WO2019204668A1 - Compositions et procédés d'inactivation de l'apo (a) par édition génique pour le traitement d'une maladie cardiovasculaire - Google Patents

Compositions et procédés d'inactivation de l'apo (a) par édition génique pour le traitement d'une maladie cardiovasculaire Download PDF

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WO2019204668A1
WO2019204668A1 PCT/US2019/028210 US2019028210W WO2019204668A1 WO 2019204668 A1 WO2019204668 A1 WO 2019204668A1 US 2019028210 W US2019028210 W US 2019028210W WO 2019204668 A1 WO2019204668 A1 WO 2019204668A1
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grna
sequence
nucleic acid
cell
seq
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Alan Richard BROOKS
Karen VO
Andrew M. Scharenberg
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Casebia Therapeutics Limited Liability Partnership
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    • 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
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    • 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
    • C12N15/87—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/53—Methods for regulating/modulating their activity reducing unwanted side-effects

Definitions

  • compositions, methods, and systems are provided for editing an LPA gene that encodes the apo(a) protein in a cell genome to modulate the expression, function, or activity of the lipoprotein particle lipoprotein(a) [Lp(a)] in the cell.
  • Some embodiments relate to methods for treating a subject with high levels of Lp(a), e.g., associated with a cardiovascular disease, or risk of developing a cardiovascular disease.
  • Lipoprotein(a) is an atherogenic lipoprotein consisting of the protein apolipoprotein(a) [apo(a)] covalently bound to the apolipoprotein B-100 (apoB) component of a low-density lipoprotein (LDL) particle.
  • the apo(a) protein is encoded by the LPA gene, made in hepatocytes and secreted into circulation.
  • LPA low-density lipoprotein
  • apo(a) has evolved from the plasminogen gene and contains related protein domains.
  • the apo(a) protein is composed of one kringle V (KV) domain, multiple copies of the kringle IV (KIV) domain, and an inactive protease-like domain, all derived from plasminogen.
  • KIV is broken down into 10 subtypes, with KIVi and KIV3-10 present in 1 copy, and KIV 2 present in 1 to greater than 40 copies.
  • the size of apo(a) varies between individual humans and is proportional to the number of copies of KIV2, which is genetically determined.
  • Plasma levels of Lp(a) are inversely correlated to the size of the apo(a) protein and this is thought to be a function of slower secretion of larger isoforms.
  • High plasma level of Lp(a) is an independent risk factor for many cardiovascular diseases, including calcific aortic valve disease, coronary heart disease, atherosclerosis, thrombosis, and stroke (reviewed in Kronenberg, F. (2016). Cardiovasc. Drugs Ther., 30( l):87- 100).
  • Lp(a) The pathogenic mechanisms of Lp(a) is mediated through its pro-atherogenic, pro- inflammatory, and pro-thrombogenic properties.
  • the combination of apo(a) and the LDL components of Lp(a) results in compounding effects on the cardiovascular system.
  • LDL alone can cause immune and inflammatory responses that characterize atherosclerosis through the entry of LDL into vessel walls where the phospholipids become oxidized.
  • Lp(a) circulates and binds to oxidized phospholipids in the plasma, which causes pro -inflammatory responses.
  • Apo(a) itself contains sites that can bind to exposed surfaces on damaged vessel walls, mediating its entry and accumulation at those locations. Small isoforms of apo(a) have been shown to promote thrombosis by inhibiting fibrinolysis.
  • Lp(a) Plasma levels of Lp(a) have been extensively examined in relation to cardiovascular disease and multiple studies have positively associated high Lp(a) levels to higher risk of cardiovascular disease (reviewed in Kronenberg, F. (2016). Cardiovasc. Drugs Ther., 30(l):87- 100). Of interest is the range of plasma Lp(a) levels in humans, which vary by 1000-fold between individuals. This broad range suggests that it may not be detrimental to significantly reduce plasma Lp(a) and therefore potential anti-Lp(a) drugs may have a wide therapeutic window. Unlike LDL, Lp(a) levels cannot be modulated by environment, diet, or existing lipid lowering drugs like statins, making it a strictly genetically-driven disease risk factor.
  • Antisense oligonucleotides against apo(B) were able to reduce Lp(a) by 25% (Santos. R. D. et al. (2015). Arterioscler Thromb Vase Biol, J5(3):689-699). Subsequently, an antisense therapy specifically against the apo(a) mRNA was tested in clinical trials and was shown to significantly decrease plasma Lp(a) levels by over 80% (Viney, N. J. et al. (2016). Lancet, 388(10057): 2239-2253). Unfortunately, antisense therapies require frequent dosing to be efficacious.
  • a system comprising a) a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding the DNA endonuclease; and b) a guide RNA (gRNA) comprising a spacer sequence complementary to a target genomic sequence within or near an apolipoprotein(a) ( LPA ) gene, or nucleic acid encoding the gRNA, wherein the DNA endonuclease and gRNA are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of promoting editing of the target genomic sequence.
  • DNA deoxyribonucleic acid
  • LPA apolipoprotein(a)
  • the gRNA comprises i) a spacer sequence complementary to a target genomic sequence within exon 3 of the LPA gene; ii) a spacer sequence complementary to a target genomic sequence within exon 2 of the LPA gene; iii) a spacer sequence complementary to a target genomic sequence within the LPA gene corresponding to a kringle IV repeat region in apo(a); or iv) a spacer sequence complementary to a target genomic sequence within a regulatory region of the LPA gene.
  • the gRNA comprises i) a spacer sequence from any one of SEQ ID NOs: 18, 13-17, and 19 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 18, 13-17, and 19; ii) a spacer sequence from any one of SEQ ID NOs: 1-12 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 1-12; iii) a spacer sequence from any one of SEQ ID NOs: 107-132 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 107-132; or iv) a spacer sequence from any one of SEQ ID NOs: 20-106 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 20-106. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 14, 15, 18, and 19.
  • the spacer sequence is 19 nucleotides in length and does not include the nucleotide at position 1 of the sequence from which it is selected. In some embodiments, the spacer sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 157-160.
  • the DNA endonuclease and gRNA are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of generating a double-strand break at the target genomic sequence in a cell to generate a genetically modified cell with reduced functional expression of apo(a) as compared to a corresponding unmodified cell.
  • the system further comprises a donor template comprising a donor cassette comprising a nucleic acid sequence encoding one or more STOP codons, and wherein the DNA endonuclease, gRNA, and donor template are configured such that a complex formed by association of the DNA
  • the endonuclease with the gRNA is capable of promoting targeted integration of the donor cassette into a target genomic locus comprising the target genomic sequence in a cell to generate a genetically modified cell with reduced functional expression of apo(a) as compared to a corresponding unmodified cell.
  • the nucleic acid sequence encoding one or more STOP codons encodes three STOP codons in each of the 3 possible translation frames in the forward orientation and/or three STOP codons in each of the 3 possible translation frames in the reverse orientation.
  • the DNA endonuclease is selected from the group consisting of a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel,
  • the DNA endonuclease is a Cas9 endonuclease.
  • the Cas9 endonuclease is from Streptococcus pyogenes (SpyCas9). In some embodiments, the Cas9 endonuclease is from Staphylococcus lugdunensis (SluCas9).
  • the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in a host cell.
  • the DNA endonuclease or nucleic acid encoding the DNA endonuclease is formulated in a liposome or lipid nanoparticle.
  • the liposome or lipid nanoparticle also comprises the gRNA or nucleic acid encoding the gRNA.
  • the system comprises the DNA endonuclease complexed with the gRNA in a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • a method of editing a genome in a cell comprising providing the following to the cell a) a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding the DNA endonuclease; and b) a guide RNA (gRNA) comprising a spacer sequence complementary to a target genomic sequence within or near an
  • DNA deoxyribonucleic acid
  • gRNA guide RNA
  • apolipoprotein(a) ( LPA ) gene in the cell or nucleic acid encoding the gRNA, wherein the DNA endonuclease and gRNA are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of promoting editing of the target genomic sequence in the cell to generate a genetically modified cell with reduced functional expression of apo(a) as compared to a corresponding unmodified cell.
  • LPA apolipoprotein(a)
  • the gRNA comprises i) a spacer sequence complementary to a target genomic sequence within exon 3 of the LPA gene; ii) a spacer sequence complementary to a target genomic sequence within exon 2 of the LPA gene; iii) a spacer sequence complementary to a target genomic sequence within the LPA gene corresponding to a kringle IV repeat region in apo(a); or iv) a spacer sequence complementary to a target genomic sequence within a regulatory region of the LPA gene.
  • the gRNA comprises i) a spacer sequence from any one of SEQ ID NOs: 13-19 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 13-19; ii) a spacer sequence from any one of SEQ ID NOs: 1-12 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 1-12; iii) a spacer sequence from any one of SEQ ID NOs: 107-132 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 107-132; or iv) a spacer sequence from any one of SEQ ID NOs: 20-106 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 20-106.
  • the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 14, 15, 18, and 19.
  • the spacer sequence is 19 nucleotides in length and does not include the nucleotide at position 1 of the sequence from which it is selected. In some embodiments, the spacer comprises the nucleotide sequence of any one of SEQ ID NOs: 157-160.
  • the DNA endonuclease and gRNA are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of generating a double-strand break at the target genomic sequence in the cell.
  • the method further comprises providing to the cell a donor template comprising a donor cassette comprising a nucleic acid sequence encoding one or more STOP codons, and the DNA endonuclease, gRNA, and donor template are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of promoting targeted integration of the donor cassette into a target genomic locus comprising the target genomic sequence in the cell.
  • the nucleic acid sequence encoding one or more STOP codons encodes three STOP codons in each of the 3 possible translation frames in the forward orientation and/or three STOP codons in each of the 3 possible translation frames in the reverse orientation.
  • the DNA endonuclease is selected from the group consisting of a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO,
  • the DNA endonuclease is a Cas9 endonuclease.
  • the Cas9 endonuclease is from Streptococcus pyogenes (SpyCas9).
  • the Cas9 endonuclease is from Staphylococcus lugdunensis (SluCas9).
  • the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in the cell.
  • the DNA endonuclease or nucleic acid encoding the DNA endonuclease is formulated in a liposome or lipid nanoparticle.
  • the liposome or lipid nanoparticle also comprises the gRNA or nucleic acid encoding the gRNA.
  • the method comprises providing to the cell the DNA endonuclease complexed with the gRNA in an RNP complex.
  • one or more additional doses of the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA are provided to the cell following a first dose of the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA.
  • the cell is a hepatocyte.
  • a genetically modified cell in which the genome of the cell has been edited by any of the methods described above.
  • a method of treating a cardiovascular disease or reducing the risk of developing a cardiovascular disease in a subject comprising providing the following to a cell in the subject a) a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding the DNA endonuclease; and b) a guide RNA (gRNA) comprising a spacer sequence complementary to a target genomic sequence within or near an apolipoprotein(a) (LPA) gene in the cell, or nucleic acid encoding the gRNA, wherein the DNA endonuclease and gRNA are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of promoting editing of the target genomic sequence in the cell to generate a genetically modified cell with reduced functional expression of apo(a) as compared to a corresponding unmodified cell.
  • DNA deoxyribonucleic acid
  • LPA apolipoprotein(a)
  • the gRNA comprises i) a spacer sequence complementary to a target genomic sequence within exon 3 of the LPA gene; ii) a spacer sequence complementary to a target genomic sequence within exon 2 of the LPA gene; iii) a spacer sequence complementary to a target genomic sequence within the LPA gene corresponding to a kringle IV repeat region in apo(a); or iv) a spacer sequence complementary to a target genomic sequence within a regulatory region of the LPA gene.
  • the gRNA comprises i) a spacer sequence from any one of SEQ ID NOs: 13- 19 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 13-19; ii) a spacer sequence from any one of SEQ ID NOs: 1-12 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 1-12; iii) a spacer sequence from any one of SEQ ID NOs: 107-132 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 107-132; or iv) a spacer sequence from any one of SEQ ID NOs: 20-106 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 20-106.
  • the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 14, 15, 18, and 19.
  • the spacer sequence is 19 nucleotides in length and does not include the nucleotide at position 1 of the sequence from which it is selected. In some embodiments, the spacer comprises the nucleotide sequence of any one of SEQ ID NOs: 157-160.
  • the DNA endonuclease and gRNA are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of generating a double-strand break at the target genomic sequence in the cell.
  • the method further comprises providing to the cell a donor template comprising a donor cassette comprising a nucleic acid sequence encoding one or more STOP codons, and wherein the DNA
  • the endonuclease, gRNA, and donor template are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of promoting targeted integration of the donor cassette into a target genomic locus comprising the target genomic sequence in the cell.
  • the nucleic acid sequence encoding one or more STOP codons encodes three STOP codons in each of the 3 possible translation frames in the forward orientation and/or three STOP codons in each of the 3 possible translation frames in the reverse orientation.
  • the DNA endonuclease is selected from the group consisting of a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3,
  • the DNA endonuclease is a Cas9 endonuclease.
  • the Cas9 endonuclease is from Streptococcus pyogenes (SpyCas9).
  • the Cas9 endonuclease is from Staphylococcus lugdunensis (SluCas9).
  • the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in the cell.
  • the DNA endonuclease or nucleic acid encoding the DNA endonuclease is formulated in a liposome or lipid nanoparticle.
  • the liposome or lipid nanoparticle also comprises the gRNA or nucleic acid encoding the gRNA.
  • providing the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA to the cell comprises administering the liposome or lipid nanoparticle to the subject.
  • providing the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA to the cell comprises administering to the subject an RNP complex comprising the DNA endonuclease and the gRNA.
  • one or more additional doses of the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA are provided to the cell following a first dose of the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA.
  • one or more additional doses of the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA are provided to the cell following a first dose of the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA until a) a target frequency of editing the target genomic sequence in a population of cells in the subject is achieved; and/or b) a target plasma level of Lp(a) in the subject is achieved.
  • the population of cells of a) and/or b) is the hepatocytes in the subject.
  • the cell is a hepatocyte.
  • kits comprising one or more elements of the system of any one of claims 1-13, and further comprising instructions for use.
  • a gRNA comprising i) a spacer sequence from any one of SEQ ID NOs: 13-19 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 13-19; ii) a spacer sequence from any one of SEQ ID NOs: 1-12 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 1-12; iii) a spacer sequence from any one of SEQ ID NOs: 107-132 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 107-132; or iv) a spacer sequence from any one of SEQ ID NOs: 20-106 or a variant thereof having no more than 3 mismatches compared to any one
  • the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 14, 15, 18, and 19.
  • the spacer sequence is 19 nucleotides in length and does not include the nucleotide at position 1 of the sequence from which it is selected.
  • the spacer comprises the nucleotide sequence of any one of SEQ ID NOs: 157-160.
  • FIG. 1 shows the genomic DNA sequence of human LPA exon 3, with unique gRNA spacer sequences indicated.
  • FIG. 2 shows INDEL analysis of 4 LPA exon 3-targeting 19 nucleotide sgRNAs in primary human hepatocyte donor HNN.
  • FIG. 3 shows TIDE analysis of cutting efficiency of LPA-T4-l9nt in primary human hepatocyte cells of a single donor.
  • FIG. 4 shows an example of a short donor DNA template containing stop codons in all three reading frames, without homology arms.
  • FIG. 5A shows INDEL analysis of LPA -targeting sgRNAs in primary human hepatocyte donor ONR.
  • FIG. 5B shows INDEL analysis of LPA-targeting sgRNAs in primary human hepatocyte donor BVI.
  • FIG. 5C shows INDEL analysis of LPA-targeting sgRNAs in primary human hepatocyte donor HJK.
  • FIG. 6 shows fold-change in LPA or PLG mRNA for PHHs from donor HJK edited by L/M -targeting sgRNAs, as determined by ddPCR.
  • High levels of Lp(a) have been associated with increased risk of cardiovascular disease.
  • the present application provides novel methods for modulating Lp(a) to reduce the risk of cardiovascular disease and/or treat cardiovascular disease.
  • the disclosures provide, inter alia, compositions and methods for editing to modulate the expression, function or activity of
  • Lipoprotein(a) [Lp(a)] in a cell by genome editing also provide, inter alia, compositions and methods for treating a patient with a cardiovascular disease (e.g., calcific aortic valve disease).
  • a cardiovascular disease e.g., calcific aortic valve disease
  • Targeted knockout of a gene can be achieved by using a sequence specific nuclease to generate a double- stranded break in the genomic DNA.
  • sequence specific nucleases with the potential to cut eukaryotic genomes, primarily at a single site, are known in the art, i.e., zinc finger nucleases, transcription activator-like effector nucleases (TALEN), MegaTal, and the CRISPR-Cas system.
  • the CRISPR-Cas system has the advantage of enabling a large number of genomic targets to be rapidly screened to identify an optimal CRISPR-Cas design.
  • the CRISPR- Cas system uses an RNA molecule referred to as a guide RNA (gRNA) (including, e.g., a single guide RNA (sgRNA)) that targets an associated Cas endonuclease (for example a Cas9 nuclease) to a specific sequence in DNA.
  • gRNA guide RNA
  • sgRNA single guide RNA
  • Cas endonuclease for example a Cas9 nuclease
  • This targeting occurs by Watson-Crick base pairing between the gRNA and the sequence of the genome corresponding to the approximately 20 bp targeting sequence of the gRNA, also referred to as the gRNA spacer.
  • a Cas endonuclease cleaves both strands of the genomic DNA, creating a double-strand break.
  • gRNA protospacer adjacent motif
  • the target sequence must contain a protospacer adjacent motif (PAM) sequence at the 3’ end of the target site that is complementary to the gRNA spacer.
  • PAM protospacer adjacent motif
  • the PAM sequence is NGG (where N is any base). Therefore, gRNA molecules that target any region of the genome can be designed in silico by locating the 20 bp sequence adjacent to all PAM motifs.
  • the NGG PAM motif occurs on average every 15 bp in the genome of eukaryotes.
  • gRNAs can be rapidly synthesized in vitro, this enables the rapid screening of all potential gRNA spacer sequences in a given genomic region to identify gRNA spacers that result in efficient cutting. In silico algorithms as well as laboratory experiments can also be used to determine the off-target potential of any given gRNA spacer. While a perfect match to the 20 bp recognition sequence of a gRNA will primarily occur only once in most eukaryotic genomes, there will be a number of additional sites in the genome with 1 or more base pair mismatches to the gRNA. These sites can be cleaved at variable frequencies, which are often not predictable based on the number or location of the mismatches.
  • Cleavage at additional off-target sites that were not identified by the in silico analysis may also occur.
  • screening a set of gRNAs in a relevant cell type to identify gRNAs that have favorable off-target profiles is a critical component of selecting a gRNA for therapeutic use.
  • a favorable off target profile will take into account not only the number of actual off-target sites and the frequency of cutting at these sites, but also the location in the genome of these sites. For example, off-target sites close to or within functionally important genes, particularly oncogenes or anti-oncogenes, would be considered as less favorable than sites in intergenic regions with no known function.
  • the identification of optimal gRNAs cannot be predicted simply by in silico analysis of the genomic sequence of an organism, but requires experimental testing.
  • INDELS are generated within the coding sequence of a gene or within an important regulatory sequence this can result in complete loss of the expression of that gene or changes in the level of expression. However, INDELS that occur in non-coding regions that also play no role in gene regulation will have no impact on genomic function. If an exogenous DNA molecule is supplied in sufficient concentration inside the nucleus of the cell in which the double-strand break occurs, the exogenous DNA may be inserted at the double-strand break during the NHEJ repair process and thus become a permanent addition to the genome. These exogenous DNA molecules are referred to as donor templates.
  • the donor template contains a coding sequence for a gene of interest together with relevant regulatory sequences such as promoters, enhancers, polyA sequences and/or splice acceptor sequences the gene of interest may be expressed from the integrated copy in the genome resulting in permanent expression for the life of the cell.
  • a stop codon may be used as part of the template to create a premature stop codon to halt the translation of the gene of interest.
  • a polyadenylation signal may be used as part of the template to force premature termination of transcription resulting in a mRNA that encodes only a portion of the protein coding sequence and thus result in a non-functional protein.
  • Particularly desirable is to create double-strand breaks to create insertions or deletions via the natural process of NHEJ and/or introduce a stop codon or a polyadenylation signal at the 5’ end of the coding sequence of the gene in order to have the largest negative impact on expression of the protein.
  • the INDELS and/or the integrated copy of the donor DNA template will be transmitted to the daughter cells when the cell divides such that the reduction in expression of the gene is heritable.
  • a gRNA e.g., an sgRNA
  • a Cas endonuclease e.g., a Cas9 endonuclease
  • the gRNA and Cas endonuclease are expressed from an AAV viral vector.
  • the transcription of the gRNA is driven off a U6 promoter and the Cas mRNA transcription is driven from either a ubiquitous promoter like EF1 -alpha or a liver specific promoter and enhancer, such as the transthyretin promoter/enhancer.
  • the size of the SpCas9 gene (4.4 Kb) precludes inclusion of the SpCas9 and the gRNA cassettes in a single AAV, thereby requiring separate AAV to deliver the gRNA and SpCas9.
  • a disadvantage of using AAV to deliver the gRNA and Cas endonuclease is that expression of the gRNA and Cas endonuclease will be long-lived in the cells that are transduced due to the persistence of AAV genomes for several years that has been demonstrated in animals and humans. Continuous long-term expression of an active nuclease presents a significant genotoxicity safety risk due to the existence of off-target cleavage events.
  • the AAV genome encoding the Cas9 and gRNA will be a template for genomic integration (e.g., via NHEJ) which is predicted to result in permanent, heritable expression of the Cas
  • gRNA endonuclease and the gRNA While the number and frequency of off-target cleavage events can be minimized by selection of optimal gRNA sequences, the genotoxic risk from off-target cleavage is amplified by long duration of activity.
  • One approach to prevent continuous expression of a Cas endonuclease or gRNA or both from an AAV vector is to incorporate sequence elements that promote self-inactivation of the viral genome. Including cleavage sites for the gRNA in the vector DNA will result in cleavage of the vector DNA in vivo.
  • Cas endonuclease expression can be limited to a shorter period of time.
  • the cleavage of the AAV episomal genome by the Cas/gRNA complex may result in degradation of the episomal AAV genomes and thus reduce the frequency at which these episomal AAV genomes are integrated into the DNA of the host cell.
  • such systems are complex, requiring, for example, that expression of the gRNA and/or Cas endonuclease is blocked in the production cell line used to produce the AAV.
  • Lipid nanoparticles are composed of an ionizable cationic lipid and 3 or more additional components, typically cholesterol, DOPE and a Polyethylene Glycol (PEG) containing lipid.
  • the cationic lipid binds to the positively charged nucleic acid forming a dense complex that protects the nucleic from degradation.
  • the components self-assemble to form particles with diameters in the size range of 50 nM to 100 nM, in which the nucleic acid is encapsulated in the core complexed with the cationic lipid and surrounded by a lipid bilayer-like structure. After injection into the circulation these particles will generally bind to apolipoprotein E (apoE).
  • ApoE is a ligand for the LDL receptor and mediates uptake into the hepatocytes of the liver via receptor-mediated endocytosis.
  • LNP of this type have been shown to efficiently deliver mRNA and siRNA to the hepatocytes of the liver of rodents, primates and humans.
  • the LNP After endocytosis, the LNP are present in endosomes.
  • the encapsulated nucleic acid undergoes a process of endosomal escape mediated by the ionizable nature of the cationic lipid. This delivers the nucleic acid into the cytoplasm where mRNA can be translated into the encoded protein.
  • Encapsulation of gRNA and mRNA encoding a Cas endonuclease into a LNP can efficiently deliver both components to the hepatocytes after IV injection.
  • the Cas endonuclease mRNA is translated into Cas protein and may form a complex with the gRNA.
  • RNA molecules in vivo is short, on the order of hours to days. Similarly, the half-life of proteins tends to be short, on the order of hours to days.
  • LNPs are less immunogenic than viral particles. While many humans have preexisting immunity to AAV, there is no pre-existing immunity to LNPs. In addition, an adaptive immune response against LNPs is unlikely to occur, which enables repeat dosing of LNPs.
  • ionizable cationic lipids have been developed for use in LNPs. These include C12-200 (Love, K. T. et al. (2010). PNAS, 107(5): 1864-1869), MC3, LN16, MD1 among others.
  • a GalNac moiety is attached to the outside of the LNP and acts as a ligand for uptake into the liver via the asialyloglycoprotein receptor. Any of these cationic lipids may be used to formulate LNPs for delivery of gRNA and Cas endonuclease mRNA to the liver.
  • ranges and amounts can be expressed as“about” a particular value or range. About also includes the exact amount. Hence“about 5 pL” means“about 5 pL” and also “5 m L.” Generally, the term“about” includes an amount that would be expected to be within experimental error such as ⁇ 10%.
  • polypeptide “polypeptide sequence,”“peptide,”“peptide sequence,” “protein,”“protein sequence” and“amino acid sequence” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds, which series may include proteins, polypeptides, oligopeptides, peptides, and fragments thereof.
  • the protein may be made up of naturally occurring amino acids and/or synthetic (e.g., modified or non-naturally occurring) amino acids.
  • “amino acid”, or“peptide residue”, as used herein means both naturally occurring and synthetic amino acids.
  • polypeptide includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, b-galactosidase, luciferase, and the like.
  • a dash at the beginning or end of an amino acid sequence indicates either a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to a carboxyl or hydroxyl end group.
  • the absence of a dash should not be taken to mean that such peptide bond or covalent bond to a carboxyl or hydroxyl end group is not present, as it is conventional in representation of amino acid sequences to omit such.
  • oligonucleotide sequence refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • this term includes, but is not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer having purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • “derivative” and“variant” refer without limitation to any compound such as nucleic acid or protein that has a structure or sequence derived from the compounds disclosed herein and whose structure or sequence is sufficiently similar to those disclosed herein such that it has the same or similar activities and utilities or, based upon such similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the referenced compounds, thereby also interchangeably referred to“functionally equivalent” or as “functional equivalents.”
  • Modifications to obtain“derivatives” or“variants” may include, for example, addition, deletion and/or substitution of one or more of the nucleic acids or amino acid residues.
  • the functional equivalent or fragment of the functional equivalent in the context of a protein, may have one or more conservative amino acid substitutions.
