WO2025171380A2 - Clivage et édition d'arn de précision à l'aide d'un ribozyme trans-clivant et d'arn guides - Google Patents

Clivage et édition d'arn de précision à l'aide d'un ribozyme trans-clivant et d'arn guides

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Publication number
WO2025171380A2
WO2025171380A2 PCT/US2025/015234 US2025015234W WO2025171380A2 WO 2025171380 A2 WO2025171380 A2 WO 2025171380A2 US 2025015234 W US2025015234 W US 2025015234W WO 2025171380 A2 WO2025171380 A2 WO 2025171380A2
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rna
trans
fusion construct
molecule
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WO2025171380A3 (fr
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Douglas Matthew ANDERSON
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University of Rochester
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University of Rochester
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/123—Hepatitis delta
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid

Definitions

  • RNA targeting, RNA-activated CRISPR-Cas13 systems are generally composed of a targeting CRISPR guide RNA (gRNA) and CRISPR associated protein, Cas13, which function as a programmable endoribonuclease (O’Connell, 2019, J Mol. Biol 431:66-87; Abudayyeh et al., 2018, Nature 550:280-84; Mohanraju et al., 2016, Science 343:aad5147).
  • Cas13 proteins have two Higher Eukaryotes and Prokaryotes Nucleotide binding (HEPN) domains which allow for cleavage of single stranded RNA.
  • HEPN Prokaryotes Nucleotide binding
  • the invention relates to a fusion construct comprising at least one guide RNA (gRNA), and at least one ribozyme or DNAzyme.
  • the fusion construct comprises at least two guide RNA (gRNA), and at least two ribozymes or DNAzymes.
  • the ribozyme or DNAzyme is specific for trans-cleavage of RNA or DNA.
  • the ribozyme or DNAzyme comprises hammerhead (HH), Hepatitis delta virus (HDV), Hairpin (HP), Varkud Satellite (VS), Twister, Twister Sister, Pistol, Hatchet, HOV, GlmS, Deoxyribozyme 8–17, Deoxyribozyme 10-23, or deoxyribozyme I- R3, or a fragment or trans-cleavage variant thereof.
  • the fusion construct comprises a trans-cleavage ribozyme.
  • the trans-cleavage ribozyme comprises a nucleotide sequence as set forth in SEQ ID NO:1-53, or a fragment or variant thereof.
  • the fusion construct further comprises at least one linker.
  • the invention relates to a fusion construct comprising at least two ribozymes or DNAzymes.
  • each of the two ribozymes or DNAzymes are specific for RNA cleavage.
  • each of the two ribozymes or DNAzymes are specific for DNA cleavage.
  • each of the at least two ribozymes or DNAzymes comprises hammerhead (HH), Hepatitis delta virus (HDV), Hairpin (HP), Varkud Satellite (VS), Twister, Twister Sister, Pistol, Hatchet, HOV, GlmS, Deoxyribozyme 8–17, Deoxyribozyme 10- 23, or deoxyribozyme I-R3, or a fragment or trans-cleavage variant thereof.
  • each of the at least two ribozymes comprises a trans- cleavage ribozyme.
  • each of the at least two ribozymes comprises a nucleotide sequence as set forth in SEQ ID NO:1-53, or a fragment or variant thereof.
  • the construct further comprises at least one linker.
  • the invention relates to a complex comprising at least one fusion construct comprising at least one guide RNA (gRNA), and at least one ribozyme or DNAzyme or a fusion construct comprising at least two ribozymes or DNAzymes.
  • the complex further comprises a CRISPR Cas protein.
  • the Cas protein is catalytically active.
  • the Cas protein comprises a mutation in Attorney Docket No.204606-0174-00WO one or both of the HEPN domains.
  • the Cas protein is catalytically dead Cas13 (dCas13).
  • the Cas protein comprises a sequence selected from SEQ ID NOs: 54-101, or a variant thereof.
  • the Cas protein further comprises a nuclear localization signal (NLS).
  • the NLS comprises a sequence selected from SEQ ID NOs: 111 - 725, or a variant thereof.
  • the invention relates to a composition comprising at least one fusion construct comprising at least one guide RNA (gRNA), and at least one ribozyme or DNAzyme.
  • the complex further comprises a CRISPR Cas protein.
  • the invention relates to a composition comprising at least one fusion construct comprising at least two ribozymes or DNAzymes. In some embodiments, each of the two ribozymes or DNAzymes are specific for RNA cleavage.
  • the invention relates to a composition comprising a complex comprising at least one fusion construct comprising at least one guide RNA (gRNA), and at least one ribozyme or DNAzyme and further comprising a CRISPR Cas protein.
  • the invention relates to a method of decreasing the number of a target RNA transcript or cleaving a target RNA transcript, the method comprising contacting the target RNA molecule with at least one fusion construct comprising at least one guide RNA (gRNA), and at least one ribozyme or DNAzyme.
  • the method is an in vitro or in vivo method.
  • the method comprises contacting the target RNA molecule with at least two fusion constructs, wherein each of the fusion constructs or complexes comprises a gRNA for targeting the fusion construct or complex to a target site on the RNA molecule for cleavage.
  • each of the fusion constructs or complexes comprises the same ribozyme.
  • each of the fusion constructs or complexes comprises a different ribozyme.
  • the method promotes splicing or ligation of the target RNA molecule.
  • the splicing or ligation is cis-splicing, trans-splicing, cis- RNA ligation or trans-RNA ligation.
  • the invention relates to a method of cleaving, splicing or ligating a target DNA molecule, the method comprising contacting the target DNA molecule with at least one fusion construct comprising at least one guide RNA (gRNA), and at least one ribozyme or DNAzyme.
  • the method is an in vitro or in vivo method.
  • the method comprises contacting the target DNA molecule with at least two fusion constructs, wherein each of the fusion constructs or complexes comprises a gRNA for targeting the fusion construct or complex to a target site on the DNA molecule for cleavage.
  • each of the fusion constructs or complexes comprises the same ribozyme or DNAzyme.
  • each of the fusion constructs or complexes comprises a different ribozyme or DNAzyme.
  • the method promotes splicing or ligation of the target DNA molecule.
  • the splicing or ligation is cis-splicing, trans-splicing, cis-DNA ligation or trans-DNA ligation.
  • the invention relates to a method of treating a disease or disorder associated with nucleic acid level or expression in a subject, the method comprising administering to the subject at least one fusion construct comprising at least one guide RNA (gRNA) and at least one ribozyme or DNAzyme or a fusion construct comprising at least two ribozymes or DNAzymes.
  • the disease or disorder is associated with the presence of an aberrant DNA molecule or RNA transcript, wherein the method promotes degradation of the aberrant DNA molecule or RNA transcript.
  • the disease or disorder is associated with the presence of a premature stop codon (PTC) in a target RNA transcript, wherein the method promotes splicing of the target RNA transcript to remove the PTC.
  • the invention relates to a method of generating a trans- ligated RNA molecule, the method comprising contacting a first RNA molecule with a first fusion construct comprising at least one guide RNA (gRNA) and at least one ribozyme or DNAzyme and a second RNA molecule with a second fusion construct comprising at least one guide RNA (gRNA) and at least one ribozyme or DNAzyme, whereby cleavage of the first and second RNA molecule and subsequent ligation of the cleaved RNA molecules generates a trans- ligated RNA molecule.
  • gRNA guide RNA
  • gRNA guide RNA
  • the first and second trans-cleaving ribozyme are connected by a flexible linker.
  • the invention relates to a method of generating a trans- ligated RNA molecule, the method comprising contacting a first RNA and a second RNA molecule with a fusion construct comprising at least two ribozymes or DNAzymes, whereby cleavage of the first and second RNA molecule and subsequent ligation of the cleaved RNA Attorney Docket No.204606-0174-00WO molecules generates a trans-ligated RNA molecule.
  • the first and second trans-cleaving ribozyme are connected by a flexible linker.
