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WO2023147558A2 - Méthodes crispr pour corriger des mutations du gène bag3 in vivo - Google Patents

Méthodes crispr pour corriger des mutations du gène bag3 in vivo Download PDF

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Publication number
WO2023147558A2
WO2023147558A2 PCT/US2023/061594 US2023061594W WO2023147558A2 WO 2023147558 A2 WO2023147558 A2 WO 2023147558A2 US 2023061594 W US2023061594 W US 2023061594W WO 2023147558 A2 WO2023147558 A2 WO 2023147558A2
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gene
bag3
sequence
nucleic acid
vector
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WO2023147558A3 (fr
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Kamel Khalili
Chen Chen
Shuren Liao
Hong Liu
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Temple Univ School of Medicine
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Temple Univ School of Medicine
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Publication of WO2023147558A3 publication Critical patent/WO2023147558A3/fr
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
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    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Dysregulation of BAG3 by mutations within the gene and/or low level expression of the protein may result in poor quality of several proteins that may cause protein aggregation and results in development of disease in brain and/or heart.
  • SUMMARY [0003] A genetic strategy based on CRISPR gene editing techniques is provided herein, for correcting mutations within the BAG3 gene to be applied in an in vivo system. Methods provided herein are noninvasive, precise, and scalable using a genetic approach. Advantages of this strategy is that it avoids risky procedures for overcoming disease, which in most cases the diseases cannot be cured.
  • a method of diagnosing and treating a subject suspected of having a myopathy comprises identifying in a subject’s biological sample, at least one Bcl2-associated athanogene 3 (BAG3) genetic mutation as compared to a control BAG3 nucleic acid sequence, wherein detection of certain genetic mutations is diagnostic of a myopathy, administering to the subject a therapeutically effective amount of a gene-editing complex, wherein the gene-editing complex corrects the bag3 mutation to a wild-type bag3, thereby, treating the subject.
  • the mutation comprises insertions, deletions, truncation, substitutions or combinations thereof.
  • the mutations encode for a mutated BAG3 polypeptide.
  • the mutation comprises an E455K.
  • the gene editing complex comprises comprising at least one isolated nucleic acid sequences wherein the isolated nucleic acid sequences encode a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and at least one guide RNA (gRNA), the gRNA being complementary to a target sequence in the bag3 gene.
  • the at least one guide RNA comprises a sequence having at least a 90% sequence identity to SEQ ID NOS: 1, 2, or 3.
  • the at least one guide RNA comprises sequences SEQ ID NOS: 1, 2, or 3.
  • a method of treating cancer comprises administering to the subject a therapeutically effective amount of an agent, wherein the agent modulates expression or amount of BAG3 molecules, proteins or peptides thereof in a target cell or tissue, as compared to a normal control, thereby treating cancer.
  • an agent modulates expression or amount of BAG3 molecules, proteins or peptides thereof in a target cell or tissue, as compared to a normal control, thereby treating cancer.
  • increased amounts of BAG3 nucleic acids and/or BAG3 peptides as compared to normal controls are diagnostic of cancer.
  • the cancer comprises melanomas, glioblastomas or adenocarcinomas.
  • the gene editing complex comprises comprising at least one isolated nucleic acid sequences wherein the isolated nucleic acid sequences encode a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)- associated endonuclease and at least one guide RNA (gRNA), the gRNA being complementary to a target sequence in the bag3 gene.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • gRNA guide RNA
  • the gene-editing agent is targeted to bag3 gene, bag3 gene regulatory elements or the combination thereof.
  • the gene regulatory elements comprise promoters, enhancers, initiation codons, stop codons, polyadenylation signals or combinations thereof.
  • a method of treating preventing or treating neurodegeneration in a subject comprises administering Bcl2-associated athanogene 3 (BAG3) polynucleotide, polypeptide and/or agents which induce BAG3 expression or function.
  • BAG3 Bcl2-associated athanogene 3
  • the agent comprises miRNA, dsDNA, lncRNA, siRNA, short hairpin RNAs (shRNAs), antisense oligonucleotide, a phosphorodiamidate morpholino oligomer (PMO), a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), ribozymes, gene-editing complexes, proteins or peptides thereof, peptidomimetics, small molecules, organic or inorganic compounds, synthetic or natural compounds, or combinations thereof.
  • the gene editing complex comprises comprising at least one isolated nucleic acid sequences wherein the isolated nucleic acid sequences encode a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and at least one guide RNA (gRNA), the gRNA being complementary to a target sequence in the bag3 gene.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • gRNA guide RNA
  • the gene- editing agent is targeted to bag3 gene, bag3 gene regulatory elements or the combination thereof.
  • the gene regulatory elements comprise promoters, enhancers, initiation codons, stop codons, polyadenylation signals or combinations thereof.
  • the agent comprises an expression vector expressing a BAG3 protein or active fragments thereof, oligonucleotides or combinations thereof.
  • the expression vector comprises a viral vector, tropic vector, plasmid, or a yeast vector.
  • a neurotropic vector, a cardiotropic vector comprises an adenovirus vector, an adeno-associated virus vector (AAV), a coxsackie virus vector, cytomegalovirus vector, Epstein-Barr virus vector, parvovirus vector, or hepatitis virus vectors.
  • a BAG3 mutation comprises an E455K mutation.
  • an SaCas9 guide comprises a mutation at amino acid 455 comprising GTGATCGAAGAGTATTTGACCAAAGAGC (SEQ ID NO: 1; 455E; underlined bases AAGAG (SEQ ID NO: 4) represents the PAM sequence), GTGATCAAAGAGTATTTGACCAAAGAGC (SEQ ID NO: 2; 455K; underlined bases AAGAG (SEQ ID NO: 4) represents the PAM sequence), TGATCAAAGAGTATTTGACCA (BAG3455K; SEQ ID NO: 3), or the combination thereof.
  • a PAM sequence comprises AAGAG (SEQ ID NO: 4).
  • neurological disorders comprise: AIDS dementia complex, Alzheimer's disease, amyotrophic lateral sclerosis, adrenoleukodystrophy, Alexander disease, Alper's disease, ataxia telangiectasia, Batten disease, bovine spongiform encephalopathy (BSE), Canavan disease, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, fatal familial insomnia, frontotemporal lobar degeneration, Huntington's disease, Kennedy's disease, Krabbe disease, Lyme disease, Machado-Joseph disease, multiple sclerosis, multiple system atrophy, neuroacanthocytosis, Niemann-Pick disease, Parkinson's disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelinoclastic sclerosis, spinocerebellar ataxia, subacute combined degeneration of spinal
  • an element means one element or more than one element.
  • a cell includes a plurality of the cells of the same type.
  • the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”
  • “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +/- 20%, +/- 10%, +/- 5%, +/- 1%, or +/- 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • AAV refers to adeno-associated virus and may be used to refer to the naturally occurring wild-type virus itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise.
  • the AAV genome is built of single stranded DNA and comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames: rep and cap, encoding replication and capsid proteins, respectively.
  • ITRs inverted terminal repeats
  • rep and cap open reading frames
  • capsid proteins encoding replication and capsid proteins, respectively.
  • a foreign polynucleotide can replace the native rep and cap genes.
  • AAVs can be made with a variety of different serotype capsids which have varying transduction profiles or as used herein, “tropism” for different tissue types.
  • serotype refers to an AAV which is identified by and distinguished from other AAVs based on capsid protein reactivity with defined antisera, e.g., AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ or DJ/8.
  • serotype AAV2 is used to refer to an AAV which contains capsid proteins encoded from the cap gene of AAV2 and a genome containing 5' and 3' ITR sequences from the same AAV2 serotype.
  • Pseudotyped AAV refers to an AAV that contains capsid proteins from one serotype and a viral genome including 5'-3' ITRs of a second serotype.
  • BAG1, BAG2, BAG3, etc. isoforms, mutants, cDNA sequences, alleles, fragments, species, coding and noncoding sequences, sense and antisense polynucleotide strands, etc.
  • BAG3 “BAG3 molecules”, “BCL2-associated athanogene 3 (BAG3) genes”, “BCL2-associated athanogene 3 (BAG3) molecules” are inclusive of all family members, isoforms, mutants, cDNA sequences, alleles, fragments, species, coding and noncoding sequences, sense and antisense polynucleotide strands, etc.
  • biological samples include solid and body fluid samples.
  • the biological samples used in the present invention can include cells, protein or membrane extracts of cells, blood or biological fluids such as ascites fluid or brain fluid (e.g., cerebrospinal fluid).
  • solid biological samples include, but are not limited to, samples taken from tissues of the central nervous system, bone, breast, kidney, cervix, endometrium, head/neck, gallbladder, parotid gland, prostate, pituitary gland, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid, heart, lung, bladder, adipose, lymph node, uterus, ovary, adrenal gland, testes, tonsils, thymus and skin, or samples taken from tumors.
  • body fluid samples include, but are not limited to blood, serum, semen, prostate fluid, seminal fluid, urine, feces, saliva, sputum, mucus, bone marrow, lymph, and tears.
  • diagnosis refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
  • detecting may also optionally encompass any of the above.
  • Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease.
  • a “biological sample obtained from the subject” may also optionally comprise a sample that has not been physically removed from the subject.
  • the guide RNA is a chimeric molecule that consists of tracrRNA and crRNA, anteceded by an 18-20-nt spacer sequence complementary to target DNA before a protospacer adjacent motif (PAM).
