WO2026035687A1 - Compositions et procédés d'expression de scn5a pleine longueur médiée par stitchr in vivo - Google Patents

Compositions et procédés d'expression de scn5a pleine longueur médiée par stitchr in vivo

Info

Publication number
WO2026035687A1
WO2026035687A1 PCT/US2025/040653 US2025040653W WO2026035687A1 WO 2026035687 A1 WO2026035687 A1 WO 2026035687A1 US 2025040653 W US2025040653 W US 2025040653W WO 2026035687 A1 WO2026035687 A1 WO 2026035687A1
Authority
WO
WIPO (PCT)
Prior art keywords
scn5a
nucleic acid
ribozyme
rna molecule
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/040653
Other languages
English (en)
Inventor
Douglas Matthew ANDERSON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Rochester
Original Assignee
University of Rochester
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Rochester filed Critical University of Rochester
Publication of WO2026035687A1 publication Critical patent/WO2026035687A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A01K2217/077—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out heterozygous knock out animals displaying phenotype
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/52—Vector systems having a special element relevant for transcription encoding ribozyme for self-inactivation

Definitions

  • the SCN5A protein is comprised of four domains in a single protein.
  • the large size of the SCN5A protein (2,016 amino acids (aa)) means the protein-coding open reading frame (>6.0 kb) greatly exceeds the packaging size limit of current non-integrating viral vectors, such as AAV ( ⁇ 4.7 kb), preventing its use in current human gene therapies.
  • CRISPR-Cas gene editing has been a recent focus for the development of therapeutic approaches to correct large gene diseases.
  • CRISPR-Cas utilizes a single guide- RNA (sgRNA) to direct Cas9 endonuclease activity to specific target sequence, where it induces double-strand DNA cleavage and triggers cellular repair pathways that can be used to generate frame-shift mutations or to insert large donor sequences through Homology Directed Repair (HDR).
  • sgRNA single guide- RNA
  • CRISPR-Cas gene editing of SCN5A mutations remains a significant challenge due to 1) the many distinct human patient-specific mutations within the SCN5A gene locus that can give rise to disease and thus require patient specific therapeutic strategies, and 2) targeted insertion of DNA sequences by CRISPR-mediated HDR is inefficient, requires donor templates containing large regions of flanking homology, and does not occur in non-dividing cells, which comprise many adult tissues, notably cardiac and skeletal muscle.
  • CRISPR-Cas Editing variations such as Based Editors or Prime Editors, can be efficient for correcting disease mutations, but are notably too large to be packaged into AAV for delivery in vivo.
  • SCN5A Sodium Voltage-Gated Channel Alpha Subunit 5
  • the invention relates to a system for generating an RNA molecule encoding voltage-gated sodium channel SCN5A comprising: a nucleic acid molecule encoding a first RNA molecule comprising a coding region encoding a first portion of SCN5A and a 3’ribozyme; and a nucleic acid molecule encoding a second RNA molecule comprising a coding region encoding a second portion of SCN5A and a 5’ribozyme.
  • the first nucleic acid molecule comprises the N- terminal coding sequence of SEQ ID NO:1, or a fragment or variant thereof
  • the second nucleic acid molecule comprises the C-terminal coding sequence of SEQ ID NO:2, or a fragment or variant thereof.
  • the first 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 encoding SCN5A.
  • the 3’ ribozyme comprises SEQ ID NO:3 or SEQ ID NO:9-61.
  • the 5’ ribozyme comprises SEQ ID NO:4 or SEQ ID NO:9-61.
  • each of the nucleic acid molecule encoding the first RNA molecule comprising the coding region encoding the first portion of SCN5A and the nucleic acid molecule encoding the second RNA molecule comprising the coding region encoding the second portion of SCN5A comprise a chicken cardiac troonin-T promoter core (cTnT) promoter sequence.
  • the cTnT promoter sequence comprises SEQ ID NO:5.
  • the first nucleic acid molecule comprises SEQ ID NO:6 and the second nucleic acid molecule comprises SEQ ID NO:7.
  • the invention relates to a method for generating an RNA molecule encoding voltage-gated sodium channel SCN5A 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 SCN5A 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 SCN5A and a 5’ribozyme.
  • the first nucleic acid molecule comprises SEQ ID NO:1
  • the second nucleic acid molecule comprises SEQ ID NO:2.
  • the 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 comprises SEQ ID NO:3 or SEQ ID NO:9-61.
  • the 5’ ribozyme comprises SEQ ID NO:4 or SEQ ID NO:9-61.
  • each of the nucleic acid molecule encoding the first RNA molecule comprising the coding region encoding the first portion of SCN5A and the nucleic acid molecule encoding the second RNA molecule comprising the coding region encoding the second portion of SCN5A comprise a chicken cardiac troonin-T promoter core (cTnT) promoter sequence.
  • the cTnT promoter sequence comprises SEQ ID NO:5.
  • the first nucleic acid molecule comprises SEQ ID NO:6 and the second nucleic acid molecule comprises SEQ ID NO:7.
  • the disease or disorder is associated with a decreased level of activity of voltage-gated sodium channel SCN5A.
  • the disease or disorder is Brugada Syndrome, Long QT Syndrome, atrial fibrillation, sick sinus syndrome or cardiomyopathy. BRIEF DESCRIPTION OF THE DRAWINGS [0029] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
  • Figure 1 depicts a diagram demonstrating that organized rhythmic contraction of the heart is precisely coordinated by ion-specific transmembrane channels and pumps in the membrane of heart cells, and that loss-of-function mutations in SCN5A disrupt this process and result in cardiac arrhythmia.