  • conservative amino acid substitution refers to substitution of an amino acid for another amino acid that has similar properties as the original amino acid.
  • the groups of conservative amino acids are as follows:
  • Conservative substitutions may be introduced in any position of a predetermined peptide or fragment thereof. It may however also be desirable to introduce non-conservative substitutions, particularly, but not limited to, a non-conservative substitution in any one or more positions.
  • a non-conservative substitution leading to the formation of a functionally equivalent fragment of the peptide would for example differ substantially in polarity, in electric charge, and/or in steric bulk while maintaining the functionality of the derivative or variant fragment.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may have additions or deletions (i.e., gaps) as compared to the reference sequence (which does not have additions or deletions) for optimal alignment of the two sequences.
  • the percentage can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., the entire polypeptide sequences or individual domains of the polypeptides), when compared and aligned for maximum correspondence over a comparison window or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be“substantially identical.” This definition also refers to the complement of a test sequence.
  • nucleic acid e.g., DNA or RNA
  • nucleic acid has a sequence of nucleotides that enables it to non-covalently bind, i.e., form Watson-Crick base pairs and/or G/U base pairs to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid).
  • standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C).
  • a DNA sequence that“encodes” a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA.
  • a DNA polynucleotide may encode an RNA (mRNA) that is translated into protein, or a DNA polynucleotide may encode an RNA that is not translated into protein (e.g., tRNA, rRNA, or a guide RNA; also called“non-coding” RNA or“ncRNA”).
  • A“protein coding sequence or a sequence that encodes a particular protein or polypeptide is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • “codon” refers to a sequence of three nucleotides that together form a unit of genetic code in a DNA or RNA molecule.
  • the term“codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide.
  • the term“codon-optimized” or“codon optimization” refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.
  • Codon usage tables are readily available, for example, at the“Codon Usage Database” available at
  • Codon-optimized coding regions can be designed by various methods known to those skilled in the art.
  • recombinant or“engineered” when used with reference, for example, to a cell, a nucleic acid, a protein, or a vector, indicates that the cell, nucleic acid, protein or vector has been modified by or is the result of laboratory methods.
  • recombinant or engineered proteins include proteins produced by laboratory methods.
  • Recombinant or engineered proteins can include amino acid residues not found within the native (non
  • recombinant or wild-type form of the protein can be include amino acid residues that have been modified, e.g., labeled.
  • the term can include any modifications to the peptide, protein, or nucleic acid sequence. Such modifications may include the following: any chemical
  • modifications of the peptide, protein or nucleic acid sequence including of one or more amino acids, deoxyribonucleotides, or ribonucleotides; addition, deletion, and/or substitution of one or more of amino acids in the peptide or protein; and addition, deletion, and/or substitution of one or more of nucleic acids in the nucleic acid sequence.
  • genomic DNA or“genomic sequence” refers to the DNA of a genome of an organism including, but not limited to, the DNA of the genome of a bacterium, fungus, archea, plant or animal.
  • “transgene,”“exogenous gene” or“exogenous sequence,” in the context of nucleic acid refers to a nucleic acid sequence or gene that was not present in the genome of a cell but artificially introduced into the genome, e.g., via genome-edition.
  • “endogenous gene” or“endogenous sequence,” in the context of nucleic acid refers to a nucleic acid sequence or gene that is naturally present in the genome of a cell, without being introduced via any artificial means.
  • vector or“expression vector” means a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e., an“insert”, may be attached so as to bring about the replication of the attached segment in a cell.
  • the term“expression cassette” refers to a vector having a DNA coding sequence operably linked to a promoter.“Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • the terms“recombinant expression vector,” or“DNA construct” are used interchangeably herein to refer to a DNA molecule having a vector and at least one insert. Recombinant expression vectors are usually generated for the purpose of expressing and/or propagating the insert(s), or for the construction of other recombinant nucleotide sequences.
  • the nucleic acid(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.
  • operably linked means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence.
  • regulatory sequence is intended to include, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel, D. V. (Ed.) (1990).
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the target cell, the level of expression desired, and the like.
  • a cell has been“genetically modified” or“transformed” or“transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell.
  • exogenous DNA e.g., a recombinant expression vector
  • the presence of the exogenous DNA results in permanent or transient genetic change.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • the genetically modified (or transformed or transfected) cells that have therapeutic activity e.g., treating cardiovascular disease, can be used and referred to as therapeutic cells.
  • concentration used in the context of a molecule such as peptide fragment refers to an amount of molecule, e.g., the number of moles of the molecule, present in a given volume of solution.
  • the terms“individual,”“subject” and“host” are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired.
  • the subject is a mammal.
  • the subject is a human being.
  • the subject is a human patient.
  • a subject is a companion animal, such as a dog, cat, or bird while in still other aspects the subject is a farm animal, such as a horse, cow, sheep, goat, or pig.
  • the subject can have or is suspected of having a cardiovascular disease and/or has one or more symptoms of a cardiovascular disease.
  • the subject is a human who is diagnosed with a risk of cardiovascular disease at the time of diagnosis or later.
  • the diagnosis with a risk of cardiovascular disease can be determined based on the presence of one or more mutations in an endogenous apolipoprotein(a) ( LPA ) gene or genomic sequence near the LPA gene in the genome that may affect the expression of the apo(a) protein.
  • LPA apolipoprotein(a)
  • treatment used referring to a disease or condition means that at least an amelioration of the symptoms associated with the condition afflicting an individual is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom, associated with the condition (e.g., a cardiovascular disease) being treated.
  • a parameter e.g., a symptom
  • treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or eliminated entirely such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition.
  • treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease.
  • an effective amount means a sufficient amount of the composition to provide the desired utility when administered to a subject having a particular condition.
  • an effective amount refers to an amount of components used for genome edition such as gRNA, donor template and/or a site-directed polypeptide (e.g., DNA
  • an appropriate“effective amount” can be determined by one of ordinary skill in the art using routine experimentation.
  • pharmaceutically acceptable excipient refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive or diluent for
  • “Pharmaceutically acceptable excipient” can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.
  • LPA gene including, e.g., LPA gene variants associated with increased cardiovascular disease risk and/or increased Lp(a) expression
  • LPA gene variants associated with increased cardiovascular disease risk and/or increased Lp(a) expression
  • LPA gene variants associated with increased cardiovascular disease risk and/or increased Lp(a) expression
  • the term“LPA gene” as used herein includes the genomic region encompassing the LPA regulatory promoters and enhancer sequences as well as the coding sequence.
  • the disclosures also provide, inter alia, systems for treating a subject having or suspected of having a disorder or health condition associated with Lp(a), employing in vivo genome editing. In some embodiments, the subject has or is suspected of having a cardiovascular disease.
  • a system comprising (a) a deoxyribonucleic acid (DNA) endonuclease (e.g., a Cas endonuclease, such as Cas9) or nucleic acid encoding the DNA endonuclease; and (b) a guide RNA (gRNA) comprising a spacer sequence complementary to a target genomic sequence within or near an apolipoprotein(a) (LPA) gene, or nucleic acid encoding the gRNA, wherein the DNA endonuclease and gRNA are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of promoting editing of the target genomic sequence, such as INDELs generated during repair (e.g., NHEJ-mediated repair) of a double-strand break introduced by the DNA endonuclease/gRNA complex, or integration of a heterologous sequence contained in a donor template (e
  • INDELs generated during repair e.
  • the system further comprises a donor template comprising a nucleic acid sequence to be inserted into the LPA gene.
  • the nucleic acid sequence to be inserted encodes one or more STOP codons, and the system is configured to insert a STOP codon in-frame into an LPA gene coding sequence such that the LPA gene is rendered non-functional.
  • the gRNA targets within or near a coding sequence in the LPA gene.
  • the gRNA targets exon 1, exon 2, or exon 3 of the LPA gene.
  • the gRNA targets a sequence in the LPA gene corresponding to a kringle IV repeat region in the apo(a) protein.
  • the gRNA targets within or near a non-coding sequence in the LPA gene. In some embodiments, the gRNA targets an LPA gene intron. In some embodiments, the gRNA targets an LPA gene regulatory region. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 1-132. In some embodiments, the DNA endonuclease is a Cas endonuclease, e.g., a Cas9 nuclease. In some embodiments, the gRNA is an sgRNA.
  • a system comprising (a) a DNA endonuclease (e.g., a Cas endonuclease, such as Cas9) or nucleic acid encoding the DNA endonuclease; and (b) a gRNA comprising i) a spacer sequence complementary to a target genomic sequence within exon 3 of an LPA gene; ii) a spacer sequence complementary to a target genomic sequence within exon 2 of an LPA gene; iii) a spacer sequence complementary to a target genomic sequence within an LPA gene corresponding to a kringle IV repeat region in apo(a); or iv) a spacer sequence complementary to a target genomic sequence within a regulatory region of an LPA gene, or nucleic acid encoding the gRNA, wherein the DNA endonuclease and gRNA are configured such that a complex formed by association of the DNA endonuclease with the gRNA
  • the gRNA comprises i) a spacer sequence from any one of SEQ ID NOs: 13-19 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 13-19; ii) a spacer sequence from any one of SEQ ID NOs: 1-12 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 1-12; iii) a spacer sequence from any one of SEQ ID NOs: 107-132 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 107-132; or iv) a spacer sequence from any one of SEQ ID NOs: 20-106 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 20- 106.
  • the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 14, 15, 18, and 19.
  • the spacer sequence is 19 nucleotides in length and does not include the nucleotide at position 1 of the sequence from which it is selected.
  • the spacer sequence is the nucleotide sequence of any one of SEQ ID NOs: 157-160.
  • the DNA endonuclease and gRNA are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of generating a double-strand break at the target genomic sequence.
  • the generation of a double strand break at the target genomic sequence in a cell can lead to the generation of a genetically modified cell with reduced functional expression of apo(a) as compared to a corresponding unmodified cell.
  • imperfect repair e.g., mediated by NHEJ
  • INDELs small nucleotide insertions or deletions
  • Introduction of INDELs into an LPA gene coding sequence can result in a frameshift mutation that renders the translation product non-functional or having reduced function as compared to the apo(a) protein encoded by the unmodified LPA gene.
  • the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 13-19 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 13-19. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 13-19. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of any one of SEQ ID NOs: 13-19. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 14 or a variant thereof having no more than 3 mismatches compared to SEQ ID NO: 14.
  • the gRNA comprises a spacer sequence from SEQ ID NO: 14. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 15 or a variant thereof having no more than 3 mismatches compared to SEQ ID NO: 15. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 15. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 18 or a variant thereof having no more than 3 mismatches compared to SEQ ID NO: 18.
  • the gRNA comprises a spacer sequence from SEQ ID NO: 18. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 19 or a variant thereof having no more than 3 mismatches compared to SEQ ID NO: 19. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 19. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of SEQ ID NO: 19.
  • the DNA endonuclease is selected from the group consisting of a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel,
  • the DNA endonuclease is a Cas9 endonuclease.
  • the Cas9 endonuclease is from Streptococcus pyogenes (SpyCas9). In some embodiments, the Cas9 endonuclease is from Staphylococcus lugdunensis (SluCas9).
  • the system comprises a nucleic acid encoding the DNA endonuclease.
  • the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in a host cell.
  • the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in a human cell.
  • the nucleic acid encoding the DNA endonuclease is DNA, such as a DNA plasmid.
  • the nucleic acid encoding the DNA endonuclease is RNA, such as mRNA.
  • the system further comprises a donor template comprising a sequence to be integrated at or near the target genomic sequence.
  • the donor template comprises a donor cassette comprising a nucleic acid sequence encoding one or more STOP codons.
  • the nucleic acid sequence encoding one or more STOP codons encodes three STOP codons in each of the 3 possible translation frames in the forward orientation and/or three STOP codons in each of the 3 possible translation frames in the reverse orientation.
  • An exemplary sequence encoding three STOP codons in each of the 3 possible translation frames in both the forward and reverse orientations is provided in SEQ ID NO: 155.
  • endonuclease, gRNA, and donor template are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of promoting targeted integration of the donor cassette into a target genomic locus (e.g., a coding region) comprising the target genomic sequence.
  • integration of the donor cassette into the target genomic locus in a cell results in the generation of a genetically modified cell with reduced functional expression of apo(a) as compared to a corresponding unmodified cell.
  • integration of the donor template into an LPA gene coding sequence such that a premature STOP codon is inserted in-frame can result in a non-functional or reduced function truncated translation product as compared to the apo(a) protein encoded by the unmodified LPA gene.
  • the donor template is configured such that the donor cassette is capable of being integrated into a genomic locus targeted by a gRNA in the system by homology directed repair (HDR).
  • HDR homology directed repair
  • the donor cassette is flanked on both sides by homology arms corresponding to sequences in the targeted genomic locus.
  • the homology arms are at least about 0.2 kb (such as at least about any of 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, or greater) in length.
  • the homology arms are at least about 0.4 kb, e.g., 0.45 kb, 0.6 kb, or 0.8 kb, in length.
  • the donor template is encoded in an Adeno Associated Virus (AAV) vector.
  • AAV vector is an AAV2, AAV5, or AAV6 vector.
  • the AAV vector is an AAV6 vector.
  • the donor template is configured such that the donor cassette is capable of being integrated into a genomic locus targeted by a gRNA in the system by non-homologous end joining (NHEJ).
  • NHEJ non-homologous end joining
  • the donor cassette is flanked on one or both sides by a gRNA target site.
  • the donor cassette is flanked on both sides by a gRNA target site.
  • the gRNA target site is a target site for a gRNA in the system.
  • the gRNA target site of the donor template is the reverse complement of a cell genome gRNA target site for a gRNA in the system.
  • the donor template is encoded in an Adeno Associated Virus (AAV) vector.
  • AAV vector is an AAV2, AAV5, or AAV6 vector.
  • the AAV vector is an AAV6 vector.
  • the DNA endonuclease or nucleic acid encoding the DNA endonuclease is formulated in a liposome or lipid nanoparticle.
  • the liposome or lipid nanoparticle also comprises the gRNA or nucleic acid encoding the gRNA.
  • the liposome or lipid nanoparticle is a lipid nanoparticle.
  • the system comprises a lipid nanoparticle comprising nucleic acid encoding the DNA endonuclease and the gRNA.
  • the nucleic acid encoding the DNA endonuclease is an mRNA encoding the DNA endonuclease.
  • the DNA endonuclease is complexed with the gRNA, forming a ribonucleoprotein (RNP) complex.
  • an LPA gene targeted for editing is a variant associated with increased cardiovascular disease risk and/or increased Lp(a) expression.
  • the present disclosure provides a genome-targeting nucleic acid that can direct the activities of an associated polypeptide (e.g., a site-directed polypeptide or DNA endonuclease) to a specific target sequence within a target nucleic acid.
  • the genome targeting nucleic acid is an RNA.
  • a genome-targeting RNA is referred to as a“guide RNA” or “gRNA” herein.
  • a guide RNA has at least i) a spacer sequence that can hybridize to a target nucleic acid sequence of interest and ii) a CRISPR repeat sequence.
  • the gRNA also has a second RNA called the tracrRNA sequence.
  • the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex.
  • the crRNA forms a duplex.
  • the duplex binds a site-directed polypeptide such that the guide RNA and site-direct polypeptide form a complex.
  • the genome-targeting nucleic acid provides target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid thus directs the activity of the site-directed polypeptide.
  • the genome-targeting nucleic acid is a double-molecule guide RNA.
  • the genome-targeting nucleic acid is a single-molecule guide RNA.
  • a double-molecule guide RNA has two strands of RNA. The first strand has in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence and a minimum CRISPR repeat sequence. The second strand has a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3’ tracrRNA sequence and an optional tracrRNA extension sequence.
  • a single-molecule guide RNA (sgRNA) in a Type II system has, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3’ tracrRNA sequence and an optional tracrRNA extension sequence.
  • the optional tracrRNA extension may have elements that contribute additional functionality (e.g., stability) to the guide RNA.
  • the single-molecule guide linker links the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure.
  • the optional tracrRNA extension has one or more hairpins.
  • a single-molecule guide RNA (sgRNA) in a Type V system has, in the 5' to 3' direction, a minimum CRISPR repeat sequence and a spacer sequence.
  • RNAs used in the CRISPR/Cas/Cpfl system can be readily synthesized by chemical means as illustrated below and described in the art. While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides.
  • HPLC high performance liquid chromatography
  • One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Cas9 or Cpfl endonuclease, are more readily generated enzymatically.
  • RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art.
  • a spacer extension sequence can modify activity, provide stability and/or provide a location for modifications of a genome targeting nucleic acid.
  • a spacer extension sequence can modify on- or off-target activity or specificity.
  • a spacer extension sequence is provided.
  • a spacer extension sequence can have a length of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, or 7000 or more nucleotides.
  • a spacer extension sequence can have a length of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, or 7000 or more nucleotides.
  • a spacer extension sequence can have a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, 7000 or more nucleotides.
  • a spacer extension sequence is less than 10 nucleotides in length.
  • a spacer extension sequence is between 10-30 nucleotides in length.
  • a spacer extension sequence is between 30-70 nucleotides in length.
  • the spacer extension sequence has another moiety (e.g., a stability control sequence, an endoribonuclease binding sequence, a ribozyme).
  • the moiety decreases or increases the stability of a nucleic acid targeting nucleic acid.
  • the moiety is a transcriptional terminator segment (i.e., a
  • the moiety functions in a eukaryotic cell. In some embodiments, the moiety functions in a prokaryotic cell. In some embodiments, the moiety functions in both eukaryotic and prokaryotic cells.
  • Non-limiting examples of suitable moieties include: a 5' cap (e.g., a 7-methylguanylate cap (m7 G)), a riboswitch sequence (e.g., to allow for regulated stability and/or regulated accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like), a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.), and/or a modification or sequence that provides a binding site for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacet
  • the spacer sequence hybridizes to a sequence in a target nucleic acid of interest.
  • the spacer of a genome-targeting nucleic acid interacts with a target nucleic acid in a sequence- specific manner via hybridization (i.e., base pairing).
  • the nucleotide sequence of the spacer thus varies depending on the sequence of the target nucleic acid of interest.
  • the spacer sequence is designed to hybridize to a target nucleic acid that is located 5' of a PAM of a Cas endonuclease used in the system.
  • the spacer can perfectly match the target sequence or can have mismatches.
  • Each Cas endonuclease has a particular PAM sequence that it recognizes in a target DNA.
  • S. pyogenes recognizes in a target nucleic acid a PAM that has the sequence 5'-NRG-3', where R has either A or G, where N is any nucleotide and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.
  • the target nucleic acid sequence has 20 nucleotides. In some embodiments, the target nucleic acid has less than 20 nucleotides. In some embodiments, the target nucleic acid has more than 20 nucleotides. In some embodiments, the target nucleic acid has at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid has at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid sequence has 20 bases immediately 5' of the first nucleotide of the PAM.
  • the target nucleic acid has the sequence that corresponds to the Ns, wherein N is any nucleotide, and the underlined NRG sequence (R is G or A) is the Streptococcus pyogenes Cas9 PAM.
  • the PAM sequence used in the compositions and methods of the present disclosure as a sequence recognized by S.p. Cas9 is NGG.
  • the spacer sequence that hybridizes to the target nucleic acid has a length of at least about 6 nucleotides (nt).
  • the spacer sequence can be at least about 6 nt, about 10 nt, about 15 nt, about 18 nt, about 19 nt, about 20 nt, about 25 nt, about 30 nt, about 35 nt or about 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50 nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, from about 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about 45 nt, from about
  • the spacer sequence has 20 nucleotides. In some embodiments, the spacer has 19 nucleotides. In some embodiments, the spacer has 18 nucleotides. In some embodiments, the spacer has 17 nucleotides. In some embodiments, the spacer has 16 nucleotides. In some embodiments, the spacer has 15
  • the percent complementarity between the spacer sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%.
  • the percent complementarity between the spacer sequence and the target nucleic acid is at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 97%, at most about 98%, at most about 99%, or 100%. In some embodiments, the percent
  • complementarity between the spacer sequence and the target nucleic acid is 100% over the six contiguous 5'-most nucleotides of the target sequence of the complementary strand of the target nucleic acid. In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is at least 60% over about 20 contiguous nucleotides. In some embodiments, the length of the spacer sequence and the target nucleic acid can differ by 1 to 6 nucleotides, which can be thought of as a bulge or bulges.
  • the spacer sequence is designed or chosen using a computer program.
  • the computer program can use variables, such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, % GC, frequency of genomic occurrence (e.g., of sequences that are identical or are similar but vary in one or more spots as a result of mismatch, insertion or deletion), methylation status, presence of SNPs, and the like.
  • a minimum CRISPR repeat sequence is a sequence with at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference CRISPR repeat sequence (e.g., crRNA from S. pyogenes).
  • a reference CRISPR repeat sequence e.g., crRNA from S. pyogenes
  • a minimum CRISPR repeat sequence has nucleotides that can hybridize to a minimum tracrRNA sequence in a cell.
  • the minimum CRISPR repeat sequence and a minimum tracrRNA sequence form a duplex, i.e., a base-paired double-stranded structure. Together, the minimum CRISPR repeat sequence and the minimum tracrRNA sequence bind to the site-directed polypeptide. At least a part of the minimum CRISPR repeat sequence hybridizes to the minimum tracrRNA sequence.
  • At least a part of the minimum CRISPR repeat sequence has at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the minimum tracrRNA sequence. In some embodiments, at least a part of the minimum CRISPR repeat sequence has at most about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the minimum tracrRNA sequence.
  • the minimum CRISPR repeat sequence can have a length from about 7 nucleotides to about 100 nucleotides.
  • the length of the minimum CRISPR repeat sequence is from about 7 nucleotides (nt) to about 50 nt, from about 7 nt to about 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25 nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to
  • the minimum CRISPR repeat sequence is approximately 12 nucleotides in length.
  • the minimum CRISPR repeat sequence is at least about 60% identical to a reference minimum CRISPR repeat sequence (e.g., wild-type crRNA from S.
  • the minimum CRISPR repeat sequence is at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical or 100% identical to a reference minimum CRISPR repeat sequence over a stretch of at least 6,
  • a minimum tracrRNA sequence is a sequence with at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., wild type tracrRNA from S. pyogenes).
  • a reference tracrRNA sequence e.g., wild type tracrRNA from S. pyogenes.
  • a minimum tracrRNA sequence has nucleotides that hybridize to a minimum CRISPR repeat sequence in a cell.
  • a minimum tracrRNA sequence and a minimum CRISPR repeat sequence form a duplex, i.e., a base-paired double-stranded structure. Together, the minimum tracrRNA sequence and the minimum CRISPR repeat bind to a site-directed polypeptide. At least a part of the minimum tracrRNA sequence can hybridize to the minimum CRISPR repeat sequence.
  • the minimum tracrRNA sequence is at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the minimum CRISPR repeat sequence.
  • the minimum tracrRNA sequence can have a length from about 7 nucleotides to about 100 nucleotides.
  • the minimum tracrRNA sequence can be from about 7 nucleotides (nt) to about 50 nt, from about 7 nt to about 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25 nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30
  • the minimum tracrRNA sequence is approximately 9 nucleotides in length. In some embodiments, the minimum tracrRNA sequence is approximately 12 nucleotides. In some embodiments, the minimum tracrRNA consists of tracrRNA nucleotides 23-48 described in Jinek, M. et al. (2012). Science, 337( 6096):816-821.
  • the minimum tracrRNA sequence is at least about 60% identical to a reference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes) sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.
  • a reference minimum tracrRNA e.g., wild type, tracrRNA from S. pyogenes
  • the minimum tracrRNA sequence is at least about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, about 99% identical or 100% identical to a reference minimum tracrRNA sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.
  • the duplex between the minimum CRISPR RNA and the minimum tracrRNA has a double helix. In some embodiments, the duplex between the minimum CRISPR RNA and the minimum tracrRNA has at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. In some embodiments, the duplex between the minimum CRISPR RNA and the minimum tracrRNA has at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.
  • the duplex has a mismatch (i.e., the two strands of the duplex are not 100% complementary). In some embodiments, the duplex has at least about 1, 2, 3, 4, or 5 or mismatches. In some embodiments, the duplex has at most about 1, 2, 3, 4, or 5 or mismatches. In some embodiments, the duplex has no more than 2 mismatches.
  • the bulge is an unpaired region of nucleotides within the duplex.
  • the bulge contributes to the binding of the duplex to the site- directed polypeptide.
  • a bulge has, on one side of the duplex, an unpaired 5'-XXXY-3' where X is any purine and Y has a nucleotide that can form a wobble pair with a nucleotide on the opposite strand, and an unpaired nucleotide region on the other side of the duplex. The number of unpaired nucleotides on the two sides of the duplex can be different.
  • the bulge has an unpaired purine (e.g., adenine) on the minimum CRISPR repeat strand of the bulge.
  • a bulge has an unpaired 5'-AAGY-3' of the minimum tracrRNA sequence strand of the bulge, where Y has a nucleotide that can form a wobble pairing with a nucleotide on the minimum CRISPR repeat strand.
  • a bulge on the minimum CRISPR repeat side of the duplex has at least 1, 2, 3, 4, or 5 or more unpaired nucleotides. In some embodiments, a bulge on the minimum CRISPR repeat side of the duplex has at most 1, 2, 3, 4, or 5 or more unpaired nucleotides. In some embodiments, a bulge on the minimum CRISPR repeat side of the duplex has 1 unpaired nucleotide.
  • a bulge on the minimum tracrRNA sequence side of the duplex has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. In some embodiments, a bulge on the minimum tracrRNA sequence side of the duplex has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. In some embodiments, a bulge on a second side of the duplex (e.g., the minimum tracrRNA sequence side of the duplex) has 4 unpaired nucleotides.
  • a bulge has at least one wobble pairing. In some embodiments, a bulge has at most one wobble pairing. In some embodiments, a bulge has at least one purine nucleotide. In some embodiments, a bulge has at least 3 purine nucleotides. In some
  • a bulge sequence has at least 5 purine nucleotides. In some embodiments, a bulge sequence has at least one guanine nucleotide. In some embodiments, a bulge sequence has at least one adenine nucleotide.
  • one or more hairpins are located 3' to the minimum tracrRNA in the 3' tracrRNA sequence.