  • the degree of complementarity 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 novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • 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
  • a guide sequence within a nucleic acid-targeting guide RNA
  • a guide sequence may direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence
  • the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid-targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by Surveyor assay as described herein.
  • preferential targeting e.g., cleavage
  • cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, 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.
  • a guide sequence, and hence a nucleic acid-targeting guide RNA may be selected to target any target nucleic acid sequence.
  • the target sequence may be DNA.
  • the target sequence may be any RNA sequence.
  • the target sequence may be a sequence within an RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), and small cytoplasmic RNA (scRNA).
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA.
  • the target sequence may be a sequence within an RNA molecule selected from the group consisting of ncRNA, and IncRNA. In some embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule. [00023]
  • complementarity refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residue in a second nucleic acid sequence.
  • “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%.97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15.16, 17, 18, 19, 20, 21, 22, 23.24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e. , rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • expression as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.
  • viruses e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses
  • hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson Crick base pairing, Hoogsteen binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these.
  • “Isolated” means altered or removed from the natural state.
  • nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • isolated nucleic acid refers to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences.
  • a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, complementary DNA (cDNA), linear or circular oligomers or polymers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.
  • the nucleic acid sequences may be “chimeric,” that is, composed of different regions.
  • chimeric compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide. These sequences typically comprise at least one region wherein the sequence is modified in order to exhibit one or more desired properties.
  • exogenous indicates that the nucleic acid or polypeptide is part of, or encoded by, a recombinant nucleic acid construct, or is not in its natural environment.
  • an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct.
  • An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism.
  • An exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct.
  • stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found.
  • PAM Protospacer adjacent motif
  • sgRNA single guide RNA
  • the PAMs are the various extended conserved bases at the 5' or 3' end of the protospacer.
  • stringent conditions for hybridization refers to conditions under which a nucleic acid having complementarily to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences.
  • target nucleic acid sequence refers to a nucleic acid (often derived from a biological sample), to which the oligonucleotide is designed to specifically hybridize.
  • the target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding oligonucleotide directed to the target.
  • the term target nucleic acid may refer to the specific subsequence of a larger nucleic acid to which the oligonucleotide is directed or to the overall sequence (e.g., gene or mRNA). The difference in usage will be apparent from context. [00033] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used, “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).
  • the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
  • parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.
  • patient or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred.
  • the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.
  • polynucleotide is a chain of nucleotides, also known as a “nucleic acid”.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art and include both naturally occurring and synthetic nucleic acids.
  • peptide “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • sequence identity or having “a sequence identity” refers to the degree of identity between any given query sequence and a subject sequence.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human, as appropriate.
  • promoter includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance.
  • promoter as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • a “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • a “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.
  • the term “transfected” or “transformed” or “transduced” means to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the transfected/transformed/transduced cell includes the primary subject cell and its progeny.
  • “Treatment” is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures. “Treatment” may also be specified as palliative care. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Accordingly, “treating” or “treatment” of a state, disorder or condition includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human or other mammal that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.
  • “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations.
  • 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 include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • vector includes an autonomously replicating plasmid or a virus.
  • the term is also 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.
  • nucleic acid sequence or an amino acid sequence is specifically referred to by a Swiss Prot. or GENBANK Accession number, the sequence is incorporated herein by reference. Information associated with the accession number, such as identification of signal peptide, extracellular domain, transmembrane domain, promoter sequence and translation start, is also incorporated herein in its entirety by reference.
  • accession number such as identification of signal peptide, extracellular domain, transmembrane domain, promoter sequence and translation start, is also incorporated herein in its entirety by reference.
  • All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice.
  • genes or gene products from a particular species are disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates.
  • the genes disclosed herein which in some embodiments relate to mammalian nucleic acid and amino acid sequences are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds.
  • the genes or nucleic acid sequences are human. Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • FIG.1 is a schematic representation of a construct of generation I BAG3 base editor.
  • FIG.2 is a schematic representation of a construct of generation I BAG3 base editor.
  • FIG.3 shows MEF cells of BAG3 E455K heterozygote mutant.
  • FIG.4 shows results from MEF cells transfected with generation I BAG3 base editor.
  • FIG.5 shows results from MEF cells transfected with generation II BAG3 base editor.
  • DETAILED DESCRIPTION [00061] The following description of certain embodiments is merely exemplary in nature and is in no way intended to limit the invention, its application or uses. Embodiments of the invention may be practiced without the theoretical aspects presented. Moreover, the theoretical aspects are presented with the understanding that Applicant does not seek to be bound by the theory presented. [00062] The human bag3 gene is located on the long arm of chromosome 10 at position 26.11 (10q26.11).
  • Mutations in the bag3 gene are associated with the development of severe diseases, such as for example, myopathies, cancer Age-Related Neurodegenerative Disorders.
  • myopathies include myofibrillar myopathy or dilated cardiomyopathy.
  • the HSP70 co-chaperone BAG3 is found to be upregulated in many human cancers of various origins, for instance in melanomas, glioblastomas, or pancreatic adenocarcinomas.
  • Age-Related Neurodegenerative Disorders such as Amyotrophic Lateral Sclerosis (also known as ALS, Lou Gehrig’s disease, or motor neuron disease), Huntington's disease (HD), Alzheimer’s disease (AD).
  • the BAG3-pathway is also able to remove disease-associated aggregation-prone proteins, such as mutated SOD1 or polyQ43-huntingtin, and is thereby linked to neuroprotection in age- related neurodegenerative disorders, like ALS, HD, or AD.
  • BAG proteins are characterized by a common conserved region located near the C terminus, termed the BAG domain (BD) that mediates direct interaction with the ATPase domain of Hsp70/Hsc70 molecular chaperones.
  • BAG family members have been identified in humans and shown to regulate, both positively and negatively, the function of Hsp70/Hsc70, and to form complexes with a range of transcription factors modulating various physiological processes including apoptosis, tumorigenesis, neuronal differentiation, stress responses, and the cell cycle.
  • BAG proteins generally differ in the N-terminal region, which imparts specificity to particular proteins and pathways. Ubiquitin-like domain at the N terminus of human BAG proteins (BAG1 and BAG6) is probably functionally relevant and conserved in yeast, plants, and worms.
  • BAG proteins regulate diverse physiological processes in animals, including apoptosis, tumorigenesis, neuronal differentiation, stress responses, and the cell cycle.
  • the six human BAG proteins identified are BAG-1 (RAP46/HAP46), BAG-2, BAG-3 (CAIR-1/Bis), BAG-4 (SODD), BAG-5, and BAG-6 (BAT3/Scythe), and all share the signature BD near the C-terminal end, with the exception of BAG 5, which contains four of such domains.
  • the NCBI reference amino acid sequence for BAG3 can be found at Genbank under accession number NP_004272.2; Public GI:14043024.
  • BAG3 The NCBI reference nucleic acid sequence for BAG3 can be found at Genbank under accession number NM_004281.3 GI:62530382.
  • Other BAG3 amino acid sequences include, for example, without limitation, 095817.3 GI:12643665; EAW49383.1 GI:119569768; EAW49382.1 GI:119569767; and CAE55998.1 GI:38502170.
  • the BAG3 polypeptide of the invention can be a variant of a polypeptide described herein, provided it retains functionality.
  • a therapeutic agent for treatment of subjects modulates the expression or amounts of BAG, e.g. BAG3 in a cell.
  • a therapeutic agent for treatment of subjects increases the expression or amounts of BAG, e.g. BAG3 in a cell.
  • compositions comprise nucleic acid sequences complementary to BCL2- associated athanogene 3 (BAG3), including without limitation, cDNA, sense and/or antisense sequences of BAG3.
  • BAG3 BCL2- associated athanogene 3
  • an oligonucleotide comprises at least five consecutive bases complementary to a nucleic acid sequence, wherein the oligonucleotide specifically hybridizes to a target sequence and modulates expression of BAG3 in vivo or in vitro.
  • the oligonucleotides include variants in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenosine, variants may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the oligonucleotide.
  • a method of diagnosing and treating a patient having cardiac disease comprises identifying in a patient sample, at least one Bcl2- associated athanogene 3 (BAG3) genetic variant as compared to a control BAG3 nucleic acid sequence, wherein detection of certain variants are predictive of whether an increase in BAG3 levels is therapeutic for the patient, and, administering to the patient identified as having such a variant, a therapeutically effective amount of an agent wherein the agent modulates expression or amount of BAG3 molecules, proteins or peptides thereof in a target cell or tissue, as compared to a normal control.
  • BAG3 Bcl2- associated athanogene 3
  • a composition comprises a gene-editing complex, wherein mutations to bag3 are edited to correct the mutations to wild type BAG3.
  • bag3 mutations include E455K.
  • the gene editing complex comprises an SaCas9 guide comprises a mutation at amino acid 455 comprising GTGATCGAAGAGTATTTGACCAAAGAGC (SEQ ID NO: 1; 455E; underlined bases AAGAG (SEQ ID NO: 4) represents the PAM sequence), GTGATCAAAGAGTATTTGACCAAAGAGC (SEQ ID NO: 2; 455K; underlined bases AAGAG (SEQ ID NO: 4) represents the PAM sequence), TGATCAAAGAGTATTTGACCA (BAG3455K; SEQ ID NO: 3), or the combination thereof.
  • RNA-guided Cas9 biotechnology induces genome editing without detectable off-target effects.
  • This technique takes advantage of the genome defense mechanisms in bacteria that CRISPR/Cas loci encode RNA-guided adaptive immune systems against mobile genetic elements (viruses, transposable elements and conjugative plasmids).