  • Figure 2 provides a diagram depicting the functional domains of SCN5A, the large cardiac sodium channel protein required for normal cardiomyocyte depolarization. Mutations in SCN5A cause multiple lethal cardiac arrhythmias, including hereditary Long QT Syndrome (LQTS) Type III and Brugada Syndrome (BrS).
  • LQTS hereditary Long QT Syndrome
  • BrS Brugada Syndrome
  • FIG 4 provides a diagram depicting the dual AAV StitchR vectors for SCN5A, the large cardiac sodium channel protein required for normal cardiomyocyte depolarization. This approach allows for AAV delivery and expression of SCN5A under the control of the cardiac-specific troponin T promoter (cTnT). Both vectors have a packaging capacity of 4.2 kb, well under the 4.7 packaging limit of a single AAV particle.
  • Figure 5 provides data demonstrating the validation of StitchR SCN5A channel activity in vitro. Patch clamp electrophysiology experiments on SCN5A channel kinetics in transiently transfected human HEK293T cells.
  • 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 Sodium Voltage-Gated Channel Alpha Subunit 5 (SCN5A).
  • the invention utilizes ribozyme-mediated trans-splicing of multiple RNA molecules to assemble a single RNA molecule encoding SCN5A. Ligation of the compatible ends of the first RNA portion encoding an N-terminal portion of SCN5A and the second RNA portion encoding a C-terminal portion of SCN5A, generates an RNA molecule encoding a full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein. [0036] 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 SCN5A and a second RNA portion encoding a C-terminal portion of SCN5A.
  • 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.
  • 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.
  • HH Hammerhead
  • HDV Hepatitis Delta Virus
  • VS Varkud Satellite
  • Sister Twister- sister
  • Hairpin Hatchet and Pistol families of ribozymes.
  • 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 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.
  • 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 SCN5A.
  • “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. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein.
  • the antisense sequence may 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.
  • 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.
  • a disease or disorder does not necessarily cause a further decrease in the animal’s state of health.
  • 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.
  • 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.
  • phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorot
  • 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.
  • 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.
  • 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.”
  • upstream sequences sequences on the DNA strand which are 3' to a reference point on the DNA
  • downstream sequences sequences on the DNA strand which are 3' to a reference point on the DNA
  • A refers to adenosine
  • C refers to cytosine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • peptide 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.
  • polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • RNA as used herein is defined as ribonucleic acid.
  • 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 a protein or fusion protein.
  • 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 protein or fusion protein of interest comprises voltage-gated sodium channel protein type 5 subunit alpha (SCN5A).
  • SCN5A voltage-gated sodium channel protein type 5 subunit alpha
  • the present invention is useful as the coding sequence for SCN5A is too large to package into a single vector.
  • the present invention can also be used to efficiently produce fusion proteins comprising SCN5A, chimeric proteins comprising SCN5A, and the like.
  • Ribozymes are small, catalytic RNA sequences which are capable of nucleotide specific self-cleavage.
  • 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 cleavage can be harnessed for the trans- ligation of independent RNA transcripts in mammalian cells, an approach named stitchR (stitch RNA) which is described in PCT publication No.
  • WO2021158964 which is incorporated herein by reference in its entirety.
  • 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.
  • Described herein is a StitchR-mediated dual AAV approach to deliver and express full-length human codon optimized SCN5A in cardiomyocytes in vivo as a novel therapeutic approach to correct Brugada Syndrome conduction defects and other defects or diseases associated with loss of function of SCN5A.
  • 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 cleavage can be harnessed for the trans- ligation of independent RNA transcripts in mammalian cells, an approach named stitchR (stitch RNA) which is described in PCT publication No. WO2021158964, which is incorporated herein by reference in its entirety. [0076] Remarkably, reconstitution of messenger RNA by stitchR allows for efficient translation and expression of full-length proteins in mammalian cells.
  • 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 SCN5A.
  • the first RNA comprises a coding region encoding a first portion of SCN5A and the second RNA comprises a coding region encoding a second portion of SCN5A, and thus the ribozyme-mediated cleavage and ligase-mediated assembly of the RNA molecules results in the production of an RNA molecule encoding a SCN5A protein having both the first and second portions.
  • the present invention can be used to produce a full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein from multiple RNAs, each comprising a coding region encoding a portion of the full-length SCN5A protein.