  • the invention relates to a method of generating a trans- ligated RNA molecule, the method comprising contacting a first RNA molecule and a second RNA molecule with a Cas protein which cleaves the first and second RNA, thereby generating compatible RNA ends, whereby cleavage of the first and second RNA molecule and subsequent ligation of the cleaved RNA molecules generates a trans-ligated RNA molecule.
  • the Cas protein is a subtype II-A or subtype II-C class Cas protein.
  • the method further comprises contacting the first and second RNA molecules with a first and second guide RNA to target the Cas protein to a specific site for cleavage.
  • Figure 1A through Figure 1G depict precision RNA cleavage, and subsequent RNA ligation in cells, can occur through multiple cleavage mechanisms.
  • Figure 1A depicts that in stitchR, encoded ribozymes, which cleave in cis, generate RNA termini which are recognized and repaired through RNA ligation either in cis or in trans.
  • Figure 1B depicts biomolecular complexes of various ribozyme sequences, such as hammerhead ribozymes, have been described which are capable of assembly in trans to form functional ribozyme sequences. This allows for targeting of endogenous cellular RNAs through delivery and expression of small target- complementary sequences.
  • Figure 1C and Figure 1D depict the stability, targeting efficiency and/or localization of trans-cleaving ribozymes may be enhanced through incorporation into the targeting guide RNAs of CRISPR-Cas systems on either 5’ or 3’ ends.
  • Figure 1E through Figure 1G depict that a similar strategy can be used for HDV trans-cleaving ribozymes, as well as many others.
  • Figure 1H depicts a diagram demonstrating that some CRISPR-Cas systems have demonstrated site-specific RNA cleavage, which when combined into multiple cleavage events, may allow for repair of sequences in cis or the combination of RNA sequences in trans, through RNA trans ligation.
  • Figure 2A and Figure 2B depicts diagrams of tandem fusion of trans-cleaving ribozymes using the same ( Figure 2A) or different ( Figure 2B) ribozymes sequences can allow for site-specific deletion and induction of RNA ligation of target sequences.
  • Figure 3 depicts sequences of the trans-cleaving hammerhead and trans-cleaving extended hammerhead with tertiary stabilizing motif (TSM).
  • TSM tertiary stabilizing motif
  • the disclosure is based on the use of trans-cleaving ribozymes or CRISPR-Cas proteins to site-specifically cleave nucleic acid molecules.
  • the invention provides methods of site-specific cleavage and subsequent ligation of nucleic acid molecules. Therefore, in some embodiments, the invention relates to methods of trans-splicing RNA molecules, trans-splicing DNA molecules, trans-ligating RNA molecules and trans-ligating DNA molecules.
  • the catalytic RNA is a trans-cleaving ribozyme.
  • the trans-cleaving ribozyme is a trans-cleaving variant of a natural or synthetic ribozyme or deoxyribozyme.
  • the trans-cleaving ribozyme is a trans- cleaving variant of hammerhead (HH), Hepatitis delta virus (HDV), Hairpin (HP), Varkud Satellite (VS), Twister, Twister Sister, Pistol, Hatchet, HOV, or GlmS.
  • the fusion construct further comprises a tertiary stabilizing motif (TSM).
  • TSM tertiary stabilizing motif
  • the invention provides fusion constructs comprising two or more linked catalytic RNA which has a trans-cleavage activity, and cleaves a target nucleic acid molecule.
  • the fusion construct comprises two or more linked catalytic RNA with trans-cleavage activity operably linked to two or more targeting gRNA.
  • the gRNA target the catalytic RNA molecules to a specific sequence for site- specific trans-cleavage of the target nucleic acid molecule(s). Subsequent repair of the cleaved nucleic acid molecule generates a spliced or ligated nucleic acid molecule.
  • the two or more catalytic RNA are trans-cleaving ribozymes.
  • each of the two catalytic RNA is a trans-cleaving ribozyme.
  • each of the two trans-cleaving ribozyme is a trans-cleaving variant of a natural or synthetic ribozyme or deoxyribozyme.
  • the trans-cleaving ribozyme is a trans- cleaving variant of hammerhead (HH), Hepatitis delta virus (HDV), Hairpin (HP), Varkud Satellite (VS), Twister, Twister Sister, Pistol, Hatchet, HOV, or GlmS.
  • the fusion construct further comprises a tertiary stabilizing motif (TSM).
  • TSM tertiary stabilizing motif
  • the invention provides fusion constructs comprising two or more linked gRNA and a CRISPR-Cas protein which can cleave one or more target nucleic acid molecule leaving compatible ends for ligation or splicing of the cleaved target nucleic acid molecule(s).
  • the fusion construct comprises one or more linked gRNA.
  • the linked gRNA fusion construct holds the cleaved ends in proximity for subsequent ligation by cellular machinery.
  • Antisense refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand.
  • an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule.
  • the antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • 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 Attorney Docket No.204606-0174-00WO 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, Attorney Docket No.204606-0174-00WO 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.
  • 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.
  • 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.
  • 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.”
  • A refers to adenosine
  • C refers to cytosine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • 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.
  • the term “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.
  • the present disclosure is based on the development of novel fusions constructs for site specific cleavage of one or more target nucleic acid molecule.
  • site specific cleavage of one or more target nucleic acid molecule using the fusion constructs of the invention generates compatible ends which are subsequently ligated by cellular machinery.
  • the invention provides methods for splicing, ligating or degrading nucleic acid molecules by contacting one or more target nucleic acid Attorney Docket No.204606-0174-00WO molecule with the fusion construct of the invention.
  • the fusion constructs are effectively delivered to a cell.
  • the fusion construct combines the catalytic activity of a trans- cleaving ribozymes and the programmable nucleic acid targeting capability of gRNA for site specific cleavage of RNA molecules.
  • the fusion construct combines the catalytic activity of a trans-cleaving ribozymes or DNAzymes and the programmable nucleic acid targeting capability of gRNA for RNA-ligation, RNA-splicing, RNA degradation, or for the generation circular RNA molecules.
  • the fusion construct combines the catalytic activity of two or more linked trans-cleaving ribozymes or DNAzymes for site specific cleavage of RNA molecules.
  • the fusion construct combines the catalytic activity of two or more linked trans-cleaving ribozymes for RNA-ligation, RNA-splicing, RNA degradation, or for the generation of circular RNA molecules.
  • the fusion construct combines the catalytic activity of two or more linked DNA-cleaving ribozymes or DNAzymes for site specific cleavage of DNA molecules. In some embodiments, the fusion construct combines the catalytic activity of two or more linked trans-cleaving ribozymes or DNAzymes for DNA-ligation, DNA-splicing, DNA degradation, or for the generation of circular DNA molecules. [0068] In one embodiment, the fusion construct combines the catalytic activity of DNA- cleaving ribozymes or DNAzymes and the programmable nucleic acid targeting capability of gRNA for site specific cleavage of DNA molecules.
  • the fusion construct combines the catalytic activity of a DNA-cleaving ribozymes or DNAzymes and the programmable nucleic acid targeting capability of gRNA for DNA-ligation, DNA-splicing, DNA degradation, or for the generation of circular DNA molecules.
  • the fusion construct combines the catalytic activity of two or more linked DNA-cleaving ribozymes or DNAzymes for site specific cleavage of DNA molecules.
  • the fusion construct combines the catalytic activity of two or more linked trans-cleaving ribozymes or DNAzymes for DNA-ligation, DNA-splicing, DNA degradation, or for the generation of circular DNA molecules.
  • the fusion construct comprises a linker.
  • the linker links a gRNA and a trans-cleaving ribozyme.
  • the linker links a Attorney Docket No.204606-0174-00WO gRNA and a DNA-cleaving ribozyme.
  • the linker links two trans-cleaving ribozymes.
  • the linker links two DNA-cleaving ribozymes.
  • the linker links two gRNAs.
  • the present invention comprises methods for site-specific deletion and subsequent ligation of target sequences using two or more fusion constructs of the invention.