  • CRISPR clusters contain spacers, the sequences complementary to antecedent mobile elements.
  • CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA).
  • Cas9 belongs to the type II CRISPR/Cas system and has strong endonuclease activity to cut target DNA.
  • Cas9 is guided by a mature crRNA that contains about 20 base pairs (bp) of unique target sequence (called spacer) and a trans-activated small RNA (tracrRNA) that serves as a guide for ribonuclease Ill-aided processing of pre-crRNA.
  • spacer base pairs
  • tracrRNA trans-activated small RNA
  • the crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called the protospacer) on the target DNA (tDNA).
  • Cas9 recognizes a trinucleotide (NGG) protospacer adjacent motif (PAM) to specify the cut site (the 3rd nucleotide from PAM).
  • the crRNA and tracrRNA can be expressed separately or engineered into an artificial fusion small guide RNA (gRNA) via a synthetic stem loop (AGAAAU) to mimic the natural crRNA/tracrRNA duplex.
  • gRNA like shRNA, can be synthesized or in vitro transcribed for direct RNA transfection or expressed from a RNA expression vector (e.g., U6 or Hl promoter-driven vectors).
  • Cas9 gRNA technology requires the expression of the Cas9 protein and gRNA, which then form a gene editing complex at the specific target DNA binding site within the target genome and inflict cleavage/mutation of the target DNA.
  • Cas9 variant system An NGG PAM at the 3 ⁇ end of the target DNA site is essential for the recognization and cleavage of the target gene by Cas9 protein.
  • NGG PAM sites other PAM sites such as NGA and NAG also exist, but their efficiency of genome editing is not high.
  • PAM sites only exist in about one-sixteenth of the human genome, thereby largely restricting the targetable genomic loci.
  • SpCas9-NG which can recognize relaxed NG PAMs (Nishimasu H., Shi X., Ishiguro S., Gao L., Hirano S. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science.2018;361:1259–1262).
  • Miller et al. developed three new SpCas9 variants recognizing non-G PAMs, such as NRRH, NRCH and NRTH PAMs (Miller S.M., Wang T., Randolph P.B., Arbab M., Shen M.W. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat Biotechnol.2020;38:471–481).
  • SpG SpCas9 variant
  • the present disclosure encompasses the use of other CRISPR-associated peptides, which can be targeted to a targeted sequence using a gRNA and can edit to target site of interest.
  • the disclosure utilizes Cpfl to edit the target site of interest.
  • the CRISPR-Cas systems of bacterial and archaeal adaptive immunity show extreme diversity of protein composition and genomic loci architecture.
  • the CRISPR-Cas system loci have more than 50 gene families and there are no strictly universal genes indicating fast evolution and extreme diversity of loci architecture. So far, adopting a multi-pronged approach, there is comprehensive Cas gene identification of about 395 profiles for 93 Cas proteins. Classification includes signature gene profiles plus signatures of locus architecture.
  • CRISPR/Cas proteins comprise at least one RNA recognition and/or RNA binding domain. RNA recognition and/or RNA binding domains interact with guide RNAs. CRISPR/Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains.
  • CRISPR-Cas systems use 2 to 4 nucleotide protospacer-adjacent motifs (PAMs) located next to target sequences for self- versus non-self-discrimination.
  • ARMAN- 1 has a strong ‘NGG’ PAM preference.
  • Cas9 also employs two separate transcripts, CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), for RNA-guided DNA cleavage. Putative tracrRNA was identified in the vicinity of both ARMAN- 1 and ARMAN-4 CRISPR-Cas9 systems (Burstein, D. et al. New CRISPR-Cas systems from uncultivated microbes.
  • the CRISPR/Cas-like protein can be a wild type CRISPR/Cas protein, a modified CRISPR/Cas protein, or a fragment of a wild type or modified CRISPR/Cas protein.
  • the CRISPR/Cas-like protein can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein.
  • nuclease i.e., DNase, RNase
  • nuclease domains of the CRISPR/Cas-like protein can be modified, deleted, or inactivated.
  • the CRISPR/Cas-like protein can be truncated to remove domains that are not essential for the function of the fusion protein.
  • the CRISPR/Cas- like protein can also be truncated or modified to optimize the activity of the effector domain of the fusion protein.
  • the term “Cas” is meant to include all Cas molecules comprising variants, mutants, orthologues, homologues, high-fidelity variants and the like.
  • CRISPR-Cas systems generally refer to an enzyme system that includes a guide RNA sequence that contains a nucleotide sequence complementary or substantially complementary to a region of a target polynucleotide, and a protein with nuclease activity.
  • CRISPR-Cas systems include Type I CRISPR-Cas system, Type II CRISPR-Cas system, Type III CRISPR-Cas system, and derivatives thereof.
  • CRISPR-Cas systems include engineered and/or programmed nuclease systems derived from naturally accruing CRISPR-Cas systems.
  • CRISPR-Cas systems contain engineered and/or mutated Cas proteins.
  • nucleases generally refer to enzymes capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids.
  • endonucleases are generally capable of cleaving the phosphodiester bond within a polynucleotide chain.
  • the CRISPR/Cas system used herein can be a type I, a type II, or a type III system.
  • Non-limiting examples of suitable CRISPR/Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, CasX, Cas ⁇ , Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl6, CsaX, Csx3, C
  • the CRISPR-Cas protein is a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cas9, Cas12 (e.g., Cas12a, Cas12b, Cas12c, Cas12d, Cas12k, Cas12j/ Cas ⁇ , Cas12L etc.
  • Cas12 e.g.
  • the CRISPR/Cas protein or endonuclease is Cas9. In some embodiments, the CRISPR/Cas protein or endonuclease is Cas 12. In certain embodiments, the Cas12 polypeptide is Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas12L or Cas12J. In some embodiments, the CRISPR/Cas protein or endonuclease is CasX. In some embodiments, the CRISPR/Cas protein or endonuclease is CasY.
  • the CRISPR/Cas protein or endonuclease is Cas ⁇ .
  • C2c2 the Class 2 type VI single-component CRISPR-Cas effector Cas 13a, previously known as C2c2 (Shmakov et al. (2015) “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”; Molecular Cell 60: 1-13; doi: dx.doi.Org/10.1016/j.molcel.2015.10.008) was characterized as an RNA-guided RNAse (Abudayyeh et al.
  • C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector”; doi: 10.1126/science.aaf5573). It was demonstrated that C2c2 (e.g. from Leptotrichia shahii) provides robust interference against RNA phage infection. Through in vitro biochemical analysis and in vivo assays, it was shown that C2c2 can be programmed to cleave ssRNA targets carrying protospacers flanked by a 3'H (non-G) PAM.
  • Cleavage is mediated by catalytic residues in the two conserved HEPN domains of C2c2, mutations in which generate a catalytically inactive RNA-binding protein.
  • C2c2 is guided by a single crRNA and can be re-programmed to deplete specific mRNAs in vivo. It was shown that LshC2c2 can be targeted to a specific site of interest and can carry out non-specific RNase activity once primed with the cognate target RNA.
  • C2c2 is now known as Cas 13a. It will be understood that the term “C2c2” herein is used interchangeably with “Cas13a”.
  • Cas13 may refer to Cas13a or Cas 13b or Cas 13c or Cas 13d or other member in the Cas 13 family.
  • the compositions comprise Cas 13.
  • the Cas 13 comprises Cas 13a.
  • the Cas 13 comprises Cas 13b.
  • the Cas 13 comprises Cas 13c.
  • the Cas 13 comprises Cas13d.
  • Cas 13a is the first naturally occurring CRISPR system that targets only RNA.
  • the Class 2 type VI-A CRISPR-Cas effector “Cast 3a” demonstrates an RNA-guided RNase function.
  • Cas13a from the bacterium Leptotrichia shahii provides interference against RNA phage.
  • In vitro biochemical analysis shows that Cas 13a is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers.
  • Cas13a can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA- binding proteins. These results demonstrate the capability of Cas13a as a new RNA -targeting tools.
  • Cas13a can be programmed to cleave particular RNA sequences in bacterial cells.
  • the Cas13 comprises a catalytically inactive Cas effector protein (e.g., dCas13).
  • the catalytically inactive Cas 13 may include truncations of Cas 13 proteins, e.g., at the C-terminus, the N-terminus, or both.
  • the dCas13 may be a catalytically inactive form of any Cas 13 subtype protein.
  • dCas13 may be dCas13a, dCas13b, dCas13c, or dCas13d.
  • the dCas13 may be modified Cas13 effector proteins from Prevotella sp. P5-125, Riemerella anatipestifer, or Porphyromonas gulae.
  • the composition comprises orthologues of Cas13.
  • the terms “orthologue” and “homologue” are well known in the art.
  • a “homologue” of a protein as used herein is a protein of the same species which performs the same or a similar function as the protein it is a homologue of. Homologous proteins may but need not be structurally related or are only partially structurally related.
  • An “orthologue” of a protein as used herein is a protein of a different species which performs the same or a similar function as the protein it is an orthologue of.
  • Orthologous proteins may but need not be structurally related or are only partially structurally related. Homologs and orthologs may be identified by homology modelling (see, e.g., Greer, Science vol. 228 (1985) 1055, and Blundell et al.
  • the Cas 13 gene is found in several diverse bacterial genomes, typically in the same locus with cas1, cas2, and cas4 genes and a CRISPR cassette (for example, FNFX1_1431- FNFX1_1428 of Francisella cf. novicida Fxl).