  • the hairpin starts at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or
  • the hairpin can start at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides 3' of the last paired nucleotide in the minimum CRISPR repeat and minimum tracrRNA sequence duplex.
  • a hairpin has at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more consecutive nucleotides. In some embodiments, a hairpin has at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more consecutive nucleotides.
  • a hairpin has a CC dinucleotide (i.e., two consecutive cytosine nucleotides).
  • a hairpin has duplexed nucleotides (e.g., nucleotides in a hairpin, hybridized together).
  • a hairpin has a CC dinucleotide that is hybridized to a GG dinucleotide in a hairpin duplex of the 3' tracrRNA sequence.
  • One or more of the hairpins can interact with guide RNA-interacting regions of a site- directed polypeptide.
  • a 3' tracrRNA sequence has a sequence with at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).
  • a reference tracrRNA sequence e.g., a tracrRNA from S. pyogenes.
  • the 3' tracrRNA sequence has a length from about 6 nucleotides to about 100 nucleotides.
  • the 3' tracrRNA sequence can have a length from about 6 nucleotides (nt) to about 50 nt, from about 6 nt to about 40 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,
  • the 3' tracrRNA sequence is at least about 60% identical to a reference 3' tracrRNA sequence (e.g., wild type 3' tracrRNA sequence from S. pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides.
  • a reference 3' tracrRNA sequence e.g., wild type 3' tracrRNA sequence from S. pyogenes
  • the 3' tracrRNA sequence is at least about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, about 99% identical, or 100% identical, to a reference 3' tracrRNA sequence (e.g., wild type 3' tracrRNA sequence from S. pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides.
  • a 3' tracrRNA sequence has more than one duplexed region (e.g., hairpin, hybridized region). In some embodiments, a 3' tracrRNA sequence has two duplexed regions.
  • the 3' tracrRNA sequence has a stem loop structure.
  • a stem loop structure in the 3' tracrRNA has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 or more nucleotides.
  • the stem loop structure in the 3' tracrRNA has at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides.
  • the stem loop structure has a functional moiety.
  • the stem loop structure can have an aptamer, a ribozyme, a protein-interacting hairpin, a CRISPR array, an intron, or an exon.
  • the stem loop structure has at least about 1, 2, 3, 4, or 5 or more functional moieties.
  • the stem loop structure has at most about 1, 2, 3, 4, or 5 or more functional moieties.
  • the hairpin in the 3' tracrRNA sequence has a P-domain.
  • the P-domain has a double- stranded region in the hairpin.
  • a tracrRNA extension sequence can be provided whether the tracrRNA is in the context of single-molecule guides or double-molecule guides.
  • a tracrRNA extension sequence has a length from about 1 nucleotide to about 400 nucleotides.
  • a tracrRNA extension sequence has a length of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 nucleotides.
  • a tracrRNA extension sequence has a length from about 20 to about 5000 or more nucleotides. In some embodiments, a tracrRNA extension sequence has a length of more than 1000 nucleotides. In some embodiments, a tracrRNA extension sequence has a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or more nucleotides. In some embodiments, a tracrRNA extension sequence can have a length of less than 1000 nucleotides.
  • a tracrRNA extension sequence has less than 10 nucleotides in length. In some embodiments, a tracrRNA extension sequence is 10-30 nucleotides in length. In some embodiments, tracrRNA extension sequence is 30-70 nucleotides in length.
  • the tracrRNA extension sequence has a functional moiety (e.g., a stability control sequence, ribozyme, endoribonuclease binding sequence).
  • a functional moiety e.g., a stability control sequence, ribozyme, endoribonuclease binding sequence.
  • the functional moiety has a transcriptional terminator segment (i.e., a transcription termination sequence).
  • the functional moiety has a total length from about 10 nucleotides (nt) to about 100 nucleotides, from about 10 nt to about 20 nt, from about 20 nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, or from about 15 nt to about 25 nt.
  • the functional moiety has a transcriptional terminator segment
  • the functional moiety functions in both eukaryotic and prokaryotic cells.
  • Non-limiting examples of suitable tracrRNA extension functional moieties include a 3' poly-adenylated tail, a riboswitch sequence (e.g., to allow for regulated stability and/or regulated accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like), a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.), and/or a modification or sequence that provides a binding site for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA
  • a tracrRNA extension sequence has a primer binding site or a molecular index (e.g., barcode sequence). In some embodiments, the tracrRNA extension sequence has one or more affinity tags.
  • the linker sequence of a single-molecule guide nucleic acid has a length from about 3 nucleotides to about 100 nucleotides.
  • An illustrative linker has a length from about 3 nucleotides (nt) to about 90 nt, from about 3 nt to about 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60 nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, from about 3 nt to about 30 nt, from about 3 nt to about 20 nt, from about 3 nt to about 10 nt.
  • the linker can have a length from about 3 nucleotides (nt) to about 90 nt, from about 3 nt to about 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60 nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, from about 3 nt to about 30 nt, from about 3 nt to about 20 nt
  • nt to about 5 nt from about 5 nt to about 10 nt, from about 10 nt to about 15 nt, from about 15 nt to about 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt, from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100 nt.
  • the linker of a single-molecule guide nucleic acid is between 4 and 40 nucleotides. In some embodiments, a linker is at least about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides. In some embodiments, a linker is at most about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.
  • Linkers can have any of a variety of sequences, although in some embodiments, the linker will not have sequences that have extensive regions of homology with other portions of the guide RNA, which might cause intramolecular binding that could interfere with other functional regions of the guide.
  • Jinek, M. et al. (2012). Science, 337(6096):8l6-82l a simple
  • the linker sequence has a functional moiety.
  • the linker sequence can have one or more features, including an aptamer, a ribozyme, a protein interacting hairpin, a protein binding site, a CRISPR array, an intron, or an exon.
  • the linker sequence has at least about 1, 2, 3, 4, or 5 or more functional moieties. In some embodiments, the linker sequence has at most about 1, 2, 3, 4, or 5 or more functional moieties.
  • a genomic location targeted by gRNAs in accordance with the preset disclosure can be at, within or near the endogenous apolipoprotein(a) ( LPA ) locus in a genome, e.g., human genome.
  • exemplary guide RNAs targeting such locations include a spacer comprising the sequence of any one of SEQ ID NOs: 1-132. As is understood by the person of ordinary skill in the art, each guide RNA is designed to include a spacer sequence
  • a spacer comprising the sequence of any one of SEQ ID NOs: 1-132 can be put into a single RNA chimera or a crRNA (along with a corresponding tracrRNA). See Jinek, M. et al. (2012). Science, 337(6096):816-821 and Deltcheva, E. et al., (2011). Nature, 471:602-601.
  • Site-directed polypeptides such as a DNA endonuclease
  • the double-strand break can stimulate a cell’s endogenous DNA-repair pathways (e.g., homology-dependent repair (HDR) or non-homologous end joining or alternative non-homologous end joining (A-NHEJ) or microhomology-mediated end joining (MMEJ).
  • HDR homology-dependent repair
  • A-NHEJ non-homologous end joining
  • MMEJ microhomology-mediated end joining
  • NHEJ can repair cleaved target nucleic acid without the need for a homologous template.
  • HDR which is also known as homologous recombination (HR) can occur when a homologous repair template, or donor, is available.
  • the homologous donor template has sequences that are homologous to sequences flanking the target nucleic acid cleavage site.
  • the sister chromatid is generally used by the cell as the repair template.
  • the repair template is often supplied as an exogenous nucleic acid, such as a plasmid, duplex oligonucleotide, single-strand oligonucleotide, double-stranded oligonucleotide, or viral nucleic acid.
  • MMEJ results in a genetic outcome that is similar to NHEJ in that small deletions and insertions can occur at the cleavage site.
  • MMEJ makes use of homologous sequences of a few base pairs flanking the cleavage site to drive a favored end joining DNA repair outcome. In some instances, it can be possible to predict likely repair outcomes based on analysis of potential microhomologies in the nuclease target regions.
  • homologous recombination is used to insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site.
  • An exogenous polynucleotide sequence is termed a donor polynucleotide (or donor or donor sequence or polynucleotide donor template) herein.
  • the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide is inserted into the target nucleic acid cleavage site.
  • the donor polynucleotide is an exogenous polynucleotide sequence, i.e., a sequence that does not naturally occur at the target nucleic acid cleavage site.
  • exogenous DNA molecule When an exogenous DNA molecule is supplied in sufficient concentration inside the nucleus of a cell in which the double-strand break occurs, the exogenous DNA can be inserted at the double-strand break during the NHEJ repair process and thus become a permanent addition to the genome.
  • exogenous DNA molecules are referred to as donor templates in some embodiments.
  • the integrated copy of the donor DNA template can be transmitted to the daughter cells when the cell divides.
  • the donor DNA template can be integrated via the HDR pathway.
  • the homology arms act as substrates for homologous recombination between the donor template and the sequences either side of the double- strand break. This can result in an error free insertion of the donor template in which the sequences either side of the double-strand break are not altered from that in the un-modified genome.
  • Supplied donors for editing by HDR vary markedly but generally contain the intended sequence with small or large flanking homology arms to allow annealing to the genomic DNA.
  • the homology regions flanking the introduced genetic changes can be 20 bp or smaller, or as large as a multi-kilobase cassette that can contain promoters, cDNAs, etc.
  • Both single- stranded and double- stranded oligonucleotide donors can be used. These oligonucleotides range in size from less than 100 nt to over many kb, though longer ssDNA can also be generated and used. Double-stranded donors are often used, including PCR amplicons, plasmids, and mini-circles.
  • an AAV vector is a very effective means of delivery of a donor template, though the packaging limits for individual donors is ⁇ 5kb. Active transcription of the donor increased HDR three-fold, indicating the inclusion of promoter can increase conversion. Conversely, CpG methylation of the donor can decrease gene expression and HDR.
  • the donor DNA can be supplied with the nuclease or
  • tethering options can be used to increase the availability of the donors for HDR in some embodiments. Examples include attaching the donor to the nuclease, attaching to DNA binding proteins that bind nearby, or attaching to proteins that are involved in DNA end binding or repair.
  • site-specific gene insertions can be conducted that use both the NHEJ pathway and HR.
  • a combination approach can be applicable in certain settings, possibly including intron/exon borders. NHEJ can prove effective for ligation in the intron, while the error-free HDR can be better suited in the coding region.
  • the methods of genome edition and compositions therefore can use a nucleic acid sequence (or oligonucleotide) encoding a site-directed polypeptide or DNA endonuclease.
  • the nucleic acid sequence encoding the site-directed polypeptide can be DNA or RNA. If the nucleic acid sequence encoding the site-directed polypeptide is RNA, it can be covalently linked to a gRNA sequence or exist as a separate sequence. In some embodiments, a peptide sequence of the site-directed polypeptide or DNA endonuclease can be used instead of the nucleic acid sequence thereof.
  • the present disclosure provides a nucleic acid having a nucleotide sequence encoding a genome-targeting nucleic acid of the disclosure, a site-directed polypeptide of the disclosure, and/or any nucleic acid or proteinaceous molecule necessary to carry out the embodiments of the methods of the disclosure.
  • a nucleic acid is a vector (e.g., a recombinant expression vector).
  • Expression vectors contemplated include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors.
  • retrovirus e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloprolif
  • vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXTl, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pCTx-l, pCTx-2, and pCTx-3. Other vectors can be used so long as they are compatible with the host cell.
  • a vector has one or more transcription and/or translation control elements.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. can be used in the expression vector.
  • the vector is a self-inactivating vector that either inactivates the viral sequences or the components of the CRISPR machinery or other elements.
  • eukaryotic promoters i.e., promoters functional in a eukaryotic cell
  • eukaryotic promoters include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-l promoter (EF1), a hybrid construct having the
  • CMV cytomegalovirus
  • CAG chicken beta-actin promoter
  • MSCV murine stem cell virus promoter
  • PGK phosphoglycerate kinase- 1 locus promoter
  • mouse metallothionein-I mouse metallothionein-I
  • RNA polymerase III promoters including for example U6 and Hl
  • descriptions of and parameters for enhancing the use of such promoters are known in art, and additional information and approaches are regularly being described; see, e.g., Ma, H. et ah, (2014). Molecular Therapy - Nucleic Acids, 3:el6l.
  • the expression vector can also contain a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector can also include appropriate sequences for amplifying expression.
  • the expression vector can also include nucleotide sequences encoding non-native tags (e.g., histidine tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the site-directed polypeptide, thus resulting in a fusion protein.
  • a promoter is an inducible promoter (e.g., a heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.).
  • a promoter is a constitutive promoter (e.g., CMV promoter, UBC promoter).
  • the promoter is a spatially restricted and/or temporally restricted promoter (e.g., a tissue specific promoter, a cell type specific promoter, etc.).
  • a vector does not have a promoter for at least one gene to be expressed in a host cell if the gene is going to be expressed, after it is inserted into a genome, under an endogenous promoter present in the genome.
  • the modifications of the target DNA due to NHEJ and/or HDR can lead to, for example, mutations, deletions, alterations, integrations, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocations and/or gene mutation.
  • the process of integrating non-native nucleic acid into genomic DNA is an example of genome editing.
  • a site-directed polypeptide is a nuclease used in genome editing to cleave DNA.
  • the site-directed polypeptide can be administered to a cell or a subject as either: one or more polypeptides, or one or more mRNAs encoding the polypeptide.
  • the term“site-directed polypeptide” is used interchangeably herein with the term“Cas endonuclease.”
  • the site-directed polypeptide can bind to a guide RNA that, in turn, specifies the site in the target DNA to which the polypeptide is directed.
  • the site-directed polypeptide is an endonuclease, such as a DNA endonuclease.
  • a site-directed polypeptide has a plurality of nucleic acid cleaving (i.e., nuclease) domains. Two or more nucleic acid-cleaving domains can be linked together via a linker.
  • the linker has a flexible linker. Linkers can have 1,
  • Naturally-occurring wild-type Cas9 enzymes have two nuclease domains, a HNH nuclease domain and a RuvC domain.
  • the“Cas9” refers to both naturally-occurring and recombinant Cas9s.
  • Cas9 enzymes contemplated herein have a HNH or HNH-like nuclease domain, and/or a RuvC or RuvC-like nuclease domain.
  • HNH or HNH-like domains have a McrA-like fold. HNH or HNH-like domains has two antiparallel b-strands and an a-helix. HNH or HNH-like domains has a metal binding site (e.g., a divalent cation binding site). HNH or HNH-like domains can cleave one strand of a target nucleic acid (e.g., the complementary strand of the crRNA targeted strand).
  • a target nucleic acid e.g., the complementary strand of the crRNA targeted strand.
  • RuvC or RuvC-like domains have an RNaseH or RNaseH-like fold. RuvC/RNaseH domains are involved in a diverse set of nucleic acid-based functions including acting on both RNA and DNA.
  • the RNaseH domain has 5 b-strands surrounded by a plurality of a-helices.
  • RuvC/RNaseH or RuvC/RNaseH-like domains have a metal binding site (e.g., a divalent cation binding site).
  • RuvC/RNaseH or RuvC/RNaseH-like domains can cleave one strand of a target nucleic acid (e.g., the non-complementary strand of a double- stranded target DNA).
  • the site-directed polypeptide has an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity to a wild-type exemplary site-directed polypeptide (e.g.,
  • the site-directed polypeptide has an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity to the nuclease domain of a wild-type exemplary site- directed polypeptide (e.g., Cas9 from S. pyogenes, supra).
  • a wild-type exemplary site- directed polypeptide e.g., Cas9 from S. pyogenes, supra.
  • a site-directed polypeptide has at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra ) over 10 contiguous amino acids. In some embodiments, a site-directed polypeptide has at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g.,
  • a site- directed polypeptide has at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a HNH nuclease domain of the site-directed polypeptide.
  • a site-directed polypeptide has at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site- directed polypeptide (e.g., Cas9 from S.
  • a site-directed polypeptide has at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site- directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a RuvC nuclease domain of the site-directed polypeptide.
  • a wild-type site- directed polypeptide e.g., Cas9 from S. pyogenes, supra
  • a site-directed polypeptide has at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site- directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a RuvC nuclease domain of the site-directed polypeptide.
  • a wild-type site- directed polypeptide e.g., Cas9 from S. pyogenes, supra
  • the site-directed polypeptide has a modified form of a wild-type exemplary site-directed polypeptide.
  • the modified form of the wild- type exemplary site- directed polypeptide has a mutation that reduces the nucleic acid-cleaving activity of the site- directed polypeptide.
  • the modified form of the wild-type exemplary site- directed polypeptide has less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity of the wild-type exemplary site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra).
  • the modified form of the site-directed polypeptide can have no substantial nucleic acid-cleaving activity.
  • a site-directed polypeptide is a modified form that has no substantial nucleic acid-cleaving activity, it is referred to herein as "enzymatically inactive.”
  • the modified form of the site-directed polypeptide has a mutation such that it can induce a single-strand break (SSB) on a target nucleic acid (e.g., by cutting only one of the sugar-phosphate backbones of a double-strand target nucleic acid).
  • the mutation results in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity in one or more of the plurality of nucleic acid-cleaving domains of the wild-type site directed polypeptide (e.g., Cas9 from S.
  • the mutation results in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the complementary strand of the target nucleic acid, but reducing its ability to cleave the non-complementary strand of the target nucleic acid. In some embodiments, the mutation results in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the non-complementary strand of the target nucleic acid, but reducing its ability to cleave the complementary strand of the target nucleic acid. For example, residues in the wild-type exemplary S.
  • pyogenes Cas9 polypeptide such as Asp 10, His840, Asn854 and Asn856, are mutated to inactivate one or more of the plurality of nucleic acid-cleaving domains (e.g., nuclease domains).
  • the residues to be mutated correspond to residues Asp 10, His840, Asn854 and Asn856 in the wild- type exemplary S. pyogenes Cas9 polypeptide (e.g., as determined by sequence and/or structural alignment).
  • Non-limiting examples of mutations include D10A, H840A, N854A or N856A.
  • mutations other than alanine substitutions are suitable.
  • a D10A mutation is combined with one or more of H840A, N854A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity.
  • a H840A mutation is combined with one or more of D10A, N854A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity.
  • a N854A mutation is combined with one or more of H840A, D10A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity.
  • a N856A mutation is combined with one or more of H840A, N854A, or D10A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity.
  • Site-directed polypeptides that have one substantially inactive nuclease domain are referred to as“nickases”.
  • variants of RNA-guided endonucleases can be used to increase the specificity of CRISPR-mediated genome editing.
  • Wild type Cas9 is typically guided by a single guide RNA designed to hybridize with a specified ⁇ 20 nucleotide sequence in the target sequence (such as an endogenous genomic locus).
  • nickase variants of Cas9 each only cut one strand, in order to create a double strand break it is necessary for a pair of nickases to bind in close proximity and on opposite strands of the target nucleic acid, thereby creating a pair of nicks, which is the equivalent of a double-strand break.
  • nickases can also be used to promote HDR versus NHEJ. HDR can be used to introduce selected changes into target sites in the genome through the use of specific donor sequences that effectively mediate the desired changes. Descriptions of various CRISPR/Cas systems for use in gene editing can be found, e.g., in international patent application publication number WO
  • the site-directed polypeptide (e.g., variant, mutated, enzymatically inactive and/or conditionally enzymatically inactive site-directed polypeptide) targets nucleic acid.
  • the site-directed polypeptide (e.g., variant, mutated, enzymatically inactive and/or conditionally enzymatically inactive endoribonuclease) targets DNA.
  • the site-directed polypeptide e.g., variant, mutated, enzymatically inactive and/or conditionally enzymatically inactive endoribonuclease) targets RNA.
  • the site-directed polypeptide has one or more non-native sequences (e.g., the site-directed polypeptide is a fusion protein).
  • the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain).
  • a Cas9 from a bacterium e.g., S. pyogenes
  • a nucleic acid binding domain e.g., S. pyogenes
  • two nucleic acid cleaving domains i.e., a HNH domain and a RuvC domain
  • the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain).
  • a Cas9 from a bacterium e.g., S. pyogenes
  • two nucleic acid cleaving domains i.e., a HNH domain and a RuvC domain.
  • the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), and two nucleic acid cleaving domains, wherein one or both of the nucleic acid cleaving domains have at least 50% amino acid identity to a nuclease domain from Cas9 from a bacterium (e.g., S. pyogenes).
  • a bacterium e.g., S. pyogenes
  • the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain), and non-native sequence (for example, a nuclear localization signal) or a linker linking the site-directed polypeptide to a non native sequence.
  • a Cas9 from a bacterium (e.g., S. pyogenes)
  • two nucleic acid cleaving domains i.e., a HNH domain and a RuvC domain
  • non-native sequence for example, a nuclear localization signal
  • the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain), wherein the site-directed polypeptide has a mutation in one or both of the nucleic acid cleaving domains that reduces the cleaving activity of the nuclease domains by at least 50%.
  • a Cas9 from a bacterium e.g., S. pyogenes
  • two nucleic acid cleaving domains i.e., a HNH domain and a RuvC domain
  • the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain), wherein one of the nuclease domains has mutation of aspartic acid 10, and/or wherein one of the nuclease domains has mutation of histidine 840, and wherein the mutation reduces the cleaving activity of the nuclease domain(s) by at least 50%.
  • a Cas9 from a bacterium e.g., S. pyogenes
  • two nucleic acid cleaving domains i.e., a HNH domain and a RuvC domain
  • the one or more site-directed polypeptides include two nickases that together effect one double-strand break at a specific locus in the genome, or four nickases that together effect two double-strand breaks at specific loci in the genome.
  • one site-directed polypeptide e.g., DNA endonuclease, affects one double- strand break at a specific locus in the genome.
  • a polynucleotide encoding a site-directed polypeptide can be used to edit a cellular genome.
  • the polynucleotide encoding a site- directed polypeptide is codon-optimized according to methods standard in the art for expression in the cell containing the target DNA of interest. For example, if the intended target nucleic acid is in a human cell, a human codon-optimized polynucleotide encoding a Cas endonuclease is contemplated for use for producing the Cas endonuclease.
  • a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic locus can be found in the genomes of many prokaryotes (e.g., bacteria and archaea). In prokaryotes, the CRISPR locus encodes products that function as a type of immune system to help defend the prokaryotes against foreign invaders, such as virus and phage. There are three stages of CRISPR locus function: integration of new sequences into the CRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreign invader nucleic acid. Five types of CRISPR systems (e.g., Type I, Type II, Type III, Type U, and Type V) have been identified.
  • a CRISPR locus includes a number of short repeating sequences referred to as “repeats.” When expressed, the repeats can form secondary hairpin structures (e.g., hairpins) and/or have unstructured single- stranded sequences.
  • the repeats usually occur in clusters and frequently diverge between species.
  • the repeats are regularly interspaced with unique intervening sequences referred to as“spacers,” resulting in a repeat- spacer-repeat locus architecture.
  • the spacers are identical to or have high homology with known foreign invader sequences.
  • a spacer-repeat unit encodes a crisprRNA (crRNA), which is processed into a mature form of the spacer-repeat unit.
  • crRNA crisprRNA
  • a crRNA has a“seed” or spacer sequence that is involved in targeting a target nucleic acid (in the naturally occurring form in prokaryotes, the spacer sequence targets the foreign invader nucleic acid).
  • a spacer sequence is located at the 5' or 3' end of the crRNA.
  • a CRISPR locus also has polynucleotide sequences encoding CRISPR Associated (Cas) genes.
  • Cas genes encode endonucleases involved in the biogenesis and the interference stages of crRNA function in prokaryotes. Some Cas genes have homologous secondary and/or tertiary structures.
  • crRNA biogenesis in a Type II CRISPR system in nature requires a trans-activating CRISPR RNA (tracrRNA).
  • the tracrRNA is modified by endogenous RNaselll, and then hybridizes to a crRNA repeat in the pre-crRNA array. Endogenous RNaselll is recruited to cleave the pre-crRNA. Cleaved crRNAs are subjected to exoribonuclease trimming to produce the mature crRNA form (e.g., 5' trimming).
  • the tracrRNA remains hybridized to the crRNA, and the tracrRNA and the crRNA associate with a site-directed polypeptide (e.g., Cas9).
  • a site-directed polypeptide e.g., Cas9
  • the crRNA of the crRNA-tracrRNA-Cas9 complex guides the complex to a target nucleic acid to which the crRNA can hybridize. Hybridization of the crRNA to the target nucleic acid activates Cas9 for targeted nucleic acid cleavage.
  • the target nucleic acid in a Type II CRISPR system is referred to as a protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • Type II systems also referred to as Nmeni or CASS4 are further subdivided into Type II-A (CASS4) and II-B (CASS4a). Jinek, M. et al. (2012). Science, 337(6096):816-821 showed that the CRISPR/Cas9 system is useful for RNA-programmable genome editing, and international patent application publication number WO 2013/176772 provides numerous examples and applications of the CRISPR/Cas
  • Type V CRISPR systems have several important differences from Type II systems.
  • Cpfl is a single RNA-guided endonuclease that, in contrast to Type II systems, lacks tracrRNA.
  • Cpfl -associated CRISPR arrays are processed into mature crRNAS without the requirement of an additional trans-activating tracrRNA.
  • the Type V CRISPR array is processed into short mature crRNAs of 42-44 nucleotides in length, with each mature crRNA beginning with 19 nucleotides of direct repeat followed by 23-25 nucleotides of spacer sequence.
  • mature crRNAs in Type II systems start with 20-24 nucleotides of spacer sequence followed by about 22 nucleotides of direct repeat.
  • Cpfl utilizes a T-rich protospacer- adjacent motif such that Cpfl-crRNA complexes efficiently cleave target DNA preceded by a short T-rich PAM, which is in contrast to the G-rich PAM following the target DNA for Type II systems.
  • Type V systems cleave at a point that is distant from the PAM
  • Type II systems cleave at a point that is adjacent to the PAM.
  • Cpfl cleaves DNA via a staggered DNA double-stranded break with a 4 or 5 nucleotide 5’ overhang.
  • Type II systems cleave via a blunt double- stranded break.
