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WO2025080780A1 - Administration et expression de systèmes crispr d'édition primaire - Google Patents

Administration et expression de systèmes crispr d'édition primaire Download PDF

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WO2025080780A1
WO2025080780A1 PCT/US2024/050689 US2024050689W WO2025080780A1 WO 2025080780 A1 WO2025080780 A1 WO 2025080780A1 US 2024050689 W US2024050689 W US 2024050689W WO 2025080780 A1 WO2025080780 A1 WO 2025080780A1
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seq
ribozyme
protein
rna
sequence
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Douglas Matthew ANDERSON
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University of Rochester
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
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    • C12N2310/30—Chemical structure
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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • Prime Editing is a variation on CRISPR-Cas genome editing which employs a Cas (WT or nickase, such as Cas9(H840A)), a Cas-tethered or untethered reverse transcriptase (such as MMLV), a prime editing guide RNA (pegRNA) which is composed of a single guide RNA with a 3’ extension to act as both a Primer Binding Site (PBS) and Reverse Transcriptase Template (RTT), and/or an additional nicking guide RNA (Anzalone et al., 2019, Nature 576, 149-157; Chen et al., 2021, Cell 184, 5635-5652 e5629; Liu et al., 2022, Nature biotechnology 40, 1388-1393; Tao et al., 2022, Signal Transduct Target Ther 7, 108; Grunewald et al., 20
  • PE has evolved through several iterations to optimize editing efficiency, (PE1-6), which include 1) optimization of Prime Editor and pegRNA sequences, 2) use of a nicking guide RNA on the opposing strand to increase editing efficiency, and 3) minimization of RT domains through deletion ( ⁇ RH domain of MMLV) or use of compact natural enzymes (such as Marathon RT).
  • Prime Editing CRISPR systems Primary editors, pegRNAs and nicking guide RNAs
  • viral vectors which greatly exceed the packaging limits of small non-integrating viral vectors, notably AAV.
  • PEmax a robust PE editor utilizing SpyCas9, the preferred Cas9 for PE, itself is 6,400 bp and exceeds the packaging capacity of a single AAV vector ( ⁇ 4,700 bp).
  • necessary transcriptional control elements such as promoters and polyadenylation elements, further increases the space required to >7.5kb, with an additional 700bp still required for peg- and nicking- guideRNAs expression cassettes.
  • the present invention comprises a system for generating an RNA molecule encoding a protein of interest 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.
  • the 3’ribozyme catalyzes itself out of the first RNA molecule, thereby generating a 3’P or 2’3’ cP end.
  • the 5’ribozyme catalyzes itself out of the second RNA molecule, thereby generating a 5’OH end.
  • the 3’P or 2’3’ cP end is ligated to the 5’OH end to form an RNA molecule comprising the coding region of the first RNA molecule and the coding region of the second RNA molecule.
  • the 3’ ribozyme is a member of the HDV family of ribozymes.
  • the 5’ ribozyme is a member of the HH family of ribozymes.
  • 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 the protein of interest; a 5’ ribozyme; and a 3’ ribozyme.
  • 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 the protein of interest; a 5’ ribozyme; and a 3’ ribozyme recognition sequence.
  • the system further comprises a ribozyme that interacts with the 3’ ribozyme recognition sequence which induces the removal of the 3’ recognition sequence.
  • the 3’ ribozyme recognition sequence comprises VS- S and wherein the ribozyme is VS-Rz.
  • the present invention relates to a method for generating an RNA molecule encoding a protein of interest comprising: administering to a cell or tissue 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 administering to a cell or tissue 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.
  • the 3’ribozyme catalyzes itself out of the first RNA molecule, thereby generating a 3’P or 2’3’ cP end.
  • the 5’ribozyme catalyzes itself out of the second RNA molecule, thereby generating a 5’OH end.
  • the 3’P or 2’3’ cP end is ligated to the 5’OH end to form an RNA molecule comprising the coding region of the first RNA molecule and the coding region of the second RNA molecule.
  • the 3’ ribozyme is a member of the HDV family of ribozymes.
  • the 5’ ribozyme is a member of the HH family of ribozymes.
  • 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 the protein of interest; a 5’ ribozyme; and a 3’ ribozyme.
  • 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 the protein of interest; a 5’ ribozyme; and a 3’ ribozyme recognition sequence.
  • 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.
  • the 3’ ribozyme recognition sequence comprises VS-S and wherein the ribozyme is VS-Rz.
  • the method further comprises administering to the cell or tissue a ligase to induce the assembly of the RNA molecule.
  • the ligase is RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase.
  • the present invention comprises an in vitro method of generating an RNA molecule encoding a protein of interest 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 the assembly of the RNA molecule from the coding region of the first RNA molecule and the coding region of the second RNA molecule.
  • the present invention comprises an in vitro method of generating an RNA molecule encoding a repeat domain protein of interest comprising the steps of: 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 molecule 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 ligate the coding region of the first RNA molecule and the coding region of the one or more additional RNA molecule; d) providing a ribozyme that recognizes the 3’ ribozyme recognition sequence and catalyzes its removal; e) repeating steps b)-d) one or more times to generate an RNA molecule encoding a plurality of repeat domains; f) providing at least one additional RNA
  • the present invention comprises a method of treating a disease or disorder in a subject caused by a mutation in a large protein of interest comprising: administering to said 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 said subject a second nucleic acid comprising a coding region encoding a second portion of the protein of interest and a 5’ribozyme.
  • the disease or disorder 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 dystrophies , DFNB9, neurosensory nonsyndromic recessive deafness, Cystic Fibrosis, Wilson Disease, Miyoshi Muscular Dystrophy and Deafness, Autosomal Recessive 9, Usher Syndrome, Type I and Deafness, Autosomal Recessive 2, Deafness, Autosomal Recessive 3 and Nonsyndromic Hearing Loss, Usher syndrome type I, autosomal recessive deafness-16 (DFNB16), Meniere's disease (MD), Deafness, Autosomal Dominant 12 and Deafness, Autosomal Recessive 21, Usher syndrome Type 1F (USH1F) and DFNB23, Deafness
  • the present invention comprises a system for generating an RNA molecule encoding a protein of interest and a circular RNA molecule comprising a nucleic acid encoding: a first portion of a protein of interest; a synthetic intron comprising a 5’ ribozyme, a cargo sequence, and a 3’ ribozyme; and a second portion of a protein of interest.
  • the protein of interest is one or more selected from the group consisting of: a therapeutic protein, a reporter protein, and a Cas9 protein.
  • 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.
  • 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 (snoRNAs), small tRNA-derived RNA (tsRNA), small rDNA-derived RNA (srRNA) and small nuclear RNA (snRNA).
  • the 3’ ribozyme of the synthetic intron is a member of the HH family of ribozymes.
  • the 5’ ribozyme of the synthetic intron is one or more selected from the group consisting of: a member of the HDV family of ribozymes, a member of the HDV family of ribozymes, and VS-S ribozyme recognition sequence.
  • the sytem further comprises one or more selected from the group consisting of: RtcB ligase and a nucleic acid encoding RtcB ligase.
  • the present invention comprises a method of delivering an RNA molecule encoding a protein of interest and a circular RNA molecule, the method 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-cleaving 5’ ribozyme, a cargo sequence and a cis- cleaving 3’ ribozyme, and a second portion of a protein of interest.
  • the protein of interest is one or more selected from the group consisting of: a therapeutic protein, a reporter protein, and a Cas9 protein.
  • 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.
  • 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 (snoRNAs), small tRNA-derived RNA (tsRNA), small rDNA-derived RNA (srRNA) and small nuclear RNA (snRNA).
  • the method further comprises administering to the cell or tissue one or more selected from the group consisting of: RtcB ligase and a nucleic acid encoding RtcB ligase.
  • RtcB ligase and a nucleic acid encoding RtcB ligase.
  • Figure 1A shows a diagram depicting the vectors encoding the N-terminal (Nt) half of GFP with 3’ HDV ribozyme and C-terminal (Ct) half of GFP with 5’ Hammerhead (HH) ribozyme.
  • Figure 1B depicts exemplary results demonstrating that co-expression of both Nt-GFP-HDV and HH-Ct-GFP in COS7 and HEK293T cells resulted in detectable GFP fluorescence, but not when transfected separately.
  • Figures 1C- 1D depict exemplary results of RT-PCR amplification ( Figure 1C) and sanger sequence analysis (Figure 1D) using primers specific to each independent RNA (G1 and G2), showing removal of the ribozymes and scar-less trans-splicing and restoration of the GFP coding sequence.
  • Figure 1E depicts exemplary Western blot results using an antibody specific to GFP showing the full-length protein size predicted for GFP.
  • Figure 2A through Figure 2E depict data demonstrating the development of a luciferase-based reporter to quantify the impact of ribozyme sequences on trans-splicing in mammalian cells.
  • Figure 2A shows a diagram depicting the vectors encoding the N-terminal (Nt) half of Luciferase with 3’ HDV ribozyme and C-terminal (Ct) half of Luciferase with 5’ Hammerhead (HH) ribozyme.
  • Figures 2B-2C depict exemplary results of RT-PCR amplification ( Figure 2B) and sanger sequence analysis ( Figure 2C) using primers specific to each independent Luc RNA (L1 and L2), showing removal of the ribozymes and scar-less trans-splicing of the luciferase open reading frame.
  • Figures 2D -2E demonstrate the impact of different HDV ( Figure 2D) and HH ( Figure 2E) ribozyme sequences on trans-splicing in mammalian cells. In addition, mutation of ribozyme catalytic nucleotides resulted in loss of luciferase activity ( Figure 2D, last column, and Figure 2E, last column).
  • Figure 3A through Figure 3D depict data demonstrating the regulation of protein expression from Nt and Ct vectors.
  • Figure 3A shows a diagram depicting placement of C- terminal protein degradation sequences which prevent expression of Nt vector encoded proteins.
  • Figure 3B depicts exemplary results demonstrating the efficiency of different protein degradation sequences at preventing GFP-HDV expression from Nt vector encoding full length GFP.
  • Figure 3C shows a diagram depicting placement of N-terminal translational control sequences to prevent translation of protein sequences in Ct vectors.
  • Figure 3D depicts exemplary results demonstrating the efficiency of different GFP sequence modifications or translational control sequences at preventing GFP fluorescence in mammalian cells.
  • Figure 4A through Figure 4D depict data demonstrating single and multiplex ribozyme-mediated trans-splicing in mammalian cells.
  • Figure 4A shows a diagram depicting vectors encoding a 4xMTS and full length GFP (no start ATG codon) with ribozymes to mediate trans-splicing and expression of a mitochondrial targeted GFP protein.
  • Figure 4B depicts exemplary results demonstrating that co-expression of these vectors results in mitochondrial localized green fluorescence which overlapped with the red fluorescence of mitotracker CMXRos.
  • Figure 4C shows a diagram depicting vectors for multiplex tran-splicing and expression of a mitochondrial targeted GFP protein (4xMTS-GFP) in reading frame 1 and a myristoylation membrane targeted red fluorescent protein (F2-Myr-RFP) in reading frame 2.
  • Figure 4D depicts exemplary results demonstrating that co-expression of all four vectors in mammalian Cos7 cells results in specific green fluorescence in mitochondrial and red fluorescence in membranes.
  • Figure 5A and Figure 5B depict data demonstrating 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 placement of chimeric splice donor (SD) and splice acceptor (SA) sequences in a generic Nt-GFP-3’Rz and 5’ Rz-Ct-GFP trans- splicing GFP reporter, wherein Rz denotes an cis-cleaving ribozyme.
  • SD chimeric splice donor
  • SA splice acceptor
  • Figure 5B depicts exemplary results of GFP fluorescence in Cos7 cells after single vector transfection (first two columns) or co-transfection (last two columns) 18 hours post-transfection (first three columns) or 36 hours (last column) post-transfection.
  • the first row depicts the use of unoptimized HH and HDV ribozymes
  • second row depicts the use of optimized Twister and RzB ribozymes
  • the last row depicts to the combination of Twister and RzB ribozymes and SD and SA sequences.
  • Figure 6A through Figure 6D depict data demonstrating ribozyme-mediated trans splicing of large protein coding genes.
  • Figure 6A shows a diagram depicting vectors encoding a split ⁇ Dystrophin-GFP fusion protein for delivery using AAV vector.
  • Figures 6B-6C depicts exemplary results of RT-PCR ( Figure 6B) and sanger sequencing (Figure 6C) analyses on cells transfected with Nt-Dys and Ct-Dys vectors showing specific trans-splicing.
  • Figure 6D depicts exemplary results of GFP fluorescence from cells transfected with both Nt and Ct Dystrophin vectors imaged using confocal microscopy showing the predicted membrane localization of Dystrophin.
  • Figure 7A through Figure 7C depict data demonstrating lentiviral delivery of ribozyme-containing RNAs for trans-splicing in target cells.
  • Figure 7A shows a diagram depicting the negative sense orientation of Nt and Ct split GFP expression cassette in the lentiviral gene transfer vector.
  • Figure 7B depicts exemplary results demonstrating that only cells co-transduced with lentivirus encoding both Nt-GFP and Ct-GFP genes show GFP fluorescence.
  • Figure 7C shows a diagram depicting the negative sense orientation of Nt and Ct split Dys expression cassette in the lentiviral gene transfer vector.
  • Figure 8A and Figure 8B depict data demonstrating ribozyme-mediated trans- splicing and expression of the toxic DTA gene.
  • Figure 8A shows a diagram depicting vectors encoding a split Nt and Ct DTA gene.
  • Figure 8B depicts exemplary results demonstrating that cells co-transfected with both Nt-DTA and Ct-DTA result in decreased expression of a co- transfected GFP reporter, consistent with the translational repressor function of DTA in mammalian cells.
  • Figure 9 depicts exemplary results demonstrating that co-expression of exogenous RNA modulating enzymes can enhance or inhibit ribozyme-mediated trans-splicing in mammalian cells.
  • Figure 10A through Figure 10D depict data demonstrating that RtcB is sufficient to catalyze ribozyme-mediated trans-splicing in vitro.
  • Figure 10A shows a diagram depicting a split luciferase trans-splicing reporter which contains an upstream T7 RNA promoter to allow for in vitro RNA transcription.
  • Figure 10B shows exemplary RT-PCR results demonstrating that in vitro trans-spliced luciferase RNA is dependent upon addition of RtcB protein (NEB) using the manufacturer’s recommended reaction conditions.
  • Figure 10C shows a diagram depicting a trans-splicing vector for conserved N-terminal (N1L) and C-terminal (N3R) domains of Spidroin.
  • Figure 10D depicts exemplary sanger sequencing results demonstrating that RtcB ligase from E.
  • Figure 11A through Figure 11C depict the in vitro directional ligation of ribozyme-catalyzed RNAs using RtcB ( Figure 11A), VS-S ( Figure 11B) and VS-Rz ( Figure 11C).
  • Figure 12A through 12D depict data demonstrating the use of trans-cleaving ribozymes for trans-splicing of RNA.
  • Figure 12A depicts secondary structures of ribozymes which cleave in cis.
  • Figure 12B depicts engineered ribozymes capable of cleaving in trans.
  • Figure 12C and Figure 12D depict diagrams demonstrating potential applications of trans- cleaving ribozymes to delete disease causing mutations, such as frame-shifting or premature stop codons, to restore protein expression and function.
  • Figure 13A and Figure 13B depict data demonstrating the secondary structures of representative ribozymes which can be utilized for scar-less trans-splicing of RNA.
  • Figure 13A depicts representative ribozymes which can be used for scar-less 5’ cleavage.
  • Figure 13B depicts representative ribozymes which can be used for scar-less 3’ cleavage.
  • N any nucleotide. Red scissors demarcate a cleavage site. Red nucleotides indicate catalytic mutations.
  • Orange nucleotides represent RNA sequence to be trans-spliced. Dark blue nucleotides indicate ribozyme sequence required to form stem. Light blue indicates tertiary stabilizing motif (TSM) in stem 1 which interacts with stem 2 loop.
  • TSM tertiary stabilizing motif
  • Figure 14A through Figure 14C depict data demonstrating scar-less cleavage and inducible RNA trans-splicing and expression with trans-activating ribozymes.
  • Figure 14A depicts a diagram showing that the VS ribozyme can be split into two components, a small VS-S stem loop, which lacks autocatalytic activity, and larger VS-Rz, which induces VS-S cleavage when delivered in trans.
  • the VS-S/VS-Rz ribozyme pair can be utilized to generate inducible scar-less trans-splicing.
  • Figure 14B shows a diagram depicting a method to utilize the VS-S/VS- Rz trans-activated ribozyme pair to generate an inducible RNA trans-splicing system. Only upon delivery or expression of VS-Rz, does the Nt-GFP-VS-S RNA generate a suitable RNA terminus that can participate in trans-splicing with the co-expressed Ct-GFP RNA.
  • Figure 14C shows a diagram depicting a method to generate an RNA with an N-terminal sequence, a variable or non- variable repeat region, and C-terminal sequence.
  • the ‘repeat’ RNA contains a 5’ autocatalytic ribozyme and a 3’ trans-activated ribozyme, such as VS-S, which allows for controlled repeat addition dependent upon the selective addition of trans-activating VS-Rz and ligase, such as RtcB.
  • Figure 15A through Figure 15E depict data demonstrating ribozyme-mediated trans-splicing with generation of stable intronic RNA sequences.
  • Figure 15A shows a diagram depicting the use of cis-cleaving ribozymes to mediate the trans-splicing of two independent RNAs.
  • Figure 15B shows a diagram depicting the use of internal cis-cleaving ribozymes to create a synthetic intron.
  • Figure 15C depicts exemplary results demonstrating efficient cis- cleavage of a synthetic intron and trans-splicing of independent RNAs to yield functional protein (GFP).
  • Figure 15D and Figure 15E show diagrams depicting the use of internal cis-cleaving ribozymes to generate a trans-spliced and translated reporter and intronic sequence, ‘cargo’, which could be any useful RNA sequence or gene expression cassette.
  • Figure 16A through Figure 16C depict exemplary results of optimized ribozyme sequences for ribozyme-mediated trans-splicing in vivo.
  • Figure 16A depicts a comparison of the relative ribozyme activity using a Luciferase trans-splicing reporter.
  • FIG. 16B depicts a comparison of HDV ribozymes (HDV68 and Genomic HDV with a Twister ribozyme (Twst).
  • Twst Twister ribozyme
  • a Twister ribozyme on the 3’ end of Nt-Luc provided the greatest luciferase activity, which was abolished with catalytic inactivating mutations (Twst mut).
  • Figure 16C depicts a comparison of Twister ribozyme sequence modifications. Shortening of the P1 stem decreased reporter activity.
  • FIG. 17A through Figure 17C depict identification of optimal ribozyme pairs for StitchR-mediated RNA trans-splicing in mammalian cells.
  • Figure 18A through Figure 18C depict data demonstrating the application of StitchR for dual AAV gene delivery and expression in mammalian cells.
  • Figure 18A depicts the design of a stitchr enabled split GFP reporter cassette, under the control of the human CMV (hCMV) promoter and bovine growth hormone poly adenylation sequence (bGH pA), subcloned into a vector flanked by AAV2 ITR sequences. Constructs were used to generate AAV2/1 serotyped virus, which only when co-transduced into human cells (HEK293T), resulted in robust full-length GFP expression as detected by epifluorescence (Figure 18B) and western blot (Figure 18C).
  • hCMV human CMV
  • bGH pA bovine growth hormone poly adenylation sequence
  • FIG 19A through Figure 19C depict data demonstrating the application of StitchR for dual AAV gene delivery and expression in vivo.
  • StitchR enabled dual AAV GFP reporter virus (2E+12 vg/ml) was injected into postnatal day 10 (P10) mice and imaged 2 months post injection using (Figure 19A) IVIS fluorescence based whole animal imaging or (Figure 19B) epifluorescence. Robust GFP fluorescence was detected in the whole body and readily observed in hindlimb musculature.
  • Figure 19C depicts data demonstrating that full-length GFP protein expression was observed only in mice injected with both GFPnt and GFPct virus, detected by western blot using an anti-GFP antibody.
  • Figure 20 depicts the subset of human, loss-of-function, monogenic diseases arising from mutations in large genes. Numbers in parentheses represent the amino acid sequence length of the adjacent disease gene. [0050] Figure 21 depicts data demonstrating that mutations in the large Dystrophin (Dys) protein can result in debilitating Duchenne Muscular Dystrophy (DMD) or less severe Becker Muscular Dystrophy (BMD).
  • DMD Duchenne Muscular Dystrophy
  • BMD Becker Muscular Dystrophy
  • Figure 22 depicts a diagram of a microDys AAV expression vector controlled by the cardiac and skeletal muscle specific CK8e promoter and bGH pA.
  • Figure 23 depicts a diagram of the dual StitchR enabled N-terminal (StitchR Dys- Nt ) and C-terminal (StitchR Dys-Ct) AAV expression vectors controlled by the cardiac and skeletal muscle specific CK8e promoter and bGH pA.
  • Figure 24 depicts a diagram of a dual AAV vector pair N-terminal (Dual AK Dys- Nt ) and C-terminal (Dual AK Dys-Ct) expression vectors utilizing the recombinogenic AK sequence and controlled by the cardiac and skeletal muscle specific CK8e promoter and bGH pA.
  • Figure 25 depicts data demonstrating that full-length miniDystrophin protein expression in human HEK293T cells transfected with either AK, StitchR or single ORF expression vectors, under the control of the core EF1a promoter, detected using Western blot with either N-terminal or C-terminal anti-Dystrophin specific antibodies.
  • StitchR technology was efficient at generating full-length protein (lane 7). This activity was dependent upon ribozyme- mediated RNA cleavage, since mutation of a single catalytic residue abolished full-length expression (lane 8).
  • StitchR was more efficient than AK recombinogenic sequence-based vectors, which did not result in detectable full-length miniDystrophin expression at this exposure level (compare lanes 4 and 7). Remarkably, StitchR was nearly as efficient in generating full- length miniDystrophin protein as a vector encoding full-length miniDystrophin in a single open reading frame (compare lanes 7 and 9).
  • Figure 26 depicts data demonstrating that full-length miniDystrophin protein expression in vivo transduced with StitchR-enabled AAV virus under the control of the core CK8e promoter, detected using Western blot.
  • FIG. 27 depicts a diagram depicting Human Dysferlin (DYSF) protein with known domains, locations of important protein interactions. Human mutations in DYSF can result in limb girdle muscular Dystrophy type 2b (LGMD2B) and Myoshi Myopathy (MM), now commonly referred to as Dysferlinopathies.
  • LGMD2B limb girdle muscular Dystrophy type 2b
  • MM Myoshi Myopathy
  • Figure 28 depicts a diagram of a StitchR enabled dual AAV vector pair to express full-length human codon optimized Dysferlin (hcoDYSF) vectors under the control of the cardiac and skeletal muscle-specific CK8e promoter and bGH pA.
  • Figure 29 depicts data demonstrating that full-length human Dysferlin protein expression in human HEK293T cells, transfected with either dual StitchR vectors or a vector encoding dysferlin in a single ORF expression vector, under the control of the core EF1a promoter, detected using Western blot with an anti-Dysferlin antibody.
  • Figure 30 depicts data demonstrating that full-length human Sterocilin (STRC) protein expression in human HEK293T cells, transfected with dual StitchR vectors, under the control of the core EF1a promoter, detected using Western blot with either an N-terminal or C- terminal anti-STRC antibody.
  • Figure 31 is a schematic depicting the ribozyme-mediated scarless cis-splicing to generate a circular RNA which can be translated via IRES-mediated translation.
  • Figure 32 depicts experimental data demonstrating the generation of GFP- encoding circular RNA and expression of GFP from the circular RNA.
  • Figure 33 is a schematic depicting the function of IRES and ribozymes for protein expression from CirculR.
  • Figure 34 depicts a lentiviral based screen for functional IRES sequences using CirculR.