  • 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 full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion 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 attached to a coding region encoding a first portion of a full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein, and at least one additional RNA molecule comprising a coding sequence encoding a second portion of a full-length SCN5A protein, a functional SCN5A fragment
  • 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 a composition comprising one or more nucleic acid molecule comprising a coding sequence encoding a portion of a SCN5A protein linked to a coding sequence of a 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. In some embodiments, said 3’ ribozyme generates a 3’P or 2’3’ cP end on the remaining one or more RNA molecule after spontaneous cis-cleavage. In some embodiments, said one or more ribozyme is a 5’ ribozyme. In some embodiments, said 5’ ribozyme generates a 5’OH end on the remaining one or more RNA molecules after spontaneous cis-cleavage. In some embodiments, 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. [0085] 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) (e.g., HDV68, HDV67, HDV56, genHDV, or antiHDV), 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 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. As such, separated RNA sequences encoding separate portions of a larger protein can be trans-spliced together in a scar-less manner to enable expression of the larger 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 SCN5A.
  • 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.
  • 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 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.
  • one or more nucleic acid of the present invention comprises a nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:7, or a fragment or variant thereof.
  • the variant nucleic acid has a degree of identity with respect to SEQ ID NO:1 or SEQ ID NO:2 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 variant of SEQ ID NO:1 or SEQ ID NO:2 encodes a portion of a functional SCN5A protein such that upon ligation of the RNA molecules a functional SCN5A protein is generated.
  • the functional SCN5A retains the ability to interact with protein partners of native SCN5A.
  • the functional SCN5A retains the ability to participate in the formation of the voltage-gated sodium channel Na V 1.5.
  • the variant of SEQ ID NO:6 or SEQ ID NO:7 comprises at least the coding sequences as set forth in SEQ ID NO:1 and SEQ ID NO:2 respectively.
  • one or more nucleic acid of the present invention comprises a nucleic acid sequence that is a portion or fragment of a nucleic acid sequence described herein.
  • the nucleic acid has a length with respect to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:7 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 fragment of SEQ ID NO:1 or SEQ ID NO:2 encodes a portion of a functional SCN5A protein such that upon ligation of the RNA molecules a functional SCN5A protein is generated.
  • the functional SCN5A retains the ability to interact with protein partners of native SCN5A.
  • the functional SCN5A retains the ability to participate in the formation of the voltage-gated sodium channel NaV1.5.
  • the fragment of SEQ ID NO:6 or SEQ ID NO:7 comprises at least the coding sequences as set forth in SEQ ID NO:1 and SEQ ID NO:2 respectively.
  • 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, NH 2 , NHR, NR 2 or ON, wherein R is C 1 -C 6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.
  • R is C 1 -C 6 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: [0104] 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.
  • said 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. In some embodiments, the present invention further comprises one or more vector comprising one or more additional RNA molecule. In some embodiments, each additional RNA molecule comprises a coding region encoding a domain of the protein of interest; a 5’ ribozyme; and a 3’ ribozyme. In some embodiments, each additional RNA molecule comprises a coding region encoding a domain of the protein of interest; a 5’ ribozyme; and a 3’ ribozyme recognition sequence. [0109] The art is replete with suitable vectors that are useful in the present invention.
  • 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.
  • the AAV serotype is myoAAV-4A.
  • 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.
  • the promoter is the human Elongation Growth Factor -1 ⁇ (EF-1 ⁇ ).
  • the promoter is the cardiac-specific cardiac Troponin-T (cTnT) promoter.
  • the cTnT promoter comprises a sequence as set forth in SEQ ID NO:5.
  • 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. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
  • 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).
  • compositions of the invention 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 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.
  • 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 SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein, as described herein.
  • 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 for delivery and expression of a full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein via cis-cleavage and trans-splicing of independent RNA molecules encoding parts of the full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein.
  • the system comprises a first vector comprising a coding sequence for the N-terminal portion of SCN5A (SEQ ID NO:1) and a second vector comprising a coding sequence for the C- terminal portion of SCN5A (SEQ ID NO:2), wherein each of the vectors further comprises a ribozyme sequence for self-cleavage of the vectors and subsequent trans-ligation for formation of an RNA encoding full length SCN5A.
  • the system comprises a first vector comprising SEQ ID NO:6 and a second vector comprising SEQ ID NO:7.