  • Cpfl contains a predicted RuvC-like endonuclease domain, but lacks a second HNH
  • Exemplary CRISPR/Cas polypeptides include the Cas9 polypeptides in Fig. 1 of Fonfara, I. et al. (2014.) Nucleic Acids Research, 42(4):2577-2590.
  • the CRISPR/Cas gene naming system has undergone extensive rewriting since the Cas genes were discovered.
  • Fig. 5 of Fonfara, I. et al. (2014.) Nucleic Acids Research, 42(4):2577-2590 provides PAM sequences for Cas9 polypeptides from various species.
  • a genome-targeting nucleic acid interacts with a site-directed polypeptide (e.g., a nucleic acid-guided nuclease such as Cas9), thereby forming a complex.
  • the genome-targeting nucleic acid e.g., gRNA
  • the site-directed polypeptide and genome targeting nucleic acid can each be administered separately to a cell or a patient.
  • the site-directed polypeptide can be pre-complexed with one or more guide RNAs, or one or more crRNA together with a tracrRNA.
  • the pre-complexed material can then be administered to a cell or a patient.
  • Such pre-complexed material is known as a ribonucleoprotein particle (RNP).
  • One approach to functionally knock-out or reduce the expression of a protein associated with a disease, such as apo(a), in an organism in need thereof is to using genome editing to target the gene in a relevant cell type in such a way that expression of the gene is functionally suppressed.
  • a method of genome editing in particular, functionally knocking out or reducing the expression of an apolipoprotein(a) [apo(a)] gene in the genome of a cell.
  • This method can be used to treat a subject, e.g., a patient with a cardiovascular disease.
  • the chromosomal DNA of relevant cells in the subject e.g., hepatocytes
  • the cardiovascular disease is stroke, myocardial infarction, atherosclerosis, familial hypercholesterolemia, atherosclerosis, thrombosis, calcific aortic valve disease, coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, congenital heart disease, or rheumatic heart disease.
  • the present disclosure provides a method of editing a genome in a cell, the method comprising: providing the following to the cell: (a) one or two gRNA(s) according to any of the gRNAs described herein or nucleic acid encoding the gRNA(s); and/or (b) a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding the DNA endonuclease.
  • the method comprises providing to the cell an AAV vector comprising the nucleic acid encoding the gRNA(s) and/or the nucleic acid encoding the DNA endonuclease. In some embodiments, the method further comprises providing (c) a donor template comprising a nucleic acid sequence encoding one or more STOP codons to the cell.
  • the spacer comprises a contiguous sequence at least 19 nucleotides in length from any one of SEQ ID NOs: 1-132 and variants thereof having at least 85% homology to a contiguous sequence at least 19 nucleotides in length from any one of SEQ ID NOs: 1-132. In some embodiments, the spacer is 20 nucleotides in length.
  • the spacer comprises a polynucleotide sequence from position 2 to position 20 of any one of SEQ ID NOs: 1-132 and variants thereof having at least 85% homology to a polynucleotide sequence from position 2 to position 20 of any one of SEQ ID NOs: 1-132.
  • a method of editing a genome in a cell comprising providing to the cell: a) a DNA endonuclease (e.g., a Cas endonuclease, such as Cas9) or nucleic acid encoding the DNA endonuclease; and b) a gRNA comprising a spacer sequence complementary to a target genomic sequence within or near an endogenous apolipoprotein(a) (LPA) gene in the cell, or nucleic acid encoding the gRNA, wherein the DNA endonuclease and gRNA are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of promoting editing of the target genomic sequence in the cell to generate a genetically modified cell with reduced expression of apo(a) as compared to a corresponding unmodified cell.
  • a DNA endonuclease e.g., a Cas endonuclease, such as Cas9
  • the cell prior to carrying out the method is an input cell that expresses apo(a) at a level greater than a reference level for the expression of apo(a) in the input cell type.
  • the genetically modified cell has reduced functional expression of apo(a) as compared to a corresponding unmodified cell.
  • the genetically modified cell has no functional expression of apo(a).
  • the method further comprises providing to the cell a donor template comprising a nucleic acid sequence to be inserted into the LPA gene.
  • the nucleic acid sequence to be inserted encodes one or more STOP codons, and the system is configured to insert a STOP codon in-frame into an LPA gene coding sequence such that the LPA gene is rendered non-functional.
  • the gRNA targets within or near a coding sequence in the LPA gene.
  • the gRNA targets exon 1, exon 2, or exon 3 of the LPA gene.
  • the gRNA targets a sequence in the LPA gene
  • the gRNA targets within or near a non-coding sequence in the LPA gene. In some embodiments, the gRNA targets an LPA gene intron. In some embodiments, the gRNA targets an LPA gene regulatory region. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 1-132. In some embodiments, the DNA endonuclease is a Cas endonuclease, e.g., a Cas9 endonuclease. In some embodiments, the gRNA is an sgRNA.
  • a method of editing a genome in a cell comprising providing to the cell: a) a DNA endonuclease (e.g., a Cas endonuclease, such as Cas9) or nucleic acid encoding the DNA endonuclease; and b) a gRNA comprising i) a spacer sequence complementary to a target genomic sequence within exon 3 of an LPA gene; ii) a spacer sequence complementary to a target genomic sequence within exon 2 of an LPA gene; iii) a spacer sequence complementary to a target genomic sequence within an LPA gene
  • the gRNA comprises i) a spacer sequence from any one of SEQ ID NOs: 13-19 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 13-19; ii) a spacer sequence from any one of SEQ ID NOs: 1-12 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 1-12; iii) a spacer sequence from any one of SEQ ID NOs: 107-132 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 107-132; or iv) a spacer sequence from any one of SEQ ID NOs: 20-106 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 20- 106.
  • the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 14, 15, 18, and 19.
  • the spacer sequence is 19 nucleotides in length and does not include the nucleotide at position 1 of the sequence from which it is selected.
  • the spacer sequence is the nucleotide sequence of any one of SEQ ID NOs: 157-160.
  • the DNA endonuclease and gRNA are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of generating a double-strand break at the target genomic sequence.
  • the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 13-19 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 13- 19. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 13-19. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of any one of SEQ ID NOs: 13-19. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 14 or a variant thereof having no more than 3 mismatches compared to SEQ ID NO: 14.
  • the gRNA comprises a spacer sequence from SEQ ID NO: 14. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 15 or a variant thereof having no more than 3 mismatches compared to SEQ ID NO: 15. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 15. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 18 or a variant thereof having no more than 3 mismatches compared to SEQ ID NO: 18.
  • the gRNA comprises a spacer sequence from SEQ ID NO: 18. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 19 or a variant thereof having no more than 3 mismatches compared to SEQ ID NO: 19. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 19. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of SEQ ID NO: 19.
  • the DNA endonuclease is a Cas endonuclease.
  • the DNA endonuclease is a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2,
  • the DNA endonuclease is a Cas9 endonuclease.
  • the Cas9 endonuclease is from Streptococcus pyogenes (SpyCas9). In some embodiments, the Cas9 endonuclease is from Staphylococcus lugdunensis (SluCas9).
  • a DNA sequence that is transcribed to the nucleic acid encoding the DNA endonuclease is codon optimized.
  • the nucleic acid sequence encoding one or more STOP codons is codon optimized.
  • the nucleic acid sequence encoding one or more STOP codons does not comprise CpG dinucleotides.
  • the nucleic acid encoding the DNA endonuclease comprises a 5’ CAP structure and 3’ polyA tail.
  • the nucleic acid encoding the DNA endonuclease is linked to the gRNA via a covalent bond.
  • the method comprises providing to the cell a nucleic acid encoding the DNA endonuclease.
  • the nucleic acid encoding the DNA endonuclease is codon- optimized for expression in the cell.
  • the nucleic acid encoding the DNA endonuclease is DNA, such as a DNA plasmid.
  • the nucleic acid encoding the DNA endonuclease is RNA, such as mRNA.
  • the nucleic acid sequence encoding one or more STOP codons is inserted into a genomic sequence of the cell. In some embodiments, the insertion is at, within, or near the endogenous apolipoprotein(a) ( LPA ) gene or LPA gene regulatory elements in the genome of the cell.
  • the donor template comprising a nucleic acid sequence encoding one or more STOP codons comprises three STOP codons in each of the 3 translation frames present in succession in the donor DNA sequence. In some embodiments, the donor DNA is delivered as a double-stranded or single-stranded oligonucleotide.
  • the single- stranded or double-stranded donor DNA contains homology arms composed of the sequences of 20 bp to 1000 bp, or more, flanking each side of the sgRNA cut site.
  • the method further comprises providing to the cell a donor template comprising a sequence to be integrated at or near the target genomic sequence.
  • the donor template comprises a donor cassette comprising a nucleic acid sequence encoding one or more STOP codons.
  • the nucleic acid sequence encoding one or more STOP codons encodes three STOP codons in each of the 3 possible translation frames in the forward orientation and/or three STOP codons in each of the 3 possible translation frames in the reverse orientation.
  • An exemplary sequence encoding three STOP codons in each of the 3 possible translation frames in both the forward and reverse orientations is provided in SEQ ID NO: 155.
  • the DNA endonuclease, gRNA, and donor template are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of promoting targeted integration of the donor cassette into a target genomic locus (e.g., a coding region) comprising the target genomic sequence.
  • integration of the donor cassette into the target genomic locus in the cell results in the generation of a genetically modified cell with reduced functional expression of apo(a) as compared to a corresponding unmodified cell.
  • integration of the donor template into an LPA gene coding sequence such that a premature STOP codon is inserted in-frame can result in a non functional or reduced function truncated translation product as compared to the apo(a) protein encoded by the unmodified LPA gene.
  • the donor template is configured such that the donor cassette is capable of being integrated into a genomic locus targeted by the gRNA by homology directed repair (HDR).
  • HDR homology directed repair
  • the donor cassette is flanked on both sides by homology arms corresponding to sequences in the targeted genomic locus.
  • the homology arms are at least about 0.2 kb (such as at least about any of 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, or greater) in length.
  • the homology arms are at least about 0.4 kb, e.g., 0.45 kb, 0.6 kb, or 0.8 kb, in length.
  • the donor template is encoded in an Adeno Associated Virus (AAV) vector.
  • AAV vector is an AAV2, AAV5, or AAV6 vector.
  • the AAV vector is an AAV6 vector.
  • the donor template is configured such that the donor cassette is capable of being integrated into a genomic locus targeted by the gRNA by non- homologous end joining (NHEJ).
  • NHEJ non- homologous end joining
  • the donor cassette is flanked on one or both sides by a gRNA target site.
  • the donor cassette is flanked on both sides by a gRNA target site.
  • the gRNA target site is a target site for the gRNA targeting the LPA gene.
  • the gRNA target site of the donor template is the reverse complement of the cell genome gRNA target site.
  • the donor template is encoded in an Adeno Associated Virus (AAV) vector.
  • AAV vector is an AAV2, AAV5, or AAV6 vector.
  • the AAV vector is an AAV6 vector.
  • the DNA endonuclease or nucleic acid encoding the DNA endonuclease is formulated in a liposome or lipid nanoparticle.
  • the liposome or lipid nanoparticle also comprises the gRNA or nucleic acid encoding the gRNA.
  • the liposome or lipid nanoparticle is a lipid nanoparticle.
  • the system comprises a lipid nanoparticle comprising nucleic acid encoding the DNA
  • the nucleic acid encoding the DNA endonuclease is an mRNA encoding the DNA endonuclease.
  • the DNA endonuclease is pre-complexed with the gRNA, forming a
  • Ribonucleoprotein (RNP) complex prior to the provision to the cell.
  • the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA are provided to the cell after the donor template is provided to the cell.
  • the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA are provided to the cell after the donor template is provided to the cell.
  • endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA are provided to the cell about 1 to 14 days after the donor template is provided to the cell.
  • one or more (such as any of one, two, three, four, five, or more) additional doses of the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA are provided to the cell following a first dose of the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA.
  • one or two gRNA(s) individually comprising a spacer complementary to a genomic sequence within or near are provided to the cell.
  • the spacer(s) are complementary to a genomic sequence within or near (for example, within any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bases from) exon 3 of the LPA gene.
  • the gRNA(s) are any one or two gRNAs comprising spacers selected from the group consisting of SEQ ID NOs: 1-19 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 1-19.
  • two gRNAs comprising spacers complementary to a genomic sequence within a transcriptional regulatory sequence of the LPA gene are provided to the cell.
  • the transcriptional regulatory sequence comprises a promoter or enhancer.
  • the gRNAs are any two gRNAs comprising spacers selected from the group consisting of SEQ ID NOs: 20-106 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 20-106.
  • a gRNA comprising a spacer complementary to a genomic sequence encoding a kringle domain in an apo(a) protein is provided to the cell.
  • the gRNA comprises a spacer selected from the group consisting of SEQ ID NOs: 107-132 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 107-132.
  • the cell prior to carrying out the method is an input cell that expresses apo(a).
  • the input cell expresses apo(a) at a level greater than a reference level for the expression of apo(a) in the input cell type.
  • the genetically modified cell has reduced functional expression of apo(a) as compared to a corresponding unmodified cell.
  • the genetically modified cell has no functional expression of apo(a).
  • the input cell is a hepatocyte.
  • the endogenous LPA gene is a variant associated with increased cardiovascular disease risk and/or increased Lp(a) expression.
  • the method is an in vivo method, and the cell is a cell in a subject. In some embodiments, the subject is human.
  • shifts in the location of the 5' boundary and/or the 3' boundary relative to particular reference loci are used to facilitate or enhance particular applications of gene editing, which depend in part on the endonuclease system selected for the editing, as further described and illustrated herein.
  • many endonuclease systems have rules or criteria that guide the initial selection of potential target sites for cleavage, such as the requirement of a PAM sequence motif in a particular position adjacent to the DNA cleavage sites in the case of CRISPR Type II or Type V endonucleases.
  • the frequency of“off-target” activity for a particular combination of target sequence and gene editing endonuclease is assessed relative to the frequency of on-target activity.
  • cells that have been correctly edited at the desired locus can have a selective advantage relative to other cells.
  • a selective advantage include the acquisition of attributes such as enhanced rates of replication, persistence, resistance to certain conditions, enhanced rates of successful engraftment or persistence in vivo following introduction into a patient, and other attributes associated with the maintenance or increased numbers or viability of such cells.
  • cells that have been correctly edited at the desired locus can be positively selected for by one or more screening methods used to identify, sort or otherwise select for cells that have been correctly edited. Both selective advantage and directed selection methods can take advantage of the phenotype associated with the correction.
  • cells can be edited two or more times in order to create a second modification that creates a new phenotype that is used to select or purify the intended population of cells.
  • a second modification could be created by adding a second gRNA for a selectable or screenable marker.
  • cells can be correctly edited at the desired locus using a DNA fragment that contains the cDNA and also a selectable marker.
  • target sequence selection is also guided by consideration of off-target frequencies in order to enhance the effectiveness of the application and/or reduce the potential for undesired alterations at sites other than the desired target.
  • the occurrence of off-target activity is influenced by a number of factors including similarities and dissimilarities between the target site and various off-target sites, as well as the particular endonuclease used.
  • Bioinformatics tools are available that assist in the prediction of off-target activity, and frequently such tools can also be used to identify the most likely sites of off-target activity, which can then be assessed in experimental settings to evaluate relative frequencies of off-target to on-target activity, thereby allowing the selection of sequences that have higher relative on-target activities.
  • Illustrative examples of such techniques are provided herein, and others are known in the art.
  • Another aspect of target sequence selection relates to homologous recombination events. Sequences sharing regions of homology can serve as focal points for homologous recombination events that result in deletion of intervening sequences. Such recombination events occur during the normal course of replication of chromosomes and other DNA sequences, and also at other times when DNA sequences are being synthesized, such as in the case of repairs of double-strand breaks (DSBs), which occur on a regular basis during the normal cell replication cycle but can also be enhanced by the occurrence of various events (such as UV light and other inducers of DNA breakage) or the presence of certain agents (such as various chemical inducers).
  • various events such as UV light and other inducers of DNA breakage
  • certain agents such as various chemical inducers
  • DSBs small insertions or deletions
  • DSBs can also be specifically induced at particular locations, as in the case of the endonucleases systems described herein, which can be used to cause directed gene modification events at selected chromosomal locations.
  • the tendency for homologous sequences to be subject to recombination in the context of DNA repair (as well as replication) can be taken advantage of in a number of circumstances, and is the basis for one application of gene editing systems, such as CRISPR, in which homology directed repair is used to insert a sequence of interest, provided through use of a“donor” polynucleotide, into a desired chromosomal location.
  • Regions of homology between particular sequences which can be small regions of “microhomology” that can have as few as ten base pairs or less, can also be used to bring about desired deletions.
  • a single DSB is introduced at a site that exhibits microhomology with a nearby sequence.
  • a result that occurs with high frequency is the deletion of the intervening sequence as a result of recombination being facilitated by the DSB and concomitant cellular repair process.
  • target sequences within regions of homology can also give rise to much larger deletions, including gene fusions (when the deletions are in coding regions), which can or cannot be desired given the particular circumstances.
  • the examples provided herein further illustrate the selection of various target regions for the creation of DSBs, as well as the selection of specific target sequences within such regions that are designed to minimize off-target events relative to on-target events.
  • the CRISPR-Cas system used in some embodiments has the advantage that a large number of genomic targets can be rapidly screened to identify an optimal CRISPR-Cas design.
  • the CRISPR-Cas system uses a RNA molecule called a single guide RNA (sgRNA) that targets an associated Cas endonuclease (for example a Cas9 nuclease) to a specific sequence in DNA. This targeting occurs by Watson-Crick based pairing between the sgRNA and the sequence of the genome within the approximately 20 bp targeting sequence of the sgRNA. Once bound at a target site, a Cas endonuclease cleaves both strands of the genomic DNA creating a double strand break.
  • sgRNA single guide RNA
  • Cas endonuclease for example a Cas9 nuclease
  • sgRNA The only requirement for designing a sgRNA to target a specific DNA sequence is that the target sequence must contain a protospacer adjacent motif (PAM) sequence at the 3’ end of the sgRNA sequence that is complementary to the genomic sequence.
  • PAM protospacer adjacent motif
  • the PAM sequence is NRG (where R is A or G and N is any base), or the more restricted PAM sequence NGG. Therefore, sgRNA molecules that target any region of the genome can be designed in silico by locating the 20 bp sequence adjacent to all PAM motifs. PAM motifs occur on average very 15 bp in the genome of eukaryotes.
  • sgRNA designed by in silico methods will generate double-strand breaks in cells with differing efficiencies and it is not possible to predict the cutting efficiencies of a series of sgRNA molecule using in silico methods. Because sgRNA can be rapidly synthesized in vitro this enables the rapid screening of all potential sgRNA sequences in a given genomic region to identify the sgRNA that results in the most efficient cutting. Typically when a series of sgRNA within a given genomic region are tested in cells a range of cleavage efficiencies between 0 and 90% is observed. In silico algorithms as well as laboratory experiments can also be used to determine the off-target potential of any given sgRNA.
  • While a perfect match to the 20 bp recognition sequence of a sgRNA will primarily occur only once in most eukaryotic genomes there will be a number of additional sites in the genome with 1 or more base pair mismatches to the sgRNA. These sites can be cleaved at variable frequencies which are often not predictable based on the number or location of the mismatches. Cleavage at additional off-target sites that were not identified by the in silico analysis can also occur. Thus, screening a number of sgRNA in a relevant cell type to identify sgRNA that have the most favorable off-target profile is a critical component of selecting an optimal sgRNA for therapeutic use.
  • a favorable off target profile will take into account not only the number of actual off-target sites and the frequency of cutting at these sites, but also the location in the genome of these sites. For example, off-target sites close to or within functionally important genes, particularly oncogenes or anti-oncogenes would be considered as less favorable than sites in intergenic regions with no known function.
  • the identification of an optimal sgRNA cannot be predicted simply by in silico analysis of the genomic sequence of an organism but requires experimental testing. While in silico analysis can be helpful in narrowing down the number of guides to test it cannot predict guides that have high on target cutting or predict guides with low desirable off-target cutting.
  • sgRNA that each has a perfect match to the genome in a region of interest varies significantly and is not predictable by any known algorithm.
  • the ability of a given sgRNA to promote cleavage by a Cas enzyme can relate to the accessibility of that specific site in the genomic DNA which can be determined by the chromatin structure in that region. While the majority of the genomic DNA in a quiescent differentiated cell, such as a hepatocyte, exists in highly condensed heterochromatin, regions that are actively transcribed exists in more open chromatin states that are known to be more accessible to large molecules such as proteins a Cas endonuclease.
  • gRNAs that can be used in the methods disclosed herein include gRNAs comprising a spacer comprising the polynucleotide sequence of any one of SEQ ID NOs: 1-132 or any derivatives thereof having at least about 85% nucleotide sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 1-132.
  • polynucleotides introduced into cells have one or more modifications that can be used individually or in combination, for example, to enhance activity, stability or specificity, alter delivery, reduce innate immune responses in host cells, or for other enhancements, as further described herein and known in the art.
  • modified polynucleotides are used in the CRISPR/Cas9/Cpfl system, in which case the guide RNAs (either single-molecule guides or double-molecule guides) and/or a DNA or an RNA encoding a Cas or Cpfl endonuclease introduced into a cell can be modified, as described and illustrated below.
  • modified polynucleotides can be used in the CRISPR/Cas9/Cpfl system to edit any one or more genomic loci.
  • modifications of guide RNAs can be used to enhance the formation or stability of the CRISPR/Cas9/Cpfl genome editing complex having guide RNAs, which can be single-molecule guides or double-molecule, and a Cas or Cpfl endonuclease.
  • Modifications of guide RNAs can also or alternatively be used to enhance the initiation, stability or kinetics of interactions between the genome editing complex with the target sequence in the genome, which can be used, for example, to enhance on-target activity.
  • Modifications of guide RNAs can also or alternatively be used to enhance specificity, e.g., the relative rates of genome editing at the on-target site as compared to effects at other (off-target) sites.
  • Modifications can also or alternatively be used to increase the stability of a guide RNA, e.g., by increasing its resistance to degradation by ribonucleases (RNases) present in a cell, thereby causing its half-life in the cell to be increased. Modifications enhancing guide RNA half- life can be particularly useful in embodiments in which a Cas or Cpfl endonuclease is introduced into the cell to be edited via an RNA that needs to be translated in order to generate
  • RNases ribonucleases
  • RNA interference including small-interfering RNAs (siRNAs), as described below and in the art, tend to be associated with reduced half-life of the RNA and/or the elicitation of cytokines or other factors associated with immune responses.
  • RNA modifications that enhance the stability of the RNA such as by increasing its degradation by RNAses present in the cell
  • modifications that enhance translation of the resulting product i.e., the endonuclease
  • modifications that decrease the likelihood or degree to which the RNAs introduced into cells elicit innate immune responses including, without limitation, modifications that enhance the stability of the RNA (such as by increasing its degradation by RNAses present in the cell), modifications that enhance translation of the resulting product (i.e., the endonuclease), and/or modifications that decrease the likelihood or degree to which the RNAs introduced into cells elicit innate immune responses.
  • modifications such as the foregoing and others, can likewise be used.
  • CRISPR/Cas9/Cpfl for example, one or more types of modifications can be made to guide RNAs (including those exemplified above), and/or one or more types of modifications can be made to RNAs encoding Cas endonuclease (including those exemplified above).
  • guide RNAs used in the CRISPR/Cas9/Cpfl system can be readily synthesized by chemical means, enabling a number of
  • modifications can be used to, e.g., enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described further below and in the art; and new types of modifications are regularly being developed.
  • modifications can have one or more nucleotides modified at the 2' position of the sugar, in some embodiments a 2'-0-alkyl, 2'-0- alkyl-O-alkyl, or 2'-fluoro-modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'-amino or 2' O-methyl modifications on the ribose of pyrimidines, a basic residues, or an inverted base at the 3' end of the RNA.
  • modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2'-deoxyoligonucleotides against a given target.
  • modified oligonucleotide include those having modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • oligonucleotides are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH 2 -NH-O-CH2, CH, ⁇ N(CH 3 ) ⁇ 0 ⁇ CH 2 (known as a methylene(methylimino) or MMI backbone), CH2 -O-N (CH 3 )-CH2, Cth -N (CH 3 )-N (CH 3 )-CH2 and O-N (CH 3 )- Cth -CH2 backbones, wherein the native phosphodiester backbone is represented as O- P- O- CH,); amide backbones (see De Mesmaeker, A. et al. (1995). Acc. Chem.
  • PNA peptide nucleic acid
  • Phosphorus -containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates,
  • phosphotriesters aminoalkylphosphotriesters, methyl and other alkyl phosphonates having 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates having 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,
  • thionoalkylphosphonates having normal 3'- 5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos.
  • Morpholino-based oligomeric compounds are described in Braasch, D. A. et al. (2002). Biochemistry, 4i(l4):4503-45l0; Genesis, Volume 30, Issue 3, (2001); Heasman, J. (2002). Dev. Biol., 243( 2):209-2l4; Nasevicius, A. et al. (2000). Nature Genetics, 26: 216-220; Lacerra, G. et al., (2000). PNAS, 97(17): 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 3 , OCH3 0(CH 2 ) n CH 3 , 0(CH 2 ) radical NH 2 , or 0(CH 2 ) n CH3, where n is from 1 to about 10; Cl to C 10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; S0 2 CH3; ON0 2 ; N0 2 ; N3; NH 2 ; heterocycloalkyl; heterocycloalkaryl;
  • a modification includes 2'- methoxyethoxy (2'-0-CH 2 CH 2 0CH 3 , also known as 2'-0-(2-methoxyethyl)) (Martin, P. et al. (1995). Helv. Chim. Acta, 78(2):486-504).
  • both a sugar and an intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an
  • guide RNAs can also include, additionally or alternatively, nucleobase (often referred to in the art simply as“base”) modifications or substitutions.
  • nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7- deazaguanine, N6 (6-aminohexyl)adenine
  • modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl,
  • nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in Kroschwitz, J. I. (Ed.) (1990). The Concise Encyclopedia of Polymer Science and Engineering , (pp. 858-859). Hoboken, N. J.: John Wiley & Sons, those disclosed in
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N- 2, N-6 and 0-6 substituted purines, having 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
  • the guide RNAs and/or mRNA (or DNA) encoding an endonuclease are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger, R. L. et al. (1989). PNAS, 86(Y1) ⁇ 6553-6556); cholic acid (Manoharan, M. et al. (1994). Bioorg. Med. Chem.