  • Figure 35A and Figure 35B depict the development of a stitchR-activity dependent gene cassette and cell lines.
  • Figure 35A Diagram depicting a stitchR-dependent ribotron interrupted HSV-TK expression cassette encoded within a lentiviral vector, in reverse orientation to enable lentiviral RNA delivery. Lentiviral delivery allows for the indelible integration of the ribotron encoded HSV-TK cassette into cells, however other methods could be used to stably express the ribotron HSV-TK transgene.
  • Figure 35B Expression of the ribozyme-activated synthetic intron (or ribotron), allows for stitchR-dependent expression of the HSV-TK cell death gene in cells, which in the presence of the non-toxic molecule ganciclovir (GCN), induces cell death.
  • GCN non-toxic molecule ganciclovir
  • Figure 36A through Figure 36J depict the discovery of ribozyme-activated RNA trans-ligation in mammalian cells.
  • Figure 36A Design of a StitchR (“Stitch RNA”) GFP reporter.
  • the open reading frame (ORF) of GFP was split between two non-overlapping N- terminal (Nt) and C-terminal (Ct) vectors. Ribozymes (Rz) were utilized on the 3' end of the Nt- GFP vector and 5' end of the Ct-GFP vector to create precise RNA termini.
  • Figure 36B Fluorescence imaging of transiently transfected Nt- and Ct-GFP vectors in HEK293T and COS7 cells. Scale bars represent 100 ⁇ m.
  • Figure 36C RT-PCR of RNA deriving from cells transfected with Nt- and Ct-GFP vectors using primers (G1 and G2) spanning the trans-ligation site.
  • FIG. 36D Sanger sequencing of the PCR band in “C.”
  • Figure 36E Western blot using a primary antibody against GFP showed full-length GFP protein was produced.
  • Figure 36F and Figure 36G RT-PCR and Sanger sequencing produced similar results for a StitchR luciferase reporter, using primers L1 and L2.
  • Figure 36H and Figure 36I StitchR luciferase assays were used to screen for optimal ribozyme sequences that produce scarless 5' cleavage and 3' cleavage.
  • Figure 36J The optimized StitchR 2.0 split-luciferase reporter manifested ⁇ 8-fold more activity than StitchR 1.0.
  • Figure 37A through Figure 37G depict the optimization of ribozyme-activated RNA trans-ligation and translation in mammalian cells.
  • Figure 37A Design of a StitchR 3.0 RNA trans-ligation reporter. The open reading frame of GFP containing an intron (splice donor (SD) and splice acceptor (SA) sequences) was split into two non-overlapping N-terminal (Nt) and C-terminal (Ct) vectors. Small catalytic ribozymes (Rz) were utilized on the 3' end of the Nt- GFP and 5' end of the Ct-GFP vectors to create precise RNA termini and scarless trans-ligation.
  • SD splice donor
  • SA splice acceptor
  • Figure 37E Confocal images of BaseScope assays, using a probe specific to the stitched seam of the reconstituted luciferase mRNA, were used to determine the location of mRNA trans-ligation in Nt- and Ct-Luc transfected COS7 cells after 24 hours, or earlier timepoints (Figure 37F). Scale bars - 10 ⁇ m.
  • Figure 37G Mean Luciferase assay values for Figure 37C, represented as fold change over empty vector.
  • Figure 38A through Figure 38F depict data demonstrating that the addition of a split intron enhances StitchR activity and allows for inclusion of non-scarless ribozymes.
  • Figure 38C Sequences and predicted 2D structures of model ribozyme family members used in the Nt- and Ct-Luc vectors described in Figure 37C. Red arrowheads indicate ribozyme cleavage sites.
  • Figure 38D Pairwise comparisons of StitchR activity, catalyzed by model ribozymes selected from the three twister ribozyme Types (P1, P3, and P5), cloned into StitchR 3.0 Nt- and Ct- luciferase (Luc) vectors and assayed in COS7 cells. Color scale represents fold Luc activity over empty vector control.
  • Figure 38E Design of a BaseScope probe used in Figure 37E and Figure 37F that is specific for the stitched full-length luciferase mRNA.
  • Figure 38F Mean Luciferase assay values for Figure 38D, represented as fold change over empty vector.
  • Figure 39A through Figure 39G depict data demonstrating that ribozyme cleavage generates RNA termini that are required for StitchR activity and recognized by the unconventional RNA ligase, RtcB.
  • Figure 39A Diagram of StitchR plasmids used in the in vitro StitchR assay. StitchR RNAs were in vitro-transcribed and then incubated with or without RtcB. Please see Materials and Methods for full reaction details.
  • FIG. 39B RT-PCR products that were generated using primers specific to the Nt- and Ct-Luc StitchR vectors, were separated using gel electrophoresis.
  • Figure 39C Western blot using an anti-Flag antibody to detect exogenous expression of Flag -tagged RtcB from bacteria, human and archea species, and Flag- tagged T4 PNK
  • Figure 40A through Figure 40I depict data demonstrating efficient expression of full-length and chimeric proteins using StitchR in mammalian cells.
  • Figure 40A Diagram of StitchR vectors that produce ACTA2 mRNA, encoding alpha smooth muscle actin ( ⁇ SMC).
  • Figure 40B Immunofluorescence staining for ⁇ SMC in wild-type HeLa cells and knockout (ACTA2-KO) HeLa cells. Scale bars - 50 ⁇ m.
  • FIG. 40C High magnification micrograph of StitchR ⁇ SMC in ACTA2 KO HeLa cells showing normal actin fiber formation (white arrowhead).
  • Figure 40D Western blot detection of ⁇ SMA protein using an N-terminal-specific primary antibody in lysates from wild-type HeLa cells and ACTA2-KO cells, transiently transfected with empty vector or with Nt- and Ct-ACTA2 vectors. Addition of a C-terminal degron in-frame with the Nt-ACTA2 vector was not required to prevent non-full-length products.
  • Figure 40E Quantification of western blot data showing StitchR restores ⁇ SMA protein to endogenous levels.
  • FIG. 41A through Figure 41J depict data demonstrating StitchR is compatible with AAV-gene delivery.
  • Figure 41A Vector diagrams for StitchR-enabled dual AAV gene delivery of a split GFP reporter with an N-terminal 3xFLAG (3F) and C-terminal 3xHA (3H) epitope tags.
  • FIG. 41B Separate StitchR Nt-GFP and Ct-GFP AAV1 serotyped virus particles were generated and used to transduce HEK293T cells. Robust GFP fluorescence was detected 48 hours post-transduction using fluorescence microscopy only in Nt-and Ct-GFP co-transduced cells. Scale bars - 50 ⁇ m.
  • Figure 41C and Figure 41D Deep sequencing of RNA transcripts from two replicate HEK293T wells transduced with both Nt- and Ct-GFP virus. Paired-end sequence reads were mapped to the full-length GFP mRNA and depth of sequence reads were quantified at the seam and flanking Nt-GFP and Ct-GFP fragments.
  • FIG. 41E In vivo imaging of GFP fluorescence in mice injected with saline, Nt-GFP, Ct- GFP, or both Nt-GFP and Ct-GFP AAV1 serotype virus, 5 months post-injection.
  • Figure 41F and Figure 41G Fluorescence whole mount imaging of leg and isolated quadriceps muscle from a StitchR injected mouse, and saline-injected control.
  • Figure 41H to Figure 41J Full-length GFP protein from quadricep muscles of StitchR injected mice, detected by Western blotting using ⁇ -GFP, ⁇ -HA and ⁇ -FLAG antibodies.
  • FIG. 41H GAPDH loading control shown in ‘ Figure 41H’ is the same for ‘ Figure 41I’ and ‘ Figure 41J.’
  • Figure 42A through Figure 42G depict data demonstrating StitchR-mediated expression of human full-length Dysferlin (DYSF) in Dysferlin-knockout mice (A/J strain).
  • Figure 42A The Dysferlin gene encodes a large membrane-associated protein in which loss-of- function mutations give rise to limb-girdle muscular dystrophy type 2B/2R, Miyoshi myopathy, and distal myopathy with anterior tibialis onset (DMAT).
  • the Dysferlin ORF exceeds the packaging capacity of a single AAV vector but can be completely packaged using a dual AAV vector approach.
  • Figure 43A through Figure 43H depict data demonstrating the design and testing of StitchR vectors for expressing full-length human Dysferlin.
  • Figure 43A Illustration of StitchR Nt and Ct AAV transfer plasmids and location of split site to express full-length human Dysferlin.
  • Figure 43B and Figure 43C Western blot and densitometry quantification of full- length Dysferlin expression in HEK293T cells transiently transfected with both Nt and Ct vectors, compared to human Dysferlin expressed from a single ORF using a primary anti- Dysferlin antibody ( ⁇ -Hamlet).
  • FIG. 43G and Figure 43H Western blot and densitometry quantification of full- length Dysferlin from quadriceps using an N-terminal specific Dysferlin ( ⁇ -Romeo) antibody showed similar results compared to a C-terminal antibody shown in Figure 42D. Vinculin loading control is the same as in Figure 42D.
  • Figure 44A through Figure 44H depict data demonstrating the StitchR-mediated therapeutic correction of the DMD mouse with a full-functional ⁇ H2-R15 Dystrophin.
  • Figure 44A Loss-of-function mutations in the large Dystrophin gene give rise to Duchenne Muscular Dystrophy.
  • FIG. 45A Illustration of StitchR Nt and Ct AAV plasmids to express human ⁇ H2-R15 Dystrophin.
  • Figure 45B and Figure 45C Western blot and densitometry quantification of Dystrophin ⁇ H2-R15 expression in HEK293T cells transiently transfected with both Nt and Ct vectors, compared with ⁇ H2-R15 Dystrophin expressed from a single ORF using a primary anti-Dystrophin antibody ( ⁇ -DysB).
  • StitchR Dystrophin ⁇ H2-R15 expression levels were 94.8% of those achieved by a single ORF (without an intron) and mutation of catalytic nucleotides in a single ribozyme abolished Dystrophin ⁇ H2-R15 expression.
  • Figure 45D Masson trichrome and
  • Figure 45E H&E staining of quadriceps and diaphragm muscle, respectively.
  • Figure 45F to Figure 45H Immunofluorescence staining for Dystrophin protein using a Dystrophin-specific antibody in quadriceps, diaphragm and heart.
  • FIG. 45I Western blot and quantification of Dystrophin levels in TA and heart.
  • Figure 45M and Figure 45N Western blot and quantification of Dystrophin levels in quadriceps muscle using an N-terminal specific antibody ( ⁇ -MANEX1101b) produced similar results as a C-terminal specific antibody shown in Figure 44E. Vinculin loading control is same as in Figure 44E.
  • Figure 46A through Figure 46H depict data demonstrating StitchR-enabled Prime Editing.
  • FIG. 46A A StitchR-enabled Prime Editor (StitchR-PE) was designed to express the Prime Editor PEmax, and Prime Editing components, including cloning sites for the pegRNA and nickase sgRNA cassettes in the Ct vector ( Figure 46B).
  • Figure 46C StitchR-PE activity was compared with PEmax expressed from a single ORF using the PEAR-GFP prime editing reporter. Scale bars - 50 ⁇ m.
  • Figure 46D Design of a luciferase-based prime editing reporter (LUPER), to precisely compare StitchR-PE and PEmax expressed from a single ORF.
  • LUPER luciferase-based prime editing reporter
  • FIG. 47A Diagram depicting an expression cassette for PEmax, and optimized SpyCas9 nickase – MMLV prime editor, which itself exceeds the packaging capacity of AAV, even in the absence of promoter or polyadenylation sequences required for gene expression.
  • Figure 47B and Figure 47C A dual StitchR-enabled Prime editor consists of an N-terminal and C-terminal vector which splits the PEmax editor within the C-terminus of the nickase SpyCas9- MMLV fusion protein. This strategy results in either vector from encoding functional elements, as well as leaves enough room in the N-terminal vector with additional room for larger promoter sequences, or fusions to the N-terminus of nCas9.
  • Cloning sites for the pegRNA and nickase sgRNA cassettes are provided in the C-terminal vector, such as only the C-terminal vector needs to be modified for use with the N-terminal vector for different targeting sequences.
  • Figure 47D Full-length PEmax encoded within a single ORF or the stitchR-enabled dual vector PEmax were compared using the PEAR-GFP prime editing reporter, which uses Prime editing to repair a 5’ splice donor site within an intron containing GFP reporter.
  • the PEAR-GFP 2in1 reporter plasmid includes expression cassettes for both PEAR and the PEAR-specific pegRNA.
  • FIG. 47G StitchR PEmax dual vectors activated the LUPER reporter at 74.4% to that observed for full- length PEmax.
  • Figure 48A through Figure 48F depict data demonstrating chemically tunable StitchR vectors for Prime Editing.
  • Figure 48A An E. coli destabilizing domain (ecDHFR) was fused to the N-terminus of the N terminal StitchR Prime editing pair. To compensate for the loss of the N-terminal NLS, a robust Ty1 nls was fused to the C-terminus of the C-terminal StitchR PEmax vector ( Figure 48B).
  • FIG. 48C Diagram depicting the predicted effect of fusing a destabilizing domain onto a PEmax editor, such that in absence of a stabilizing small molecules (Such as TMP), the PEmax protein will be degraded. However, in the presence of stabilizing molecule (such as TMP), PEmax protein will be stabilized ( Figure 48D).
  • Figure 48E In the absence of TMP (DMSO control), only a few PEAR-GFP positive cells were observed, indicating disruption of prime editing due to protein destabilization. In the presence of 1 ⁇ M TMP, robust PEAR-GFP expressing cells were observed, which corresponded to a 37-fold increase in our LUPER prime editing luciferase reporter (Figure 48F).
  • FIG. 49A through Figure 49G depict data demonstrating prime editing strategy for disruption of the myostatin (MSTN) gene to treat muscle atrophy or sarcopenia.
  • the myostatin (MSTN) gene is a member of the TGFb superfamily that is expressed in muscle, and functions as an inhibitor of muscle growth. Disruption of Myostatin in multiple species results in a double-muscled phenotype, characterized by significantly large muscles and enhanced performance.
  • Figure 49A MSTN is encoded by three exons, with the mature functional domain encoded within the terminal exon.
  • Figure 49G High conservation at the Splice Donor region of Exon2-Intron2 allows for identical, or highly similar pegRNA and nicking sgRNAs to be used to disrupt MSTN across multiple species, which could have therapeutic or useful applications across numerous species.
  • Figure 50A through Figure 50C depict data demonstrating PE-mediated insertion of a minimal polyA site to prevent microsatellite RNA repeat expression for the treatment of Myotonic Dystrophy Type 1 (DM1).
  • Figure 50A Previous studies have identified the minimal sequence requirements to recapitulate polyadenylation in mammalian cells, and naturally occurring small polyA sites, such as that found in the HSV TK gene.
  • FIG. 50B Microsatellite repeat expansion in the 3’UTR of the human DMPK gene, when transcribed into RNA, forms toxic nuclear RNA foci, which binds and sequesters important splicing factors and gives rise to abnormal splicing. This also reduces DMPK protein expression due to sequestration of DMPK RNAs within the nucleus.
  • Figure 50C PE-mediated insertion of a small polyadenylation site upstream of the microsatellite CTG repeat expansion would serve to both 1) prematurely polyadenylate and terminate transcription upstream of the repeat sequence, and 2) allow for DMPK protein expression.
  • Antisense refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand.
  • an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule.
  • the antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.
  • nucleic acid molecules to a solid support
  • attachment as used herein is intended to encompass direct or indirect, covalent or non-covalent attachment, unless indicated otherwise, either explicitly or by context.
  • microspheres as used herein interchangeably, “beads” or grammatical equivalents thereof describe small discrete particles capable of acting a solid support for attachment of a biomolecule (e.g., a nucleic acid molecule).
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • 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 state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health. [0091] A disease or disorder is “alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • the terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal or cell whether in vitro or in vivo, amenable to the methods described herein.
  • the subjects include 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, cynomologous monkeys, spider monkeys, and macaques (e.g., Rhesus). Rodents include, but are not limited to, mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cat), canine species (e.g., dog, fox, wolf), avian species (e.g., chicken, emu, ostrich), and fish (e.g., zebrafish, trout, catfish and salmon).
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the patient, subject or individual is a human.
  • an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • a “coding region” of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.
  • a “coding region” of a mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anti-codon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon.
  • the coding region may thus include nucleotide residues comprising codons for amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).
  • “Complementary” as used herein to refer to a nucleic acid refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil.
  • a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In one embodiment, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • DNA as used herein is defined as deoxyribonucleic acid.
  • expression as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are 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, and the like.
  • Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.
  • wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
  • 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 Package of the 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.
  • “Isolated” means altered or removed from the natural state.
  • nucleic acid or a peptide naturally present in its normal context in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • isolated when used in relation to a nucleic acid, as in “isolated oligonucleotide” or “isolated polynucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant with which it is ordinarily associated in its source.
  • an isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature.
  • non-isolated nucleic acids e.g., DNA and RNA
  • a given DNA sequence e.g., a gene
  • RNA sequences e.g., a specific mRNA sequence encoding a specific protein
  • isolated nucleic acid includes, by way of example, such nucleic acid in cells ordinarily expressing that nucleic acid where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.
  • the isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form.
  • the oligonucleotide When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide contains at a minimum, the sense or coding strand (i.e., the oligonucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double- stranded).
  • isolated when used in relation to a polypeptide, as in “isolated protein” or “isolated polypeptide” refers to a polypeptide that is identified and separated from at least one contaminant with which it is ordinarily associated in its source.
  • 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, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages.
  • nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).
  • nucleic acid typically refers to large polynucleotides. [0108] 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.
  • RNA transcripts The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the “coding strand”; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as “downstream sequences.”
  • expression cassette is meant a nucleic acid molecule comprising a coding sequence operably linked to promoter/regulatory sequences necessary for transcription and, optionally, translation of the coding sequence.
  • operably linked refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
  • the term also refers to the linkage of sequences encoding amino acids in such a manner that a functional (e.g., enzymatically active, capable of binding to a binding partner, capable of inhibiting, etc.) protein or polypeptide is produced.
  • promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulator sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a n inducible manner.
  • stringent conditions for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme.
  • a sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence.
  • An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced substantially only when an inducer which corresponds to the promoter is present.
  • a “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • the term “polynucleotide” as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.”
  • the monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
  • nucleic acid bases In the context of the present invention, the following abbreviations for the commonly occurring 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. [0119] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • RNA as used herein is defined as ribonucleic acid.
  • ribozyme refers to an RNA molecule capable of acting as an enzyme. For example, some ribozymes are capable of cleaving RNA molecules. RNA cleaving ribozymes typically consist at least of a catalytic domain and a recognition sequence that is recognized by the catalytic domain. The catalytic domain can be a part of the same RNA molecule as the recognition sequence, and thus mediate cis-cleavage.
  • the catalytic domain can be a separate RNA molecule from the RNA molecule comprising the recognition sequence, and thus mediate trans-cleavage.
  • “Recombinant polynucleotide” refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell. [0123] A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
  • a non-coding function e.g., promoter, origin of replication, ribosome-binding site, etc.
  • polypeptide as used herein is defined as a polypeptide produced by using recombinant DNA methods.
  • solid surface As used herein, the terms “solid surface,” “solid support” and other grammatical equivalents thereof refer to any material that is appropriate for or can be modified to be appropriate for the attachment of a biomolecule (e.g., a nucleic acid molecule).
  • tag refers to any chemical modification of a biomolecule (e.g., a nucleic acid molecule) that provides additional functionality (e.g., attachment to a solid support, fluorescence visualization, etc.).
  • Variant is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.
  • a “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • vector includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • Ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the present invention provides compositions and methods for efficiently and reliably ligating two or more individual RNA molecules to produce a larger single RNA molecule that encodes proteins and fusion proteins.
  • the invention utilizes ribozyme-mediated trans-splicing of multiple RNA molecules to assemble a single RNA molecule encoding a protein or fusion protein of interest.
  • the present invention can be used to efficiently produce fusion proteins, chimeric proteins, and the like.
  • the present invention is useful in producing large expression products (e.g., proteins or fusion proteins) whose coding sequence may be too large to package into a single vector.
  • the full- length expression product is great than 1000 amino acid residues in length.
  • the full-length expression product is great than 2000 amino acid residues in length. In some embodiments, the full-length expression product is great than 3000 amino acid residues in length.
  • the technology of the present invention also allows for the rapid and easy combination of two different sequences, which could have a multiplier effect for generating novel protein combinations or library sequences. This may be particularly useful, for example, for generating synthetic antibodies (like nanobodies) or for functional selection of enzymes. [0131]
  • the present invention also provides compositions and methods for efficiently delivering one or more RNA molecule with a ribozyme-flanked synthetic intron.
  • the ribozyme- flanked synthetic intron can be placed between a first RNA portion encoding an N-terminal portion of a protein of interest and a second RNA portion encoding a C-terminal portion of a protein of interest.
  • the ribozyme-flanked synthetic intron can comprise 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 generate three RNA fragments: 1) the first RNA portion encoding an N-terminal portion of a protein of interest, 2) the ribozyme-flanked synthetic intron, and 3) second RNA portion encoding a C-terminal portion of a protein of interest.
  • Said cis- splicing generates compatible ends for ligation.
  • Ligation of the compatible ends of the cis-spliced synthetic intron generates a circular RNA molecule, more resistant to degradation than a linear RNA molecule.
  • Ligation of the compatible ends of the first RNA portion encoding an N-terminal portion of a protein of interest and the second RNA portion encoding a C-terminal portion of a protein of interest generates an RNA molecule encoding a full-length protein of interest.
  • the full-length protein of interest can be, for example, a therapeutic protein, CRISPR-Cas protein, or reporter protein to provide a proxy indicator for delivery and expression of the cargo sequence in the circular RNA molecule comprising the ribozyme-flanked synthetic intron.
  • the present invention provides one or more nucleic acid molecules encoding two or more RNA molecules.
  • one or more of the RNA molecules comprise a ribozyme.
  • one or more of the RNA molecules comprise a coding region and a ribozyme.
  • the ribozyme self-cleaves out of the RNA molecule leaving the coding region.
  • Exemplary ribozymes that may be used in the context of the present invention include, but is not limited to, members of the Hammerhead (HH), Hepatitis Delta Virus (HDV), Varkud Satellite (VS), Sister, Twister-sister, Hairpin, Hatchet, Pistol, HOV Linc, or lantern families of ribozymes.
  • the present invention is not limited to any particular ribozyme, but rather encompasses members of all known endogenous ribozyme families and any potential artificial ribozymes. That is, the described ribozymes, all known endogenous ribozymes, and potential artificial ribozymes can be used in the ligation of multiple RNAs, transplicing, and circularization, as described elsewhere herein.
  • the composition comprises a nucleic acid molecule encoding a first RNA molecule, where the first RNA molecule comprises a coding region and a 3’ ribozyme, where the 3’ ribozyme is able to catalyze itself out of the RNA molecule leaving the coding region with a 3’P or 2’3’ cyclic phosphate (cP) end.
  • the 3’ ribozyme comprises an HDV ribozyme.
  • the composition comprises a nucleic acid molecule encoding a second RNA molecule, where the second RNA molecule comprises a coding region and a 5’ ribozyme, where the 5’ ribozyme is able to catalyze itself out of the RNA molecule leaving the coding region with a 5’OH end.
  • the 5’ ribozyme comprises an HH ribozyme.
  • a ligase joins the coding region of the first RNA molecule to the coding region of the second RNA molecule together to form a longer RNA molecule encoding a protein of interest.
  • the composition comprises a first RNA molecule, where the first RNA molecule comprises a coding region and a 3’ ribozyme, where the 3’ ribozyme is able to catalyze itself out of the RNA molecule leaving the coding region with a 3’P or 2’3’ cyclic phosphate (cP) end.
