of AAV serotype, promoter and delivery method, the expression of the specific gene contained in the AAV vector can be specifically targeted to one or more types of cells.

[0161] AAV vectors can have a complete or partial deletion of one or more AAV wild-type genes (preferably the rep and / or cap genes), but retain functional flanking ITR sequences. Despite the high degree of homology, different serotypes have tropisms for different tissues. The receptor for AAV1 is unknown; however, it is known that AAV1 transduces skeletal and cardiac muscle more efficiently than AAV2. Since most studies have been done with pseudotyped vectors, in which vector DNA flanked by AAV2 ITRs is packaged into capsids of the alternate serotype, it is clear that the biological differences are capsid-related rather than genome-related. Recent evidence suggests that DNA expression cassettes packaged in AAV 1 capsids are at least 1 log10 more efficient at transducing cardiomyocytes than those packaged in AAV2 capsids. In one embodiment, the viral delivery system is an adeno-associated viral delivery system. The adeno-associated virus can be serotype 1 (AAV 1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8) or serotype 9 (AAV9).

[0162] Ideal AAV fragments for assembly into vectors include cap proteins (including vp1, vp2, vp3 and hypervariable regions), rep proteins (including rep 78, rep 68, rep 52 and rep 40), and sequences encoding these proteins. These fragments can be easily used in various vector systems and host cells. Such fragments can be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, but are not limited to, AAVs with non-naturally occurring capsid proteins. Such artificial capsids can be produced by any suitable technique using a combination of selected AAV sequences (e.g., fragments of vp1 capsid proteins) and heterologous sequences, which can be obtained from different selected AAV serotypes, non-contiguous parts of the same AAV serotype, non-AAV viral sources, or non-viral sources. Artificial AAV serotypes can be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Thus, exemplary AAVs or artificial AAVs suitable for expressing one or more proteins include AAV2 / 8 (see U.S. Pat. No. 7,282,199), AAV2 / 5 (available from the National Institutes of Health), AAV2 / 9 (International Patent Publication No. WO2005 / 033321), AAV2 / 6 (U.S. Pat. No. 6,156,303), and AAVrh8 (International Patent Publication No. WO2003 / 042397), among others.

[0163] In one embodiment, the composition comprises a lentiviral vector to deliver one or more nucleic acids of the present invention. In one embodiment, the present invention includes a lentiviral vector comprising one or more RNA molecules encoding one or more target proteins. For example, vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of transgenes and propagation of transgenes in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncometaviruses (such as murine leukemia viruses) because they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0164] In certain embodiments, the vector further comprises conventional control elements that are operably linked to the transgene in a manner that allows it to be transcribed, translated, and / or expressed in cells transfected with the plasmid vector or infected with the virus produced by the present invention. As used herein, "operably linked" sequences include expression control sequences that are adjacent to the gene of interest and expression control sequences that act in trans or remotely to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, where desired, sequences that enhance secretion of the encoded product. A large number of expression control sequences, including natural, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be used.

[0165] Additional promoter elements (such as enhancers) regulate the frequency of transcription initiation. Usually, these are located in the region of 30-110bp upstream of the start site, but recently it has been shown that many promoters also contain functional elements located downstream of the start site. The spacing between the promoter elements is normally flexible, so when elements are inverted or moved relative to each other, promoter function is retained. In the thymidine kinase (tk) promoter, before activity began to decline, the spacing between the promoter elements could be increased to 50bp. Depending on the promoter, it seems that each element can work together or independently to activate transcription.

[0166] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive the high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus (Epstein-Barr virus) immediate early promoter, Rous sarcoma (Roussarcoma) virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turn off expression when such expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0167] Enhancer sequences found on vectors also regulate the expression of genes contained therein. Typically, enhancers are combined with protein factors to enhance the transcription of the gene. The enhancer may be located upstream or downstream of the gene it regulates. The enhancer may also be tissue-specific, to enhance the transcription in a particular cell or tissue type. In one embodiment, vectors of the present invention comprise one or more enhancers to promote the transcription of the gene present in the vector.

[0168] In order to assess the expression of fusion protein of the present invention, the expression vector to be introduced into the cell can also contain selectable marker gene or reporter gene or both, to promote identifying and selecting expressing cells from the cell mass seeking to be transfected by viral vector or infected. In other aspects, selectable marker can be carried on an independent DNA fragment and be used in the cotransfection program. Both selectable marker and reporter gene can be flanked by suitable regulatory sequences to enable it to be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo etc.

[0169] Reporter gene is used to identify the functionality of the cell that may be transfected and assessment control sequence. Generally speaking, reporter gene is such gene, and this gene is not present in receptor organism or tissue or is not expressed by receptor organism or tissue, and encodes such polypeptide, and the expression of this polypeptide can be proved by some easily detectable characteristics (such as enzymatic activity). The expression of reporter gene is measured at the appropriate time after DNA is introduced into the receptor cell. Suitable reporter gene can include the gene (for example, Ui-Tei et al., 2000FEBS Letters 479:79-82) of coding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene. Suitable expression system is well-known and can use known technology to prepare or commercially obtain. Generally speaking, the construct with the minimum 5 ' flanking region that demonstrates the highest level of reporter gene expression is accredited as promoter. This type of promoter region can be connected with reporter gene and can be used to the ability of transcription driven by assessment agent regulation promoter.

[0170] protein

[0171] In some embodiments, the present invention includes a composition comprising a ligase. In some embodiments, the ligase mediates the ligation of the 3'P or 2'3'cP end of an RNA molecule to the 5'OH end of an RNA molecule. In some embodiments, the ligase is RNA 2',3'-cyclic phosphate and 5'-OH (RtcB) ligase. In some embodiments, the RtcB ligase is from one or more domains of an organism selected from the group consisting of: eukaryotes, bacteria, and archaea. In some embodiments, the organism is selected from the group consisting of: humans, Escherichia coli, Deinococcus radiodurans, Pyrococcus horikoshii, Pyrococcus species ST04, and Thermococcus species EP. In some embodiments, the ligase comprises one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91.

[0172] In some embodiments, one or more proteins of the present invention comprise an amino acid sequence that is substantially homologous to an amino acid sequence described herein. For example, in some embodiments, the protein has a degree of identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% relative to the original amino acid sequence.

[0173] In some embodiments, one or more proteins of the present invention comprise an amino acid sequence that is a portion of an amino acid sequence described herein. For example, in some embodiments, the protein has a length of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% relative to the original amino acid sequence.

[0174] In some embodiments, one or more proteins of the present invention comprise an amino acid sequence that is a portion of an amino acid sequence described herein and is substantially homologous to an amino acid sequence described herein. For example, in some embodiments, the protein has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% homology to the original amino acid sequence. The degree of homology may be determined and / or the protein may have a length of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% relative to the original amino acid sequence.

[0175] Pharmaceutical composition

[0176] The present invention also encompasses the use of pharmaceutical compositions of the present invention or salts thereof for implementing the methods of the present invention. Such pharmaceutical compositions may be composed of at least one nucleic acid of the present invention or a salt thereof in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one nucleic acid of the present invention or a salt thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination thereof. The nucleic acids of the present invention may be present in the form of physiologically acceptable salts, for example in pharmaceutical compositions in combination with physiologically acceptable cations or anions as known in the art.

[0177] In one embodiment, the pharmaceutical composition useful for practicing the methods of the present invention can be administered to deliver a dose between 1 ng / kg / day and 100 mg / kg / day. In another embodiment, the pharmaceutical composition useful for practicing the methods of the present invention can be administered to deliver a dose between 1 ng / kg / day and 500 mg / kg / day.

[0178] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any other ingredients in the pharmaceutical compositions of the present invention will vary depending on the identity, size, and condition of the subject being treated and further on the route of administration of the composition. For example, the composition may contain between 0.1% and 100% (w / w) active ingredient.

[0179] Pharmaceutical compositions useful in the methods of the present invention may be suitably developed for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ocular administration or another route of administration. Compositions useful in the methods of the present invention may be applied directly to the skin or any other tissue of a mammal. Other contemplated formulations include liposomal formulations, resealed erythrocytes containing the active ingredient, and immunologically based formulations. The route or routes of administration will be readily apparent to those skilled in the art and will depend on a number of factors, including the type and severity of the disease being treated, the type and age of the veterinary or human subject being treated, and the like.

[0180] The formulations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology or later developed in the art of pharmacology. Generally speaking, such preparation methods include the steps of combining the active ingredient with a carrier or one or more other auxiliary ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single or multi-dose units.

[0181] As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject or a convenient fraction of such a dose, such as, for example, one-half or one-third of such a dose. The unit dosage form can be a single daily dose, or a single dose of multiple daily doses (e.g., about 1 to 4 times or more per day). When multiple daily doses are used, the unit dosage form of each dose can be the same or different.

[0182] In one embodiment, the compositions of the present invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions of the present invention comprise a therapeutically effective amount of the nucleic acid of the present invention and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, physiological saline, ethanol, and other pharmaceutically acceptable saline solutions, such as phosphates and organic acid salts. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0183] The carrier can be a solvent or dispersion medium, and it contains for example water, ethanol, polyol (for example glycerol, propylene glycol and liquid polyethylene glycol etc.), their suitable mixture, and vegetable oil.For example, by using a coating such as lecithin, by keeping required particle size in the case of dispersion, and by using a surfactant, suitable fluidity can be maintained. The prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal etc. In many cases, isotonic agents are included in the compositions, such as sugar, sodium chloride or polyol, such as mannitol and sorbitol. The absorption of the compositions can be extended by including the agent that delays absorption in the injectable composition, such as aluminum monostearate or gelatin. In one embodiment, the pharmaceutically acceptable carrier is not independent DMSO.

[0184] The preparations can be mixed with conventional excipients (i.e., pharmaceutically acceptable organic or inorganic carrier materials suitable for oral, vaginal, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration known in the art). The pharmaceutical formulations can be sterilized and, if necessary, mixed with adjuvants (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances). They can also be combined with other active agents, such as other analgesics, when necessary.

[0185] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulating and disintegrants; binders; lubricants; sweeteners; flavorings; colorants; preservatives; physiologically degradable compositions, such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersants or wetting agents; emulsifiers, demulcents; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0186] The compositions of the present invention may contain from about 0.005% to 2.0% of a preservative, based on the total weight of the composition. Preservatives are used to prevent deterioration due to exposure to environmental contaminants. Examples of preservatives useful according to the present invention include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidoureas, and combinations thereof. An exemplary preservative is a combination of from about 0.5% to 2.0% benzyl alcohol and from 0.05% to 0.5% sorbic acid.

[0187] In one embodiment, the composition comprises an antioxidant and a chelating agent that inhibits nucleic acid degradation. Exemplary antioxidants for some compounds are BHT, BHA, α-tocopherol, and ascorbic acid in the range of about 0.01% to 0.3% by weight of the total weight of the composition, and BHT in the range of 0.03% to 0.1% by weight. In one embodiment, the chelating agent is present in an amount of 0.01% to 0.5% by weight of the total weight of the composition. Exemplary chelating agents include edetates (e.g., disodium edetate) and citric acid in the range of about 0.01% to 0.20% by weight. In some embodiments, the chelating agent is in the range of 0.02% to 0.10% by weight of the total weight of the composition. Chelating agents can be used to chelate metal ions that may be harmful to the shelf life of the preparation in the chelating composition. Although BHT and disodium edetate are exemplary antioxidants and chelating agents for some compounds, other suitable equivalent antioxidants and chelating agents can therefore be substituted, as known to those skilled in the art.

[0188] Conventional methods can be used to prepare liquid suspensions to realize the suspension of active ingredients in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethanol, vegetable oils (such as peanut oil, olive oil, sesame oil or coconut oil), fractionated vegetable oils and mineral oils (such as liquid paraffin). Liquid suspensions can further include one or more additional ingredients, which include but are not limited to suspending agents, dispersants or wetting agents, emulsifiers, moderators, preservatives, buffers, salts, flavorings, coloring agents and sweeteners. Oily suspensions can further include thickening agents. Known suspending agents include but are not limited to sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinyl pyrrolidone, tragacanth gum, gum arabic and cellulose derivatives (such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose). Known dispersants or wetting agents include, but are not limited to, naturally occurring phospholipids (such as lecithin); condensation products of alkylene oxides with fatty acids, with long-chain aliphatic alcohols, with partial esters derived from fatty acids and hexitol, or with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifiers include, but are not limited to, lecithin and gum arabic. Known preservatives include, but are not limited to, methylparaben, ethylparaben, or n-propylparaben, ascorbic acid, and sorbic acid. Known sweeteners include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickeners for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0189] The liquid solution of active component in aqueous or oily solvent can be prepared in the mode substantially the same as liquid suspension, and major difference is that active component is dissolved in solvent, rather than being suspended in solvent.As used herein, " oily " liquid is a kind of liquid that comprises carbon-containing liquid molecule and shows polarity characteristic less than water.The liquid solution of pharmaceutical composition of the present invention can comprise each component in the component described for liquid suspension, and it should be understood that suspending agent will not necessarily contribute to the dissolving of active component in solvent.Aqueous solvent comprises for example water and isotonic saline.Oily solvent comprises for example almond oil, oily ester, ethanol, vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil), fractionated vegetable oil and mineral oil (such as liquid paraffin).

[0190] The powder and granular formulations of the pharmaceutical formulations of the present invention can be prepared using known methods. Such formulations can be directly administered to a subject and used, for example, to form tablets, fill capsules, or to prepare aqueous or oily suspensions or solutions by adding aqueous or oily vehicles thereto. Each of these formulations can further comprise one or more of a dispersant or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweeteners, flavorings, or coloring agents, can also be included in these formulations.

[0191] Pharmaceutical composition of the present invention can also be prepared, packaged or sold in the form of oil-in-water emulsion or water-in-oil emulsion.The oil phase can be vegetable oil (such as olive oil or peanut oil), mineral oil (such as liquid paraffin), or these combinations.This type of composition can further comprise one or more emulsifying agents, such as naturally occurring gum (such as gum arabic or gum tragacanth), naturally occurring phosphatide (such as soybean or lecithin phosphatide), ester or partial ester (such as sorbitan monooleate) derived from the combination of fatty acid and hexitol anhydride, and the condensation product (such as monooleic acid polyoxyethylene sorbitan ester) of this type of partial ester and ethylene oxide.These emulsions can also comprise additional ingredients, comprise for example sweetener or flavoring.

[0192] Methods of impregnating or coating materials with chemical compositions are known in the art and include, but are not limited to, methods of depositing or binding the chemical composition onto a surface, methods of incorporating the chemical composition into the structure of the material during its synthesis (i.e., such as with physiologically degradable materials), and methods of absorbing aqueous or oily solutions or suspensions into an absorbent material (with or without subsequent drying).

[0193] The administration regimen can affect what constitutes an effective amount. The therapeutic formulation can be administered to the subject before or after diagnosis of the disease. Further, several divided doses and staggered doses can be administered daily or sequentially, or the dose can be a continuous infusion or a bolus injection. Further, the dose of the therapeutic formulation can be proportionally increased or decreased according to the urgency of the therapeutic or preventive situation.

[0194] Known procedures can be used to administer the present compositions to subjects (including mammals, such as humans) at dosages and time periods that effectively prevent or treat diseases. The effective amount of the nucleic acid required to achieve the therapeutic effect can vary according to factors such as the following: the activity of the specific nucleic acid used; the time of administration; the excretion rate of the nucleic acid; the duration of treatment; other drugs, compounds, or materials used in combination with the nucleic acid; the state, age, sex, weight, condition, general health, and past medical history of the disease or illness of the subject being treated; and similar factors well-known in the medical field. The dosage regimen can be adjusted to provide the best therapeutic response. For example, several separate doses can be administered daily or the dosage can be proportionally reduced according to the urgency of the treatment situation. A non-limiting example of the effective dose range of the nucleic acid compound of the present invention is about 1 to 5,000 mg / kg body weight / day. One of ordinary skill in the art will be able to study the relevant factors and determine the effective amount of therapeutic nucleic acid without the need for excessive experiments.

[0195] In some embodiments, the nucleic acid of the present invention can be administered to the subject in a manner that is consistent with the present invention.This nucleic acid can be applied to the subject by the frequency of several times every day, or can be applied by lower frequency, for example, once a day, once a week, once every two weeks, once a month, or by even lower frequency application, for example, once every several months or even once a year or longer.Should be understood that, in non-limiting examples, the amount of nucleic acid administered every day can be applied every day, every other day, every 2 days, every 3 days, every 4 days or every 5 days.For example, with regard to application every other day, 5mg / day dosage can be started on Monday, then the first follow-up 5mg / day dosage is applied on Wednesday, then the second follow-up 5mg / day dosage is applied on Friday, and the rest can be deduced by analogy.Dosage frequency will be apparent to technical personnel, and depends on many factors, such as but not limited to the type of the disease treated and the order of severity, the type and the age of the animal etc.

[0196] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

[0197] A physician with ordinary skill in the art, such as a physician or veterinarian, can readily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start with a dosage of the nucleic acid of the invention used in the pharmaceutical composition that is lower than the level required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0198] In certain embodiments, it is particularly advantageous to formulate the nucleic acid in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as single dosages for a subject to be treated; each unit contains a predetermined quantity of a therapeutic nucleic acid in combination with the required pharmaceutical vehicle calculated to produce the desired therapeutic effect. The dosage unit form of the present invention is determined by and directly dependent on: (a) the unique characteristics of the nucleic acid and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such nucleic acids for treating a disease in a subject.

[0199] In one embodiment, the present composition is applied to the subject at a dosage of 1 to 5 times or more every day. In another embodiment, the present composition is applied to the subject with a dosage range including but not limited to once a day, once every two days, once every three days to once a week, and once every two weeks. For those skilled in the art, it is apparent that the frequency of application of the various combination compositions of the present invention will vary depending on the subject, depending on many factors, including but not limited to age, disease to be treated or illness, sex, overall health and other factors. Therefore, the present invention should not be interpreted as being limited to any specific dosage regimen, and will be determined by the attending physician to give the subject accurate dosage and composition when considering all other factors relevant to any subject.

[0200] The compositions of the present invention for administration may range from about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0201] In some embodiments, the dosage of the compositions of the invention is from about 1 mg to about 2,500 mg. In some embodiments, the dosage of the compositions of the invention used in the compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of the second composition as described herein (i.e., a drug used to treat the same disease as the disease treated by the composition of the invention or another disease) is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0202] In one embodiment, the invention relates to a packaged pharmaceutical composition comprising a container containing a therapeutically effective amount of a nucleic acid of the invention, alone or in combination with a second agent; and instructions for using the nucleic acid to treat, prevent, or alleviate one or more symptoms of a disease in a subject.

[0203] The term "container" includes any container for holding a pharmaceutical composition. For example, in one embodiment, the container is a packaging containing a pharmaceutical composition. In other embodiments, the container is not a packaging containing a pharmaceutical composition, that is, the container is a vessel containing a packaged pharmaceutical composition or an unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition, such as a box or a bottle. In addition, packaging technology is well known in the art. It should be understood that the instructions for use of the pharmaceutical composition can be included in the packaging containing the pharmaceutical composition, so that the instructions have formed an enhanced functional relationship with the packaged product. However, it should be understood that the instructions can contain information relevant to the ability of the nucleic acid to perform its intended function (for example, to treat or prevent a disease in a subject, or to deliver an imaging agent or a diagnostic agent to a subject).

[0204] Routes of administration for any of the compositions of the present invention include oral, nasal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, buccal, and intranasal), intravesical, intraduodenal, intragastric, rectal, intraperitoneal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, or administration.

[0205] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, sera, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powders or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, etc. It should be understood that the formulations and compositions useful in the present invention are not limited to the specific formulations and compositions described herein.

[0206] system

[0207] In some embodiments, the present invention relates to a system for cis-cleavage and trans-splicing of independent RNA molecules. In some embodiments, the present invention relates to a system for cis-cleavage and trans-splicing of a single RNA molecule. In some embodiments, cis-cleavage and trans-splicing of independent RNA molecules or single RNA molecule fragments produce a single RNA molecule encoding a full-length protein of interest as described herein. In some embodiments, the system comprises a ligase as described herein or a nucleic acid encoding a ligase (such as RtcB).

[0208] In one embodiment, the present invention relates to an inducible system for producing a single RNA encoding a full-length protein from two separate RNA molecules encoding a first portion and a second portion of the full-length protein by cis-cleavage by a ribozyme and trans-splicing of the two independent RNA molecules. In some embodiments, the system comprises a ribozyme recognition sequence and a ribozyme as described herein. In some embodiments, the system comprises a ligase as described herein or a nucleic acid encoding a ligase.

[0209] In one embodiment, the present invention relates to a system for assembling a full-length RNA viral genome. Exemplary RNA viruses include, but are not limited to, coronaviruses, paramyxoviruses, orthomyxoviruses, retroviruses, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles viruses, Newcastle disease viruses, and picornaviruses. In one embodiment, the system comprises a first nucleic acid encoding a first portion of the RNA viral genome and encoding a 3' ribozyme. In one embodiment, the system comprises a second nucleic acid encoding a second portion of the RNA viral genome and encoding a 5' ribozyme. In one embodiment, the system comprises the first portion of the RNA viral genome and a 3' ribozyme. In one embodiment, the system comprises the second portion of the RNA viral genome and a 5' ribozyme. In one embodiment, the system comprises a nucleic acid encoding a ligase or a ligase. In one embodiment, after cis-cleavage by the 3' and 5' ribozymes, the first portion of the RNA viral genome and the second portion of the RNA viral genome are ligated together, thereby producing a full-length RNA viral genome.

[0210] in vivo

[0211] In one embodiment, the present invention relates to a system for delivering and expressing a full-length protein by cis-cleavage and trans-splicing of independent RNA molecules encoding a portion of one or more full-length proteins. In some embodiments, the system allows for the delivery and expression of large proteins that exceed the packaging size of traditional vectors (e.g., dystrophin that exceeds the packaging size of AAV vectors), synthetic repeat domain proteins (e.g., synthetic spider silk) in which the nucleic acid construct is difficult to synthesize in vitro, or toxic / antiviral proteins (e.g., DTA). In one embodiment, the present invention includes an AAV system for delivering and expressing one or more full-length proteins of interest. In some embodiments, the system comprises a ligase or a nucleic acid encoding a ligase as described herein.

[0212] In one embodiment, the present invention includes a lentiviral delivery system for delivering one or more nucleic acid molecules encoding one or more proteins of interest. In one aspect, the lentiviral delivery system comprises (1) a packaging plasmid, (2) an envelope plasmid, and (3) a transfer plasmid. In one embodiment, the transfer plasmid encodes a first RNA molecule and a second RNA molecule.

[0213] In one embodiment, the present invention includes a dual lentiviral delivery system comprising a first lentiviral vector and a second lentiviral vector. In one embodiment, the first lentiviral vector system comprises (1) a packaging plasmid, (2) an envelope plasmid, and (3) a first transfer plasmid. In one embodiment, the second lentiviral vector system comprises (1) a packaging plasmid, (2) an envelope plasmid, and (3) a second transfer plasmid. In one embodiment, the first transfer plasmid encodes a first RNA molecule. In one embodiment, the second transfer plasmid encodes a second RNA molecule.

[0214] In one embodiment, the packaging plasmid comprises a nucleic acid sequence encoding a gag-pol polyprotein. In one embodiment, the gag-pol polyprotein comprises a catalytically dead integrase. In one embodiment, the gag-pol polyprotein comprises a D116N integrase mutation.