  • the two or more fusion constructs comprise the same ribozyme. In some embodiments, the two or more fusion constructs comprise different ribozymes. In some embodiments, a first fusion construct comprises a ribozyme linked to a gRNA targeting a region upstream of a target nucleotide sequence to be edited or deleted and a second fusion construct comprises a ribozyme linked to a gRNA targeting a region downstream of a target nucleotide sequence to be edited or deleted. Co-administration of the two fusion constructs facilitates deletion of an intervening sequence, or splicing, of the targeted nucleic acid molecule.
  • a first fusion construct comprises a ribozyme linked to a gRNA targeting a target nucleotide sequence on a first nucleic acid molecule and a second fusion construct comprises a ribozyme linked to a gRNA targeting a target nucleotide sequence on a second nucleic acid molecule.
  • Co-administration of the two fusion constructs facilitates site specific cleavage and subsequent ligation to generate a ligated nucleic acid molecule. Therefore, in one embodiment, the present invention relates to methods for trans-ligating RNA or DNA molecules, comprising contacting at least two different RNA or DNA molecules in a cell or in vitro with one or more fusion construct.
  • nucleic acid molecules that can be ligated, degraded or spliced according to the methods of the invention include, but are not limited to, genomic DNA, extrachromosomal DNA, plasmid DNA, viral DNA, viral RNA, mRNA, lncRNA, non-coding RNA, or circular RNA molecules.
  • cleavage of the RNA generates at least one RNA molecule with a ‘3’-phosphate or 2’,3’-cyclic phosphate termini and at least one RNA molecule with a 5’ hydroxyl RNA termini.
  • the method further comprises contacting said at least one RNA molecule with a ‘3’-phosphate or 2’,3’-cyclic phosphate termini and at least one RNA molecule with a 5’ hydroxyl RNA termini with RtcB ligase or a nucleic acid encoding RtcB ligase.
  • RtcB ligase or a nucleic acid encoding RtcB ligase.
  • cis-cleaving ribozymes can be engineered to cleave in trans, such that target RNAs can be cleaved in a nucleotide specific manner, resulting in similar RNA termini.
  • the present invention comprises a composition comprising a single nucleic acid molecule encoding a single RNA molecule comprising a trans- cleaving engineered ribozyme.
  • said trans-cleaving engineered ribozyme is capable of trans-cleaving a separate RNA molecule.
  • said trans-cleaving engineered ribozyme recognizes a specific nucleic acid sequence in the separate RNA molecule.
  • the trans-cleaving engineered ribozyme targets a disease causing mutation for deletion.
  • the disease causing mutation is in an exon.
  • the disease causing mutation is in an intron.
  • the composition comprises two trans-cleaving engineered ribozymes, targeted upstream and downstream of the disease causing mutation.
  • trans-cleavage upstream and downstream of the disease causing mutation results in removal of the disease causing mutation.
  • the remaining portions of the gene are trans-spliced together after trans-cleavage of the disease causing mutation.
  • the trans-spliced gene is expressed as a functional protein.
  • said one or more ribozyme of the composition is capable of spontaneously trans-cleaving one or more target RNA molecule.
  • one or more ribozyme is a 3’ ribozyme.
  • the 3’ ribozyme generates a 3’- phosphate (3’P) or 2’,3’-cyclic phosphate (2’3’ cP) end on the remaining one or more RNA molecule after spontaneous trans-cleavage.
  • one or more ribozyme is a 5’ ribozyme.
  • the 5’ ribozyme generates a 5’OH end on the remaining one or more RNA molecules after spontaneous trans-cleavage.
  • the fusion construct comprises one or more trans-cleaving ribozyme.
  • the trans-cleaving ribozyme is a trans-cleaving hammerhead (HH) ribozyme, or a trans-cleaving Hepatitis delta virus (HDV) ribozyme.
  • the fusion construct comprises at least two linked trans-cleaving hammerhead (HH) ribozymes.
  • the fusion construct comprises at least two linked trans-cleaving Hepatitis Attorney Docket No.204606-0174-00WO delta virus (HDV) ribozymes.
  • the fusion construct comprises a trans- cleaving hammerhead (HH) ribozyme linked to a trans-cleaving Hepatitis delta virus (HDV) ribozyme.
  • the fusion construct comprises a 3’ ribozyme.
  • the 3’ ribozyme is a trans-cleaving variant of a natural or synthetic ribozyme or deoxyribozyme.
  • said 3’ ribozyme is from one or more family selected from the group consisting of: hammerhead (HH), Hepatitis delta virus (HDV), Hairpin (HP), Varkud Satellite (VS), Twister, Twister Sister, Pistol, Hatchet, HOV, or GlmS, or a variant or fragment thereof that has trans-cleaving functionality.
  • the 3’ ribozyme comprises an overhang of one or more nucleotides.
  • the overhang comprises a nucleotide sequence that targets the ribozyme to a sequence upstream of a target cleavage site within a target RNA molecule.
  • the overhang comprises a gRNA.
  • the fusion construct comprises a 5’ ribozyme.
  • the 5’ ribozyme is a trans-cleaving variant of a natural or synthetic ribozyme or deoxyribozyme.
  • said 3’ ribozyme is from one or more family selected from the group consisting of: hammerhead (HH), Hepatitis delta virus (HDV), Hairpin (HP), Varkud Satellite (VS), Twister, Twister Sister, Pistol, Hatchet, HOV, or GlmS, or a variant or fragment thereof that has trans-cleaving functionality.
  • the 5’ ribozyme comprises an overhang of one or more nucleotides.
  • the trans-cleaving HH ribozyme comprises the nucleic acid sequence of SEQ ID NO: 2. In one embodiment, the trans-cleaving HH ribozyme comprises the nucleic acid sequence of SEQ ID NO: 3. In one embodiment, the fusion construct comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 operably linked to a gRNA. In one embodiment, the fusion construct comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 operably linked to one or more additional trans-cleaving ribozyme.
  • the fusion construct comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 operably linked to one or more additional trans-cleaving ribozyme and one or more gRNA sequence.
  • the HH ribozyme is modified to include a tertiary stabilizing motif (TSM).
  • TSM tertiary stabilizing motif
  • one or more ribozyme is a HDV ribozyme, or a variant or fragment thereof that is capable of trans-cleavage.
  • the trans-cleaving HDV ribozyme of the composition comprises SEQ ID NO:4, or a variant or fragment thereof.
  • the fusion construct comprises SEQ ID NO:4 operably linked to a gRNA. In one embodiment, the fusion construct comprises SEQ ID NO:4 operably linked to one or more additional trans-cleaving ribozyme. In one embodiment, the fusion construct comprises SEQ ID NO:4 operably linked to one or more additional trans-cleaving ribozyme and one or more gRNA sequence. In one embodiment, the HDV ribozyme is modified to include a tertiary stabilizing motif (TSM). [0081] In one embodiment, the fusion construct of the composition comprises a sequence of a ribozyme as set forth in any one of SEQ ID NO:1-53 or a variant or fragment thereof that is capable of trans-cleavage.
  • TSM tertiary stabilizing motif
  • the fusion construct comprises at least one of SEQ ID NO: 1-53, or a variant or fragment thereof that is capable of trans-cleavage operably linked to a gRNA. In one embodiment, the fusion construct comprises at least one of SEQ ID NO: 1-53, or a variant or fragment thereof that is capable of trans-cleavage operably linked to one or more additional trans-cleaving ribozyme. In one embodiment, the fusion construct comprises at least one of SEQ ID NO: 1-53, or a variant or fragment thereof that is capable of trans-cleavage operably linked to one or more additional trans-cleaving ribozyme and one or more gRNA sequence.
  • the fusion construct comprises a trans-cleavage ribozyme that has been modified to include a tertiary stabilizing motif (TSM).
  • TSM tertiary stabilizing motif
  • the 3’P or 2’3’ cP end and the 5’OH end of nucleic acid molecules that have undergone ribozyme-mediated cleavage can be ligated together.
  • separated nucleic acid sequences on the same or on separate nucleic acid molecules can be trans- spliced or trans-ligated together.
  • the trans-spliced or trans-ligated nucleic acid molecule expresses a longer protein or fusion protein.