  • sugars and other moieties can be used to target proteins and complexes having nucleotides, such as cationic polysomes and liposomes, to particular sites.
  • nucleotides such as cationic polysomes and liposomes
  • hepatic cell directed transfer can be mediated via asialoglycoprotein receptors
  • these targeting moieties or conjugates can include conjugate groups covalently bound to functional groups, such as primary or secondary hydroxyl groups.
  • Conjugate groups of the disclosure include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence- specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present disclosure. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl- 5 -tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium l,2-di-0-hexadecyl-rac- glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a
  • Longer polynucleotides that are less amenable to chemical synthesis and are typically produced by enzymatic synthesis can also be modified by various means. Such modifications can include, for example, the introduction of certain nucleotide analogs, the incorporation of particular sequences or other moieties at the 5' or 3' ends of molecules, and other modifications.
  • the mRNA encoding Cas9 is approximately 4 kb in length and can be synthesized by in vitro transcription.
  • Modifications to the mRNA can be applied to, e.g., increase its translation or stability (such as by increasing its resistance to degradation with a cell), or to reduce the tendency of the RNA to elicit an innate immune response that is often observed in cells following introduction of exogenous RNAs, particularly longer RNAs such as that encoding Cas9.
  • TriLink Biotech AxoLabs, Bio-Synthesis Inc., Dharmacon and many others.
  • TriLink for example, 5-methyl-CTP can be used to impart desirable characteristics, such as increased nuclease stability, increased translation or reduced interaction of innate immune receptors with in vitro transcribed RNA.
  • 5-Methylcytidine-5'-triphosphate (5-methyl-CTP), N6- methyl-ATP, as well as pseudo-UTP and 2-thio-UTP have also been shown to reduce innate immune stimulation in culture and in vivo while enhancing translation, as illustrated in publications by Kormann et al. and Warren et ah, referred to below.
  • Biotechnology 29.T54-157 Such modifications can be used, for example, to increase the stability of the RNA molecule and/or reduce its immunogenicity.
  • chemical modifications such as pseudo-U, N6-methyl-A, 2-Thio-U and 5-methyl-C, it was found that substituting just one quarter of the uridine and cytidine residues with 2-thio-U and 5-methyl-C respectively resulted in a significant decrease in toll-like receptor (TLR) mediated recognition of the mRNA in mice.
  • TLR toll-like receptor
  • these modifications can be used to effectively increase the stability and longevity of the mRNA in vivo, see, e.g., Kormann, M. S. D. et al. (2011). Nature Biotechnology 29.T54- 157.
  • iPSCs induced pluripotency stem cells
  • ARC A Anti Reverse Cap Analog
  • polynucleotides described in the art include, for example, the use of polyA tails, the addition of 5' cap analogs (such as m7G(5’)ppp(5’)G (mCAP)), modifications of 5' or 3' untranslated regions (UTRs), or treatment with phosphatase to remove 5' terminal phosphates - and new approaches are regularly being developed.
  • 5' cap analogs such as m7G(5’)ppp(5’)G (mCAP)
  • UTRs untranslated regions
  • treatment with phosphatase to remove 5' terminal phosphates - and new approaches are regularly being developed.
  • RNA interference including small-interfering RNAs (siRNAs).
  • siRNAs present particular challenges in vivo because their effects on gene silencing via mRNA interference are generally transient, which can require repeat administration.
  • siRNAs are double- stranded RNAs (dsRNA) and mammalian cells have immune responses that have evolved to detect and neutralize dsRNA, which is often a by-product of viral infection.
  • dsRNA double- stranded RNAs
  • mammalian cells have immune responses that have evolved to detect and neutralize dsRNA, which is often a by-product of viral infection.
  • PKR dsRNA-responsive kinase
  • RAG-I retinoic acid-inducible gene I
  • TLR3, TLR7 and TLR8 Toll-like receptors
  • TLR7/TLR8 interaction while generally preserving silencing activity; see, e.g., Judge, A. D. et al. (2006). Mol. Ther., 73:494-505; and Cekaite, L. et al., (2007). J. Mol. Biol., 3 ⁇ 55(l):90-l08. Additional modifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine have also been shown to minimize the immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko, K. et al. (2005). Immunity, 23(2): 165- 175.
  • RNAs can enhance their delivery and/or uptake by cells, including for example, cholesterol, tocopherol and folic acid, lipids, peptides, polymers, linkers and aptamers; see, e.g., the review by Winkler, J. (2013). Ther. Deliv.,
  • any nucleic acid molecules used in the methods provided herein e.g., a nucleic acid encoding a genome-targeting nucleic acid of the disclosure and/or a site- directed polypeptide are packaged into or on the surface of delivery vehicles for delivery to cells.
  • Delivery vehicles contemplated include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles.
  • a variety of targeting moieties can be used to enhance the preferential interaction of such vehicles with desired cell types or locations.
  • Introduction of the complexes, polypeptides, and nucleic acids of the disclosure into cells can occur by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI) -mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.
  • PEI polyethyleneimine
  • guide RNA polynucleotides RNA or DNA
  • endonuclease polynucleotide(s) RNA or DNA
  • viral or non- viral delivery vehicles known in the art.
  • endonuclease polypeptide(s) can be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles.
  • the DNA endonuclease can be delivered as one or more polypeptides, either alone or pre-complexed with one or more guide RNAs, or one or more crRNA together with a tracrRNA.
  • polynucleotides can be delivered by non-viral delivery vehicles including, but not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes.
  • non-viral delivery vehicles including, but not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes.
  • polynucleotides such as guide RNA, sgRNA, and mRNA encoding an endonuclease
  • LNP lipid nanoparticle
  • Lipid nanoparticles are generally composed of an ionizable cationic lipid and 3 or more additional components, typically cholesterol, DOPE and a polyethylene glycol (PEG) containing lipid, see, e.g., Example 3 A.
  • the cationic lipid can bind to the positively charged nucleic acid forming a dense complex that protects the nucleic from degradation.
  • the components self-assemble to form particles in the size range of 50 to 150 nM in which the nucleic acid is encapsulated in the core complexed with the cationic lipid and surrounded by a lipid bilayer like structure.
  • these particles can bind to apolipoprotein E (apoE).
  • ApoE is a ligand for the LDL receptor and mediates uptake into the hepatocytes of the liver via receptor mediated endocytosis.
  • LNP of this type have been shown to efficiently deliver mRNA and siRNA to the hepatocytes of the liver of rodents, primates and humans. After endocytosis, the LNP are present in endosomes.
  • the encapsulated nucleic acid undergoes a process of endosomal escape mediate by the ionizable nature of the cationic lipid. This delivers the nucleic acid into the cytoplasm where mRNA can be translated into the encoded protein.
  • encapsulation of gRNA and mRNA encoding a Cas endonuclease (e.g., a Cas9 endonuclease) into a LNP is used to efficiently deliver both components to the hepatocytes after IV injection. After endosomal escape the Cas endonuclease mRNA is translated into the Cas endonuclease and can form a complex with the gRNA.
  • inclusion of a nuclear localization signal into the Cas endonuclease sequence promotes translocation of the Cas endonuclease/gRNA complex to the nucleus.
  • the small gRNA crosses the nuclear pore complex and forms complexes with Cas endonuclease in the nucleus.
  • the gRNA/Cas endonuclease complexes scan the genome for homologous target sites and generate double-strand breaks preferentially at the desired target site in the genome.
  • the half-life of RNA molecules in vivo is short on the order of hours to days. Similarly, the half-life of proteins tends to be short, on the order of hours to days.
  • LNPs are generally less immunogenic than viral particles. While many humans have preexisting immunity to AAV there is no pre-existing immunity to LNPs. In addition, an adaptive immune response against LNPs is unlikely to occur, which enables repeat dosing of LNPs.
  • ionizable cationic lipids have been developed for use in LNPs. These include C12-200 (Love, K. T. et al (2010). PNAS 107(5): 1864-1869), MC3, LN16, MD1 among others.
  • a GalNac moiety is attached to the outside of the LNP and acts as a ligand for uptake into the liver via the asialyloglycoprotein receptor. Any of these cationic lipids can be used to formulate LNPs for delivery of gRNA and Cas endonuclease mRNA to the liver.
  • an LNP has a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.
  • a nanoparticle can range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.
  • LNPs can be made from cationic, anionic, or neutral lipids.
  • Neutral lipids such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as 'helper lipids' to enhance transfection activity and nanoparticle stability.
  • Limitations of cationic lipids include low efficacy owing to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses.
  • LNPs can also have hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.
  • lipids used to produce LNPs are: DOTMA, DOSPA, DOTAP, DMRIE, DC- cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE- polyethylene glycol (PEG).
  • cationic lipids are: 98N12-5, C 12-200, DLin-KC2- DMA (KC2), DLin-MC3 -DMA (MC3), XTC, MD1, and 7C1.
  • neutral lipids are: DPSC, DPPC, POPC, DOPE, and SM.
  • PEG-modified lipids are: PEG-DMG, PEG- CerCl4, and PEG-CerC20.
  • the lipids can be combined in any number of molar ratios to produce a LNP.
  • the polynucleotide(s) can be combined with lipid(s) in a wide range of molar ratios to produce a LNP.
  • the site-directed polypeptide and genome-targeting nucleic acid can each be administered separately to a cell or a patient.
  • the site-directed polypeptide can be pre-complexed with one or more guide RNAs, or one or more crRNA together with a tracrRNA.
  • the pre-complexed material can then be administered to a cell or a patient.
  • Such pre-complexed material is known as a ribonucleoprotein particle (RNP).
  • RNA is capable of forming specific interactions with RNA or DNA. While this property is exploited in many biological processes, it also comes with the risk of promiscuous interactions in a nucleic acid-rich cellular environment.
  • One solution to this problem is the formation of ribonucleoprotein particles (RNPs), in which the RNA is pre-complexed with an endonuclease.
  • RNPs ribonucleoprotein particles
  • Another benefit of the RNP is protection of the RNA from degradation.
  • the endonuclease in the RNP can be modified or unmodified.
  • the gRNA, crRNA, tracrRNA, or sgRNA can be modified or unmodified. Numerous modifications are known in the art and can be used.
  • the endonuclease and sgRNA can be generally combined in a 1:1 molar ratio.
  • the endonuclease, crRNA and tracrRNA can be generally combined in a 1:1:1 molar ratio.
  • a wide range of molar ratios can be used to produce a RNP.
  • a recombinant adeno-associated virus (AAV) vector can be used for delivery.
  • Techniques to produce rAAV particles, in which an AAV genome to be packaged that includes the polynucleotide to be delivered, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions.
  • the AAV rep and cap genes can be from any AAV serotype for which recombinant virus can be derived, and can be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-l, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-l 1, AAV-12, AAV-13 and AAV rh.74.
  • Production of pseudotyped rAAV is disclosed in, for example, international patent application publication number WO 01/83692. See Table 1.
  • a method of generating a packaging cell involves creating a cell line that stably expresses all of the necessary components for AAV particle production.
  • a plasmid (or multiple plasmids) having a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell.
  • AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski, R. J. et al.
  • the packaging cell line is then infected with a helper virus, such as adenovirus.
  • a helper virus such as adenovirus.
  • the advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV.
  • Other examples of suitable methods employ adenovirus or baculovirus, rather than plasmids, to introduce rAAV genomes and/or rep and cap genes into packaging cells.
  • AAV vector serotypes can be matched to target cell types.
  • the following exemplary cell types can be transduced by the indicated AAV serotypes among others.
  • the serotypes of AAV vectors suitable to liver tissue/cell type include, but not limited to, AAV3, AAV5, AAV8 and AAV9.
  • other viral vectors can be used.
  • Such viral vectors include, but are not limited to, lentivirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein Barr virus, papovavirusr, poxvirus, vaccinia virus, and herpes simplex virus.
  • Cas9 mRNA, sgRNA targeting one or two loci in an LPA gene, and donor DNA are each separately formulated into lipid nanoparticles, or are all co-formulated into one lipid nanoparticle, or co-formulated into two or more lipid nanoparticles.
  • Cas endonuclease e.g., Cas9 endonuclease
  • mRNA is formulated in a lipid nanoparticle, while gRNA (e.g., sgRNA) and DNA donor are delivered in an AAV vector.
  • gRNA e.g., sgRNA
  • Cas endonuclease mRNA and gRNA are co-formulated in a lipid nanoparticle, while donor DNA is delivered in an AAV vector.
  • RNA can be expressed from the same DNA, or can also be delivered as an RNA.
  • the RNA can be chemically modified to alter or improve its half-life, or decrease the likelihood or degree of immune response.
  • the endonuclease protein can be complexed with the gRNA prior to delivery. Viral vectors allow efficient delivery; split versions of a Cas
  • endonuclease and smaller Cas endonuclease orthologs can be packaged in AAV, as can donors for HDR.
  • a range of non- viral delivery methods also exist that can deliver each of these components, or non- viral and viral methods can be employed in tandem.
  • nanoparticles can be used to deliver a Cas endonuclease and guide RNA, while AAV can be used to deliver a donor DNA.
  • a CRISPR-Cas system described herein comprises a gRNA (e.g., an sgRNA) directed to a DNA sequence within or near (for example, within any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bases from) an LPA gene together with a Cas endonuclease (e.g., a Cas9 endonuclease).
  • the Cas endonuclease is delivered as an mRNA encoding the Cas endonuclease operably fused to one or more nuclear localization signals (NLS).
  • NLS nuclear localization signals
  • the gRNA and Cas endonuclease mRNA are delivered to hepatocytes by packaging into a lipid nanoparticle.
  • the lipid nanoparticle contains the lipid C 12-200 (Love, K. T. et al. (2010). PNAS, 107(5): 1864-1869).
  • the ratio of the gRNA to the Cas endonuclease mRNA that is packaged in the LNP is 1:1 (mass ratio) to result in maximal DNA cleavage in vivo in mice.
  • different mass ratios of the gRNA to the Cas endonuclease mRNA that is packaged in the LNP can be used, for example, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1 or 2:1 or reverse ratios.
  • the Cas endonuclease mRNA and the gRNA are packaged into separate LNP formulations and the Cas endonuclease mRNA-containing LNP is delivered to a subject about 1 to about 8 hours before the LNP containing the gRNA to allow time for the Cas endonuclease mRNA to be translated prior to delivery of the gRNA.
  • an LNP formulation encapsulating a gRNA (e.g., sgRNA) and a Cas endonuclease (e.g., Cas9 endonuclease) mRNA is administered to a subject, e.g., a patient, that previously was administered a DNA donor template packaged into an AAV.
  • the LNP-nuclease formulation is administered to the patient within 1 day to 28 days, within 7 days to 28 days, or within 7 days to 14 days after administration of the AAV-donor DNA template.
  • the optimal timing of delivery of the LNP- nuclease formulation relative to the AAV-donor DNA template can be determined using the techniques known in the art, e.g., studies done in animal models including mice and monkeys.
  • a DNA donor template that encodes a series of stop codons or a polyadenylation signal is delivered to hepatocytes of a subject, e.g., a patient, using a non- viral delivery method. While some subjects (generally 30%) have pre-existing neutralizing antibodies directed to most commonly used AAV serotypes that prevents efficacious gene delivery by the AAV, this is not a barrier for non-viral delivery methods. Several non-viral delivery
  • LNPs lipid nanoparticles
  • lipid nanoparticles are known to efficiently deliver their encapsulated cargo to the cytoplasm of hepatocytes after intravenous injection in animals and humans.
  • LNPs are actively taken up by the liver through a process of receptor mediated endocytosis, resulting in preferential uptake into the liver.
  • a DNA sequence that can promote nuclear localization of plasmids e.g., a 366 bp region of the simian virus 40 (SV40) origin of replication and early promoter, can be added to the donor template.
  • SV40 simian virus 40
  • Other DNA sequences that bind to cellular proteins can also be used to improve nuclear entry of DNA.
  • the level of Lp(a) is measured in the blood of a subject, e.g., a patient, following the first administration of an LNP-nuclease formulation, e.g., containing gRNA and Cas9 nuclease, with the goal of reducing the levels of Lp(a) . If the reduction in the levels of Lp(a) is not deemed sufficient to have a clinical benefit, for example, reducing LP(a) levels to less than 55 mg/dL, then a second or third administration of the LNP-nuclease formulation can be given to promote additional targeted disruption of the LPA gene.
  • an LNP-nuclease formulation e.g., containing gRNA and Cas9 nuclease
  • LNP-nuclease formulation The feasibility of using multiple doses of the LNP-nuclease formulation to obtain the desired reduction in levels of Lp(a)can be tested and optimized using techniques known in the field, e.g., tests using animal models, including mouse models and non-human primate models.
  • a gene therapy approach for treating a cardiovascular disease in a patient or reducing the risk of developing a cardiovascular disease in a patient by editing the genome of the patient functionally knocks out an LPA gene in the genome of a relevant cell type in patients and this can provide a permanent cure for the cardiovascular disease by permanently reducing the levels of Lp(a) in the blood.
  • a cell type subject to the gene therapy approach in which to functionally knock out an LPA gene is the hepatocyte because these cells express and secrete apo(a).
  • the cardiovascular disease is stroke, myocardial infarction, atherosclerosis, familial hypercholesterolemia, atherosclerosis, thrombosis, calcific aortic valve disease, coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, congenital heart disease, or rheumatic heart disease.
  • cellular and in vivo methods for using genome engineering tools to create permanent changes to the genome by functionally knocking-out an LPA gene in the genome of a cell use endonucleases, such as CRISPR-associated (CRISPR/Cas9, Cpfl and the like) nucleases, to permanently delete, insert, edit, correct, or replace any sequences from a genome or insert an exogenous sequence.
  • CRISPR-associated (CRISPR/Cas9, Cpfl and the like) nucleases to permanently delete, insert, edit, correct, or replace any sequences from a genome or insert an exogenous sequence.
  • the examples set forth in the present disclosure functionally knockout an LPA gene with a single treatment or a limited number of treatments (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatments, rather than requiring repetitive therapies for the lifetime of the patient).
  • an in vivo based therapy is employed.
  • the chromosomal DNA of the cells in the patient is edited using the materials and methods described herein.
  • the cells are hepatocytes.
  • the present disclosure provides a method of treating a cardiovascular disease or reducing the risk of developing a cardiovascular disease in a subject in need thereof comprising: providing (i) a gRNA according to any of the gRNAs described herein or nucleic acid encoding the gRNA; and (ii) a deoxyribonucleic acid (DNA) endonuclease or nucleic acid encoding the DNA endonuclease to a cell in the subject, thereby genetically modifying the cell.
  • a gRNA according to any of the gRNAs described herein or nucleic acid encoding the gRNA
  • the method comprises providing to the cell an AAV vector comprising the nucleic acid encoding the gRNA and/or nucleic acid encoding the DNA endonuclease.
  • the subject is a patient having or is suspected of having cardiovascular disease.
  • the subject is diagnosed with a risk of cardiovascular disease.
  • the cell is a hepatocyte.
  • the cardiovascular disease is selected from the group consisting of stroke, myocardial infarction, atherosclerosis, familial hypercholesterolemia, atherosclerosis, thrombosis, calcific aortic valve disease, coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, congenital heart disease, and rheumatic heart disease.
  • the subject is human.
  • a method of treating a cardiovascular disease or reducing the risk of developing a cardiovascular disease in a subject comprising providing to a cell in the subject: a) a DNA endonuclease (e.g., a Cas endonuclease, such as Cas9) or nucleic acid encoding the DNA endonuclease; and b) a gRNA comprising a spacer sequence
  • the cell prior to carrying out the method is an input cell that expresses apo(a) at a level greater than a reference level for the expression of apo(a) in the input cell type.
  • the genetically modified cell has reduced functional expression of apo(a) as compared to a corresponding unmodified cell.
  • the genetically modified cell has no functional expression of apo(a).
  • the method further comprises providing to the cell a donor template comprising a nucleic acid sequence to be inserted into the LPA gene.
  • the nucleic acid sequence to be inserted encodes one or more STOP codons, and the system is configured to insert a STOP codon in-frame into an LPA gene coding sequence such that the LPA gene is rendered non functional.
  • the gRNA targets within or near a coding sequence in the LPA gene. In some embodiments, the gRNA targets exon 1, exon 2, or exon 3 of the LPA gene.
  • the gRNA targets a sequence in the LPA gene corresponding to a kringle IV repeat region in the apo(a) protein. In some embodiments, the gRNA targets within or near a non-coding sequence in the LPA gene. In some embodiments, the gRNA targets an LPA gene intron. In some embodiments, the gRNA targets an LPA gene regulatory region. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 1-132. In some embodiments, the DNA endonuclease is a Cas endonuclease, e.g., a Cas9 endonuclease. In some embodiments, the gRNA is an sgRNA.
  • the subject has a cardiovascular disease
  • the method is a method of treating the cardiovascular disease.
  • the subject is diagnosed with or suspected of having the cardiovascular disease.
  • the subject is at risk for developing a cardiovascular disease
  • the method is a method of reducing the risk of the subject developing the cardiovascular disease.
  • the subject is diagnosed with or suspected of being at risk for developing the cardiovascular disease.
  • the endogenous LPA gene is a variant associated with increased cardiovascular disease risk and/or increased Lp(a) expression.
  • a method of treating a cardiovascular disease or reducing the risk of developing a cardiovascular disease in a subject comprising providing to a cell in the subject: a) a DNA endonuclease (e.g., a Cas endonuclease, such as Cas9) or nucleic acid encoding the DNA endonuclease; and b) a gRNA comprising i) a spacer sequence complementary to a target genomic sequence within exon 3 of an LPA gene; ii) a spacer sequence complementary to a target genomic sequence within exon 2 of an LPA gene; iii) a spacer sequence complementary to a target genomic sequence within an LPA gene corresponding to a kringle IV repeat region in apo(a); or iv) a spacer sequence complementary to a target genomic sequence within a regulatory region of an LPA gene, or nucleic acid encoding the gRNA, wherein the DNA endonuclease (e.g., a Cas end
  • the gRNA comprises i) a spacer sequence from any one of SEQ ID NOs: 13-19 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 13-19; ii) a spacer sequence from any one of SEQ ID NOs: 1-12 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 1-12; iii) a spacer sequence from any one of SEQ ID NOs: 107-132 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 107-132; or iv) a spacer sequence from any one of SEQ ID NOs: 20-106 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 20- 106.
  • the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 14, 15, 18, and 19.
  • the spacer sequence is 19 nucleotides in length and does not include the nucleotide at position 1 of the sequence from which it is selected.
  • the spacer sequence is the nucleotide sequence of any one of SEQ ID NOs: 157-160.
  • the DNA endonuclease and gRNA are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of generating a double-strand break at the target genomic sequence.
  • the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 13-19 or a variant thereof having no more than 3 mismatches compared to any one of SEQ ID NOs: 13-19. In some embodiments, the gRNA comprises a spacer sequence from any one of SEQ ID NOs: 13-19. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of any one of SEQ ID NOs: 13-19.
  • the gRNA comprises a spacer sequence from SEQ ID NO: 14 or a variant thereof having no more than 3 mismatches compared to SEQ ID NO: 14. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 14. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 15 or a variant thereof having no more than 3 mismatches compared to SEQ ID NO: 15. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 15. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of SEQ ID NO: 15.
  • the gRNA comprises a spacer sequence from SEQ ID NO: 18 or a variant thereof having no more than 3 mismatches compared to SEQ ID NO: 18. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 18. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 19 or a variant thereof having no more than 3 mismatches compared to SEQ ID NO: 19. In some embodiments, the gRNA comprises a spacer sequence from SEQ ID NO: 19. In some embodiments, the gRNA comprises a spacer having the nucleotide sequence of SEQ ID NO: 19.
  • the spacer comprises a contiguous sequence at least 19 nucleotides in length from any one of SEQ ID NOs: 1-132 and variants thereof having at least 85% homology to a contiguous sequence at least 19 nucleotides in length from any one of SEQ ID NOs: 1-132. In some embodiments, the spacer is 20 nucleotides in length.
  • the spacer comprises a polynucleotide sequence from position 2 to position 20 of any one of SEQ ID NOs: 1-132 and variants thereof having at least 85% homology to a polynucleotide sequence from position 2 to position 20 of any one of SEQ ID NOs: 1-132.
  • one or two gRNA(s) individually comprising a spacer complementary to a genomic sequence within or near for example, within any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bases from) exon 1, 2, or 3 of the LPA gene are provided to the cell.
  • the spacer(s) are complementary to a genomic sequence within or near (for example, within any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bases from) exon 3 of the LPA gene.
  • a genomic sequence within or near (for example, within any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bases from) exon 3 of the LPA gene.
  • the gRNA(s) are any one or two gRNAs comprising spacers selected from the group consisting of SEQ ID NOs: 1-19 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 1-19.
  • two gRNAs comprising spacers complementary to a genomic sequence within a transcriptional regulatory sequence of the LPA gene are provided to the cell.
  • the transcriptional regulatory sequence comprises a promoter or enhancer.
  • the gRNAs are any two gRNAs comprising spacers selected from the group consisting of SEQ ID NOs: 20-106 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 20-106.
  • a gRNA comprising a spacer complementary to a genomic sequence encoding a kringle domain in an apo(a) protein is provided to the cell.
  • the gRNA comprises a spacer selected from the group consisting of SEQ ID NOs: 107-132 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 107-132.
  • the DNA endonuclease is selected from the group consisting of a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf
  • the DNA endonuclease is Cas9.
  • the Cas9 endonuclease is from Streptococcus pyogenes (SpyCas9).
  • the Cas9 endonuclease is from Staphylococcus lugdunensis (SluCas9).
  • a DNA sequence that is transcribed to the nucleic acid encoding the DNA endonuclease is codon optimized.
  • the nucleic acid encoding the DNA endonuclease comprises a 5’ CAP structure and 3’ polyA tail.
  • the nucleic acid encoding the DNA endonuclease is linked to the gRNA via a covalent bond.
  • the method comprises providing to the cell a nucleic acid encoding the DNA endonuclease.