[0215] In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an HIV envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding a vesicular stomatitis virus g-protein (VSV-g) envelope protein. In one embodiment, the envelope protein can be selected based on the desired cell type.

[0216] In one embodiment, the first RNA molecule of a single transfer plasmid comprises a protein coding region and a 3' ribozyme encoding the first portion of a protein of interest. In one embodiment, the second RNA molecule of a single transfer plasmid comprises a protein coding region and a 5' ribozyme encoding the second portion of a protein of interest. In one embodiment, the transfer plasmid comprises a 5' long terminal repeat (LTR) sequence and a 3' LTR sequence. In one embodiment, the 3' LTR is a self-inactivating (SIN) LTR. Therefore, in one embodiment, the 5' LTR comprises a U3 sequence, an R sequence, and a U5 sequence, and the 3' LTR comprises an R sequence and a U5 sequence, but does not comprise a U3 sequence. In one embodiment, the 5' LTR and the 3' LTR are flanked by sequences encoding the first portion of the protein of interest and the second portion of the protein of interest.

[0217] In one embodiment, the first RNA molecule of the first transfer plasmid comprises a protein coding region and a 3' ribozyme encoding the first portion of the target protein. In one embodiment, the second RNA molecule of the second transfer plasmid comprises a protein coding region and a 5' ribozyme encoding the second portion of the target protein. In one embodiment, the first and second transfer plasmids comprise a 5' long terminal repeat (LTR) sequence and a 3' LTR sequence. In one embodiment, the 3' LTR is a self-inactivating (SIN) LTR. Therefore, in one embodiment, the 5' LTR comprises a U3 sequence, an R sequence, and a U5 sequence, and the 3' LTR comprises an R sequence and a U5 sequence, but does not comprise a U3 sequence. In one embodiment, the 5' LTR and 3' LTR of the first transfer plasmid flank the sequence of the first portion of the target protein and the 3' ribozyme. In one embodiment, the 5' LTR and 3' LTR of the second transfer plasmid flank the sequence of the second portion of the target protein and the 5' ribozyme.

[0218] In one embodiment, a packaging plasmid, an envelope plasmid, and a transfer plasmid are introduced into a cell. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding a gag-pol protein to produce a gag-pol polyprotein. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding an envelope protein to produce an envelope protein. In one embodiment, the cell transcribes a single transfer plasmid to provide a first RNA molecule and a second RNA molecule. In one embodiment, the cell transcribes the first transfer plasmid to provide the first RNA molecule and transcribes the second transfer plasmid to provide the second RNA molecule. In one embodiment, the gag-pol protein, the envelope polyprotein, the first RNA molecule, and the second RNA molecule are packaged into viral particles. In one embodiment, the viral particles are collected from the cell culture medium. In one embodiment, the viral particle transduces a target cell, wherein the 3' ribozyme catalyzes its own detachment from the first RNA molecule, thereby generating a 3'P or 2'3'cP terminus, the 5' ribozyme catalyzes its own detachment from the second RNA molecule, thereby generating a 5'OH terminus, an endogenous RNA 2',3'-cyclic phosphate, and 5'-OH (RtcB) ligase ligates the 3'P or 2'3'cP terminus to the 5'OH terminus, thereby generating a complete RNA molecule encoding a protein of interest, and the cell translates the protein of interest.

[0219] In one embodiment, a packaging plasmid, an envelope plasmid, and a first transfer plasmid are introduced into a cell. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding a gag-pol protein to produce a gag-pol polyprotein. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding an envelope protein to produce an envelope protein. In one embodiment, the cell transcribes the first transfer plasmid to provide a first RNA molecule. In one embodiment, the gag-pol protein, envelope polyprotein, and first RNA molecule are packaged into a first viral particle. In one embodiment, the first viral particle is collected from a cell culture medium.

[0220] In one embodiment, a packaging plasmid, an envelope plasmid, and a second transfer plasmid are introduced into a cell. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding a gag-pol protein to produce a gag-pol polyprotein. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding an envelope protein to produce an envelope protein. In one embodiment, the cell transcribes the second transfer plasmid to provide a second RNA molecule. In one embodiment, the gag-pol protein, envelope polyprotein, and second RNA molecule are packaged into a second viral particle. In one embodiment, the second viral particle is collected from the cell culture medium.

[0221] In one embodiment, the first viral particle and the second viral particle transduce a target cell, wherein the 3' ribozyme catalyzes its own detachment from the first RNA molecule, thereby producing a 3'P or 2'3'cP end, the 5' ribozyme catalyzes its own detachment from the second RNA molecule, thereby producing a 5'OH end, an endogenous RNA 2',3'-cyclic phosphate, and a 5'-OH (RtcB) ligase ligates the 3'P or 2'3'cP end to the 5'OH end, thereby producing a complete RNA molecule encoding a protein of interest, and the cell translates the protein of interest. In one embodiment, the present invention relates to a system for preventing unwanted incomplete protein expression from a split precursor RNA molecule. In one embodiment, the system comprises a translation controller that incorporates a protein degradation sequence into a split precursor RNA molecule as described herein.

[0222] In one embodiment, the present invention relates to a system for expressing two or more proteins of interest from two or more pairs of independent RNA molecules encoding portions of the proteins of interest by cis-cleavage by ribozymes and trans-splicing of the paired independent RNA molecules. In one embodiment, each individual pair of independent RNA molecules has a separate reading frame such that trans-splicing of unwanted pairs does not result in translation of a full-length functional protein as described herein. In some embodiments, the system comprises a ligase as described herein or a nucleic acid encoding a ligase.

[0223] In one embodiment, the present invention includes a system for delivering and expressing a full-length protein of interest and a cargo sequence. In one embodiment, the system comprises a first portion of an RNA encoding a first portion of the protein of interest, the first portion of the RNA being linked at its 3' end to a synthetic intron; and a second portion of an RNA encoding a second portion of the protein of interest, the second portion of the RNA being linked at its 5' end to the synthetic intron. In one embodiment, the synthetic intron is flanked on either side by a 5' ribozyme sequence and a 3' ribozyme sequence. In one embodiment, the synthetic intron comprises a cargo sequence positioned between the 5' and 3' ribozyme sequences. In one embodiment, self-cleavage of the 5' and 3' ribozyme sequences produces three separate RNA molecules: 1) a first segment comprising the first portion of the RNA encoding the first portion of the protein of interest; 2) a second segment comprising the synthetic intron; and 3) a third segment comprising the second portion of the RNA encoding the second portion of the protein of interest. In one embodiment, the compatible ends of the second segment are joined to produce a circular RNA molecule comprising the synthetic intron containing the cargo sequence. In embodiments, the first and third segments are joined together to produce a single, full-length linear RNA molecule. In one embodiment, the full-length protein of interest includes a therapeutic protein, a reporter protein, a recombinase, an antibiotic resistance gene product, an antibody, or a Cas9 protein. In one embodiment, the cargo sequence includes a therapeutic nucleic acid sequence (e.g., a miRNA sequence or a CRISPR guide RNA sequence) or encodes a therapeutic protein. In some embodiments, the full-length protein of interest includes Cas9 and the cargo sequence includes a guide RNA sequence, thereby targeting Cas9 to a specific genomic sequence for editing. In some embodiments, the system includes a ligase as described herein or a nucleic acid encoding a ligase.

[0224] In one embodiment, the present invention includes a system for gene editing, which comprises one or more trans-cutting engineered ribozymes. In some embodiments, the system comprises two trans-cutting engineered ribozymes that target the upstream and downstream of the pathogenic mutation. In some embodiments, trans-cutting upstream and downstream of the pathogenic mutation leads to the removal of the pathogenic mutation. In some embodiments, after trans-cutting of the pathogenic mutation, the remainder of the gene is trans-spliced ​​together. In some embodiments, the trans-spliced ​​gene is expressed as a functional protein. In some embodiments, the system comprises a ligase as described herein or a nucleic acid encoding a ligase.

[0225] in vitro

[0226] In one embodiment, the present invention includes an in vitro system for producing an RNA molecule encoding a protein of interest. In one embodiment, the system comprises at least two RNA molecules. In one embodiment, the at least two RNA molecules comprise a first RNA molecule and a second RNA molecule.

[0227] In one embodiment, the first RNA molecule comprises a coding region encoding a first portion of a protein of interest. In one embodiment, the first RNA molecule comprises a 3' ribozyme. In one embodiment, the first RNA molecule comprises a coding region encoding a first portion of a protein of interest and a 3' ribozyme as described herein.

[0228] In one embodiment, the second RNA molecule comprises a coding region encoding a second portion of a protein of interest. In one embodiment, the second RNA molecule comprises a 5' ribozyme. In one embodiment, the second RNA molecule comprises a coding region encoding a second portion of a protein of interest as described herein and a 5' ribozyme.

[0229] In one embodiment, the in vitro system for producing an RNA molecule encoding a protein of interest further comprises a ligase. In one embodiment, the ligase induces assembly of an RNA molecule from the coding region of a first RNA molecule and the coding region of a second RNA molecule. In one embodiment, the ligase is an RNA 2', 3'-cyclic phosphate and 5'-OH (RtcB) ligase as described herein.

[0230] In one embodiment, the invention includes an in vitro system for producing an RNA molecule encoding a repeat domain protein of interest. In one embodiment, the system comprises a first RNA molecule, one or more additional RNA molecules, and a final RNA molecule.

[0231] In one embodiment, the first RNA molecule comprises a coding region encoding a first portion of a protein of interest. In one embodiment, the first RNA molecule comprises a 3' ribozyme. In one embodiment, the first RNA molecule comprises a coding region encoding a first portion of a protein of interest and a 3' ribozyme. In one embodiment, the 3' ribozyme catalyzes its own detachment from the first RNA molecule, thereby producing a 3'P or 2'3'cP end. In one embodiment, the first RNA molecule further comprises a 5' tag. In one embodiment, the 5' tag mediates attachment of the first RNA molecule to a solid support.

[0232] In one embodiment, the one or more additional RNA molecules comprise a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme recognition sequence. In one embodiment, the 5' ribozyme cleaves itself to produce a 5' OH terminus. In one embodiment, the 3' ribozyme recognition sequence comprises a VS-S sequence as described herein.

[0233] In one embodiment, the last RNA molecule comprises a coding region encoding the last portion of the protein of interest. In one embodiment, the last RNA molecule comprises a 5' ribozyme. In one embodiment, the last RNA molecule comprises a coding region encoding the last portion of the protein of interest and a 5' ribozyme. In one embodiment, the 5' ribozyme cleaves itself to produce a 5' OH terminus.

[0234] In one embodiment, the system further comprises a ribozyme. In one embodiment, the ribozyme comprises a VS-Rz as described herein. In one embodiment, the VS-Rz recognizes VS-S and mediates cleavage of the VS-S from one or more additional RNA molecules, as described herein. In one embodiment, the cleavage produces a 3'P or 2'3'cP terminus.

[0235] In one embodiment, the system comprises a ligase. In some embodiments, the ligase ligates the 3'P or 2'3'cP end of the first RNA molecule to the 5'OH end of the one or more additional RNA molecules. In some embodiments, the ligase ligates the 3'P or 2'3'cP end of the one or more additional RNA molecules to the 5'OH end of the last RNA molecule. In some embodiments, the ligase ligates the 3'P or 2'3'cP end of the first RNA molecule to the 5'OH end of the one or more additional RNA molecules, and the 3'P or 2'3'cP end of the one or more additional RNA molecules to the 5'OH end of the last RNA molecule, thereby generating a complete RNA molecule encoding an N-terminal domain, one or more additional domains, and a C-terminal domain. In some embodiments, the ligase is an RNA 2', 3'-cyclic phosphate and 5'-OH (RtcB) ligase as described herein.

[0236] method

[0237] In some embodiments, the present invention relates to methods for cis-cleavage and trans-splicing of independent RNA molecules. In some embodiments, the present invention relates to methods for cis-cleavage and trans-splicing of a single RNA molecule. In some embodiments, cis-cleavage and trans-splicing of independent RNA molecules or single RNA molecule fragments produces a single RNA molecule encoding a full-length protein of interest as described herein. In some embodiments, the method comprises administering a ligase as described herein or a nucleic acid encoding a ligase.

[0238] In one embodiment, the present invention relates to an inducible method for producing a single RNA encoding a full-length protein from two separate RNA molecules encoding a first portion and a second portion of the full-length protein by cis-cleavage by a ribozyme and trans-splicing of the two independent RNA molecules. In some embodiments, the method comprises a ribozyme recognition sequence and a ribozyme as described herein. In some embodiments, the method comprises administering a ligase as described herein or a nucleic acid encoding a ligase.

[0239] in vivo

[0240] In one embodiment, the present invention includes a method for producing an RNA molecule encoding a protein of interest. In some embodiments, the method includes administering at least two nucleic acid molecules to a cell or tissue. In one embodiment, the at least two nucleic acid molecules include a first RNA molecule and a second RNA molecule. In some embodiments, the at least two nucleic acid molecules encode the first RNA molecule and the second RNA molecule.

[0241] In one embodiment, the first RNA molecule comprises a coding region encoding a first portion of a protein of interest. In one embodiment, the first RNA molecule comprises a 3' ribozyme. In one embodiment, the first RNA molecule comprises a coding region encoding a first portion of a protein of interest and a 3' ribozyme. In one embodiment, the 3' ribozyme catalyzes its own detachment from the first RNA molecule, thereby generating a 3'P or 2'3'cP terminus. In one embodiment, the 3' ribozyme is a member of the HDV ribozyme family.

[0242] In one embodiment, the second RNA molecule comprises a coding region encoding a second portion of a protein of interest. In one embodiment, the second RNA molecule comprises a 5' ribozyme. In one embodiment, the second RNA molecule comprises a coding region encoding a second portion of a protein of interest and a 5' ribozyme. In one embodiment, the 5' ribozyme catalyzes its own detachment from the second RNA molecule, thereby generating a 5' OH terminus. In one embodiment, the 5' ribozyme is a member of the HH ribozyme family.

[0243] In one embodiment, the 3'P or 2'3'cP terminus is linked to the 5'OH terminus to form an RNA molecule comprising the coding region of the first RNA molecule and the coding region of the second RNA molecule.

[0244] In one embodiment, the method comprises administering to a cell or tissue one or more additional nucleic acid molecules encoding one or more additional RNA molecules, each comprising a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme.

[0245] In one embodiment, the method comprises administering to a cell or tissue one or more additional nucleic acid molecules encoding one or more additional RNA molecules, each of which comprises a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme recognition sequence. In one embodiment, the 3' ribozyme recognition sequence comprises VS-S. In one embodiment, the ribozyme is VS.

[0246] In one embodiment, the method comprises administering to the cell or tissue one or more selected from the group consisting of: a nucleic acid molecule encoding a ligase and a ligase. In one embodiment, the ligase induces assembly of an RNA molecule from the coding region of a first RNA molecule and the coding region of a second RNA molecule. In one embodiment, the ligase is an RNA 2', 3'-cyclic phosphate and 5'-OH (RtcB) ligase.

[0247] In some embodiments, the method comprises administering to a cell or tissue at least one AAV vector encoding a first RNA molecule comprising a protein coding region encoding a first portion of a protein of interest and a 3' ribozyme, and a second RNA molecule comprising a protein coding region encoding a second portion of a protein of interest and a 5' ribozyme. In some embodiments, the method comprises administering a ligase or a nucleic acid encoding a ligase as described herein.

[0248] In some embodiments, the method comprises administering at least two AAV vectors, including a first AAV vector and a second AAV vector. In one embodiment, the first AAV vector encodes a first RNA molecule comprising a protein coding region encoding a first portion of a protein of interest and a 3' ribozyme. In one embodiment, the second AAV vector encodes a second RNA molecule comprising a protein coding region encoding a second portion of a protein of interest and a 5' ribozyme to a cell or tissue. In some embodiments, the method comprises administering a ligase or a nucleic acid encoding a ligase as described herein.

[0249] In some embodiments, the method comprises administering to a cell or tissue at least one lentiviral vector encoding a first RNA molecule and a second RNA molecule, wherein the first RNA molecule comprises a protein coding region encoding a first portion of a protein of interest and a 3' ribozyme, and the second RNA molecule comprises a protein coding region encoding a second portion of a protein of interest and a 5' ribozyme. In some embodiments, the method comprises administering a ligase or a nucleic acid encoding a ligase as described herein.

[0250] In some embodiments, the method comprises administering at least two lentiviral vectors, including a first lentiviral vector and a second lentiviral vector. In one embodiment, the first lentiviral vector encodes a first RNA molecule comprising a protein coding region encoding a first portion of a protein of interest and a 3' ribozyme. In one embodiment, the second lentiviral vector encodes a second RNA molecule comprising a protein coding region encoding a second portion of a protein of interest and a 5' ribozyme to the cell or tissue. In some embodiments, the method comprises administering a ligase or a nucleic acid encoding a ligase as described herein.

[0251] In some embodiments, the method comprises administering to a cell or tissue at least one lentiviral vector delivery system to provide a first RNA molecule comprising a protein coding region encoding a first portion of a protein of interest and a 3' ribozyme, and a second RNA molecule comprising a protein coding region encoding a second portion of a protein of interest and a 5' ribozyme. In some embodiments, the method comprises administering a ligase or a nucleic acid encoding a ligase as described herein.

[0252] In some embodiments, the method comprises administering at least two lentiviral vector delivery systems, including a first lentiviral vector delivery system and a second lentiviral vector delivery system. In one embodiment, the first lentiviral vector delivery system provides a first RNA molecule comprising a protein coding region encoding a first portion of a protein of interest and a 3' ribozyme. In one embodiment, the second lentiviral vector delivery system provides a second RNA molecule to the cell or tissue, comprising a protein coding region encoding a second portion of a protein of interest and a 5' ribozyme. In some embodiments, the method comprises administering a ligase or a nucleic acid encoding a ligase as described herein.

[0253] In some embodiments, the method includes administering two or more delivery vehicles selected from the group consisting of: AAV vectors, lentiviral vectors, lentiviral vector delivery systems, or combinations thereof. In one embodiment, the two or more delivery vehicles include a first delivery vehicle and a second delivery vehicle. In one embodiment, the first delivery vehicle provides a first RNA molecule comprising a protein coding region and a 3' ribozyme encoding a first portion of a protein of interest. In one embodiment, the second delivery vehicle provides a second RNA molecule to a cell or tissue comprising a protein coding region and a 5' ribozyme encoding a second portion of a protein of interest. In some embodiments, the method includes administering a ligase as described herein or a nucleic acid encoding a ligase.

[0254] Methods for introducing genes into cells and expressing genes in cells are known in the art. In the case of expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred to host cells by physical, chemical, or biological means.

[0255] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). An exemplary method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0256] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and in particular retroviral vectors, have become the most widely used method for inserting genes into mammalian, such as human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0257] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0258] In the case of using non-viral delivery system, exemplary delivery vehicle is liposome. Consider using lipid preparation to introduce described nucleic acid into host cell (in vitro, in vitro or in vivo). In another aspect, described nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of liposome, dispersed in the lipid bilayer of liposome, attached to liposome by the connecting molecule associated with liposome and oligonucleotide, embedded in liposome, compounded with liposome, dispersed in the solution containing lipid, mixed with lipid, combined with lipid, included in lipid as suspension, included or compounded together with micelle, or otherwise associated with lipid. The composition associated with lipid, lipid / DNA or lipid / expression vector is not limited to any specific structure in solution. For example, they may be present in double-layer structure in micellar form, or exist with " collapse " structure. They can also be simply dispersed in solution, thereby may form aggregates of uneven size or shape. Lipid is a fatty substance, and it can be naturally occurring lipid or synthetic lipid. For example, lipids include the fat droplets naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0259] Suitable lipids can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dioctyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term that covers a variety of unilamellar and multilamellar lipid vehicles formed by creating closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure containing a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure and entrain water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having structures different from normal vesicle structures in solution are also encompassed. For example, lipids may present micellar structures or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0260] Regardless of the method used to introduce exogenous nucleic acid into the host cell, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blotting) or by assays described herein to identify agents falling within the scope of the present invention.

[0261] In one embodiment, the present invention relates to a method for expressing two or more proteins of interest from two or more pairs of independent RNA molecules encoding a portion of the protein of interest by cis-cleavage of a ribozyme and trans-splicing of the paired independent RNA molecules. In one embodiment, the method includes administering one, two, or three pairs of nucleic acid molecules encoding or comprising RNA molecules, wherein each single pair of independent RNA molecules has a separate reading frame so that the trans-splicing of unwanted pairs does not result in translation of full-length functional proteins. In one embodiment, the method further includes administering to cells or tissues one or more of the following groups: nucleic acid molecules encoding a ligase and a ligase. In one embodiment, the ligase is an RNA 2', 3'-cyclic phosphate and a 5'-OH (RtcB) ligase as described herein.

[0262] In one embodiment, the present invention includes a method for delivering and expressing a full-length protein of interest and a cargo sequence. In one embodiment, the method comprises administering to a cell or tissue a first portion of an RNA encoding a first portion of the protein of interest, the first portion of the RNA being linked at its 3' end to a synthetic intron; and a second portion of an RNA encoding a second portion of the protein of interest, the second portion of the RNA being linked at its 5' end to the synthetic intron. In one embodiment, the synthetic intron is flanked on either side by a 5' ribozyme sequence and a 3' ribozyme sequence. In one embodiment, the synthetic intron comprises a cargo sequence positioned between the 5' and 3' ribozyme sequences. In one embodiment, self-cleavage of the 5' and 3' ribozyme sequences produces three separate RNA molecules: 1) a first segment comprising the first portion of the RNA encoding the first portion of the protein of interest; 2) a second segment comprising the synthetic intron; and 3) a third segment comprising the second portion of the RNA encoding the second portion of the protein of interest. In one embodiment, the compatible ends of the second segment are joined to produce a circular RNA molecule comprising the synthetic intron containing the cargo sequence. In one embodiment, the first and third segments are joined together to produce a single, full-length linear RNA molecule. In one embodiment, the full-length protein of interest includes a therapeutic protein, a reporter protein, a recombinase, an antibiotic resistance gene product, an antibody, or a Cas9 protein. In one embodiment, the cargo sequence includes a therapeutic nucleic acid sequence (e.g., a miRNA sequence or a CRISPR guide RNA sequence) or encodes a therapeutic protein. In some embodiments, the full-length protein of interest includes Cas9 and the cargo sequence includes a guide RNA sequence, thereby targeting Cas9 to a specific genomic sequence for editing. In some embodiments, the method includes administering a ligase as described herein or a nucleic acid encoding a ligase to a cell or tissue.

[0263] In one embodiment, the present invention includes a gene editing method comprising one or more trans-cutting engineered ribozymes. In some embodiments, the method comprises administering a first trans-cutting engineered ribozyme and a second trans-cutting engineered ribozyme, wherein the first trans-cutting engineered ribozyme targets the upstream of the pathogenic mutation and the second trans-cutting engineered ribozyme targets the downstream of the pathogenic mutation. In some embodiments, trans-cutting upstream and downstream of the pathogenic mutation leads to the removal of the pathogenic mutation. In some embodiments, after trans-cutting of the pathogenic mutation, the remainder of the gene is trans-spliced ​​together. In some embodiments, the trans-spliced ​​gene is expressed as a functional protein.