  • the trans-cleaving ribozyme comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least Attorney Docket No.204606-0174-00WO 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:1-53.
  • the trans- cleaving ribozyme comprises a sequence of SEQ ID NO:1. In one embodiment, the trans- cleaving ribozyme comprises a sequence of SEQ ID NO:2. In one embodiment, the trans- cleaving ribozyme comprises a sequence of SEQ ID NO:3. In one embodiment, the trans- cleaving ribozyme comprises a sequence of SEQ ID NO:4.
  • the nucleic acid sequence encoding a fusion construct comprises a nucleic acid sequence comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-53 operably linked to second nucleic acid sequence comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least
  • the trans-cleaving ribozyme protein is a fragment of a trans- cleaving ribozyme.
  • the fragment of the trans-cleaving ribozyme is capable of being complemented with a second fragment of the trans-cleaving ribozyme in trans providing inducible catalytic activity.
  • the fragment of the trans-cleaving ribozyme is a fragment of a trans-cleaving hammerhead (HH) ribozyme, or trans-cleaving ribozyme Hepatitis delta virus (HDV) ribozyme.
  • HH trans-cleaving hammerhead
  • HDV Hepatitis delta virus
  • the fragment of the trans-cleaving ribozyme comprises at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at Attorney Docket No.204606-0174-00WO least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the full length of SEQ ID NO:1-4.
  • the fusion construct comprises one or more DNAzyme (also known as Deoxyribozyme, or DNA enzyme) that cleaves RNA molecules.
  • DNAzyme also known as Deoxyribozyme, or DNA enzyme
  • Exemplary RNA cleaving DNAzymes that can be incorporated into the fusion construct of the invention include, but are not limited to DNAzymes described in Breaker et al., (1994, Chem Biol 1, 223-229) and Wang et al., (2019, Scientific reports 9, 8224.)
  • the DNAzyme is Deoxyribozyme 8–17 or Deoxyribozyme 10-23 or a fragment or variant thereof.
  • the fusion construct comprises one or more DNAzyme that cleaves DNA molecules.
  • Exemplary DNA cleaving DNAzymes that can be incorporated into the fusion construct of the invention include, but are not limited to DNAzymes described in Gu et al., (2013, Journal of the American Chemical Society 135, 9121-9129.)
  • the DNAzyme is deoxyribozyme I-R3 or a fragment or variant thereof.
  • RNA Stabilization [0089]
  • the ribozyme (or DNAzyme) or the fusion construct may contain a modification to increase the stability of the ribozyme (or DNAzyme) or fusion construct.
  • the modification comprises inclusion of a tertiary stabilizing motif (TSM).
  • TSM tertiary stabilizing motif
  • the TSM comprises an amendment to at least one stem loop of a ribozyme (or DNAzyme) that alters the stability of the stem loop.
  • the TSM increases hybridization of the ribozyme or the fusion construct comprising the ribozyme to a target nucleic acid molecule.
  • the ribozyme (or DNAzyme) or the fusion construct may contain a modification which holds two complementary RNA motifs in proximity.
  • the fusion construct comprises a linker.
  • the linker links the gRNA and catalytic RNA.
  • the linker is connected to the 5’ end of the gRNA and to the 3’ end of the catalytic RNA. In one embodiment, the linker is connected to the 3’ end of the catalytic RNA and to the 5’ end of the gRNA.
  • Linkers can be flexible linkers or more rigid linkers.
  • the recombinant expression vectors of the disclosure comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids.
  • the recombinant expression vectors of the invention can be designed for production of RNA molecules in prokaryotic or eukaryotic cells.
  • fusion constructs of the invention can be expressed in bacterial cells such as Escherichia coli, insect cells (using Attorney Docket No.204606-0174-00WO baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
  • a fusion construct of the disclosure is expressed in mammalian cells using a mammalian expression vector.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells and many others.
  • Examples of mammalian expression vectors include pCDM8 (Seed, 1987.
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, adenovirus 2, cytomegalovirus, Rous Sarcoma Virus, and simian virus 40.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987.
  • lymphoid-specific promoters Calame and Eaton, 1988. Adv. Immunol.43: 235-275
  • promoters of T cell receptors Winoto and Baltimore, 1989.
  • EMBO J.8: 729-733 promoters of T cell receptors
  • immunoglobulins Bonerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748
  • neuron-specific promoters e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.
  • pancreas-specific promoters Eslund, et al., 1985. Science 230: 912-916
  • mammary gland-specific promoters e.g., milk whey promoter; U.S. Pat. No.4,873,316 and European Application Publication No.264,166
  • Developmentally- Attorney Docket No.204606-0174-00WO regulated promoters are also encompassed, e.g., the murinehox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev.3: 537-546).
  • the fusion constructs of the invention include targeting nucleic acids, including CRISPR guide RNAs (gRNAs) for targeting the fusion construct or a complex comprising the fusion construct to a target RNA.
  • the targeting nucleic acids is a gRNA.
  • the gRNA comprises guide sequence.
  • the gRNA comprises a direct repeat (DR) sequence.
  • the gRNA comprises a direct repeat sequence and a guide sequence fused or linked to a guide sequence or spacer sequence.
  • the direct repeat sequence may be located upstream (i.e., 5′) from the guide sequence or spacer sequence.
  • the direct repeat sequence may be located downstream (i.e., 3′) from the guide sequence or spacer sequence.
  • the gRNA comprises a stem loop.
  • the gRNA comprises a single stem loop.
  • the direct repeat sequence forms a stem loop.
  • the direct repeat sequence forms a single stem loop.
  • the gRNA is complementary to a target RNA.
  • the fusion construct comprises a catalytic RNA capable of cleaving ssRNA and the gRNA guide sequence comprises a sequence having sufficient complementarity to a sequence adjacent to the target sequence.
  • the RNase is capable of cleaving ssRNA and the crRNA guide sequence comprises a sequence having sufficient complementarity to the target sequence and creating a bulge ssRNA at the target site.
  • the spacer length of the guide RNA is from 15 to 35 nt. In one embodiment, the spacer length of the guide RNA is at least 15 nucleotides.
  • the spacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific cleavage of the polynucleotide.
  • the degree of complementarity between a guide sequence and its corresponding target sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • Burrows-Wheeler Transform e.g. the Burrows Wheeler Aligner
  • ClustalW Clustal X
  • BLAT Novoalign
  • ELAND Illumina, San Diego, Calif.
  • SOAP available at soap.genomics.org.cn
  • Maq available at maq.sourceforge.net.
  • a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Preferably the guide sequence is 1030 nucleotides long. The ability of a guide sequence to direct sequence-specific cleavage by a fused catalytic RNA may be assessed by any suitable assay.
  • the components of a system including the guide sequence to be tested may be provided to a host cell having the corresponding target sequence, followed by an assessment of preferential cleavage of a polynucleotide comprising the target sequence.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a system, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • Other assays are possible, and will occur to those skilled in the art.
  • the degree of complementarity between a guide sequence and its corresponding target sequence can be about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%;
  • a guide or RNA or sgRNA can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length; or guide or RNA or sgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length; and advantageously tracr RNA Attorney Docket No.204606-0174-00WO is 30 or 50 nucleotides in length.
  • an aspect of the invention is to reduce off-target interactions, e.g., reduce the guide interacting with a target sequence having low complementarity.
  • the invention involves mutations that result in the CRISPR-Cas system being able to distinguish between target and off-target sequences that have greater than 80% to about 95% complementarity, e.g., 83%-84% or 88-89% or 94-95% complementarity (for instance, distinguishing between a target having 18 nucleotides from an off-target of 18 nucleotides having 1, 2 or 3 mismatches).
  • the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or 99% or 99.5% or 99.9%, or 100%.
  • Off target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% complementarity between the sequence and the guide, with it advantageous that off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity between the sequence and the guide.
  • the isolated nucleic acid sequences of the disclosure can be obtained using any of the many recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.
  • the isolated nucleic acid 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 protein of the disclosure.
  • the composition comprises an isolated RNA molecule encoding a fusion of the disclosure, 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 Attorney Docket No.204606-0174-00WO 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.