  • the nucleic acid encoding the DNA endonuclease is codon-optimized for expression in the cell.
  • the nucleic acid encoding the DNA endonuclease is DNA, such as a DNA plasmid.
  • the nucleic acid encoding the DNA endonuclease is RNA, such as mRNA.
  • the method further comprises providing (iii) a donor template comprising a nucleic acid sequence encoding one or more STOP codons.
  • the donor template comprising a nucleic acid sequence encoding one or more STOP codons comprises three STOP codons in each of the 3 translation frames present in succession in the donor DNA sequence.
  • the nucleic acid sequence encoding one or more STOP codons is codon optimized.
  • the nucleic acid sequence encoding one or more STOP codons does not comprise CpG dinucleotides.
  • the nucleic acid sequence encoding one or more STOP codons is inserted into a genomic sequence of the cell.
  • the insertion is at, within, or near the endogenous apolipoprotein(a) ( LPA ) gene or LPA gene regulatory elements in the genome of the cell.
  • the donor DNA is delivered as a double-stranded or single-stranded oligonucleotide.
  • the single- stranded or double- stranded donor DNA contains homology arms composed of the sequences of 20 bp to 1000 bp, or more, flanking each side of the sgRNA cut site.
  • the method further comprises providing to the cell a donor template comprising a sequence to be integrated at or near the target genomic sequence.
  • the donor template comprises a donor cassette comprising a nucleic acid sequence encoding one or more STOP codons.
  • the nucleic acid sequence encoding one or more STOP codons encodes three STOP codons in each of the 3 possible translation frames in the forward orientation and/or three STOP codons in each of the 3 possible translation frames in the reverse orientation.
  • the DNA endonuclease, gRNA, and donor template are configured such that a complex formed by association of the DNA endonuclease with the gRNA is capable of promoting targeted integration of the donor cassette into a target genomic locus (e.g., a coding region) comprising the target genomic sequence.
  • integration of the donor cassette into the target genomic locus in the cell results in the generation of a genetically modified cell with reduced functional expression of apo(a) as compared to a corresponding unmodified cell.
  • integration of the donor template into an LPA gene coding sequence such that a premature STOP codon is inserted in-frame can result in a non-functional or reduced function truncated translation product as compared to the apo(a) protein encoded by the unmodified LPA gene.
  • the donor template is configured such that the donor cassette is capable of being integrated into a genomic locus targeted by the gRNA by homology directed repair (HDR).
  • HDR homology directed repair
  • the donor cassette is flanked on both sides by homology arms corresponding to sequences in the targeted genomic locus.
  • the homology arms are at least about 0.2 kb (such as at least about any of 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, or greater) in length.
  • the homology arms are at least about 0.4 kb, e.g., 0.45 kb, 0.6 kb, or 0.8 kb, in length.
  • the donor template is encoded in an Adeno Associated Virus (AAV) vector.
  • AAV vector is an AAV2, AAV5, or AAV6 vector.
  • the AAV vector is an AAV6 vector.
  • the donor template is configured such that the donor cassette is capable of being integrated into a genomic locus targeted by the gRNA by non-homologous end joining (NHEJ).
  • NHEJ non-homologous end joining
  • the donor cassette is flanked on one or both sides by a gRNA target site.
  • the donor cassette is flanked on both sides by a gRNA target site.
  • the gRNA target site is a target site for the gRNA targeting the LPA gene.
  • the gRNA target site of the donor template is the reverse complement of the cell genome gRNA target site.
  • the donor template is encoded in an Adeno Associated Virus (AAV) vector.
  • AAV vector is an AAV2, AAV5, or AAV6 vector.
  • the AAV vector is an AAV6 vector.
  • the DNA endonuclease or nucleic acid encoding the DNA endonuclease is formulated in a liposome or lipid nanoparticle.
  • the liposome or lipid nanoparticle also comprises the gRNA or nucleic acid encoding the gRNA.
  • the liposome or lipid nanoparticle is a lipid nanoparticle.
  • the system comprises a lipid nanoparticle comprising nucleic acid encoding the DNA endonuclease and the gRNA.
  • the nucleic acid encoding the DNA endonuclease is an mRNA encoding the DNA endonuclease.
  • the DNA endonuclease is pre-complexed with the gRNA, forming a Ribonucleoprotein (RNP) complex, prior to the provision to the cell.
  • RNP Ribonucleoprotein
  • the method employs a donor template, the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA are provided to the cell after the donor template is provided to the cell.
  • the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA are provided to the cell about 1 to 14 days after the donor template is provided to the cell.
  • one or more (such as any of one, two, three, four, five, or more) additional doses of the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA are provided to the cell following a first dose of the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA.
  • one or more additional doses of the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA are provided to the cell following a first dose of the DNA endonuclease or nucleic acid encoding the DNA endonuclease and the gRNA or nucleic acid encoding the gRNA until a) a target frequency of editing the target genomic sequence in a population of cells in the subject is achieved; and/or b) a target plasma level of Lp(a) in the subject is achieved.
  • the population of cells of a) is the hepatocytes in the subject.
  • the cell prior to carrying out the method is an input cell that expresses apo(a).
  • the input cell expresses apo(a) at a level greater than a reference level for the expression of apo(a) in the input cell type.
  • the genetically modified cell has reduced functional expression of apo(a) as compared to a corresponding unmodified cell.
  • the genetically modified cell has no functional expression of apo(a).
  • the input cell is a hepatocyte.
  • ex vivo cell therapy typically uses a patient’s own cells, which are isolated, manipulated and returned to the same patient.
  • the subject has high levels of Lp(a) (e.g., plasma Lp(a)).
  • a subject with high levels of Lp(a) can include, e.g., subjects with greater Lp(a) levels than 90% of the human population.
  • the subject has symptoms of a cardiovascular disease.
  • the subject does not have symptoms of a cardiovascular disease.
  • the subject is at risk of developing a cardiovascular disease.
  • the subject is suspected of having a cardiovascular disease.
  • the subject has plasma Lp(a) levels in excess of about 30 mg/dL (such as in excess of about any of 40 mg/dL, 50 mg/dL, 60 mg/dL, 70 mg/dL, 80 mg/dL, 100 mg/dL, 200 mg/dL, 300 mg/dL, or greater).
  • the subject has one or more genetic markers (e.g., deletion, insertion, and/or mutation) in the endogenous LPA gene or its regulatory sequences such that the activity, including the expression level or functionality, of the apo(a) protein is substantially increased compared to a normal, healthy subject.
  • the subject who is in need of the treatment method accordance with the disclosures is a patient having high levels of Lp(a) as defined as Lp(a) levels higher than 90% of the human population (e.g., higher than 60 mg/dL) and symptoms of a cardiovascular disease.
  • the subject can be a human suspected of having the
  • the subject can be a human diagnosed with a risk of the cardiovascular disease due to the presence of Lp(a) levels in excess of 60 mg/dL or 70 mg/dL or 80 mg/dL or 100 mg/dL or 200 mg/dL or 300 mg/dL.
  • the subject who is in need of the treatment can have one or more genetic differences (e.g., deletion, insertion and/or mutation) in the endogenous LPA gene or its regulatory sequences such that the activity including the expression level or functionality of the apo(a) protein is substantially increased compared to a normal, healthy subject.
  • the frequency of editing the target genomic sequence in a population of cells in the subject is greater than about 10% (such as greater than about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater).
  • the population of cells in the subject is the liver cells in the subject.
  • the population of cells in the subject is the hepatocytes in the subject.
  • the plasma Lp(a) level in the subject following carrying out the method is reduced to about 50 mg/dL or lower (such to about any of 40 mg/dL, 30 mg/dL, 20 mg/dL, or lower). In some embodiments, the plasma Lp(a) level in the subject following carrying out the method is reduced to about 40 mg/dL or lower. In some embodiments, the plasma Lp(a) level in the subject following carrying out the method is reduced to about 30 mg/dL or lower. In some embodiments, the plasma Lp(a) level in the subject following carrying out the method is reduced to about 20 mg/dL or lower.
  • the subject is human.
  • compositions for carrying out the methods disclosed herein can include one or more of the following: a genome targeting nucleic acid (e.g., gRNA); a site-directed polypeptide (e.g., DNA endonuclease) or a nucleotide sequence encoding the site-directed polypeptide; and a polynucleotide to be inserted (e.g., a donor template) to affect the desired genetic modification of the methods disclosed herein.
  • a genome targeting nucleic acid e.g., gRNA
  • a site-directed polypeptide e.g., DNA endonuclease
  • a polynucleotide to be inserted e.g., a donor template
  • a composition has a nucleotide sequence encoding a genome targeting nucleic acid (e.g., gRNA).
  • gRNA genome targeting nucleic acid
  • a composition has a site-directed polypeptide (e.g., DNA endonuclease). In some embodiments, a composition has a nucleotide sequence encoding the site-directed polypeptide.
  • a composition has a polynucleotide (e.g., a donor template) to be inserted into a genome.
  • a polynucleotide e.g., a donor template
  • a composition has (i) a nucleotide sequence encoding a genome targeting nucleic acid (e.g., gRNA) and (ii) a site-directed polypeptide (e.g., DNA endonuclease) or a nucleotide sequence encoding the site-directed polypeptide.
  • a composition has (i) a nucleotide sequence encoding a genome targeting nucleic acid (e.g., gRNA) and (ii) a polynucleotide (e.g., a donor template) to be inserted into a genome.
  • a composition has (i) a site-directed polypeptide (e.g., DNA endonuclease) or a nucleotide sequence encoding the site-directed polypeptide and (ii) a polynucleotide (e.g., a donor template) to be inserted into a genome.
  • a site-directed polypeptide e.g., DNA endonuclease
  • a polynucleotide e.g., a donor template
  • a composition has (i) a nucleotide sequence encoding a genome targeting nucleic acid (e.g., gRNA), (ii) a site-directed polypeptide (e.g., DNA endonuclease) or a nucleotide sequence encoding the site-directed polypeptide and (iii) a polynucleotide (e.g., a donor template) to be inserted into a genome.
  • a genome targeting nucleic acid e.g., gRNA
  • a site-directed polypeptide e.g., DNA endonuclease
  • a polynucleotide e.g., a donor template
  • the composition has a single molecule guide genome-targeting nucleic acid. In some embodiments of any of the above compositions, the composition has a double-molecule genome-targeting nucleic acid. In some embodiments of any of the above compositions, the composition has two or more double molecule guides or single-molecule guides. In some embodiments, the composition has a vector that encodes the nucleic acid targeting nucleic acid. In some embodiments, the genome-targeting nucleic acid is a DNA endonuclease, in particular, Cas9.
  • a composition can contain composition that includes one or more gRNA that can be used for genome-edition.
  • the gRNA for the composition can target a genomic site at, within, or near the endogenous LPA gene. Therefore, in some embodiments, the gRNA can have a spacer sequence complementary to a genomic sequence at, within, or near the LPA gene.
  • a gRNA for a composition comprises a spacer comprising the sequence of any one of SEQ ID NOs: 1-132 and variants thereof having at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% identity or homology to the sequence of any one of SEQ ID NOs: 1-132.
  • the variants of the spacer have at least about 85% homology to the sequence of any one of SEQ ID NOs: 1-132.
  • a gRNA for a composition has a spacer that is complementary to a target site in the genome.
  • the spacer sequence is 15 bases to 20 bases in length.
  • a complementarity between the spacer sequence to the genomic sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100%.
  • a composition can have a DNA endonuclease or an
  • the DNA endonuclease and/or a donor template comprising multiple stop codons and a functional polyadenylation signal.
  • the DNA endonuclease and/or a donor template comprising multiple stop codons and a functional polyadenylation signal.
  • endonuclease is DNA or RNA.
  • one or more of any oligonucleotides or nucleic acid sequences for the kit can be encoded in an Adeno Associated Virus (AAV) vector. Therefore, in some embodiments, a gRNA can be encoded in an AAV vector. In some embodiments, a nucleic acid encoding a DNA endonuclease can be encoded in an AAV vector. In some embodiments, a donor template can be encoded in an AAV vector. In some embodiments, two or more oligonucleotides or nucleic acid sequences can be encoded in a single AAV vector. Thus, in some embodiments, a gRNA sequence and a DNA endonuclease-encoding nucleic acid can be encoded in a single AAV vector.
  • AAV Adeno Associated Virus
  • a composition can have a liposome or a lipid nanoparticle.
  • any compounds (e.g., a DNA endonuclease or a nucleic acid encoding the DNA endonuclease, gRNA and donor template) of the composition can be formulated in a liposome or lipid nanoparticle.
  • one or more such compounds are associated with a liposome or lipid nanoparticle via a covalent bond or non- covalent bond.
  • any of the compounds can be separately or together contained in a liposome or lipid nanoparticle.
  • each of a DNA endonuclease or a nucleic acid encoding the DNA endonuclease, gRNA and donor template is separately formulated in a liposome or lipid nanoparticle.
  • a DNA endonuclease is formulated in a liposome or lipid nanoparticle with gRNA.
  • a DNA endonuclease or a nucleic acid encoding the DNA endonuclease, gRNA and donor template are formulated in a liposome or lipid nanoparticle together.
  • a composition described above further has one or more additional reagents, where such additional reagents are selected from a buffer, a buffer for introducing a polypeptide or polynucleotide into a cell, a wash buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of the polypeptide from DNA, adaptors for sequencing and the like.
  • a buffer can be a stabilization buffer, a reconstituting buffer, a diluting buffer, or the like.
  • a composition can also include one or more components that can be used to facilitate or enhance the on-target binding or the cleavage of DNA by the endonuclease, or improve the specificity of targeting.
  • any components of a composition are formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending upon the particular mode of administration and dosage form.
  • pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc.
  • guide RNA compositions are generally formulated to achieve a physiologically compatible pH, and range from a pH of about 3 to a pH of about 11, about pH 3 to about pH 7, depending on the formulation and route of administration.
  • the pH is adjusted to a range from about pH 5.0 to about pH 8.
  • the composition has a therapeutically effective amount of at least one compound as described herein, together with one or more pharmaceutically acceptable excipients.
  • the composition can have a combination of the compounds described herein, or can include a second active ingredient useful in the treatment or prevention of bacterial growth (for example and without limitation, anti-bacterial or anti microbial agents), or can include a combination of reagents of the disclosure.
  • a second active ingredient useful in the treatment or prevention of bacterial growth for example and without limitation, anti-bacterial or anti microbial agents
  • gRNAs are formulated with one or more other nucleic acids, e.g., nucleic acid encoding a DNA endonuclease and/or a donor template.
  • nucleic acid encoding a DNA endonuclease and a donor template separately or in combination with other nucleic acids
  • oligonucleotides are formulated with the method described above for gRNA formulation.
  • Suitable excipients can include, for example, carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.
  • Other exemplary excipients include antioxidants (for example and without limitation, ascorbic acid), chelating agents (for example and without limitation, EDTA), carbohydrates (for example and without limitation, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (for example and without limitation, oils, water, saline, glycerol and ethanol), wetting or emulsifying agents, pH buffering substances, and the like.
  • any compounds (e.g., a DNA endonuclease or a nucleic acid encoding encoding the DNA endonuclease, gRNA and donor template) of a composition can be delivered via transfection such as electroporation.
  • a DNA endonuclease can be precomplexed with a gRNA, forming a Ribonucleoprotein (RNP) complex, prior to the provision to the cell and the RNP complex can be electroporated.
  • the donor template can be delivered via electroporation.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
  • aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • the amount of an active compound used in the cell compositions that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • kits that contains any of the above-described
  • compositions e.g., a composition for genome edition or a therapeutic cell composition and one or more additional components.
  • kits can have one or more additional therapeutic agents that can be administered simultaneously or in sequence with the composition for a desired purpose, e.g., genome edition or cell therapy.
  • a kit can further include instructions for using the components of the kit to practice the methods.
  • the instructions for practicing the methods are generally recorded on a suitable recording medium.
  • the instructions can be printed on a substrate, such as paper or plastic, etc.
  • the instructions can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging), etc.
  • the instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the Internet), can be provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.
  • nucleases engineered to target specific sequences there are four major types of nucleases: meganucleases and their derivatives, zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and CRISPR-Cas9 nuclease systems.
  • the nuclease platforms vary in difficulty of design, targeting density and mode of action, particularly as the specificity of ZFNs and TALENs is through protein-DNA interactions, while RNA-DNA interactions primarily guide Cas9.
  • Cas9 cleavage also requires an adjacent motif, the PAM, which differs between different CRISPR systems.
  • Streptococcus pyogenes cleaves using a NRG PAM
  • CRISPR from Neisseria meningitidis can cleave at sites with PAMs including NNNNGATT, NNNNNGTTT and NNNNGCTT.
  • a number of other Cas9 orthologs target protospacer adjacent to alternative PAMs.
  • CRISPR endonucleases such as a Cas9 endonuclease
  • teachings described herein, such as therapeutic target sites could be applied to other forms of endonucleases, such as ZFNs, TALENs, HEs, or MegaTALs, or using combinations of nucleases.
  • endonucleases such as ZFNs, TALENs, HEs, or MegaTALs, or using combinations of nucleases.
  • Additional binding domains can be fused to a Cas endonuclease (e.g., a Cas9 endonuclease) to increase specificity.
  • the target sites of these constructs would map to the identified gRNA target site, but would require additional binding motifs, such as for a zinc finger domain.
  • a meganuclease can be fused to a TALE DNA-binding domain.
  • the meganuclease domain can increase specificity and provide the cleavage.
  • inactivated or dead Cas dCas
  • dCas inactivated or dead Cas
  • dCas can be fused to a cleavage domain and require the sgRNA/Cas target site and adjacent binding site for the fused DNA-binding domain. This likely would require protein engineering of the dCas (e.g., dCas9), in addition to the catalytic inactivation, to decrease binding without the additional binding site.
  • compositions and methods of editing a genome in accordance with the present disclosures can utilize or be done using any of the following approaches.
  • Zinc Finger Nucleases Zinc Finger Nucleases
  • Zinc finger nucleases are modular proteins having an engineered zinc finger DNA binding domain linked to the catalytic domain of the type II endonuclease Fokl. Because Fokl functions only as a dimer, a pair of ZFNs must be engineered to bind to cognate target “half-site” sequences on opposite DNA strands and with precise spacing between them to enable the catalytically active Fokl dimer to form. Upon dimerization of the Fokl domain, which itself has no sequence specificity per se, a DNA double-strand break is generated between the ZFN half- sites as the initiating step in genome editing.
  • each ZFN typically has 3-6 zinc fingers of the abundant Cys2-His2 architecture, with each finger primarily recognizing a triplet of nucleotides on one strand of the target DNA sequence, although cross-strand interaction with a fourth nucleotide also can be important. Alteration of the amino acids of a finger in positions that make key contacts with the DNA alters the sequence specificity of a given finger. Thus, a four-finger zinc finger protein will selectively recognize a 12 bp target sequence, where the target sequence is a composite of the triplet preferences contributed by each finger, although triplet preference can be influenced to varying degrees by neighboring fingers.
  • ZFNs can be readily re-targeted to almost any genomic address simply by modifying individual fingers, although considerable expertise is required to do this well.
  • proteins of 4-6 fingers are used, recognizing 12-18 bp respectively.
  • a pair of ZFNs will typically recognize a combined target sequence of 24-36 bp, not including the 5-7 bp spacer between half-sites.
  • the binding sites can be separated further with larger spacers, including 15- 17 bp.
  • a target sequence of this length is likely to be unique in the human genome, assuming repetitive sequences or gene homologs are excluded during the design process.
  • TALENs Transcription Activator-Like Effector Nucleases
  • TALENs represent another format of modular nucleases whereby, as with ZFNs, an engineered DNA binding domain is linked to the Fokl nuclease domain, and a pair of TALENs operate in tandem to achieve targeted DNA cleavage.
  • the major difference from ZFNs is the nature of the DNA binding domain and the associated target DNA sequence recognition properties.
  • the TALEN DNA binding domain derives from TALE proteins, which were originally described in the plant bacterial pathogen Xanthomonas sp.
  • TALEs have tandem arrays of 33-35 amino acid repeats, with each repeat recognizing a single base pair in the target DNA sequence that is typically up to 20 bp in length, giving a total target sequence length of up to 40 bp.
  • Nucleotide specificity of each repeat is determined by the repeat variable diresidue (RVD), which includes just two amino acids at positions 12 and 13.
  • RVD repeat variable diresidue
  • the bases guanine, adenine, cytosine and thymine are predominantly recognized by the four RVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly, respectively.
  • ZFNs the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs have also benefitted from the use of obligate heterodimer variants of the Fokl domain to reduce off- target activity.
  • Fokl domains have been created that are deactivated in their catalytic function. If one half of either a TALEN or a ZFN pair contains an inactive Fokl domain, then only single-strand DNA cleavage (nicking) will occur at the target site, rather than a DSB. The outcome is comparable to the use of CRISPR/Cas9/Cpfl“nickase” mutants in which one of the Cas cleavage domains has been deactivated. DNA nicks can be used to drive genome editing by HDR, but at lower efficiency than with a DSB.
  • Homing endonucleases are sequence- specific endonucleases that have long recognition sequences (14-44 base pairs) and cleave DNA with high specificity - often at sites unique in the genome.
  • HEs can be used to create a DSB at a target locus as the initial step in genome editing.
  • some natural and engineered HEs cut only a single strand of DNA, thereby functioning as site-specific nickases.
  • the large target sequence of HEs and the specificity that they offer have made them attractive candidates to create site-specific DSBs.
  • the MegaTAL platform and Tev-mTALEN platform use a fusion of TALE DNA binding domains and catalytically active HEs, taking advantage of both the tunable DNA binding and specificity of the TALE, as well as the cleavage sequence specificity of the HE; see, e.g., Boissel, S. et al. (2014). Nucleic Acids Research,
  • the MegaTev architecture is the fusion of a meganuclease (Mega) with the nuclease domain derived from the GIY-YIG homing endonuclease I-Tevl (Tev).
  • the two active sites are positioned ⁇ 30 bp apart on a DNA substrate and generate two DSBs with non-compatible cohesive ends; see, e.g., Wolfs, J. M. et al. (2014). Nucleic Acids Research, 42(13): 8816-8829. It is anticipated that other combinations of existing nuclease-based approaches will evolve and be useful in achieving the targeted genome modifications described herein. dCas9-FokI or dCpfl -Fokl and Other Nucleases
  • the CRISPR genome editing system typically uses a single Cas9 endonuclease to create a DSB.
  • the specificity of targeting is driven by a nucleotide sequence in the guide RNA that undergoes Watson-Crick base-pairing with the target DNA (plus an additional 2 bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 from S. pyogenes).
  • fusion of the TALE DNA binding domain to a catalytically active HE takes advantage of both the tunable DNA binding and specificity of the TALE, as well as the cleavage sequence specificity of I-Tevl, with the expectation that off-target cleavage can be further reduced.
  • Embodiment 1 A guide RNA (gRNA) comprising a spacer that is complementary to a genomic sequence within or near an endogenous apolipoprotein(a) ( LPA ) gene locus.
  • gRNA guide RNA
  • Embodiment 2 The gRNA of embodiment 1, wherein the spacer comprises a contiguous sequence at least 19 nucleotides in length from any one of SEQ ID NOs: 1-132 and variants thereof having at least 85% homology to a contiguous sequence at least 19 nucleotides in length from any one of SEQ ID NOs: 1-132.
  • Embodiment 3 The gRNA of embodiment 2, wherein the spacer is 20 nucleotides in length.
  • Embodiment 4 The gRNA of embodiment 2, wherein the spacer comprises a polynucleotide sequence from position 2 to position 20 of any one of SEQ ID NOs: 1-132 and variants thereof having at least 85% homology to a polynucleotide sequence from position 2 to position 20 of any one of SEQ ID NOs: 1-132.
  • Embodiment 5 A composition comprising one or two of the gRNAs of any one of embodiments 1-4 or nucleic acid encoding the one or two gRNAs.
  • Embodiment 6 The composition of embodiment 5, comprising an Adeno Associated Virus (AAV) vector comprising the nucleic acid encoding the one or two gRNAs.
  • AAV Adeno Associated Virus
  • Embodiment 7 The composition of embodiment 5, further comprising a
  • DNA deoxyribonucleic acid
  • Embodiment 8 The composition of embodiment 7, comprising an AAV vector comprising the nucleic acid encoding the DNA endonuclease.
  • Embodiment 9 The composition of embodiment 7, comprising an AAV vector comprising the nucleic acid encoding the one or two gRNAs and the nucleic acid encoding the DNA endonuclease.
  • Embodiment 10 The composition of any one of embodiments 7-9, further comprising a donor template comprising a nucleic acid sequence encoding one or more STOP codons.
  • Embodiment 11 The composition of any one of embodiments 7-10, wherein the DNA endonuclease is selected from the group consisting of a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Cs
  • Embodiment 12 The composition of any one of embodiments 7-11, wherein the DNA endonuclease is Cas9.
  • Embodiment 13 The composition of any one of embodiments 7-12, wherein the nucleic acid encoding the DNA endonuclease is codon optimized.
  • Embodiment 14 The composition of any one of embodiments 10-13, wherein the nucleic acid sequence encoding one or more STOP codons is codon optimized.
  • Embodiment 15 The composition of any one of embodiments 10-14, wherein the nucleic acid sequence encoding one or more STOP codons does not comprise CpG dinucleotides.
  • Embodiment 16 The composition of any one of embodiments 10-15, wherein the nucleic acid encoding the DNA endonuclease is a deoxyribonucleic acid (DNA) sequence.
  • DNA deoxyribonucleic acid
  • Embodiment 17 The composition of any one of embodiments 7-15, wherein the nucleic acid encoding the DNA endonuclease is a ribonucleic acid (RNA) sequence.
  • RNA ribonucleic acid
  • Embodiment 18 The composition of embodiment 17, wherein the RNA sequence encoding the DNA endonuclease is linked to the gRNA via a covalent bond.
  • Embodiment 19 The composition of any one of embodiments 5-18, further comprising a liposome or lipid nanoparticle.
  • Embodiment 20 The composition of any one of embodiments 7-19, wherein the nucleic acid encoding the DNA endonuclease is formulated in a liposome or lipid nanoparticle.
  • Embodiment 21 The composition of embodiment 20, wherein the liposome or lipid nanoparticle encapsulates the gRNA.