  • a CRISPR cassette for example, FNFX1_1431- FNFX1_1428 of Francisella cf. novicida Fxl.
  • the layout of this putative novel CRISPR- Cas system appears to be similar to that of type II-B.
  • the Cas13 protein contains a readily identifiable C-terminal region that is homologous to the transposon ORF-B and includes an active RuvC-like nuclease, an arginine-rich region, and a Zn finger (absent in Cas9).
  • Cas13 is also present in several genomes without a CRISPR-Cas context and its relatively high similarity with ORF-B suggests that it might be a transposon component. It was suggested that if this was a genuine CRISPR-Cas system and Cas13 is a functional analog of Cas9 it would be a novel CRISPR-Cas type, namely type V (See Annotation and Classification of CRISPR-Cas Systems. Makarova K S, Koonin E V. Methods Mol Biol. 2015; 1311 :47- 75). [00088] Cas13 has several advantages over other Cas proteins, e.g. Cas9.
  • RNA editing doesn't require homology-directed repair (HDR) machinery and could thus Cas 13 can be used in non-dividing cells.
  • Cas 13 enzymes also do not require a PAM sequence at the target locus, making them more flexible than Cas9/Cpfl.
  • Some Cas13 enzymes prefer targets with a given single base protospacer flanking site (PFS) sequence, but orthologues like LwaCas13a do not require a specific PFS.
  • PFS single base protospacer flanking site
  • the Cas 13 protein is from an organism from a genus comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Leptospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium,
  • the Cas 13 protein is from an organism comprising S. mutans, S. agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C. coli; N. salsuginis, N. tergarcus; S. auricularis, S. carnosus; N. meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C. botulinum, C. difficile, C. tetani, C. sordellii, L inadai, F. tularensis 1, P. albensis, L. bacterium, B.
  • the Cas 13 may comprise a chimeric protein comprising a first fragment from a first protein (e.g., a Cas 13) orthologue and a second fragment from a second (e.g., a Cas13) protein orthologue, and wherein the first and second protein orthologues are different.
  • a first protein e.g., a Cas 13
  • a second protein orthologue e.g., a Cas13
  • At least one of the first and second protein (e.g., a Cas13) orthologues may comprise a Cast 3 from an organism comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus
  • a chimeric protein comprising a first fragment and a second fragment wherein each of the first and second fragments is selected from a Cas13 of an organism comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus,
  • sordellii Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC20171, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW201 l_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp.
  • the Cast 3 protein may be an orthologue of an organism of a genus which includes, but is not limited to Acidaminococcus sp, Lachnospiraceae bacterium or Moraxella bovoculi.
  • the type V Cas protein may be an orthologue of an organism of a species which includes but is not limited to Acidaminococcus sp. BV3L6; Lachnospiraceae bacterium ND2006 (LbCas13) or Moraxella bovoculi 237.
  • the homologue or orthologue of Cas 13 as referred to herein has a sequence homology or identity of at least 80%, or at least 85%, or at least 90%, such as for instance at least 95% with the wild type FnCas13, AsCas13 or LbCas13.
  • the Cas peptide is Cas9.
  • the CRISPR-associated endonuclease Cas9 nuclease can have a nucleotide sequence identical to the wild type Streptococcus pyogenes sequence.
  • the CRISPR-associated endonuclease may be a sequence from other species, for example other Streptococcus species, such as thermophiles.
  • the Cas9 nuclease sequence can be derived from other species including, but not limited to: Nocardiopsis Sanonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens , Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans , Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldiculosiruptor becscii, Candidat
  • Pseudomonas aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microorganisms may also be a source of the Cas9 sequence utilized in the embodiments disclosed herein.
  • the wild type Streptococcus pyogenes Cas9 sequence can be modified.
  • the nucleic acid sequence can be codon optimized for efficient expression in plant cells
  • the Cas9 nuclease sequence can be for example, the sequence contained within a commercially available vector such as PX330 or PX260 from Addgene (Cambridge, MA).
  • the Cas9 endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers KM099231.1 GI:669193757; KM099232.1 GI:669193761; or KM099233.1 GI:669193765 or Cas9 amino acid sequence of PX330 or PX260 (Addgene, Cambridge, MA).
  • the Cas9 nucleotide sequence can be modified to encode biologically active variants of Cas9, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type Cas9 by virtue of containing one or more mutations (e.g., an addition, deletion, or substitution mutation or a combination of such mutations).
  • One or more of the substitution mutations can be a substitution e.g., a conservative amino acid substitution).
  • a biologically active variant of a Cas9 polypeptide can have an amino acid sequence with at least or about 50% sequence identity e.g., at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity) to a wild type Cas9 polypeptide.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.
  • the Cas9 nuclease sequence can be a mutated sequence.
  • the Cas9 nuclease can be mutated in the conserved HNH and RuvC domains, which are involved in strand specific cleavage.
  • an aspartate-to-alanine (D10A) mutation in the RuvC catalytic domain allows the Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA to yield single- stranded breaks, and the subsequent preferential repair through HDR can potentially decrease the frequency of unwanted indel mutations from off-target double-stranded breaks.
  • the Cas9 can be an orthologous. Six smaller Cas9 orthologues have been used and reports have shown that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter.
  • embodiments of the disclosure also encompass CRISPR systems including newly developed “enhanced-specificity” S. pyogenes Cas9 variants (eSpCas9), which dramatically reduce off target cleavage.
  • eSpCas9 variants eSpCas9 variants
  • These variants are engineered with alanine substitutions to neutralize positively charged sites in a groove that interacts with the nontarget strand of DNA. This aim of this modification is to reduce interaction of Cas9 with the non-target strand, thereby encouraging re- hybridization between target and non-target strands.
  • SpCas9 variants comprise one or more point mutations, including, but not limited to R780A, K810A, K848A, K855A, H982A, K1003A, and R1060A (Slaymaker et al., 2016, Science, 351(6268): 84-88).
  • SpCas9 variants comprise DI 135E point mutation (Kleinstiver et al., 2015, Nature, 523(7561): 481-485). In some embodiments, SpCas9 variants comprise one or more point mutations, including, but not limited to N497A, R661A, Q695A, Q926A, DI 135E, L169A, and Y450A (Kleinstiver et al., 2016, Nature, doi:10.1038/naturel6526). In some embodiments, SpCas9 variants comprise one or more point mutations, including but not limited to M495A, M694A, and M698A. Y450 is involved with hydrophobic base pair stacking.
  • SpCas9 variants comprise one or more point mutations at one or more of the following residues: R780, K810, K848, K855, H982, KI 003, R1060, DI 135, N497, R661, Q695, Q926, L169, Y450, M495, M694, and M698.
  • SpCas9 variants comprise one or more point mutations selected from the group of: R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, DI 135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A, and M698A.
  • the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of N497A, R661A, Q695A, and Q926A.
  • the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of N497A, R661A, Q695A, Q926A, and DI 135E. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of N497A, R661A, Q695A, Q926A, and L169A. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of N497A, R661A, Q695A, Q926A, and Y450A.
  • the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of N497A, R661A, Q695A, Q926A, and M495A. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of N497A, R661A, Q695A, Q926A, and M694A. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of N497A, R661A, Q695A, Q926A, and H698A.
  • the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of N497A, R661A, Q695A, Q926A, DI 135E, and L169A. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of N497A, R661A, Q695A, Q926A, DI 135E, and Y450A. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of N497A, R661A, Q695A, Q926A, DI 135E, and M495A.
  • the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, ofN497A, R661A, Q695A, Q926A, D1135E, and M694A. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of N497A, R661A, Q695A, Q926A, DI 135E, and M698A. [00099] In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of R661A, Q695A, and Q926A.
  • the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of R661A, Q695A, Q926A, and DI 135E. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of R661A, Q695A, Q926A, and L169A. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of R661A, Q695A, Q926A, and Y450A.
  • the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of R661A, Q695A, Q926A, and M495A. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of R661A, Q695A, Q926A, and M694A. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of R661A, Q695A, Q926A, and H698A.
  • the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of R661A, Q695A, Q926A, DI 135E, and L169A. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of R661A, Q695A, Q926A, DI 135E, and Y450A. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of R661A, Q695A, Q926A, DI 135E, and M495A.
  • the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of R661A, Q695A, Q926A, DI 135E, and M694A. In some embodiments, the SpCas9 variant comprises the point mutations, relative to wildtype SpCas9, of R661A, Q695A, Q926A, DI 135E, and M698A.
  • SpCas9(K855A), SpCas9(K810A/K1003A/R1060A) (a.k.a.
  • the mutant Cas9 comprises one or more mutations that alter PAM specificity (Kleinstiver et al., 2015, Nature, 523(7561):481 -485; Kleinstiver et al., 2015, Nat Biotechnol, 33(12): 1293-1298).
  • the CRISPR systems comprise phage encoded CRISPR systems.
  • the CRISPR systems comprise phage encoded CRISPR systems.
  • metagenomic analysis of microbial samples isolated from soil, aquatic, human, and animal microbiomes the widespread occurrence of diverse, compact CRISPR-Cas systems encoded in phage genomes, was reported demonstrating an unexpected biological reservoir of anti-viral machinery within infectious agents (Basem Al- Shayeb et al., Diverse virus-encoded CRISPR-Cas systems include streamlined genome editors, Cell, Volume 185, Issue 24, 2022, Pages 4574-4586.e16, ISSN 0092-8674, doi.org/10.1016/j.cell.2022.10.020).