  • nucleic acid molecule may contain at least one modified nucleotide analogue.
  • the ends may be stabilized by incorporating modified nucleotide analogues.
  • nucleotide analogues include sugar- and/or backbone- modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone).
  • the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom.
  • the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group.
  • the 2’ OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.
  • 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.
  • 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 Attorney Docket No.204606-0174-00WO 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: [0111] 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.
  • nucleic acid of the invention may be present at one or more of, a phosphate group, a sugar group, backbone, N-terminus, C-terminus, or nucleobase.
  • the present invention also includes a vector in which the isolated nucleic acid of the present invention is inserted. The art is replete with suitable vectors that are useful in the present invention.
  • the expression of natural or synthetic nucleic acids encoding a protein of the disclosure is typically achieved by operably linking a nucleic acid encoding the protein of the disclosure 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. Nos.5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties.
  • 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. Viral vector technology is well known in the art and is described, for example, in Sambrook et al.
  • 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. No.6,326,193).
  • the nucleic acid encoding one or more fusion construct of the present invention comprises a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 linked to a gRNA.
  • 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.
  • nucleic acids may Attorney Docket No.204606-0174-00WO be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • 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.
  • Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates.
  • Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium.
  • Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a Attorney Docket No.204606-0174-00WO micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated. [0125] Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed.
  • Nanoparticles 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.
  • Nanoparticles [0126]
  • the present disclosure provides fusion constructs, nucleic acids, or a combination thereof of any of the preceding paragraphs formulated in a
  • the , nucleic acids, or a combination thereof is formulated in a lipid nanoparticle.
  • the fusion constructs, nucleic acids, or a combination thereof is formulated in a lipid-polycation complex, referred to as a cationic lipid nanoparticle.
  • the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine.
  • the fusion constructs, nucleic acids, or a combination thereof is formulated in a lipid nanoparticle that includes a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidyl-ethanolamine (DOPE).
  • DOPE dioleoyl phosphatidyl-ethanolamine
  • the lipid nanoparticle comprises at least one ionizable cationic lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)-modified lipid.
  • a lipid nanoparticle formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size.
  • the lipid nanoparticle formulation is composed of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.
  • changing the composition of the cationic lipid can more Attorney Docket No.204606-0174-00WO effectively deliver siRNA to various antigen presenting cells (Basha et al.
  • lipid nanoparticle formulations may comprise 35 to 45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipid and/or 55% to 65% cationic lipid.
  • the ratio of lipid to RNA (e.g., mRNA) in lipid nanoparticles may be 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1 and/or at least 30:1.
  • the ratio of PEG in the lipid nanoparticle formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the lipid nanoparticle formulations.
  • lipid nanoparticle formulations may contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0% and/or 3.0% to 6.0% of the lipid molar ratio of PEG-c-DOMG (R-3-[( ⁇ -methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2- dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) as compared to the cationic lipid, DSPC and cholesterol.
  • PEG-c-DOMG R-3-[( ⁇ -methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2- dimyristyloxypropyl-3-amine
  • the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG (1,2- Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).
  • the cationic lipid may be selected from any lipid known in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.
  • the fusion constructs, nucleic acids, or a combination thereof is formulated as a nanoparticle that comprises at least one lipid selected from, but not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids.
  • the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D- DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids.
  • the amino alcohol cationic lipid may be the lipids described in and/or made by the methods described in U.S. Patent Publication No. US20130150625, herein incorporated by reference in its entirety.
  • the cationic lipid may be 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1- yloxy]-2- ⁇ [(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl ⁇ propan-1-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2 ⁇ [(9Z)-octadec-9-en-1- yloxy]methyl ⁇ propan-1-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca- Attorney Docket No.204606-0174-00WO 9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (Compound 3 in US20130150625); and 2- (dimethyla
  • Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)- non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and further comprise a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid.
  • an ionizable cationic lipid for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-
  • a lipid nanoparticle formulation consists essentially of (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl- [1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of 20-60% cationic
  • a lipid nanoparticle formulation includes 25% to 75% on a molar basis of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)- non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., 35 to 65%, 45 to 65%, 60%, 57.5%, 50% or 40% on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethyl
  • a lipid nanoparticle formulation includes 0.5% to 15% on a molar basis of the neutral lipid, e.g., 3 to 12%, 5 to 10% or 15%, 10%, or 7.5% on a molar basis.
  • neutral lipids include, without limitation, DSPC, POPC, DPPC, DOPE and SM.
  • the formulation includes 5% to 50% on a molar basis of the sterol (e.g., 15 to 45%, 20 to 40%, 40%, 38.5%, 35%, or 31% on a molar basis.
  • a non-limiting example of a sterol is cholesterol.
  • a lipid nanoparticle formulation includes 0.5% to 20% on a molar basis of the PEG or PEG-modified lipid (e.g., 0.5 to 10%, 0.5 to 5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% on a molar basis.
  • a PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da.
  • a Attorney Docket No.204606-0174-00WO PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of less than 2,000, for example around 1,500 Da, around 1,000 Da, or around 500 Da.
  • PEG-modified lipids include PEG-distearoyl glycerol (PEG-DMG) (also referred herein as PEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005) the contents of which are herein incorporated by reference in their entirety).
  • PEG-DMG PEG-distearoyl glycerol
  • PEG-cDMA further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005) the contents of which are herein incorporated by reference in their entirety.
  • the molar lipid ratio is 50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG- DPG), 57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g., DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG- modified lipid, e.g., PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationic lipid/neutral lipid
  • lipid nanoparticle formulations may comprise a cationic lipid, a PEG lipid and a structural lipid and optionally comprise a non-cationic lipid.
  • a lipid nanoparticle may comprise 40-60% of cationic lipid, 5-15% of a non- cationic lipid, 1-2% of a PEG lipid and 30-50% of a structural lipid.
  • the lipid nanoparticle may comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid.
  • a lipid nanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5% structural lipid.
  • the cationic lipid may be any cationic lipid described herein such as, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319. Attorney Docket No.204606-0174-00WO [0139]
  • the lipid nanoparticle formulations described herein may be 4 component lipid nanoparticles.
  • the lipid nanoparticle may comprise a cationic lipid, a non- cationic lipid, a PEG lipid and a structural lipid.
  • the lipid nanoparticle may comprise 40-60% of cationic lipid, 5-15% of a non-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structural lipid.
  • the lipid nanoparticle may comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid.
  • the lipid nanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5% structural lipid.
  • the cationic lipid may be any cationic lipid described herein such as, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.
  • the lipid nanoparticle formulations described herein may comprise a cationic lipid, a non-cationic lipid, a PEG lipid and a structural lipid.
  • the lipid nanoparticle comprise 50% of the cationic lipid DLin-KC2-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of the structural lipid cholesterol.
  • the lipid nanoparticle comprise 50% of the cationic lipid DLin-MC3-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of the structural lipid cholesterol.
  • the lipid nanoparticle comprise 50% of the cationic lipid DLin-MC3-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DMG and 38.5% of the structural lipid cholesterol.
  • the lipid nanoparticle comprise 55% of the cationic lipid L319, 10% of the non-cationic lipid DSPC, 2.5% of the PEG lipid PEG-DMG and 32.5% of the structural lipid cholesterol.
  • a nanoparticle e.g., a lipid nanoparticle
  • a nanoparticle e.g., a lipid nanoparticle
  • the present disclosure includes the use of one or more Cas protein which cleaves RNA to promote ligation or splicing of RNA molecules.
  • the invention relates to use of one or more Cas protein which cleaves RNA resulting in Attorney Docket No.204606-0174-00WO compatible ends for ligation of the cleaved RNA.
  • one or more Cas protein or one or more nucleic acid molecules encoding one or more Cas protein are effectively delivered to a cell in combination with two or more gRNA to provide targeted RNA cleavage and subsequent RNA ligation.
  • the invention provides for cleavage and subsequent RNA ligation of two or more RNA molecules. In some embodiments, the invention provides for cleavage and subsequent ligation of a single RNA molecule, thereby providing a method for generating a spliced RNA molecule.