[0264] In one embodiment, the present invention relates to an in vivo method for assembling a full-length RNA viral genome. Exemplary RNA viruses include, but are not limited to, coronaviruses, paramyxoviruses, orthomyxoviruses, retroviruses, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles viruses, Newcastle disease viruses, and picornaviruses. In one embodiment, the method comprises administering to a cell or tissue a first nucleic acid encoding a first portion of an RNA viral genome and encoding a 3' ribozyme. In one embodiment, the method comprises administering to a cell or tissue a second nucleic acid encoding a second portion of an RNA viral genome and encoding a 5' ribozyme. In one embodiment, the method comprises administering to a cell or tissue a first RNA molecule comprising the first portion of an RNA viral genome and a 3' ribozyme. In one embodiment, the method comprises administering to a cell or tissue a second RNA molecule comprising the second portion of an RNA viral genome and a 5' ribozyme. In one embodiment, the method comprises administering to a cell or tissue a nucleic acid encoding a ligase as described herein or a ligase. In one embodiment, after cis-cleavage by the 3' and 5' ribozymes, the first portion of the RNA viral genome and the second portion of the RNA viral genome are ligated together, thereby producing a full-length RNA viral genome.

[0265] in vitro

[0266] In one embodiment, the present invention includes an in vitro method for producing an RNA molecule encoding a protein of interest. In one embodiment, the method includes the step of providing at least two RNA molecules. In one embodiment, the step includes providing a first RNA molecule and a second RNA molecule.

[0267] In one embodiment, the first RNA molecule comprises a coding region encoding a first portion of a target protein. In one embodiment, the first RNA molecule comprises a 3' ribozyme. In one embodiment, the first RNA molecule comprises a coding region encoding a first portion of a target protein and a 3' ribozyme.

[0268] In one embodiment, the second RNA molecule comprises a coding region encoding a second portion of the target protein. In one embodiment, the second RNA molecule comprises a 5' ribozyme. In one embodiment, the second RNA molecule comprises a coding region encoding a second portion of the target protein and a 5' ribozyme.

[0269] In one embodiment, the in vitro method for producing an RNA molecule encoding a protein of interest further comprises providing a ligase. In one embodiment, the ligase induces assembly of an RNA molecule from the coding region of a first RNA molecule and the coding region of a second RNA molecule. In one embodiment, the ligase is an RNA 2', 3'-cyclic phosphate and 5'-OH (RtcB) ligase as described herein.

[0270] In one embodiment, the invention includes an in vitro method for producing an RNA molecule encoding a multi-domain protein of interest. In one embodiment, the method comprises the steps of: a) providing a first RNA molecule, b) providing one or more additional RNA molecules, c) providing a ribozyme, and d) providing a final RNA molecule.

[0271] In one embodiment, the first RNA molecule of step a) comprises a coding region encoding a first portion of a protein of interest. In one embodiment, the first RNA molecule comprises a 3' ribozyme. In one embodiment, the first RNA molecule comprises a coding region encoding a first portion of a protein of interest and a 3' ribozyme. In one embodiment, the 3' ribozyme catalyzes its own detachment from the first RNA molecule, thereby producing a 3'P or 2'3'cP end. In one embodiment, the first RNA molecule further comprises a 5' tag. In one embodiment, the 5' tag mediates attachment of the first RNA molecule to a solid support.

[0272] In one embodiment, the one or more additional RNA molecules of step b) comprise a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme recognition sequence. In one embodiment, the 5' ribozyme cleaves itself to produce a 5' OH terminus. In one embodiment, a ligase is provided to catalyze the attachment of the first RNA molecule to the one or more additional RNA molecules. In one embodiment, the ligase is an RNA 2', 3'-cyclic phosphate and 5'-OH (RtcB) ligase as described herein. In one embodiment, the 3' ribozyme recognition sequence comprises a VS-S sequence as described herein.

[0273] In one embodiment, the ribozyme of step c) comprises a VS-Rz as described herein. In one embodiment, the VS-Rz recognizes VS-S and mediates cleavage of the VS-S from the one or more additional RNA molecules. In one embodiment, the cleavage produces a 3'P or 2'3'cP terminus. In one embodiment, steps b) to c) are repeated at least once to generate an RNA molecule encoding multiple domains. In one embodiment, the VS-Rz is removed prior to repeating step b).

[0274] In one embodiment, the last RNA molecule of step d) comprises a coding region encoding the last portion of the protein of interest. In one embodiment, the last RNA molecule comprises a 5' ribozyme. In one embodiment, the last RNA molecule comprises a coding region encoding the last portion of the protein of interest and a 5' ribozyme. In one embodiment, the 5' ribozyme catalyzes its own separation from the last RNA molecule, thereby producing a 5' OH terminus. In one embodiment, a ligase is provided to catalyze the attachment of the one or more additional RNA molecules to the last RNA molecule, thereby producing a complete RNA molecule encoding an N-terminal domain, one or more additional domains, and a C-terminal domain. In one embodiment, the ligase is an RNA 2', 3'-cyclic phosphate and 5'-OH (RtcB) ligase as described herein.

[0275] Any RNA molecule disclosed herein can be transcribed in vitro from a template DNA (referred to as an "in vitro transcription template"). The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, a synthetic DNA sequence, or any other suitable DNA source. In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a polyA tail. The specific nucleic acid sequence composition and length of the in vitro transcription template will depend on the mRNA encoded by the template.

[0276] In one embodiment, the length of the 5'UTR is between 0 and 3000 nucleotides. The length of the 5' and 3'UTR sequences to be added to the coding region can be varied by various methods, including but not limited to designing PCR primers that anneal to different regions of the UTR. Using this method, one of ordinary skill in the art can modify the 5' and 3'UTR lengths required for optimal translation efficiency after transfection of the transcribed RNA.

[0277] The 5' and 3' UTRs can be naturally occurring endogenous 5' and 3' UTRs of the target gene. Alternatively, a UTR sequence that is not endogenous to the target gene can be added by incorporating the UTR sequence into the forward and reverse primers or by any other modification of the template. The use of a UTR sequence that is not endogenous to the target gene can be used to modify the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in the 3' UTR sequence can reduce the stability of the mRNA. Therefore, the 3' UTR can be selected or designed to increase the stability of the transcribed RNA based on the properties of the UTRs well known in the art.

[0278] In one embodiment, the 5'UTR may contain a Kozak sequence of an endogenous gene. Alternatively, when a 5'UTR that is not endogenous to the target gene is added by PCR as described above, a consensus Kozak sequence can be redesigned by adding a 5'UTR sequence. The Kozak sequence can improve the translation efficiency of certain RNA transcripts, but it does not seem that all RNAs need to achieve efficient translation. The requirement of many mRNAs for Kozak sequences is known in the art. In other embodiments, the 5'UTR may be derived from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs can be used in the 3' or 5'UTR to prevent exonuclease degradation of the mRNA.

[0279] In order to synthesize RNA from a DNA template, a transcription promoter should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that acts as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, the promoter is a T7 RNA polymerase promoter as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of T7, T3, and SP6 promoters are known in the art.

[0280] In one embodiment, the mRNA has a cap at the 5' end and a 3' poly (A) tail, which determines ribosome binding, translation initiation, and mRNA stability in the cell. On a circular DNA template (e.g., plasmid DNA), RNA polymerase produces long tandem products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA that is linearized at the 3' UTR end produces normal-sized mRNA that, when polyadenylated after transcription, is effective in eukaryotic transfection.

[0281] On a linear DNA template, bacteriophage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).

[0282] The conventional method for integrating polyA / T stretches into DNA templates is molecular cloning. However, polyA / T sequences integrated into plasmid DNA can lead to plasmid instability, which can be improved by using recombination-incompetent bacterial cells for plasmid propagation.

[0283] After in vitro transcription, the poly(A) tail of the RNA can be further extended using poly(A) polymerase (e.g., E. coli polyA polymerase (E-PAP) or yeast polyA polymerase). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300 to 400 nucleotides results in an approximately two-fold increase in the translation efficiency of the RNA. In addition, attaching different chemical groups to the 3' end can increase mRNA stability. Such attachments can contain modified nucleotides / artificial nucleotides, aptamers, and other compounds. For example, poly(A) polymerase can be used to incorporate ATP analogs into the poly(A) tail. ATP analogs can further increase the stability of the RNA.

[0284] The 5' cap also provides stability for the mRNA molecule. In one embodiment, the RNA produced by the method includes a 5' cap 1 structure. Such a cap 1 structure can be produced using vaccinia capping enzyme and 2'-O-methyltransferase (CellScript, Madison, WI). Alternatively, a 5' cap is provided using techniques known in the art and described herein (Cougot et al., Trends in Biochem. Sci., 29: 436-444 (2001); Stepinski et al., RNA, 7: 1468-95 (2001); Elango et al., Biochim. Biophys. Res. Commun., 330: 958-966 (2005)).

[0285] Certain embodiments of the present invention can use a solid support consisting of an inert substrate or matrix (e.g., a glass slide, polymer beads, etc.), which has been functionalized, for example, by applying a layer or coating of an intermediate material comprising reactive groups that allows covalent attachment to biomolecules (e.g., polynucleotides). Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate (e.g., glass), particularly polyacrylamide hydrogels as described in WO 2005 / 065814 and US2008 / 0280773 (the contents of which are incorporated herein by reference in their entirety). In such embodiments, biomolecules (e.g., polynucleotides) can be directly covalently attached to the intermediate material (e.g., hydrogel), but the intermediate material itself can be non-covalently attached to a substrate or matrix (e.g., a glass substrate). The term "covalently attached to a solid support" should be correspondingly interpreted as including this type of arrangement.

[0286] As will be appreciated by those skilled in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon, TM etc.), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glasses, plastics, optical fiber bundles and a wide variety of other polymers.

[0287] In some embodiments, the solid support comprises microspheres or beads. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, latex or cross-linked dextran (such as agarose), cellulose, nylon, cross-linked micelles and polytetrafluoroethylene, as well as any other material for solid supports outlined herein can be used. "Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind. is a useful guide. In certain embodiments, the microsphere is a magnetic microsphere or bead.

[0288] The beads need not be spherical; irregular particles may be used. Alternatively or additionally, the beads may be porous. Bead sizes range from nanometers, i.e., 100 nm, to millimeters, i.e., 1 mm, with beads of about 0.2 micrometers to about 200 micrometers being preferred, and beads of about 0.5 micrometers to about 5 micrometers being particularly preferred, although smaller or larger beads may be used in some embodiments.

[0289] In one embodiment, the present invention relates to an in vitro method for assembling a full-length RNA viral genome. Exemplary RNA viruses include, but are not limited to, coronaviruses, paramyxoviruses, orthomyxoviruses, retroviruses, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles viruses, Newcastle disease viruses, and picornaviruses. In one embodiment, the method comprises providing a first RNA molecule comprising a first portion of an RNA viral genome and a 3' ribozyme. In one embodiment, the method comprises providing a second RNA molecule comprising a second portion of an RNA viral genome and a 5' ribozyme. In one embodiment, after cis-cleavage of the 3' and 5' ribozymes, as described herein, the first portion of the RNA viral genome and the second portion of the RNA viral genome have compatible ends for ligation. In one embodiment, the method comprises contacting the first RNA molecule and the second RNA molecule with a ligase as described herein, thereby producing a full-length RNA viral genome.

[0290] Treatment and uses

[0291] The present invention provides methods for treating a disease or condition in a subject, alleviating the symptoms of a disease or condition in a subject, and / or reducing the risk of a subject developing a disease or condition. For example, in one embodiment, the methods of the present invention treat a disease or condition in a mammal, alleviating the symptoms of a disease or condition in a mammal, and / or reducing the risk of a mammal developing a disease or condition. In one embodiment, the methods of the present invention treat a disease or condition in a plant, alleviating the symptoms of a disease or condition in a plant, and / or reducing the risk of a plant developing a disease or condition. In one embodiment, the methods of the present invention treat a disease or condition in a yeast organism, alleviating the symptoms of a disease or condition in a yeast organism, and / or reducing the risk of a yeast organism developing a disease or condition.

[0292] In one embodiment, the subject is a cell. In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the cell is a plant, animal, or fungal cell. In some embodiments, the cell is a plant cell. In one embodiment, the cell is an animal cell. In some embodiments, the cell is a yeast cell.

[0293] In one embodiment, the subject is a mammal. For example, in one embodiment, the subject is a human, a non-human primate, a dog, a cat, a horse, a cow, a goat, a sheep, a rabbit, a pig, a rat, or a mouse. In one embodiment, the subject is a non-mammalian subject. For example, in one embodiment, the subject is a zebrafish, a fruit fly, or a roundworm.

[0294] In one embodiment, the disease or condition is caused by a missing or defective protein whose nucleic acid sequence exceeds the packaging size of the viral vector. Therefore, in one embodiment, the compositions, systems and methods of the present invention can be used to treat, alleviate, or reduce the risk of the disease or condition. Therefore, in one embodiment, the method comprises administering one or more compositions of the present invention to a subject. Further, in one embodiment, the method comprises utilizing one or more systems of the present invention to treat a disease or condition in a subject, alleviate the symptoms of a disease or condition in a subject, and / or reduce the risk of a subject developing a disease or condition.

[0295] In one embodiment, the disease or condition is one or more selected from the group consisting of: Duchenne muscular dystrophy; autosomal recessive polycystic kidney disease; hemophilia A; Stargardt macular degeneration; limb-girdle muscular dystrophy; DFNB9; neurosensory non-syndromic recessive hearing loss; cystic fibrosis; Wilson's disease; autosomal recessive Miyoshi muscular dystrophy and hearing loss type 9; Usher syndrome type 1 and autosomal recessive hearing loss type 2; autosomal recessive hearing loss type 3 and non-syndromic hearing loss; Usher syndrome type 1; autosomal recessive hearing loss type 16 (DFNB16); Meniere's disease (MD); autosomal dominant hearing loss type 12 and autosomal recessive hearing loss type 21; Usher syndrome type 1F (USH1F) and DFNB23; autosomal recessive hearing loss type 28 and non-syndromic hearing loss; autosomal recessive hearing loss type 30 and non-syndromic hearing loss; autosomal recessive otospondylosis and autosomal recessive epiphyseal dysplasia Somatic dominant epiphyseal dysplasia with hypertrophic auricles; autosomal recessive deafness type 77 and autosomal recessive nonsyndromic sensorineural hearing loss type Dfnb; autosomal recessive nonsyndromic hearing impairment DFNB84; autosomal recessive deafness type 84B and rare hereditary hearing loss; autosomal dominant peripheral neuropathy, myopathy, hoarseness, and hearing loss, and deafness type 4A; congenital thrombocytopenia; sensorineural hearing loss; DFNA56; HXB; autosomal dominant Deafness 56; six-arm protein; epileptic encephalopathy; Timothy syndrome and long Qt syndrome type 8; X-linked retinal disorders; hyperaldosteronism; spinocerebellar ataxia type 42; primary aldosteronism; seizures and nervous system abnormalities and sinus node dysfunction and deafness; neurodevelopmental disorders; hypokalemic periodic paralysis; epilepsy; developmental and epileptic encephalopathy; Brody myopathy; Darier's disease / heart disease; von Willebrand disease and Zellweger syndrome. In one embodiment, the disease or disorder is any disease or disorder caused by a genetic mutation suitable for CRISPR-Cas9-mediated editing.

[0296] In one embodiment, the method of the invention comprises administering to a subject having Duchenne muscular dystrophy a composition comprising a first nucleic acid comprising a coding region encoding a first portion of a dystrophin protein and a 3' ribozyme, and a second nucleic acid comprising a coding region encoding a second portion of a dystrophin protein and a 5' ribozyme, wherein the first nucleic acid transcribes a first RNA molecule and the second nucleic acid transcribes a second RNA molecule, and wherein cis-cleavage by the 3' and 5' ribozymes and trans-splicing of the coding region encoding the first portion of the dystrophin protein and the coding region encoding the second portion of the dystrophin protein produces a single RNA molecule encoding full-length dystrophin protein.

[0297] In one embodiment, the method of the invention comprises administering to a subject having Duchenne muscular dystrophy a composition comprising a first nucleic acid encoding the nucleic acid sequence of SEQ ID NO: 129 and a second nucleic acid encoding the nucleic acid sequence of SEQ ID NO: 130, wherein the first nucleic acid transcribes a first RNA molecule and the second nucleic acid transcribes a second RNA molecule, and wherein cis cleavage by the 3' and 5' ribozymes and trans-splicing of the first RNA molecule and the second RNA produces a single RNA molecule encoding full-length dystrophin.

[0298] In one embodiment, the method of the invention comprises administering to a subject suffering from Duchenne muscular dystrophy a composition comprising a first nucleic acid encoding the nucleic acid sequence of SEQ ID NO: 22 and a second nucleic acid encoding the nucleic acid sequence of SEQ ID NO: 23, wherein the first nucleic acid transcribes a first RNA molecule and the second nucleic acid transcribes a second RNA molecule, and wherein cis-cleavage by the 3' and 5' ribozymes and trans-splicing of the first and second RNA molecules produces a single RNA molecule encoding a full-length dystrophin protein with a C-terminal GFP reporter protein. In one embodiment, the second nucleic acid encodes a fragment of SEQ ID NO: 23, wherein the fragment does not include the coding sequence for the C-terminal GFP reporter protein.

[0299] In one embodiment, the method comprises administering to a subject having Duchenne muscular dystrophy a composition comprising a first RNA molecule encoding a first portion of a dystrophin protein and comprising a 3' ribozyme, and a second RNA molecule encoding a second portion of a dystrophin protein and comprising a 5' ribozyme, wherein cis-cleavage by the 3' and 5' ribozymes and trans-splicing of the first and second RNA molecules produces a single RNA molecule encoding full-length dystrophin protein.

[0300] In one embodiment, the method comprises administering to a subject having Duchenne muscular dystrophy a composition comprising a first RNA molecule comprising the nucleic acid sequence of SEQ ID NO: 129 and a second RNA molecule comprising the nucleic acid sequence of SEQ ID NO: 130, wherein cis-cleavage by the 3' and 5' ribozymes and trans-splicing of the first and second RNA molecules produces a single RNA molecule encoding a full-length dystrophin protein.

[0301] In one embodiment, the method comprises administering to a subject suffering from Duchenne muscular dystrophy a composition comprising a first RNA molecule comprising the nucleic acid sequence of SEQ ID NO: 22 and a second RNA molecule comprising the nucleic acid sequence of SEQ ID NO: 23, wherein cis-cleavage by the 3' and 5' ribozymes and trans-splicing of the first and second RNA molecules produces a single RNA molecule encoding a full-length dystrophin protein with a C-terminal GFP reporter protein. In one embodiment, the second nucleic acid encodes a fragment of SEQ ID NO: 23, wherein the fragment does not include the coding sequence for the C-terminal GFP reporter protein.

[0302] In one embodiment, the method of the present invention comprises administering a composition to a subject having one or more diseases selected from Table 1, the composition comprising a first nucleic acid comprising a coding region encoding a first portion of a therapeutic protein corresponding to a relevant disease in Table 1 and a 3' ribozyme, and a second nucleic acid comprising a coding region encoding a second portion of a therapeutic protein corresponding to a relevant disease in Table 1 and a 5' ribozyme, wherein the first nucleic acid transcribes a first RNA molecule and the second nucleic acid transcribes a second RNA molecule, and wherein cis-cleavage by the 3' and 5' ribozymes and trans-splicing of the coding region encoding the first portion of the therapeutic protein and the coding region encoding the second portion of the therapeutic protein produces a single RNA molecule encoding the full-length therapeutic protein.

[0303] In one embodiment, the method comprises administering to a subject having one or more diseases selected from Table 1 a composition comprising a first RNA molecule encoding a first portion of a therapeutic protein corresponding to a disease of interest in Table 1 and comprising a 3' ribozyme, and a second RNA molecule encoding a second portion of a therapeutic protein corresponding to a disease of interest in Table 1 and comprising a 5' ribozyme, wherein cis-cleavage by the 3' and 5' ribozymes and trans-splicing of the first and second RNA molecules produces a single RNA molecule encoding the full-length therapeutic protein.

[0304] Table 1. List of monogenic diseases caused by large gene mutations (including protein size (number of amino acids), gene symbol, protein name, and disease name).

[0305]

[0306]

[0307]

[0308]

[0309] Experimental Examples

[0310] The present invention is further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and, unless otherwise indicated, are not intended to be limiting. Therefore, the present invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations that become apparent as a result of the teachings provided herein.

[0311] Without further description, it is believed that one of ordinary skill in the art can use the preceding description and the following illustrative examples to make and utilize the present invention and practice the claimed methods. Therefore, the following working examples should not be construed as limiting the remainder of the disclosure in any way.

[0312] Example 1: Ribozyme-mediated RNA assembly and expression in mammalian cells

[0313] Ribozymes (Rz) are small, catalytic RNA sequences capable of nucleotide-specific self-cleavage (Doherty and Doudna 2000). Ribozyme-mediated RNA cleavage generates distinct 3'-phosphate and 5'-hydroxyl termini that resemble substrates for ubiquitous RNA repair pathways across all three kingdoms of life. As demonstrated here, ribozyme-mediated cis-cleavage can be exploited for trans-splicing of independent RNA transcripts in mammalian cells, a method termed stitchR (stitch RNA). Remarkably, remodeling messenger RNA with stitchR allows for efficient translation and expression of full-length proteins in mammalian cells. As demonstrated, stitchR can be used to assemble protein-encoding functional domains or to deliver and express large protein-coding sequences via viral vectors. Furthermore, overexpression of the RNA 2',3'-cyclic phosphate and 5'-OH (RtcB) ligase enhanced stitchR activity in mammalian cells and was sufficient to catalyze stitchR activity in vitro. These data demonstrate a novel approach for scarless trans-splicing of functional RNAs in cells using ribozymes, which could be used for numerous research and therapeutic applications.

[0314] Autocatalytic RNA sequences are widespread in nature and catalyze a diverse range of biological processes, including intron splicing, rolling circle viral genome replication, and peptide bond formation (Weinberg et al., 2019). At least seven major ribozyme families with distinct sequence and structural features have been identified, including hammerhead (HH) ribozymes, hepatitis delta virus (HDV) ribozymes, Varkud satellite (VS) ribozymes, Sister ribozymes, Twister-sister ribozymes, hairpin ribozymes, Hatchet ribozymes, and Pistol ribozymes. The most extensively studied are members of the HH, HDV, and Twister ribozyme families, which, due to their small size and cleavage properties, have been used in vitro and in vivo to generate RNAs with precise ends without ribozyme sequences ( Figure 13 )(Ferre-D'Amare and Doudna 1996; Avis et al. 2012; Zhang et al. 2017).

[0315] In both prokaryotes and eukaryotes, most cellular RNAs, including messenger and long noncoding RNAs, are synthesized and spliced ​​with 5'-phosphate (P) and 3'-hydroxyl (OH) termini. In contrast, unconventional cis-splicing of many tRNAs and the mRNA encoding the ER stress response protein XBP1 is catalyzed by enzymatic pathways that generate unique 5'-OH and 3'-P or 2'3' cyclic phosphate (cP) termini. Recent findings suggest that the ubiquitous RNA 2',3'-cyclic phosphate and 5'-OH (RtcB) ligase in mammals catalyzes unconventional RNA cis-splicing. Furthermore, RtcB and several other enzyme families may function to repair host cell RNAs damaged by stress or exogenous nuclear toxins. Because ribozyme-mediated cleavage results in similar terminal ends, ribozyme-cleaved RNAs may be subject to trans-splicing via endogenous RNA repair pathways.

