In vivo nickase-based LPA gene editing for the treatment of cardiovascular disease

Abstract

This specification provides gene editing systems and compositions aimed at achieving in vivo editing of the LPA gene. Treatment or prevention of cardiovascular disease by gene editing interference with apo(a) production and reduction of blood lipoprotein(a) [Lp(a)] concentration is disclosed herein. A nickase-based gene editing system designed to achieve insertion and / or deletion (indel variant) and / or non-synonymous variant introduction in the coding sequence of LPA is disclosed. A nickase-based gene editing system generally comprises one or more nickases and one or more mRNAs encoding multiple guide oligonucleotides (e.g., gRNAs), and may be delivered intravenously in vivo to a mammalian subject requiring it as a potentially one-time treatment via a suitable delivery system such as lipid nanoparticles (LNPs) (with or without a GalNAc targeting moiety), or administered by other means. The manufacture, use, and formulation of gene editing systems and compositions are also disclosed.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 453,207, filed on 20 March 2023, and U.S. Provisional Patent Application No. 63 / 554,838, filed on 16 February 2024, which are incorporated herein by reference in their entirety. [Background technology]

[0002] Lipoprotein(a) [Lp(a)] is a low-density lipoprotein (LDL) particle containing apolipoprotein(a) [apo(a)] covalently bound to apolipoprotein B-100 (apoB-100), the major protein component of LDL particles. The apo(a) protein is encoded by the LPA gene, which is specifically expressed in hepatocytes of the liver of humans and certain non-human primates, and is secreted into the bloodstream to become a component of Lp(a). LDL cholesterol (quantified total cholesterol content of LDL particles circulating in the bloodstream) is a well-established incidental risk factor for atherosclerotic cardiovascular disease (ASCVD), which remains a leading cause of death worldwide despite the availability of approved long-term treatments, including statins, ezetimibe, and proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors. Blood Lp(a) levels are established as a specific risk factor for ASCVD, and there are no approved therapies that specifically target LPA, apo(a), or Lp(a). Furthermore, unlike LDL cholesterol, Lp(a) is established as a risk factor for calcified aortic valve disease, a distinct type of cardiovascular disease characterized by hardening or thickening of the aortic valve. [Overview of the project]

[0003] This application discloses novel gene editing technologies, including, individually and / or in combination, gene editing material (editor) mRNA, guide oligonucleotides (e.g., guide RNA or "gRNA"), gene editing and delivery systems, and their components, formulations and pharmaceutical compositions, which constitute distinct aspects of the subject matter of the Invention disclosed herein. The disclosed gene editing technologies enable in vitro and in vivo editing of the LPA gene, including the LPA gene found in human hepatocytes of the liver. Various embodiments are disclosed that target inactivating the LPA gene through the introduction of loss-of-function LPA variants for the substantial benefit of patients, such as patients with pre-existing cardiovascular disease or patients at risk of developing cardiovascular disease, by interfering with the production of apo(a) protein and / or Lp(a) protein, thereby reducing blood apo(a) and Lp(a) concentrations.

[0004] Accordingly, the gene editing and delivery systems described herein, their respective components and elements, and the manufacture and / or use of such systems and components, individually and / or in any combination, constitute individual aspects of the subject matter of the Invention disclosed herein. Accordingly, the use of the compositions and methods disclosed herein for interfering with the production of apo(a) protein and / or Lp(a) protein and / or for preventing and / or treating cardiovascular disease constitutes a separate aspect of the subject matter of the Invention disclosed herein. The location, nature and / or degree of interference with the LPA gene and / or the corresponding interference with the production of apo(a) and Lp(a) further constitute aspects of the subject matter of the Invention disclosed herein.