  • the system or composition comprises a delivery vehicle or a combination of delivery vehicles for delivery of at least one nucleic acid molecule for the in vivo generation and expression of a full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A or a SCN5A fusion protein to a subject.
  • the invention provides a single delivery vehicle for delivery of a first and second RNA molecule, wherein each of the first and second RNA molecule comprises a coding sequence for a portion of a full length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein.
  • the invention provides a combination of two or more delivery vehicles for delivery of a first and second nucleic acid molecule, wherein the first delivery vehicle comprises or encapsulates a nucleic acid molecule comprises a coding sequence for a first portion of a full length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein, and the second delivery vehicle comprises or encapsulates a nucleic acid molecule comprises a coding sequence for a second portion of a full length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein.
  • lipid nanoparticle refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids.
  • lipid nanoparticles comprise a cationic lipid and at least one excipient.
  • excipients include, but are not limited to, neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid).
  • the at least one agent or polynucleotide is encapsulated in the lipid portion of the LNP or an aqueous space enveloped by some or all of the lipid portion of the LNP, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.
  • the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm
  • the lipid nanoparticles are substantially non-toxic.
  • the genome editing complex when present in the lipid nanoparticles, is resistant in aqueous solution to degradation by intra- or intercellular enzymes.
  • the LNP may comprise any lipid capable of forming a particle to which the at least one agent is attached, or in which the at least one agent is encapsulated.
  • lipid refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents.
  • Lipids are usually divided into at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.
  • the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.
  • the LNP comprises an ionizable cationic lipid.
  • the term “ionizable cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids.
  • the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.
  • the ionizable cationic lipid comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH.
  • Such lipids include, but are not limited to, N,N-dioleyl-N,N- dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3- (N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1-(2,3- dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DO
  • cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)- N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.).
  • LIPOFECTIN® commercially available cationic liposomes compris
  • delivery of a vector for in vivo generation and expression of a full length SCN5A protein, a functional SCN5A fragment, a functional SCN5A or a SCN5A fusion protein comprises any suitable delivery method, including exemplary transfection methods described elsewhere herein.
  • delivery of a vector to a subject comprises mixing the vector with a transfection reagent prior to the step of contacting.
  • a method of present invention further comprises administering the vector together with the transfection reagent.
  • the transfection reagent is a cationic lipid reagent.
  • the transfection reagent is a cationic polymer reagent.
  • the transfection reagent is a lipid-based transfection reagent.
  • the transfection reagent is a protein-based transfection reagent.
  • the transfection reagent is a carbohydrate-based transfection reagent.
  • the transfection reagent is a cationic lipid-based transfection reagent.
  • the transfection reagent is a cationic polymer-based transfection reagent.
  • the transfection reagent is a polyethyleneimine based transfection reagent.
  • the transfection reagent is calcium phosphate.
  • the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®.
  • the transfection reagent is any other transfection reagent known in the art.
  • the transfection reagent forms a liposome.
  • Liposomes in another embodiment, increase intracellular stability, increase uptake efficiency and improve biological activity.
  • liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids, which make up the cell membrane. They have, in another embodiment, an internal aqueous space for entrapping water-soluble compounds and range in size from 0.05 to several microns in diameter.
  • liposomes can deliver nucleic acid molecules (e.g., DNA or RNA) to cells in a biologically active form.
  • compositions of the pharmaceutical 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.
  • pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to subjects of all sorts.
  • compositions suitable for administration to humans in order to render the compositions suitable for administration to various subjects is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.
  • compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration.
  • Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient, which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • 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.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.
  • Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue- penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.
  • a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline.
  • a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline.
  • Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi dose containers containing a preservative.
  • Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g. sterile pyrogen free water