  • Phage-encoded CRISPR-Cas systems include members of all six CRISPR types (types I–VI) as defined by bacterially encoded examples. New evidence was found for new or alternative modes of nucleic acid interference involving phage-encoded type I, III, IV, and VI systems. In addition, the phage and phage-like sequences result in a several-fold expansion of CRISPR-Cas9 and -Cas12 enzymes belonging to the type II and type V families that are widely deployed for genome editing applications. Cas ⁇ , one of the most divergent in sequence of the phage-encoded type V enzymes identified, was found to have robust biochemical activity as an RNA-guided double-stranded DNA (dsDNA) cutter.
  • dsDNA RNA-guided double-stranded DNA
  • cryo-EM cryoelectron microscopy
  • RNA-targeting type III and type VI systems recognize abundant or essential transcripts of competing phages, and type III systems retain the catalytic residues required to cleave ssDNA and enable nicking of the target DNA, but lack components used for non-specific transcript cleavage following RNA target recognition.
  • the minimal trans-cutting of ssDNA and RNA observed for Cas ⁇ implies limited ability to target single-stranded replication intermediates of MGEs.
  • a second important difference between cellular and phage-encoded CRISPR systems is the absence of a processive nuclease, such as Cas3, in some phage type I systems. Together with the presence of CysH, which may be recruited as a putative effector in type IV systems, these observations suggest alternate outcomes to nucleic acid-targeting by these phage-encoded pathways.
  • the lack of a Cas3 nuclease in type I systems targeting plasmid-like elements suggests a gene silencing mechanism that precludes DNA cutting.
  • Such phage-based type I systems could assist the activity of the co- occurring Cas ⁇ systems that are found in the same genome.
  • Phage genomes are a natural reservoir of miniature single-effector CRISPR-Cas systems, including DNA targeting type II and type V enzymes belonging to the Cas9 and Cas12 superfamilies.
  • Cas ⁇ Some of these, such as Cas ⁇ bear sufficient sequence-level divergence to cluster separately from Cas12 and Cas9 systems and obscure a direct evolutionary relationship with known Cas superfamilies. Nonetheless, Cas ⁇ s structure, domain composition, and biochemical mechanism are similar to other type V enzymes. This finding implies that within phage genomes, distinct type V nucleases may have evolved multiple times from ancestral transposon-encoded TnpB families, which also function as RNA-guided nucleases. Despite being from different clades of phages and having divergent sequences and domain organizations, a convergent evolution of Cas12-like architecture in the Cas ⁇ and Cas ⁇ protein structures was observed.
  • both can process their own pre-crRNA and rely on the same RuvC active site used for DNA cleavage for this activity.
  • This extreme compression of enzymatic activities within one active site has not been observed for bacterially encoded CRISPR-Cas proteins. Nonetheless, the phage-encoded enzymes diverge functionally from one another in other ways, including guide RNA structure and maturation process. This may reflect the interplay between rapid phage evolution, which generates diversity, and selective pressure to maintain CRISPR compatibility in a variety of host environments over time, which favors pathway conservation.
  • Base editing is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations in the DNA without generating DSBs.
  • Two major classes of base editors have been developed: cytidine base editors or CBEs allowing C>T conversions and adenine base editors or ABEs allowing A>G conversions.
  • RNA editing system In addition to editing DNA, CRISPR-Cas systems can also edit RNA. Class 2 Type VI CRISPR-Cas13 systems contain a single RNA-guided Cas13 protein with ribonuclease activity, which can bind to target single-stranded RNA (ssRNA) and specifically cleave the target (Abudayyeh O.O., et al. RNA targeting with CRISPR-Cas13. Nature.2017;550:280–284).
  • ssRNA target single-stranded RNA
  • Cas13a also known as C2c2
  • Cas13b also known as C2c2
  • Cas13c and Cas13d
  • O'Connell M.R Molecular mechanisms of RNA targeting by Cas13-containing Type VI CRISPR-Cas systems. J Mol Biol. 2019;431:66–87). They have successfully been applied in RNA knockdown, transcript labeling, splicing regulation and virus detection. Later, Feng Zhang et al.
  • RNA base editing systems REPAIR system, enables A-to-I (G) replacement; RESCUE system, enables C-to-U replacement
  • REPAIR catalytically inactivated Cas13
  • ADAR2 adenosine deaminase acting on RNA type 2
  • ADAR2 adenosine deaminase acting on RNA type 2
  • Cox D.B.T. et al. RNA editing with CRISPR-Cas13. Science.2017;358:1019–1027.
  • Abudayyeh O.O. et al. A cytosine deaminase for programmable single-base RNA editing. Science.2019;365:382–386).
  • RNA editing Compared with DNA editing, RNA editing has the advantages of high efficiency and high specificity. Furthermore, it can make temporary, reversible genetic edits to the genome, avoiding the potential risks and ethical issues caused by permanent genome editing. At present, RNA editing has been widely used for pre-clinical studies of various diseases, which opens a new era for RNA level research, diagnosis and treatment.
  • CRISPR-Cas systems in gene therapy Gene therapy refers to the introduction of foreign genes into target cells to treat specific diseases caused by mutated or defective genes (Naldini L. Gene therapy returns to centre stage. Nature.2015;526:351–360). Target cells of gene therapy are mainly divided into two categories: somatic cells and germ line cells.
  • a gRNA includes a mature crRNA that contains about 20 base pairs (bp) of unique target sequence (called spacer) and a trans-activated small RNA (tracrRNA) that serves as a guide for ribonuclease Ill-aided processing of pre-crRNA.
  • spacer base pairs
  • tracrRNA trans-activated small RNA
  • the crRNA:tracrRNA duplex directs endonuclease to target sequence via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target sequence.
  • the crRNA and tracrRNA can be expressed separately or engineered into an artificial fusion gRNA via a synthetic stem loop (AGAAAU) to mimic the natural crRNA/tracrRNA duplex.
  • gRNA can be synthesized or in vitro transcribed for direct RNA transfection or expressed from U6 or Hl -promoted RNA expression vector.
  • the disclosure encompasses an isolated nucleic acid (e.g., gRNA) having substantial homology to a nucleic acid disclosed herein.
  • the isolated nucleic acid has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with a nucleotide sequence of a gRNA described elsewhere herein.
  • each gRNA includes a sequence that is complementary to a target sequence in bag3.
  • the exemplary target is bag3.
  • a gRNA sequence has at least a 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to each of SEQ ID NOS: 1, 2, or 3.
  • a gRNA sequence has at least a 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity sequence identity to the complementary sequence of to each of SEQ ID NOS: 1, 2, or 3.
  • the gRNAs comprise SEQ ID NOS: 1, 2, or 3.
  • the gRNAs are complementary to SEQ ID NOS: 1, 2, or 3.
  • Guide RNA sequences according to the present disclosure can be sense or antisense sequences. The specific sequence of the gRNA may vary, but, regardless of the sequence, useful guide RNA sequences will be those that minimize off-target effects while achieving high efficiency and specificity of the target gene.
  • the guide RNA sequence can be configured as a single sequence or as a combination of one or more different sequences, e.g., a multiplex configuration. Multiplex configurations can include combinations of two, three, four, five, six, seven, eight, nine, ten, or more different guide RNAs.
  • the guide RNAs can be encoded by a single vector.
  • multiple vectors can be engineered to each include two or more different guide RNAs. Useful configurations will result in the excision of bag3 sequences between cleavage sites.
  • the excised region can vary in size from a single nucleotide to several thousand nucleotides.
  • the length of the guide RNA sequence can vary from about 20 to about 60 or more nucleotides, for example about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, about 60 or more nucleotides.
  • the sequence of the gRNA that is substantially complementary to the target is about 10-30 nucleotides in length.
  • the gRNA comprises a nucleotide sequence that binds to the desired target sequence in the sample.
  • the gRNA comprises a nucleotide sequence that is substantially complementary to the target sequence, and thus binds to the target sequence.
  • the target DNA typically immediately precedes a 5’-NGG proto-spacer adjacent motif (PAM).
  • PAM proto-spacer adjacent motif
  • Other Cas9 orthologs may have different PAM specificities.
  • Cas9 from S. thermophilus requires 5’-NNAGAA for CRISPR-1 and 5’-NGGNG for CRISPR-3 and Neisseria meningiditis requires 5’- NNNNGATT).
  • the specific sequence of the guide RNA may vary, but, regardless of the sequence, useful guide RNA sequences will be those that minimize off-target effects while achieving high efficiency editing of bag3 target sequence(s).
  • the specific sequence of the guide RNA may vary, but, regardless of the sequence, useful guide RNA sequences will be those that minimize off-target effects while achieving high efficiency editing of bag3.
  • the length of the guide RNA sequence can vary from about 20 to about 60 or more nucleotides, for example about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, about 60 or more nucleotides.
  • Useful selection methods identify regions having extremely low homology between the foreign viral genome and host cellular genome, include bioinformatic screening using target sequence+NGG target- selection criteria to exclude off-target human transcriptome or (even rarely) untranslated- genomic sites, and WGS, Sanger sequencing and SURVEYOR assay, to identify and exclude potential off-target effects.
  • Algorithms such as CRISPR Design Tool (CRISPR Genome Engineering Resources; Broad Institute) can be used to identify target sequences with or near requisite PAM sequences as defined by the type of Cas peptide (e.g. Cas13, Cas9, Cas9 variant, Cpfl) used.
  • the composition comprises multiple different gRNAs, each targeted to a different target sequence.