  • the 3’ ribozyme comprises an HDV ribozyme.
  • the composition comprises a second RNA molecule, where the second RNA molecule comprises a coding region and a 5’ ribozyme, where the 5’ ribozyme is able to catalyze itself out of the RNA molecule leaving the coding region with a 5’OH end.
  • the 5’ ribozyme comprises an HH ribozyme.
  • a ligase joins the coding region of the first RNA molecule to the coding region of the second RNA molecule together to form a longer RNA molecule encoding a protein of interest.
  • the first RNA comprises a coding region encoding a first portion of the protein of interest and the second RNA comprises a coding region encoding a second portion of the protein of interest, and thus the ribozyme-mediated cleavage and ligase- mediated assembly of the RNA molecules results in the production of an RNA molecule encoding a protein having both the first and second portions.
  • the present invention can be used to produce full-length proteins from multiple RNAs, each comprising a coding region encoding a portion of the full-length protein. Further, the present invention can be used to produce fusion proteins comprising multiple domains, where each RNA molecule comprises a coding region encoding a domain of the fusion protein.
  • the present invention can be used to generate an RNA molecule encoding a protein having a leader sequence, N-terminal tag, C- terminal tag, or the like by assembling an RNA from a first RNA comprising a coding sequence encoding the leader sequence, N-terminal tag, or C-terminal tag, and a second RNA molecule comprising a coding sequence encoding the protein.
  • the invention relates to formation of a single RNA molecule from three or more individual RNA molecules.
  • the composition comprise a nucleic acid molecule encoding a first RNA molecule, where the first RNA molecule comprises a coding region encoding the N-terminal region of a protein; a nucleic acid molecule encoding a second RNA molecule, where the second RNA molecule comprises a coding region encoding the C-terminal region of a protein; and one or more nucleic acid molecules encoding one or more additional RNA molecules, each comprising a coding region encoding a protein domain (e.g., repeat domain).
  • a protein domain e.g., repeat domain
  • the first RNA molecule comprises a coding region encoding the N-terminal region and a 3’ ribozyme, where the 3’ ribozyme is able to catalyze itself out of the RNA molecule leaving the coding region with a 3’P or 2’3’ cyclic phosphate (cP) end.
  • the 3’ ribozyme comprises an HDV ribozyme.
  • the second RNA molecule comprises a coding region encoding the C-terminal region and a 5’ ribozyme, where the 5’ ribozyme is able to catalyze itself out of the RNA molecule leaving the coding region with a 5’OH end.
  • the 5’ ribozyme comprises an HH ribozyme.
  • the additional RNA molecules each comprise a coding region encoding a protein domain, a 3’ ribozyme and a 5’ ribozyme.
  • the 3’ribozyme is an HDV ribozyme.
  • the 5’ribozyme is an HH ribozyme.
  • the 3’ribozyme is able to catalyze itself out of the RNA molecule and the 5’ribozyme is able to catalyze itself out of the RNA molecule leaving the coding region with a 5’OH and a 3’P or 2’3’ cP end.
  • the additional RNA molecules each comprise a coding region encoding a protein domain, a 5’ ribozyme and a 3’ ribozyme recognition sequence.
  • the 5’ribozyme is able to catalyze itself out of the RNA molecule leaving the coding region with a 5’OH end; and the 3’ribozyme recognition sequence interacts with a ribozyme to induce the splicing of the 3’ribozyme recognition sequence out of RNA molecule leaving coding region with and a 3’P or 2’3’ cP end.
  • the 3’ribozyme recognition sequence comprises a Vsv1 sequence that interacts with a VS ribozyme.
  • This technique can be used to generate RNA molecules encoding a protein with multiple repeat domains by sequentially adding coding regions encoding a repeat domain by sequentially providing a ribozyme (e.g. VS ribozyme) to interact with a 3’ ribozyme recognition sequence to generate a 3’P or 2’3’ cP end and ligating the coding region to the 5’OH end of another coding region encoding a repeat domain.
  • the sequential addition of repeat domains can be performed on a solid substrate or support, where the first RNA molecule encoding the N- terminal region is bound to the substrate or support.
  • the multiple RNA molecules are ligated together after ribozyme-mediated generation of the 5’OH and 3’P or 2’3’cP ends.
  • the RNA molecules are ligated together by an endogenous ligase that exists in the native cell or tissue in which the RNA assembly is taking place.
  • the method of the present invention comprises the step of adding an exogenous ligase to induce the ligation of the processed RNA molecules together.
  • the ligase is RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase.
  • the present invention relates to the use of deoxyribozymes, enzymes or DNAases for cleaving of single stranded DNA sequences for trans-splicing or editing in trans.
  • deoxyribozymes self cleaving DNA sequences which leave 3’-P (or 2’3’-cP) and 5’-OH ends
  • enzymes or DNAses which cleave and leave those same ends can be used on single stranded DNA substrates for trans splicing or editing in trans with trans cleaving deoxyriboyme sequences.
  • RTCB can be used to act upon single stranded DNA substrates with 3’-P (or 2’3’-cP) and 5’-OH ends generated by the deoxyribozymes, enzymes or DNAases.
  • the present invention relates to compositions and methods for prime editing using ribozyme-activated RNA cleavage and trans-ligation for the efficient expression prime editors (e.g, nCas9(H840A) – MMLV fusions) and associated pegRNA and/or nickase sgRNA.
  • the present invention relates to a composition comprising one or more nucleic acid molecule encoding one or more ribozyme.
  • the present invention comprises one or more RNA molecule comprising one or more ribozyme.
  • the one or more RNA molecule comprises at least a first RNA molecule and a second RNA molecule.
  • said one or more ribozyme of the composition is capable of spontaneously cis-cleaving from said one or more RNA molecule.
  • said one or more ribozyme is a 3’ ribozyme.
  • said 3’ ribozyme generates a 3’P or 2’3’ cP end on the remaining one or more RNA molecule after spontaneous cis-cleavage.
  • said one or more ribozyme is a 5’ ribozyme.
  • said 5’ ribozyme generates a 5’OH end on the remaining one or more RNA molecules after spontaneous cis-cleavage.
  • said 3’P or 2’3’ cP end and said 5’OH end can be ligated together.
  • said first RNA molecule comprises a 3’ ribozyme.
  • said 3’ ribozyme is from one or more family selected from the group consisting of: Hammerhead (HH), Hepatitis Delta Virus (HDV), Varkud Satellite (VS), Twister (Twst), Sister, Twister-sister (TS), Hairpin, Hatchet, Pistol, HOV Linc or a variant or fragment thereof that maintains cis-cleaving functionality.
  • the 3’ ribozyme comprises the lantern ribozyme (Zhou et al.2003, Research Square; DOI: 10.21203/rs.3.rs-2567304/v1).
  • the present invention is not limited to any particular ribozyme, but rather encompasses members of all known endogenous ribozyme families and any potential artificial ribozymes.
  • the 3’ ribozyme comprises a Type P1 Twister, a Type P3 Twister, or a Type P5 Twister.
  • the 3’ribozyme comprises a Type P1 Twister.
  • the 3’ ribozyme comprises Type P1 Twister from rice (Oryza sativa, Osa).
  • the 3’ ribozyme comprises an overhang of one or more nucleotides.
  • the overhang comprises a nucleotide sequence that hybridizes to a sequence upstream of said 3’ ribozyme within the first RNA molecule. In some embodiments, the overhang improves efficiency of spontaneous cis-cleavage. [0143] In some embodiments, said second RNA molecule comprises a 5’ ribozyme.
  • said 5’ ribozyme is from one or more family selected from the group consisting of: Hammerhead (HH), Hepatitis Delta Virus (HDV), Varkud Satellite (VS), Twister (Twst), Sister, Twister-sister (TS), Hairpin, Hatchet, Pistol, HOV Linc or a variant or fragment thereof that maintains cis-cleaving functionality.
  • the 5’ ribozyme comprises the lantern ribozyme (Zhou et al., 2023, Research Square; DOI: 10.21203/rs.3.rs-2567304/v1).
  • the present invention is not limited to any particular ribozyme, but rather encompasses members of all known endogenous ribozyme families and any potential artificial ribozymes.
  • the 5’ ribozyme comprises a Type P1 Twister, a Type P3 Twister, or a Type P5 Twister.
  • the 5’ribozyme comprises a Type P1 Twister.
  • the 5’ ribozyme comprises Type P1 Twister from rice (Oryza sativa, Osa).
  • the 5’ ribozyme comprises an overhang of one or more nucleotides.
  • the overhang comprises a nucleotide sequence that hybridizes to a sequence downstream of said 5’ ribozyme within the second RNA molecule. In some embodiments, the overhang improves efficiency of spontaneous cis-cleavage.
  • the HDV ribozyme of the composition comprises one or more selected from the group consisting of: HDV, HDV68, HDV67, HDV56, genHDV, and antiHDV, or a variant or fragment thereof.
  • HDV68 comprises the nucleic acid sequence of SEQ ID NO: 9.
  • HDV67 comprises the nucleic acid sequence of SEQ ID NO: 10.
  • HDV56 comprises the nucleic acid sequence of SEQ ID NO: 11.
  • genHDV comprises the nucleic acid sequence of SEQ ID NO: 12.
  • antiHDV comprises the nucleic acid sequence of SEQ ID NO: 13.
  • the HH ribozyme comprises one or more nucleotides in a stem 1 overhang that hybridize with nucleotides of the sequence upstream or downstream of said HH ribozyme.
  • 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 nucleotide, 12 or more nucleotides, 14 or more nucleotides, 16 or more nucleotides, 18 or more nucleotides, or 20 or more nucleotides.
  • the HH ribozyme comprising one or more nucleotide stem 1 overhang 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 nucleotides designated as N correspond to nucleotides that hybridize with nucleotides of the sequence downstream of said HH ribozyme.
  • the HH ribozyme has one or more nucleotide in a stem 3 overhang.
  • the HH ribozyme has a 5 nucleotide stem 3 overhang.
  • the HH ribozyme comprises the nucleic acid sequence of SEQ ID NO: 105, wherein nucleotides designated as N correspond to nucleotides that hybridize with nucleotides of the sequence upstream of said HH ribozyme.
  • the HH ribozyme is modified in the stem 2 loop.
  • the HH ribozyme with 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 nucleotides designated as N correspond to nucleotides that hybridize with nucleotides of the sequence downstream of said HH ribozyme.
  • the HH ribozyme is modified in stem 1 to include a tertiary stabilizing motif (TSM).
  • TSM tertiary stabilizing motif
  • the HH ribozyme is modified in the stem 2 loop and is modified in stem 1 to include a tertiary stabilizing motif (TSM).
  • TSM tertiary stabilizing motif
  • the modified HH ribozyme cis-cleaves more efficiently than HH ribozyme.
  • the modified HH ribozyme is RzB.
  • RzB comprises the nucleic acid sequence of SEQ ID NO: 125, wherein nucleotides designated as N correspond to nucleotides that hybridize with nucleotides of the sequence downstream of said HH ribozyme.
  • the Twister ribozyme comprises the nucleic acid sequence of SEQ ID NO: 32.
  • the Twister ribozyme comprises one or more nucleotide in a P1 stem overhang.
  • 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.
  • the Twister ribozyme comprising one or more nucleotide P1 stem overhang 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 nucleotides designated as N correspond to nucleotides that hybridize with nucleotides of the sequence downstream of said Twister ribozyme.
  • said one or more ribozyme of the composition is composed of first part and a second part. In some embodiments, the first part is incorporated into said one or more RNA molecule.
  • the first part is a ribozyme recognition sequence.
  • said second part is introduced separately.
  • cis-cleavage of the first part from said one or more RNA molecule only occurs if the first part and the second part are brought into contact with one another.
  • said one or more ribozyme is VS ribozyme.
  • the VS ribozyme comprises the nucleic acid sequence of SEQ ID NO: 14.
  • the first part is VS ribozyme stem loop (VS-S).
  • VS-S comprises the nucleic acid sequence of SEQ ID NO: 15.
  • the second part is the remaining portion of VS without the stem loop (VS-Rz).
  • VS-Rz comprises the nucleic acid sequence of SEQ ID NO: 16.
  • Ribozymes are autocatalytic RNAs which cleave in cis, to produce unique RNA 3’ and 5’ termini, as described herein.
  • cis-cleaving ribozymes can be engineered to cleave in trans, such that target RNAs can be cleaved in a nucleotide specific manner, resulting in similar RNA termini.
  • the present invention comprises a composition comprising a single nucleic acid molecule encoding a single RNA molecule comprising a trans- cleaving engineered ribozyme.
  • the trans-cleaving engineered ribozyme is capable of trans-cleaving a separate RNA molecule. In one embodiment, the trans-cleaving engineered ribozyme recognizes a specific nucleic acid sequence in the separate RNA molecule. In some embodiments, the trans-cleaving engineered ribozyme targets a disease causing mutation for deletion. In some embodiment, the disease causing mutation is in an exon. In some embodiment, the disease causing mutation is in an intron. In some embodiments, the composition comprises two trans-cleaving engineered ribozymes, targeted upstream and downstream of the disease causing mutation. In some embodiments, trans-cleavage upstream and downstream of the disease causing mutation results in removal of the disease causing mutation.
  • the remaining portions of the gene are trans-spliced together after trans-cleavage of the disease causing mutation.
  • the trans-spliced gene is expressed as a functional protein.
  • the 3’P or 2’3’ cP end and the 5’OH end of RNA molecules that have undergone ribozyme-mediated cleavage can be ligated together.
  • separated RNA sequences encoding separate portions of a larger full-length protein can be trans-spliced together in a scar-less manner to enable expression of the full-length protein.
  • the present invention relates to a composition comprising one or more nucleic acid molecule encoding two or more portions of a protein of interest and encoding one or more ribozyme. In one embodiment, the present invention relates to a composition comprising one or more RNA molecule encoding two or more portions protein of interest and comprising one or more ribozyme.
  • the one or more nucleic acid molecules encoding two or more portions of a protein of interest comprise a first nucleic acid molecule encoding a first portion of a protein of interest and a second nucleic acid molecule encoding a second portion of a protein of interest.
  • the first nucleic acid comprises a first RNA molecule.
  • the second nucleic acid comprises a second RNA molecule.
  • the first RNA molecule is linked at the 3’ end to a 3’ ribozyme.
  • the second RNA molecule is linked at the 5’ end to a 5’ ribozyme.
  • the 3’P or 2’3’ cP end of first RNA molecule is ligated to the 5’OH end of the second RNA molecule, thereby generating a single RNA molecule encoding a full-length protein of interest.
  • the full-length protein of interest functions identically to an endogenously expressed full-length protein of the same sequence.
  • the full-length protein of interest comprises a therapeutic protein.
  • 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, CDH23, STRC, OTOG, TECTA, PCDH15, TRIOBP, MYO3A, COL11A2, LOXHD1, PTPRQ, OTOGL, MYH14, MYH9, TNC, CACNA1A, CACNA1C, CACNA1F, CACNA1H, CACNA1G, CACNA1D, CACNA1B, CA
  • the full-length protein of interest is a recombinase.
  • the recombinase is one or more selected from the group consisting of, but not limited to: CRE recombinase, FLP recombinase.
  • the full-length protein of interest is a eukaryotic/prokaryotic antibiotic resistance gene product.
  • 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.
  • the full-length protein of interest is an antibody.
  • the antibody is capable of binding to a target protein of interest.
  • the antibody is an antibody fragment, synthetic antibody, nanobody, or a fragment or variant thereof that maintains the ability to bind to the target protein.
  • the full-length protein of interest comprises a synthetic repeat protein, including, but not limited to, those composing hydrogels, synthetic spider silks, and collagens.
  • the synthetic repeat protein comprises one or more selected from the group consisting of, but not limited to: Spidroin, Silk, Keratin, Collagen, Elastin, Resilin, and Squid Ring Teeth, beta- solenoid proteins, Zinc Finger Nucleases (ZFNs, and Tal effector nucleases (TALENs).
  • the full-length protein of interest comprises a toxic protein or an antiviral protein, which may inhibit generation of lentiviral particles in mammalian packing cells.
  • the toxic protein is a cell suicide gene.
  • 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, Conopeptides, Abrin, Verotoxin, Tetanospasmin, Botulinum toxin, pseudomonas exotoxin A, anthrax, saporin, and pokeweed antiviral protein (PAP).
  • DTA diphtheria toxin A
  • HSV-tk HSV-tk
  • Ricin Cholera toxin
  • Major Prion Protein Pertussis toxin
  • Ectatomin Ectatomin
  • Conopeptides Abrin
  • 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.
  • the full-length protein of interest comprises a prime editor.
  • the full-length protein of interest comprises a prime editor comprising a fusion protein comprising a nickase and a reverse transcriptase (RT).
  • RT reverse transcriptase
  • An exemplary nickase includes, but is not limited to nCas9(H840A).
  • An exemplary RT includes, but is not limited to MMLV.
  • the full-length protein comprises a prime editor comprising a fusion protein comprising nCas9(H840A) and MMLV.
  • the prime editor comprises the PE1, PE2, PE3, PE4, PE5, PE6, or PEmax editor.
  • the fusion protein further comprises one or more NLS sequences, such as a TY1 NLS.
  • the fusion protein comprises a degradation or destabilization domain, such as E coli dihydrofolate reductase (ecDHFR).
  • the composition of the present invention comprises a first nucleic acid molecule comprising or encoding an RNA encoding a first portion of the prime editor fusion protein and a 3’ ribozyme (such as any of the ribozymes described herein), and a second nucleic acid molecule comprising or encoding an RNA encoding a 5’ ribozyme (such as any of the ribozymes described herein) and a second portion of the prime editor fusion protein.
  • the prime editor comprising a fusion protein comprising a nickase and a reverse transcriptase (RT).
  • An exemplary nickase includes, but is not limited to nCas9(H840A).
  • An exemplary RT includes, but is not limited to MMLV.
  • the prime editor comprising a fusion protein comprising nCas9(H840A) and MMLV.
  • the prime editor comprises the PE1, PE2, PE3, PE4, PE5, PE6, or PEmax editor.
  • the fusion protein further comprises one or more NLS sequences, such as a TY1 NLS.
  • the fusion protein comprises a degradation or destabilization domain, such as E coli dihydrofolate reductase (ecDHFR).
  • the first nucleic acid molecule and/or the second nucleic acid molecule further comprise one or more sequences comprising or encoding a prime editing guide RNA (pegRNA) and/or nickase sgRNA.
  • pegRNA prime editing guide RNA
  • the first nucleic acid molecule comprises or encodes the first portion of the prime editor; and the second nucleic acid molecule comprises or encodes the second portion of the prime editor and further comprise or encodes one more pegRNA and/or nickase sgRNA.
  • the first nucleic acid molecule comprises or encodes the first portion of the prime editor, wherein the first portion of the prime editor further comprises a degradation or destabilization domain (e.g., ecDHFR).
  • ecDHFR degradation or destabilization domain
  • the first nucleic acid molecule comprises or encodes the first portion of the prime editor, wherein the first portion of the prime editor further comprises a degradation or destabilization domain (e.g., ecDHFR) at the N-terminus of the prime editor.
  • a degradation or destabilization domain e.g., ecDHFR
  • N-terminal or C-terminal RNA molecules encoding a portion of a protein of interest could be subject to translation prior to ribozyme-mediated cleavage, or when expressed separately, potentially resulting in unwanted or truncated protein expression.
  • translational control of protein degradation sequences can be utilized to limit this unwanted expression.
  • the one or more RNA molecule of the composition comprises a nucleic acid sequence encoding a translational control of protein degradation sequence.
  • the first RNA molecule comprises a nucleic acid sequence encoding a translational control of protein degradation sequence.
  • the second RNA molecule comprises a nucleic acid sequence encoding a translational control of protein degradation sequence.
  • said translational control of protein degradation sequences prevent partial expression of protein prior to cleavage of ribozyme sequences and splicing.
  • the translational control of protein degradation sequences comprise one or more selected from the group consisting of: a hCL1-PEST sequence, an E1A-PEST sequence, removal of the nucleic acid’s poly(A) sequence, simulated translation through a poly A tail to generate a poly K tail, deletion of the ATG stop codon, silent mutations within N-terminal NTG codons, a 5’ UTR of yeast GCN4 sequence encoding four small upstream ORFs that function as translation inhibitors, a small internal fragment of a 5’ UTR of yeast GCN4 sequence.
  • the translational control of protein degradation sequences comprise one or more nucleic acid sequence 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.
  • the translational control of protein degradation sequences comprise one or more amino acid sequence 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.
  • RNA nuclear localization signals may be useful to prevent cytosolic export and translation of un- spliced RNA molecules.
  • the one or more RNA molecule of the composition comprises a nucleic acid sequence encoding an RNA nuclear localization sequence.
  • the first RNA molecule comprises a nucleic acid sequence encoding an RNA nuclear localization sequence.
  • the second RNA molecule comprises a nucleic acid sequence encoding an RNA nuclear localization sequence.
  • the RNA nuclear localization sequences prevent cytosolic RNA export and translation of partial protein prior to cleavage of ribozyme sequences and splicing.
  • the RNA nuclear localization sequences comprise one or more nucleic acid sequence selected from the group consisting of: SEQ ID NO: 50, and SEQ ID NO: 51.
  • the composition further comprises one or more additional RNA molecule, each additional RNA molecule comprising a coding region encoding a domain of the protein of interest; a 5’ ribozyme; and a 3’ ribozyme.
  • the system further comprises one or more additional nucleic acid molecule encoding one or more additional RNA molecule, each additional RNA molecule comprising a coding region encoding a domain of the protein of interest; a 5’ ribozyme; and a 3’ ribozyme.
  • the composition further comprises one or more additional RNA molecule, each additional RNA molecule comprising a coding region encoding a domain of the protein of interest; a 5’ ribozyme; and a 3’ ribozyme recognition sequence.
  • the system further comprises one or more additional nucleic acid molecule encoding one or more additional RNA molecule, each additional RNA molecule comprising a coding region encoding a domain of the protein of interest; a 5’ ribozyme; and a 3’ ribozyme recognition sequence.
  • Pre-mRNA splicing by the spliceosome has been shown to enhance mRNA translation, either through deposition of factors which promote a pioneer round of translation or through promoting RNA processing and export to the cytoplasm.
  • the addition of a chimeric cis- splicing intron within a transgene has also been shown to promote transgene protein expression.
  • the addition of splice donor and splice acceptor sites recognized and cis-spliced by the spliceosome may enhance protein expression from split precursor RNA molecules.
  • the composition comprises one or more RNA molecule comprising a splice donor or a splice acceptor sequence.
  • the first RNA molecule of the composition comprises splice donor sequence. In one embodiment, the splice donor sequence is linked to the 3’ end of the first RNA molecule following 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, inclusion of the splice donor and splice acceptor sequences enhances protein expression following ribozyme- mediated trans-splicing.
  • the composition of the present invention comprises at least four nucleic acid molecules comprising at least two pairs of nucleic acid molecules.
  • each pair of nucleic acid molecules encodes at least two portions of a protein of interest and encodes at least two ribozymes.
  • the composition comprises at least four RNA molecules comprising at least two pairs of RNA molecules.
  • each pair of RNA molecules encodes at least two portions of a protein of interest and comprises at least two ribozymes [0161]
  • the at least two pairs of RNA molecules comprises a first pair of RNA molecules and second pair of RNA molecules.
  • the first pair of RNA molecules comprises a first RNA molecule and a second RNA molecule.
  • the second pair of RNA molecules comprises a third RNA molecule and fourth RNA molecule.
  • said third RNA molecule and said fourth RNA molecule have different open reading frame the first RNA molecule and the second RNA molecule, such that, upon spontaneous cis-cleavage, ligation of either the first RNA molecule or the second RNA molecule with either the third RNA molecule or fourth RNA molecule cannot translate a full-length functional protein product.
  • the at least two pairs of RNA molecules further comprises a third pair of RNA molecules.