  • Cas proteins that can be delivered according to the methods of the invention include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2.
  • the Cas protein has DNA or RNA cleavage activity. In some embodiments, the Cas protein directs cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the Cas protein directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. [0144] In one embodiment, the Cas protein has RNA binding activity. In some embodiments, the Cas protein is catalytically active.
  • the Cas protein comprises a mutation in one or both of the HEPN domains. In some embodiments, the Cas protein is catalytically dead. In some embodiments, the catalytically dead Cas protein retains its pre-cRNA processing RNAse activity, and therefore is capable of generating the necessary ends for RNA stitching. [0145] In one embodiment, Cas protein is Cas13.
  • the Cas protein is PspCas13b, PspCas13b Truncation, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, Attorney Docket
  • Additional Cas proteins are known in the art (e.g., Konermann et al., Cell, 2018, 173:665-676 e14, Yan et al., Mol Cell, 2018, 7:327-339 e5; Cox, D.B.T., et al., Science, 2017, 358: 1019-1027; Abudayyeh et al., Nature, 2017, 550: 280-284, Gootenberg et al., Science, 2017, 356: 438-442; and East-Seletsky et al., Mol Cell, 2017, 66: 373-383 e3, which are herein incorporated by reference).
  • the Cas protein is a subtype II-A or subtype II-C Cas protein.
  • Exemplary subtype II-A or subtype II-C Cas proteins include, but are not limited to S. aureus Cas9 (SauCas9) and C. jejuni Cas9 (CjeCas9).
  • the nucleic acid sequence encoding a Cas protein comprises a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 54-101.
  • the nucleic acid sequence encoding a Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of a variant of one of SEQ ID NOs: 54-101, wherein the variant renders the Cas protein catalytically inactive.
  • the catalytically dead Cas protein retains its pre-cRNA processing RNAse activity, and therefore is capable of generating the necessary ends for RNA stitching.
  • the nucleic acid sequence encoding a Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 54-101 having one or more insertions, deletions or substitutions, wherein the one or more insertions, deletions or substitutions renders the Cas protein catalytically inactive.
  • the catalytically dead Cas protein retains its pre-cRNA processing RNAse activity, and therefore is capable of generating the necessary ends for RNA stitching.
  • the nucleic acid sequence encoding a Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 54-101.
  • Localization Signals [0149]
  • the Cas protein is operably linked to a localization signal, such as a nuclear localization signal (NLS), nuclear export signal (NES) or other localization signals to localize to organelles, such as mitochondria.
  • the localization signal localizes the Cas protein to the site in which the target RNA is located.
  • the Cas protein is operably linked to a NLS.
  • the NLS is a retrotransposon NLS.
  • the NLS is derived from Ty1, yeast GAL4, SKI3, L29 or histone H2B proteins, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, Adenovirus El a or DBP protein, influenza virus NS1 protein, hepatitis vims core antigen or the mammalian lamin, c-myc, max, c- myb, p53, c-erbA, jun, Tax, steroid receptor or Mx proteins, Nucleoplasmin (NPM2), Nucleophosmin (NPM1), or simian vims 40 (“SV40”) T-antigen.
  • NPM2 Nucleoplasmin
  • NPM1 Nucleophosmin
  • SV40 simian vims 40
  • the NLS is a Ty1 or Ty1-derived NLS, a Ty2 or Ty2-derived NLS or a MAK11 or MAK11-derived NLS.
  • the NLS comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 111 - 725.
  • the NLS protein comprises a sequence of one of SEQ ID NOs: 111 - 725.
  • Attorney Docket No.204606-0174-00WO Nuclear Export Signal [0152]
  • the Cas protein is operably linked to a Nuclear Export Signal (NES).
  • the NES is attached to the N-terminal end of the Cas protein.
  • the NES localizes the Cas protein to the cytoplasm for targeting cytoplasmic RNA.
  • Organelle Localization Signal [0153]
  • the Cas protein is operably linked to a localization signal that localizes the Cas protein to an organelle.
  • the localization signal localizes the protein to the nucleolus, ribosome, vesicle, rough endoplasmic reticulum, Golgi apparatus , cytoskeleton, smooth endoplasmic reticulum, mitochondria, vacuole, cytosol, lysosome, or centriole.
  • a number of localization signals are known in the art.
  • the Cas protein is operably linked to a localization signal that localizes the Cas protein to an organelle or extracellularly.
  • the present invention provides systems for site specific cleavage of nucleic acid molecules, splicing nucleic acid molecules, ligating nucleic acid molecules, for decreasing the numbers of an RNA transcript in a subject, or any combination thereof.
  • the system comprises a fusion construct comprising one or more ribozyme or DNAzyme.
  • the system comprises one or more fusion construct comprising a gRNA and one or more ribozyme or DNAzyme, wherein the gRNA substantially hybridizes to a target nucleotide sequence.
  • the system comprises a CRISPR-Cas system at least two gRNA and further comprises a Cas protein which cleaves RNA to generate compatible ends for ligation.
  • the CRISPR-Cas system gRNA substantially hybridizes to a target RNA sequence in an RNA transcript.
  • the nucleic acid sequence Attorney Docket No.204606-0174-00WO encoding the Cas protein and the nucleic acid sequence encoding a gRNA are in the same vector.
  • the nucleic acid sequence encoding the Cas protein and the nucleic acid sequence encoding a gRNA are in different vectors.
  • the nucleic acid sequence encoding a fusion construct comprises a nucleic acid sequence comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-4 operably linked to a gRNA.
  • the system comprises, in one or more vectors, a) a nucleic acid sequence encoding a fusion construct, wherein the fusion construct comprises a CRISPR- Cas system gRNA and a catalytic RNA, and optionally a TSM, a linker or a combination thereof and b) a nucleic acid sequence encoding a CRISPR-Cas protein optionally operably linked to a localization signal such as an NLS or NES.
  • the CRISPR-Cas system gRNA substantially hybridizes to a target RNA sequence.
  • the system comprises, in one or more vectors, a nucleic acid sequence encoding a fusion construct, wherein the fusion construct comprises at least two linked gRNA molecules and optionally a linker, a TSM or a combination thereof, a localization signal such as an NLS or NES, and b) a nucleic acid sequence encoding a CRISPR-Cas protein and optionally a linker or a combination thereof and b) a nucleic acid sequence encoding a CRISPR- Cas protein optionally operably linked to a localization signal such as an NLS or NES.
  • each of the two or more CRISPR-Cas system gRNA substantially hybridizes to a target RNA sequence.
  • the two or more target RNA sequences are on the same RNA molecule.
  • the two or more target RNA sequences are on separate RNA molecules.
  • the composition comprises a fusion construct, wherein the fusion construct comprises at least one catalytic RNA, and optionally a gRNA, a tertiary stabilizing motif (TSM), a linker or any combination thereof.
  • the gRNA substantially hybridizes to a target RNA sequence in an RNA transcript.
  • the gRNA substantially hybridizes to a target DNA sequence in a DNA molecule.
  • the composition comprises a fusion construct, wherein the fusion construct comprises two linked catalytic RNA, and optionally a gRNA, a tertiary stabilizing motif (TSM), or a combination thereof.
  • compositions of the disclosure also encompasses the use of pharmaceutical compositions of the disclosure to practice the methods of the disclosure.
  • a pharmaceutical composition may consist of at least one modulator (e.g., inhibitor or activator) composition 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 modulator (e.g., inhibitor or activator) composition 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 compound of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
  • the pharmaceutical compositions useful for practicing the methods of the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In another embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.
  • the relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • compositions that are useful in the methods of the invention may be suitably developed for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration.
  • a composition useful within the methods of the invention may be directly administered to the skin, or any other tissue of a mammal.
  • Other contemplated formulations include liposomal preparations, resealed erythrocytes containing the Attorney Docket No.204606-0174-00WO active ingredient, and immunologically-based formulations.
  • the route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human subject being treated, and the like.
  • the formulations 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.