  • this multiplexed strategy provides for increased efficacy.
  • the compositions described herein utilize about 1 gRNA to about 6 gRNAs. In some embodiments, the compositions described herein utilize at least about 1 gRNA. In some embodiments, the compositions described herein utilize at most about 6 gRNAs.
  • the compositions described herein utilize about 1 gRNA to about 2 gRNAs, about 1 gRNA to about 3 gRNAs, about 1 gRNA to about 4 gRNAs, about 1 gRNA to about 5 gRNAs, about 1 gRNA to about 6 gRNAs, about 2 gRNAs to about 3 gRNAs, about 2 gRNAs to about 4 gRNAs, about 2 gRNAs to about 5 gRNAs, about 2 gRNAs to about 6 gRNAs, about 3 gRNAs to about 4 gRNAs, about 3 gRNAs to about 5 gRNAs, about 3 gRNAs to about 6 gRNAs, about 4 gRNAs to about 5 gRNAs, about 4 gRNAs to about 6 gRNAs, or about 5 gRNAs to about 6 gRNAs.
  • the compositions described herein utilize about 1 gRNA, about 2 gRNAs, about 3 gRNAs, about 4 gRNAs, about 5 gRNAs, or about 6 gRNAs.
  • the CRISPR endonuclease can be encoded by the same nucleic acid or vector as the guide RNA sequences. Alternatively, or in addition, the CRISPR endonuclease can be encoded in a physically separate nucleic acid from the guide RNA sequences or in a separate vector.
  • the RNA molecules e.g.
  • crRNA, tracrRNA, gRNA are engineered to comprise one or more modified nucleobases.
  • known modifications of RNA molecules can be found, for example, in Genes VI, Chapter 9 (“Interpreting the Genetic Code”), Lewis, ed. (1997, Oxford University Press, New York), and Modification and Editing of RNA, Grosjean and Benne, eds. (1998, ASM Press, Washington DC).
  • Modified RNA components include the following: 2'-O- methylcytidine; N 4 -methylcytidine; N 4 -2'-O-dimethylcytidine; N 4 - acetylcytidine; 5- methylcytidine; 5, 2'-O-dimethyl cytidine; 5 -hydroxymethyl cytidine; 5- formylcytidine; 2'- O-methyl-5-formaylcytidine; 3 -methylcytidine; 2-thiocytidine; lysidine; 2'- O- methyluridine; 2-thiouridine; 2-thio-2'-O-methyluridine; 3,2'-O-dimethyluridine; 3-(3- amino- 3- carboxypropyl)uridine; 4-thiouridine; ribosylthymine; 5,2'-O-dimethyluridine; 5- methyl-2- thiouridine; 5 -hydroxyuridine; 5-methoxyuridine; uridine 5-oxy
  • Isolated nucleic acid molecules can be produced by standard techniques. For example, PCR techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein, including nucleotide sequences encoding a polypeptide described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995.
  • sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified.
  • Various PCR strategies also are available by which site specific nucleotide sequence modifications can be introduced into a template nucleic acid.
  • Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides.
  • one or more pairs of long oligonucleotides can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed.
  • DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.
  • Isolated nucleic acids of the disclosure also can be obtained by mutagenesis of, e.g., a naturally occurring portion of a Cas13 -encoding DNA.
  • the gRNA is a synthetic oligonucleotide.
  • the synthetic nucleotide comprises a modified nucleotide. Modification of the inter-nucleoside linker (i.e. backbone) can be utilized to increase stability or pharmacodynamic properties. For example, inter-nucleoside linker modifications prevent or reduce degradation by cellular nucleases, thus increasing the pharmacokinetics and bioavailability of the gRNA.
  • a modified inter-nucleoside linker includes any linker other than other than phosphodiester (PO) liners, that covalently couples two nucleosides together.
  • the modified inter-nucleoside linker increases the nuclease resistance of the gRNA compared to a phosphodiester linker.
  • the inter- nucleoside linker includes phosphate groups creating a phosphodiester bond between adjacent nucleosides.
  • the gRNA comprises one or more inter-nucleoside linkers modified from the natural phosphodiester.
  • inter-nucleoside linkers of the gRNA, or contiguous nucleotide sequence thereof are modified.
  • the inter- nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage.
  • S Sulphur
  • Modifications to the ribose sugar or nucleobase can also be utilized herein.
  • a modified nucleoside includes the introduction of one or more modifications of the sugar moiety or the nucleobase moiety.
  • the gRNAs comprise one or more nucleosides comprising a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety when compared to the ribose sugar moiety found in deoxyribose nucleic acid (DNA) and RNA.
  • DNA deoxyribose nucleic acid
  • RNA RNA-derived nucleic acid
  • Numerous nucleosides with modification of the ribose sugar moiety can be utilized, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or stability. Such modifications include those where the ribose ring structure is modified.
  • HNA hexose ring
  • LNA locked nucleic acids
  • UNA unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons
  • Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids or tricyclic nucleic acids.
  • Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.
  • Sugar modifications also include modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2'-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2', 3', 4' or 5' positions.
  • Nucleosides with modified sugar moieties also include 2' modified nucleosides, such as 2' substituted nucleosides.
  • a 2' sugar modified nucleoside is a nucleoside that has a substituent other than H or — OH at the 2' position (2' substituted nucleoside) or comprises a 2' linked biradicle and includes 2' substituted nucleosides and LNA (2'-4' biradicle bridged) nucleosides.
  • 2' substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O- methoxyethyl- RNA (MOE), 2'-amino-DNA, 2'-Fluoro-RNA, and 2'-F-ANA nucleoside.
  • the modification in the ribose group comprises a modification at the 2' position of the ribose group.
  • the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'- deoxy, and 2'-O-(2-methoxyethyl).
  • the gRNA comprises one or more modified sugars. In some embodiments, the gRNA comprises only modified sugars. In certain embodiments, the gRNA comprises greater than 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2'-O-methoxyethyl group.
  • the gRNA comprises both inter- nucleoside linker modifications and nucleoside modifications.
  • Target specificity can be used in reference to a guide RNA, or a crRNA specific to a target polynucleotide sequence or region (e.g, SEQ ID NOS: 1, 2, or 3) and further includes a sequence of nucleotides capable of selectively annealing/hybridizing to a target (sequence or region) of a target polynucleotide (e.g. corresponding to a target), e.g., a target DNA.
  • a crRNA or the derivative thereof contains a target-specific nucleotide region complementary to a region of the target DNA sequence.
  • a crRNA or the derivative thereof contains other nucleotide sequences besides a target-specific nucleotide region.
  • the other nucleotide sequences are from a tracrRNA sequence.
  • gRNAs are generally supported by a scaffold, wherein a scaffold refers to the portions of gRNA or crRNA molecules comprising sequences which are substantially identical or are highly conserved across natural biological species (e.g. not conferring target specificity). Scaffolds include the tracrRNA segment and the portion of the crRNA segment other than the polynucleotide -targeting guide sequence at or near the 5' end of the crRNA segment, excluding any unnatural portions comprising sequences not conserved in native crRNAs and tracrRNAs.
  • the crRNA or tracrRNA comprises a modified sequence. In certain embodiments, the crRNA or tracrRNA comprises at least 1, 2, 3, 4, 5, 10, or 15 modified bases (e.g. a modified native base sequence).
  • Complementary generally refers to a polynucleotide that includes a nucleotide sequence capable of selectively annealing to an identifying region of a target polynucleotide under certain conditions.
  • the term “substantially complementary” and grammatical equivalents is intended to mean a polynucleotide that includes a nucleotide sequence capable of specifically annealing to an identifying region of a target polynucleotide under certain conditions.
  • Annealing refers to the nucleotide base-pairing interaction of one nucleic acid with another nucleic acid that results in the formation of a duplex, triplex, or other higher-ordered structure.
  • the primary interaction is typically nucleotide base specific, e.g., A:T, A:U, and G:C, by Watson-Crick and Hoogsteen-type hydrogen bonding.
  • base-stacking and hydrophobic interactions can also contribute to duplex stability.
  • Hybridization generally refers to process in which two single- stranded polynucleotides bind non-covalently to form a stable double stranded polynucleotide.
  • a resulting double-stranded polynucleotide is a “hybrid” or “duplex.”
  • 100% sequence identity is not required for hybridization and, in certain embodiments, hybridization occurs at about greater than 70%, 75%, 80%, 85%, 90%, or 95% sequence identity.
  • sequence identity includes in addition to non-identical nucleobases, sequences comprising insertions and/or deletions.
  • the nucleic acid of the disclosure including the RNA (e.g., crRNA, tracrRNA, gRNA) or nucleic acids encoding the RNA, may be produced by standard techniques.
  • PCR polymerase chain reaction
  • isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid e.g., using automated DNA synthesis in the 3’ to 5’ direction using phosphoramidite technology) or as a series of oligonucleotides.
  • Isolated nucleic acids of the disclosure also can be obtained by mutagenesis of, e.g., a naturally occurring portion crRNA, tracrRNA, RNA- encoding DNA, or of a Cas -encoding DNA.
  • the isolated RNA are synthesized from an expression vector encoding the RNA molecule, as described in detail elsewhere herein.
  • Nucleic Acids and Vectors [000129]
  • the composition of the disclosure comprises an isolated nucleic acid encoding one or more elements of the CRISPR-Cas system described herein.
  • the composition comprises an isolated nucleic acid encoding at least one guide nucleic acid (e.g., gRNA).