[0005] In some embodiments, the present invention provides pharmaceutical compositions for in vivo editing of the LPA gene in mammalian subjects, comprising an engineered, naturally occurring non-natural gene editing system and a delivery system. In embodiments, the gene editing system comprises one or more polynucleotides (e.g., mRNA) encoding one or more CRISPR Cas nickases, a first guide oligonucleotide (e.g., gRNA), and a second guide oligonucleotide (e.g., gRNA). The first guide oligonucleotide comprises a first spacer sequence and a first scaffold region. The first spacer sequence is complementary to the first strand of the LPA gene at a first target sequence. The first scaffold region functions as a binding scaffold for at least one of one or more Cas nickases. The second guide oligonucleotide comprises a second spacer sequence and a second scaffold region. The second spacer sequence is complementary to the second strand of the LPA gene at a second target sequence. The second scaffold region functions as a binding scaffold for at least one of one or more Cas nickases. The delivery system is engineered to deliver one or more polynucleotides encoding one or more CRISPR Cas nickases, a first guide oligonucleotide, and / or a second guide oligonucleotide to the liver individually and / or collectively. The first guide oligonucleotide and at least one of the one or more Cas nickases are engineered to cause at least one of the one or more Cas nickases to nick either the first or second strand of the LPA gene at a first position between positions 160,664,275 and 160,531,482 of human chromosome 6. The second guide oligonucleotide and at least one of the one or more Cas nickases are engineered to cause at least one of the Cas nickases to nick either the first or second strand of the LPA gene at a second position between positions 160,664,275 and 160,531,482 of chromosome 6.

[0006] In some embodiments, the expression of an edited LPA gene results in a decrease or absence of apo(a) protein production in cells in which the LPA gene has been edited. In some embodiments, the expression of an edited LPA gene results in a decrease or absence of apo(a) protein production in hepatocytes. In some embodiments, the hepatocytes are primary hepatocytes.

[0007] In some embodiments, the expression of an edited LPA gene results in a decrease in the amount of RNA, such as LPA mRNA or premRNA, in cells in which the LPA gene has been edited. While not intended to be theoretically bound, it is thought that the transcription of an edited LPA gene may, in some embodiments, result in nonsense mutation-dependent degradation of LPA RNA.

[0008] In some embodiments, the expression of the edited LPA gene results in reduced or absent production of apo(a) protein, or production of non-functional apo(a) protein, leading to a decrease in blood Lp(a) concentration. In some embodiments, blood Lp(a) concentration is reduced to a degree sufficient to treat ASCVD and / or calcified aortic valve disease. In embodiments, plasma Lp(a) concentration is reduced. In embodiments, serum blood concentration is reduced.

[0009] Embodiments of the present invention relate to a pharmaceutical composition comprising one or more components (e.g., mRNA, gRNA) of an LPA gene editing system, including the pharmaceutical composition that enables in vivo delivery. In an exemplary embodiment, the delivery system for the LPA gene editing system comprises a lipid nanoparticle (LNP) comprising (a) one or more ionizable lipids, (b) cholesterol, (c) one or more PEG-lipids, and (d) phospholipids, and optionally comprising a targeting moiety such as a GalNAc lipid.

[0010] In some embodiments, the Cas nickase is Cas9 nickase. In some embodiments, the Cas9 nickase is engineered to puncture the opposite strand of the LPA gene into which the operable guide hybridizes when it operably interacts with a first or second guide oligonucleotide (e.g., gRNA). In some embodiments, the Cas9 nickase is engineered to puncture the same strand of the LPA gene into which the operable guide oligonucleotide (e.g., gRNA) hybridizes when it operably interacts with a first or second guide oligonucleotide.

[0011] In exemplary embodiments, Cas nickase includes Streptococcus pyogenes Cas9 nickase having a D10A mutation encoded within a polynucleotide (e.g., mRNA).

[0012] In some embodiments, the first guide oligonucleotide (e.g., gRNA) has a first spacer sequence substantially identical to a first protospacer sequence adjacent to a first protospacer-adjacent motif (PAM) sequence on one strand of the LPA gene, and the second guide oligonucleotide (e.g., gRNA) has a second spacer sequence substantially identical to a second protospacer sequence adjacent to a second protospacer-adjacent motif (PAM) sequence on the other strand of the LPA gene, where the Cas9 nickase having the D10A mutation works together with the first and second guide oligonucleotides in a "PAM-out" configuration to introduce two nicks on opposing strands of the LPA gene (e.g., antisense and sense strands) between the first and second protospacer-adjacent motifs (PAMs) (see, for example, Figure 1A).