  • the third pair of RNA molecules comprises a fifth RNA molecule and a sixth RNA molecule.
  • said fifth RNA molecule and said sixth RNA molecule have different open reading frame the first pair of RNA molecules and the second pair of RNA molecules, such that, upon spontaneous cis-cleavage, only ligation of the first pair, second pair or third pair of RNA molecules can translate a full-length functional protein product.
  • Ribozyme-mediated trans-splicing between two independent RNAs can occur when one RNA contains a 3’ ribozyme and another contains 5’ ribozyme, as described herein. However, when transcribed in cis within the same RNA molecule, two ribozymes can mediate their own scar-less removal.
  • 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 ribozyme. In one embodiment, the present invention relates to a composition comprising a single RNA molecule encoding two or more portions protein of interest and comprising one or more ribozyme.
  • the single nucleic acid molecule encodes a first portion of RNA, a synthetic intron, and a second portion of RNA.
  • the synthetic intron comprises a 5’ ribozyme and a 3’ ribozyme.
  • the first portion of RNA encodes a first portion of a protein of interest.
  • the second portion of RNA encodes a second portion of a protein of interest.
  • the single nucleic acid comprises a sequence linked in the order: (first portion of RNA encoding first portion of protein of interest)-(5’ ribozyme of synthetic intron)-(3’ ribozyme of synthetic intron)-(second portion of RNA encoding second portion of protein of interest).
  • the first portion of the protein of interest is the N-terminal portion of GFP.
  • the 5’ ribozyme of the synthetic intron comprises HDV.
  • the first portion of RNA and the 5’ ribozyme of the synthetic intron comprise the nucleic acid sequence of SEQ ID NO: 127, wherein lowercase letters designate the 5’ ribozyme sequence and uppercase letters designate the sequence encoding the N-terminal portion of GFP (See Example 4, “GFP with internal synthetic ribozyme intron with and without cargo”).
  • the second portion of the protein of interest is the C-terminal portion of GFP.
  • the 3’ ribozyme of the synthetic intron comprises HH.
  • the second portion of RNA and the 3’ ribozyme of the synthetic intron comprise the nucleic acid sequence of SEQ ID NO: 128, wherein lowercase letters designate the 3’ ribozyme sequence and uppercase letters designate the sequence encoding the C-terminal portion of GFP. (See Example 4, “GFP with internal synthetic ribozyme intron with and without cargo”).
  • the synthetic intron comprises a cargo sequence placed between said 5’ ribozyme and said 3’ ribozyme.
  • the single nucleic acid comprises a sequence linked in the order: (first portion of RNA encoding first portion of protein of interest)-(5’ ribozyme of synthetic intron)-(cargo sequence)-(3’ ribozyme of synthetic intron)- (second portion of RNA encoding second portion of protein of interest).
  • the 5’ ribozyme sequence of the synthetic intron does not require bilateral flanking sequences for activity.
  • circular RNA generated from the ligation of the ends of the synthetic intron comprising a 5’ ribozyme sequence that does not require bilateral flanking sequences for activity can exist in both circular and re-cleaved linear forms.
  • the ribozyme sequence is a HDV ribozyme.
  • the 5’ ribozyme sequence of the synthetic intron does require bilateral flanking sequences for activity.
  • circular RNA generated from ligation of the ends of the synthetic intron comprising a 5’ ribozyme sequence that does require bilateral flanking sequences for activity can exist only in circular form.
  • the ribozyme sequence is a HH ribozyme.
  • the 5’ ribozyme sequence of the synthetic intron is a ribozyme recognition sequence.
  • the ribozyme recognition sequence requires the addition of a trans-cleaving ribozyme for inducible cleavage.
  • the ribozyme recognition sequence comprises VS-S.
  • VS-S is encoded by a nucleic acid sequence comprising SEQ ID NO: 15.
  • the trans-cleaving ribozyme comprises VS-Rz.
  • VS-Rz is encoded by a nucleic acid sequence comprising SEQ ID NO: 16.
  • self-cleavage of the 5’ ribozyme sequence and the 3’ ribozyme sequence generates three separate RNA molecules: 1) a first fragment comprising the first portion of RNA encoding a first portion of a protein of interest, 2) a second fragment comprising the synthetic intron, 3) a third fragment comprising the second portion of RNA encoding a second portion of a protein of interest.
  • the compatible ends of the second fragment are ligated to generate a circular RNA molecule comprising the synthetic intron comprising the cargo sequence.
  • the first fragment and third fragment are ligated together to generate a single full-length linear RNA molecule.
  • 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.
  • 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 (snoRNAs), small tRNA-derived RNA (tsRNA), small rDNA- derived RNA (srRNA) and small nuclear RNA (snRNA).
  • the single full-length linear RNA molecule encodes a full- length protein of interest.
  • the full-length protein of interest is a therapeutic protein.
  • 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, Retinal-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, CACNA
  • the full-length protein of interest is a recombinase.
  • the recombinase is one or more selected from the group consisting of, but not limited to: CRE recombinase, FLP recombinase.
  • the full-length protein of interest is a eukaryotic/prokaryotic antibiotic resistance gene product.
  • 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.
  • the full-length protein of interest is a reporter protein.
  • the reporter protein is one or more selected from the group consisting of: green fluorescent protein (GFP), red fluorescent protein (RFP), and luciferase (Luc).
  • the reporter protein is used as a proxy indicator to assess delivery and expression of the cargo sequence.
  • the full-length protein of interest is an antibody.
  • the antibody is capable of binding to a target protein of interest.
  • the antibody is an antibody fragment, synthetic antibody, nanobody, or a fragment or variant thereof that maintains the ability to bind to the target protein.
  • the full-length protein of interest comprises a toxic protein or an antiviral protein, which may inhibit generation of lentiviral particles in mammalian packing cells.
  • the toxic protein is a cell suicide gene.
  • 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, Conopeptides, Abrin, Verotoxin, Tetanospasmin, Botulinum toxin, pseudomonas exotoxin A, anthrax, saporin, and pokeweed antiviral protein (PAP).
  • DTA diphtheria toxin A
  • HSV-tk Ricin
  • Cholera toxin Major Prion Protein
  • Pertussis toxin Ectatomin
  • Conopeptides Abrin
  • Verotoxin Verotoxin
  • Tetanospasmin Botulinum toxin
  • pseudomonas exotoxin A anthrax
  • saporin saporin
  • PAP pokeweed
  • 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.
  • MxA Interferon-induced GTP-binding protein
  • MPO Myeloperoxidase
  • the present invention provides compositions and methods for generating a circular RNA molecule encoding a protein of interest comprising a nucleic acid encoding: a 5’ ribozyme, a first portion of a protein of interest, an IRES sequence, a second portion of a protein of interest, and a 3’ribozyme.
  • the present invention provides compositions and methods for generating a circular RNA molecule encoding a prime editor comprising a nucleic acid encoding: a 5’ ribozyme, a first portion of a prime editor, an IRES sequence, a second portion of a prime editor, and a 3’ribozyme.
  • the prime editor comprises a fusion protein comprising a nickase and a reverse transcriptase (RT).
  • RT reverse transcriptase
  • An exemplary nickase includes, but is not limited to nCas9(H840A).
  • An exemplary RT includes, but is not limited to MMLV.
  • the prime editor comprises a fusion protein comprising nCas9(H840A) and MMLV.
  • the prime editor comprises the PE1, PE2, PE3, PE4, PE5, PE6, or PEmax editor.
  • the fusion protein further comprises one or more NLS sequences, such as a TY1 NLS.
  • the fusion protein comprises a degradation or destabilization domain, such as E coli dihydrofolate reductase (ecDHFR).
  • ecDHFR E coli dihydrofolate reductase
  • the technology of the present invention can be used to assemble a full-length RNA virus genome.
  • the one or more nucleic acid molecule encoding one or more ribozyme of the present invention encodes one or more portion of an RNA virus genome. In one embodiment, the one or more RNA molecule comprising one or more ribozyme of the present invention comprises one or more portion of an RNA virus genome. [0176] In one embodiment, the one or more nucleic acid molecule comprises a first nucleic acid molecule encoding a first portion of the RNA virus genome and encoding a 3’ ribozyme. In one embodiment, the one or more nucleic acid molecule comprises a second nucleic acid encoding a second portion of the RNA virus genome and encoding a 5’ ribozyme.
  • the one or more RNA molecule comprises a first RNA molecule comprising a first portion of the RNA virus genome and a 3’ ribozyme. In one embodiment, the said one or more RNA molecule comprises a second RNA molecule comprising a second portion of the RNA virus genome and a 5’ ribozyme. In one embodiment, the composition comprises a nucleic acid encoding a ligase or a ligase. In one embodiment, upon cis-cleavage of the 3’ and 5’ ribozymes, the first portion of the RNA virus genome and the second portion of the RNA virus genome are ligated together, thereby generating a full-length RNA virus genome.
  • RNA viruses include, but are not limited to: coronaviruses, paramyxoviruses, orthomyxoviruses, retroviruses, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles viruses, Newcastle disease viruses, and picornaviruses.
  • the present invention comprises a composition comprising a nucleic acid encoding a ligase.
  • the ligase mediates ligation of the 3’P or 2’3’ cP end and the 5’OH end.
  • the ligase is RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase.
  • the RtcB ligase is from one or more domain of organism selected from the group consisting of: Eukarya, Bacteria, and Archaea.
  • the organism is selected from the group consisting of: human, E. coli, Deinococcus radiodurans, Pyrococcus horikoshii, Pyrococcus sp. ST04, and Thermococcus sp. EP.
  • the nucleic acid sequence encoding a 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.
  • the nucleic acid sequence encoding a ligase encodes one or more amino acid sequence 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.
  • the composition comprises at least one linear or circular DNA molecule or RNA molecule comprising a coding region encoding a first portion of the protein of interest (N-terminal coding sequence), 5’ ribozyme, an intervening sequence to be removed, and a 3’ribozyme directly linked to a coding region encoding a second portion of the protein of interest (C-terminal coding sequence).
  • N-terminal coding sequence is directly linked to the 5’ ribozyme sequence and the 3’ribozyme is directly linked to the C-terminal coding sequence.
  • each of the 3’ ribozyme and the 5’ ribozyme comprises a sequence of SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:
  • the nucleic acid molecule comprises a splice donor or splice acceptor sequence.
  • the splice donor comprises SEQ ID NO:245 or SEQ ID NO:246.
  • the splice acceptor comprises SEQ ID NO:247.
  • the nucleic acid molecule comprising the N-terminal coding sequence comprises a splice donor (SD) sequence
  • the nucleic acid molecule comprising the C-terminal coding sequence comprises the splice acceptor (SA) sequence.
  • Ribozymes can either follow (placed 3’) to a SD sequence for an N-terminal mRNA half or precede (placed 5’) to a SA for a C-terminal mRNA half. Ribozyme does not need to be scarless when reconstituting an Intron sequence (SD-SA pair), since the SD-SA pair will recreate the mRNA reading frame.
  • the provided is a combination of nucleic acid molecules encoding a prime editor.
  • the dual StitchR-enabled Prime editor consists of an N-terminal and C-terminal vector which splits the PEmax editor within the C-terminus of the nickase SpyCas9-MMLV fusion protein.
  • the dual StitchR-enabled Prime editor comprises a first nucleic acid molecule comprising SEQ ID NO:269, and a second nucleic acid molecule comprising SEQ ID NO:270.
  • the nucleic acid molecule comprising the prime editing N-terminal vector comprises a 5’ ribozyme sequence, a splice donor sequence, or a combination thereof and the C-terminal vector comprises a 3’ ribozyme sequence, a splice acceptor sequence, or a combination thereof.
  • nucleic acid molecules which function as dual StitchR-enabled Prime editors include, but are not limited to, SEQ ID NO:300, and SEQ ID NO:301, SEQ ID NO:304 and SEQ ID NO:305, and SEQ ID NO:304 and SEQ ID NO:306.
  • the provided is a vector encoding a prime editor and comprising a combination of a 5’ ribozyme and 3’ ribozyme, which is sufficient for circularization in cells (or in vitro when incubated with RTCB ligase) and translation.
  • the RNA molecule is encoded downstream of a promoter, which will produce circular RNA for expression of PEmax.
  • one or more nucleic acid of the present invention comprises a nucleic acid sequence that is substantially homologous to a nucleic acid sequence described herein.
  • the nucleic acid has a degree of identity with respect to the original nucleic acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.
  • one or more nucleic acid of the present invention comprises a nucleic acid sequence that is a portion of a nucleic acid sequence described herein.
  • the nucleic acid has a length with respect to the original nucleic acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.
  • one or more nucleic acid of the present invention comprises 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.
  • the nucleic acid has a degree of identity with respect to the original nucleic acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.
  • nucleic acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.
  • the nucleic acid of the present invention may comprise any type of nucleic acid, including, but not limited to DNA and RNA.
  • the composition comprises an isolated DNA molecule, including for example, an isolated cDNA molecule, encoding a fusion protein of the invention.
  • the composition comprises an isolated RNA molecule encoding a fusion protein of the invention, or a functional fragment thereof.
  • the nucleic acid molecules of the present invention can be modified to improve stability in serum or in growth medium for cell cultures. Modifications can be added to enhance stability, functionality, and/or specificity and to minimize immunostimulatory properties of the nucleic acid molecule of the invention.
  • the 3’- residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine by 2’- deoxythymidine is tolerated and does not affect function of the molecule.
  • the nucleic acid molecule may contain at least one modified nucleotide analogue.
  • the ends may be stabilized by incorporating modified nucleotide analogues.
  • Non-limiting examples of nucleotide analogues include sugar- and/or backbone- modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone).
  • the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom.
  • the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group.
  • 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 C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.
  • R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.
  • Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase.
  • modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined.
  • the nucleic acid molecule comprises at least one of the following chemical modifications: 2’-H, 2’-O-methyl, or 2’-OH modification of one or more nucleotides.
  • a nucleic acid molecule of the invention can have enhanced resistance to nucleases.
  • a nucleic acid molecule can include, for example, 2’-modified ribose units and/or phosphorothioate linkages.
  • the 2’ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.
  • the nucleic acid molecules of the invention can include 2’-O-methyl, 2’-fluorine, 2’-O-methoxyethyl, 2’-O-aminopropyl, 2’-amino, and/or phosphorothioate linkages.
  • LNA locked nucleic acids
  • ENA ethylene nucleic acids
  • 2’-4’-ethylene-bridged nucleic acids e.g., 2’-4’-ethylene-bridged nucleic acids
  • certain nucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, can also increase binding affinity to a target.
  • the nucleic acid molecule includes a 2’-modified nucleotide, e.g., a 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-dimethylaminoethyloxyethyl (2’-O-DMAEOE), or 2’-O-N- methylacetamido (2’-O-NMA).
  • a 2’-modified nucleotide e.g., a 2’-deoxy, 2’-deoxy-2’-fluoro, 2’-O-methyl, 2’-O-methoxyethyl (2’-O-MOE
  • the nucleic acid molecule includes at least one 2’-O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule include a 2’-O-methyl modification.
  • the nucleic acid molecule of the invention has one or more of the following properties: [0194] Nucleic acid agents discussed herein include otherwise unmodified RNA and DNA as well as RNA and DNA that have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates.
  • Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, or as occur naturally in the human body.
  • the art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196).
  • Such rare or unusual RNAs, often termed modified RNAs are typically the result of a post-transcriptional modification and are within the term unmodified RNA as used herein.
  • Modified RNA refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, or different from that which occurs in the human body. While they are referred to as “modified RNAs” they will of course, because of the modification, include molecules that are not, strictly speaking, RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone.
  • the present invention also includes a composition comprising one or more vector in which one or more nucleic acid molecule of the present invention is inserted.
  • the vector encodes at least two RNA molecules.
  • the vector comprises at least two RNA molecules.
  • the at least two RNA molecules are encoded by the same vector.
  • the at least two RNA molecules are contained within the same vector.
  • the at least two RNA molecules comprise a first RNA molecule and a second RNA molecule.
  • the present invention comprises at least two vectors encoding at least two RNA molecules.
  • the at least two vectors comprise at least two RNA molecules.
  • the at least two vectors encode separate RNA molecules.
  • the at least two vectors comprise separate RNA molecules.
  • the at least two separate RNA molecules comprise a first RNA molecule and a second RNA molecule.
  • the first RNA molecule is encoded by a first vector and the second RNA molecule is encoded by a second vector.
  • the first RNA molecule comprises a first vector and the second RNA molecule comprises a second vector.
  • the present invention further comprises a vector encoding one or more additional RNA molecule.
  • the present invention further comprises one or more vector comprising one or more additional RNA molecule.
  • each additional RNA molecule comprises a coding region encoding a domain of the protein of interest; a 5’ ribozyme; and a 3’ ribozyme.
  • each additional RNA molecule comprises a coding region encoding a domain of the protein of interest; a 5’ ribozyme; and a 3’ ribozyme recognition sequence.
  • the expression of natural or synthetic nucleic acids encoding a fusion protein of the invention is typically achieved by operably linking a nucleic acid encoding the fusion protein of the invention or portions thereof to a promoter, and incorporating the construct into an expression vector.
  • the vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the vectors of the present invention may 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, e.g., U.S. Pat.
  • the invention provides a gene therapy vector.
  • the isolated nucleic acid of the invention can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the vector may be provided to a cell in the form of a viral vector.
  • Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • the composition includes a vector derived from an adeno- associated virus (AAV).
  • AAV adeno- associated virus
  • AAV vector means a vector derived from an adeno- associated virus serotype, including without limitation, 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 the treatment of various disorders.
  • AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.
  • AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Despite the high degree of homology, the different serotypes have tropisms for different tissues. The receptor for AAV1 is unknown; however, AAV1 is known to transduce skeletal and cardiac muscle more efficiently than AAV2. Since most of the studies have been done with pseudotyped vectors in which the vector DNA flanked with AAV2 ITR is packaged into capsids of alternate serotypes, it is clear that the biological differences are related to the capsid rather than to the genomes.
  • the viral delivery system is an adeno-associated viral delivery system.
  • the adeno-associated virus can be of 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).
  • Desirable AAV fragments for assembly into vectors include the cap proteins, including the vp1, vp2, vp3 and hypervariable regions, the rep proteins, including rep 78, rep 68, rep 52, and rep 40, and the sequences encoding these proteins. These fragments may be readily utilized in a variety of vector systems and host cells. Such fragments may 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.
  • artificial AAV serotypes include, without limitation, AAV with a non-naturally occurring capsid protein.
  • Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vp1 capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV serotype, non-contiguous portions of the same AAV serotype, from a non-AAV viral source, or from a non-viral source.
  • An artificial AAV serotype may be, without limitation, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid.
  • exemplary AAVs, or artificial AAVs, suitable for expression of one or more proteins include AAV2/8 (see U.S. Pat.
  • the composition comprises a lentiviral vector to deliver one or more nucleic acid of the present invention.
  • the present invention comprises a lentiviral vector comprising one or more RNA molecule encoding one or more protein of interest.
  • vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • the vector also includes conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention.
  • operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance 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 enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • polyA polyadenylation
  • a great number of expression control sequences including promoters which are native, constitutive, inducible and/or tissue- specific, are known in the art and may be utilized.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor -1 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters.
  • Inducible promoters are also contemplated as part of the invention.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • Enhancer sequences found on a vector also regulates expression of the gene contained therein. Typically, enhancers are bound with protein factors to enhance the transcription of a gene. Enhancers may be located upstream or downstream of the gene it regulates.
  • Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type.
  • the vector of the present invention comprises one or more enhancers to boost transcription of the gene present within the vector.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.
  • the present invention comprises a composition comprising a ligase.
  • the ligase mediates ligation of the 3’P or 2’3’ cP end of an RNA molecule and the 5’OH end of an RNA molecule.
  • the ligase is RNA 2',3'- Cyclic Phosphate and 5'-OH (RtcB) ligase.
  • the RtcB ligase is from one or more domain of organism selected from the group consisting of: Eukarya, Bacteria, and Archaea. In some embodiments, the organism is selected from the group consisting of: human, E.
  • the ligase comprises one or more amino acid sequence 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.
  • one or more protein of the present invention comprises an amino acid sequence that is substantially homologous to an amino acid sequence described herein.
  • the protein has a degree of identity with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.
  • one or more protein of the present invention comprises an amino acid sequence that is a portion of an amino acid sequence described herein.
  • the protein has a length with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.
  • one or more protein of the present invention comprises 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.
  • the protein has a degree of identity with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 81%, of at least 82%, of at least 83%, of at least 84%, of at least 85%, of at least 86%, of at least 87%, of at least 88%, of at least 89%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5% and/or has a length with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of
  • compositions of the invention also encompasses the use of pharmaceutical compositions of the invention or salts thereof to practice the methods of the invention.
  • a pharmaceutical composition may consist of at least one nucleic acid of the 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 invention or a salt thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these.
  • the nucleic acid of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
  • the pharmaceutical compositions useful for practicing the methods of the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In another embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.
  • the relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • compositions that are useful in the methods of the invention may be suitably developed for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration.
  • a composition useful within the methods of the invention may be directly administered to the skin, or any other tissue of a mammal.
  • Other contemplated formulations include liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
  • the route(s) of administration will be readily apparent to the skilled artisan and will depend upon any 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.
  • compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a nucleic acid of the invention and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers that are useful include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol are included in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
  • the pharmaceutically acceptable carrier is not DMSO alone.
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, vaginal, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • compositions of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition.
  • the preservative is used to prevent spoilage in the case of exposure to contaminants in the environment.
  • preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof.
  • an exemplary preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.
  • the composition includes an anti-oxidant and a chelating agent that inhibits the degradation of the nucleic acid.
  • exemplary antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the range of about 0.01% to 0.3% and BHT in the range of 0.03% to 0.1% by weight by total weight of the composition.
  • the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition.
  • Exemplary chelating agents include edetate salts (e.g.
  • disodium edetate and citric acid in the weight range of about 0.01% to 0.20%.
  • the chelating agent is in the range of 0.02% to 0.10% by weight by total weight of the composition.
  • the chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidants and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.
  • Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle.
  • Aqueous vehicles include, for example, water, and isotonic saline.
  • Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents.
  • Oily suspensions may further comprise a thickening agent.
  • suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose.
  • Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively).
  • Known emulsifying agents include, but are not limited to, lecithin, and acacia.
  • Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid.
  • Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.
  • Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.
  • an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.
  • Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent.
  • Aqueous solvents include, for example, water, and isotonic saline.
  • Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto.
  • Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
  • a pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion.
  • the oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these.
  • Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.
  • These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
  • Methods for impregnating or coating a material with a chemical composition include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the subject either prior to or after a diagnosis of disease.
  • compositions of the present invention may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • Administration of the compositions of the present invention to a subject include a mammal, for example a human, may be carried out using known procedures, at dosages and for periods of time effective to prevent or treat disease.
  • An effective amount of the nucleic acid necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular nucleic acid employed; the time of administration; the rate of excretion of the nucleic acid; the duration of the treatment; other drugs, compounds or materials used in combination with the nucleic acid; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a non-limiting example of an effective dose range for a nucleic acid of the invention is from about 1 and 5,000 mg/kg of body weight/per day.
  • the nucleic acid may be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less.
  • the amount of nucleic acid dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
  • a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.
  • compositions of this 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.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the nucleic acid of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic nucleic acid calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the nucleic acid and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a nucleic acid for the treatment of a disease in a subject.
  • the compositions of the invention are administered to the subject in dosages that range from one to five times per day or more.
  • compositions of the invention are administered to the subject in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any subject will be determined by the attending physical taking all other factors about the subject into account.
  • compositions of the invention for administration may be in the range of 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 there between.
  • the dose of a composition of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a composition of the invention used in 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.
  • a dose of a second composition 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.
  • the present invention is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a nucleic acid of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the nucleic acid to treat, prevent, or reduce one or more symptoms of a disease in a subject.