  • the pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • sterile injectable formulations may be prepared using a non toxic parenterally acceptable diluent or solvent, such as water or 1,3 butane diol, for example.
  • a non toxic parenterally acceptable diluent or solvent such as water or 1,3 butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono or di- glycerides.
  • Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers.
  • the formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 1 to about 6 nanometers.
  • compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container.
  • a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container.
  • such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. In some embodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers.
  • dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition.
  • the propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (in some instances having a particle size of the same order as particles comprising the active ingredient).
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g., sterile pyrogen free water
  • the pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non toxic parenterally acceptable diluent or solvent, such as water or 1,3 butane diol, for example.
  • a non toxic parenterally acceptable diluent or solvent such as water or 1,3 butane diol
  • Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono or di- glycerides.
  • Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • the present invention relates to methods of cis- cleavage and trans-splicing of independent RNA molecules.
  • cis- cleavage and trans-splicing of independent RNA molecules results in a single RNA molecule encoding a full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein, 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 SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein, from two separate RNA molecules encoding a first part and a second part of the SCN5A protein via cis-cleavage of ribozymes and trans-splicing of the two independent RNA molecules.
  • the present invention relates to in vivo methods of generating an RNA molecule encoding a full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein.
  • 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 portion of a SCN5A protein and a second portion of a SCN5A protein.
  • 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.
  • 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’ ribozyme comprises SEQ ID NO:3 or SEQ ID NO:9-61.
  • the 5’ ribozyme comprises SEQ ID NO:4 or SEQ ID NO:9-61.
  • 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 a full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein; a 5’ ribozyme; and a 3’ ribozyme.
  • the method comprises administering to the cell or tissue two or more RNA molecules encoding two or more portions of a full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein.
  • 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.
  • the RNA molecule can be administered as a linear or circular RNA molecule.
  • 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 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 a first RNA molecule and the coding region of one or more additional RNA molecule.
  • trans-ligation of the coding sequences for the first and second (or more) portion of a full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein occurs in a scarless manner, such that there is no intervening sequence between the two or more portions of the full-length SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein after translation.
  • the ligase is RNA 2',3'-Cyclic Phosphate and 5'- OH (RtcB) ligase.
  • 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.
  • 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.
  • 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.
  • 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. [0171] Lipids suitable for use can be obtained from commercial sources.
  • 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.
  • Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • Treatment and Use provides methods of treating, reducing the symptoms of, and/or reducing the risk of developing a disease or disorder associated with reduced levels of SCN5A activity.
  • 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.
  • 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 method of the present invention comprises administering to a subject having Brugada Syndrome, Long QT Syndrome, atrial fibrillation, sick sinus syndrome or cardiomyopathy due to an absent or defective SCN5A a composition comprising a first nucleic acid comprising a coding region encoding a first portion of SCN5A and a 3’ ribozyme, and a second nucleic acid comprising a coding region encoding a second portion of SCN5A 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 SCN5A and the coding region encoding the second portion of SCN5A, generates a single RNA molecule encoding a full-length SCN
  • the method of the present invention comprises administering to a subject having Brugada Syndrome, Long QT Syndrome, atrial fibrillation, sick sinus syndrome or cardiomyopathy due to an absent or defective SCN5A a composition comprising a first nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 1, or a fragment or variant thereof, and a second nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 2, or a fragment or variant thereof, 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 SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein.
  • the method of the present invention comprises administering to a subject having Brugada Syndrome, Long QT Syndrome, atrial fibrillation, sick sinus syndrome or cardiomyopathy due to an absent or defective SCN5A a composition comprising a first nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 6, or a fragment or variant thereof, and a second nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 7, or a fragment or variant thereof, 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 SCN5A protein, a functional SCN5A fragment, a functional SCN5A variant or a SCN5A fusion protein.