[0013] In some embodiments, the first guide oligonucleotide (e.g., gRNA) has a first spacer sequence substantially identical to a first protospacer sequence adjacent to a first protospacer-adjacent motif (PAM) sequence on one strand of the LPA gene, and the second guide oligonucleotide (e.g., gRNA) has a second spacer sequence substantially identical to a second protospacer sequence adjacent to a second protospacer-adjacent motif (PAM) sequence on the other strand of the LPA gene, where the Cas9 nickase having the D10A mutation works together with the first and second guide oligonucleotides in the "PAM-in" configuration to introduce two nicks on opposing strands of the LPA gene (e.g., antisense and sense strands) outside the first and second protospacer-adjacent motifs (PAMs) (see, for example, Figure 1C).

[0014] In some embodiments, Cas nickase includes Streptococcus pyogenes Cas9 nickase having the H840A mutation encoded within a polynucleotide (e.g., mRNA).

[0015] In some embodiments, the first guide oligonucleotide (e.g., gRNA) has a first spacer sequence substantially identical to a first protospacer sequence adjacent to a first protospacer-adjacent motif (PAM) sequence on one strand of the LPA gene, and the second guide oligonucleotide (e.g., gRNA) has a second spacer sequence substantially identical to a second protospacer sequence adjacent to a second protospacer-adjacent motif (PAM) sequence on the other strand of the LPA gene, where the Cas9 nickase having the H840A mutation works together with the first and second guide oligonucleotides in a "PAM-out" configuration to introduce two nicks on the first and second protospacer sequences on opposing strands of the LPA gene (e.g., antisense and sense strands) between the first and second protospacer-adjacent motifs (PAMs) (see, for example, Figure 1B).

[0016] In some embodiments, the first guide oligonucleotide (e.g., gRNA) has a first spacer sequence substantially identical to a first protospacer sequence adjacent to a first protospacer-adjacent motif (PAM) sequence on one strand of the LPA gene, and the second guide oligonucleotide (e.g., gRNA) has a second spacer sequence substantially identical to a second protospacer sequence adjacent to a second protospacer-adjacent motif (PAM) sequence on the other strand of the LPA gene, where the Cas9 nickase having the H840A mutation works together with the first and second guide oligonucleotides in the "PAM-in" configuration to introduce two nicks on the first and second protospacer sequences on the opposing strands of the LPA gene (e.g., sense and antisense strands) outside the first and second protospacer-adjacent motifs (PAMs) (see, for example, Figure 1D).

[0017] In some embodiments, Cas9 nickase, acting with a first and second guide nucleotide, nicks the DNA, creating nicks on opposing strands and resulting in a 5' overhang (e.g., as shown in Figures 1A and 1D). In some embodiments, Cas9 nickase, acting with a first and second guide nucleotide, nicks the DNA, creating nicks on opposing strands and resulting in a 3' overhang (e.g., as shown in Figures 1B and 1C). The overhang can have any length suitable for enabling non-homologous end joining. The length of the overhang is determined by the distance between nicks in the DNA strand. It will be understood that the suitable overhang length for enabling non-homologous end joining can vary depending on several factors. In some embodiments, the overhang has a length of 1 to 200 nucleotides, e.g., 10 to 150 nucleotides, 15 to 100 nucleotides, or 20 to 50 nucleotides. In some embodiments, the overhang has a length of 10 nucleotides or more, for example, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, or 50 nucleotides or more.In some embodiments, the overhang has a length of 200 nucleotides or less, for example, 190 nucleotides, 180 nucleotides, 170 nucleotides, 160 nucleotides, 150 nucleotides, 140 nucleotides, 130 nucleotides, 120 nucleotides, 110 nucleotides, 100 nucleotides, 90 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, or 50 nucleotides or less. In some embodiments, the overhang has a length of 20 to 50 nucleotides, for example, 23 to 45 nucleotides, 30 to 40 nucleotides, 31 to 40 nucleotides, 32 to 40 nucleotides, 33 to 40 nucleotides, 31 to 39 nucleotides, 32 to 39 nucleotides, 33 to 39 nucleotides, or 34 to 38 nucleotides.