  • a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound or conjugate of the invention and a pharmaceutically acceptable carrier.
  • compositions that are useful, include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • Attorney Docket No.204606-0174-00WO isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol are included in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
  • the pharmaceutically acceptable carrier is not DMSO alone.
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, vaginal, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like.
  • active agents e.g., other analgesic agents.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • compositions of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition.
  • the preservative is used to prevent spoilage in the case of exposure to contaminants in the environment.
  • preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof.
  • an exemplary preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.
  • the composition includes an anti-oxidant and a chelating agent that inhibits the degradation of the compound.
  • exemplary antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the range of about 0.01% to 0.3% and BHT in the range of 0.03% to 0.1% by weight by total weight of the composition.
  • the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition.
  • Exemplary chelating agents include edetate salts (e.g.
  • disodium edetate and citric acid in the weight range of about 0.01% to 0.20%.
  • the chelating agent is in the range of 0.02% to 0.10% by weight by total weight of the composition.
  • the chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidants and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.
  • Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle.
  • Aqueous vehicles include, for example, water, and isotonic saline.
  • Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents.
  • Oily suspensions may further comprise a thickening agent.
  • suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose.
  • Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively).
  • Known emulsifying agents include, but are not limited to, lecithin, and acacia.
  • Known preservatives include, but are not limited to, Attorney Docket No.204606-0174-00WO methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid.
  • Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.
  • Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.
  • an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.
  • Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent.
  • Aqueous solvents include, for example, water, and isotonic saline.
  • Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto.
  • Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
  • a pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion.
  • the oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these.
  • Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene Attorney Docket No.204606-0174-00WO sorbitan monooleate.
  • These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
  • Methods for impregnating or coating a material with a chemical composition include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the subject either prior to or after a diagnosis of disease.
  • compositions of the present invention may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • Administration of the compositions of the present invention to a subject include a mammal, for example a human, may be carried out using known procedures, at dosages and for periods of time effective to prevent or treat disease.
  • an effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day.
  • the compound may be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two Attorney Docket No.204606-0174-00WO weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
  • a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • compositions of the invention are administered to the subject in dosages that range from one to five times per day or more.
  • compositions of the invention are administered to the subject in range of dosages that include, Attorney Docket No.204606-0174-00WO but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks.
  • Compounds of the invention for administration may be in the range of from about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments there between.
  • the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • the present invention is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound or conjugate of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound or conjugate to treat, prevent, or reduce one or more symptoms of a disease in a subject.
  • the term “container” includes any receptacle for holding the pharmaceutical composition.
  • the container is the packaging that contains the pharmaceutical composition.
  • the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition.
  • packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound’s ability to perform its intended function, e.g., treating or preventing a disease in a subject, or delivering an imaging or diagnostic agent to a subject.
  • Routes of administration of any of the compositions of the invention include oral, nasal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, and (intra)nasal,), intravesical, intraduodenal, intragastrical, rectal, intra-peritoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, or administration.
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.
  • the invention provides methods of site-specific cleavage of nucleic acid molecules, splicing of nucleic acid molecules, ligation of nucleic acid molecules and degradation of nucleic acid molecules.
  • the method comprises administering to a subject a fusion construct of the disclosure comprising at least one catalytic RNA molecule.
  • the method comprises administering to a subject a fusion construct of the disclosure comprising at least one gRNA molecule and at least one CRISPR-Cas protein, or nucleic acid molecule encoding at least one CRISPR-Cas protein for site-specific cleavage of one or more target nucleic acid molecule.
  • the method comprises administering to a subject or a cell at least one fusion construct of the disclosure, or a composition comprising at least one fusion construct of the disclosure.
  • the method is used for site-specific cleavage of cytoplasmic RNA, nuclear RNA, lncRNA, mRNA, genomic DNA, extrachromosomal DNA, or plasmid DNA.
  • the method is useful for generating a spliced nucleic acid molecule.
  • Exemplary spliced nucleic acid molecules may be linear or circular.
  • the spliced nucleic acid molecule is a spliced RNA molecule.
  • the spliced nucleic acid molecule is a spliced DNA molecule.
  • the method is useful for generating a ligated nucleic acid molecule.
  • Exemplary ligated nucleic acid molecules may be linear or circular.
  • the ligated nucleic acid molecule is a ligated RNA molecule.
  • the ligated nucleic acid molecule is a ligated DNA molecule.
  • the fusion construct is administered to a cell.
  • the cell is a prokaryotic cell or eukaryotic cell.
  • the cell is a eukaryotic cell.
  • the cell is a plants, animals, or fungi cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is an animal cell. In one embodiment, the cell is a yeast cell.
  • the subject is a mammal. For example, in one embodiment, the subject is a human, non-human primate, dog, cat, horse, cow, goat, sheep, rabbit, pig, rat, or mouse. In one embodiment, the subject is a non-mammalian subject. For example, in one embodiment, the subject is a zebrafish, fruit fly, or roundworm. Attorney Docket No.204606-0174-00WO [0197] In one embodiment, the amount of nuclear RNA is reduced in vitro.
  • the present invention provides methods of treating a subject with a disease or disorder.
  • the method comprises administering to the subject a fusion construct of the invention comprising at least one catalytic RNA and optionally a gRNA, a TSM or any combination thereof.
  • the method further comprises administering at least one CRISPR-Cas protein to the subject.
  • the CRISPR- Cas protein is operably linked to a localization sequence, such as an NLS or NES.
  • the present invention provides methods of treating a subject with a disease or disorder associated with abnormal nuclear RNA.
  • the disease or disorder associated with abnormal nuclear RNA is selected from the group consisting of Myotonic Dystrophy type 2 (DM2), Amyotrophic lateral sclerosis (ALS), Huntington’s disease- like 2 (HDL2), Spinocerebellar ataxias 8, 31 and 10 (SCA8, -31, -10) and fragile X-associated tremor ataxia syndrome (FXTAS).
  • DM2 Myotonic Dystrophy type 2
  • ALS Amyotrophic lateral sclerosis
  • HDL2 Huntington’s disease- like 2
  • SCA8 -31, -10) Spinocerebellar ataxias 8, 31 and 10
  • FXTAS fragile X-associated tremor ataxia syndrome
  • the abnormal nuclear RNA is toxic nuclear RNA foci.
  • the disease or disorder associated with toxic nuclear RNA foci Myotonic Dystrophy type 1.
  • the targeting nucleotide sequence comprises a sequence complementary to a CTG repeat expansion in the 3’UTR of the human dystrophia myotonica- protein kinase (DMPK) gene.
  • DMPK human dystrophia myotonica- protein kinase
  • the present invention provides methods of treating a disease or disorder associated with increased gene expression.
  • the method comprises administering to the subject administering to the subject a fusion construct of the invention comprising at least one catalytic RNA and optionally a gRNA, a localization sequence, such as an NLS or NES, a TSM or any combination thereof.
  • the guide nucleic acid molecule comprises a targeting nucleotide sequence complimentary to a target RNA sequence in the RNA transcript of the gene or a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the RNA transcript of the gene.
  • the method further comprises administering at least one Cas protein.
  • the present invention provides methods of treating a disease or disorder associated with increased gene expression.
  • the method comprises administering to the subject administering to the subject a fusion construct comprising at least Attorney Docket No.204606-0174-00WO two gRNA molecules.
  • the guide nucleic acid molecule comprises a targeting nucleotide sequence complimentary to a target RNA sequence in the RNA transcript of the gene or a guide nucleic acid molecule comprising a targeting nucleotide sequence complimentary to a target RNA sequence in the RNA transcript of the gene.
  • the method further comprises administering at least one Cas protein which cleaves the RNA transcript thereby preventing translation and protein expression.
  • the present invention provides methods of treating a disease or disorder associated with RNA.
  • the invention provides a method of treating an RNA virus infection.
  • the catalytic RNA cleaves the viral RNA sequence thereby preventing translation and expression of viral protein.
  • the present invention provides methods of treating a disease or disorder associated with DNA.
  • the invention provides a method of treating an DNA virus infection.