  • the method of the invention comprises systemic administration of the subject, including for example enteral or parenteral administration.
  • the method comprises intradermal delivery of the composition.
  • the method comprises intravenous delivery of the composition.
  • the method comprises intramuscular delivery of the composition.
  • the method comprises subcutaneous delivery of the composition.
  • the method comprises inhalation of the composition.
  • the method comprises intranasal delivery of the composition.
  • composition may be administered to a mammal 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, several years, or even less frequently, such as every 10-20 years, 15-30 years, or even less frequently, such as every 50-100 years.
  • 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 mammal, etc.
  • a research plan was developed to validate StitchR- mediated vectors for expressing full-length SCN5A in vivo for POC studies in mice in a mouse model of BrS.
  • Described herein is 1) the design, generation and validation of StitchR- mediated vectors for expressing full-length SCN5A in cells and 2) the generation of AAV virus and determination of the efficacy of this approach for expression of full-length SCN5A in a mouse model of BrS in vivo.
  • the full-length cardiac-expressed isoform of the human SCN5A gene encodes a single large protein composed of 4 domains, required for cardiac cell depolarization and normal heart rhythm.
  • the approach and vectors express a full-length human codon optimized SCN5A protein, could be utilized to correct all human loss-of-function mutations which give rise to BrS.
  • Vector design and construction [0189] Vectors have been designed, constructed and validated for stitchR-mediated expression of a split, codon-optimized SCN5A gene pair using transient transfection in cells.
  • the vector encoding full-length SCN5A could never be packaged into an AAV particle, but serves here in plasmid transfection-based assays as a positive control. All SCN5A ORFs were fully sequenced using Sanger sequencing and full plasmid sequence using Oxford Nanopore sequencing.
  • the human EF1a promoter strong ubiquitous expression was utilized for testing in vitro to validate vector constructs in vitro, but can be replaced with the cardiac-specific cardiac Troponin-T (cTnT) promoter for in vivo studies.
  • StitchR-mediated trans-ligation vs other dual expression approaches, such as INTEINS - whereby two protein halves are spliced together, is that StitchR allows for protein expression from a single reconstituted full-length mRNA. Therefore, StitchR-mediated SCN5A expressed protein behaves identically to SCN5A expressed from a single plasmid.
  • patch clamp electrophysiology is used to measure and compare channel kinetics of SCN5A in transiently transfected HEK293T cells ( Figure 5). AAV virus generation.
  • High titer AAV virus is generated to generate myoAAV-4A serotyped virus, an AAV9 evolved variant, which shows the greatest tropism for both human and mouse cardiac tissues.
  • High titer ( ⁇ 5e13gc/ml to ⁇ 2e14 gc/ml) AAV viral preps are received for large gene inserts.
  • 1E+12 vg of both AAVs (2E+12 total) is injected intraperitoneally into 6 male and 6 female Scn5a+/- heterozygous mice, with comparable number of male and female wild type and Scn5a+/- heterozygous mice injected with saline as control. Measurements are made at 10 weeks of age.
  • RNA isolation and SCN5A trans-ligation validation [0194] Heart tissues snap frozen in liquid nitrogen are pulverized using a Bessman tissue pulverizer to obtain whole tissue homogenates. For RNA isolation, pulverized heart tissue homogenates are added to 1 ml of ice-cold TRIzol and homogenized in a VWR Beadmill using 2.8mm ceramic beads. Total RNA is extracted by isopropanol precipitation and cDNA are generated using SuperScript III (Invitrogen) for qRTPCR sequence analyses using primers specific to the SCN5A trans-ligation junction. Cardiac function assessment using non-invasive cardiac monitoring.
  • Heart function is measured and compared in all AAV treated mice (12 male and 12 females for each AAV vector pair) at 8 weeks of age by two-dimensional echocardiography using the SIG Visual Sonics Vevo 2100. Measurements of heart rate (HR), fractional shortening (FS) and ejection fraction (EF), and left ventricular dimensions are recorded and compared. Additionally, non-invasive ECG measurements are recorded on anesthetized mice using an AdInstruments BioAmp ECG apparatus. ECG recordings are captured for 10 minutes for each animal and analyzed using AdInstruments LabChart7 software. Tissue harvest and histological analyses.
  • Protein lysates generated above are compared by western blot for SCN5A expression in wild type mice.
  • Successful in vivo demonstration of full-length SCN5A protein expression at levels comparable to endogenous levels, and phenotypic rescue lead to experiments assessing the therapeutic efficacy of this approach in large animal models (ex. Pig) of BrS.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Veterinary Medicine (AREA)
  • Biomedical Technology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des compositions, des systèmes et des méthodes utilisant le cis-clivage et le trans-épissage de molécules d'ARN médiés par des ribozymes pour exprimer un SCN5A fonctionnel, ainsi que des procédés d'utilisation associés pour traiter le syndrome de Brugada et d'autres maladies associées à la perte de fonction du SCN5A.
PCT/US2025/040653 2024-08-05 2025-08-05 Compositions et procédés d'expression de scn5a pleine longueur médiée par stitchr in vivo Pending WO2026035687A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463679363P 2024-08-05 2024-08-05
US63/679,363 2024-08-05