[0018] In some embodiments, the arrangement of the first and second scaffold regions of the first and second guide oligonucleotides is the same. In some embodiments, the arrangement of the first and second scaffold regions is different.

[0019] In some embodiments, the in vivo LPA gene editing system comprises one polynucleotide (e.g., mRNA) encoding one CRISPR Cas9 nickase. In some embodiments, the in vivo LPA gene editing system further comprises one or more polynucleotides (e.g., mRNA) each encoding a CRISPR Cas nickase.

[0020] In some embodiments, the in vivo LPA gene editing system comprises a Cas nickase manipulated by a first guide oligonucleotide and a second guide oligonucleotide (e.g., gRNA) to direct to a protospacer sequence located within or adjacent to exon 20 of the LPA gene, extending from position 160,599,659 to position 160,599,500 of chromosome 6. In some such embodiments, the gene editing system is configured to introduce an indel variant and / or non-synonymous variant into the LPA gene.

[0021] In some embodiments, the in vivo LPA gene editing system comprises a Cas nickase manipulated by a first guide oligonucleotide and a second guide oligonucleotide (e.g., gRNA) to direct to a protospacer sequence located within or adjacent to exon 23 of the LPA gene, extending from position 160,591,101 to position 160,590,944 of chromosome 6. In some such embodiments, the gene editing system is configured to introduce an indel variant and / or non-synonymous variant into the LPA gene.

[0022] In some embodiments, the in vivo LPA gene editing system comprises a Cas nickase manipulated by a first guide oligonucleotide and a second guide oligonucleotide (e.g., gRNA) to direct to a protospacer sequence located within or adjacent to exon 25 of the LPA gene, extending from position 160,586,630 to position 160,586,449 of chromosome 6. In some such embodiments, the gene editing system is configured to introduce an indel variant and / or non-synonymous variant into the LPA gene.

[0023] In some embodiments, the in vivo LPA gene editing system comprises a Cas nickase manipulated by a first guide oligonucleotide and a second guide oligonucleotide (e.g., gRNA) to direct to a protospacer sequence located within or adjacent to exon 19 of the LPA gene, extending from position 160,601,098 to position 160,600,917 of chromosome 6. In some such embodiments, the gene editing system is configured to introduce an indel variant and / or non-synonymous variant into the LPA gene.

[0024] In some embodiments, the in vivo LPA gene editing system comprises a Cas nickase manipulated by a first guide oligonucleotide and a second guide oligonucleotide (e.g., gRNA) to direct to a protospacer sequence located within or adjacent to exon 31 of the LPA gene, extending from position 160,548,659 to position 160,548,478 of chromosome 6. In some such embodiments, the gene editing system is configured to introduce an indel variant and / or non-synonymous variant into the LPA gene.

[0025] In some embodiments, a plurality of different guide oligonucleotides (e.g., gRNAs) are used to achieve the desired editing of the LPA gene. In some embodiments, different guide oligonucleotides (e.g., gRNAs) are delivered simultaneously in vivo to target human liver cells / tissues and act to achieve editing of the LPA gene in human liver cells. In some embodiments, different guide oligonucleotides (e.g., gRNAs) are delivered simultaneously in vitro to target human liver cells / tissues (e.g., HuH-7 cells) and act to achieve editing of the LPA gene in human liver cells. In some embodiments, one or more of the guide oligonucleotides (e.g., gRNAs) are delivered to target liver tissue and liver cells via lipid nanoparticles (LNPs), which may include ionizable lipids, cholesterol, PEG-lipids, and phospholipids, and may also include a targeting moiety such as GalNAc lipids. In some embodiments, the phospholipids include distearoylphosphatidylcholine (DSPC). The same or different LNPs may also function to deliver mRNA encoding a gene editing substance (editor), i.e., Cas nickase (e.g., Cas9 nickase). In embodiments, the mRNA and / or guide oligonucleotide (e.g., gRNA) are selected from those specified herein. In embodiments, the Cas nickase (gene editing substance (editor)) and guide oligonucleotide (e.g., gRNA) may be selected from the gene editing systems and configurations illustrated in Figures 1A to 1D.