[0316] Ribozyme-cleaved mRNA is trans-spliced ​​and translated in mammalian cells

[0317] To determine whether ribozymes can be used for scarless trans-splicing of RNA in mammalian cells, two expression plasmids were designed containing non-overlapping N-terminal (Nt) and C-terminal (Ct) fragments of the fluorescent reporter gene GFP (Nt-GFP and Ct-GFP, respectively). Ribozymes were designed to catalyze their own removal from adjacent nucleotides of the GFP fragment, including the 3'HDV ribozyme on Nt-GFP and the 5'HH ribozyme on Ct-GFP ( Figure 1 A). Expression of either Nt or Ct GFP ribozyme RNA alone resulted in no detectable GFP fluorescence when transfected into mammalian COS-7 or HEK293T cells ( Figure 1 B). Notably, co-expression of RNA encoding Nt and Ct-GFP produced green fluorescence after 48 hours ( Figure 1 B). RT-PCR analysis and Sanger sequencing revealed that trans-splicing of separate Nt- and Ct-GFP RNAs occurred between the predicted ribozyme-catalyzed cleavage sites ( Figure 1 C and Figure 1 D). Furthermore, full-length GFP protein was detected in co-transfected cells by Western blotting ( Figure 1 E) These data demonstrate that the endogenous mammalian cellular RNA repair pathway is sufficient to catalyze the trans-splicing of independently ribozyme-processed RNAs, which are efficiently translated into full-length proteins. This RNA trans-splicing method has been named stitchR.

[0318] Effects of ribozyme sequence and type on ribozyme-mediated trans-splicing

[0319] To precisely quantify the relative amount of functional full-length protein produced by ribozyme-mediated trans-splicing in cells, two non-overlapping halves of firefly luciferase were used to generate a reporter gene ( Figure 2 A). Consistent with our previous findings, only co-transfection of the RNA encoding Nt-luciferase ribozyme and Ct-luciferase ribozyme resulted in trans-splicing and luciferase activity in cells ( Figure 2 B and Figure 2 C). Using this assay, we further characterized the effects of different HH and HDV ribozyme sequences on trans-splicing activity in mammalian cells. A 6-base pair (bp) overlap in the stem 1 HH ribozyme provided maximal luciferase activity and mutation of the HH catalytic residue abolished activity, consistent with previous reports on HH ribozyme activity characterized in vitro ( Figure 2 D). Furthermore, the luciferase activities of the genomic and antigenomic HDV ribozyme sequences were comparable, except for the smallest 56-nucleotide HDV ribozyme (HDV56), which had significantly lower activity ( Figure 2 E). Also consistent with previous reports, a C to U mutation of a nucleotide required for HDV catalysis resulted in a complete loss of luciferase activity ( Figure 2 E) These findings demonstrate that ribozyme-mediated trans-splicing activity is dependent on ribozyme cleavage in mammalian cells.

[0320] Use translational controllers and / or protein degradation sequences to prevent unwanted or truncated proteins from Nt or Ct vectors Express

[0321] The Nt or Ct RNA may undergo translation prior to ribozyme-mediated cleavage or, when expressed alone, may result in unwanted or truncated protein expression. To restrict expression of unspliced ​​Nt or Ct vectors, the efficacy of previously characterized translational controllers of protein degradation sequences on the stability of vectors encoding full-length GFP was tested. Addition of the HDV ribozyme to the 3' end of GFP did not appear to alter GFP fluorescence ( Figure 3 A and 3B). To selectively block GFP expression, the effects of removing the protein degradation sequences hCL1-PEST, E1A-PEST, the vector's poly(A) sequence, or mimicking translation with a poly A tail to generate a poly K tail were tested ( Figure 3A and 3B). All degradation sequences were cloned in-frame with the GFP open reading frame, allowing translation via the HDV ribozyme sequence. Incorporation of hCL1-PEST showed a significant reduction in GFP fluorescence, while EF1a PEST did not. Deletion of the vector poly(A) sequence from the expression vector resulted in a complete loss of GFP expression, while translation via multiple poly(A) sequences to generate a poly K tail also resulted in reduced fluorescence.

[0322] For the Ct-encoded GFP reporter gene, inclusion of the 5′ HH ribozyme and deletion of the GFP start codon (ATG) still resulted in weak but detectable GFP expression despite the lack of a predicted upstream alternative ATG ( Figure 3 C and D). Further silent mutations within the N-terminal NTG codon in GFP (GFPcdn) further reduced GFP detection; however, weak fluorescence was still evident. Inclusion of the 5'UTR of the yeast GCN4 gene, encoding four small upstream ORFs that act as translational inhibitors, eliminated detectable GFP fluorescence. A smaller internal fragment of the GCN4 5'UTR encoding only these four uORFs was equally effective in preventing GFP expression. These data demonstrate that translational control of protein degradation sequences can be used to prevent unwanted protein expression from a single Nt or Ct vector.

[0323] These translational controllers or protein degradation sequences can be used in other dual-vector applications where it is desirable to limit the expression of unwanted or truncated proteins, such as dual AAV vector strategies that rely on homologous recombination to generate large protein-coding open reading frames.

[0324] Single and multiple trans-splicing of functional protein-coding RNAs

[0325] To determine whether ribozyme-mediated trans-splicing could be used to combine protein-coding functional domains in cells, RNA encoding four copies of the mitochondrial targeting sequence (Nt-4xMTS) and an open reading frame encoding full-length GFP lacking the ATG start codon (Ct-GFP) were generated ( Figure 4 A). Co-expression of these two independent RNAs resulted in strong expression of mitochondrial-localized GFP, which overlapped with the red fluorescent mitochondrial marker MitoTracker Red CMXRos ( Figure 4 B) These findings demonstrate that ribozyme-mediated trans-splicing can be used to rapidly combine two independent RNAs to express a specific functional fusion protein in cells.

[0326] Due to the three open reading frames in which proteins are translated, ribozyme-mediated trans-splicing and expression of multiple different functional proteins simultaneously may also be possible. By exploiting this property, trans-splicing of RNAs located in three different compatible open reading frames can be used to generate functional proteins. To demonstrate this functionality, an additional ribozyme pair was designed in reading frame 2 (F2), which encodes a myristoylated membrane targeting sequence (Nt-F2-Myr) and a red fluorescent protein (Ct-F2-RFP) ( Figure 4 C). These Nt and Ct vector pairs also include an hCL1-PEST protein degradation sequence and a GCN4 translation inhibition sequence to limit expression of truncated proteins from individual Nt and Ct vectors, respectively. In co-transfected cells, GFP fluorescence is highly specific to mitochondria, while RFP fluorescence is highly specific to membranes ( Figure 4 D), demonstrating the ability of this RNA trans-splicing method to generate diverse functional proteins in cells.

[0327] Optimized ribozymes enhance protein expression in ribozyme-mediated trans-splicing

[0328] Small sequence modifications can profoundly affect the catalytic activity of ribozymes by altering secondary structure, stability, or binding to metal ion cofactors. Using our trans-splicing luciferase reporter assay, we identified improved ribozyme types and sequence modifications that enhance trans-splicing luciferase reporter activity in mammalian cells ( Figure 16 The hammerhead variant ribozyme of RzB containing the tertiary stabilization motif (TSM) showed higher activity than the ribozyme without TSM ( Figure 16 A). Furthermore, when cloned 3' to Nt-Luc, the Twister (twst) ribozyme exhibited greater activity than the HDV ribozyme. Catalytic mutations within the twister ribozyme can similarly abolish luciferase activity ( Figure 16 B) and is dependent on the formation of the P1 stem ( Figure 16 C). Since Twister ribozymes require a U at position 1, this requirement may limit the design of scarless trans-splicing to sequences ending in U. Therefore, we tested whether nucleotide substitutions at position 1 could be tolerated and found that U1A did not show significantly different activity, while substitutions with U1C or U1G retained activity, but with reduced activity ( Figure 16 C).

[0329] Optimized splice donor and acceptor sequences enhance protein expression in ribozyme-mediated trans-splicing

[0330] Pre-mRNA splicing by the spliceosome has been shown to enhance mRNA translation by depositing factors that promote the first round of translation or by promoting RNA processing and export to the cytoplasm. Adding chimeric cis-splicing introns within transgenes has also been shown to promote transgenic protein expression. It was then investigated whether the trans-spliced ​​RNA could undergo cis-splicing by the spliceosome and whether this would affect the translation and expression of the trans-spliced ​​mRNA. To test this, splice donor (SD) and splice acceptor (SA) sequences were incorporated into the trans-splicing GFP reporter gene so that the trans-spliced ​​RNA would reconstruct the chimeric intron ( Figure 5 A). Notably, the addition of SD and SA sequences robustly enhanced GFP fluorescence compared to the trans-spliced ​​GFP reporter without SD or SA sequences ( Figure 5 B). RT-PCR and Sanger sequencing showed that Nt-GFP and Ct-GFP RNA containing SD and SA sequences were both trans- and cis-spliced, resulting in the recovery of the normal GFP open reading frame (data not shown). These data indicate that trans-splicing may occur in the nucleus and that subsequent cis-splicing is a useful strategy for enhancing expression of trans-spliced ​​RNA.

[0331] Ribozyme-mediated trans-splicing and expression of large gene sequences for delivery using viral therapeutic vectors

[0332] Ribozyme-mediated trans-splicing can be used to deliver and express large protein-coding mRNAs that exceed the packaging size limit of therapeutic viral gene therapy vectors such as AAV. Figure 6 A). This may help restore expression of large genes that are mutated in many human monogenic diseases, such as dystrophin (Dys) in Duchenne muscular dystrophy (DMD), CFTR in cystic fibrosis (CF), and factor VIII (F8) in hemophilia A. In a cell-based transfection assay, vectors encoding Nt and Ct split μdystrophin co-expressed with a C-terminal GFP tag were trans-spliced ​​in mammalian cells ( Figure 6 B and Figure 6 C) and is localized to the membrane ( Figure 6 D) These data demonstrate the feasibility of reconstructing and expressing large protein-coding genes using ribozyme-mediated trans-splicing.

[0333] Lentiviral delivery of ribozyme-enabled RNA for trans-splicing in cells deliver

[0334] The autocatalytic self-cleavage of ribozymes may hinder the packaging of ribozyme-encoding RNA by positive-sense RNA viruses (such as commonly used gamma-retroviruses and lentiviral vectors). To circumvent this potential problem, the GFP expression cassette with Nt and Ct split was encoded on the negative strand of the third-generation lentiviral vector backbone ( Figure 7 A). Lentiviral particles were generated for Nt and Ct vectors, respectively, and then used to transduce HEK293T cells. Cells transduced with both Nt-GFP and Ct-GFP showed green fluorescence expression, while cells transduced with either Nt-GFP or Ct-GFP alone showed no detectable fluorescence ( Figure 7 B) These data demonstrate that lentiviral vectors are capable of delivering and expressing ribozymes encoding RNAs for trans-splicing.

[0335] This approach can also be used to deliver large gene sequences that exceed the packaging size of these viral vectors, such as Dys( Figure 7 C). Ribozyme-mediated trans-splicing may also allow the safe manipulation or reconstruction of viral genomes, such as lentiviral or large coronavirus RNA genomes.

[0336] Safe manipulation, delivery, and expression of toxic or antiviral genes using viral vectors

[0337] Ribozyme-mediated trans-splicing can also allow for the safe manipulation or reconstitution of toxic or antiviral proteins that might inhibit the production of lentiviral particles in mammalian packaging cells. These include a number of cell suicide genes, such as the translation inhibitory diphtheria toxin A (DTA) ( Figure 8 A). We demonstrate that vectors encoding split DTA sequences, upon trans-splicing and expression, inhibit co-expression of a CS2GFP reporter construct, consistent with a translational repressive role for DTA in mammalian cells ( Figure 8 B).

[0338] Enzymes for enhancing or inhibiting ribozyme-mediated trans-splicing

[0339] Many families of enzymes have been suggested to link 5'-OH and 3'-P or 2'3' cyclic phosphate (cP) termini, the most notable being RtcB, which has been found to be conserved across all three domains of life. Human codon-optimized RtcB orthologs from eukaryotic (H. sapiens), bacterial (E. coli), and archaeal (P. horikoshii) species were cloned and co-expressed to measure their effects on the activity of a trans-splicing luciferase reporter gene. Interestingly, co-expression of RtcB from P. horikoshii resulted in an enhancement of luciferase activity (4.5-fold), whereas the human and bacterial orthologs showed modest or no enhancement ( Figure 9 ).

[0340] Other enzyme families have been shown to regulate these RNA ends. Interestingly, expression of T4 polynucleotide kinase (T4 PNK), which functions as a 5'-hydroxykinase, 3'-phosphatase, and 2',3'-cyclic phosphodiesterase, significantly inhibited luciferase activity ( Figure 9These data indicate that coexpression of exogenous enzymes can enhance or inhibit ribozyme-mediated trans-splicing in mammalian cells.

[0341] RtcB is sufficient to catalyze ribozyme-mediated RNA trans-splicing in vitro

[0342] Due to their nucleotide-specific cleavage, ribozymes have been widely used to generate precise RNA ends in vitro. We next sought to determine whether ribozymes could be used to direct trans-splicing of independently synthesized RNA in vitro. In vitro RNA transcription of Nt- and Ct-luciferase-ribozyme reporter gene constructs using T7 RNA polymerase revealed that the addition of recombinant E. coli RtcB was both necessary and sufficient to catalyze trans-splicing detected using RT-PCR. Figure 10 A and Figure 10 B). Similarly, RNA encoding the domain of the spider silk protein Spidroin was designed ( Figure 10 C). Spidroin is the main component of spider dragline silk, a material highly prized for its tensile properties, but difficult to synthesize in heterologous systems due to the protein's highly repetitive nature. Spidroin naturally consists of multiple A and Q repeats flanked by conserved N-terminal (N1L) and C-terminal (N3R) domains. Following in vitro synthesis of spidroin RNA using T7 polymerase, addition of recombinant RtcB ligase from Escherichia coli was found to be sufficient to catalyze trans-ligation of RNAs encoding the ribozyme-cleaved N1L and N3R, as detected by RT-PCR and Sanger sequencing ( Figure 10 D).

[0343] Controlled tandem trans-splicing of RNAs encoding multidomain proteins

[0344] We next examined whether the addition of a third RNA encoding an AQ fusion domain with flanking ribozymes would result in tandem repeat assembly, albeit uncontrolled ( Figure 11 A). Although directed trans-splicing between each individual RNA could be detected, assembly of three or more independent RNA fragments could not be detected (data not shown). This is likely due to the rapid circularization of RNAs containing ends compatible with RtcB ligation. As an alternative, the use of transactivated VS ribozymes has the potential to allow the sequential and controlled assembly of RNA sequences in vitro ( Figure 11 B and Figure 11C). In this approach, the 3'-terminal RNA ribozyme is only suitable for ligation after addition and trans-cleavage by VS-Rz. Because the VS-Rz transactivating ribozyme RNA is not covalently attached, the stepwise addition of stitchR-compatible RNA, VS-Rz, and RtcB ligase allows for controlled tandem assembly of RNA sequences, which may facilitate the assembly of repetitive RNAs encoding biologically or industrially important proteins (e.g., synthetic spider silk, elastin, collagen, etc.).

[0345] Trans-splicing of endogenous RNA using trans-cleaving ribozymes: therapeutic applications for correcting disease-causing mutations

[0346] Ribozymes are autocatalytic RNAs that cleave in cis to generate unique RNA ends that we have shown are trans-spliced ​​and subsequently expressed in mammalian cells ( Figure 12 A). Notably, cis-cleaving ribozymes can be engineered to cleave in trans, allowing the target RNA to be cleaved in a nucleotide-specific manner, generating similar RNA ends ( Figure 12 B)(Carbonell et al. 2011; Webb and Luptak 2018). Thus, trans-cleaving ribozymes can be used to catalyze scarless trans-splicing of RNA in cells or in vitro. This approach can be used for numerous applications, a major one of which is the deletion of disease-causing mutations in gene transcripts by targeting mutation flanking sequences in exonic or intronic sequences ( Figure 12 C and Figure 12 D).

[0347] In summary, this article demonstrates that ribozyme-mediated cleavage of independent RNAs expressed in cells is efficiently assembled and can be translated in mammalian cells. This method, referred to herein as stitchR, can be used as a new method for the combinatorial assembly of functional RNAs and proteins for basic and therapeutic applications. Due to the autocatalytic nature of ribozymes and the endogenous RNA repair pathways present in cells, stitchR only requires the expression of a single RNA for trans-splicing and translation to occur in cells. In vitro, the RtcB ligase has been shown to be sufficient for trans-splicing, and since RtcB is ubiquitous and widely expressed in all three kingdoms of life, stitchR has the potential to become a useful method in many different organisms.

[0348] The robustness of this system relies on the efficient and precise nature of ribozyme-mediated RNA cleavage, which produces reliable and accurate nucleotide-specific termini that are crucial for restoring protein-coding open reading frames. Furthermore, the ability to generate RNA using ribozymes that completely catalyze their own removal allows for scarless assembly, resulting in RNA that is essentially indistinguishable from its native counterpart.

[0349] While ribozyme cleavage has been extensively studied in vitro, in vivo ribozyme cleavage is less understood and is thought to be influenced by the availability of metal ions required for folding and catalysis through interactions with RNA-binding proteins. StitchR serves as an indirect readout for ribozyme-mediated cleavage, and interestingly, we found that it is significantly affected by variations in ribozyme sequence and structure. This suggests that optimization of ribozyme cleavage may be a useful approach for enhancing stitchR activity in vivo. Further analysis of the influence of RNA repair pathway components such as RtcB, RtcA, and Archease may also serve as important factors in regulating stitchR activity.

[0350] Ribozymes have naturally evolved to function in cis to promote their self-cleavage, however, many ribozyme families (particularly HDV and HH) have been engineered to cleave target RNAs in trans. It is noteworthy here that combining trans-cleaving ribozymes with stitchR can further allow for a robust RNA cleavage and repair method to be performed in cells or in vitro. This approach can be used as a nucleotide-specific "cut and paste" method for RNA, which may help generate RNA diversity or help remove certain harmful mutations in pathogenic RNAs.

[0351] Example 2: Trans-splicing and expression of RNA can be induced using transactivated ribozymes

[0352] Most ribozymes are autocatalytic and require only metal ions as cofactors, which are readily found in the biological environment, which facilitate folding and chemical catalysis. Varkud satellite (VS) ribozymes can be used for scarless trans-splicing if the donor RNA ends with a G nucleotide. Interestingly, the VS ribozyme can be modified to allow transactivation of the ribozyme to induce catalysis (Guo and Collins 1995; Ouellet et al. 2009). When split into two components, the small VS stem-loop (VS-S) is insufficient to induce cis-cleavage alone, however, the addition of the remaining sequence VS-Rz promotes efficient cleavage of VS-S ( Figure 14 A). This transactivation feature may allow for inducible ribozyme-mediated trans-cleavage, where the addition of a VS-Rz sequence is required to enable VS-S cleavage on an Nt donor RNA, which is then suitable for trans-splicing with a Ct acceptor RNA containing a 5'-OH terminus ( Figure 14 B) The VS-Rz sequence, which contains typical 5'-P- and 3'-OH RNA ends, cannot participate in trans-splicing and therefore can serve as a multi-turnover catalyst for the reaction.

[0353] The ability to control ribozyme-mediated cleavage, such as by the addition of a desired transactivation sequence (such as VS-Rz), could allow for the controlled addition of variable or non-variable RNA sequences to generate synthetic repetitive RNAs ( Figure 14C). One approach is to generate RNAs with unique N-terminal domains, unique C-terminal domains, and internal variable or non-variable "repeat" domains. This approach requires that both the N-terminal and C-terminal RNAs contain a single ribozyme at the 3' and 5' termini, respectively. Internally repeated RNAs require ribozymes at both the 5' and 3' termini to function as both acceptors and donors during trans-splicing. However, the addition of ribozymes to both ends of the RNA or to RNAs with both a 3'-P and a 5'-OH leads to cyclization by ligases such as RtcB (Desai et al., 2015), thereby preventing participation in the growing linear chain. However, the use of inducible transactivating ribozymes can control RNA domain synthesis by adding and removing VS-Rz and RtcB ligases to gradually ligate the 5' and 3' termini ( Figure 14 C). This approach can be used to generate highly repetitive RNA sequences that can then be translated to produce synthetic repetitive proteins (such as those that make up hydrogels, synthetic spider silk, or collagen), which are difficult to produce and encode as DNA due to recombinant methods. These methods may be useful for drug delivery, biomaterials, or the production of industrial materials (Chambre et al., 2020).

[0354] Example 3: Generation of Stable Synthetic Intron Sequences Using Ribozymes

[0355] When one RNA contains a 3' ribozyme and the other contains a 5' ribozyme, ribozyme-mediated trans-splicing can occur between the two independent RNAs ( Figure 15 A). However, it has been demonstrated that both ribozymes can mediate their own scarless removal when transcribed in cis within the same RNA ( Figure 15 B). This approach also generates two independent RNAs with 3'-P and 5'OH ends, which can undergo trans-splicing and translation in cells ( Figure 15 B) This can also be achieved in vitro by adding a ligase such as RtcB.

[0356] The intronic sequences produced by the ribozyme also contain compatible 5'-OH and 3'-P termini and can be cis-spliced ​​or circularized, which is a common readout of RtcB ligase activity in vitro. Unlike the rapidly degradable lariat RNA produced by the spliceosome during exon splicing, RNA loops are considered highly stable because they no longer contain 5' or 3' termini and are therefore not degraded by RNA exonucleases. A cargo sequence that can contain any number of functional or useful RNAs (such as microRNAs, CRISPR guide RNAs, etc.) or gene expression sequences can be inserted as "cargo" between the two ribozymes ( Figure 15C). This approach can be used to co-deliver and express useful RNA sequences during ribozyme-mediated trans-splicing and expression. If one of the internal ribozymes does not require bilateral flanking sequences for activity, such as for the 5' HDV ribozyme, the RNA loop can exist in both circular and re-cleaved linear forms ( Figure 15 C). When VS-S is used instead of HDV, the system can be made inducible, requiring delivery or expression of VS-Rz. Using ribozymes that require bilateral flanking sequences for cleavage (such as the HH ribozyme), the cleavage can be designed so that RNA circularization of the cargo RNA is unidirectional ( Figure 15 D).