  • the term “container” includes any receptacle for holding the pharmaceutical composition.
  • the container is the packaging that contains the pharmaceutical composition.
  • the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition.
  • packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the nucleic acid’s ability to perform its intended function, e.g., treating or preventing a disease in a subject, or delivering an imaging or diagnostic agent to a subject.
  • Routes of administration of any of the compositions of the invention include oral, nasal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, and (intra)nasal,), intravesical, intraduodenal, intragastrical, rectal, intra-peritoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, or administration.
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.
  • the present invention relates to systems for cis-cleavage and trans-splicing of independent RNA molecules. In some embodiments, the present invention relates to systems cis-cleavage and trans-splicing of a single RNA molecule. In some embodiments, cis-cleavage and trans-splicing of independent RNA molecules or fragments of a single RNA molecule results in a single RNA molecule encoding a full-length protein of interest, as described herein. In some embodiments, the system comprises a ligase or a nucleic acid encoding a ligase, such as RtcB, as described herein.
  • the present invention relates to an inducible system for generating a single RNA encoding a full-length protein from two separate RNA molecules encoding a first part and a second part of the full-length protein via cis-cleavage of ribozymes and trans-splicing of the two independent RNA molecules.
  • the system comprises a ribozyme recognition sequence and a ribozyme, as described herein.
  • the system comprises a ligase or a nucleic acid encoding a ligase, as described herein.
  • the present invention relates to a system of assembling a full- length RNA virus genome.
  • RNA viruses include, but are not limited to: coronaviruses, paramyxoviruses, orthomyxoviruses, retroviruses, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles viruses, Newcastle disease viruses, and picornaviruses.
  • the system comprises a first nucleic acid encoding a first portion of the RNA virus genome and encoding a 3’ ribozyme.
  • the system comprises a second nucleic acid encoding a second portion of the RNA virus genome and encoding a 5’ ribozyme.
  • the system comprises a first portion of the RNA virus genome and a 3’ ribozyme. In one embodiment, the system comprises a second portion of the RNA virus genome and a 5’ ribozyme. In one embodiment, the system comprises a nucleic acid encoding a ligase or a ligase. In one embodiment, upon cis-cleavage of the 3’ and 5’ ribozymes, the first portion of the RNA virus genome and the second portion of the RNA virus genome are ligated together, thereby generating a full-length RNA virus genome.
  • the present invention relates to a system for delivery and expression of one or more full-length protein via cis-cleavage and trans-splicing of independent RNA molecules encoding parts of the full-length protein.
  • the system allows for the delivery and expression of large proteins that exceed the package size of traditional vectors (for example, dystrophin that exceeds the packaging size of AAV vectors), synthetic repeat domain proteins whose nucleic acid constructs are difficult to synthesize in vitro (for example, synthetic spider silk), or toxic/antiviral proteins (for example, DTA).
  • the present invention comprises an AAV system for delivery and expression of one or more full-length protein of interest.
  • the system comprises a ligase or a nucleic acid encoding a ligase, as described herein.
  • the invention comprises a lentiviral delivery system to deliver one or more nucleic acid molecule encoding one or more protein of interest.
  • the lentiviral delivery system comprises (1) a packaging plasmid, (2) an envelope plasmid, and (3) a transfer plasmid.
  • the transfer plasmid encodes a first RNA molecule and a second RNA molecule.
  • the invention comprises a dual lentiviral delivery system, comprising a first lentiviral vector and a second lentiviral vector.
  • the first lentiviral vector system comprises (1) a packaging plasmid, (2) an envelope plasmid, and (3) a first transfer plasmid.
  • the second lentiviral vector system comprises (1) a packaging plasmid, (2) an envelope plasmid, and (3) a second transfer plasmid.
  • the first transfer plasmid encodes a first RNA molecule.
  • the second transfer plasmid encodes a second RNA molecule.
  • the packaging plasmid comprises a nucleic acid sequence encoding a gag-pol polyprotein.
  • the gag-pol polyprotein comprises catalytically dead integrase.
  • the gag-pol polyprotein comprises the D116N integrase mutation.
  • the envelope plasmid comprises a nucleic acid sequence encoding an envelope protein.
  • the envelope plasmid comprises a nucleic acid sequence encoding an HIV envelope protein.
  • the envelope plasmid comprises a nucleic acid sequence encoding a vesicular stomatitis virus g-protein (VSV-g) envelope protein.
  • VSV-g vesicular stomatitis virus g-protein
  • the envelope protein can be selected based on the desired cell type.
  • the first RNA molecule of the single transfer plasmid comprises a protein coding region encoding a first portion of the protein of interest and a 3’ ribozyme.
  • the second RNA molecule of the single transfer plasmid comprises a protein coding region encoding a second portion of the protein of interest and a 5’ ribozyme.
  • the transfer plasmid comprises a 5’ long terminal repeat (LTR) sequence and a 3’ LTR sequence.
  • the 3’ LTR is a Self-inactivating (SIN) LTR.
  • 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.
  • the 5’LTR and 3’LTR flank the sequence encoding the first portion of the protein of interest and the second portion of the protein of interest.
  • the first RNA molecule of the first transfer plasmid comprises a protein coding region encoding a first portion of the protein of interest and a 3’ ribozyme.
  • the second RNA molecule of the second transfer plasmid comprises a protein coding region encoding a second portion of the protein of interest and a 5’ ribozyme.
  • the first and second transfer plasmids comprise a 5’ long terminal repeat (LTR) sequence and a 3’ LTR sequence.
  • the 3’ LTR is a Self- inactivating (SIN) LTR.
  • 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.
  • the 5’LTR and 3’LTR of the first transfer plasmid flank the sequence encoding the first portion of the protein of interest and the 3’ ribozyme.
  • the 5’LTR and 3’LTR of the second transfer plasmid flank the sequence encoding the second portion of the protein of interest and the 5’ ribozyme.
  • the packaging plasmid, the envelope plasmid, and the transfer plasmid are introduced into a cell.
  • the cell transcribes and translates the nucleic acid sequence encoding the gag-pol protein to produce the gag-pol polyprotein.
  • the cell transcribes and translates the nucleic acid sequence encoding the envelope protein to produce the envelope protein.
  • the cell transcribes the single transfer plasmid to provide the first RNA molecule and the second RNA molecule.
  • the cell transcribes the first transfer plasmid to provide the first RNA molecule and the second transfer plasmid to provide the second RNA molecule.
  • the gag- pol protein, envelope polyprotein, first RNA molecule and second RNA molecule are packaged into a viral particle.
  • the viral particles are collected from the cell media.
  • the viral particles transduce a target cell, wherein the 3’ribozyme catalyzes itself out of the first RNA molecule, thereby generating a 3’P or 2’3’ cP end, the 5’ribozyme catalyzes itself out of the second RNA molecule, thereby generating a 5’OH end, endogenous RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase ligates the 3’P or 2’3’ cP end to the 5’OH end, thereby generating a complete RNA molecule encoding the protein of interest, and the cell translates the protein of interest.
  • the 3’ribozyme catalyzes itself out of the first RNA molecule, thereby generating a 3’P or 2’3’ cP end
  • the 5’ribozyme catalyzes itself out of the second RNA molecule, thereby generating a 5’OH end
  • the packaging plasmid, the envelope plasmid, and the first transfer plasmid are introduced into a cell.
  • the cell transcribes and translates the nucleic acid sequence encoding the gag-pol protein to produce the gag-pol polyprotein.
  • the cell transcribes and translates the nucleic acid sequence encoding the envelope protein to produce the envelope protein.
  • the cell transcribes the first transfer plasmid to provide the first RNA molecule.
  • the gag-pol protein, envelope polyprotein, first RNA molecule are packaged into a first viral particle.
  • the first viral particles are collected from the cell media.
  • the packaging plasmid, the envelope plasmid, and the second transfer plasmid are introduced into a cell.
  • the cell transcribes and translates the nucleic acid sequence encoding the gag-pol protein to produce the gag-pol polyprotein.
  • the cell transcribes and translates the nucleic acid sequence encoding the envelope protein to produce the envelope protein.
  • the cell transcribes the second transfer plasmid to provide the second RNA molecule.
  • the gag-pol protein, envelope polyprotein, second RNA molecule are packaged into a second viral particle.
  • the second viral particles are collected from the cell media.
  • the first viral particle and the second viral particle transduce a target cell, wherein the 3’ribozyme catalyzes itself out of the first RNA molecule, thereby generating a 3’P or 2’3’ cP end, the 5’ribozyme catalyzes itself out of the second RNA molecule, thereby generating a 5’OH end, endogenous RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase ligates the 3’P or 2’3’ cP end to the 5’OH end, thereby generating a complete RNA molecule encoding the protein of interest, and the cell translates the protein of interest.
  • the present invention relates to a system of preventing unwanted partial protein expression from a split precursor RNA molecule.
  • the system comprises incorporating translational control of protein degradation sequences in the split precursor RNA molecule, as described herein.
  • the present invention relates to a system for expression of two or more proteins of interest from two or more pairs of independent RNA molecules encoding parts of the proteins of interest via cis-cleavage of ribozymes and trans-splicing of the pairs of independent RNA molecules.
  • each individual pair of independent RNA molecules has a separate reading frame, such that trans-splicing of undesired pairs does not result in translation of a full-length functional protein, as described herein.
  • the system comprises a ligase or a nucleic acid encoding a ligase, as described herein.
  • the present invention comprises a system for delivery and expression of a full-length protein of interest and a cargo sequence.
  • the system comprises a first portion of RNA encoding a first portion of the protein of interest linked at its 3’ end to a synthetic intron and a second portion of RNA encoding a second portion of the protein of interest linked at its 5’ end to a synthetic intron.
  • the synthetic intron is flanked on either side by a 5’ ribozyme sequence and a 3’ ribozyme sequence.
  • the synthetic intron comprises a cargo sequence placed between said 5’ ribozyme sequence and 3’ ribozyme sequence.
  • self-cleavage of the 5’ ribozyme sequence and the 3’ ribozyme sequence generates three separate RNA molecules: 1) a first fragment comprising the first portion of RNA encoding a first portion of a protein of interest, 2) a second fragment comprising the synthetic intron, 3) a third fragment comprising the second portion of RNA encoding a second portion of a protein of interest.
  • the compatible ends of the second fragment are ligated to generate a circular RNA molecule comprising the synthetic intron comprising the cargo sequence.
  • the first fragment and third fragment are ligated together to generate a single full-length linear RNA molecule.
  • the full-length protein of interest comprises a therapeutic protein, a reporter protein, a recombinase, an antibiotic resistance gene product, antibody, or Cas9 protein.
  • the cargo sequence comprises a therapeutic nucleic acid sequence (for example, a miRNA sequence or a CRISPR guide RNA sequence) or encodes a therapeutic protein.
  • the full-length protein of interest comprises Cas9 and the cargo sequence comprises a guide RNA sequence, thereby targeting Cas9 to a particular genomic sequence for editing.
  • the system comprises a ligase or a nucleic acid encoding a ligase, as described herein.
  • the present invention comprises a system for gene editing, comprising one or more trans-cleaving engineered ribozymes.
  • the system comprises two trans-cleaving engineered ribozymes, targeted upstream and downstream of the disease causing mutation.
  • trans-cleavage upstream and downstream of the disease causing mutation results in removal of the disease causing mutation.
  • the remaining portions of the gene are trans-spliced together after trans-cleavage of the disease causing mutation.
  • the trans-spliced gene is expressed as a functional protein.
  • the system comprises a ligase or a nucleic acid encoding a ligase, as described herein.
  • the present invention comprises an in vitro system for generating an RNA molecule encoding a protein of interest.
  • the system comprises at least two RNA molecules.
  • the at least two RNA molecules comprises a first RNA molecule and a second RNA molecule.
  • the first RNA molecule comprises a coding region encoding a first portion of the protein of interest.
  • the first RNA molecule comprises a 3’ribozyme.
  • the first RNA molecule comprises a coding region encoding a first portion of the protein of interest and a 3’ribozyme, as described herein.
  • the second RNA molecule comprises a coding region encoding a second portion of the 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 the protein of interest and a 5’ribozyme, as described herein. [0270] In one embodiment, the in vitro system for generating an RNA molecule encoding a protein of interest further comprises a ligase. In one embodiment, the ligase induces the assembly of the RNA molecule from the coding region of the first RNA molecule and the coding region of the second RNA molecule.
  • the ligase is RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase, as described herein.
  • the present invention comprises an in vitro system for generating an RNA molecule encoding repeat domain protein of interest.
  • the system comprises a first RNA molecule and at least one additional RNA molecule.
  • the first RNA molecule comprises a coding region encoding a first portion of the protein of interest.
  • the first RNA molecule comprises a 3’ribozyme.
  • the first RNA molecule comprises a coding region encoding a first portion of the protein of interest and a 3’ribozyme.
  • the 3’ ribozyme catalyzes itself out of the first RNA molecule, thereby generating a 3’P or 2’3’ cP end.
  • the first RNA molecule further comprises a 5’ tag.
  • the 5’ tag mediates attachment of said first RNA molecule to a solid support.
  • the one or more additional RNA molecule comprises a coding region encoding a domain of the protein of interest; a 5’ ribozyme; and a 3’ ribozyme recognition sequence.
  • the 5’ ribozyme cleaves itself to generate a 5’OH end.
  • the 3’ ribozyme recognition sequence comprises a VS-S sequence, as described herein.
  • at least one additional RNA molecule comprises a coding region encoding at least one additional portion of the protein of interest.
  • at least one additional RNA molecule comprises a 5’ribozyme.
  • at least one additional RNA molecule comprises a coding region encoding at least one additional portion of the protein of interest and a 5’ribozyme.
  • the 5’ ribozyme cleaves itself to generate a 5’OH end.
  • the system further comprises a ribozyme.
  • the ribozyme comprises VS-Rz, as described herein.
  • the VS- Rz recognizes VS-S, as described herein, and mediates its cleavage from the one or more additional RNA molecule.
  • the cleavage generates a 3’P or 2’3’ cP end.
  • the system comprises a ligase.
  • 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 molecule.
  • the ligase ligates the 3’P or 2’3’ cP end of the one or more additional RNA molecule to the 5’OH end of at least one additional 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 molecule, and ligates the 3’P or 2’3’ cP end of the one or more additional RNA molecule to the 5’OH end of at least one additional RNA molecule, thereby generating a complete RNA molecule encoding an N-terminal domain, one or more additional domain, and a C-terminal domain.
  • the ligase is RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase, as described herein.
  • Methods [0277] the present invention relates to methods of cis-cleavage and trans-splicing of independent RNA molecules. In some embodiments, the present invention relates to methods of cis-cleavage and trans-splicing of a single RNA molecule. In some embodiments, cis-cleavage and trans-splicing of independent RNA molecules or fragments of a single RNA molecule results in a single RNA molecule encoding a full-length protein of interest, as described herein.
  • the method comprises administering ligase or a nucleic acid encoding a ligase, as described herein.
  • the present invention relates to an inducible method for generating a single RNA encoding a full-length protein from two separate RNA molecules encoding a first part and a second part of the full-length protein via cis-cleavage of ribozymes and trans-splicing of the two independent RNA molecules.
  • the method comprises a ribozyme recognition sequence and a ribozyme, as described herein.
  • the method comprises administering ligase or a nucleic acid encoding a ligase, as described herein.
  • the present invention comprises a method of generating an RNA molecule encoding a protein of interest.
  • the method comprises administering at least two nucleic acid molecules to a cell or tissue.
  • the at least two nucleic acid molecules comprise a first RNA molecule and a second RNA molecule.
  • the at least two nucleic acid molecules encode a first RNA molecule and a second RNA molecule.
  • the first RNA molecule comprises a coding region encoding a first portion of the protein of interest.
  • the first RNA molecule comprises a 3’ribozyme.
  • the first RNA molecule comprises a coding region encoding a first portion of the protein of interest and a 3’ribozyme.
  • the 3’ribozyme catalyzes itself out of the first RNA molecule, thereby generating a 3’P or 2’3’ cP end.
  • the 3’ ribozyme is a member of the HDV family of ribozymes [0281]
  • the second RNA molecule comprises a coding region encoding a second portion of the protein of interest.
  • the second RNA molecule comprises a 5’ribozyme.
  • the second RNA molecule comprises a coding region encoding a second portion of the protein of interest and a 5’ribozyme.
  • the 5’ribozyme catalyzes itself out of the second RNA molecule, thereby generating a 5’OH end.
  • the 5’ ribozyme is a member of the HH family of ribozymes.
  • the 3’P or 2’3’ cP end is ligated to the 5’OH end to form an RNA molecule comprising the coding region of the first RNA molecule and the coding region of the second RNA molecule.
  • trans-ligation of the coding sequences for the first and second portion of the protein of interest occurs in a scarless manner, such that there is no intervening sequence between the first and second portion of the protein of interest after translation.
  • the method 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 the protein of interest; a 5’ ribozyme; and a 3’ ribozyme.
  • the method 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 the protein of interest; a 5’ ribozyme; and a 3’ ribozyme recognition sequence.
  • the 3’ ribozyme recognition sequence comprises VS-S.
  • the ribozyme is VS.
  • 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.
  • the ligase induces the assembly of the RNA molecule from the coding region of the first RNA molecule and the coding region of the second RNA molecule.
  • trans-ligation of the coding sequences for the first and second portion of the protein of interest occurs in a scarless manner, such that there is no intervening sequence between the first and second portion of the protein of interest after translation.
  • the ligase is RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase.
  • the method comprises administering at least one AAV vector encoding a first RNA molecule comprising a protein coding region encoding a first portion of the protein of interest and a 3’ ribozyme, and a second RNA molecule comprising a protein coding region encoding a second portion of the protein of interest and a 5’ ribozyme to a cell or tissue.
  • the method comprises administering ligase or a nucleic acid encoding a ligase, as described herein.
  • the method comprises administering at least two AAV vectors, comprising a first AAV vector and a second AAV vector.
  • the first AAV vector encodes a first RNA molecule comprising a protein coding region encoding a first portion of the protein of interest and a 3’ ribozyme.
  • the second AAV vector encodes a second RNA molecule comprising a protein coding region encoding a second portion of the protein of interest and a 5’ ribozyme to a cell or tissue.
  • the method comprises administering ligase or a nucleic acid encoding a ligase, as described herein.
  • trans-ligation of the coding sequences for the first and second portion of the protein of interest occurs in a scarless manner, such that there is no intervening sequence between the first and second portion of the protein of interest after translation.
  • the method comprises administering at least one lentiviral vector, encoding a first RNA molecule comprising a protein coding region encoding a first portion of the protein of interest and a 3’ ribozyme, and a second RNA molecule comprising a protein coding region encoding a second portion of the protein of interest and a 5’ ribozyme to a cell or tissue.
  • the method comprises administering ligase or a nucleic acid encoding a ligase, as described herein.
  • the method comprises administering at least two lentiviral vectors, comprising a first lentiviral vector and a second lentiviral vector.
  • the first lentiviral vector encodes a first RNA molecule comprising a protein coding region encoding a first portion of the protein of interest and a 3’ ribozyme.
  • the second lentiviral vector encodes a second RNA molecule comprising a protein coding region encoding a second portion of the protein of interest and a 5’ ribozyme to a cell or tissue.
  • the method comprises administering ligase or a nucleic acid encoding a ligase, as described herein.
  • the method comprises administering at least one lentiviral vector delivery system to provide a first RNA molecule comprising a protein coding region encoding a first portion of the protein of interest and a 3’ ribozyme, and a second RNA molecule comprising a protein coding region encoding a second portion of the protein of interest and a 5’ ribozyme to a cell or tissue.
  • the method comprises administering ligase or a nucleic acid encoding a ligase, as described herein.
  • the method comprises administering at least two lentiviral vector delivery systems, comprising a first lentiviral vector delivery system and a second lentiviral vector delivery system.
  • the first lentiviral vector delivery system provides a first RNA molecule comprising a protein coding region encoding a first portion of the protein of interest and a 3’ ribozyme.
  • the second lentiviral vector delivery system provides a second RNA molecule comprising a protein coding region encoding a second portion of the protein of interest and a 5’ ribozyme to a cell or tissue.
  • the method comprises administering ligase or a nucleic acid encoding a ligase, as described herein.
  • the method comprises administering two or more delivery vehicles selected from the group consisting of: an AAV vector, a lentiviral vector, a lentiviral vector delivery system, or a combination thereof.
  • the two or more delivery vehicles comprises a first delivery vehicle and a second delivery vehicle.
  • the first delivery vehicle provides a first RNA molecule comprising a protein coding region encoding a first portion of the protein of interest and a 3’ ribozyme.
  • the second delivery vehicle provides a second RNA molecule comprising a protein coding region encoding a second portion of the protein of interest and a 5’ ribozyme to a cell or tissue.
  • the method comprises administering ligase or a nucleic acid encoding a ligase, as described herein.
  • the present invention comprises a method of generating a circular RNA molecule encoding a protein of interest.
  • the method comprises administering at least one nucleic acid molecule to a cell or tissue, wherein the nucleic acid molecule comprises at least a 5’ ribozyme directly linked to the sequence encoding the C- terminal or N-terminal portion of the protein of interest and a 3’ ribozyme directly linked to the sequence encoding the C-terminal or N-terminal portion of the protein of interest.
  • the 3’ribozyme catalyzes itself out of the RNA molecule, thereby generating a 3’P or 2’3’ cP end.
  • the 3’ ribozyme is a member of the HDV family of ribozymes.
  • the 5’ribozyme catalyzes itself out of the RNA molecule, thereby generating a 5’OH end.
  • the 5’ ribozyme is a member of the HH family of ribozymes.
  • the 3’P or 2’3’ cP end is ligated to the 5’OH end to form a circular RNA molecule in which the coding sequence of the N-terminus and C-terminus of the protein of interest are operably linked.
  • circularization occurs in a scarless manner, such that there is no intervening sequence between the N-terminus and C- terminus after translation.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising 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).
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule 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).
  • an exemplary delivery vehicle is a liposome.
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. [0299] Lipids suitable for use can be obtained from commercial sources.
  • dimyristyl phosphatidylcholine can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL).
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated.
  • the present invention relates to a method of expressing two or more proteins of interest from two or more pairs of independent RNA molecules encoding parts of the proteins of interest via cis-cleavage of ribozymes and trans-splicing of the pairs of independent RNA molecules.
  • the method comprises administering one, two, or three pairs of nucleic acid molecules encoding or comprising RNA molecules, wherein each individual pair of independent RNA molecules has a separate reading frame, such that trans- splicing of undesired pairs does not result in translation of a full-length functional protein.
  • the method further 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.
  • the ligase is RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase, as described herein.
  • the present invention comprises a method of delivery and expression of a full-length protein of interest and a cargo sequence.
  • the method comprises administering to a cell or tissue a first portion of RNA encoding a first portion of the protein of interest linked at its 3’ end to a synthetic intron and a second portion of RNA encoding a second portion of the protein of interest linked at its 5’ end to a synthetic intron.
  • the synthetic intron is flanked on either side by a 5’ ribozyme sequence and a 3’ ribozyme sequence.
  • the synthetic intron comprises a cargo sequence placed between said 5’ ribozyme sequence and 3’ ribozyme sequence.
  • self- cleavage of the 5’ ribozyme sequence and the 3’ ribozyme sequence generates three separate RNA molecules: 1) a first fragment comprising the first portion of RNA encoding a first portion of a protein of interest, 2) a second fragment comprising the synthetic intron, 3) a third fragment comprising the second portion of RNA encoding a second portion of a protein of interest.
  • the compatible ends of the second fragment are ligated to generate a circular RNA molecule comprising the synthetic intron comprising the cargo sequence.
  • the first fragment and third fragment are ligated together to generate a single full-length linear RNA molecule.
  • the full-length protein of interest comprises a therapeutic protein, a reporter protein, a recombinase, an antibiotic resistance gene product, antibody, or Cas9 protein.