[0026] In some embodiments, an in vivo gene editing system includes mRNA encoding a Cas nickase (a gene editing substance (editor)) and one or more guide oligonucleotides (e.g., gRNAs). The first guide oligonucleotide may include a first spacer sequence and a first scaffold region. The second guide oligonucleotide may include a second spacer sequence and a scaffold region different from or the same as the first scaffold region. The first spacer sequence may correspond to a first protospacer sequence and may be designed to be complementary to a strand complementary to the first protospacer, or otherwise hybridize, to facilitate nicking by a nickase (e.g., Cas nickase) on one or the other DNA strand. The second spacer sequence may correspond to a second protospacer sequence located on the opposite chain of the first protospacer sequence and operably adjacent to the first protospacer sequence, and the second spacer sequence is designed to be complementary to a chain complementary to the second protospacer, or otherwise hybridize, in order to facilitate nicking by a nickase on a chain that is not nicked via the action of a nickase associated with the first guide oligonucleotide.

[0027] In embodiments, the present invention provides isolated oligonucleotides (e.g., gRNAs) or nucleic acids encoding them. Each isolated oligonucleotide (e.g., gRNA) comprises (i) a spacer sequence that is identical or substantially identical to a target protospacer sequence adjacent to a protospacer-adjacent motif (PAM) sequence in the LPA gene, and contains about 15 to about 26 nucleotides, for example, about 17 to about 23 nucleotides, and (ii) a scaffold region. The isolated oligonucleotide functions as a guide nucleic acid for instructing a gene editing substance (editor) to bring about editing in the LPA gene. In embodiments, the edit(s) are configured to modify the LPA gene to produce an indel variant or a non-synonymous variant.

[0028] In some embodiments, the first protospacer is located on the sense strand of the LPA gene, and the second protospacer is located on the antisense strand.

[0029] In some embodiments, the mRNA encoding Cas nickase (the gene-editing substance (editor)) includes (a) a 5' untranslated region (UTR), (b) a 3'UTR region, (c) a poly(A) tail adjacent to the 3'UTR compared to the 5'UTR, comprising a chain of 80 to 150 nucleotides containing adenine nucleotides, and (d) a gene-editing substance (editor) coding region located between the 5'UTR and the 3'UTR, encoding a damaged CRISPR Cas endonuclease domain. In some embodiments, the gene-editing substance (editor) coding region also encodes a polymerase domain.

[0030] In this embodiment, the mRNA has 90% or more sequence identity with any of the mRNA sequences listed in Table 1. For example, the mRNA may have 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with any of the mRNA sequences listed in Table 1.

[0031] In this embodiment, Cas nickase is selected from the Streptococcus pyogene Cas9 variant, the Staphylococcus aureus Cas9 variant, or the Cas12a / Cpf1 variant.

[0032] In embodiments, this disclosure describes a method for causing a modification to the LPA gene resulting in loss of function. This method includes delivering a pharmaceutical composition in vivo to human liver cells or administering it to a mammalian subject. The pharmaceutical composition comprises a polynucleotide encoding Cas nickase (e.g., mRNA), a first guide oligonucleotide and a second guide oligonucleotide (e.g., gRNA), and a delivery system. In some embodiments, the delivery system is configured to deliver the polynucleotide encoding Cas nickase, the first guide oligonucleotide, and / or the second guide oligonucleotide individually or together to the liver. In some embodiments, the delivery system includes an LNP. In other embodiments, this method includes delivering a pharmaceutical composition in vitro to human liver cells (e.g., HuH-7 cells) or administering it to a mammalian subject.