Publications (1)

Publication Number Publication Date
WO2026035687A1 true WO2026035687A1 (fr) 2026-02-12

Family

ID=97027779

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/040653 Pending WO2026035687A1 (fr) 2024-08-05 2025-08-05 Compositions et procédés d'expression de scn5a pleine longueur médiée par stitchr in vivo

Country Status (1)

Country Link
WO (1) WO2026035687A1 (fr)

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5350674A (en) 1992-09-04 1994-09-27 Becton, Dickinson And Company Intrinsic factor - horse peroxidase conjugates and a method for increasing the stability thereof
US5399346A (en) 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5585362A (en) 1989-08-22 1996-12-17 The Regents Of The University Of Michigan Adenovirus vectors for gene therapy
US6156303A (en) 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
WO2001029058A1 (fr) 1999-10-15 2001-04-26 University Of Massachusetts Genes de voies d'interference d'arn en tant qu'outils d'interference genetique ciblee
US6326193B1 (en) 1999-11-05 2001-12-04 Cambria Biosciences, Llc Insect control agent
WO2001096584A2 (fr) 2000-06-12 2001-12-20 Akkadix Corporation Matieres et procedes de lutte contre les nematodes
WO2003042397A2 (fr) 2001-11-13 2003-05-22 The Trustees Of The University Of Pennsylvania Methode de detection et/ou d'identification de sequences de virus associes aux adenovirus (aav) et d'isolation de nouvelles sequences ainsi identifiees
WO2005033321A2 (fr) 2003-09-30 2005-04-14 The Trustees Of The University Of Pennsylvania Variantes des virus associes aux adenovirus (aav), sequences, vecteurs les contenant, et leur utilisation
US7282199B2 (en) 2001-12-17 2007-10-16 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing same, and uses therefor
US20120276209A1 (en) 2009-11-04 2012-11-01 The University Of British Columbia Nucleic acid-containing lipid particles and related methods
WO2021158964A1 (fr) 2020-02-07 2021-08-12 University Of Rochester Assemblage et expression d'arn à médiation par ribozyme
CN118127080A (zh) * 2024-02-09 2024-06-04 劲帆生物医药科技（武汉）有限公司 一种用于在宿主细胞中表达目的基因的双aav载体系统

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5589466A (en) 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence
US5399346A (en) 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
US5585362A (en) 1989-08-22 1996-12-17 The Regents Of The University Of Michigan Adenovirus vectors for gene therapy
US5350674A (en) 1992-09-04 1994-09-27 Becton, Dickinson And Company Intrinsic factor - horse peroxidase conjugates and a method for increasing the stability thereof
US6156303A (en) 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
WO2001029058A1 (fr) 1999-10-15 2001-04-26 University Of Massachusetts Genes de voies d'interference d'arn en tant qu'outils d'interference genetique ciblee
US6326193B1 (en) 1999-11-05 2001-12-04 Cambria Biosciences, Llc Insect control agent
WO2001096584A2 (fr) 2000-06-12 2001-12-20 Akkadix Corporation Matieres et procedes de lutte contre les nematodes
WO2003042397A2 (fr) 2001-11-13 2003-05-22 The Trustees Of The University Of Pennsylvania Methode de detection et/ou d'identification de sequences de virus associes aux adenovirus (aav) et d'isolation de nouvelles sequences ainsi identifiees
US7282199B2 (en) 2001-12-17 2007-10-16 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing same, and uses therefor
WO2005033321A2 (fr) 2003-09-30 2005-04-14 The Trustees Of The University Of Pennsylvania Variantes des virus associes aux adenovirus (aav), sequences, vecteurs les contenant, et leur utilisation
US20120276209A1 (en) 2009-11-04 2012-11-01 The University Of British Columbia Nucleic acid-containing lipid particles and related methods
WO2021158964A1 (fr) 2020-02-07 2021-08-12 University Of Rochester Assemblage et expression d'arn à médiation par ribozyme
CN118127080A (zh) * 2024-02-09 2024-06-04 劲帆生物医药科技（武汉）有限公司 一种用于在宿主细胞中表达目的基因的双aav载体系统