  • Embodiment 22 The composition of any one of embodiments 5-21, wherein the DNA endonuclease is pre-complexed with the gRNA, forming a Ribonucleoprotein (RNP) complex.
  • RNP Ribonucleoprotein
  • Embodiment 23 The composition of any one of embodiments 5-22, wherein the composition comprises one or two gRNA(s) individually comprising a spacer complementary to a genomic sequence within or near exon 1, 2, or 3 of the LPA gene.
  • Embodiment 24 The composition of embodiment 23, wherein the gRNA(s)
  • Embodiment 25 The composition of embodiment 23 or embodiment 24, wherein the gRNA(s) are any one or two gRNA(s) comprising spacers selected from the group consisting of SEQ ID NOs: 1-19 or variants thereof having at least 85% homology to the spacer(s) of SEQ ID NOs: 1-19.
  • Embodiment 26 The composition of any one of embodiments 5-22, wherein the composition comprises two gRNAs comprising spacers complementary to a genomic sequence within a transcriptional regulatory sequence of the LPA gene.
  • Embodiment 27 The composition of embodiment 26, wherein the transcriptional regulatory sequence comprises a promoter or enhancer.
  • Embodiment 28 The composition of embodiment 26 or embodiment 27, wherein the gRNAs are any two gRNAs comprising spacers selected from the group consisting of SEQ ID NOs: 20-106 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 20-106.
  • Embodiment 29 The composition of any one of embodiments 5-22, wherein the composition comprises a gRNA comprising a spacer complementary to a genomic sequence encoding a kringle domain in an apo(a) protein.
  • Embodiment 30 The composition of embodiment 29, wherein the gRNA comprises a spacer selected from the group consisting of SEQ ID NOs: 107-132 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 107-132.
  • Embodiment 31 The composition of any one of embodiments 10-22, wherein the donor template comprising a nucleic acid sequence encoding one or more STOP codons comprises three STOP codons in each of the 3 translation frames present in succession in the donor DNA sequence.
  • Embodiment 32 The composition of embodiment 31, wherein the donor DNA is delivered as a double-stranded or single-stranded oligonucleotide.
  • Embodiment 33 The composition of embodiment 32, wherein the single- stranded or double-stranded donor DNA contains homology arms composed of the sequences of 20 bp to 1000 bp, or more, flanking each side of the sgRNA cut site.
  • Embodiment 34 The composition of any one of embodiments 31-33, wherein the composition comprises one or two gRNA(s) individually comprising a spacer complementary to a genomic sequence within or near exon 1, 2, or 3 of the LPA gene.
  • Embodiment 35 The composition of embodiment 34, wherein the spacer(s) are complementary to a genomic sequence within or near exon 3 of the LPA gene.
  • Embodiment 36 The composition of embodiment 34 or embodiment 35, wherein the gRNA(s) are any one or two gRNAs comprising spacers selected from the group consisting of SEQ ID NOs: 1-19 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 1-19.
  • Embodiment 37 A kit comprising the composition of any one of embodiments 5-36, further comprising instructions for use.
  • Embodiment 38 A method of editing a genome in a cell, the method comprising: providing the following to the cell:
  • Embodiment 39 The method of embodiment 38, comprising providing to the cell an AAV vector comprising the nucleic acid encoding the gRNA(s) and/or the nucleic acid encoding the DNA endonuclease.
  • Embodiment 40 The method of embodiment 38 or embodiment 39, further comprising providing (c) a donor template comprising a nucleic acid sequence encoding one or more STOP codons to the cell.
  • Embodiment 41 The method of any one of embodiments 38-40, wherein the spacer comprises a contiguous sequence at least 19 nucleotides in length from any one of SEQ ID NOs: 1-132 and variants thereof having at least 85% homology to a contiguous sequence at least 19 nucleotides in length from any one of SEQ ID NOs: 1-132.
  • Embodiment 42 The method of embodiment 41, wherein the spacer is 20 nucleotides in length.
  • Embodiment 43 The method of embodiment 41, wherein the spacer comprises a polynucleotide sequence from position 2 to position 20 of any one of SEQ ID NOs: 1-132 and variants thereof having at least 85% homology to a polynucleotide sequence from position 2 to position 20 of any one of SEQ ID NOs: 1-132.
  • Embodiment 44 The method of any one of embodiments 38-43, wherein the DNA endonuclease is selected from the group consisting of a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cpfl endonucle
  • Embodiment 45 The method of any one of embodiments 38-44, wherein the DNA endonuclease is Cas9.
  • Embodiment 46 The method of any one of embodiments 38-45, wherein a DNA sequence that is transcribed to the nucleic acid encoding the DNA endonuclease is codon optimized.
  • Embodiment 47 The method of any one of embodiments 38-46, wherein the nucleic acid sequence encoding one or more STOP codons is codon optimized.
  • Embodiment 48 The method of any one of embodiments 38-47, wherein the nucleic acid sequence encoding one or more STOP codons does not comprise CpG dinucleotides.
  • Embodiment 50 The method of any one of embodiments 38-48, wherein the nucleic acid encoding the DNA endonuclease comprises a 5’ CAP structure and 3’ polyA tail.
  • Embodiment 51 The method of any one of embodiments 38-49, wherein the nucleic acid encoding the DNA endonuclease is linked to the gRNA via a covalent bond.
  • Embodiment 52 The method of any one of embodiments 40-51, wherein one or more of (a), (b) and (c) are formulated in a liposome or lipid nanoparticle.
  • Embodiment 53 The method of any one of embodiments 38-52, wherein the DNA endonuclease is formulated in a liposome or lipid nanoparticle.
  • Embodiment 54 The method of embodiment 53, wherein the liposome or lipid nanoparticle encapsulates the gRNA.
  • Embodiment 55 The method of any one of embodiments 38-54, wherein the DNA endonuclease is pre-complexed with the gRNA, forming a Ribonucleoprotein (RNP) complex, prior to the provision to the cell.
  • RNP Ribonucleoprotein
  • Embodiment 56 The method of any one of embodiments 40-55, wherein (a) and (b) are provided to the cell after (c) is provided to the cell.
  • Embodiment 57 The method of any one of embodiments 40-55, wherein (a) and (b) are provided to the cell about 1 to 14 days after (c) is provided to the cell.
  • Embodiment 58 The method of any one of embodiments 40-57, wherein the nucleic acid sequence encoding one or more STOP codons is inserted into a genomic sequence of the cell.
  • Embodiment 59 The method of embodiment 58, wherein the insertion is at, within, or near the endogenous apolipoprotein(a) ( LPA ) gene or LPA gene regulatory elements in the genome of the cell.
  • LPA endogenous apolipoprotein(a)
  • Embodiment 60 The method of embodiment 58 or embodiment 59, wherein the donor template comprising a nucleic acid sequence encoding one or more STOP codons comprises three STOP codons in each of the 3 translation frames present in succession in the donor DNA sequence.
  • Embodiment 61 The method of any one of embodiments 58-60, wherein the donor DNA is delivered as a double-stranded or single-stranded oligonucleotide.
  • Embodiment 62 The method of embodiment 61, wherein the single- stranded or double-stranded donor DNA contains homology arms composed of the sequences of 20 bp to 1000 bp, or more, flanking each side of the sgRNA cut site.
  • Embodiment 63 The method of any one of embodiments 58-62, wherein one or two gRNA(s) individually comprising a spacer complementary to a genomic sequence within or near exon 1, 2, or 3 of the LPA gene are provided to the cell.
  • Embodiment 64 The method of embodiment 63, wherein the spacer(s) are
  • Embodiment 65 The method of embodiment 63 or embodiment 64, wherein the gRNA(s) are any one or two gRNAs comprising spacers selected from the group consisting of SEQ ID NOs: 1-19 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 1-19.
  • Embodiment 66 The method of any one of embodiments 38 or 41-55, wherein one or two gRNA(s) individually comprising a spacer complementary to a genomic sequence within or near exon 1, 2, or 3 of the LPA gene are provided to the cell.
  • Embodiment 67 The method of embodiment 66, wherein the spacer(s) are
  • Embodiment 68 The method of embodiment 66 or embodiment 67, wherein the gRNA(s) are any one or two gRNAs comprising spacers selected from the group consisting of SEQ ID NOs: 1-19 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 1-19.
  • Embodiment 69 The method of any one of embodiments 38 or 41-55, wherein two gRNAs comprising spacers complementary to a genomic sequence within a transcriptional regulatory sequence of the LPA gene are provided to the cell.
  • Embodiment 70 The method of embodiment 69, wherein the transcriptional regulatory sequence comprises a promoter or enhancer.
  • Embodiment 71 The method of embodiment 69 or embodiment 70, wherein the gRNAs are any two gRNAs comprising spacers selected from the group consisting of SEQ ID NOs: 20-106 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 20-106.
  • Embodiment 72 The method of any one of embodiments 38 or 41-55, wherein a gRNA comprising a spacer complementary to a genomic sequence encoding a kringle domain in an apo(a) protein is provided to the cell.
  • Embodiment 73 The method of embodiment 72, wherein the gRNA comprises a spacer selected from the group consisting of SEQ ID NOs: 107-132 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 107-132.
  • Embodiment 74 The method of any one of embodiments 38-73, wherein the cell is a hepatocyte.
  • Embodiment 75 A genetically modified cell wherein the genome of the cell is edited by the method of any one of embodiments 38-74.
  • Embodiment 76 A genetically modified cell, wherein the cell comprises a non functional apolipoprotein(a) (LPA) gene.
  • LPA apolipoprotein(a)
  • Embodiment 77 The cell of embodiment 76, wherein the LPA gene (i) lacks a nucleic acid encoding an exon or (ii) comprises a missense or nonsense mutation in an exon.
  • Embodiment 78 The cell of embodiment 77, wherein the exon is the third exon.
  • Embodiment 79 The cell of any one of embodiments 75-78, wherein the LPA gene comprises one or more premature STOP codons.
  • Embodiment 80 The cell of embodiment 79, wherein the LPA gene comprises a premature STOP codon in the third exon.
  • Embodiment 81 The cell of embodiment 76, wherein the promoter of the LPA gene comprises one or more non-functional transcriptional regulatory elements.
  • Embodiment 82 The cell of embodiment 81, wherein the transcriptional regulatory elements comprise a promoter or an enhancer.
  • Embodiment 83 The cell of embodiment 76, wherein the LPA gene lacks one or more nucleic acid sequences encoding one or more kringle domains of the apo(a) protein.
  • Embodiment 84 The cell of any one of embodiment 76-83, wherein the cell is a hepatocyte.
  • Embodiment 85 A method of treating a cardiovascular disease in a subject in need thereof comprising: administering the genetically modified cell of any one of embodiments 75- 84 to the subject.
  • Embodiment 86 The method of embodiment 85, wherein the subject is a patient having or is suspected of having cardiovascular disease.
  • Embodiment 87 The method of embodiment 85, wherein the subject is diagnosed with a risk of cardiovascular disease.
  • Embodiment 88 The method of any one of embodiments 85-87, wherein the genetically modified cell is autologous.
  • Embodiment 89 The method of any one of embodiments 85-87, wherein the genetically modified cell is allogenic.
  • Embodiment 90 The method of any one of embodiments 85-89, wherein the cell is a hepatocyte.
  • Embodiment 91 The method of any one of embodiments 85-89, further comprising: obtaining a biological sample from the subject wherein the biological sample comprises a cell; and editing the genome of the cell according to the method of any one of embodiments 36-71, thereby producing the genetically modified cell.
  • Embodiment 92 The method of embodiment 81, wherein the cell is a hepatocyte.
  • Embodiment 93 A method of treating a cardiovascular disease in a subject in need thereof comprising:
  • Embodiment 94 The method of embodiment 93, comprising providing to the cell an AAV vector comprising the nucleic acid encoding the gRNA and/or the nucleic acid encoding the DNA endonuclease.
  • Embodiment 95 The method of embodiment 93 or embodiment 94, wherein the method further comprises providing (iii) a donor template comprising a nucleic acid sequence encoding one or more STOP codons.
  • Embodiment 96 The method of any one of embodiments 93-95, wherein the subject is a patient having or is suspected of having cardiovascular disease.
  • Embodiment 97 The method of any one of embodiments 93-95, wherein the subject is diagnosed with a risk of cardiovascular disease.
  • Embodiment 98 The method of any one of embodiments 93-97, wherein the genetically modified cell is autologous.
  • Embodiment 99 The method of any one of embodiments 93-97, wherein the genetically modified cell is allogenic.
  • Embodiment 100 The method of any one of embodiments 93-99, wherein the spacer comprises a contiguous sequence at least 19 nucleotides in length from any one of SEQ ID NOs: 1-132 and variants thereof having at least 85% homology to a contiguous sequence at least 19 nucleotides in length from any one of SEQ ID NOs: 1-132.
  • Embodiment 101 The method of embodiment 100, wherein the spacer is 20 nucleotides in length.
  • Embodiment 102 The method of embodiment 100, wherein the spacer comprises a polynucleotide sequence from position 2 to position 20 of any one of SEQ ID NOs: 1-132 and variants thereof having at least 85% homology to a polynucleotide sequence from position 2 to position 20 of any one of SEQ ID NOs: 1-132.
  • Embodiment 103 The method of any one of embodiments 93-102, wherein the DNA endonuclease is selected from the group consisting of a Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslOO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cpfl endon
  • Embodiment 105 The method of any one of embodiments 93-104, wherein a DNA sequence that is transcribed to the nucleic acid encoding the DNA endonuclease is codon optimized.
  • Embodiment 106 The method of any one of embodiments 95-105, wherein the nucleic acid sequence encoding one or more STOP codons is codon optimized.
  • Embodiment 107 The method of any one of embodiments 95-106, wherein the nucleic acid sequence encoding one or more STOP codons does not comprise CpG dinucleotides.
  • Embodiment 108 The method of any one of embodiments 93-107, wherein the nucleic acid encoding the DNA endonuclease comprises a 5’ CAP structure and 3’ polyA tail.
  • Embodiment 109 The method of any one of embodiments 93-107, wherein the nucleic acid encoding the DNA endonuclease is linked to the gRNA via a covalent bond.
  • Embodiment 110 The method of any one of embodiments 95-106, wherein one or more of (i), (ii) and (ii) are formulated in a liposome or lipid nanoparticle.
  • Embodiment 111 The method of any one of embodiments 93-110, wherein the DNA endonuclease is formulated in a liposome or lipid nanoparticle.
  • Embodiment 112. The method of embodiment 111, wherein the liposome or lipid nanoparticle encapsulates the gRNA.
  • Embodiment 113 The method of any one of embodiments 93-112, wherein the DNA endonuclease is precomplexed with the gRNA, forming a Ribonucleoprotein (RNP) complex, prior to the provision to the cell.
  • RNP Ribonucleoprotein
  • Embodiment 114 The method of any one of embodiments 93-113, wherein (i) and (ii) are provided to the cell after (iii) is provided to the cell.
  • Embodiment 115 The method of any one of embodiments 114, wherein (i) and (ii) are provided to the cell about 1 to 14 days after (iii) is provided to the cell.
  • Embodiment 116 The method of any one of embodiments 95-115, wherein the nucleic acid sequence encoding one or more STOP codons is inserted into a genomic sequence of the cell.
  • Embodiment 117 The method of embodiment 116, wherein the insertion is at, within, or near the endogenous apolipoprotein(a) ( LPA ) gene or LPA gene regulatory elements in the genome of the cell.
  • Embodiment 118 The method of any one of embodiments 95-117, wherein the donor template comprising a nucleic acid sequence encoding one or more STOP codons comprises three STOP codons in each of the 3 translation frames present in succession in the donor DNA sequence.
  • Embodiment 119 The method of any one of embodiments 93-118, wherein the donor DNA is delivered as a double-stranded or single-stranded oligonucleotide.
  • Embodiment 120 The method of embodiment 93-119, wherein the single- stranded or double-stranded donor DNA contains homology arms composed of the sequences of 20 bp to 1000 bp, or more, flanking each side of the sgRNA cut site.
  • Embodiment 121 The method of any one of embodiments 93-120, wherein one or two gRNA(s) individually comprising a spacer complementary to a genomic sequence within or near exon 1, 2, or 3 of the LPA gene are provided to the cell.
  • Embodiment 122 The method of embodiment 121, wherein the spacer(s) are complementary to a genomic sequence within or near exon 3 of the LPA gene.
  • Embodiment 123 The method of embodiment 121 or embodiment 122, wherein the gRNA(s) are any one or two gRNAs comprising spacers selected from the group consisting of SEQ ID NOs: 1-19 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 1-19.
  • Embodiment 124 The method of any one of embodiments 93-94 or 96-113, wherein one or two gRNA(s) individually comprising a spacer complementary to a genomic sequence within or near exon 1, 2, or 3 of the LPA gene are provided to the cell.
  • Embodiment 125 The method of embodiment 124, wherein the spacer(s) are complementary to a genomic sequence within or near exon 3 of the LPA gene.
  • Embodiment 126 The method of embodiment 124 or embodiment 125, wherein the gRNA(s) are any one or two gRNAs comprising spacers selected from the group consisting of SEQ ID NOs: 1-19 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 1-19.
  • Embodiment 127 The method of any one of embodiments 93-94 or 96-113, wherein two gRNAs comprising spacers complementary to a genomic sequence within a transcriptional regulatory sequence of the LPA gene are provided to the cell.
  • Embodiment 128 The method of embodiment 127, wherein the transcriptional regulatory sequence comprises a promoter or enhancer.
  • Embodiment 129. The method of embodiment 127 or embodiment 128, wherein the gRNAs are any two gRNAs comprising spacers selected from the group consisting of SEQ ID NOs: 20-106 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 20-106.
  • Embodiment 130 The method of any one of embodiments 93-94 or 96-113, wherein a gRNA comprising a spacer complementary to a genomic sequence encoding a kringle domain in an apo(a) protein is provided to the cell.
  • Embodiment 131 The method of embodiment 130, wherein the gRNA comprises a spacer selected from the group consisting of SEQ ID NOs: 107-132 or variants thereof having at least 85% homology to the spacers of SEQ ID NOs: 107-132.
  • Embodiment 132 The method of any one of embodiments 93-131, wherein the cardiovascular disease is selected from the group consisting of stroke, myocardial infarction, atherosclerosis, familial hypercholesterolemia, atherosclerosis, thrombosis, calcific aortic valve disease, coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, congenital heart disease, and rheumatic heart disease.
  • the cardiovascular disease is selected from the group consisting of stroke, myocardial infarction, atherosclerosis, familial hypercholesterolemia, atherosclerosis, thrombosis, calcific aortic valve disease, coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease,
  • Embodiment 133 The method of any one of embodiments 93-132, wherein the subject is human.
  • Embodiment 134 A syringe comprising the genetically modified cell of any one of embodiments 75-84.
  • Embodiment 135. A catheter comprising the genetically modified cell of any one of embodiments 75-84.
  • Embodiment 136 A method of treating a cardiovascular disease in a subject in need thereof comprising: administering the gRNA of any one of embodiments 1-4 or the composition of any one of embodiments 5-36 to the subject.
  • Embodiment 137 The method of embodiment 136, wherein the cardiovascular disease is selected from the group consisting of stroke, myocardial infarction, atherosclerosis, familial hypercholesterolemia, atherosclerosis, thrombosis, calcific aortic valve disease, coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, congenital heart disease, and rheumatic heart disease.
  • Embodiment 138 The method of embodiment 136 or embodiment 137, wherein the subject is human.
  • Example 1 In si/ ' ico identification of guide RNA spacers targeting the LPA gene to achieve functional reduction of apo(a) protein
  • gRNA spacer sequences that target Cas9-mediated cleavage at the human LPA gene
  • an in silico algorithm based upon the CCTop algorithm was used to identify all possible gRNA target sites for Cas endonucleases that utilize a PAM with the sequence NGG (NGG PAM) in the -200,000 bp region on chromosome 6q25.3-q26 that includes the immediate upstream region of the LPA gene, the entire LPA gene, and the immediate downstream region of the LPA gene.
  • LPA gene includes the genomic region encompassing the LPA regulatory promoters and enhancer sequences as well as the coding sequence.
  • the in silico analysis resulted in identification of over 20,000 candidate spacers. Sequence homology of candidate spacers to other sites in the human reference genome was also provided by the analysis algorithm and non unique spacer sequences with at least one perfect match to a different site in the human genome were removed from the list of candidates.
  • Example 1A Identification of guide RNA spacers targeting exon 3 of the LPA gene
  • Table 2 Sequences of four Exon3Gs that do not have perfect matches to other sites in the human genome, and have a perfect match in the LPA gene of non-human primate Macaca fascicularis.
  • MM Mismatch. Refers to the number of sites in the genome with either 0, 1, 2, 3, or 4 mismatches to the spacer.
  • sgRNA/Cas9-mediated editing by measuring the frequency of introduction of small insertions and deletions (INDELs) was performed using sgRNAs containing one of the 4 unique spacers from Table 2 and Cas9 from S. pyogenes (SpCas9).
  • the sgRNAs contained the respective spacer sequence followed by the tracrRNA scaffold sequence of SEQ ID NO: 161
  • the same tracrRNA sequence was used for all sgRNAs described in Examples 1-6.
  • cryopreserved primary hepatocytes were plated at a density of 3.5 x 10 5 cells per well in a 24-well collagen I-coated plate. The cells were cultured in InVitroGROTM CP Medium supplemented with TorpedoTM Antibiotic Mix (Bio IVT).
  • a pair of primers flanking the target site was used in a polymerase chain reaction (PCR) using a 55 °C annealing temperature to amplify an 812 bp region from the genomic DNA.
  • PCR polymerase chain reaction
  • the PCR product was purified and sequenced using Sanger sequencing with a sequencing primer (LPA TIDE F; 5’- CAAGAGAAGAGAATGGTCG-3’ (SEQ ID NO: 137)).
  • sequence data was analyzed by an algorithm called Tracking of Indels by Decomposition (TIDE) to determine the frequency of insertions and deletions (INDELs) present at the predicted cut sites for each of the sgRNA/Cas9 complexes (Brinkman el al.
  • TIDE Tracking of Indels by Decomposition
  • the most common INDEL is a 1 bp insertion, which would result in a frame shift and create a stop codon at a location downstream of the gRNA cut site and thus result in the edited LPA gene encoding a predicted truncated apo(a) protein.
  • the 20-nt spacer forms of these four sgRNAs were tested in the same experimental system to determine if a longer form of the sgRNA spacer results in improved on-target cutting (Example 4A).
  • Example IB Identification of guide RNA spacers targeting regulatory regions and exon 2 of the LPA gene
  • One approach to downregulate apo(a) expression involves the use of two gRNAs with target sites in the LPA gene that flank one or more positive regulatory regions, such as an enhancer or promoter, or portions thereof to delete the intervening region between the target sites.
  • a set of unique gRNA spacers (SEQ ID NOs: 20-106) for the purpose of removing or otherwise mutating a regulatory region or portion thereof was identified by narrowing down the set of unique gRNA spacers described above to only the gRNA spacers that target the 4000 bp region upstream of the transcription initiation site of the LPA gene. This set of spacers is referred to herein as regulatory LPA spacers, or RegGs.
  • disruption of the 5’ untranslated region (5’ UTR) and the start of the signal peptide that are located around exon 2 by unique gRNA spacers may also result in a functional reduction in apo(a) expression.
  • Example 1C Identification of guide RNA spacers targeting kringle domains of the LPA gene [0500]
  • a single gRNA that targets a repetitive kringle domain can be used to target multiple Cas-mediated double-strand breaks at the LPA locus, potentially resulting in deletion of the regions between the double-strand breaks that leads to functional reduction in apo(a) expression.
  • a set of gRNA spacers (SEQ ID NOs: 107-132) that are unique to the LPA kringle domains was identified by narrowing down the set of unique gRNA spacers described above to only the gRNA spacers that target the kringle domains of LPA and that have low predicted off-target cleavage in other regions of the genome.
  • This set of guides is referred to herein as kringle-domain guides (KDGs)
  • Functional reduction of apo(a) may also be achieved by introducing into a coding region of the LPA gene (e.g., by NHEJ or HDR) a short DNA donor template that codes for one or more stop codons (e.g., three or more stop codons in each of the three translation frames present in one or both of the forward and reverse orientations).
  • a short DNA donor template that codes for one or more stop codons (e.g., three or more stop codons in each of the three translation frames present in one or both of the forward and reverse orientations).
  • the N-terminal exonic region of the LPA gene targeted in Example 1A one can be used.
  • gRNAs having any one of the four gRNA spacers identified in Example 1A that are unique to LPA exon 3 (Tl, T2, T4, and T5) are used.
  • the DNA donor template is delivered as a double- stranded or single- stranded oligonucleotide, and can be configured for integration by NHEJ or HDR.
  • the single-stranded or double-stranded donor DNA may contain homology arms corresponding to sequences from about 20 bp to about 1000 bp, or more, flanking each side of the sgRNA cut site.
  • a sequence present in the DNA donor template having stop codons in each of the three translation frames in both orientations is integrated at the cut site, early termination of translation will result even after a random INDEL event.
  • An example of such a DNA donor template is shown in FIG. 4.
  • EXAMPLE 2 In vitro validation of LPA -targeted gRNA spacers
  • sequences representing all 140 spacer sequences described above from the in silico analysis were synthesized by in vitro transcription (IVT gRNA) and evaluated in a system using transfection into a human embryonic kidney (HEK) cell line engineered to constitutively express the SpCas9 nuclease.
  • IVT gRNA in vitro transcription
  • HEK human embryonic kidney
  • PCR 7 tol3 amplify different areas in the repeat regions of the apo(a) kringle IV domain.
  • the guide RNA molecules that target this region will have multiple cut sites.
  • NHP non-human primate
  • Y 100% match to Macaca fascicularis .
  • the cutting efficiency of the sgRNAs ranged from 0% to 30%. Eighteen of the sgRNA spacer sequences have 100% identity with the LPA gene sequence of non-human primate (Macaca fascicularis). This is important because non-human primates (NHP) are used for a number of experiments in testing gene therapies for use in humans. Moreover, rodent species (mice and rats) lack a LPA gene and therefore don’t make the apo(a) protein, precluding their use as models for evaluating knockdown of apo(a), whereas NHP are known to express the LPA gene and have circulating Lp(a).