[0033] In embodiments, LNPs are formulated in a weight ratio of mRNA to guide oligonucleotide (e.g., gRNA) of 1:1+ / -5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of mRNA or guide oligonucleotide, or in any therapeutically effective ratio. The 1:1+ / -100% weight ratio includes weight ratios of 1:2 to 2:1. The 1:1 mRNA-to-guide oligonucleotide weight ratio refers to the cumulative weight ratio of all mRNA and all guide oligonucleotides. For example, if a single mRNA is contained in the formulation and two guide oligonucleotides are contained in the formulation, the weight ratio of mRNA to guide oligonucleotides is the weight of the single mRNA relative to the total weight of the two gRNAs in the formulation.

[0034] In the embodiment, the LNP has an N / P ratio of about 4 to about 7, about 4, about 4.5, about 5, about 5.5, or about 6, about 6.5, or about 7, with each ratio being + / - 5 to 20%.

[0035] In the embodiment, the buffer solution containing LNP has a pH of about 7.5 ± 1.5 and contains tris and / or sucrose.

[0036] In practice, LNPs have average diameters of approximately 70nm+ / -20nm, 70nm+ / -10nm, 70nm+ / -5nm; 60nm+ / -20nm, 60nm+ / -10nm, 60nm+ / -5nm; 50nm+ / -20nm, 50nm+ / -10nm, 50nm+ / -5nm; 45nm+ / -20nm, 45nm+ / -10nm, 45nm+ / -5nm.

[0037] In embodiments, this disclosure describes a method for in vivo editing of the LPA gene in a mammalian subject of interest, comprising administering a pharmaceutical composition to the subject. The pharmaceutical composition is (i) A polynucleotide (e.g., mRNA) that encodes CRISPR-Cas nickase, (ii) A first guide oligonucleotide (e.g., gRNA) comprising a first spacer sequence and a scaffold region, (iii) A second guide oligonucleotide (e.g., gRNA) comprising a second spacer sequence and a scaffold region, (iv) A delivery system is engineered to deliver mRNA, the first gRNA and / or the second gRNA individually and / or together to the liver, thereby, during operation, Cas nickase nicks the first and second strands of the LPA gene at positions 160,664,275 to 160,531,482 of chromosome 6, respectively.

[0038] In some embodiments, the mammalian subject is human.

[0039] In one embodiment, the subject has a high blood Lp(a) concentration. In another embodiment, the subject has an inversely correlated apo(a) concentration.

[0040] In one embodiment, the subject has a cardiovascular disease associated with high blood Lp(a) concentration. In another embodiment, the subject has a cardiovascular disease associated with inversely correlated blood apo(a) concentration.

[0041] In one embodiment, in vivo editing of the LPA gene results in a reduction of blood Lp(a) concentration. In another embodiment, in vivo editing of the LPA gene results in an inversely correlated blood apo(a) concentration.

[0042] In some embodiments, the present invention provides a method for treating and preventing cardiovascular disease by inactivating the LPA gene in vivo in a mammalian subject, the method comprising the step of administering the subject a pharmaceutical composition of the present invention.

[0043] In some embodiments, the present invention provides a method for treating and preventing cardiovascular disease by reducing blood Lp(a) concentration or inversely correlated apo(a) concentration in a mammalian subject, the method comprising the step of administering the subject a pharmaceutical composition of the present invention.

[0044] In some embodiments, the present invention provides a method for treating and / or preventing cardiovascular disease associated with the LPA gene in a mammalian subject, comprising the step of administering a pharmaceutical composition of the present invention to the subject.

[0045] In some embodiments, the present invention provides a gene editing system for editing the LPA gene. The gene editing system is constructed by expressing one or more exogenous polynucleotides (e.g., mRNAs) encoding one or more CRISPR Cas nickases in a cell and introducing a first gRNA and a second gRNA into the cell. The first guide oligonucleotide (e.g., gRNA) includes (i) a first spacer sequence complementary to the first strand of the LPA gene at a first target sequence, and (ii) a first scaffold region that functions as a binding scaffold for at least one of one or more Cas nickases. The second guide oligonucleotide (e.g., gRNA) includes (i) a second spacer sequence complementary to the second strand of the LPA gene at a second target sequence, and (ii) a second scaffold region that functions as a binding scaffold for at least one of one or more Cas nickases. The first gRNA and at least one of the one or more Cas nicksases are engineered to cause at least one of the one or more Cas nicksases to insert a nick into either the first or second strand of the LPA gene at the first position, between positions 160,664,275 and 160,531,482 on chromosome 6. The second gRNA and at least one of the one or more Cas nicksases are engineered to cause at least one of the one or more Cas nicksases to insert a nick into either the first or second strand of the LPA gene at the second position, between positions 160,664,275 and 160,531,482 on chromosome 6.