  • the catalytic RNA cleaves the viral DNA sequence thereby preventing translation and expression of viral protein.
  • the present invention provides methods of treating, reducing the symptoms of, and/or reducing the risk of developing a disease or disorder in a subject.
  • methods of the invention can be used to treat, reduce the symptoms of, and/or reduce the risk of developing a disease or disorder in a mammal.
  • the methods of the invention can be used to treat, reduce the symptoms of, and/or reduce the risk of developing a disease or disorder in a plant.
  • the methods of the invention can be used treat, reduce the symptoms of, and/or reduce the risk of developing a disease or disorder in a yeast organism.
  • the subject is a cell.
  • the cell is a prokaryotic cell or eukaryotic cell.
  • the cell is a eukaryotic cell.
  • the cell is a plant, animal, or fungi cell.
  • the cell is a plant cell.
  • the cell is an animal cell.
  • the cell is a yeast cell.
  • the subject is a mammal.
  • the subject is a human, non-human primate, dog, cat, horse, cow, goat, sheep, rabbit, pig, rat, or mouse.
  • the subject is a non-mammalian subject.
  • the subject is a zebrafish, fruit fly, or roundworm.
  • Attorney Docket No.204606-0174-00WO [0208]
  • the disease or disorder is caused by one or more mutations in a genomic locus.
  • the disease or disorder may be treated, reduced, or the risk can be reduced via an element that prevents or reduces mRNA transcript, or prevents or reduces translation of the protein.
  • the method comprises manipulation of an RNA transcript.
  • the disease or disorder is caused by abnormal RNA.
  • the disease or disorder may be treated, reduced, or the risk can be reduced via an element that prevents or reduces RNA transcript.
  • the method comprises manipulation of an RNA transcript.
  • the disease or disorder is associated with abnormal RNA or increased RNA transcription.
  • the disease or disorder is an endocrine disease.
  • endocrine diseases include but are not limited to, ⁇ -thalassemias, neonatal diabetes, IPEX syndrome, Mayer–Rokitanski–Küster– Hausersyndrome, Hypothalamic-pituitary-adrenal axis dysregulation, Adrenal dysfunction, Gonadal dysfunction, Ectopic Cushing syndrome, Pre-eclampsia, Diabetic nephropathy, Type I diabetes, Type II diabetes, and IGF-1 deficiency.
  • the disease or disorder is a tumorigenic disease.
  • tumorigenic diseases include but are not limited to, mantle cell lymphoma, hereditary & sporadic parathyroid tumors, Medullary thyroid carcinoma, poliverative conditions, colorectal cancer, gliblastoma, Chronic lymphocytic leukemia, and Breast cancer.
  • the disease or disorder is a neurological disease or disorder.
  • neurological diseases include but are not limited to, Parkinsons diseases, Oculopharyngeal muscular dystrophy, Huntington’s disease, Fabry disease, Fragile X syndrome, spinal muscular atrophy, Amyotrophic Lateral Sclerosis, Spinocerebellar ataxia Spinocerebellar ataxia 1, Spinocerebellar ataxia 2, Spinocerebellar ataxia 3, Spinocerebellar ataxia 6, Spinocerebellar ataxia 7, Spinocerebellar ataxia 8, Spinocerebellar ataxia 10, Spinocerebellar ataxia 17, Spinocerebellar ataxia 31, and Alzheimer’s disease, .
  • the disease or disorder is a hematological disease or disorder.
  • hematological diseases include but are not limited to, ⁇ - Thalassemia, and ⁇ -Thalassemia.
  • Attorney Docket No.204606-0174-00WO [0214]
  • the disease or disorder is an infection or immunological disease or disorder.
  • infection or immunological diseases include but are not limited to, B-cell differentiation, T-cell activation, systemic lupus erythematosus, Wiskott-Aldrich syndrome, Osteoarthritis, scleroderma, and IPEX syndrome.
  • the disease or disorder is a musculoskeletal disease or disorder.
  • infection or immunological diseases include Myotonic dystrophy type 1, Spinal and bulbar muscular atrophy, and Dentatorubral- pallidoluysian atrophy.
  • Exemplary diseases or disorders and corresponding targets include, but are not limited to those listed in Table 1. Additional diseases and disorders and corresponding genes are known in the art, for example in Rehfeld et al., Alternations in Polyadenylation and its Implications for Endocrine Disease, Front.
  • the disease or disorder is may be treated, reduced, or the risk can be reduced via an element that prevents or reduces viral mRNA transcript, or prevents or reduces translation of viral protein.
  • the method comprises manipulation of a viral RNA transcript.
  • the virus is an RNA virus.
  • the virus produces RNA during its lifecycle.
  • the virus is a human virus, a plant virus Attorney Docket No.204606-0174-00WO or an animal virus.
  • viruses include, but are not limited to, viruses of families Adenoviridae, Adenoviridae, Alphaflexiviridae, Anelloviridae, Arenavirus, Arteriviridae, Asfarviridae, Astroviridae, Benyviridae, Betaflexiviridae, Birnaviridae, Bornaviridae, Bromoviridae, Caliciviridae, Caulimoviridae, Circoviridae, Closteroviridae, Coronaviridae, Filoviridae, Flaviviridae, Geminiviridae, Hantaviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Kitaviridae, Luteoviridae, Nairoviridae, Nanoviridae, Nimaviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picornaviridae, Polyomaviridae,
  • Example 1 Precision RNA cleavage and editing using trans-cleaving ribozyme and CRISPR-Cas systems
  • CRISPRase catalytically dead CRISPR-CAS systems fused to active RNAase domains
  • RNA editing applications using either trans-cleaving ribozymes (Huang et al., 2019, Nucleic acids research 47, 2514-2522; Fauzi et al., 1997, Nucleic acids research 25, 3124-3130; Roth et al., 2014, Nat Chem Biol 10, 56-60), trans-cleaving ribozymes as fusions to CRISPR-Cas guide-RNAs, or CRISPR-Cas systems alone.
  • trans-cleaving ribozymes Huang et al., 2019, Nucleic acids research 47, 2514-2522; Fauzi et al., 1997, Nucleic acids research 25, 3124-3130; Roth et al., 2014, Nat Chem Biol 10, 56-60
  • trans-cleaving ribozymes as fusions to CRISPR-Cas guide-RNAs, or CRISPR-Cas systems alone.
  • Cas enzymes such as Cas9 from both subtypes II-A and II-C, recognize and cleave single-stranded RNA (ssRNA) site-specifically, which themselves could be used to induce RNA trans-ligation when targeted to multiple sequences in cis or trans (Strutt et al., 2018, Elife 7, doi:10.7554/eLife.32724).
  • ssRNA single-stranded RNA
  • Sequences S EQ ID NO Description Sequence 1 Trans ⁇ cleaving 3’nnnnnnnnndwaagcaggcgauggccugaguagucnnnnnnnn5’ Hammerhead 2 Trans ⁇ cleaving Extended 3’nnnnnnnndwaagcagcguaagucgcugaguagucnnnnnaatnnnn5’ Hammerhead with TSM (tertiary stabilizing motif) and PLMV catalytic sequence 3 Trans ⁇ cleaving Extended 3’nnnnnnnnndwaagcagcguagggucgcugaguagucnnnnnaaunnnnn5’ Hammerhead with TSM Attorney Docket No.204606-0174-00WO (tertiary stabilizing motif) and RzB catalytic sequence Trans ⁇ cleaving HDV 3’gggccccaggguaagcgguacggcuucguacaacgggunnnnnnn

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Abstract

L'invention concerne des compositions pour le clivage, l'épissage et la ligature spécifiques à un site de molécules d'acide nucléique comprenant de l'ARN et de l'ADN, et des procédés d'utilisation des compositions pour générer des molécules d'acide nucléique épissées ou ligaturées et pour le traitement de maladies.
PCT/US2025/015234 2024-02-09 2025-02-10 Clivage et édition d'arn de précision à l'aide d'un ribozyme trans-clivant et d'arn guides Pending WO2025171380A2 (fr)

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