  • the cargo sequence comprises a therapeutic nucleic acid sequence (for example, an miRNA sequence or a CRISPR guide RNA sequence) or encodes a therapeutic protein.
  • the full-length protein of interest comprises Cas9 and the cargo sequence comprises a guide RNA sequence, thereby targeting Cas9 to a particular genomic sequence for editing.
  • the method comprises administering to the cell or tissue a ligase or a nucleic acid encoding a ligase, as described herein.
  • the present invention comprises a method of gene editing, comprising one or more trans-cleaving engineered ribozymes.
  • the method comprises administering a first trans-cleaving engineered ribozyme and a second trans-cleaving engineered ribozyme, wherein the first trans-cleaving engineered ribozyme targets upstream and the second trans-cleaving engineered ribozyme downstream of a disease-causing mutation.
  • trans-cleavage upstream and downstream of the disease-causing mutation results in removal of the disease causing mutation.
  • the remaining portions of the gene are trans-spliced together after trans-cleavage of the disease-causing mutation.
  • the trans-spliced gene is expressed as a functional protein.
  • the present invention relates to in vivo methods of assembling a full-length RNA virus genome.
  • RNA viruses include, but are not limited to: coronaviruses, paramyxoviruses, orthomyxoviruses, retroviruses, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles viruses, Newcastle disease viruses, and picornaviruses.
  • the method comprises administering to a cell or tissue a first nucleic acid encoding a first portion of the RNA virus genome and encoding a 3’ ribozyme.
  • the method comprises administering to the cell or tissue a second nucleic acid encoding a second portion of the RNA virus genome and encoding a 5’ ribozyme. In one embodiment, the method comprises administering to the cell or tissue a first RNA molecule comprising a first portion of the RNA virus genome and a 3’ ribozyme. In one embodiment, the method comprises administering to the cell or tissue a second RNA molecule comprising a second portion of the RNA virus genome and a 5’ ribozyme. In one embodiment, the method comprises administering to the cell or tissue a nucleic acid encoding a ligase or a ligase, as described herein.
  • the present invention relates to in vivo methods of generating a circular RNA molecule encoding a protein of interest.
  • the method comprises the step of administering a DNA molecule or an in vitro transcribed linear RNA molecule comprising a coding region encoding a first portion of the protein of interest (N- terminal coding sequence), 5’ ribozyme, an intervening sequence to be removed, and a 3’ribozyme directly linked to a coding region encoding a second portion of the protein of interest (C-terminal coding sequence).
  • N-terminal coding sequence is directly linked to the 5’ ribozyme sequence and the 3’ribozyme is directly linked to the C-terminal coding sequence.
  • the coding sequences for the N-terminus and C- terminus are in reverse orientation in the linear RNA molecule.
  • each of the 3’ ribozyme and the 5’ ribozyme comprises a sequence of SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:
  • the 5’ ribozyme is SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ
  • the 3’ ribozyme is SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ
  • the 5’ ribozyme comprises SEQ ID NO:174 and the 3’ ribozyme comprises SEQ ID NO:175. In some embodiments, the 5’ ribozyme comprises SEQ ID NO:176 and the 3’ ribozyme comprises SEQ ID NO:172.
  • the coding sequence of the protein of interest is in the reverse orientation in the linear RNA molecule. In some embodiments, the linear RNA molecule further comprises an IRES, a polyadenylation sequence, an AK recombinogenic sequence (SEQ ID NO: 180) or any combination thereof.
  • the method comprises the step of providing a linear RNA molecule comprising a 3’ ribozyme, a coding region encoding a second portion of the protein of interest (C-terminal coding sequence), a coding region encoding a first portion of the protein of interest (N-terminal coding sequence), and a 5’ribozyme.
  • the N-terminal coding sequence is directly linked to the 5’ ribozyme sequence and the 3’ribozyme is directly linked to the C-terminal coding sequence.
  • the coding sequences for the N- terminus and C-terminus are in reverse orientation in the linear RNA molecule.
  • each of the 3’ ribozyme and the 5’ ribozyme comprises a sequence of SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:
  • the 3’ ribozyme comprises SEQ ID NO:171 and the 5’ ribozyme comprises SEQ ID NO:170. In some embodiments, the 3’ ribozyme comprises SEQ ID NO:169 and the 5’ ribozyme comprises SEQ ID NO:168.
  • the coding sequence of the protein of interest is in the reverse orientation in the linear RNA molecule.
  • the linear RNA molecule further comprises an IRES, a polyadenylation sequence, an AK recombinogenic sequence (SEQ ID NO: 180) or any combination thereof. [0307] In some embodiments, the linear RNA molecule comprises a splice donor or splice acceptor sequence.
  • the splice donor comprises SEQ ID NO:245 or SEQ ID NO:246. In some embodiments, the splice acceptor comprises SEQ ID NO:247.
  • the present invention comprises an in vitro method of generating an RNA molecule encoding a protein of interest. In one embodiment, the method comprises the step of providing at least two RNA molecules. In one embodiment, the step comprises providing a first RNA molecule and a second RNA molecule. [0309] In one embodiment, the first RNA molecule comprises a coding region encoding a first portion of the protein of interest. In one embodiment, the first RNA molecule comprises a 3’ribozyme.
  • the first RNA molecule comprises a coding region encoding a first portion of the protein of interest and a 3’ribozyme.
  • the second RNA molecule comprises a coding region encoding a second portion of the protein of interest.
  • the second RNA molecule comprises a 5’ribozyme.
  • the second RNA molecule comprises a coding region encoding a second portion of the protein of interest and a 5’ribozyme.
  • the in vitro method of generating an RNA molecule encoding a protein of interest further comprises providing a ligase.
  • the ligase induces the assembly of the RNA molecule from the coding region of the first RNA molecule and the coding region of the second RNA molecule.
  • the ligase is RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase, as described herein.
  • the present invention comprises an in vitro method of generating 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 molecule, c) providing a ribozyme, and d) providing a last RNA molecule.
  • the first RNA molecule of step a) comprises a coding region encoding a first portion of the protein of interest.
  • the first RNA molecule comprises a 3’ribozyme.
  • the first RNA molecule comprises a coding region encoding a first portion of the protein of interest and a 3’ribozyme.
  • the 3’ ribozyme catalyzes itself out of the first RNA molecule, thereby generating a 3’P or 2’3’ cP end.
  • the first RNA molecule further comprises a 5’ tag.
  • the 5’ tag mediates attachment of said first RNA molecule to a solid support.
  • At least one additional RNA molecule of step b) comprises a coding region encoding a domain of the protein of interest; a 5’ ribozyme; and a 3’ ribozyme recognition sequence.
  • the 5’ ribozyme cleaves itself to generate a 5’OH end.
  • a ligase is provided to catalyze ligation of the first RNA molecule to the one or more additional RNA molecule.
  • the ligase is RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase, as described herein.
  • the 3’ ribozyme recognition sequence comprises a VS-S sequence, as described herein.
  • the ribozyme of step c) comprises VS-Rz, as described herein.
  • the VS-Rz recognizes VS-S, and mediates its cleavage from the one or more additional RNA molecule.
  • the cleavage generates a 3’P or 2’3’ cP end.
  • steps b) through c) are repeated at least one time to generate an RNA molecule encoding a plurality of domains.
  • the VS-Rz is removed prior to repeating step b).
  • the RNA molecule of step d) comprises a coding region encoding a last portion of the protein of interest.
  • the RNA molecule comprises a 5’ribozyme.
  • the 5’ ribozyme catalyzes itself out of the RNA molecule, thereby generating a 5’OH end.
  • a ligase is provided to catalyze ligation of the one or more additional RNA molecule to the RNA molecule, thereby generating a complete RNA molecule encoding an N-terminal domain, one or more additional domain, and a C-terminal domain.
  • the ligase is RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase, as described herein.
  • the present invention comprises an in vitro method of generating a circular RNA molecule encoding a protein of interest.
  • the method comprises the step of providing a plasmid or vector comprising a coding region encoding a first portion of the protein of interest (N-terminal coding sequence), 5’ ribozyme, an intervening sequence to be removed, and a 3’ribozyme directly linked to a coding region encoding a second portion of the protein of interest (C-terminal coding sequence).
  • the N-terminal coding sequence is directly linked to the 5’ ribozyme sequence and the 3’ribozyme is directly linked to the C-terminal coding sequence.
  • the coding sequences for the N-terminus and C-terminus are in reverse orientation in the linear RNA molecule.
  • each of the 3’ ribozyme and the 5’ ribozyme comprises a sequence of SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175 or SEQ ID NO:176.
  • the 5’ ribozyme is SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:174 or SEQ ID NO:176.
  • the 3’ ribozyme is SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:172 or SEQ ID NO:174.
  • the 5’ ribozyme comprises SEQ ID NO:174 and the 3’ ribozyme comprises SEQ ID NO:175.
  • the 5’ ribozyme comprises SEQ ID NO:176 and the 3’ ribozyme comprises SEQ ID NO:172.
  • the coding sequence of the protein of interest is in the reverse orientation in the linear RNA molecule.
  • the linear RNA molecule further comprises an IRES, a polyadenylation sequence, an AK recombinogenic sequence (SEQ ID NO: 180) or any combination thereof.
  • the method comprises the step of providing a linear RNA molecule comprising a 3’ ribozyme, a coding region encoding a second portion of the protein of interest (C-terminal coding sequence), a coding region encoding a first portion of the protein of interest (N-terminal coding sequence), and a 5’ribozyme.
  • the N-terminal coding sequence is directly linked to the 5’ ribozyme sequence and the 3’ribozyme is directly linked to the C-terminal coding sequence.
  • each of the 3’ ribozyme and the 5’ ribozyme comprises a sequence of SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:192, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:
  • the 5’ ribozyme is SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ
  • the 3’ ribozyme is SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ
  • the 3’ ribozyme comprises SEQ ID NO:171 and the 5’ ribozyme comprises SEQ ID NO:170. In some embodiments, the 3’ ribozyme comprises SEQ ID NO:169 and the 5’ ribozyme comprises SEQ ID NO:168.
  • the coding sequence of the protein of interest is in the reverse orientation in the linear RNA molecule. In some embodiments, the linear RNA molecule further comprises an IRES, a polyadenylation sequence, an AK recombinogenic sequence (SEQ ID NO: 180) or any combination thereof.
  • RNA molecule of the present disclosure may be transcribed in vitro from 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, synthetic DNA sequence or any other appropriate source of DNA.
  • an in vitro transcription template encodes a 5′ untranslated (UTR) region, contains an open reading frame, and encodes a 3′ UTR and a polyA tail.
  • an in vitro transcription template lacks the polyA tail.
  • the particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA, or fragment thereof (e.g., N-terminal or C-terminal fragment) encoded by the template.
  • the 5’ UTR is between zero and 3000 nucleotides in length.
  • the length of 5’ and 3’ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.
  • the 5’ and 3’ UTRs can be the naturally occurring, endogenous 5’ and 3’ UTRs for the gene of interest.
  • UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
  • the use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3’ UTR sequences can decrease the stability of mRNA. Therefore, 3’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5’ UTR can contain the Kozak sequence of the endogenous gene.
  • a consensus Kozak sequence can be redesigned by adding the 5’ UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art.
  • the 5’ UTR can be derived from an RNA virus whose RNA genome is stable in cells.
  • RNA polymerase promoter a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
  • the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • the promoter is a T7 RNA polymerase promoter, as described elsewhere herein.
  • RNA polymerase promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
  • the mRNA has both a cap on the 5’ end and a 3’ poly(A) tail which determine ribosome binding, initiation of translation and stability of mRNA in the cell.
  • RNA polymerase produces a long concatameric product, which is not suitable for expression in eukaryotic cells.
  • phage 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)).
  • the conventional method of integration of polyA/T stretches into a DNA template is molecular cloning.
  • Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP) or yeast polyA polymerase.
  • E-PAP E. coli polyA polymerase
  • yeast polyA polymerase E. coli polyA polymerase
  • increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
  • the attachment of different chemical groups to the 3’ end can increase mRNA stability.
  • RNAs produced by the methods to include a 5’ cap1 structure can be generated using Vaccinia capping enzyme and 2’-O-methyltransferase enzymes (CellScript, Madison, WI).
  • 5’ cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem.
  • Certain embodiments of the invention may make use of solid supports comprised of an inert substrate or matrix (e.g. glass slides, polymer beads etc.) which has been functionalized, for example by application of a layer or coating of an intermediate material comprising reactive groups which permit covalent attachment to biomolecules, such as polynucleotides.
  • an inert substrate or matrix e.g. glass slides, polymer beads etc.
  • an intermediate material comprising reactive groups which permit covalent attachment to biomolecules, such as polynucleotides.
  • Such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, particularly polyacrylamide hydrogels as described in WO 2005/065814 and US 2008/0280773, the contents of which are incorporated herein in their entirety by reference.
  • the biomolecules e.g. polynucleotides
  • the intermediate material e.g. the hydrogel
  • the intermediate material may itself be non-covalently attached to the substrate or matrix (e.g. the glass substrate).
  • covalent attachment to a solid support is to be interpreted accordingly as encompassing this type of arrangement.
  • Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonTM, 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 variety of other polymers.
  • 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 dextrans such as Sepharose, cellulose, nylon, cross-linked micelles and teflon, as well as any other materials outlined herein for solid supports may all be used.
  • “Microsphere Detection Guide” from Bangs Laboratories, Fishers Ind. is a helpful guide.
  • the microspheres are magnetic microspheres or beads. [0332] The beads need not be spherical; irregular particles may be used.
  • the beads may be porous.
  • the bead sizes range from nanometers, i.e.100 nm, to millimeters, i.e.1 mm, with beads from about 0.2 micron to about 200 microns being preferred, and from about 0.5 to about 5 micron being particularly preferred, although in some embodiments smaller or larger beads may be used.
  • the present invention relates to in vitro methods of assembling a full-length RNA virus genome.
  • RNA viruses include, but are not limited to: coronaviruses, paramyxoviruses, orthomyxoviruses, retroviruses, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles viruses, Newcastle disease viruses, and picornaviruses.
  • the method comprises providing a first RNA molecule comprising a first portion of the RNA virus genome and a 3’ ribozyme.
  • the method comprises providing a second RNA molecule comprising a second portion of the RNA virus genome and a 5’ ribozyme.
  • the method comprises contacting the first RNA molecule and the second RNA molecule with a ligase, as described herein, thereby generating a full-length RNA virus genome.
  • the methods of the invention of treat, reduce the symptoms of, and/or reduce the risk of developing a disease or disorder in a plant. In one embodiment, the methods of the invention of treat, reduce the symptoms of, and/or reduce the risk of developing a disease or disorder in a yeast organism.
  • the subject is a cell. In one embodiment, the cell is a prokaryotic cell or eukaryotic cell. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the cell is a plants, animals, or fungi cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is an animal cell. In one embodiment, the cell is a yeast cell.
  • the subject is a mammal.
  • the subject is a human, non-human primate, dog, cat, horse, cow, goat, sheep, rabbit, pig, rat, or mouse.
  • the subject is a non-mammalian subject.
  • the subject is a zebrafish, fruit fly, or roundworm.
  • the disease or disorder is caused by an absent or defective protein, the nucleic acid sequence of which exceeds the packaging size of a viral vector.
  • the disease or disorder may treated, reduced, or the risk can be reduced using the compositions, systems and methods of the present invention.
  • the method comprises administering to the subject one or more composition of the present invention. Further, in one embodiment, the method comprises utilizing one or more system of the present invention to treat, reduce the symptoms of, and/or reduce the risk of developing a disease or disorder in a subject.
  • the disease or disorder is Duchenne Muscular Dystrophy, Becker Muscular Dystrophy (BMD), autosomal recessive polycystic kidney disease, hemophilia A, Stargardt macular degeneration, limb-girdle muscular dystrophies, autosomal recessive profound prelingual deafness, autosomal recessive nonsyndromic hearing loss (ARNSHL), sensorineural deafness, cystic fibrosis, Wilson Disease, Miyoshi myopathy, autosomal recessive deafness-9 (DFNB9), Usher Syndrome Type I, GJB2-related autosomal recessive non-syndromic hearing loss (GJB2-AR NSHL), Autosomal recessive cerebelloparenchymal disorder type 3, non-syndromic hearing loss, autosomal recessive deafness-16 (DFNB16), Meniere's disease (MD), autosomal dominant non-syndromic sensorineural deafness
  • the disease or disorder is any caused by a genetic mutation that is amenable CRISPR-Cas9 mediated editing.
  • the method of the present 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 Dystrophin and a 3’ ribozyme, and a second nucleic acid comprising a coding region encoding a second portion of Dystrophin 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 of the 3’ and 5’ ribozymes and trans-splicing of the coding region encoding the first portion of Dystrophin and the coding region encoding the second portion of Dystrophin, generates a single RNA molecule encoding
  • the method of the present 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 transcription of the first nucleic acid results in a first RNA molecule and transcription of the second nucleic acid results in a second RNA molecule, and wherein cis-cleavage of the 3’ and 5’ ribozymes and trans-splicing of the first RNA molecule and second RNA molecule, generates a single RNA molecule encoding a full- length Dystrophin protein.
  • the method of the present 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: 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 of the 3’ and 5’ ribozymes and trans-splicing of the first RNA molecule and second RNA molecule, generates a single RNA molecule encoding a full-length Dystrophin protein with a C-terminal GFP reporter.
  • 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.
  • the method comprises administering to a subject having Duchenne Muscular Dystrophy a composition comprising a first RNA molecule encoding a first portion of Dystrophin and comprising a 3’ ribozyme, and a second RNA molecule encoding a second portion of Dystrophin and comprising a 5’ ribozyme, wherein cis-cleavage of the 3’ and 5’ ribozymes and trans-splicing of the first and second RNA molecules generates a single RNA molecule encoding a full-length Dystrophin protein.
  • 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 of the 3’ and 5’ ribozymes and trans- splicing of the first and second RNA molecules generates a single RNA molecule encoding a full-length Dystrophin protein.
  • 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: 22, and a second RNA molecule comprising the nucleic acid sequence of SEQ ID NO: 23, wherein cis-cleavage of the 3’ and 5’ ribozymes and trans- splicing of the first and second RNA molecules generates a single RNA molecule encoding a full-length Dystrophin protein with a C-terminal GFP reporter.
  • 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.
  • the method of the present invention comprises administering to a subject having one or more disease selected from Table 1 a composition comprising a first nucleic acid comprising a coding region encoding a first portion of a therapeutic protein corresponding to the related 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 the related 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 of the 3’ and 5’ ribozymes and trans-splicing of the coding region encoding a first portion of the therapeutic protein and the coding region encoding the second portion of the therapeutic protein, generates a single RNA molecule encoding the full-length therapeutic protein [0346] In one embodiment, the method
  • the prime editor for the editing of nucleic acid sequences.
  • the prime editor delivered via the compositions described herein, can be used for the correction of disease-causing SNPs.
  • the prime editor, delivered via the compositions described herein can be used for modifying genome sequences to enhance or prevent age-related or microsatellite repeat-induced diseases.
  • the method can be used to disrupt the expression of myostatin (MSTN), a negative regulator of muscle growth.
  • MSTN myostatin
  • the method can be used to insert small sequences for the prevention of the expression of toxic RNA sequences, such as the microsatellite repeat causing myotonic dystrophy type 1 (DM1).
  • toxic RNA sequences such as the microsatellite repeat causing myotonic dystrophy type 1 (DM1).
  • DM1 myotonic dystrophy type 1
  • Ribozyme-mediated RNA Assembly and Expression in Mammalian Cells [0352] Ribozymes (Rzs) are small catalytic RNA sequences which are capable of nucleotide-specific self-cleavage (Doherty and Doudna 2000).
  • Ribozyme-mediated RNA cleavage generates unique 3’ phosphate and 5’-hydroxy termini, which resemble substrates for ubiquitous RNA repair pathways present in all three kingdoms of life.
  • ribozyme-mediated cis-cleavage can be harnessed for the trans-splicing of independent RNA transcripts in mammalian cells, an approach named stitchR (stitch RNA).
  • stitchR switch RNA
  • reconstitution of messenger RNA by stitchR allowed for efficient translation and expression of full-length proteins in mammalian cells.
  • stitchR can be harnessed for the combination of protein coding functional domains or for the delivery and expression of large protein coding sequences by viral vectors.
  • RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) Ligase enhances stitchR activity in mammalian cells and is sufficient for catalyzing stitchR activity in vitro.
  • RtcB 5'-OH
  • ribozyme families have been identified with distinct sequence and structural features, including Hammerhead (HH), Hepatitis Delta Virus (HDV), Varkud Satellite (VS), Sister, Twister-sister, Hairpin, Hatchet and Pistol. Most widely studied are the HH, HDV, and Twister family members, which due to their small size and cleavage characteristics, have been utilized in vitro and in vivo to generate RNAs with precise termini devoid of ribozyme sequences ( Figure 13) (Ferre-D'Amare and Doudna 1996; Avis et al.2012; Zhang et al.2017).
  • RNAs are synthesized and spliced with 5’-phosphate (P) and 3’-hydroxyl (OH) termini, including messenger and long noncoding RNA.
  • P 5’-phosphate
  • OH 3’-hydroxyl
  • unconventional cis-splicing of many tRNAs and the mRNA encoding the ER stress-responsive protein XBP1 are catalyzed by enzymatic pathways which result in unique 5’- OH and either 3’-P or 2’3’ cyclic Phosphate (cP) ends.
  • RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase in mammals. Additionally, RtcB and several other enzyme families may function to repair host cell RNAs which have been damaged by stress or exogenous ribotoxins. Since ribozyme-mediated cleavage results in similar terminal ends, ribozyme-cleaved RNAs could be subject to trans-splicing by endogenous RNA repair pathways.
  • Ribozyme-cleaved mRNAs are trans-spliced and translated in mammalian cells [0355] To determine whether ribozymes could be utilized for scar-less 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 GFP (Nt-GFP and Ct- GFP, respectively). Ribozymes were designed to catalyze their own removal from adjacent nucleotides of the GFP fragments, including a 3’ HDV ribozyme on Nt-GFP and a 5’ HH ribozyme on Ct-GFP ( Figure 1A).
  • Nt or Ct RNAs could be subject to translation prior to ribozyme-mediated cleavage, or when expressed separately, potentially resulting in unwanted or truncated protein expression.
  • the efficacy of previously characterized translational control of protein degradation sequence on the stability of vectors encoding full-length GFP was tested. Addition of an HDV ribozyme on the 3’ end of GFP did not appear to alter GFP fluorescence ( Figure 3A and B).
  • RNAs encoding 4 copies of a mitochondrial targeting sequence (Nt-4xMTS) and an open reading frame encoding full-length GFP, lacking its ATG start codon (Ct-GFP), were generated ( Figure 4A). Co-expression of these two independent RNAs resulted in robust expression of mitochondrial-localized GFP, which overlapped with the red fluorescent mitochondrial marker MitoTracker Red CMXRos ( Figure 4B).
  • Ribozyme mediated trans-splicing can be used to rapidly combine two independent RNAs to express specific functional fusion proteins in cells.
  • Ribozyme mediated trans-splicing and expression of multiple different functional proteins at the same time may also be possible due to the three open reading frames in which proteins are translated.
  • functional proteins can be generated using trans-splicing of RNAs which are in compatible three different open reading frames.
  • an additional ribozyme pair in reading frame 2 F2 which encoded a myristoylation membrane targeting sequence (Nt-F2-Myr) and red fluorescent protein (Ct-F2-RFP) were designed (Figure 4C).
  • Nt and Ct vector pairs also included the hCL1- PEST protein degradation sequence and GCN4 translational inhibitory sequences to limit truncated protein expression from individual Nt and Ct vectors, respectively.
  • GFP fluorescence was highly specific to mitochondria and RFP fluorescence was highly specific to membranes ( Figure 4D), demonstrating the ability of this approach for trans-splicing of RNA to generate different functional proteins in cells.