[0357] Example 4: Sequence

[0358] Nucleic acid sequence encoding trans-splicing protein

[0359] Nt-GFP (SEQ ID NO: 1)

[0360] AUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCA CCGGCAAGCUGCCCGGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUU

[0361] Ct-GFP (SEQ ID NO: 2)

[0362] CAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAGUAA

[0363] Nt - Luciferase (SEQ ID NO:3)

[0364] AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUGGAAGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCUGGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGGACAUCACUUACGCUGAGUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAUAUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCAAUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCCGCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGGGCAUUUCGCAGCCUACCGUGGUGUUCGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGUGCAAAAAAAGCUCCCAAUCAUCCAAAAAAUUAUUAUCAUGGAUUCUAAAACGGAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCCCGGUUUUAAUGAAUACGAUUUUGUGCCAGAGUCCUUCGAUAGGGACAAGACAAUUGCACUGAUCAUGAACUCCUCUGGAUCUACUGGUCUGCCUAAAGGUGUCGCUCUGCCUCAUAGAACUGCCUGCGUGAGAUUCUCGCAUGCCAGAGAUCCUAUUUUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCCAUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCGAGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUCUGAGGAGCCUU

[0365] Ct - luciferase (SEQ ID NO:4)

[0366] CAGGAUUACAAGAUUCAAAGUGCGCUGCUGGUGCCAACCCUAUUCUCCUUCUUCGCCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGCUUCUGGUGGCGCUCCCCUCUCUAAGGAAGUCGGGGAAGCGGUUGCCAAGAGGUUCCAUCUGCCAGGUAUCAGGCAAGGAUAUGGGCUCACUGAGACUACAUCAGCUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGUUGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUGGGCGUUAAUCAAAGAGGCGAACUGUGUGUGAGAGGUCCUAUGAUUAUGUCCGGUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUGGCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUCGUUGACCGCCUGAAGUCUCUGAUUAAGUACAAAGGCUAUCAGGUGGCUCCCGCUGAAUUGGAAUCCAUCUUGCUCCAACACCCCAACAUCUUCGACGCAGGUGUCGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUUUGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAGUCAAGUAACAACCGCGAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAAGUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCCUCAUAAAGGCCAAGAAGGGCGGAAAGAUCGCCGUGUAGUAA

[0367] N1L(SEQ ID NO:5)

[0368] ATGGGTCAGGCCAATACGCCCTGGAGCAGTAAGGCAAACGCGGATGCCTTTATAAATTCATTCATCAGTGCAGCATCCAATACTGGTTCCTTCTCTCAAGACCAAATGGAGGACATGTCACTCATCGGCAATACTCTGATGGCTGCCATGGACAATATGGGAGGCCGCATAACACCATCTAAGTTGCAGGCGTTGGATATGGCCTTCGCATCATCAGTGGCCGAGATCGCGGCTAGTGAGGGCGGCGACTTGGGAGTCACTACCAACGCGATCGCGGATGCCCTCACTTCTGCTTTTTATCAAACGACCGGGGTTGTCAATTCACGATTCATATCTGAGATCAGGAGCCTCATAGGAATGTTCGCGCAGGCTTCCGCAAATGACGTTTATGCATCTGCTGGCTCTGGCAGCGGGGGTGGTGGGTATGGAGCCAGCTCAGCATCTGCGGCTTCTGCAAGTGCTGCTGCCCCGAGTGGCGTAGCTTATCAGGCTCCTGCTCAGGCTCAAATCAGTTTTACGTTGCGAGGGCAACAACCTGTTTCC

[0369] AQ(SEQ ID NO:6)

[0370] GGTCCTTATGGACCCGGTGCTAGCGCTGCGGCAGCAGCCGCTGGCGGTTATGGCCCAGGTTCAGGGCAACAGGGGCCTGGGCAACAAGGACCTGGCCAACAAGGTCCTGGTCAGCAGGGTCCAGGGCAGCAG

[0371] NR3(SEQ ID NO:7)

[0372] GGCGCTGCTTCCGCTGCAGTATCAGTAGGTGGCTATGGACCTCAATCTAGTAGCGCCCCTGTTGCCTCTGCCGCCGCATCTCGACTTTCAAGTCCCGCCGCTAGTTCCAGGGTCAGTTCCGCGGTATCTAGCTTGGTAAGTAGCGGACCCACTAATCAAGCGGCACTTTCAAACACAATATCCTCAGTAGTCAGTCAAGTAAGCGCATCAAACCCTGGCTTGTCAGGGTGTGACGTTCTGGTTCAGGCACTTCTGGAAGTTGTCTCAGCGTTGGTAAGCATCCTGGGTAGCTCCTCCATAGGTCAAATTAATTATGGCGCGAGCGCCCAATACACACAAATGGTGGGTCAGAGTGTGGCGCAGGCACTCGCAGGCGACTACAAGGATCATGACGGAGACTATAAGGATCATGATATAGATTACAAGGACGATGATGACAAGGCCTAGTAA

[0373] Nt-4xMTS(SEQ ID NO:8)

[0374] AUGAGUGUGUUGACGCCGUUGCUUCUGCGAGGGCUUACCGGGUCUGCUAGAAGACUUCCGGUCCCCAGGGCCAAGAUACAUAGCCUCGGAGACCCGAUGUCUGUGCUCACUCCUCUGCUUUUGCGAGGACUGACUGGGUCCGCCAGACGACUCCCGGUGCCGAGAGCUAAAAUCCAUAGCCUGGGAAAAUUGGCAACUAUGUCAGUCCUGACGCCGCUUCUUCUCCGGGGUCUUACAGGGUCUGCAAGAAGGCUGCCUGUACCUCGGGCGAAAAUUCAUAGCUUGGGCGACCCGAUGAGUGUAUUGACGCCCCUGUUGCUGAGAGGAUUGACUGGGUCAGCGCGCCGGCUCCCUGUCCCCCGAGCUAAGAUUCACUCCCUUGGUAAGCUGAGAAUCCUCCAAUCAACGGUUCCGAGAGCAAGAGAUCCGCCGGUCGCCACGAGGCCUCUCGAG

[0375] Nt-DTA(SEQ ID NO:17)

[0376] AUGGACCCCGACGACGUGGUGGACAGCAGCAAGAGCUUCGUGAUGGAGAACUUCAGCAGCUACCACGGCACCAAGCCCGGCUACGUGGACAGCAUCCAGAAGGGCAUCCAGAAGCCCAAGAGCGGCACCCAGGGCAACUACGACGACGACUGGAAGGGCUUCUACAGCACCGACAACAAGUACGACGCUGCCGGCUACAGCGUGGACAACGAGAACCCCCUGAGCGGCAAGGCCGGCGGCGUGGUGAAGGUGACCUACCCCGGCCUGACCAAGGUGCUGGCCCUGAAGGUG

[0377] Ct-DTA(SEQ ID NO:18)

[0378] GACAAUGCCGAGACCAUCAAGAAGGAGCUGGGCCUGAGCCUGACCGAGCCCCUGAUGGAGCAGGUGGGCACCGAGGAGUUCAUCAAGAGAUUCGGCGACGGCGCCAGCAGAGUGGUGCUGAGCCUGCCCUUCGCCGAGGGCAGCAGCAGCGUGGAGUACAUCAACAACUGGGAGCAGGCCAAGGCCCUGAGCGUGGAGCUGGAGAUCAACUUCGAGACCAGAGGCAAGAGAGGCCAGGACGCCAUGUACGAGUACAUGGCCCAGGCUUGCGCCGGCAACAGAGUGAGAAGAUAGUAA

[0379] GFPcdn(no start ATG codon)(SEQ ID NO:19)

[0380] GUUAGCAAGGGCGAGGAGCUCUUCACCGGGGUCGUCCCCAUCCUCGUCGAGCUCGACGGCGACGUAACGGCCACAAGUUCAGGCGUCUCCGGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUCACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCU CGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCGACCACAUGAAGCAGCACGACUUCUAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUCAAGGACGACGGCAACUACAAGACCCGGCCCGAGGUGAAGUUCGAGGGCGACACCCUGG UGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUCAACUACAACAGCCAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCACAACAUCGAGGACGGCAGCGUGCAGCUCGCC GACCACUACCAGCAGAACACCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAG

[0381] F2-Myr(SEQ ID NO:20)

[0382] AUGGGGUUGUUGUUUCAGCAAGACAGCGGCGAAGGUGAAGCAGCAGCAGAAAGACCAGGCGAGGCUGCGGUAGCAUCAAGUCCCUCCAAGGCUAAUGGGCAGGAACACGUCAAAGUUGGAAGCGU

[0383] F2-RFP(SEQ ID NO:21)

[0384] AGCCAUCAUCAAGGAGUUCAUGCGCUUCAAGGUGCACAUGGAGGGCUCCGUGAACGGCCACGAGUUCGAGAUCGAGGGCGAGGGCGAGGGCCGCCCCUACGAGGGCACCCAGACCGCCAAGCUGAAGGUGACCAAGGGUGGCCCCCUGCCCUUCGCCUGGGACAUCCUGUCCCCUCAGUUCAUGUACGGCUCCAAGGCCUACGUGAAGCACCCCGCCGACAUCCCCGACUACUUGAAGCUGUCCUUCCCCGAGGGCUUCAAGUGGGAGCGCGUGAUGAACUUCGAGGACGGCGGCGUGGUGACCGUGACCCAGGACUCCUCCCUGCAGGACGGCGAGUUCAUCUACAAGGUGAAGCUGCGCGGCACCAACUUCCCCUCCGACGGCCCCGUAAUGCAGAAGAAGACCAUGGGCUGGGAGGCCUCCUCCGAGCGGAUGUACCCCGAGGACGGCGCCCUGAAGGGCGAGAUCAAGCAGAGGCUGAAGCUGAAGGACGGCGGCCACUACGACGCUGAGGUCAAGACCACCUACAAGGCCAAGAAGCCCGUGCAGCUGCCCGGCGCCUACAACGUCAACAUCAAGUUGGACAUCACCUCCCACAACGAGGACUACACCAUCGUGGAACAGUACGAACGCGCCGAGGGCCGCCACUCCACCGGCGGCAUGGACGAGCUGUACAAGUAGUAA

[0385] Nt-uDys(SEQ ID NO:22)

[0386]

[0387] Ct-uDys-GFP(SEQ ID NO:23)

[0388]

[0389] Nt-miniDys(ΔH2-R15)(SEQ ID NO:129)

[0390]

[0391] Ct-miniDys(ΔH2-R15)(SEQ ID NO:130)

[0392]

[0393] Ribozyme nucleic acid sequences for scarless 3' RNA cleavage

[0394] HDV68 (SEQ ID NO:9)

[0395] GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGGCGAAUGGGAC

[0396] HDV68 Catalytic Mutant (SEQ ID NO:24)

[0397] 5'-GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGGUGAAUGGGAC-3'

[0398] HDV67 (SEQ ID NO:10)

[0399] GGGUCGGCAUGGCAUCUCCACCUCCUCGCGGUCCGACCUGGGCUACUUCGGUAGGCUAAGGGAGAAG

[0400] HDV56 (SEQ ID NO:11)

[0401] GAGGGAUAGUACAGAGCCUCCCCGUGGCUCCCUUGGAUAACCAACUGAUACUGUAC

[0402] Genomic HDV (genHDV) (SEQ ID NO:12)

[0403] GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUUCCGAGGGGACCGUCCCCUCGGUAAUGGCGAAUGGGACCCA

[0404] Antigenomic HDV (anti-HDV) (SEQ ID NO:13)

[0405] GGGUCGGCAUGGCAUCUCCACCUCCUCGCGGUCCGACCUGGGCAUCCGAAGGAGGACGCACGUCCACUCGGAUGGCUAAGGGAGAGCCACU

[0406] VS Ribozyme (SEQ ID NO:14)[[ID= forty-two]]

[0407] GCGGUAGUAAGCAGGGAACUCACCUCCAAUUUCAGUACUGAAAUUGUCGUAGCAGUUGACUACUGUUAUGUGAUUGGUAGAGGCUAAGUGACGGUAUUGGCGUAAGUCAGUAUUGCAGCACAGCACAAGCCCGCUUGCGAGAAU

[0408] VS-S (SEQ ID NO: 15)

[0409] GAAGGGCGUCGUCGCCCCGAG

[0410] VS-Rz (SEQ ID NO: 16)

[0411] GCGGUAGUAAGCAGGGAACUCACCUCCAAUUUCAGUACUGAAAUUGUCGUAGCAGUUGACUACUGUUAUGUGAUUGGUAGAGGCUAAGUGACGGUAUUGGCGUAAGUCAGUAUUGCAGCACAGCACAAGCCCGCUUGCGAGAAU

[0412] Hammerhead ribozyme with stem 3 overhang specific for Nt-Luc (SEQ ID NO: 25)

[0413] 5'-GAGCCUUACCGGAUGUGUUUUCCGGUCUGAUGAGUCCGGUAGCGGACGAAAGGCUC-3'

[0414] Twister ribozyme with 5nt P1 stem for Ct-Luc (SEQ ID NO: 26)

[0415] 5'-AGCCUUAACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGAGGCU-3'

[0416] Twister ribozyme with 5nt P1 stem for Ct-Luc and T6A mutations (SEQ ID NO: 27)

[0417] 5'-AGCCUAAACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGAGGCU-3'

[0418] Twister ribozyme mutant with 5nt P1 stem for Ct-Luc (SEQ ID NO: 28)

[0419] 5'-AGCCUUAACUCUUCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGAGGCU-3'

[0420] Twister ribozyme with 5 nt P1 stem for Ct-Luc (SEQ ID NO: 29) 5'-AGCCUUAACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGAGGCU-3'

[0421] Twister ribozyme with 2nt P1 stem for Ct-Luc (SEQ ID NO: 30)

[0422] 5'-AGCCUUAACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGAG-3'

[0423] Twister ribozyme with 1 nt P1 stem for Ct-Luc (SEQ ID NO: 31) 5'-AGCCUUAACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGG-3'

[0424] Twister ribozyme without the P1 stem for Ct-Luc (SEQ ID NO: 32) 5'-AGCCUUAACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGG-3'

[0425] Hammerhead (HH) ribozyme for 3' (SEQ ID NO: 105)

[0426] 5'NNNNDWHACCGGAUGUGUUUUCCGGUCUGAUGAGUCCGGUAGCGGACGAAWHNNNN 3'

[0427] Twister ribozyme WT with 5nt P1 stem (SEQ ID NO: 106)

[0428] 5'NNNNNUAACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGNNNNN 3'

[0429] Twister ribozyme mutant with a 5nt P1 stem (SEQ ID NO: 107)

[0430] 5'NNNNNUAACUCUUCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGNNNNN 3'

[0431] Twister ribozyme with a 5nt P1 stem with a U1A mutation (SEQ ID NO: 108)

[0432] 5'NNNNNAAACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGNNNNN 3'

[0433] Twister ribozyme with a 5nt P1 stem with a U1C mutation (SEQ ID NO: 109)

[0434] 5'NNNNNNCAACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGNNNNN 3'

[0435] Twister ribozyme with a 5nt P1 stem with a U1G mutation (SEQ ID NO: 110)

[0436] 5'NNNNNGAACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGNNNNN 3'

[0437] Ribozyme nucleic acid sequences for scarless 5' RNA cleavage

[0438] Hammerhead (HH) ribozyme with stem 1 overhang specific for Ct-Luc

[0439] 16HH (SEQ ID NO: 33)

[0440] 5'-GAAUCUUGUAAUCCUGCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC-3'

[0441] 14HH (SEQ ID NO: 34)

[0442] 5'-AUCUUGUAAUCCUGCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC-3'

[0443] 12HH (SEQ ID NO: 35)

[0444] 5'-CUUGUAAUCCUGCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC-3'

[0445] 8HH (SEQ ID NO: 36)

[0446] 5'-UAAUCCUGCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC-3'

[0447] 6HH (SEQ ID NO: 37)

[0448] 5'-AUCCUGCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC-3'

[0449] 6HH mutant (SEQ ID NO: 38)

[0450] 5'-AUCCUGCUGAUGAGUCCGUGAGGACGAGACGAGUAAGCUCGUC-3'

[0451] 4HH (SEQ ID NO: 39)

[0452] 5'-CCUGCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC-3'

[0453] Hammerhead ribozyme 4nt overhang for 5' (SEQ ID NO: 111)

[0454] 5'NNNNCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC3'

[0455] Hammerhead ribozyme 6 nt overhang for 5' (SEQ ID NO: 112)

[0456] 5'NNNNNNCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC 3'

[0457] Hammerhead ribozyme 8 nt overhang for 5' (SEQ ID NO: 113)

[0458] 5'NNNNNNNNCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC 3'

[0459] Hammerhead ribozyme 10 nt overhang for 5' (SEQ ID NO: 114)

[0460] 5'NNNNNNNNNNCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC 3'

[0461] Hammerhead ribozyme 12 nt overhang for 5' (SEQ ID NO: 115)

[0462] 5'NNNNNNNNNNNNCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC 3'

[0463] Hammerhead ribozyme 14 nt overhang for 5' (SEQ ID NO: 116)

[0464] 5'NNNNNNNNNNNNNNCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC 3'

[0465] Hammerhead ribozyme 16 nt overhang for 5' (SEQ ID NO: 117)

[0466] 5'NNNNNNNNNNNNNNNNCUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC 3'

[0467] TX2 hammerhead ribozyme 4nt overhang for 5' (Huang et al. 2019) (SEQ ID NO: 118)

[0468] 5'NNNNCUGAUGAGUCCGGUAGCGGACGAAACGCGCUUCGGUGCGUC 3'

[0469] TX2 hammerhead ribozyme 6 nt overhang for 5' (Huang et al., 2019) (SEQ ID NO: 119)

[0470] 5'NNNNNNCUGAUGAGUCCGGUAGCGGACGAAACGCGCUUCGGUGCGUC 3'

[0471] TX2 hammerhead ribozyme 8 nt overhang for 5' (Huang et al. 2019) (SEQ ID NO: 120)

[0472] 5'NNNNNNNNCUGAUGAGUCCGGUAGCGGACGAAACGCGCUUCGGUGCGUC 3'

[0473] TX2 hammerhead ribozyme 10 nt overhang for 5' (Huang et al. 2019) (SEQ ID NO: 121)

[0474] 5'NNNNNNNNNNCUGAUGAGUCCGGUAGCGGACGAAACGCGCUUCGGUGCGUC 3'

[0475] TX2 hammerhead ribozyme 12 nt overhang for 5' (Huang et al. 2019) (SEQ ID NO: 122)

[0476] 5'NNNNNNNNNNNNCUGAUGAGUCCGGUAGCGGACGAAACGCGCUUCGGUGCGUC 3'

[0477] TX2 hammerhead ribozyme 14 nt overhang for 5' (Huang et al. 2019) (SEQ ID NO: 123)

[0478] 5'NNNNNNNNNNNNNNCUGAUGAGUCCGGUAGCGGACGAAACGCGCUUCGGUGCGUC 3'

[0479] TX2 hammerhead ribozyme 16 nt overhang for 5' (Huang et al. 2019) (SEQ ID NO: 124)

[0480] 5'NNNNNNNNNNNNNNNNCUGAUGAGUCCGGUAGCGGACGAAACGCGCUUCGGUGCGUC 3'

[0481] RzB hammerhead ribozyme for 5' (Saksmerprome et al., 2004) (SEQ ID NO: 125)

[0482] 5'NNNNNNUAANNNNNCUGAUGAGUCGCUGGGAUGCGAGAAACGCCUUCGGGCGUC 3'

[0483] RzB (Saksmerprome et al., 2004), with a stem 1 overhang specific for Ct-Luc (SEQ ID NO: 40)

[0484] 5'-UUGUAAUAAUCCUGCUGAUGAGUCGCUGGGAUGCCGACGAAACGCCUUCGGGCGUC-3'

[0485] Splice donor sequence of Nt vector (SEQ ID NO:41)

[0486] 5'-GUAAGUAUCAAGGUUACAAGACAGGUUUAAGGAGACCAAUAGAAACUGGGCU-3'

[0487] Splice acceptor sequence of the Ct vector (SEQ ID NO: 42)

[0488] 5’-UGUCGAGACAGAGAAGACUCUUGCGUUUCUGAUAGGCACCUAUUGGUCUUACUGACAUCCACUUUGCCUUUCUCUCCACAG-3’

[0489] Translational regulatory sequence of Ct vector

[0490] GCN4 5'UTR uORF (Zhang and Hinnebusch 2011) (SEQ ID NO: 43)

[0491] 5’-AAACAAAAACUCACAACACAGGUUACUCUCCCCCCUAAAUUCAAAUUUUUUUUGCCCAUCAGUUUCACUAGCGAAUUAUACAACUCACCAGCCACACAGCUCACUCAUCUACUUCGCAAUCAAAACAAAAUAUUUUAUUUUAGUUCAGUUUAUUAAGUUAUUAUCAGUAUCGUAUUAAAAAAUUAAAGAUCAUUGAAAAAUGGCUUGCUAAACCGAUUAUAUUUUGUUUUUAAAGUAGAUUAUUAUUAGAAAAUUAUUAAGAGAAUUAUGUGUUAAAUUUAUUGAAAGAGAAAAUUUAUUUUCCCUUAUUAAUUAAAGUCCUUUACUUUUUUUGAAAACUGUCAGUUUUUUGAAGAGUUAUUUGUUUUGUUACCAAUUGCUAUCAUGUACCCGUAGAAUUUUAUUCAAGAUGUUUCCGUAACGGUUACCUUUCUGUCAAAUUAUCCAGGUUUACUCGCCAAUAAAAAUUUCCCUAUACUAUCAUUAAUUAAAUCAUUAUUAUUACUAAAGUUUUGUUUACCAAUUUGUCUGCUCAAGAAAAUAAAUUAAAUACAAAUAAA-3’

[0492] sGCN4 5'UTR uORF (SEQ ID NO: 104)

[0493] UUAAAGAUCAUUGAAAAAUGGCUUGCUAAACCGAUUAUAUUUUGUUUUUAAAGUAGAUUAUUAUUAGAAAAUUAUUAAGAGAAUUAUGUGUUAAAUUUAUUGAAAGAGAAAAUUUAUUUUCCCUUAUUAAUUAAAGUCCUUUACUUUUUUUGAAAACUGUCAGUUUUUUGAAGAGUUAUUUGUUUUGUUACCAAUUGCUAUCAUGUACCCGUAGAAUUUUAUUCAAGAUGUUUCCGUAACGGUUACCU

[0494] SRY 5'UTR uORF (Calvo et al., 2009) (SEQ ID NO:44)

[0495] 5’-GUUGAGGGGGUGUUGAGGGCGGAGAAAUGCAAGUUUCAUUACAAAAGUUAACGUAACAAAGAAUCUGGUAGAAAUGAGUUUUGGAUAGUAAAAUAAGUUUCGAACUCUGGCACCUUUCAAUUUUGUCGCACUCUCCUUGUUUUUGACA-3’

[0496] Hoxa9 TIE (Leppek et al., 2020) (SEQ ID NO:45)

[0497] 5'-GAAAAAACAGAAGAGGGAAGGAUACCAGAGCGGUUCAUACAGGGCCCAGAAACUAGGCGAGGUGACCCCUCAGCAAGACAAACACCUCUUGAUGUUGACUGGCGAUUUUCCCCAUCUCCAGUCUGGGGAGCGGGACUAGGCAUACAGAUGAUGGAGCUUAGAACCCGCUGGCUAGGGAAUAAAAUUCGCUGGGCAGUUUGUGCUCAAAGAAGUGGGCCAGGGCGCUUGUGACACAAUCAGGGCGUUUGUGACACAAACCCUUGAGGGUUGGCAGUUCUCUCCUUGGCGGUUGCUCUGGUUGCUCUGUGGGGCCUUCCCUGUGGAGCAAGGGUGAUCUGGCCGA-3'

[0498] Hoxa3 TIE (Leppek et al., 2020) (SEQ ID NO:46)

[0499] 5’-AGGACAAUUCGUCUCUUGGGCUGCCGAAGCGACAGCUGUCAGAGAGGCAGAAGCUUCUGGGAGCCGCGGUCUGAAGGCUACGUGUGCUGCCUGGUCAUUCAAAGUGUCAAUUUUAGGUCCAGAAGUGUCCAAACCACAAGUUCUCAAAACUCUGAAAAAUGGCUCCCUCC-3’