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
AKINC ET AL., MOL THER., vol. 18, no. 7, 2010, pages 1357 - 1364
BASHA ET AL., MOL THER, vol. 19, no. 12, 2011, pages 2186 - 2200
BELLIVEAU ET AL., MOL THER NUCLEIC ACIDS, vol. 1, 2012, pages e37
DOISNE NICOLAS ET AL: "In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant", FRONTIERS IN PHYSIOLOGY, vol. 12, 28 May 2021 (2021-05-28), CH, pages 1 - 13, XP093323639, ISSN: 1664-042X, DOI: 10.3389/fphys.2021.661413 *
GHOSH ET AL., GLYCOBIOLOGY, vol. 5, 1991, pages 505 - 10
JAYARAMAN ET AL., ANGEW CHEM INT ED ENGL., vol. 51, no. 34, 2012, pages 8529 - 8533
LEE ET AL., INT J CANCER., vol. 131, no. 5, 2012, pages E781 - 90
LEUNG ET AL., J PHYS CHEM C NANOMATER INTERFACES, vol. 116, no. 34, 2012, pages 18440 - 18450
LIMBACH ET AL., NUCLEIC ACIDS RES., vol. 22, 1994, pages 2183 - 2196
MAIER ET AL., MOL THER., vol. 21, no. 8, 2013, pages 1570 - 1578
MUI ET AL., MOL THER NUCLEIC ACIDS., vol. 2, 2013, pages e139
SAMBROOK ET AL.: "A Laboratory Approach", 2012, COLD SPRING HARBOR LABORATORY, article "Molecular Cloning"
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY
SEMPLE ET AL., NAT BIOTECHNOL., vol. 28, no. 2, 2010, pages 172 - 176
TAM ET AL., NANOMEDICINE, vol. 9, no. 5, 2013, pages 665 - 74
UI-TEI ET AL., FEBS LETTERS, vol. 479, 2000, pages 79 - 82
ZHOU ET AL., RESEARCH SQUARE, 2003
ZHOU ET AL., RESEARCH SQUARE, 2023

Similar Documents

Publication Publication Date Title
JP7583732B2 (ja) フェニルアラニンヒドロキシラーゼ（ｐａｈ）治療薬を発現するための非ウイルス性ｄｎａベクターおよびその使用
EP4244342B1 (fr) Méganucléases modifiées ayant une spécificité pour les séquences de reconnaissance du gène de la dystrophine
CA3208153A1 (fr) Compositions et methodes de traitement de la maladie de fabry
EP4463555A1 (fr) Système rapporteur d'édition génique et arn guide et composition associée ; composition et procédé pour éliminer l'adn avec plus de deux arng ; édition génique dans l'oeil ; et édition génique utilisant des éditeurs de bases
JP2025172742A (ja) トランスサイレチン遺伝子における認識配列に対する特異性を有する操作されたメガヌクレアーゼ
JP2023542131A (ja) フェニルアラニンヒドロキシラーゼ（ｐａｈ）を発現させるための閉端ｄｎａベクター及びその使用
US11504388B2 (en) Trans-splicing ribozyme targeting rhodopsin transcript and uses thereof
US20240084276A1 (en) Engineered meganucleases that target human mitochondrial genomes
KR20250158036A (ko) 리보자임-매개 rna 조립 및 발현
US20240200046A1 (en) Engineered meganucleases that target human mitochondrial genomes
US20250382641A1 (en) Polypeptide linkers for use in engineered meganucleases
JP2023513211A (ja) タンパク質合成を増強するためのＣＲＩＳＰＲ－Ｃａｓ１３による標的ＲＮＡ翻訳
WO2025233894A1 (fr) Méganucléases modifiées ciblant des génomes mitochondriaux humains
WO2026028165A1 (fr) Méganucléases modifiées ayant une spécificité pour les séquences de reconnaissance dans le gène c9orf72
WO2025072604A1 (fr) Approches de thérapie génique d'édition d'arn pour le traitement de la dystrophie myotonique de type 1 (dm1)

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25765416

Country of ref document: EP

Kind code of ref document: A1