  • Optimized ribozymes enhance protein expression in ribozyme-mediated trans-splicing [0362] Small sequence modifications can profoundly impact ribozyme catalytic activity by altering secondary structure, stability or binding to metal ion cofactors.
  • Ribozyme-mediated trans-splicing and expression of large gene sequences for delivery using viral therapeutic vectors Ribozyme-mediated trans-splicing could be harnessed for the delivery and expression of large protein coding mRNAs which exceed the packaging size limit for therapeutic viral gene therapy vectors, such as AAV ( Figure 6A). This could be useful to restore expression of large genes mutated in numerous human monogenic diseases, such as Dystrophin (Dys) in Duchenne Muscular Dystrophies (DMDs), CFTR in Cystic Fibrosis (CF), Factor VIII (F8) in Hemophilia A, etc.
  • Dystrophin Dystrophin
  • DMDs Duchenne Muscular Dystrophies
  • CFTR Cystic Fibrosis
  • F8 Factor VIII
  • Ribozyme-mediated trans splicing could also allow for the safe handling or reconstitution of toxic or antiviral proteins which may inhibit generation of lentiviral particles in mammalian packaging cells.
  • These include a number of cell suicide genes, such as the translational inhibitory diptheria toxin A (DTA) ( Figure 8A).
  • DTA translational inhibitory diptheria toxin A
  • Figure 8B Vectors encoding a split DTA sequence, upon trans-splicing and expression, inhibit the co-expression of a CS2GFP reporter construct, consistent with the translational inhibitory role of DTA in mammalian cells.
  • Enzymes to enhance or inhibit ribozyme-mediated trans-splicing [0368] A number of enzyme families have been suggested to ligate 5’-OH and either 3’-P or 2’3’ cyclic Phosphate (cP) ends, most notably RtcB which is found conserved in all three domains of life. Human codon optimized RtcB orthologs from Eukarya (H. sapiens), Bacteria (E. coli) and Archaea (P. horikoshii) species were cloned and co-expressed to measure their effects on the activity of the trans-splicing luciferase reporter. Interestingly, co-expression of RtcB from P.
  • RtcB is sufficient to catalyze ribozyme-mediated RNA trans-splicing in vitro [0370] Due to their nucleotide-specific cleavage, ribozymes have been utilized extensively in vitro to generate precise RNA ends. It was next sought to determine if ribozymes could be used for directional trans-splicing of independently synthesized RNAs in vitro. Using in vitro RNA transcription of the Nt- and Ct-Luciferase-ribozyme reporter constructs using T7 RNA polymerase, it was found that the addition of recombinant E. coli RtcB was both necessary and sufficient to catalyze the trans-splicing, detected using RT-PCR ( Figure 10 A and Figure 10B).
  • RNAs encoding domains of the spider protein Spidroin were designed (Figure 10C).
  • Spidroin is the major component of spider dragline silk, a material revered for its tensile properties, but which has been difficult to synthesize in heterologous systems due to the highly repetitive nature of the protein.
  • Spidroin naturally consists of multiple A and Q repeats, flanked by conserved N-terminal (N1L) and C-terminal (N3R) domains.
  • N1L N-terminal
  • N3R C-terminal
  • RNAs which contain termini which are both compatible for ligation by RtcB.
  • utilization of a trans-activated VS ribozyme has the potential to allow for the sequential and controlled assembly of RNAs sequences in vitro ( Figure 11B and Figure 11C).
  • the 3’ terminal RNA ribozyme is only suitable for ligation by RtcB upon the addition and trans- cleavage by VS-Rz.
  • stepwise addition of stitchR compatible RNAs, VS-Rz and RtcB ligase could allow for the controlled tandem assembly of RNA sequences, which may be useful for the assembly of repeat RNAs encoding biologically or industrially important proteins, such as synthetic spider silks, elastins, collagens, etc.
  • Ribozymes are autocatalytic RNAs which cleave in cis, to produce unique RNA termini that are trans-spliced and subsequently expressed in mammalian cells ( Figure 12A).
  • cis-cleaving ribozymes can be engineered to cleave in trans, such that target RNAs can be cleaved in a nucleotide specific manner, resulting in similar RNA termini ( Figure 12B) (Carbonell et al.2011; Webb and Luptak 2018).
  • trans-cleaving ribozymes could be utilized to catalyze scarless trans-splicing of RNA in cells or in vitro.
  • This approach could be useful for myriad applications, one major one being the deletion of disease-causing mutations in gene transcripts by targeting mutation flanking sequences in either exon or intron sequences ( Figure 12C and Figure 12D).
  • stitchR ribozyme-mediated cleavage of independent RNAs expressed in cells are efficiently assembled and capable of translation in mammalian cells.
  • stitchR has the ability to function as a novel method for the combinatorial assembly of functional RNA and proteins for both basic and therapeutic applications.
  • stitchR Due to the autocatalytic nature of ribozymes and the endogenous RNA repair pathways present in cells, stitchR only requires the expression of separate RNAs for trans- splicing and translation to occur in cells. In vitro, it is demonstrated that the RtcB ligase was sufficient for trans-splicing, and due to the ubiquitous and widespread expression of RtcB across all three kingdoms of life, stitchR has the potential to be a useful approach in many diverse organisms. [0374] The robust nature of this system relies on the efficient and precise nature of ribozyme-mediated RNA cleavage, which produces reliable and precise nucleotide specific ends essential for the restoration of protein coding open reading frames.
  • RNAs which are essentially indistinguishable from their natural counterparts.
  • StitchR serves as an indirect readout of ribozyme mediated cleavage, which interestingly was found herein to significantly influenced by changes in ribozyme sequence and structure.
  • Ribozymes have naturally evolved to function in cis to promote their self- cleavage, however, a number of ribozyme families (notably HDV and HH) have been engineered to cleave target RNAs in trans. It is noted herien that combining trans-cleaving ribozymes with stitchR may further allow for a powerful RNA cleavage and repair method in cells or in vitro.
  • RNA-specific ‘cut and paste’ approach for RNA which may be useful for generating RNA diversity or for removing certain deleterious mutations in disease causing RNAs.
  • Example 2 Inducible trans-splicing and expression of RNA using trans-activated ribozymes [0377] Most ribozymes are autocatalytic and only require metal ions as cofactors, readily found in biological environments, which aid in folding and chemical catalysis. The Varkud Satellite (VS) ribozyme can be utilized for scar-less trans-splicing, if the donor RNA ends in a G nucleotide.
  • VS Varkud Satellite
  • the VS ribozyme can be modified to allow for trans-activation of the ribozyme to induce catalysis (Guo and Collins 1995; Ouellet et al.2009).
  • the small VS stem loop (VS-S) is not alone sufficient to induce cis-cleavage, however, the addition of the remaining sequence, VS-Rz, promote efficient cleavage of the VS-S ( Figure 14A).
  • This trans-activation feature could allow for inducible ribozyme-mediated trans- cleavage, where addition of VS-Rz sequence is required for VS-S cleavage on an Nt donor RNA, which could then be suitable for trans-splicing with an Ct acceptor RNA containing a 5’-OH termini ( Figure 14B).
  • the VS-Rz sequence which contains typical 5’-P- and 3’-OH RNA termini, cannot participate in trans-splicing, and thus may function as a multi-turnover catalyst of the reaction.
  • RNA sequences may allow for the controlled addition of variable or non-variable RNA sequences to generate synthetic repeat RNAs ( Figure 14C).
  • One approach is to generate an RNA with a unique N-terminal domain, a unique C-terminal domain, and an internal variable or non-variable ‘repeat’ domain. This approach would require both the N-terminal and C-terminal RNAs to contain a single ribozyme on the 3’ and 5’ ends, respectively.
  • the internal repeat RNA would require ribozymes on both 5’ and 3’ ends, to allow it to function as both an acceptor and donor during trans-splicing.
  • the addition of ribozymes on both termini of an RNA, or an RNA with both 3’-P and 5’-OH leads to circularization by ligases, such as RtcB (Desai et al.2015), preventing participation in a growing linear chain.
  • RtcB Desai et al.2015
  • the utilization of an inducible trans-activated ribozyme could allow for step-wise ligation of 5’ and 3’ ends through addition and removal of both VS-Rz and RtcB ligase, leading to controlled RNA domain synthesis ( Figure 14C).
  • RNA sequences which could be subsequently translated to create synthetic repeat proteins, such as those composing hydrogels, synthetic spider silks, or collagens, etc, which can be difficult to generate and encode as DNA due to recombination.
  • These approaches may be useful for drug delivery, generation of biomaterials or industrial materials (Chambre et al.2020).
  • Example 3 Generation of stable synthetic intronic sequences using ribozymes [0379] Ribozyme-mediated trans-splicing between two independent RNAs can occur when one RNA contains a 3’ ribozyme and another contains 5’ ribozyme ( Figure 15A).
  • RNA circles are thought to highly stable, since they no longer contain 5’ or 3’ ends and thus cannot be degraded by RNA exonucleases.
  • Cargo sequences which could include any number of functional or useful RNAs (such as microRNA, CRISPR guide RNA, etc), or gene expression sequences, could be inserted as ‘cargo’ between the two ribozymes ( Figure 15C). This approach could be useful for the co-delivery and expression of useful RNA sequences during ribozyme-mediated trans-splicing and expression.
  • RNA circle can exist in both circular and re-cleaved linear forms ( Figure 15C).
  • VS-S in place of HDV
  • the system could be made inducible, requiring the delivery or expression of VS-Rz.
  • Use of ribozymes which require bilateral flanking sequences for cleavage, such as an HH ribozyme, cleavage can be designed such that RNA circularization of the cargo RNA is unidirectional ( Figure 15D).
  • Coli RtcB protein sequence 84 E. Coli RtcB human codon optimized nucleic acid sequence 85 Deinococcus radiodurans RtcB protein sequence 86 Deinococcus radiodurans RtcB human codon optimized nucleic acid sequence 87 Pyrococcus horikoshii RtcB protein sequence 88 Pyrococcus horikoshii RtcB human codon optimized nucleic acid sequence 89 Pyrococcus sp. ST04 RtcB protein sequence 90 Pyrococcus sp. ST04 RtcB human codon optimized nucleic acid sequence 91 Thermococcus sp. EP1 RtcB protein sequence 92 Thermococcus sp.
  • EP1 RtcB human codon optimized nucleic acid sequence 93 Human Archease protein sequence 94 Human Archease human codon optimized nucleic acid sequence 95 Pyrococcus horikoshii Archease protein sequence 96 Pyrococcus horikoshii Archease human codon optimized nucleic acid sequence 97 T4 Polynucleotide Kinase (T4 PNK) protein sequence 98 T4 PNK human codon optimized nucleic acid sequence 99 E. Coli thpR protein sequence 100 E.
  • Coli thpR human codon optimized nucleic acid sequence 101 Human PNKP protein sequence 1 02 Human PNKP human codon optimized nucleic acid sequence GFP with internal synthetic ribozyme intron with and without cargo
  • SEQ ID NO Sequence Name 103 NtGFP-HDV-HH-CtGFP 126 NtGFP-HDV-CARGO-HH-CtGFP 127 NtGFP-HDV 128 HH-CtGFP
  • Example 4 Identification of optimal Ribozyme Pairs for enhancing StitchR trans-splicing and expression in mammalian cells [0381] Experiments were conducted to identify optimal ribozyme pairs for StitchR mediated RNA trans-splicing in mammalian cells.
  • a StitchR-enabled luciferase-based reporter was utilized to screen the relative activity of a representative ribozyme sequence from each of the major ribozyme families, including Twister, Twister Sister, Hammerhead, HDV, Pistol, Varkud Satellite (VS), Hairpin, and Hovlinc (Hov) in mammalian cells ( Figure 17A and Figure 17B).
  • SD Splice Donor
  • SA Splice Acceptor
  • Twister ribozyme in both Luc Nt and Luc Ct vectors ( Figure 17B), which remarkably approximated the expression observed from a vector encoding luciferase in a single open reading frame (ORF).
  • ORF open reading frame
  • different subtypes of Twister ribozymes were assayed to determine their relative activity in promoting mRNA stitchr activity and expression in mammalian cells ( Figure 17C). It was observed that using Twister (Osa) in both Luc Nt and Luc Ct vectors produced the highest expression.
  • Ribozymes can either follow (placed 3’) to a SD sequence for an N-terminal mRNA half or precede (placed 5’) to a SA for a C-terminal mRNA half.
  • Ribozyme does not need to be scarless when reconstituting an Intron sequence (SD-SA pair), since the SD-SA pair will recreate the mRNA reading frame.
  • P1 Type Twister Ribozymes SEQ ID NO Sequence Name 131 Twister (Osa) 132 Twister (Dre) 133 Twister (Nvi) 1 34 Twister (Sbi) P3 Type Twister Ribozymes SEQ ID NO Sequence Name 1 35 Twister (Env1) P5 Type Twister Ribozymes SEQ ID NO Sequence Name 136 Twister (Spu) 1 37 Twister (Cpa) Ribozyme Family Sequences SEQ ID NO Sequence Name 138 Twister Sister 139 HDV (antigenomic) 140 Hammerhead (RzB) 141 Pistol 142 Varkud Satellite 143 Hatchet 144 Hairpin 145 Hovlinc
  • Example 5 Application of StitchR for dual AAV gene delivery and expression in mammalian cells in in vivo [0383]
  • Figure 18 demonstrates the application of StitchR for dual AAV gene delivery and expression in mammalian cells.
  • Figure 18A depicts the design of a stitchr enabled split GFP reporter cassette, under the control of the human CMV (hCMV) promoter and bovine growth hormone poly adenylation sequence (bGH pA), subcloned into a vector flanked by AAV2 ITR sequences. Constructs were used to generate AAV2/1 serotyped virus, which only when co-transduced into human cells (HEK293T), resulted in robust full-length GFP expression as detected by epifluorescence (Figure 18B) and western blot (Figure 18C).
  • hCMV human CMV
  • bGH pA bovine growth hormone poly adenylation sequence
  • Figure 19 demonstrates the application of StitchR for dual AAV gene delivery and expression in vivo.
  • StitchR enabled dual AAV GFP reporter virus (2E+12 vg/ml) was injected into postnatal day 10 (P10) mice and imaged 2 months post injection using
  • Figure 19A IVIS fluorescence based whole animal imaging or
  • Figure 19B epifluorescence.
  • Robust GFP fluorescence was detected in the whole body and readily observed in hindlimb musculature.
  • C Full-length GFP protein expression was observed only in mice injected with both GFPnt and GFPct virus, detected by western blot using an anti-GFP antibody.
  • AAV StitchR Split GFP Vector Sequences AAV hCMV GFPnt (SEQ ID NO:146) AAV ITR-hCMV-Kozak-3xHA-GFP Nt-SD-Rz-bGH pA-AAV ITR 3xHA-GFP Nt is bolded; Ribozyme is underlined, italicized and bolded 5’-CCTGCAGGCAGCTGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGT CGCCCGGCCTCAGTGAGCGAGCGAGCGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGC GGTACCAAGAATTCGCTAGCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCAT AGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCC CATTGACGTCAATAATA
  • Figure 20 provides a schematic of disorders and associated proteins/genes. Delivery and Expression of a Fully Functional miniDystrophin gene using StitchR- activated dual AAV gene therapy [0386] Experiments were conducted to examine the ability of StitchR to deliver dystrophin and functional dystrophin variants. Mutations in the large Dystrophin (Dys) protein can result in debilitating Duchenne Muscular Dystrophy (DMD) or less severe Becker Muscular Dystrophy (BMD).
  • DMD Duchenne Muscular Dystrophy
  • BMD Becker Muscular Dystrophy
  • Figure 21 depicts the characterized Human Dystrophin (Dys) protein domains, locations of important protein interactions, sequence deletions which are found in mild Becker Muscular Dystrophy (BMD), and engineered therapeutic dystrophins which can either fit into a single AAV (microDys) or a dual AAV vector pair (StitchR miniDys).
  • Figure 22 depicts a diagram of a microDys AAV expression vector controlled by the cardiac and skeletal muscle specific CK8e promoter and bGH pA.
  • Figure 23 depicts a diagram of the dual StitchR enabled N-terminal (StitchR Dys-Nt ) and C-terminal (StitchR Dys-Ct) AAV expression vectors controlled by the cardiac and skeletal muscle specific CK8e promoter and bGH pA.
  • Figure 24 depicts a diagram of a dual AAV vector pair N-terminal (Dual AK Dys-Nt ) and C-terminal (Dual AK Dys-Ct) expression vectors utilizing the recombinogenic AK sequence and controlled by the cardiac and skeletal muscle specific CK8e promoter and bGH pA.
  • StitchR was more efficient than AK recombinogenic sequence-based vectors, which did not result in detectable full-length miniDystrophin expression at this exposure level (compare lanes 4 and 7 of Figure 25). Remarkably, StitchR was nearly as efficient in generating full-length miniDystrophin protein as a vector encoding full-length miniDystrophin in a single open reading frame (compare lanes 7 and 9). In these experiments, twister (osa) was used as the ribozyme in both the Dys-N-terminal and Dys-C-terminal StitchR vectors. Experiments were also conducted to compare expression of miniDystrophin in vivo.
  • Figure 26 depicts full-length miniDystrophin protein expression in vivo transduced with StitchR-enabled AAV virus under the control of the core CK8e promoter, detected using Western blot.
  • StitchR technology was efficient at generating full-length miniDystrophin protein, which approximated the levels of endogenous mouse Dystrophin protein, in skeletal muscle (quadriceps) and heart. MiniDystrophin was not detected in the liver, demonstrating the muscle specificity of the CK8e promoter.
  • twister osa was used as the ribozyme in both the Dys-N-terminal and Dys-C-terminal StitchR vectors.
  • FIG. 28 is a diagram of a StitchR enabled dual AAV vector pair to express full-length human codon optimized Dysferlin (hcoDYSF) vectors under the control of the cardiac and skeletal muscle-specific CK8e promoter and bGH pA.
  • hcoDYSF human codon optimized Dysferlin
  • Figure 29 depicts Full-length human Dysferlin protein expression in human HEK293T cells, transfected with either dual StitchR vectors or a vector encoding dysferlin in a single ORF expression vector, under the control of the core EF1a promoter, detected using Western blot with an anti-Dysferlin antibody.
  • AAV EF1a Dysferlin Nt (SEQ ID NO:157)
  • AAV ITR-EF1a-Kozak-Dysferlin Nt-SD-Rz-Degron-bGH pA-AAV ITR Dysferlin-Nt is bolded; ribozyme is underlined, italicized and bolded
  • Figure 30 depicts Full-length human Sterocilin (STRC) protein expression in human HEK293T cells, transfected with dual StitchR vectors, under the control of the core EF1a promoter, detected using Western blot with either an N-terminal or C-terminal anti-STRC antibody.
  • STRC Sterocilin
  • Figure 32 demonstrates that circular RNA encoding GFP can be generated from a split GFP construct, which is mediated by ribozyme cleavage, as inclusion of mutant ribozymes failed produce a circular RNA.
  • IRES and ribozymes are critical for circulR cargo protein expression (Figure 33).
  • the circulR technology can be used to perform a lentiviral based screen for functional IRES sequences ( Figure 34).
  • Ribozymes can be encoded in RNA viruses, such as Lentivirus, if encoded in reverse. The use of 161 nt library ⁇ 750,000 unique clones. In initial screening ⁇ 50 clones were recovered.
  • Example 8 StitchR ‘ribotron’ and CirculR HSV TK selection vector
  • ribozyme-mediated cleavage produces RNA termini which are subject to RNA trans-ligation in cells (StitchR activity).
  • a ribotron-regulated cell death gene (Herpes Simplex Virus Thymidine Kinase (HSV TK)) cassette encoded within a lentiviral vector (in reverse orientation) was generated ( Figure 35A), which when integrated into target cells, allows for expression of HSV-TK and sensitization to ganciclovir (GCN) ( Figure 35B).
  • HSV TK Herpes Simplex Virus Thymidine Kinase
  • This reporter may be useful for identifying cellular enzymes which enable stitchR activity, for example, using a CRISPR-Cas genome-wide mutagenesis library. In this approach, CRISPR-mediated knockdown or knockout of stitchR-related enzymes would prevent ribotron removal and subsequent activation of HSV TK-mediated cell death.
  • Reorientation of the ribozyme sequences to generate a split circulR expression vector could also be used to enable a stitchR-dependent HSV-TK cell death gene cassette.
  • Example 9 StitchR ‘ribotron’ and CirculR HSV TK selection vector
  • a ‘Ribotron’ HSV TK expression cassette was cloned into reverse orientation in Lentiviral vector Sequences: (SEQ ID NO:162) EF1a-kozak-NT HSV TK-Rz-degron-Rz-Ct HSV TK-RBGpA-minCMV-kozak-BLAST-Histone3’end TAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAG AAGTTGGGGGGAGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGT CGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTT TTTCGCAACGGGTTTGCCGCCAGAACACAGGGGATCCCGCCACCATGGCCTC
  • AK AK
  • AK AK
  • HH Hammerhead
  • HDV Hepatitis Delta Virus
  • VS Varkud Satellite
  • Twister Twister-sister
  • Hairpin Hatchet and Pistol.
  • HH, HDV and Twister family members that, due to their small size and robust cleavage characteristics, have been utilized both in vitro and in vivo to generate RNAs with precise termini devoid of ribozyme sequences (Ferre- D'Amare et al., 1996, Nucleic Acids Res 24, 977-978; Avis et al., 2012, Methods Mol Biol 941, 83-98; Zhang et al., 2017, Bio Protoc 7; Roth et al., 2014, Nat Chem Biol 10, 56-60).
  • RNAs are synthesized and processed through 5'-phosphate (5'-P) and 3'-hydroxyl (3'-OH) termini, including mRNAs, long noncoding RNAs, and most small noncoding RNAs (Padgett et al., 1986, Annu Rev Biochem 55, 1119- 1150).
  • 5'-P 5'-phosphate
  • 3'-OH 3'-hydroxyl
  • RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase in metazoa Li et al., 2014, Mol Cell 55, 758-770; Desai et al., 2012, Biochemistry 51, 1333-1335; Kosmaczewski et al., 2014, EMBO Rep 15, 1278-1285.
  • RtcB and several other enzyme families also function to repair host cellular RNAs that have been damaged by stress or cleavage by exogenous ribotoxins (Manwar et al., 2020, Sci China Life Sci 63, 251-258; Shigematsu et al., 2018, Front Genet 9, 562).
  • RNA cleavage results in the same 5'-OH and 2',3'-cP RNA termini and has been used to circularize RNA in cells through cis- or trans-ligation when aided by stem-forming sequences (Fedor et al., 2009, Annu Rev Biophys 38, 271-299; Litke et al., 2021, Methods 196, 104-112; Litke et al., 2019, Nat Biotechnol 37, 667-675).
  • stem-forming sequences Fedor et al., 2009, Annu Rev Biophys 38, 271-299; Litke et al., 2021, Methods 196, 104-112; Litke et al., 2019, Nat Biotechnol 37, 667-675.
  • linear mRNAs are subject to trans-ligation by endogenous unconventional RNA repair pathways in eukaryotic cells is unknown.
  • Ribozymes were designed to catalyze their complete removal from adjacent nucleotides of the GFP fragments, including initially a 3'- HDV ribozyme on the Nt-GFP-encoding mRNA and a 5'-HH ribozyme on the Ct-GFP mRNA.
  • StitchR 3.0 The intron-enhanced approach was named StitchR 3.0.
  • the inclusion of a split intron at the StitchR seam also allowed for the use of ribozymes that are not completely scarless, since any remaining ribozyme sequences will be removed through splicing of the stitched intron.
  • StitchR 3.0 split- luciferase reporter the relative efficiency of a member of each ribozyme family to catalyze StitchR activity in either the Nt or Ct position was compared ( Figure 37C, and Figure 38C). All ribozymes tested displayed some level of StitchR activity over an empty vector control.