[0500] NRAS 5'UTR G-quadruplex (Kumari et al., 2007) (SEQ ID NO:47)

[0501] 5'-CGUCCCGUGUGGGAGGGGCGGGUCUGGGUGCGGCCUGC-3'

[0502] Human IFNG 5'UTR pseudoknot (Kaempfer 2006) (SEQ ID NO:48)

[0503] CACAUUGUUCUGAUCAUCUGAAGAUCAGCUAUUAGAAGAGAAAGAUCAGUUAAGUCCUUUGGACCUGAUCAGCUUGAUACAAGAACUACUGAUUUCAACUUCUUUGGCUUAAUUCUCUCGGAAACG

[0504] Rat ODC 5'UTR (Manzella and Blackshear 1990) (SEQ ID NO:49)

[0505] 5'-UGUCAGUCCCUGCAGCCGCCGCCGCCGGCCGCCUUCAGUCAGCAGCUCGGCGCCACCUCCGGUCGGCGACUGCGGCGGGCUCGACGAGGCGGCUGACGGGGCGGCGGCGGGAAGACGGCCGGGUGCGCCUUG-3'

[0506] RNA nuclear localization signal

[0507] SIRLOIN RNA nuclear localization signal (Lubelsky and Ulitsky 2018) (SEQ ID NO:50)

[0508] 5'-CGCCUCCCGGGUUCAAGCGAUUCUCCUGCCUCAGCCUCCCGAGUAGCUG-3'

[0509] BORG lncRNA NLS(Zhang Co.,2014)(SEQ ID NO:51)

[0510] 5'-ACCCAGAAUCUACAAGUCAGCCCCAAUUAAAUGUUUUUUA-3

[0511] Protein degradation amino acid sequence

[0512] N-terminal and C-terminal protein degradation sequences of Nt or Ct vectors

[0513] FKBP DD(Banaszynski Publication,2006)(SEQ ID NO:52)

[0514] MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKPE

[0515] C-terminal protein degradation sequence

[0516] PEST(Old ODC PEST)(Li, 1998)(SEQ ID NO:53)

[0517] SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV*

[0518] ODC PEST(vaccine)(Rogers Drug,1986)(SEQ ID NO:54)

[0519] SHGFPPEVEEQDDGTLPMSCAQESGMDRHPACASARINV*

[0520] ODC PEST(S)(SEQ ID NO:55)

[0521] NPDFPPEVEEQDASTLPVSCAWESGMKRHRAACASASINV*

[0522] CL1(Gilon Protein,1998)(SEQ ID NO:56)

[0523] ACKNWFSSLSHFVIHLNSHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV*

[0524] CL1-PEST(SEQ ID NO:57)

[0525] ACKNWFSSLSHFVIHLNSHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV*

[0526] E1A PEST(Rogers Infection,1986)(SEQ ID NO:58)

[0527] SRECNSSTDSCDSGPSNTPPEIHPVVPLCPIKPVAVRVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP*

[0528] C-myc PEST (Rogers Syndrome, 1986) (SEQ ID NO:59)

[0529] LHEETPPTTSSDSEEEQEDEEIDVVVKR

[0530] c-Fos PEST(Rogers Laboratory,1986)(SEQ ID NO:60)

[0531] AAHRKGSSSNEPSSDSLSSPTLLAL

[0532] v-Myb PEST(Rogers Code,1986)(SEQ ID NO:61)

[0533] PSPPVDHGCLPEESASPARCMIVHQS

[0534] NPDC1 PEST(SEQ ID NO:62)

[0535] PPKELDTASSDEENEDGDFTVYECPGLAPTSAVERPLFDHALSAPLPAPSSPPALP

[0536] IkBa PEST (Shumway Healthcare, 1999) (SEQ ID NO:63)

[0537] PESEDEESYDTESEFTEFTEDELPYDDCVFGGQRLTL

[0538] mmAZIN2 PEST(Lambertos&Penafiel 2019)(SEQ ID NO:64)

[0539] GQLLPAEEDQDAEGVCKPLSCGWEITDTLCVGPVFTPASIM*

[0540] xlAZIN2 PEST(Lambertos&Penafiel 2019)(SEQ ID NO:65)

[0541] VQLLQRGLQQTEEKENVCTPMSCGWEISDSLCFTRTFAATSII*

[0542] C-terminal degrons directed by the CRL2 ubiquitin ligase (Lin et al., 2018)

[0543] NS1(SEQ ID NO:66)

[0544] TSLYKKVGMGRK*

[0545] NS6(SEQ ID NO:67)

[0546] SLYKKWGTMAAG*

[0547] NS7(SEQ ID NO:68)

[0548] YKKVGTMRGRGL*

[0549] NS12(SEQ ID NO:69)

[0550] ERAPTGRWGRRG*

[0551] NS15(SEQ ID NO:70)

[0552] EGPLWHPRICGS*

[0553] SELK(SEQ ID NO:71)

[0554] LRGPSPPPMAGG*

[0555] SELS(SEQ ID NO:72)

[0556] WRPGRRGPSSGG*

[0557] C-terminal degrons directed by E3 ubiquitin ligases (Koren et al., 2018)

[0558] EMID1(SEQ ID NO:73)

[0559] RDERG*

[0560] IRX6(SEQ ID NO:74)

[0561] GAEAG*

[0562] Ubiquitin Degrons (Chassin et al., 2019)

[0563] UbVR (SEQ ID NO: 75)

[0564] QIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGVRASAS

[0565] 2xUbVR (SEQ ID NO: 76)

[0566] TSQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGVRASASQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGVRASAS

[0567] Sequences that mimic translation through polyA tails

[0568] 12x poly K coding tail sequence (SEQ ID NO: 77)

[0569] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0570] Translation product 12x poly K (SEQ ID NO: 78)

[0571] KKKKKKKKKKKK*

[0572] 16x poly K coding tail sequence (SEQ ID NO: 79)

[0573] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATAA

[0574] Translation product 16x poly K (SEQ ID NO: 80)

[0575] KKKKKKKKKKKKKKK*

[0576] Enzymes for enhancing or suppressing ribozyme-mediated trans-splicing

[0577] Human RtcB protein sequence (SEQ ID NO:81)

[0578] MSRSYNDELQFLEKINKNCWRIKKGFVPNMQVEGVFYVNDALEKLMFEELRNACRGGGVGGFLPAMKQIGNVAALPGIVHRSIGLPDVHSGYGFAIGNMAAFDMNDPEAVVSPGGVGFDINCGVRLLRTNLDESDVQPVKEQLAQAMFDHIPVGVGSKGVIPMNAKDLEEALEMGVDWSLREGYAWAEDKEHCEEYGRMLQADPNKVSARAKKRGLPQLGTLGAGNHYAEIQVVDEIFNEYAAKKMGIDHKGQVCVMIHSGSRGLGHQVATDALVAMEKAMKRDKIIVNDRQLACARIASPEGQDYLKGMAAAGNYAWVNRSSMTFLTRQAFAKVFNTTPDDLDLHVIYDVSHNIAKVEQHVVDGKERTLLVHRKGSTRAFPPHHPLIAVDYQLTGQPVLIGGTMGTCSYVLTGTEQGMTETFGTTCHGAGRALSRAKSRRNLDFQDVLDKLADMGIAIRVASPKLVMEEAPESYKNVTDVVNTCHDAGISKKAIKLRPIAVIKG*

[0579] Human RtcB human codon-optimized nucleic acid sequence (SEQ ID NO:82)

[0580]

[0581] Escherichia coli RtcB protein sequence (SEQ ID NO: 83)

[0582] MNYELLTTENAPVKMWTKGVPVEADARQQLINTAKMPFIFKHIAVMPDVHLGKGSTIGSVIPTKGAIIPAAVGVDIGCGMNALRTALTAEDLPENLAELRQA IETAVPHGRTTGRCKRDKGAWENPPVNVDAKWAELEAGYQWLTQKYPRFLNTNNYKHLGTLGTGNHFIEICLDESDQVWIMLHSGSRGIGNAIGNAIGTYFIDLAQ KEMQETLETLPSRDLAYFMEGTEYFDDYLKAVAWAQLFASLNRDAMMENVVTALQSITQKTVRQPQTLAMEEINCHHNYVQKEQHFGEEIYVTRKGAVSARA GQYGIIPGSMGAKSFIVRGLGNEESFCSCSHGAGRVMSRTKAKKLFSVEDQIRATAHVECRKDAEVIDEIPMAYKDIDAVMAAQSDLVEVIYTLRQVVCVKG

[0583] Escherichia coli RtcB human codon-optimized nucleic acid sequence (SEQ ID NO: 84)

[0584]

[0585] Deinococcus radiodurans RtcB protein sequence (SEQ ID NO: 85)

[0586] MNGKHITKLGFEGKAVGLALSAAGLREDAGVSRGDILDELRSVQNYPEQYQGGGVYADLATHLIEQQAAQQTRQSAKLRAAPLPYRTWGEDLIEPGAHRQMDVAMQLPISRAGALMP DAHVGYGLPIGGVLATENAVIPYGVGVDIGCSMMLSVFPVAATGLSVDEARSLLLKHTRFGAGVGFEKRRDRLDHPVLAEATWDEQPLLRHLFDKAAGQIGSSGSGNHFVEFGTFTLAQ ADPQLEGLLDPGEYLAVLSHSGSRGFGAQVAGHFTNLAQRLWPALDKEAQKLAWLPLDSEAGQAYWQAMNLAGRYALANHEQIHARLARALGEKPLLRAQNSHNLAWKQQVNGQELIV HRKGATPAEAGQLGLIPGSMADPGYLVRGRGNPEALASASHGAGRQLGRKAAERSLAKKDVQAYLKDRGVTLIGGGIDEAPQAYKRIEDVIARQRDLVDVLGEFRPRVVRMDTGSEDV

[0587] Deinococcus radiodurans RtcB human codon-optimized nucleic acid sequence (SEQ ID NO: 86)

[0588]

[0589] Pyrococcus horikoshii RtcB protein sequence (SEQ ID NO: 87)

[0590] MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLV DTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGSGNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLG HQVASDYLRIMERAIRKYRIPWPDRELLVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGH EAVPRLYRDVGQPVLIPGSMGTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYKNVDNVVKVVSEAGIAKLVARMRPIGVAKG*

[0591] Pyrococcus horikoshii RtcB human codon-optimized nucleic acid sequence (SEQ ID NO: 88)

[0592]

[0593] Pyrococcus sp. ST04 RtcB protein sequence (SEQ ID NO: 89)

[0594] MTVPLKRIDRIRWEIPKFDKRMRVPGRVYADEVLIEKMRSDRTLEQAANVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPKIKQLV DTLFKNVPSGVGSQGRIRLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPDAVSQRAKQRGAPQLGSLGSGNHFLEVQVVDKIYDEEVAKAYGLFEGQVVVMVHTGSRGLG HQVASDYLRIMERAIRKYRIPWPDRELLVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFRQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKKVTVIVHRKGATRAFPPGH EAIPRIYRDVGQPVLIPGSMGTASYVLAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRNELLQRGIYVRAASMRVVAEEAPGAYKNVDNVVKVVSEAGIAKLVARMRPIGVAKG*

[0595] Pyrococcus sp. ST04 RtcB human codon-optimized nucleic acid sequence (SEQ ID NO: 90)

[0596]

[0597] Thermococcus sp. EP1 RtcB protein sequence (SEQ ID NO: 91)

[0598] MEIPLKRLDKIRWEIPKFNRRMRVPGRVYADDTLLQKMRQDKTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGVGYDINCGVRLIRTNLVEKEVRPKIKQLI DTLFKNVPSGLGSKGRIRLHWTQLDDVLADGAKWAVDNGYGWKDDLEHLEEGGRMEGANPNAVSQKAKQRGAPQLGSLGSGNHFLEIQVVDKVFNEEIAKAYGLFEGQIVVMVHTGSRGL GHQVASDYLRIMEKANRKYNVPWPDRELVSVPFQTEEGQRYFSAMKAAANFAWANRQMITHWVRESFEEVFKQKAEDLGMHIVYDVAHNIAKVEEHEVNGRKIKVVVHRKGATRAFPAGH EAIPKAYRDVGQPVLIPGSMGTASYVLAGAEGSMRETFGSTCHGAGRVLSRHAATRQFRGDRLRNELMQRGIYIRAASMRVVAEEAPGAYKNVDNVVRVVHEAGIANLVARMRPIGVAKG*

[0599] Pyrococcus sp. EP1 RtcB human codon-optimized nucleic acid sequence (SEQ ID NO: 92)

[0600]

[0601] Archaease protein sequence of human (SEQ ID NO:93)

[0602] MAQEEEDVRDYNLTEEQKAIKAKYPPVNRKYEYLDHTADVQLHAWGDTLEEAFEQCAMAMFGYMTDTGTVEPLQTVEVETQGDDLQSLLFHFLDEWLYKFSADEFFIPREVKVLSIDQRNFKLRSIGWGEEFSLSKHPQGTEVKAITYSAMQVYNEENPEVFVIIDI*

[0603] Nucleic acid sequence of codon-optimized Archaease of human (SEQ ID NO:94)

[0604] AGGAACAAAAGGCCATCAAAGCGAAATATCCGCCTGTAAACCGAAAGTATGAGTACCTGGATCACACTGCGGACGTCCAGTTGCATGCCTGGGGCGACACTCTGGAGGAGGCATTCGAACAATGTGCAATGGCAATGTTTGGCTACATGACTGATACAGGCACAGTGGAGCCCCTTCAAACGGTAGAGGTAGAAACTCAGGGAGAtGATCTTCAGAGCTTGCTCTTCCATTTTCTCGACGAATGGTTGTATAAGTTCAGTGCCGACGAGTTcTTCATTCCACGCGAAGTGAAAGTGCTGAGTATTGATCAGAGAAACTTTAAACTTAGGTCTATTGGGTGGGGTGAAGAGTTCTCTTTGTCTAAACACCCTCAAGGAACTGAGGTAAAGGCGATAACTTACTCAGCCATGCAGGTATATAACGAGGAGAATCCTGAGGTTTTCGTAATCATTGATATATAG

[0605] Archaease protein sequence of Pyrococcus horikoshii (SEQ ID NO:95)

[0606] MKKWEHYEHTADIGYGDSLEEAFEAVAIALFDVMVNVNKVEKVREIEVEAEDLEALLYSFLEELLVIHDIEGLVFRDFEVKIERVNGKYRLRAKAYGEKLDLKKHEPKEEVKAITYHDMKIERLPNGKWMAQLVPDI*

[0607] Archease-derived cytokines (SEQ ID NO:96)

[0608] ATGAAGAAATGGGAGCACTATGAGCATACTGCCGACATTGGTATTCGGGGATATGGGGATAGCCTTGAGGAGGCATTCGAAGCAGTAGCCATCGCGCTCTTTGATGT AATGGTGAACGTGAATAAAGTCGAGAAGAAGGAAGTCCGAGAAATTGAAGTGGAGGCAGAAGATTTGGAGGCCCTCCTTTATTCATTCCTGGAAGAACTGTTGGTTA TTCATGATATAGAGGGACTGGTTTTCAGGGACTTTGAAGTTAAGATAGAGAGAGTAAATGGCAAATACCGACTTCGAGCGAAAGCCTACGGTGAGAAGCTTCGACCTC AAGAAGCACGAACCGAAAGAGGAAGTAAGGCGATAACCTACCATGATATGAAATTGAACGGTTGCCCAATGGAAAGTGGATGGCTCAACTCGTTCCAGATATTTAG

[0609] T4 PNK (SEQ ID NO:97)

[0610] MKKIILTIGCPGSGKSTWAREFIAKNPGFYNINRDDYRQSIMAHEERDEYKYTKKKEGIVTGMQFDTAKSILYGGDSVKGVIISDTNLNPERRLAWETFAKEYGWKVEHKVFDVPWTELVKRNSKRGTKAVPIDVLRSMYKSMREYLGLPVYNGTPGKPKAVIFDVDGTLAKMNGRGPYDLEKCDTDVINPMVVELSKMYALMGYQIVVVSGRESGTKEDPTKYYRMTRKWVEDIAGVPLVMQCQREQGDTRKDDVVKEEIFWKHIAPHFDVKLAIDDRTQVVEMWRRIGVECWQVASGDF*

[0611] Nucleic acid sequence optimized for codons of T4 PNK (SEQ ID NO:98)

[0612] ATGAAGAAAATTATACTTACAATCGGATGCCCTGGTAGTGGTAAGAGCACTTGGGCGAGGGAATTTATTGCGAAgAACCCtGGATTTTATAATATCAATCGAGACGACTACCGGCAGTCTATTATGGCCCACGAGGAACGAGACGAATACAAGTATACCAAGAAGAAAGAAGGGATTGTCACGGGTATGCAATTTGACACCGCCAAATCAATACTGTACGGAGGTGATTCAGTCAAAGGCGTTATCATATCAGACACTAACCTCAATCCTGAACGCCGATTGGCATGGGAAACATTTGCGAAGGAATACGGTTGGAAGGTTGAACACAAGGTGTTCGATGTCCCGTGGACCGAACTGGTAAAACGCAATTCTAAACGAGGCACTAAAGCTGTGCCCATTGACGTACTTCGAAGTATGTACAAGTCCATGAGAGAGTACCTGGGGCTTCCCGTCTATAACGGTACGCCGGGCAAACCGAAGGCGGTGATCTTTGACGTAGATGGGACTCTGGCGAAGATGAATGGTCGCGGACCATACGATTTGGAAAAATGTGACACAGATGTAATCAACCCAATGGTAGTAGAGCTTAGCAAGATGTACGCATTGATGGGcTACCAAATTGTCGTGGTGTCCGGGCGGGAGTCAGGCACAAAAGAAGATCCGACGAAGTATTATCGCATGACACGGAAATGGGTCGAAGATATAGCCGGGGTgCCTCTCGTTATGCAATGTCAACGAGAACAGGGCGACACACGGAAGGATGACGTAGTGAAGGAGGAAATTTTCTGGAAGCATATAGCGCCACACTTTGACGTTAAGCTCGCCATCGACGACCGAACTCAGGTGGTCGAGATGTGGCGACGAATTGGCGTAGAGTGTTGGCAAGTTGCATCTGGAGATTTTTAG

[0613] Escherichia coli thpR protein sequence (SEQ ID NO:99)

[0614] MSEPQRLFFAIDLPAEIREQIIHWRATHFPPEAGRPVAADNLHLTLAFLGEVSAEKEKALSLLAGRIRQPGFTLTLDDAGQWLRSRVVWLGMRQPPRGLIQLANMLRSQAARSGCFQSNRPFHPHITLLRDASEAVTIPPPGFNWSYAVTEFTLYASSFARGRTRYTPLKRWALTQ*

[0615] Nucleic acid sequence of Escherichia coli thpR optimized for human codons (SEQ ID NO:100)

[0616] ATGAGTGAGCCTCAACGATTGTTCTTTGCCATAGATTTGCCTGCTGAAATTAGAGAGCAAATTATCCATTGGAGAGCCACCCATTTCCCCCCAGAAGCTGGACGACCAGTCGCAGCGGACAACCTCCACCTTACACTGGCGTTCTTGGGTGAAGTGAGCGCCGAGAAAGAGAAAGCTCTCTCACTTCTGGCTGGGAGGATTCGGCAGCCGGGCTTTACCCTTACTCTGGATGATGCCGGCCAGTGGCTGAGGTCCAGGGTTGTCTGGCTCGGAATGAGGCAACCACCTAGGGGGCTCATCCAGCTCGCCAATATGCTGAGATCCCAGGCCGCAAGGTCTGGCTGCTTCCAATCAAACAGGCCATTCCACCCGCATATTACCTTGCTCAGAGATGCCTCCGAGGCAGTAACTATTCCACCTCCCGGCTTTAACTGGAGTTACGCCGTCACAGAATTTACTCTGTACGCCTCCAGCTTCGCCCGAGGGAGAACCAGGTACACGCCTTTGAAGCGGTGGGCCTTGACCCAGTAG

[0617] Human PNKP protein sequence (SEQ ID NO:101)

[0618] MGEVEAPGRLWLESPPGGAPPIFLPSDGQALVLGRGPLTQVTDRKCSRTQVELVADPETRTVAVKQLGVNPSTTGTQELKPGLEGSLGVGDTLYLVNGLHPLTLRWEETRTPESQPDTPPGTPLVSQDEKRDAELPKKRMRKSNPGWENLEKLLVFTAAGVKPQGKVAGFDLDGTLITTRSGKVFPTGPSDWRILYPEIPRKLRELEAEGYKLVIFTNQMSIGRGKLPAEEFKAKVEAVVEKLGVPFQVLVATHAGLYRKPVTGMWDHLQEQANDGTPISIGDSIFVGDAAGRPANWAPGRKKKDFSCADRLFALNLGLPFATPEEFFLKWPAAGFELPAFDPRTVSRSGPLCLPESRALLSASPEVVVAVGFPGAGKSTFLKKHLVSAGYVHVNRDTLGSWQRCVTTCETALKQGKRVAIDNTNPDAASRARYVQCARAAGVPCRCFLFTATLEQARHNNRFREMTDSSHIPVSDMVMYGYRKQFEAPTLAEGFSAILEIPFRLWVEPRLGRLYCQFSEG*

[0619] Human PNKP human codon-optimized nucleic acid sequence (SEQ ID NO: 102)

[0620]

[0621] GFP with an internally synthesized ribozyme intron with and without cargo

[0622] NtGFP-HDV-HH-CtGFP (SEQ ID NO:103)

[0623] AUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUggccggcauggucccagccuccucgcuggcgccggcugggcaacaugcuucggcauggcgaaugggaccccgggacauaacuaguuaaaccaaauccuugcugaugaguccgugaggacgaaacgaguaagcucgucCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAG

[0624] NtGFP-HDV-CARGO-HH-CtGFP (SEQ ID NO:126)

[0625] AUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUggccggcauggucccagccuccucgcuggcgccggcugggcaacaugcuucggcauggcgaaugggacNuccuugcugaugaguccgugaggacgaaacgaguaagcucgucCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAG

[0626] NtGFP-HDV (SEQ ID NO: 127)

[0627] AUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUggccggcauggucccagccuccucgcuggcgccggcugggcaacaugcuucggcauggcgaaugggac

[0628] HH-CtGFP (SEQ ID NO: 128)

[0629] uccuugcugaugaguccgugaggacgaaacgaguaagcucgucCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAG

[0630] The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be interpreted as including all such embodiments and equivalent variations. Sequence Listing <110> University of Rochester Douglas Matthew Anderson <120> Ribozyme-mediated RNA assembly and expression <130> 204606-0127-00WO <150> US 62 / 971,356 <151> 2020-02-07 <160> 130 <170> PatentIn version 3.5 <210> 1 <211> 302 <212> RNA <213> Artificial sequence <220> <223> Nt-GFP <400> 1 auggugagca agggcgagga gcuguucacc gggguggugc ccauccuggu cgagcuggac 60 ggcgacguaa acggccacaa guucagcgug uccggcgagg gcgagggcga ugccaccuac 120 ggcaagcuga cccugaaguu caucugcacc accggcaagc ugcccgugcc cuggcccacc 180 cucgugacca cccugaccua cggcgugcag ugcuucagcc gcuaccccga ccacaugaag 240 cagcacgacu ucuucaaguc cgccaugccc gaaggcuacg uccaggagcg caccaucuuc 300 uu 302 <210> 2 <211> 421 <212> RNA <213> Artificial sequence <220> <223> Ct-GFP <400> 2 caaggacgac ggcaacuaca agacccgcgc cgaggugaag uucgagggcg acacccuggu 60 gaaccgcauc gagcugaagg gcaucgacuu caaggaggac ggcaacaucc uggggcacaa 120 gcuggaguac aacuacaaca gccacaacgu cuauaucaug gccgacaagc agaagaacgg 180 caucaaggug aacuucaaga uccgccacaa caucgaggac ggcagcgugc agcucgccga 240 ccacuaccag cagaacaccc ccaucggcga cggccccgug cugcugcccg acaaccacua 300 ccugagcacc caguccgccc ugagcaaaga ccccaacgag aagcgcgauc acaugguccu 360 gcuggaguuc gugaccgccg ccgggaucac ucucggcaug gacgagcugu acaaguagua 420 a 421 <210> 3 <211> 831 <212> RNA <213> Artificial sequence <220> <223> Nt-Luciferase <400> 3 auggaagacg ccaaaaacau aaagaaaggc ccggcgccau ucuauccgcu ggaagaugga 60 accgcuggag agcaacugca uaaggcuaug aagaguaacg cccugguucc uggaacaauu 120 gcuuuuacag augcacauau cgagguggac aucacuuacg cugaguacuu cgaaaugucc 180 guucgguugg cagaagcuau gaacgauau gggcugaaua caaaucacag aaucgucgua 240 ugcagugaaa acucucuuca auucuuuaug ccgguguugg gcgcguuauu uaucggaguu 300 cgaguugcgc ccgcgaacga cauuuaau gaacgugaau ugcucaacag uaugggcauu 360 ucgcagccua ccgugguguu cguuuccaaa aagggguugc aaaaauuuu gaacgugcaa 420 aaaaagcucc caaucaucca aaaaauuauu aucauggauu cuaaaacgga uuaccaggga 480 540 uuugugccag agucuucga uagggacaag acaauugcac ugaucaugaa cuccucugga 600 660 caugccagag auccuauuuu uggcaaucaa aucauuccgg auacugcgau uuuaaguguu 720 guuccauucc aucacgguuu ugggaauguuu acuacacucg gauauuugau auguggauuu 780 cgagucgucu uaauguauag auuugaagaa gagcuguuuc ugaggagccu u 831 <210> 4 <211> 825 <212> RNA <213> Artificial sequence <220> <223> Ct-luciferase[[ID=X]] <400> 4 caggauuaca agauucaaag ugcgcugcug gugccaaccc uauucuccuu cuucgccaaa 60 agcacucuga uugacaaaua cgauuuaucu aauuuacacg aaauugcuuc ugguggcgcu 120 ccccucucua aggaagucgg ggaagcgguu gccaagaggu uccaucugcc agguaucagg 180 caaggauaug ggcucacuga gacuacauca gcuauucuga uuacacccga gggggaugau 240 aaaccgggcg cggucgguaa aguuguucca uuuuuugaag cgaagguugu ggaucuggau 300 accgggaaaa cgcugggcgu uaaucaaaga ggcgaacugu gugugagagg uccuaugauu 360 auguccgguu auguaaacaa uccggaagcg accaacgccu ugauugacaa ggauggaugg 420 cuacauucug gagacauagc uuacugggac gaagacgaac acuucuucau cguugaccgc 480 cugaagucuc ugauuaagua caaaggcuau cagguggcuc ccgcugaauu ggaauccauc 540 It should be noted that in the original text, there is an unclear "X" in the translation of line 13 which needs to be further confirmed in the original context. Also, the translation strictly adheres to the requirements of preserving tags and line breaks as per the instructions.uugcuccaac accccaacau cuucgacgca ggugucgcag gucuucccga cgaugacgcc 600 ggugaacuuc ccgccgccgu uguuguuuug gagcacggaa agacgaugac ggaaaaagag 660 aucguggauu acgucgccag ucaaguaaca accgcgaaaa aguugcgcgg aggaguugug 720 uuuguggacg aaguaccgaa aggucuuacc ggaaaacucg acgcaagaaa aaucagagag 780 auccucauaa aggccaagaa gggcggaaag aucgccgugu aguaa 825 <210> 5 <211> 543 <212> DNA <213> Artificial sequence <220> <223> N1L <400> 5 atgggtcagg ccaatacgcc ctggagcagt aaggcaaacg cggatgcctt tataaattca 60 ttcatcagtg cagcatccaa tactggttcc ttctctcaag accaaatgga ggacatgtca 120 ctcatcggca atactctgat ggctgccatg gacaatatgg gaggccgcat aacaccatct 180 aagttgcagg cgttggatat ggccttcgca tcatcagtgg ccgagatcgc ggctagtgag 240 ggcggcgact tgggagtcac taccaacgcg atcgcggatg ccctcacttc tgctttttat 300 caaacgaccg gggttgtcaa ttcacgattc atatctgaga tcaggagcct cataggaatg 360 ttcgcgcagg cttccgcaaa tgacgtttat gcatctgctg gctctggcag cgggggtggt 420 gggtatggag ccagctcagc atctgcggct tctgcaagtg ctgctgcccc gagtggcgta 480 gcttatcagg ctcctgctca ggctcaaatc agttttacgt tgcgagggca acaacctgtt 540 tcc 543 <210> 6 <211> 132 <212> DNA <213> Artificial Sequence <220> <223> AQ <400> 6 ggtccttatg gacccggtgc tagcgctgcg gcagcagccg ctggcggtta tggcccaggt 60 tcagggcaac aggggcctgg gcaacaagga cctggccaac aaggtcctgg tcagcagggt 120 ccagggcagc ag 132 <210> 7 <211> 450 <212> DNA <213> Artificial Sequence <220> <223> NR3 <400> 7 ggcgctgctt ccgctgcagt atcagtaggt ggctatggac ctcaatctag tagcgcccct 60 gttgcctctg ccgccgcatc tcgactttca agtcccgccg ctagttccag ggtcagttcc 120 gcggtatcta gcttggtaag tagcggaccc actaatcaag cggcactttc aaacacaata 180 tcctcagtag tcagtcaagt aagcgcatca aaccctggct tgtcagggtg tgacgttctg 240 gttcaggcac ttctggaagt tgtctcagcg ttggtaagca tcctgggtag ctcctccata 300 ggtcaaatta attatggcgc gagcgcccaa tacacacaaa tggtgggtca gagtgtggcg 360 caggcactcg caggcgacta caaggatcat gacggagact ataaggatca tgatatagat 420 tacaaggacg atgatgacaa ggcctagtaa 450 <210> 8 <211> 4535] <212> RNA <213> Artificial sequence <220> <223> Nt-InvMTS <400> 8 augagugugu ugacgccguu gcuucugcga gggcuuaccg ggucugcuag aagacuuccg 60 guccccaggg ccaagauaca uagccucgga gacccgaugu cugugcucac uccucugcuu 120 uugcgaggac ugacuggguc cgccagacga cucccggugc cgagagcuaa aauccauagc 180 cugggaaaau uggcaacuau gucaguccug acgccgcuuc uucuccgggg ucuuacaggg 240 ucugcaagaa ggcugccugu accucgggcg aaaauucaua gcuugggcga cccgaugagu 300 guauugacgc cccuguugcu gagaggauug acugggucag cgcgccggcu cccugucccc 360 cgagcuaaga uucacucccu ugguaagcug agaauccucc aaucaacggu uccgagagca 420 agagauccgc cggucgccac gaggccucuc gag 453 <210> 9 <211> 68 <212> RNA <213> Artificial sequence <220> <223> HDV68 <400> 9 ggccggcaug gucccagccu ccucgcuggc gccggcuggg caacaugcuu cggcauggcg 60 aaugggac 68 <210> 10 <211> 67 <212> RNA <213> Artificial sequence <220> <223> HDV67 <400> 10 gggucggcau ggcaucucca ccuccucgcg guccgaccug ggcuacuucg guaggcuaag 60 ggagaag 67 <210> 11 <211> 56 <212> RNA <213> Artificial sequence <220> <223> HDV56 <400> 11 gagggauagu acagagccuc cccguggcuc ccuuggauaa ccaacugaua cuguac 56 <210> 12 <211> 87 <212> RNA <213> Artificial sequence <220> <223> Genomic HDV <400> 12 ggccggcaug gucccagccu ccucgcuggc gccggcuggg caacauuccg aggggaccgu 60 ccccucggua auggcgaaug ggaccca 87 <210> 13 <211> 91 <212> RNA <213> Artificial sequence <220> <223> Antigenomic HDV <400> 13 gggucggcau ggcaucucca ccuccucgcg guccgaccug ggcauccgaa ggaggacgca 60 cguccacucg gauggcuaag ggagagccac u 91 <210> 14 <211> 144 <212> RNA <213> Artificial sequence <220> <223> VS ribozyme <400> 14 gcgguaguaa gcagggaacu caccuccaau uucaguacug aaauugucgu agcaguugac 60 uacuguuaug ugauugguag aggcuaagug acgguauugg cguaagucag uauugcagca 120 cagcacaagcccgcuugcgagaau 144 <210> 15 <211> 21 <212> RNA <213> Artificial sequence <220> <223> VS-S <400> 15 gaagggcguc gucgccccga g 21 <210> 16 <211> 144 <212> RNA <213> Artificial sequence <220> <223> VS-Rz <400> 16 gcgguaguaa gcagggaacu caccuccaau uucaguacug aaauugucgu agcaguugac 60 uacuguuaug ugauugguag aggcuaagug acgguauugg cguaagucag uauugcagca 120 cagcacaagc ccgcuugcga gaau 144 <210> 17 <211> 291 <212> RNA <213> Artificial sequence <220> <223> Nt-DTA <400> 17 auggaccccg acgacguggu ggacagcagc aagagcuucg ugauggagaa cuucagcagc 60 uaccacggca ccaagcccgg cuacguggac agcauccaga agggcaucca gaagcccaag 120 agcggcaccc agggcaacua cgacgacgac uggaagggcu ucuacagcac cgacaacaag 180 uacgacgcug ccggcuacag cguggacaac gagaaccccc ugagcggcaa ggccggcggc 240 guggugaagg ugaccuaccc cggccugacc aaggugcugg cccugaaggu g 291 <210> 18 <211> 297 <212> RNA <213> Artificial sequence <220> <223> Ct-DTA <400> 18 gacaaugccg agaccaucaa gaaggagcug ggccugagcc ugaccgagcc ccugauggag 60 caggugggca ccgaggaguu caucaagaga uucggcgacg gcgccagcag aguggugcug 120 agccugcccu ucgccgaggg cagcagcagc guggaguaca ucaacaacug ggagcaggcc 180 aaggcccuga gcguggagcu ggagaucaac uucgagacca gaggcaagag aggccaggac 2 S gccauguacg aguacauggc ccaggcuugc gccggcaaca gagugagaag auaguaa 297 <210> 19 <211> 717 <212> RNA <213> Artificial sequence <220> <223> GFPcdn (without the start ATG codon) <400> 19 guuagcaagg gcgaggagcu cuucaccggg gucgucccca uccucgucga gcucgacggc 60 gacguaaacg gccacaaguu cagcgucucc ggcgagggcg agggcgaugc caccuacggc 120 aagcucaccc ugaaguucau cugcaccacc ggcaagcugc ccgugcccug gcccacccuc 180 gugaccaccc ugaccuacgg cgugcagugc uucagccgcu accccgacca caugaagcag 240 cacgacuucu ucaaguccgc caugcccgaa ggcuacgucc aggagcgcac caucuucuuc 300 aaggacgacg gcaacuacaa gacccgcgcc gaggugaagu ucgagggcga cacccuggug 360 aaccgcaucg agcugaaggg caucgacuuc aaggaggacg gcaacauccu ggggcacaag 420 cuggaguaca acuacaacag ccacaacguc uauaucaugg ccgacaagca gaagaacggc 480 aucaagguga acuucaagau ccgccacaac aucgaggacg gcagcgugca gcucgccgac 540 cacuaccagc agaacacccc caucggcgac ggccccgugc ugcugcccga caaccacuac 600 cugagcaccc aguccgcccu gagcaaagac cccaacgaga agcgcgauca caugguccug 660 cuggaguucg ugaccgccgc cgggaucacu cucggcaugg acgagcugua caaguag 717 <210> 20 <211> 131 <212> RNA <213> Artificial Sequence <220> <223> F2-Myr <400> 20 auggguuguu guuucagcaa gacagcggcg aaaggugaag cagcagcaga aagaccaggc 60 gaggcugcgg uagcaucaag ucccuccaag gcuaaugggc aggaaaacgg acacgucaaa 120 guuggaagcg u 131 <210> 21 <211> 685 <212> RNA <213> Artificial sequence <220> <223> F2-RFP <400> 21 agccaucauc aaggaguuca ugcgcuucaa ggugcacaug gagggcuccg ugaacggcca 60 cgaguucgag aucgagggcg agggcgaggg ccgccccuac gagggcaccc agaccgccaa 120 gcugaaggug accaagggug gcccccugcc cuucgccugg gacauccugu ccccucaguu 180 cauguacggc uccaaggccu acgugaagca ccccgccgac auccccgacu acuugaagcu 240 guccuucccc gagggcuuca agugggagcg cgugaugaac uucgaggacg gcggcguggu 300 gaccgugacc caggacuccu cccugcagga cggcgaguuc aucuacaagg ugaagcugcg 360 cggcaccaac uuccccuccg acggccccgu aaugcagaag aagaccaugg gcugggaggc 420 cuccuccgag cggauguacc ccgaggacgg cgcccugaag ggcgagauca agcagaggcu 480 gaagcugaag gacggcggcc acuacgacgc ugaggucaag accaccuaca aggccaagaa 540 gcccgugcag cugcccggcg ccuacaacgu caacaucaag uuggacauca ccucccacaa 600 cgaggacuac accaucgugg aacaguacga acgcgccgag ggccgccacu ccaccggcgg 660 cauggacgag cuguacaagu aguaa 685 <210> 22 <211> 2337 <212> RNA <213> Artificial sequence <220> <223> Nt-uDys <400> 22 augcuuuggu gggaagaagu agaggacugu uaugaaagag aagauguuca aaagaaaaca 60 uucacaaaau ggguaaaugc acaauuuucu aaguuuggga agcagcauau ugagaaccuc 120 uucagugacc uacaggaugg gaggcgccuc cuagaccucc ucgaaggccu gacagggcaa 180 aaacugccaa aagaaaaagg auccacaaga guucaugccc ugaacaaugu caacaaggca 240 cugcggguuu ugcagaacaa uaauguugau uuagugaaua uuggaaguac ugacaucgua 300 gauggaaauc auaaacugac ucuugguuug auuuggaaua uaauccucca cuggcagguc 360 aaaaauguaa ugaaaaaauu cauggcugga uugcaacaaa ccaacaguga aaagauuucuc 420 cugagcuggg uccgacaauc aacucguaau uauccacagg uuaauguaau caacuucacc 480 540 uuugacugga auguguggu uugccagcag ucagccacac aacgacugga acaugcauuc 600 660 accuauccag auaagaaguc caucuuaaug uacaucacau cacucuucca aguuuugccu 720 caacaaguga caauugaagc cauccaggaa guggaaugu ugccaaggcc accuaaagug 780 acuaaagaag acauuuuca guuacauucau caaaugcacu auucucaaca gaucacgguc 840 900 960 cauuuggaag cuccugaaga caucacuuu ggcaguucau ugaugggagag ugaaguaaac 1020 cuggaccguu aucaaacagc uuuagaagaa guauuaucgu ggcuucuuuc ugcugaggac 1080 acauugcaag cacaggaga gauuucuau gauguggaag uggugaaga cacauucau 1140 acucaugagg gguacaugau ggauuugaca gcccaucagg gccggguugg uaauauucua 1200 caauuggaa guaagcugau uggagau aauuucag agoagaaga aacugaagua 1260 shakeaaaaaaaaghaaaaaaaaaaaaugccaaaaaaaaaggaaaaaaggc 1320 aaaaaaaaaaaacauag aguuuaaaug gaucuccaga aucagaaacu gaagagoug 1380 aaugacuggc uaaaaaaaac agagaaaaaaaaaaaagaaga gccucuugga 1440 ccugaucuug agaccuaa acgccaagua cicacaua aggugcuuca agagaucua 1500 gaacagaac aagucagggu caaucucuc acucacaugg ugguggugu ugaugaaucu 1560 aguggagauc acgcaacugc ugcuuuggaa gaacaua agguauuggg agaucgaugg 1620 gcaacaucu guagauggac agaagaccgc uggguucuu uacaagacau cuucucau 1680 uggcaacguc uacugaga acagugccuu uuuagugcau ggcuuucaga aaagaagau 1740 gcagugaca agaucacac aacuggcuuu aaagaucaa augaauguau aucaagucuu 1800 caaaaacugg ccguuuuaaa agcggaucua gaaaagaaaa agcaauccau gggcaaacug 1860 uauucacuca aacaagaucu ucuuucaaca cugaagaaua agucagugac ccagaagacg 1920 gaagcauggc uggauaacuu ugcccggugu ugggauaauu uaguccaaaa acuugaaaag 1980 aguacagcac agauuucaca ggcugucacc accacucagc caucacuaac acagacaacu 2040 guaauggaaa caguaacuac ggugaccaca agggaacaga uccugguaaa gcaugcucaa 2100 gaggaacuuc caccaccacc uccccaaaag aagaggcaga uuacugugga ucuugaaaga 2160 cuccaggaac uucaagaggc cacggaugag cuggaccuca agcugcgcca agcugaggug 2220 aucaagggau ccuggcagcc cgugggcgau cuccucauug acucucucca agaucaccuc 2280 gagaaaguca aggcacuucg aggagaaauu gcgccucuga aagagaacgu gagccac 2337 <210> 23 <211> 1974 <212> RNA <213> Artificial sequence <220> <223> Ct-uDys-GFP <400> 23 gucaaugacc uugcucgcca gcuuaccacu uugggcauuc agcucucacc guauaaccuc 60 agcacucugg aagaccugaa caccagaugg aagcuucugc agguggccgu cgaggaccga 120 gucaggcagc ugcaugaagc ccacagggac uuugguccag caucucagca cuuucuuucc 180 acgucugucc agggucccug ggagagagcc aucucgccaa acaaagugcc cuacuauauc 240 aaccacgaga cucaaacaac uugcugggac caucccaaaa ugacagagcu cuaccagucu 300 uaagcugacc ugaauaaugu cagauucuca guuauagga cugccaugaa acuccgaaga 360 cugcagaagg cccuuugcuu ggaucucuug agccugucag cugcauguga ugccuuggac 420 cagcacaacc ucaagcaaaa ugaccagccc auggauaucc ugcagauuau uaauuguuug 480 accacuauuu augaccgccu ggagcaagag cacaacaauu uggucaacgu cccucucugc 540 gggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg cguguccugu cuuuuaaaac uggcaucauu ucccugugua aagcacauuu ggagacaag 660 uacagauacc uuuucaagca aguggcaagu ucaacagggau uuugugacca gcgcaggcug 720 ggccuccuuc ugcaugauuc uauccaaauu ccaagacagu ugggugaagu ugcauccuu 780 gggggcagua acauugagcc aaguguccgg agcugcuucc aauuugcuaa uaauaagcca 840 gagaucgaag cggcccuuu ccuagacugg augacugg aaccccaguc caugguggugg 900 cugcccgucc ugcacagagu ggcugcugca gaaacugcca agcaucaggc caaauguaac 960 aucugcaaag aguguccaau cauuggauuc agguacagga gucuaaagca cuuuaaauuau 1020 gacaucugcc aaagcugcuu uuuuucuggu cgaguugcaa aaggccauaa aaugcacuau 1080 cccauggugg aauauugcac uccgacuaca ucagggaaag auguucgaga cuuugccaag 1140 guacuaaaaa acaaauuucg aaccaaaaagg uauuuugcga agcauccccg aaugggcuac 1200 cugccagugc agacugucuu agaggggac aacauggaa cugacacaau ucuagaggug 1260 agcaagggcg aggagcuguu caccggggug gugcccaucc uggucgagcu ggacggcgac 1320 guaaacggcc aaagucag cguguccggc gagggcgagg gcgaugccac cuacggcaag 1380 cugacccuga aguucaucug caccaccggc aagcugcccg ugcccuggcc cacccucug 1440 accacccuga ccuacggcgu gcagugcuuc agccgcuacc ccgaccacau gaagcagcac 1500 gacuucuuca aguccgccau gcccgaaggc uacguccagg agcgcaccau cuucuucaag 1560 gacgacggca acuacaagac ccgcgccgag gugaaguucg agggcgacac ccuggugaac 1620 cgcaucgagc ugaagggcau cgacuucaag gaggacggca acauccuggg gcacaagcug 1680 gaguacaacu acaacagcca caacgucuau aucauggccg acaagcagaa gaacggcauc 1740 aaggugaacu ucaagauccg ccacaacauc gaggacggca gcgugcagcu cgccgaccac 1800 uaccagcaga acacccccau cggcgacggc cccgugcugc ugcccgacaa ccacuaccug 1860 agcacccagu ccgcccugag caaagacccc aacgagaagc gcgaucacau gguccugcug 1920 gaguucguga ccgccgccgg gaucacucuc ggcauggacg agcuguacaa guaa 1974 <210> 24 <211> 68 <212> RNA <213> Artificial sequence <220> <223> HDV68 catalytic mutant <400> 24 ggccggcaug gucccagccu ccucgcuggc gccggcuggg caacaugcuu cggcauggug 60 aaugggac 68 <210> 25 <211> 56 <212> RNA <213> Artificial sequence <220> <223> Hammerhead ribozyme with stem 3 overhang specific for Nt-Luc <400> 25 gagccuuacc ggauguguuu uccggucuga ugaguccggu agcggacgaa aggcuc 56 <210> 26 <211> 54 <212> RNA <213> Artificial sequence <220> <223> Twisted ribozyme with a 5 nt P1 stem for Ct-Luc <400> 26 agccuuaaca cugccaaugc cggucccaag cccggauaaa aguggaggga ggcu 54 <210> 27 <211> 54 <212> RNA <213> Artificial sequence <220> <223> Twisted ribozymes with 5 nt P1 stem for Ct-Luc and T6A mutations <400> 27 agccuaaaca cugccaaugc cggucccaag cccggauaaa aguggaggga ggcu 54 <210> 28 <211> 54 <212> RNA <213> Artificial sequence <220> <223> Twisted ribozyme mutant with a 5 nt P1 stem for Ct-Luc <400> 28 agccuuaacu cuuccaaugc cggucccaag cccggauaaa aguggaggga ggcu 54 <210> 29 <211> 54 <212> RNA <213> Artificial sequence <220> <223> Twisted ribozyme with a 5 nt P1 stem for Ct-Luc <400> 29 agccuuaaca cugccaaugc cggucccaag cccggauaaa aguggaggga ggcu 54 <210> 30 <211> 51 <212> RNA <213> Artificial sequence <220> <223> Twisted ribozyme with a 2 nt P1 stem for Ct-Luc <400> 30 agccuuaaca cugccaaugc cggucccaag cccggauaaa aguggaggga g 51 <210> 31 <211> 49 <212> RNA <213> Artificial sequence <220> <223> Twisted ribozyme with 1 nt P1 stem for Ct-Luc <400> 31 agccuuaaca cugccaaugc cgguccccaag cccggauaaa aguggaggg 49 <210> 32 <211> 49 <212> RNA <213> Artificial sequence <220> <223> Twisted ribozyme without P1 stem for Ct-Luc <400> 32 agccuuaaca cugccaaugc cgguccccaag cccggauaaa aguggaggg 49 <210> 33 <211> 53 <212> RNA <213> Artificial sequence <220> <223> 16 HH Stem 1 overhang specific for Ct-Luc <400> 33 gaaucuugua auccugcuga ugaguccgug aggacgaaac gaguaagcuc guc 53 <210> 34 <211> 51 <212> RNA <213> Artificial sequence <220> <223> 14 HH stem 1 overhang specific for Ct-Luc <400> 34 aucuuguaau ccugcugaug aguccgugag gacgaaacga guaagcucgu c 51 <210> 35 <211> 49 <212> RNA <213> Artificial sequence <220> <223> 12 HH stem 1 overhang specific for Ct-Luc <400> 35 cuuguaaucc ugcugaugag uccgugagga cgaaacgagu aagcucguc 49 <210> 36 <211> 45 <212> RNA <213> Artificial sequence <220> <223> 8 HH stem 1 overhang specific for Ct-Luc <400> 36 uaauccugcu gaugaguccg ugaggacgaa acgaguaagc ucguc 45 <210> 37 <211> 43 <212> RNA <213> Artificial sequence <220> <223> 6 HH stem 1 overhang specific for Ct-Luc <400> 37 auccugcuga ugaguccgug aggacgaaac gaguaagcuc guc 43 <210> 38 <211> 43 <212> RNA <213> Artificial sequence <220> <223> 6 HH mutant stem 1 overhang specific for Ct-Lu <400> 38 auccugcuga ugaguccgug aggacgagac gaguaagcuc guc 43 <210> 39 <211> 41 <212> RNA <213> Artificial sequence <220> <223> 4 HH stem 1 overhang specific for Ct-Luc <400> 39 ccugcugaug aguccgugag gacgaaacga guaagcucgu c 41 <210> 40 <211> 55 <212> RNA <213> Artificial sequence <220> <223> RzB stem 1 overhang specific for Ct-Luc <400> 40 uuguaauaau ccugcugaug agucgcuggg augcgacgaa acgccuucgg gcguc 55 <210> 41 <211> 52 <212> RNA <213> Artificial sequence <220> <223> Splice donor sequence of Nt vector <400> 41 guaaguauca agguuacaag acagguuuaa ggagaccaau agaaacuggg cu 52 <210> 42 <211> 81 <212> RNA <213> Artificial sequence <220> <223> Splice acceptor sequence of Ct vector <400> 42 ugucgagaca gagaagacuc uugcguuucu gauaggcacc uauuggucuu acugacaucc 60 acuuugccuu ucucuccaca g 81 <210> 43 <211> 560 <212> RNA <213> Artificial sequence <220> <223> GCN4 5' UTR uORF <400> 43 aaacaaaaac ucacaacaca gguuacucuc cccccuaaau ucaaauuuuu uuugcccauc 60 aguuucacua gcgaauuaua caacucacca gccacacagc ucacucaucu acuucgcaau 120 caaaacaaaa uauuuuauuu uaguucaguu uauuaaguua uuaucaguau cguauuaaaa 180 aauuaaagau cauugaaaaa uggcuugcua aaccgauuau auuuuguuuu uaaaguagau 240 uauuauuaga aaauuauuaa gagaauuaug uguuaaauuu auugaaagag aaaauuuauu 300 uucccuuauu aauuaaaguc cuuuacuuuu uuugaaaacu gucaguuuuu ugaagaguua 360 uuuguuuugu uaccaauugc uaucauguac ccguagaauu uuauucaaga uguuuccgua 420 acgguuaccu uucugucaaa uuauccaggu uuacucgcca auaaaaauuu cccuauacua 480 ucauuaauua aaucauuauu auuacuaaag uuuuguuuac caauuugucu gcucaagaaa 540 auaaauuaaa uacaaauaaa 560 <210> 44 <211> 148 <212> RNA <213> Artificial sequence <220> <223> SRY 5' UTR uORF <400> 44 guugaggggg uguugagggc ggagaaaugc aaguuucauu acaaaaguua acguaacaaa 60 gaaucuggua gaaaugaguu uuggauagua aaauaaguuu cgaacucugg caccuuucaa 120 uuuugucgca cucuccuugu uuuugaca 148 <210> 45 <211> 343 <212> RNA <213> Artificial sequence <220> <223> Hoxa9 TIE <40s> 45 gaaaaaacag aagagggaag gauaccagag cgguucauac agggcccaga aacuaggcga 60 ggugaccccu cagcaagaca aacaccucuu gauguugacu ggcgauuuuc cccaucucca 120 gucuggggag cgggacuagg cauacagaug auggagcuua gaacccgcug gcuagggaau 180 It should be noted that there may be an error in the "40s" in line . It is likely supposed to be "400".aaaauucgcu gggcaguuug ugcucaaaga agugggccag ggcgcuugug acacaaucag 240 ggcguuugug acacaaaccc uugaggguug gcaguucucu ccuuggcggu ugcucugguu 300 gcucuguggg gccuucccug uggagcaagg gugaucuggc cga 343 <210> 46 <211> 170 <212> RNA <213> Artificial sequence <220> <223> Hoxa3 TIE <400> 46 aggacaauuc gucucuuggg cugccgaagc gacagcuguc agagaggcag aagcuucugg 60 gagccgcggu cugaaggcua cgugugcugc cuggucauuc aaagugucaa uuuuaggucc 120 agaagugucc aaaccacaag uucucaaaac ucugaaaaau ggcucccucc 170 <210> 47 <211> 38 <212> RNA <213> Artificial sequence <220> <223> NRAS 5'UTR G-pseudoknot <400> 47 cgucccgugu gggaggggcg ggucugggug cggccugc 38 <210> 48 <211> 126 <212> RNA <213> Artificial sequence <220> <223> Human IFNG 5' UTR pseudoknot <400> 48 cacauuguuc ugaucaucug aagaucagcu auuagaagag aaagaucagu uaaguccuuu 60 ggaccugauc agcuugauac aagaacuacu gauuucaacu ucuuuggcuu aauucucucg 120 gaaacg 126 <210> 49 <211> 132 <212> RNA <213> Artificial sequence <220> <223> Rat ODC 5'UTR <400> 49 ugucaguccc ugcagccgcc gccgccggcc gccuucaguc agcagcucgg cgccaccucc 60 ggucggcgac ugcggcgggc ucgacgaggc ggcugacggg gcggcggcgg gaagacggcc 120 gggugcgccu ug 132 <210> 50 <211> 49 <212> RNA <213> Artificial sequence <220> <223> SIRLOIN RNA nuclear localization signal <400> 50 cgccucccgg guucaagcga uucuccugcc ucagccuccc gaguagcug 49 <210> 51 <211> 42 <212> RNA <213> Artificial sequence <220> <223> BORG lncRNA NLS <400> 51 accucagaau cuacaaguca gccccaauua aauguuguuu ua 42 <210> 52 <211> 108 <212> PRT <213> Artificial sequence <220> <223> FKBP DD <400> 52 Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 15 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30 Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 50 55 60 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp 65 70 75 80 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 85 90 95 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Pro Glu 100 105 <210> 53 <211> 40 <212> PRT <213> Artificial sequence <220> <223> PEST (Enhanced ODC PEST) <400> 53 Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Ala Ala Gly Thr Leu 1 5 10 15 Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala Ala 20 25 30 Cys Ala Ser Ala Arg Ile Asn Val 35 40 <210> 54 <211> 40 <212> PRT <213> Synthetic Sequence <220> <223> ODC PEST (Yeast) <400> 54 Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Asp Asp Gly Thr Leu 1 5 10 15 Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala Ala 20 25 30[[ID=三十三]] Cys Ala Ser Ala Arg Ile Asn Val 35 40 <210> 55 <211> 40 <212> PRT <213> Synthetic Sequence <220> <223> ODC PEST (Human) <400> 55 Asn Pro Asp Phe Pro Pro Glu Val Glu Glu Gln Asp Ala Ser Thr Leu 1 5 10 15 Pro Val Ser Cys Ala Trp Glu Ser Gly Met Lys Arg His Arg Ala Ala It should be noted that in the translation of the amino acid sequence part, it is translated according to the common amino acid name translation method. And for the tags related to sequence information, they are retained as they are to maintain the integrity of the original patent text information. If there are any specific requirements or corrections for this translation, please feel free to let me know.20 25 30 Cys Ala Ser Ala Ser Ile Asn Val 35 40 <210> 56 <211> 57 <212> PRT <213> Synthetic sequence <220> <223> CL1 <400> 56 Ala Cys Lys Asn Trp Phe Ser Ser Leu Ser His Phe Val Ile His Leu 1 5 10 15 Asn Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Ala Ala Gly Thr 20 25 30 Leu Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala 35 40 45 Ala Cys Ala Ser Ala Arg Ile Asn Val 50 55 <210> 57 <211> 57 <212> PRT <213> Synthetic sequence <220> <223> CL1-PEST <400> 57 Ala Cys Lys Asn Trp Phe Ser Ser Leu Ser His Phe Val Ile His Leu 1 5 10 15 Asn Ser His Gly Phe Pro Pro Glu Val Glu Glu Gln Ala Ala Gly Thr 20 25 30 Leu Pro Met Ser Cys Ala Gln Glu Ser Gly Met Asp Arg His Pro Ala 35 40 45 Ala Cys Ala Ser Ala Arg Ile Asn Val 50 55 <210> 58 <211> 68 <212> PRT <213> Artificial sequence <220> <223> E1A PEST <400> 58 Ser Arg Glu Cys Asn Ser Ser Thr Asp Ser Cys Asp Ser Gly Pro Ser 1 5 10 15 Asn Thr Pro Pro Glu Ile His Pro Val Val Pro Leu Cys Pro Ile Lys 20 25 30 Pro Val Ala Val Arg Val Gly Gly Arg Arg Gln Ala Val Glu Cys Ile 35 40 45 Glu Asp Leu Leu Asn Glu Pro Gly Gln Pro Leu Asp Leu Ser Cys Lys 50 55 60 Arg Pro Arg Pro 65 <210> 59 <211> 31 <212> PRT <213> Artificial sequence <220> <223> C-myc PEST <400> 59 Leu His Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu 1 5 10 15 Gln Glu Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg 20 25 30 <210> 60 <211> 25 <212> PRT <213> Artificial sequence <220> <223> c-Fos PEST <400> 60 Ala Ala His Arg Lys Gly Ser Ser Ser Asn Glu Pro Ser Ser Asp Ser 1 5 10 15 Leu Ser Ser Pro Thr Leu Leu Ala Leu 20 25 <210> 61 <211> 26 <212> PRT <213> Artificial sequence <220> <223> v-Myb PEST <400> 61 Pro Ser Pro Pro Val Asp His Gly Cys Leu Pro Glu Glu Ser Ala Ser 1 5 10 15 Pro Ala Arg Cys Met Ile Val His Gln Ser 20 25 <210> 62 <211> 59 <212> PRT <213> Artificial sequence <220> <223> NPDC1 PEST <400> 62 Pro Pro Lys Glu Leu Asp Thr Ala Ser Ser Asp Glu Glu Asn Glu Asp 1 5 10 15 Gly Asp Phe Thr Val Tyr Glu Cys Pro Gly Leu Ala Pro Thr Gly Glu 20 25 30 Met Glu Val Arg Asn Pro Leu Phe Asp His Ala Ala Leu Ser Ala Pro 35 40 45 Leu Pro Ala Pro Ser Ser Pro Pro Ala Leu Pro 50 55 <210> 63 <211> 37 <212> PRT <213> Artificial sequence <220> <223> IkBa PEST <400> 63 Pro Glu Ser Glu Asp Glu Glu Ser Tyr Asp Thr Glu Ser Glu Phe Thr 1 5 10 15 Glu Phe Thr Glu Asp Glu Leu Pro Tyr Asp Asp Cys Val Phe Gly Gly 20 25 30 Gln Arg Leu Thr Leu 35 <210> 64 <211> 41 <212> PRT <213> Artificial sequence <220> <223> m.m. AZIN2 PEST <400> 64 Gly Gln Leu Leu Pro Ala Glu Glu Asp Gln Asp Ala Glu Gly Val Cys 1 5 10 15 Lys Pro Leu Ser Cys Gly Trp Glu Ile Thr Asp Thr Leu Cys Val Gly 20 25 30 Pro Val Phe Thr Pro Ala Ser Ile Met 35 40 <210> 65 <211> 43 <212> PRT <213> Artificial sequence <220> <223> xl AZIN2 PEST <400> 65 Val Gln Leu Leu Gln Arg Gly Leu Gln Gln Thr Glu Glu Lys Glu Asn 1 5 10 15 Val Cys Thr Pro Met Ser Cys Gly Trp Glu Ile Ser Asp Ser Leu Cys 20 25 30 Phe Thr Arg Thr Phe Ala Ala Thr Ser Ile Ile 35 40 <210> 66 <211> 12 <212> PRT <213> Artificial sequence <220> <223> NS1 <400> 66 Thr Ser Leu Tyr Lys Lys Val Gly Met Gly Arg Lys 1 5 10 <210> 67 <211> 12 <212> PRT <213> Artificial sequence <220> <223> NS6 <400> 67 Ser Leu Tyr Lys Lys Val Gly Thr Met Ala Ala Gly 1 5 10 <210> 68 <211> 12 <212> PRT <213> Artificial sequence <220> <223> NS7 <400> 68 Tyr Lys Lys Val Gly Thr Met Arg Gly Arg Gly Leu 1 5 10 <210> 69 <211> 12 <212> PRT <213> Artificial sequence <220> <223> NS12 <400> 69 Glu Arg Ala Pro Thr Gly Arg Trp Gly Arg Arg Gly 1 5 10 <210> 70 <211> 12 <212> PRT <213> Artificial sequence <220> <223> NS15 <400> 70 Glu Gly Pro Leu Trp His Pro Arg Ile Cys Gly Ser 1 5 10 <210> 71 <211> 12 <212> PRT <213> Artificial sequence <220> <223> SELK <400> 71 Leu Arg Gly Pro Ser Pro Pro Pro Met Ala Gly Gly 1 5 10 <210> 72 <211> 12 <212> PRT <213> Artificial sequence <220> <223> SELS <400> 72 Trp Arg Pro Gly Arg Arg Gly Pro Ser Ser Gly Gly 1 5 10 <210> 73 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> EMID1 <400> 73 Arg Asp Glu Arg Gly 1 5 <210> 74 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> IRX6 <400> 74 Gly Ala Glu Ala Gly 1 5 <210> 75 <211> 80 <212> PRT <213> Artificial Sequence <220> <223> UbVR <400> 75 Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val 1 5 10 15 Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys 20 25 30 Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln 35 40 45 Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser 50 55 60 Thr Leu His Leu Val Leu Arg Leu Arg Gly Val Arg Ala Ser Ala Ser 65 70 75 80 <210> 76 <211> 162 <212> PRT <213> Artificial Sequence <220> <223> 2xUbVR <400> 76 Thr Ser Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu 1 5 10 15 Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln 20 25 30 Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly 35 40 45 Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys 50 55 60 Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Val Arg Ala Ser 65 70 75 80 Ala Ser Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu 85 90 95 Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln 100 105 110 Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly 115 120 125 Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys 130 135 140 Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Val Arg Ala Ser 145 150 155 160 Ala Ser <210> 77 <211> 39 <212> DNA <213> Artificial sequence <220> <223> 12x poly K coding tail sequence <400> 77 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaataa 39 <210> 78 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Translation product 12x poly K <400> 78 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 <210> 79 <211> 51 <212> DNA <213> Artificial sequence <220> <223> 16x poly-K coding tail sequence <400> 79 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaata a 51 <210> 80 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Translation product 16x poly K <400> 80 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 <210> 81 <211> 505 <212> PRT <213> Artificial sequence <220> <223> Human RtcB protein sequence <400> 81 Met Ser Arg Ser Tyr Asn Asp Glu Leu Gln Phe Leu Glu Lys Ile Asn 1 5 10 15 Lys Asn Cys Trp Arg Ile Lys Lys Gly Phe Val Pro Asn Met Gln Val 20 25 30 Glu Gly Val Phe Tyr Val Asn Asp Ala Leu Glu Lys Leu Met Phe Glu 35 40 45 ...