  • NHP non-human primates
  • the INDEL frequency estimated from this experiment may be an underestimate because the experiment was performed using sgRNA molecules synthesized by in vitro transcription wherein the amount of full-length, high-quality sgRNA molecules may be suboptimal.
  • the top seven performing sgRNAs were chemically synthesized with the base modifications described in Example 1A (Synthego Corp, Menlo Park, CA), and cleavage efficiency was assessed in the human liver cell line, HepG2.
  • the average result of three experiments show two- to six-fold higher INDEL frequency compared to the experiment done in HEK cells using IVT guide RNA molecules (Table 4).
  • Table 4 Cleavage efficiency of high INDEL-generating sgRNA molecules targeting the LPA gene in HepG2 cells.
  • EXAMPLE 3A Evaluation of cleavage efficiency of LPA gRNA in vivo in mice
  • lipid nanoparticle (LNP) delivery vehicle is used to deliver Cas9 and the gRNA molecules targeting human LPA to the hepatocytes of mice engrafted with primary human hepatocytes (e.g., obtained from Phoenix Bio).
  • LNP lipid nanoparticle
  • the gRNA is chemically synthesized incorporating chemically modified nucleotides to improve resistance to nucleases.
  • the SpCas9 mRNA is designed to encode the SpCas9 protein fused to a nuclear localization domain (NLS) that is required to transport the SpCas9 protein into the nuclear compartment where cleavage of genomic DNA can occur.
  • NLS nuclear localization domain
  • Additional components of the Cas9 mRNA include a KOZAK sequence at the 5’ end prior to the first codon to promote ribosome binding, and a polyA tail at the 3’ end composed of a series of A residues.
  • An example of the sequence of an SpCas9 mRNA with NLS sequences is provided in SEQ ID NO: 138.
  • the mRNA can be produced by different methods well known in the art.
  • One of such methods used herein is in vitro transcription using T7 polymerase in which the sequence of the mRNA is encoded in a plasmid that contains a T7 polymerase promoter. Briefly, upon incubation of the plasmid in an appropriate buffer containing T7 polymerase and ribonucleotides a RNA molecule is produced that encodes the amino acid sequence of the desired protein.
  • Either natural ribonucleotides or chemically modified ribonucleotides in the reaction mixture are used to generate mRNA molecules with either natural chemical structure or with modified chemical structures that may have advantages in terms of expression, stability or immunogenicity.
  • sequence of the SpCas9 coding sequence can be optimized for codon usage by utilizing the most frequently used codon for each amino acid. Additionally, the coding sequence can be optimized to remove cryptic ribosome binding sites and upstream open reading frames in order to promote the most efficient translation of the mRNA into SpCas9 protein.
  • a primary component of an LNP used in these studies is the lipid C 12-200 (Love, K. T. et al (2010). PNAS, 107(5):1864-1869).
  • the C12-200 lipid forms a complex with the highly- charged RNA molecules.
  • the C 12-200 is combined with L2-dioleoyI-sn-glycero-3- phosphoethano!arnine (DOPE), DMPE-mPEG2000 and cholesterol.
  • DOPE L2-dioleoyI-sn-glycero-3- phosphoethano!arnine
  • DOPE L2-dioleoyI-sn-glycero-3- phosphoethano!arnine
  • DMPE-mPEG2000 L2-dioleoyI-sn-glycero-3- phosphoethano!arnine
  • cholesterol L2-dioleoyI-sn-glycero-3- phosphoethano!arnine
  • gRNA and the Cas9 mRNA in the LNP are pipetted into glass vials as appropriate.
  • the ratio of C 12-200 to DOPE, DMPE-mPEG2000 and cholesterol is adjusted to optimize the formulation.
  • a typical ratio is composed of C 12-200, DOPE, cholesterol and mPEG2000-DMG at a molar ratio of 50:10:38.5:1.5.
  • the gRNA and mRNA are diluted in 100 mM Na citrate pH 3.0 and 300 mM NaCl in RNase free tubes.
  • the NanoAssemblr cartridge (Precision NanoSystems) is washed with ethanol on the lipid side and with water on the RNA side.
  • the working stock of lipids is pulled into a syringe, air removed from the syringe and the working stock is inserted in the cartridge.
  • the same procedure is used for loading a syringe with the mixture of gRNA and Cas9 mRNA.
  • the Nanoassemblr run is then performed under standard conditions.
  • the LNP suspension is then dialyzed using a 20 Kd cutoff dialysis cartridges in 4 liters of PBS for four hours and then concentrated using centrifugation through 20 Kd cutoff spin cartridges (Amicon) including washing three times in PBS during centrifugation. Finally, the LNP suspension is sterile filtered through 0.2 mM syringe filter.
  • Endotoxin levels are checked using commercial endotoxin kit (LAL assay) and particle size distribution is determined by dynamic light scattering.
  • the concentration of encapsulated RNA is determined using a RiboGreen assay (Thermo Fisher).
  • the gRNA and the Cas9 mRNA are formulated separately into LNP and then mixed together prior to treatment of cells in culture or injection into animals.
  • Using separately formulated gRNA and Cas9 mRNA allows specific ratios of gRNA and Cas9 mRNA to be tested.
  • LNP encapsulating the gRNA molecule and Cas9 mRNA are mixed at a 1:1 mass ratio of the RNA.
  • Alternative LNP formulations that utilized alternative cationic lipid molecules are also used for in vivo delivery of the gRNA and Cas9 mRNA.
  • Freshly prepared LNP encapsulating the gRNA molecule and Cas9 mRNA are injected into the tail vein (TV injection) of humanized liver mice.
  • the LNP is dosed by retro orbital (RO) injection.
  • the dose of LNP given to mice ranges from 0.5 to 2 mg of RNA per kg of body weight.
  • Human LP(a) levels and/or apo(a) levels are monitored in the serum before and after treatment. Successive dosing regimens of LNP can be used to increase the percentage of hepatocytes that have been gene edited and thus increase the degree of reduction in LP(a) levels.
  • mice After injection of the LNP (e.g., three days post- injection) the mice are sacrificed and a piece of the left and right lobes of the liver and a piece of the spleen are collected and genomic DNA is purified from each. The genomic DNA is then subjected to TIDES analysis to measure the cutting frequency and cleavage profile at the target site in the LPA gene.
  • EXAMPLE 3B Evaluation of cleavage efficiency of apo(a) gRNA in vivo in non-human primates
  • lipid nanoparticle (LNP) delivery vehicle To deliver Cas9 and the gRNA molecules targeting exon 3 of human LPA, or other regions of the LPA gene, to the hepatocytes of non-human primates that express apo(a), such as Macaca fascicularis , a lipid nanoparticle (LNP) delivery vehicle is used as described in Example 3A.
  • LNP lipid nanoparticle
  • Freshly prepared LNP encapsulating the gRNA molecule and Cas9 mRNA are injected into the non-human primate. Lp(a) levels are monitored in the plasma or serum before and after treatment. Successive dosing regimens of LNP can be used to increase the percentage of hepatocytes that have been gene edited.
  • EXAMPLE 4 Evaluation of on-target cleavage by gRNA/Cas9 in human primary hepatocytes
  • liver cell lines derived from tumors are convenient cell culture models for evaluating different gRNA molecules, yet these cells contain numerous genetic changes compared to normal hepatocytes. While the gene expression profile of liver cancer cell lines such as HuH7 and HepG2 generally reflect those of normal hepatocytes, there are numerous differences. In particular, differences in the chromatin organization of cancer cell lines compared to normal tissues is to be expected and may influence the accessibility of Cas9 to genomic targets. To select gRNA sequences to be used in humans, a normal human cell representative of the cell type being targeted may be used where possible. In the case of the gene editing strategy described herein, one of the most relevant cell types is normal hepatocytes obtained from humans. Such cells are referred to as primary human hepatocytes and are obtained from individual donors.
  • gRNA/Cas9 Primary human hepatocytes are one of the most relevant cell types for evaluation of potency and off-target cleavage of a gRNA/Cas9 that will be delivered to the liver of patients. These cells are grown in culture as adherent monolayers for a limited duration. Methods have been established for transfection of adherent cells with mRNA, for example MessengerMaxTM (Invitrogen, cat # LMRNA0015). After transfection with a mixture of Cas9 mRNA and gRNA the on-target cleavage efficiency is measured using TIDES analysis.
  • MessengerMaxTM Invitrogen, cat # LMRNA0015
  • Cryopreserved human hepatocytes are plated on tissue culture plates in optimized media and transfected the same day with a mixture of Cas9 mRNA and synthetic gRNA using the MessengerMaxTM reagent (Invitrogen, cat # LMRNA0015). Genomic DNA is extracted from the cells 48 hours later and the on-target cutting frequency is measured by determining the INDEL frequency using TIDES analysis. Using this approach additional guides can be identified that are candidates for gene editing at the LPA gene in vivo in patients.
  • Example 4A Evaluation of on-target cleavage at exon 3 of the human EPA gene by gRNA/Cas9 in human primary hepatocytes
  • Cryopreserved PHH from three different donors were plated on tissue culture plates coated with collagen type I (Corning; Corning, NY; cat # 354408) in optimized media (BioIVT; Westbury, NY; cat # INVITROGRO CP) and transfected the same day with a mixture of 0.6 pg Cas9 mRNA (TriLink BioTechnologies; San Diego, CA; cat # L-7206) and 0.2 pg of synthetic guide RNA (Synthego Corp) using the lipid-based MessengerMaxTM reagent (Invitrogen, cat # LMRNA0015).
  • the chemically synthesized sgRNAs contained base-specific 2’-0-methyl and 3’ phosphorothioate modifications in the first and last 3 nucleotides that provided enhanced stability, that may enable improved editing efficiency.
  • Genomic DNA was extracted from the cells 48 hours later and the on-target cutting frequency was measured by determining the INDEL frequency using TIDES analysis.
  • the PCR primers used to amplify the genomic DNA for TIDE analysis of the five sgRNAs are shown in Table 5.
  • Table 5 Sequences of PCR primers used to perform TIDE analysis of the L/ -targeting sgRNAs with spacers Tl, T2, T3894, T4 and T5
  • the INDEL frequency of the five tested sgRNA molecules with 20-nt spacer sequences targeting human EPA ranged from 9% to 45% in PHH cells from three donors (FIGS. 5A-5C and summarized in Table 6).
  • the corresponding sgRNAs with l9-nt spacer sequences (T1-19, T2-19, T4-19 and T5-19) exhibited lower INDEL frequencies ranging from 10-30% (FIG. 2 and summarized in Table 6), indicating that for these sgRNAs, a one nucleotide shorter spacer sequence was less efficient.
  • Example 4B Evaluation of on-target cleavage at regulatory regions, exon 2, and kringle IV repeat regions of the human LPA gene by gRNA/Cas9 in human primary hepatocytes
  • TIDE primer F Sequences of PCR primers and sequencing primer (referred to as“TIDE primer F”) used to perform TIDE analysis of the LPA sgRNAs targeting regulatory regions, exon 2, and kringle IV repeat regions
  • the INDEL frequency of the RegGs, Exon2Gs and KDGs ranged from 8% to 62% in PHH from several donors (FIGS. 5A-5C, Table 8). Apart from the sgRNA with spacer T221, for which the INDEL frequency was 8%, the other RegGs, Exon2Gs and KDGs in this example resulted in INDEL frequencies that were generally higher than Exon3Gs (Example 4A-4C), suggesting that the frequency of DNA disruption is higher using individual guides from the RegGs, Exon2Gs and KDGs that target LPA regulatory regions, exon 2, or kringle IV repeats, respectively. These higher frequencies of DNA disruption from using the RegGs, Exon2Gs and KDGs may result in a higher chance of functional knockdown of apo(a) protein.
  • Table 8 Cleavage efficiency of sgRNAs targeting regulatory regions, exon 2, and kringle IV repeat regions of the LPA gene in PHH. Guides are sorted by 5’ to 3’ location on the gene
  • Example 5 Evaluation of mRNA changes after genomic DNA disruption at the LPA gene.
  • Mutations in DNA may affect the levels of messenger RNA (mRNA) via the nonsense- mediated decay mechanism, which detects premature stop codons and other disruptions in mRNA and tags it for degradation (Isken, O., & Maquat, L. E. (2007). Genes &
  • ddPCRTM an advanced microfluidic based technology that achieves partitioning of samples at a massive scale, was performed using the Bio-Rad QX200 Droplet Digital PCR System.
  • the Qx200 System consists of two instruments: a droplet generator and a droplet reader.
  • the automated droplet generator creates about 20,000 highly uniform nanoliter- sized droplets per sample.
  • the nucleic acid, along with the primer and probes, is distributed randomly in the droplets.
  • the droplets are then subjected to the manufacturer’s optimized end-point PCR in a PCR plate using a deep well thermocycler.
  • the L/ -specific region on the cDNA was amplified using primers TTTCTGAACAAGCACCAACG (SEQ ID NO: 162) and
  • the PrimePCRTM ddPCRTM Expression Probe Assay detecting the human gene HPRT1 was used as a sample reference control (BioRad, cat # dHsaCPE5192872). As a control to evaluate the specificity of the guides, changes in the levels of mRNA encoding plaminogen ( PLG ), a gene which contains greater than 85% sequence homology to LPA was performed.
  • the PrimePCRTM ddPCRTM Expression Probe Assay detecting the human PLG gene was used (Biorad, cat # dHsaCPE503l038).
  • PHH samples edited with an unrelated sgRNA spacer sequence GGGGCCACTAGGGACAGGAT (SEQ ID NO: 165) targeting the Adeno-associated virus integration site 1 (AAVS1) locus in the first intron of the protein phosphatase 1 regulatory subunit 12C gene were used as controls for LPA and PLG mRNA expression changes.
  • the PCR plate was transferred to a droplet reader where the positive and negative droplets for the probes against LPA, PLG and HPRT were read and analyzed using the QuantaSoftTM Software. To account for sample- specific variations, the ratio of positive LPA or PLG readings to the positive readings of the reference control HPRT1 were calculated.
  • the LPA to HPRT1 or PLG to HPRT1 ratios of samples edited with the ZT -specific gRNA spacers were then divided by the LPA to HPRT1 or PLG to HPRT1 ratios of the samples edited with the AAVS 1 gRNA spacer to get LPA or PLG mRNA fold change data. These data are plotted on two graphs in FIG. 6. mRNA levels of PHHs treated with RegGs and Exon3Gs showed no significant change in LPA mRNA while samples edited with Exon2Gs and KDGs had significant decreases in LPA mRNA (FIG. 6). On the other hand, only one sample, T10461, had significantly decreased PLG mRNA levels (FIG. 6).
  • Example 6 Evaluation of off-target cleavage of selected guide RNA molecules targeting the human LPA gene
  • An additional criterion for the selection of a gRNA for therapeutic use is determination of off-target sites and frequencies. While in silico prediction algorithms can be helpful in identifying potential gRNA molecules, data generated in a relevant cell may be more
  • relevant cell systems for evaluation of off-target cleavage include HepG2 cells and primary human hepatocytes (PHHs).
  • HepG2 cells can be nucleofected with the selected guide RNA and Cas9 protein in a
  • ribonucleoprotein (RNP) complex resulting in on-target cleavage.
  • RNP ribonucleoprotein
  • genomic DNA is isolated from the cells and on-target cleavage is measured using the same TIDES-based methodology described above.
  • the same genomic DNA is subjected to the GUIDE-seq analysis approach (described in Tsai, S. Q. et al. (2015). Nature Biotechnology, 33: 187-197).
  • This method relies on the integration of the double- stranded oligonucleotide at sites of double-strand breaks. After random shearing of the genomic DNA and ligation of linkers, PCR using primers complementary to the linker and the integrated oligonucleotide is used to amplify the integration sites which are then sequenced. Once the sites of double-strand breaks at off-target sites are identified, whole genome sequencing can be performed to determine the frequency of off-target cleavage at each of these sites.
  • Example 6A Analysis of off-target sites for LPA-targeted gRNAs in human cells
  • GUIDE-seq (Tsai, S. Q. et al. (2015). Nature Biotechnology, 33(2): 187- 197) is an empirical method used to identify cleavage sites.
  • GETIDE-seq relies on the spontaneous capture of an oligonucleotide at the site of a double-strand break in chromosomal DNA.
  • genomic DNA is purified from the cells, sonicated, and a series of adapter ligations are performed to create a library.
  • the oligonucleotide-containing libraries are subjected to high-throughput DNA sequencing, and the output is processed using the default GETIDE-seq software to identify sites of oligonucleotide capture.
  • the double-stranded GETIDE-seq oligonucleotide (GETIDE-seq ODN) was generated by annealing two complementary single-stranded oligonucleotides by heating to 95 °C then cooling slowly to room temperature.
  • RNPs were prepared by mixing 240 pmol of sgRNA (Synthego Corp) and 48 pmol of 20 mM Cas9 TrueCut V2 (Invitrogen, cat # A36498) in a final volume of 4.8 pl.
  • 4 pl of the 10 mM GETIDE-seq ODN was mixed with 1.2 m ⁇ of the RNP mix, then added to a nucleofection cassette (Lonza).
  • HepG2 cells grown as adherent cultures were treated with trypsin to release them from the plate, the trypsin was deactivated, cells were pelleted and resuspended at 12.5 x 10 6 cells/ml in SF Cell Line NucleofectorTM Solution (Lonza, cat # V4XC-2032), and 20 m ⁇ cell suspension (2.5 x 10 5 cells) was added to each nucleofection cuvette. Nucleofection was performed with the EH- 100 cell program in the 4-D NucleofectorTM Unit (Lonza).
  • PCR reactions were performed with the Platinum PCR SuperMix High Fidelity reagent (Invitrogen), using 35 cycles of PCR and an annealing temperature of 55 °C.
  • PCR products were first analyzed by agarose gel electrophoresis to confirm that the correct products were generated, then directly sequenced using the TIDE primer shown in Table 5 located at the 5’ end of the PCR product. Sequence data were then analyzed using Tsunami, a modified version of the TIDES algorithm (Brinkman, E. K. et al. (2014). Nucleic Acids Research, 42(22):el68). This determined the frequency of INDELs present at the predicted cut site for the guide
  • GUIDE-seq was performed with 40 pmol (-1.67 mM) of the GUIDE-seq ODN to increase the sensitivity of off-target cleavage site identification.
  • the capture of the GUIDE-seq ODN at the on-target sites in HepG2 cells as measured by TIDE analysis is shown in Table 9.
  • the average INDEL frequency ranged from about 20% up to greater than about 50%, and average GUIDE-seq oligo integration rates ranged from about 3% up to about 30%.
  • Table 9 Frequency of total INDEL and capture of the GUIDE-seq ODN at the on-target site for human L/ -targeting sgRNAs with spacers Tl, T2, T4 and T5 in HepG2 cells
  • the R 2 value is a measure of the quality of the TIDES analysis with higher values indicative of higher quality data.
  • R 2 values above 0.95 are considered to be of high quality, and therefore guide RNA molecules with high cutting efficiencies and R 2 values above 0.95 can be useful in protocols for cleavage of the human LPA gene.
  • Genomic DNA extracted from the HepG2 cells that were nucleofected with RNP and the GUIDE-seq ODN was quantified using a Qubit fluorometer (ThermoFisher Scientific) and all samples were normalized to 400 ng in 120 pl volume of TE buffer.
  • the genomic DNA was sheared to an average length of 200 bp according to the standard operating procedure for the Covaris S220 sonicator. To confirm average fragment length, 1 pl of the sample was analyzed on a TapeStation (Agilent) according to manufacturer’s protocol.
  • Samples of sheared DNA were cleaned using AMPure XP SPRI beads according to the manufacturer’s protocol and eluted in 17 pl of TE buffer.
  • the end repair reaction was performed on the genomic DNA by mixing 1.2 pl of dNTP mix (5 mM each dNTP), 3 m ⁇ of 10 x T4 DNA ligase buffer, 2.4 pl of End-Repair Mix, 2.4 pl of lOx Platinum Taq Buffer (Mg 2+ free), and 0.6 pl of Taq Polymerase (non-hotstart) and 14 pl sheared DNA sample (from previous step) for a total volume of 22.5 m ⁇ per tube and incubated in a thermocycler (12 °C, 15 minutes; 37 °C, 15 minutes; 72 °C, 15 minutes; 4 °C hold).
  • a reaction was prepared containing 14 pl nuclease-free H 2 0, 3.6 m ⁇ 10 x Platinum Taq Buffer, 0.7 pl dNTP mix (10 mM each), 1.4 pl MgCl 2 , 50 mM, 0.36 m ⁇ Platinum Taq Polymerase, 1.2 pl sense or antisense gene specific primer (10 pM), 1.8 m ⁇ TMAC (0.5 M), 0.6 m ⁇ P5_l (10 mM) and 10 m ⁇ of the sample from the previous step.
  • This mix was incubated in a thermocycler (95 °C, 5 minutes, then 15 cycles of 95 °C, 30 seconds; 70 °C (minus 1 °C per cycle) for 2 minutes; 72 °C, 30 seconds; followed by 10 cycles of 95 °C, 30 seconds; 55 °C, 1 minute; 72 °C, 30 seconds; followed by 72 °C, 5 minutes).
  • the PCR reaction was cleaned using AMPure XP SPRI beads according to manufacturer protocol and eluted in 15 pl of TE Buffer. 1 pl of sample was checked on TapeStation according to manufacturer’s protocol to track sample progress.
  • a second PCR was performed by mixing 6.5 m ⁇ Nuclease-free H 2 0, 3.6 m ⁇ lOx Platinum Taq Buffer (Mg 2+ free), 0.7 pl dNTP mix (10 mM each), 1.4 pl MgCl 2 (50 mM), 0.4 pl Platinum Taq Polymerase, 1.2 pl of Gene Specific Primer (GSP) 2 (sense: +, or antisense: -), 1.8 m ⁇ TMAC (0.5 M), 0.6 m ⁇ P5_2 (10 mM) and 15 m ⁇ of the PCR product from the previous step.
  • GSP Gene Specific Primer
  • GSP1+ was used in the first PCR then GSP2+ was used in PCR2. If GSP1- primer was used in the first PCR reaction, then GSP2- primer was used in this second PCR reaction. After adding 1.5 m ⁇ of P7 (10 mM) the reaction was incubated in a thermocycler with the following program: 95 °C, 5 minutes; then 15 cycles of 95 °C, 30 seconds; 70 °C (minus 1 °C per cycle) for 2 minutes; 72 °C, 30 seconds; followed by 10 cycles of 95 °C, 30 seconds; 55 °C,
  • the PCR reaction was cleaned up using AMPure XP SPRI beads according to manufacturer protocol and eluted in 30 m ⁇ of TE Buffer and 1 m ⁇ analyzed on a TapeS tation according to manufacturer protocol to confirm amplification.
  • the library of PCR products was quantitated using Kapa Biosystems kit for Illumina Library Quantification, according to manufacturer supplied protocol and subjected to next generation sequencing on the Illumina system to determine the sites at which the oligonucleotide had become integrated.
  • GUIDE-seq was completed on three independent cell sample replicates (from three independent transfections) for each sgRNA and the results are shown in Tables 10 and 11.
  • the GETIDE-seq approach resulted in a 3% to 30% frequency of on-target oligo capture (Table 9) in HepG2 cells for sgRNAs with spacers Tl, T2, T4, and T5, indicating that this method is appropriate in this cell type.
  • On-target read counts met the internal pre-set criteria of 10,000 on- target reads for all four spacers.
  • Table 10 Summary of GETIDE-seq results for sgRNAs with spacers Tl, T2, T4, and T5 in HepG2 cells
  • Position refers to the genomic location in Genome Reference Consortium Human Build 38 (hg38). The NCBI Genome Data Viewer was used to annotate each position
  • the mismatch score is the sum of each non-complementary Watson-Crick nucleotide pair or gap where a nucleotide residue is missing in either the off-target site or the on-target site.
  • the mismatch score provides an indication of the degree of homology of the guide spacer target sequence to the off-target site in the genome, with higher numbers indicating less homology.
  • the percentage of off-target to on-target reads provides an overall representation of whether a gRNA is specific to its intended target, other factors may be involved. For example, an off-target site for a candidate gRNA in an exon of an essential gene required for survival of an organism could render the gRNA unsuitable for use in the clinic.
  • an off-target site in a non-coding or intronic region may pose less concern.
  • Considerations useful for evaluating a gRNA intended for therapeutic use include 1) the number of off-target sites, 2) the location of the off-target sites, 3) the frequency of off-target editing compared to on-target editing, and 4) the degree of homology of the off-target site to the gRNA spacer sequence.
  • the off-target sequences on chr 12: 107027942 and chr2:213057028 have high mismatches to the Tl spacer sequence, with six and three mismatches in the seven-nucleotide seed region, respectively (Table 11), indicative that these off-target sites are artifactual.

Abstract

L'invention concerne des matériaux et des procédés pour traiter une maladie cardiovasculaire chez un sujet. L'invention concerne également des matériaux et des procédés d'inactivation fonctionnelle d'un gène de LPA dans un génome.
PCT/US2019/028210 2018-04-18 2019-04-18 Compositions et procédés d'inactivation de l'apo (a) par édition génique pour le traitement d'une maladie cardiovasculaire WO2019204668A1 (fr)

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WO2020093025A1 (fr) * 2018-11-01 2020-05-07 Synthego Corporation Procédés d'inactivation d'une séquence cible par l'introduction d'un codon d'arrêt prématuré
CN111763715A (zh) * 2020-08-17 2020-10-13 上海韦翰斯生物医药科技有限公司 脂蛋白a亚型kiv-2结构域的拷贝数量检测试剂盒
US11345932B2 (en) 2018-05-16 2022-05-31 Synthego Corporation Methods and systems for guide RNA design and use
WO2023109940A1 (fr) * 2021-12-16 2023-06-22 上海拓界生物医药科技有限公司 Arnsi ciblant le lpa et conjugué
WO2023180904A1 (fr) * 2022-03-21 2023-09-28 Crispr Therapeutics Ag Méthodes et compositions pour traiter les maladies liées aux lipoprotéines
CN117568313A (zh) * 2024-01-15 2024-02-20 上海贝斯昂科生物科技有限公司 基因编辑组合物及其用途

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