  • T4 polynucleotide kinase which acts as a 5'-hydroxyl kinase, a 3'-phosphatase and a 2',3'-cyclic phosphodiesterase, significantly abrogated StitchR activity in mammalian cells, further suggesting that the unique RNA termini are necessary for StitchR activity (Figure 39D). Since RtcB functions in the pathways required for unconventional intron splicing of pre-tRNA and XBP1 mRNA through cis-ligation, it was tested whether chimeric RNAs generated between StitchR-expressed RNAs and either XBP1 mRNA or tRNA could be detect.
  • Pre-tRNA-Tyr-GTA-2-1 was selected because all tyrosine pre-tRNAs contain an intron and tRNA-Tyr-GTA-2-1 is abundant in HEK293T cells (Torres et al., 2019, Proc Natl Acad Sci U S A 116, 8451-8456). RT-PCR with primers specific to each possible ligation product were unable to detect chimeric ligation products between StitchR and either tRNA or XBP1 mRNA ( Figure 39E to Figure 39G). [0405] Given the robust activity of the optimized StitchR reporters, it was investigated whether StitchR could efficiently reconstitute the expression of an endogenous cellular protein.
  • a StitchR 3.0 Nt and Ct vector pair was designed to express ⁇ -Smooth Muscle Actin ( ⁇ SMA), encoded by the ACTA2 gene, which is normally expressed in HeLa cells but disrupted in the commercially available ACTA2-knockout HeLa cell line (ACTA2-KO) ( Figure 40A).
  • ⁇ SMA ⁇ -Smooth Muscle Actin
  • ACTA2-KO commercially available ACTA2-knockout HeLa cell line
  • StitchR can also be used to generate novel chimeric proteins by trans-ligating two different protein functional domains.
  • StitchR could be multiplexed to generate two different chimeric proteins at the same time.
  • a StitchR Nt vector was developed, encoding a cell membrane targeting myristylation sequence (Nt-F2-Myr) and a Ct vector, encoding full-length RFP minus a start codon (Ct-F2-flRFP), in a different reading frame than the previous 4xMTS-flGFP vectors.
  • Nt-F2-Myr cell membrane targeting myristylation sequence
  • Ct-F2-flRFP start codon
  • AAV1-serotyped virus particles were generated for both Nt- and Ct-GFP vectors and transduced HEK293T cells in culture, which resulted in robust GFP fluorescence for cells transduced with both Nt and Ct virus (Figure 41B).
  • Deep sequencing of total RNA from StitchR GFP transduced HEK293T cells showed sequencing reads that mapped across the entire trans-ligated GFP mRNA, including across the seam (Figure 41C).
  • StitchR trans-ligation efficiency was estimated by quantifying sequencing depth for reads which crossed the seam, compared to the average depth of sequencing reads for the N-terminus and C-terminus of the GFP mRNA.
  • a StitchR 4.0 Nt and Ct vector pair was generated to express full-length human Dysferlin (2,080 amino acid, 6,240 ORF isoform).
  • co-transfection of the Dysferlin StitchR 4.0 pair resulted in 79.9% of the expression of full-length Dysferlin expressed from a single ORF and for which expression was abolished by mutation of a ribozyme catalytic residue ( Figure 43A to Figure 43C).
  • mice were injected at postnatal day 8 (P8) with 1E+12 vg of each StitchR Nt- and Ct virus (2E+12 total), serotyped with the muscle-trophic AAV9 capsid and under the control of the muscle-specific CK8e promoter (Coley et al., 2016, Hum Mol Genet 25, 130-145; Goncalves et al., 2011, Mol Ther 19, 1331-1341; Zincarelli et al., 2008, Mol Ther 16, 1073-1080).
  • Dystrophin the gene mutated in Duchenne Muscular Dystrophy (DMD) encodes a protein of 3,685 amino acids from an ORF that exceeds the packaging limit of AAV genomes by more than 2-fold (Figure 44A)(Koenig et al., 1988, Cell 53, 219-228; Gao et al., 2015, Compr Physiol 5, 1223-1239).
  • Figure 44A Large naturally occurring deletions in the Dystrophin gene, which can result in loss of almost half of the Dystrophin protein, can manifest in a much milder disease called Becker Muscular Dystrophy (BMD) ( Figure 44A)(England et al., 1990, Nature 343, 180-182).
  • Dystrophin-KO mice which display an age-dependent disease phenotype characterized by degenerating and regenerating myofibers, were injected at postnatal day 8 (P8) intraperitoneally with 3E+12 vg of each StitchR Nt- and Ct virus (6E+12 total), serotyped with the muscle-trophic AAV9 capsid and under the control of the muscle-specific CK8e promoter (31-33).
  • a firefly luciferase-based prime editing reporter was generated similar in design to PEAR-GFP, which was named LUPER (Luciferase Prime Editing Reporter) ( Figure 46D).
  • StitchR-PE resulted in approximately 82% the activity of a single ORF-encoded PEmax ( Figure 46E).
  • StitchR-PE was also twice as effective as a previously reported split intein vector pair for expressing PEmax ( Figure 46E), a protein trans-splicing technology(Davis et al., 2024, Nat Biotechnol 42, 253- 264).
  • Stitched mRNAs appear to behave essentially indistinguishably from their natural full- length counterparts, in that they can be spliced using conventional introns and translated into full-length proteins.
  • This technology was developed and optimized for use in mammalian cells by identifying optimal ribozyme sequences (StitchR 2.0) and then adding a split intron to further enhance expression and allow for non-scarless ribozymes to be utilized (StitchR 3.0).
  • StitchR 4.0 that utilized a Type P1 Twister ribozyme in both Nt and Ct vectors, was ⁇ 900 fold enhanced over StitchR 1.0 and approached the expression levels observed for single ORF vectors.
  • Dual AAV approaches based on other DNA, RNA and protein trans-splicing technologies have been in development for over the past 20 years, highlighting the significant clinical need and challenges that remain for these efforts.
  • Dual AAV approaches can be complicated by efficiency and/or the production of non-full-length protein products, or in the case of inteins, be dependent upon on their production (Riedmayr et al., 2023, Nat Commun 14, 6578; Lai et al., 2005, Nat Biotechnol 23, 1435-1439; Sun et al., 2000, Nat Med 6, 599-602).
  • the data show that StitchR-mediated dual vector approaches are capable of restoring large therapeutic proteins to endogenous levels and correcting disease pathology in vivo.
  • StitchR ribozymes function simply as a means to an ‘end:’ as RNA “molecular scissors” that generate the unique 5'-OH and 2',3'-cP termini that are required to activate mRNA trans-ligation in cells. All ribozymes tested here were capable of StitchR activity and catalyzed the trans-ligation of diverse RNA sequences, suggesting that StitchR activity is not a sequence-specific phenomenon.
  • StitchR is a highly programmable approach for trans-ligation and translation of chimeric or full-length mRNAs that can be utilized for myriad research and therapeutic applications.
  • mice used in these studies were: C57BL/6J (strain#:000664), A/J (strain#:000646), D2-mdx (strain#:013141), and Charles River: C57BL/6 (strain#:027). Mice were housed in a 12:12 hour light:dark cycle in a temperature-controlled URMC animal housing room, with ad lib access to water and food.
  • Mammalian cell lines consisting of human HEK293T (Takara, 632180), human HeLa (Abcam, ACTA2-KO, ab264014; wild-type ab271142), and primate COS7, were maintained in DMEM (Gibco, 11965-092) supplemented with 10% Fetal Bovine Serum (FBS, Gemini Bio) and penicillin/streptomycin, at 37°C in an atmosphere of 5% CO2.
  • HeLa culture medium was additionally supplemented with 1 mM sodium pyruvate.
  • HEK293T, HeLa and COS7 cells were seeded and transiently transfected using Fugene6 (Promega), according to manufacturer’s protocol. Plasmids were transfected in equimolar concentrations. For StitchR dual vector comparisons to single ORF vectors, transfections were balanced with empty vector to maintain the same concentration of DNA per transfection. Eighteen to forty-eight hours after transfection, cells were processed for one of the following: RNA extraction, protein lysate preparation, live cell imaging, or fixation followed by confocal microscopy.
  • StitchR Nt and Ct vectors used in cell-based assays were constructed in the expression plasmid CS2, under the control of the simian CMV IE94 promoter. Sequence inserts were cloned using synthetic DNA (IDT, gBlock or custom gene synthesis) and either restriction digest followed by ligation (T4 DNA Ligase, NEB) or Gibson assembly (HiFi DNA Assembly, NEB).
  • Nt- Luc and Ct-Luc vectors were first generated lacking ribozyme or intron sequences (Nt-Luc- Destination and Ct-Luc-Destination). Upon digestion with BsmBI, inserts were cloned using either annealed complementary oligonucleotides, or restriction-digested synthetic double stranded DNAs (gBlock, IDT). [0419] A custom AAV transfer plasmid was generated by subcloning AAV2 ITRs into a synthetic backbone containing only a bacterial origin of replication and ampicillin resistance gene (gBlock, IDT).
  • AAV virus production and delivery [0420] AAV virus was generated using Boston Children’s Hospital Viral Core service. Briefly, AAV particles were produced with either AAV1 or AAV9 serotype capsids, as described in the text, and purified by iodixonal gradient ultracentrifugation.
  • AAVs were diluted in sterile saline solution (Medline, RDI30296) to 100 ⁇ l volume. Diluted AAVs were injected intraperitoneally into postnatal day-8 (P8) or -10 (P10) mouse pups using a 31G insulin syringe (BD, 328438).
  • StitchR Nt-GFP and Ct-GFP AAVs 1E+12 vector genomes of each virus were injected together into each animal.
  • human ⁇ H2-R15 Dystrophin 3E+12 viral particles of each Nt and Ct AAVs were injected per mouse.
  • human Dysferlin Nt and Ct AAVs, 1E+12vector genomes of each virus were injected together into each mouse.
  • DNase I TURBO DNase, Invitrogen
  • Superscript III Invitrogen
  • RT-PCR of tissue-derived RNA dissected tissues were first snap-frozen in liquid nitrogen, and subsequently pulverized using a Bessman Tissue Pulverizer. Samples were processed immediately or stored at -80°C. Pulverized samples were further processed in a beadmill with ceramic beads (VWR) in Trizol, and then purified according to manufacturer protocols.
  • Luciferase Assays [0422] Whole-cell lysates of transiently transfected cells were prepared in 1x Passive Lysis Buffer (Promega). Luciferase activity was measured using a FluoStar OPTIMA microplate reader (BMG Labtech) and Luciferase Assay Substrate (Promega, E1501), and normalized to beta-galactosidase activity from co-transfection of a lacZ expressing vector, which was determined using the FluoReporter LacZ/Galactosidase Quantitation Kit (Invitrogen).
  • BaseScope RNA in situ Hybridization was performed using custom probes and transiently- transfected COS7 cells plated on chamber slides, and hybridization was detected using Fast Red, following manufacturer protocols (Advanced Cell Diagnostics, 323900-USM). For fluorescent confocal imaging, mounting medium containing DAPI (Invitrogen, P36941) was used in place of counterstaining with Hematoxylin.
  • DAPI Invitrogen, P36941
  • skeletal muscle tissues were first flash-frozen in liquid nitrogen and then pulverized using a Bessmen Tissue Pulverizer. Protein lysates were prepared in skeletal muscle lysis buffer (10% SDS, 62.5 mM Tris (pH 6.8), 1 mM EDTA, and protease inhibitor) and a bead homogenizer for 30 seconds. Lysates were incubated at room temperature (RT) for 10 minutes, then centrifuged at 10,000 rpm for 5 minutes at RT. Supernatants were collected and protein concentration was measured using a BCA assay kit (Thermo Scientific).
  • Protein lysates were prepared in 1x Laemmli Sample Buffer (800 ⁇ l of 4x Laemmli Buffer + 100 ⁇ l ⁇ - mercaptoethanol + 100 ⁇ l DTT).
  • 50 ⁇ g of total protein for each sample was loaded on to a Tris-acetate 3-8% gradient gel (Bio-Rad), and subjected to 100 volts for 20 minutes and, subsequently 200 V for 35 minutes.
  • proteins were transferred to polyvinylidene difluoride (PVDF) membranes for one hour and 40 minutes at 80V and 4oC.
  • PVDF polyvinylidene difluoride
  • Membranes were blocked with 5% skimmed milk in TBST for one hour at RT, then incubated on rocking shaker with primary antibodies dissolved in either 1% skimmed milk or 4% BSA overnight at 4°C. Membranes were washed 3 times in 1x TBST for 10 minutes each, and then incubated with secondary antibodies at room temperature for one hour. Membranes were developed using super signal ECL reagent (Bio-Rad Laboratories). Serum creatine kinase assay [0427] For measurement of serum creatine kinase, blood was drawn retro-orbitally from anesthetized mice and transferred to 1.5 mL Eppendorf tubes for each treatment group.
  • CK Creatine kinase
  • RNA trans-ligation assay with RtcB ligase [0429] For the CS2 plasmids encoding StitchR Nt-Luc- and Ct-Luc, the 5' SP6 RNA polymerase promoter was replaced with a T7 RNA polymerase promoter (CS2 T7, unpublished). For in vitro-transcription, plasmid templates were linearized using SnabI restriction digest and gel-purified. Approximately 500 ng of linear plasmid DNA was used in each transcription reaction (Maxiscript, Ambion). Reactions were performed for 1 hour at 37oC, and then treated with DNase I (TURBO DNase, Invitrogen).
  • mice were euthanized, and their skin pulled back, then imaged using the manufacturer GFP protein- detection default settings and an exposure time of 10 seconds. The fluorescence intensity was quantified using Living Image® 4.5.4 Software.
  • Immunocytochemistry [0431] Transiently transfected HeLa cells on chamber slides were fixed in 4% formaldehyde in DPBS for 20 minutes, blocked in 3% Bovine Serum Albumin (BSA), and incubated with primary anti-alpha smooth muscle Actin antibody (Abcam, EPR5368) at 1:250 in 1% BSA for 1 hour at room temperature.
  • BSA Bovine Serum Albumin
  • RNA Sequencing [0433] Total RNA concentrations were determined using a NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with an Agilent Bioanalyzer (Agilent, Santa Clara, CA).
  • TruSeq Stranded Total Sample Preparation Kit (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer’s protocols. Briefly, ribosomal-depletion was performed on 200 nanograms (ng) total RNA with biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads, followed by RNA fragmentation. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis using dUTP incorporation for strand marking. End repair and 3 ⁇ adenylation was then performed on double stranded cDNA.
  • Illumina adaptors were ligated to both ends of the cDNA and amplified with PCR primers specific to the adaptor sequences to generate cDNA amplicons of approximately 200-500bp in size.
  • the amplified libraries were sequenced using a NovaSeq 6000 (Illumina, San Diego, CA). Paired-end reads of 50 basepairs (bp) were generated for each sample. Demultiplexing, QC, Alignment, and Analysis of RNA Sequencing Data [0434]
  • Raw reads generated from the Illumina basecalls were demultiplexed using bcl2fastq version 2.19.1.
  • Samtools (1.9) view was used to filter for mapped reads (parameters: -F 4) and bam files (Danecek et al., 2021, Gigascience 10).
  • Deeptools (3.5.1) bamCoverage was used to generate bigWig files normalized using counts per million (parameters: -bs 1 --normalizeUsing CPM) (Ramirez et al., 2016, Nucleic Acids Res 44, W160- 165).
  • IGV was used to visualize the aligned bigWig files (Robinson et al., 2011, Nat Biotechnol 29, 24-26).
  • Bedtools (2.30.0) intersect was used to identify and filter reads that crossed the seam using a mapped bam file and bed file containing the seam position (-wa -abam BAM -b BED-F 1.0) (Quinlan et al., 2010, Bioinformatics 26, 841-842).
  • Samtools depth was used to quantify the depth across the GFP sequence (paremeters: -b ⁇ BED ⁇ ⁇ BAM ⁇ ) (Danecek et al., 2021, Gigascience 10). The depth was quantified for both the unfiltered bam file containing the full GFP sequence (Nt-GFP and Ct-GFP) and filtered bam file containing only reads at the seam.
  • Sequences BaseScope probe design SEQ ID Sequence Name NO 2 20 BA-Luciferase-1zz-st-C1 RT-PCR Primers SEQ ID Sequence Name NO 221 trans-ligated Luciferase mRNA seam (L1- Forward primer) 222 trans-ligated Luciferase mRNA seam (L2- Reverse primer) 223 trans-ligated eGFP mRNA seam (G1-Forward primer) 224 trans-ligated eGFP mRNA seam (G2-Reverse primer) 225 XBP1 (X1-Forward primer) 226 XBP1 (X2-Reverse primer) 227 Tyr-GTA-2-1 (T1-Forward primer) 2 28 Tyr-GTA-2-1 (T2-Reverse primer) StitchR 1.0 Ribozyme Sequences SEQ ID Sequence Name NO 229
  • PE has evolved through several iterations to optimize editing efficiency, (PE1-6), which include 1) optimization of Prime Editor and pegRNA sequences, 2) use of a nicking guide RNA on the opposing strand to increase editing efficiency, and 3) minimization of RT domains through deletion ( ⁇ RH domain of MMLV) or use of compact natural enzymes (such as Marathon RT).
  • Prime Editing CRISPR systems Primary editors, pegRNAs and nicking guide RNAs
  • viral vectors which greatly exceed the packaging limits of small non-integrating viral vectors, notably AAV.
  • PEmax a robust PE editor utilizing SpyCas9, the preferred Cas9 for PE, itself is 6,400 bp and exceeds the packaging capacity of a single AAV vector ( ⁇ 4,700 bp) ( Figure 47A).
  • Inclusion of necessary transcriptional control elements, such as promoters and polyadenylation elements, further increases the space required to >7.5kb, with an additional 700bp still required for peg- and nicking- guideRNAs expression cassettes.
  • Figure 47A depicts an expression cassette for PEmax, and optimized SpyCas9 nickase – MMLV prime editor, which itself exceeds the packaging capacity of AAV, even in the absence of promoter or polyadenylation sequences required for gene expression.
  • Figure 47B and Figure 47C depicts dual StitchR-enabled Prime editor comprising of an N-terminal and C- terminal vector which splits the PEmax editor within the C-terminus of the nickase SpyCas9- MMLV fusion protein. This strategy results in either vector from encoding functional elements, as well as leaves enough room in the N-terminal vector with additional room for larger promoter sequences, or fusions to the N-terminus of nCas9.
  • FIG. 47D depicts the results of experiments where full-length PEmax encoded within a single ORF or the stitchR-enabled dual vector PEmax were compared using the PEAR-GFP prime editing reporter, which uses Prime editing to repair a 5’ splice donor site within an intron containing GFP reporter.
  • the PEAR-GFP 2in1 reporter plasmid includes expression cassettes for both PEAR and the PEAR-specific pegRNA.
  • StitchR PEmax dual vectors activated the LUPER reporter at 74.4% to that observed for full-length PEmax.
  • the StitchR PEmax vector system can encode all the necessary components for performing Prime Editing in vivo, including expression cassettes for the PEmax editor , pegRNA, and a nicking guide RNA, well within the packaging capacity of AAV. Additional space remains for inclusion of larger regulatory sequences, such as tissue-specific promoters which can be used for tissue-specific PE.
  • the current StitchR-PEmax system can be further compacted through 1) reduction of the MMLV size (for example, ⁇ RH domain of MMLV), or 2) inclusion of a compact WPRE and polyA sequence (such as W3SL), or 3) use of a more robust nuclear localization sequence (such as TY1 NLS).
  • This additional space could be devoted to inclusion of larger promoter sequences, fluorescent reporters (such as GFP), selection genes (such as puromycin), or additional peg or nick sgRNA sequences.
  • a chemically stabilized degron sequence E.
  • ecDHFR coli dihydrofolate reductase
  • myostatin a negative regulator of muscle growth
  • MSTN myostatin
  • a negative regulator of muscle growth has been viewed as a potential target for treating muscle atrophy or sarcopenia, or age-related loss of muscle mass and strength
  • MSTN myostatin
  • Natural mutations in myostatin have been identified in cattle, sheep, dogs and human patients which all present with extraordinarily large muscles (Schuelke et al., 2004, The New England journal of medicine 350, 2682-2688; McPherron et al., 1997, Proceedings of the National Academy of Sciences of the United States of America 94, 12457-12461).
  • dogs with natural disruption of a single copy of MSTN are significantly faster and have larger muscles than dogs with a WT copy (Mosher et al., 2007, PLoS genetics 3, e79).
  • Genetic disruption of MSTN in developing or mature mice results in enhanced muscle growth (McPherron et al., 1997, Nature 387, 83-90; Welle et al., 2009, Physiological genomics 38, 342-350).
  • Prime editing could be used to full disrupt MSTN expression through disruption of a MSTN splice donor in a conserved region of the myostatin gene locus (Figure 49B- Figure 49D), or introduction of a premature stop codon (Figure 49E and Figure 49F), with either approach predicted to prevent expression of the mature domain of the myostatin protein located in the terminal exon.
  • Disruption of only a single allele (heterozygous) would be predicted to be beneficial, which may occur from incomplete PE editing within a single cell, mosaic editing within a tissue, or controlled by targeting allele-specific SNPs in a patient’s genome.
  • Figure 49 depicts the prime Editing strategy for disruption of the myostatin (MSTN) gene to treat muscle atrophy or sarcopenia.
  • the myostatin (MSTN) gene is a member of the TGFb superfamily that is expressed in muscle, and functions as an inhibitor of muscle growth. Disruption of Myostatin in multiple species results in a double-muscled phenotype, characterized by significantly large muscles and enhanced performance.
  • MSTN is encoded by three exons, with the mature functional domain encoded within the terminal exon.
  • Figure 49G depicts high conservation at the Splice Donor region of Exon2-Intron2 allows for identical, or highly similar pegRNA and nicking sgRNAs to be used to disrupt MSTN across multiple species, which could have therapeutic or useful applications across numerous species.
  • PE editors can be used to insert small sequences ( ⁇ 50 nucleotides), which could be beneficial for prevention of expression of toxic RNA sequences, such as the microsatellite repeat which can cause Myotonic Dystrophy Type 1 (DM1).
  • DM1 Myotonic Dystrophy Type 1
  • Microsatellite repeat of a CTG expansion in the DMPK UTR results in expression of CUG RNA repeats which form toxic RNA foci and widespread splicing defects resulting from the sequestration of the splicing factors, such as MBNL1.
  • Prime Editing insertion of a synthetic polyA sequence upstream of the microsatellite expansion would serve to both prevent CUG RNA expression, and preserve the normal expression of the DMPK protein.
  • PE-mediated insertion of a small polyadenylation site upstream of the microsatellite CTG repeat expansion would serve to both 1) prematurely polyadenylate and terminate transcription upstream of the repeat sequence, and 2) allow for DMPK protein expression (Figure 50C).
  • RNA which is sufficient for circularization in cells (or in vitro when incubated with RTCB ligase) and translation. When encoded downstream of a promoter, will produce circular RNA for expression of PEmax.
  • SEQ ID NO:309 T7-Rz-CVB3ires-NLS-SpyCas9(H840A)-linker-NLS-MMLV-NLS-NLS-Rz TAATACGACTCACTATAGGGCTGTCTTAACGACACTGATGAGTCGCTGGGATGCGACGAAACGCCTTCGGGC GTCTGTCGAGACAGCCCGGGTTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGT ATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGC GTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGGG

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Abstract

La présente invention concerne des compositions, des systèmes et des procédés d'utilisation de cis-clivage et de trans-épissage médiés par ribozyme de molécules d'ARN pour exprimer des composants et des systèmes d'édition primaire.
PCT/US2024/050689 2023-10-10 2024-10-10 Administration et expression de systèmes crispr d'édition primaire Pending WO2025080780A1 (fr)

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