Claims

1. A composition for producing an RNA molecule encoding a target protein, comprising: a nucleic acid molecule encoding a first RNA molecule comprising a coding region encoding a first portion of a protein of interest and a 3' ribozyme; and a nucleic acid molecule encoding a second RNA molecule, wherein the second RNA molecule comprises a coding region encoding a second portion of a target protein and a 5' ribozyme; wherein the 3' ribozyme is a member of the HDV ribozyme family and the 5' ribozyme is a member of the HH ribozyme family.

2. The composition of claim 1, wherein the composition: a) further comprising one or more additional nucleic acid molecules encoding one or more additional RNA molecules, each of which comprises a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme; or b) further comprising one or more additional nucleic acid molecules encoding one or more additional RNA molecules, each of which comprises a coding region encoding a domain of a target protein, a 5' ribozyme, and a 3' ribozyme recognition sequence.

3. The composition of claim 2, wherein the composition further comprises a ribozyme that can interact with the 3' ribozyme recognition sequence, wherein the ribozyme induces the removal of the 3' recognition sequence.

4. The composition of claim 2, wherein the 3' ribozyme recognition sequence comprises VS-S and wherein the ribozyme is VS-Rz.

5. Use of a nucleic acid molecule encoding a first RNA molecule and a nucleic acid molecule encoding a second RNA molecule in the preparation of a medicament for producing an RNA molecule encoding a target protein, wherein the first RNA molecule comprises a coding region encoding a first portion of the target protein and a 3' ribozyme, and the second RNA molecule comprises a coding region encoding a second portion of the target protein and a 5' ribozyme, wherein the producing comprises: administering the nucleic acid molecule encoding the first RNA molecule to a cell or tissue; and administering the nucleic acid molecule encoding the second RNA molecule to the cell or tissue; wherein the 3' ribozyme is a member of the HDV ribozyme family and the 5' ribozyme is a member of the HH ribozyme family.

6. The use of claim 5, wherein the producing further comprises: a) administering to the cell or tissue one or more additional nucleic acid molecules encoding one or more additional RNA molecules, each of which comprises a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme; or b) administering to the cell or tissue one or more additional nucleic acid molecules encoding one or more additional RNA molecules, each of which comprises a coding region encoding a domain of a protein of interest, a 5' ribozyme, and a 3' ribozyme recognition sequence; and / or c) administering a ligase to the cell or tissue to induce assembly of the RNA molecule.

7. The use of claim 6, wherein the production further comprises administering to the cell or tissue a ribozyme that interacts with the 3' ribozyme recognition sequence, the ribozyme inducing the removal of the 3' recognition sequence.

8. The use of claim 6, wherein the 3' ribozyme recognition sequence comprises VS-S and wherein the ribozyme is VS-Rz.

9. The use according to claim 6, wherein the ligase is RNA 2',3'-cyclic phosphate and 5'-OH (RtcB) ligase.

10. An in vitro method for producing an RNA molecule encoding a target protein, comprising: Providing a first RNA molecule, wherein the first RNA molecule comprises a coding region encoding a first portion of a target protein and a 3' ribozyme; providing a second RNA molecule, the second RNA molecule comprising a coding region encoding a second portion of a target protein and a 5' ribozyme; and providing a ligase to induce assembly of the RNA molecule from the coding region of the first RNA molecule and the coding region of the second RNA molecule; wherein the 3' ribozyme is a member of the HDV ribozyme family and the 5' ribozyme is a member of the HH ribozyme family.

11. An in vitro method for producing an RNA molecule encoding a repeat domain protein of interest, comprising: a) providing a first RNA molecule, wherein the first RNA molecule comprises a coding region encoding a first part of a target protein and a 3' ribozyme; b) providing one or more additional RNA molecules, wherein the one or more additional RNA molecules comprise a coding region encoding a domain of a target protein, a 5' ribozyme, and a 3' ribozyme recognition sequence; c) providing a ligase to ligate the coding region of the first RNA molecule and the coding region of the one or more additional RNA molecules; d) providing a ribozyme that recognizes the 3' ribozyme recognition sequence and catalyzes the removal of the 3' ribozyme recognition sequence; e) repeating steps b)-d) one or more times to generate RNA molecules encoding multiple repeat domains; f) providing a final RNA molecule, the final RNA molecule comprising a coding region encoding the last part of the target protein and a 5' ribozyme; and g) providing a ligase to ligate the coding regions of the one or more additional RNA molecules to the coding region of the final RNA molecule, thereby producing a complete RNA molecule encoding the repeat domain protein; wherein the 3' ribozyme is a member of the HDV ribozyme family and the 5' ribozyme is a member of the HH ribozyme family.

12. A composition for producing an RNA molecule encoding a target protein and a circular RNA molecule comprising a nucleic acid encoding: The first part of the target protein; a synthetic intron comprising a 5' ribozyme, a cargo sequence, and a 3' ribozyme; and The second part of the target protein; wherein the 3' ribozyme is a member of the HDV ribozyme family, and the 5' ribozyme is a member of the HH ribozyme family; wherein the cargo sequence is one or more selected from the group consisting of: a sequence encoding a therapeutic protein of interest, a CRISPR guide RNA sequence, a small RNA sequence, and a trans-cleaving ribozyme sequence.

13. The composition of claim 12, wherein the small RNA sequence comprises one or more selected from the group consisting of microRNA (miRNA), Piwi-interacting RNA (piRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small tRNA-derived RNA (tsRNA), small rDNA-derived RNA (srRNA), and small nuclear RNA (snRNA).

14. The composition of claim 12 or 13, wherein a) the target protein is one or more selected from the group consisting of: a therapeutic protein, a reporter protein and a Cas9 protein; and / or b) further comprises one or more selected from the group consisting of: RtcB ligase and a nucleic acid encoding RtcB ligase.

15. A method for delivering RNA molecules and circular RNA molecules encoding target proteins for non-therapeutic purposes, comprising: administering to a cell or tissue a nucleic acid encoding: a first portion of a protein of interest; a synthetic intron comprising a cis-cutting 5' ribozyme, a cargo sequence, and a cis-cutting 3' ribozyme; and a second portion of the protein of interest; wherein the 3' ribozyme is a member of the HDV ribozyme family, and the 5' ribozyme is a member of the HH ribozyme family; wherein the cargo sequence is one or more selected from the group consisting of: a sequence encoding a therapeutic protein of interest, a CRISPR guide RNA sequence, a small RNA sequence, and a trans-cleaving ribozyme sequence.

16. The method of claim 15, wherein the small RNA sequence comprises one or more selected from the group consisting of microRNA (miRNA), Piwi-interacting RNA (piRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small tRNA-derived RNA (tsRNA), small rDNA-derived RNA (srRNA), and small nuclear RNA (snRNA).

17. The method according to claim 15 or 16, a) wherein the target protein is one or more selected from the group consisting of: a therapeutic protein, a reporter protein and a Cas9 protein; and / or b) further comprising administering to the cell or tissue one or more selected from the group consisting of: RtcB ligase and a nucleic acid encoding RtcB ligase.

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