  • the composition comprises an isolated nucleic acid encoding a Cas peptide, or functional fragment or derivative thereof.
  • the composition comprises an isolated nucleic acid encoding at least one guide nucleic acid (e.g., gRNA) and encoding a Cas peptide, or functional fragment or derivative thereof.
  • the composition comprises an isolated nucleic acid encoding at least one guide nucleic acid (e.g., gRNA) and further comprises an isolated nucleic acid encoding a Cas peptide, or functional fragment or derivative thereof.
  • the composition comprises at least one isolated nucleic acid encoding a gRNA, where the gRNA is substantially complementary to a target sequence of the bag3, as described elsewhere herein.
  • the composition comprises at least one isolated nucleic acid encoding a gRNA, where the gRNA is complementary to a target sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to a target sequence described herein.
  • the composition comprises at least one isolated nucleic acid encoding a Cas peptide described elsewhere herein, or a functional fragment or derivative thereof.
  • the composition comprises at least one isolated nucleic acid encoding a Cas peptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence homology with a Cas peptide described elsewhere herein.
  • 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, including for example, an isolated cDNA, encoding a gRNA or peptide of the disclosure, or functional fragment thereof.
  • the composition comprises an isolated RNA encoding a peptide of the disclosure, or a functional fragment thereof.
  • the isolated nucleic acids may be synthesized using any method known in the art.
  • the present disclosure can comprise use of a vector in which the isolated nucleic acid described herein is inserted.
  • the art is replete with suitable vectors that are useful in the present disclosure.
  • Vectors include, for example, viral vectors (such as adenoviruses (“Ad”), adeno-associated viruses (AAV), and vesicular stomatitis virus (VSV) and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell.
  • Ad adenoviruses
  • AAV adeno-associated viruses
  • VSV vesicular stomatitis virus
  • retroviruses vesicular stomatitis virus
  • Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells.
  • Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.
  • Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities.
  • Other vectors include those described by Chen et al. BioTechniques, 34: 167-171 (2003). A large variety of such vectors is known in the art and is generally available.
  • the expression of natural or synthetic nucleic acids encoding an RNA and/or peptide is typically achieved by operably linking a nucleic acid encoding the RNA and/or peptide 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, enhancers and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the vectors of the present disclosure 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 disclosure provides a gene therapy vector. [000135]
  • the isolated nucleic acid of the disclosure 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. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
  • 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).
  • a number of viral based systems have been developed for gene transfer into mammalian cells.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • the composition includes a vector derived from an adeno-associated virus (AAV).
  • AAV adeno-associated viral vectors have become powerful gene delivery tools for the treatment of various disorders.
  • AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner.
  • AAV vectors comprising nucleic acids encoding the CRISPR-Cas systems described herein.
  • an AAV vector includes to any vector that comprises or derives from components of AAV and is suitable to infect mammalian cells, including human cells, of any of a number of tissue types, such as brain, heart, lung, skeletal muscle, liver, kidney, spleen, or pancreas, whether in vitro or in vivo.
  • an AAV vector includes an AAV type viral particle (or virion) comprising a nucleic acid encoding a protein of interest (e.g. CRISPR-Cas systems described herein).
  • the AAVs disclosed herein are be derived from various serotypes, including combinations of serotypes (e.g., “pseudotyped” AAV) or from various genomes (e.g., single-stranded or self-complementary).
  • the AAV vector is a human serotype AAV vector.
  • a human serotype AAV is derived from any known serotype, e.g., from AAV1, AAV2, AAV4, AAV6, or AAV9.
  • the serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVDJ, or AAVDJ/8.
  • the composition includes a vector derived from an adeno- associated virus (AAV).
  • AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.
  • a variety of different AAV capsids have been described and can be used, although AAV which preferentially target the liver and/or deliver genes with high efficiency are particularly desired.
  • the sequences of the AAV8 are available from a variety of databases. While the examples utilize AAV vectors having the same capsid, the capsid of the gene editing vector and the AAV targeting vector are the same AAV capsid.
  • Another suitable AAV is, e.g., rh10 (WO 2003/042397).
  • Still other AAV sources include, e.g., AAV9 (see, for example, U.S. Pat. No.7,906,111; US 2011-0236353-Al), and/or hu37 (see, e.g., U.S. Pat.
  • Still other AAV can be selected, optionally taking into consideration tissue preferences of the selected AAV capsid.
  • AAV vectors disclosed herein include a nucleic acid encoding a CRISPR-Cas systems described herein.
  • the nucleic acid also includes one or more regulatory sequences allowing expression and, in some embodiments, secretion of the protein of interest, such as e.g., a promoter, enhancer, polyadenylation signal, an internal ribosome entry site (“IRES”), a sequence encoding a protein transduction domain (“PTD”), and the like.
  • the nucleic acid comprises a promoter region operably linked to the coding sequence to cause or improve expression of the protein of interest in infected cells.
  • Such a promoter can be ubiquitous, cell- or tissue-specific, strong, weak, regulated, chimeric, etc., for example, to allow efficient and stable production of the protein in the infected tissue.
  • the promoter is homologous to the encoded protein, or heterologous, although generally promoters of use in the disclosed methods are functional in human cells.
  • regulated promoters include, without limitation, Tet on/off element containing promoters, rapamycin- inducible promoters, tamoxifen-inducible promoters, and metallothionein promoters.
  • other promoters used include promoters that are tissue specific for tissues such as kidney, spleen, and pancreas.
  • the recombinant AAV vector comprises packaged within an AAV capsid, a nucleic acid, generally containing a 5' AAV ITR, the expression cassettes described herein and a 3' AAV ITR.
  • an expression cassette contains regulatory elements for an open reading frame(s) within each expression cassette and the nucleic acid optionally contains additional regulatory elements.
  • the AAV vector in some embodiments, comprises a full-length AAV 5' inverted terminal repeat (ITR) and a full-length 3' ITR.
  • ITR inverted terminal repeat
  • AITR A shortened version of the 5' ITR, termed AITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted.
  • trs terminal resolution site
  • the abbreviation “sc” refers to self-complementary.
  • Self- complementary AAV refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra- molecular double-stranded DNA template.
  • scAAV double stranded DNA
  • the two complementary halves of scAAV Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription
  • dsDNA double stranded DNA
  • scAAV Self-complementary recombinant adeno-associated virus
  • the ITRs are selected from a source which differs from the AAV source of the capsid.
  • AAV2 ITRs are selected for use with an AAV capsid having a particular efficiency for a selected cellular receptor, target tissue or viral target.
  • the ITR sequences from AAV2, or the deleted version thereof (AITR) are used for convenience and to accelerate regulatory approval (i.e. pseudotyped).
  • a single-stranded AAV viral vector is used.
  • a producer cell line is transiently transfected with a construct that encodes the transgene flanked by ITRs and a construct(s) that encodes rep and cap.
  • a packaging cell line that stably supplies rep and cap is transfected (transiently or stably) with a construct encoding the transgene flanked by ITRs.
  • AAV virions are produced in response to infection with helper adenovirus or herpesvirus, requiring the separation of the rAAVs from contaminating virus.
  • helper adenovirus or herpesvirus More recently, systems have been developed that do not require infection with helper virus to recover the AAV - the required helper functions (i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied, in trans, by the system.
  • helper functions can be supplied by transient transfection of the cells with constructs that encode the required helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or posttranscriptional level.
  • the transgene flanked by ITRs and rep/cap genes are introduced into insect cells by infection with baculovirus-based vectors.
  • the CRISPR-Cas systems, and/or any of the present RNAs, for instance a guide RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other viral vector types, or combinations thereof.
  • AAV adeno associated virus
  • an endonuclease and one or more guide RNAs can be packaged into one or more viral vectors.
  • the viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the viral delivery is via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery can be either via a single dose, or multiple doses.
  • the actual dosage to be delivered herein can vary greatly depending upon a variety of factors, such as the vector chose, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc.
  • Pox viral vectors introduce the gene into the cells cytoplasm.
  • Avipox virus vectors result in only a short term expression of the nucleic acid.
  • Adenovirus vectors, adeno- associated virus vectors and herpes simplex virus (HSV) vectors may be an indication for some embodiments.
  • the adenovirus vector results in a shorter term expression (e.g., less than about a month) than adeno-associated virus, in some embodiments, may exhibit much longer expression.
  • the particular vector chosen will depend upon the target cell and the condition being treated.
  • the vector also includes conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the disclosure.
  • operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • polyA polyadenylation
  • a great number of expression control sequences, including promoters which are native, constitutive, inducible and/or tissue- specific, are known in the art and may be utilized.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
  • promoters typically contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • the Rous sarcoma virus (RSV) and MMT promoters may also be used.
  • Certain proteins can be expressed using their native promoter.
  • Other elements that can enhance expression can also be included such as an enhancer or a system that results in high levels of expression such as a tat gene and tar element.
  • This cassette can then be inserted into a vector, e.g., a plasmid vector such as, pUC19, pUCl 18, pBR322, or other known plasmid vectors, that includes, for example, an E. coli origin of replication.
  • a vector e.g., a plasmid vector such as, pUC19, pUCl 18, pBR322, or other known plasmid vectors, that includes, for example, an E. coli origin of replication.
  • a suitable promoter is Elongation Growth Factor -la (EF-la).
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatinine kinase promoter. Further, the disclosure should not be limited to the use of constitutive promoters.
  • Inducible promoters are also contemplated as part of the disclosure.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • Enhancer sequences found on a vector also regulates expression of the gene contained therein. Typically, enhancers are bound with protein factors to enhance the transcription of a gene. Enhancers may be located upstream or downstream of the gene it regulates.
  • Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type.
  • the vector of the present disclosure comprises one or more enhancers to boost transcription of the gene present within the vector.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, micro injection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
  • a preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • an exemplary delivery vehicle is a liposome.
  • the use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/ expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources.
  • dimyristyl phosphatidylcholine can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL).
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated.
  • compositions comprise, 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 disclosure.
  • “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 disclosure.
  • ELISAs and Western blots immunological means
  • assays described herein to identify agents falling within the scope of the disclosure.
  • the cell may be transfected or transformed with one or more vectors comprising an isolated nucleic acid sequence encoding a gRNA and/or a Cas peptide.
  • the cell can be the subject’s cells or they can be haplotype matched or a cell line.
  • the cells can be irradiated to prevent replication.
  • the cells are human leukocyte antigen (HLA)-matched, autologous, cell lines, or combinations thereof.
  • the cells can be a stem cell.
  • Embryonic stem cells and artificial pluripotent stem cells (induced pluripotent stem cell, iPS cells) have been established from many animal species, including humans. These types of pluripotent stem cells would be the most useful source of cells for regenerative medicine because these cells are capable of differentiation into almost all of the organs by appropriate induction of their differentiation, with retaining their ability of actively dividing while maintaining their pluripotency.
  • iPS cells in particular, can be established from self-derived somatic cells, and therefore are not likely to cause ethical and social issues, in comparison with ES cells which are produced by destruction of embryos. Further, iPS cells, which are a selfderived cell, make it possible to avoid rejection reactions, which are the biggest obstacle to regenerative medicine or transplantation therapy.
  • Delivery vehicles as used herein include any types of molecules for delivery of the compositions embodied herein, both for in vitro or in vivo delivery. Examples, include, without limitation: expression vectors, nanoparticles, colloidal compositions, lipids, liposomes, nanosomes, carbohydrates, organic or inorganic compositions and the like. Any suitable method can be used to deliver the compositions to the subject.
  • the nucleases e.g. CRISPR/Cas, or the genes encoding the nuclease may be delivered to systematic circulation or may be delivered or otherwise localized to a specific tissue type.
  • a delivery vehicle is an expression vector, wherein the expression vector encodes a desired nucleic acid sequence.
  • the vector comprises an isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and at least one guide RNA (gRNA), the gRNA being complementary to a target nucleic acid sequence in subject’s genome.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • gRNA guide RNA
  • the compositions of the disclosure can be formulated as a nanoparticle, for example, nanoparticles comprised of a core of high molecular weight linear polyethylenimine (LPEI) complexed with DNA and surrounded by a shell of polyethyleneglycol modified (PEGylated) low molecular weight LPEI.
  • LPEI high molecular weight linear polyethylenimine
  • PEGylated polyethyleneglycol modified
  • L-PEI has been used to efficiently deliver genes in vivo into a wide range of organs such as lung, brain, pancreas, retina, bladder as well as tumor.
  • liposomes are used to effectuate transfection into a cell or tissue.
  • the pharmacology of a liposomal formulation of nucleic acid is largely determined by the extent to which the nucleic acid is encapsulated inside the liposome bilayer. Encapsulated nucleic acid is protected from nuclease degradation, while those merely associated with the surface of the liposome is not protected.
  • Encapsulated nucleic acid shares the extended circulation lifetime and biodistribution of the intact liposome, while those that are surface associated adopt the pharmacology of naked nucleic acid once they disassociate from the liposome.
  • Nucleic acids may be entrapped within liposomes with conventional passive loading technologies, such as ethanol drop method (as in SALP), reverse-phase evaporation method, and ethanol dilution method (as in SNALP).
  • SALP ethanol drop method
  • SNALP ethanol dilution method
  • Liposomal delivery systems provide stable formulation, provide improved pharmacokinetics, and a degree of 'passive' or 'physiological' targeting to tissues.
  • Encapsulation of hydrophilic and hydrophobic materials, such as potential chemotherapy agents, are known. See for example U.S. Pat.
  • the complexes of the disclosure are coupled to or encapsulated in polymersomes.
  • polymersomes are tiny hollow spheres that enclose a solution, made using amphiphilic synthetic block copolymers to form the vesicle membrane.
  • Common polymersomes contain an aqueous solution in their core and are useful for encapsulating and protecting sensitive molecules, such as drugs, enzymes, other proteins and peptides, and DNA and RNA fragments.
  • the polymersome membrane provides a physical barrier that isolates the encapsulated material from external materials, such as those found in biological systems.
  • Non- viral vectors are modified to effectuate targeted delivery and transfection.
  • PEGylation i.e. modifying the surface with polyethyleneglycol
  • PEGylation is the predominant method used to reduce the opsonization and aggregation of non- viral vectors and minimize the clearance by reticuloendothelial system, leading to a prolonged circulation lifetime after intravenous (i.v.) administration.
  • compositions of the compounds of the disclosure typically comprise a compound of the instant disclosure and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the type of carrier can be selected based upon the intended route of administration.
  • the carrier is suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal or oral administration.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, micro emulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can 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.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
  • the compounds can be administered in a time release formulation, for example in a composition which includes a slow release polymer, or in a fat pad described herein.
  • the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG).
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • certain methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the compound may be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate the agent.
  • the compound can be administered to a subject in an appropriate carrier or diluent co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluoro-phosphate (DEP) and trasylol.
  • Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • the active agent in the composition e.g., CRISPR/Cas
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result to thereby influence the therapeutic course of a particular disease state.
  • a therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.
  • the active agent is formulated in the composition in a prophylactically effective amount.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
  • the prophylactically effective amount will be less than the therapeutically effective amount.
  • the amount of active compound in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the instant disclosure is dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the compound(s) of the instant disclosure can be administered in a manner that prolongs the duration of the bioavailability of the compound(s), increases the duration of action of the compound(s) and the release time frame of the compound by an amount selected from the group consisting of at least 3 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks, and at least a month, over that of the compound(s) in the absence of the duration-extending administration.
  • the duration of any or all of the preceding effects is extended by at least 30 minutes, at least an hour, at least 2 hours, at least 3 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weeks, at least 3 weeks or at least a month.
  • a compound of the instant disclosure can be formulated into a pharmaceutical composition wherein the compound is the only active agent therein.
  • the pharmaceutical composition can contain additional active agents.
  • two or more compounds of the instant disclosure may be used in combination.
  • the present disclosure provides a method of treating or preventing myopathies, cancer, neurological diseases or disorders.
  • the method comprises administering to a subject in need thereof, an effective amount of a composition comprising at least one of a guide nucleic acid and a Cas peptide, or functional fragment or derivative thereof.
  • the method comprises administering a composition comprising an isolated nucleic acid encoding at least one of: the guide nucleic acid and a Cas peptide, or functional fragment or derivative thereof.
  • the method comprises administering a composition described herein to a subject diagnosed with a myopathies, cancer, neurological diseases or disorders, at risk for developing a myopathies, cancer, neurological diseases or disorders and the like.
  • a composition described herein to a subject diagnosed with a myopathies, cancer, neurological diseases or disorders, at risk for developing a myopathies, cancer, neurological diseases or disorders and the like.
  • methods of modifying and/or editing a bag3 sequence in the genome of a cell comprises contacting a cell, or providing to the cell, a CRISPR- Cas system or composition targeting one or more regions in the bag3 gene.
  • the methods comprise removing or excising a sequence from a genome of the cell.
  • FIG.1 is a schematic representation of a construct of generation I BAG3 base editor.
  • FIG.3 shows the BAG3 E455K Heterozygote Mutant MEF cells (top panel) and the genotyping of these mutant cells (bottom panel) confirmed that half of DNA has a ”A” at the position in the sequence, which is translated into a mutant amino acid, Lys, while the other half has a “G” at the same position in the sequence, which is translated into the wild type amino acid, Glu.
  • FIG.4 shows results from BAG3455K base editing with Generation I editor. Right panel shows the reaction of MEF Heterozygote mutant cells are edited with generation I editor, Left panel shows the genotyping of edited cells.
  • FIG.5 shows results from BAG3455K base editing with Generation II editor.
  • Right panel shows the reaction of MEF Heterozygote mutant cells are edited with generation II editor
  • Left panel shows the genotyping of edited cells.

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Abstract

L'invention concerne des compositions comprenant des complexes d'édition de gènes pour le traitement de myopathies, du cancer et de maladies neurodégénératives.
PCT/US2023/061594 2022-01-28 2023-01-30 Méthodes crispr pour corriger des mutations du gène bag3 in vivo Ceased WO2023147558A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119842664A (zh) * 2025-03-19 2025-04-18 浙江工业大学 基于大肠杆菌内源性EcoTnpB蛋白的基因编辑系统及其应用

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EP3812473A1 (fr) * 2014-01-31 2021-04-28 Temple University Of The Commonwealth System Of Higher Education Bag3 comme cible pour le traitement de l'insuffisance cardiaque
US20180084767A1 (en) * 2016-09-21 2018-03-29 Recombinetics, Inc. Animal models for cardiomyopathy
BR112020025071A2 (pt) * 2018-06-08 2021-03-23 Temple University - Of The Commonwealth System Of Higher Education terapia de otimização de gene bag3

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119842664A (zh) * 2025-03-19 2025-04-18 浙江工业大学 基于大肠杆菌内源性EcoTnpB蛋白的基因编辑系统及其应用

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