[0046] In some embodiments, the present invention provides a gene editing system comprising (i) means for expressing one or more CRISPR Cas nickases in a cell, and (ii) means for directing one or more Cas nickases to a first and second position in the LPA gene, introducing a nick into the first strand of the LPA gene and introducing a nick into the second strand of the LPA gene.

[0047] In some embodiments, the present invention provides a pharmaceutical composition for in vivo editing of the LPA gene in a mammalian subject, comprising: an engineered, non-naturally occurring gene editing system; and a population of lipid nanoparticles encapsulating the gene editing system. The gene editing system comprises (i) nickase or one or more polynucleotides (mRNAs) encoding nickase; (ii) a first guide oligonucleotide (gRNA) comprising a first spacer sequence including a region complementary to the first strand of the LPA gene at a first target sequence, and a first scaffold region functioning as a binding scaffold for nickase; and (ii) a second guide oligonucleotide (gRNA) comprising a second spacer sequence including a region complementary to the second strand of the LPA gene at a second target sequence, and a second scaffold region functioning as a binding scaffold for nickase. The first and second strands are opposing strands. The first spacer has an array that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% similar to or identical to the Guide 1 Protospacer listed in Table 2 or Table 5, and the second spacer has an array that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% similar to or identical to the Guide 2 Protospacer listed in Table 2 or Table 5.

[0048] Further aspects and advantages of the Disclosure will be readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the Disclosure. As will be understood, other different embodiments are possible, and some of their details can be modified in various obvious ways without departing from the Disclosure. Therefore, the drawings and description should be considered as illustrative and not limiting in any way. [Brief explanation of the drawing]

[0049] [Figure 1A]This is a schematic diagram illustrating a nickase-based editing system, specifically a double nickase editing system and its configuration. The components of the gene editing substance (editor) and guide oligonucleotide (indicated as gRNA) are identified and described operationally. In each configuration, the editing system introduces a single nick on opposing DNA strands, which mobilizes DNA repair enzymes to facilitate non-homologous end joining (NHEJ) repair and produce editing (e.g., producing an indel variant or non-synonymous variant). Figure 1A shows one system and configuration using a nickase Cas9 protein with a RuvC domain mutation, exemplified by Streptococcus pyogenes (S. pyogenes) Cas9 with the D10A mutation, together with two guide oligonucleotides. The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the lower strand, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the upper strand, with the 5' ends of the first spacer sequence and the 5' ends of the second spacer sequence being close to each other compared to the 3' ends of the spacers. Cas9 nickase with the D10A mutation, in conjunction with the first and second guide oligonucleotides, in a "PAM-out" configuration, inserts two nicks on the target sequences on the upper and lower strands of the gene (e.g., LPA) between the first protospacer-adjacent motif (PAM) and the second protospacer-adjacent motif (PAM), respectively, where the two protospacer-adjacent motifs (PAMs) of the two protospacer sequences are separated by the length of the region covered by the protospacer sequences. That is, in a "PAM-out" configuration, the two PAMs are distal to each other and adjacent to the two protospacer sequences. Figure 1B shows a system and configuration using a nickase Cas9 protein with a mutation in the HNH domain, exemplified by Streptococcus pyogenes Cas9 with the H840A mutation, along with two guide oligonucleotides.The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the lower strand, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the upper strand, with the 5' ends of the first spacer sequence and the 5' ends of the second spacer sequence being closer to each other than the 3' ends of the spacers. In the "PAM-out" configuration, Cas9 nickase with the H840A mutation works together with the first and second guide oligonucleotides to introduce a first nick on the first protospacer sequence and a second nick on the second protospacer sequence between the first protospacer-adjacent motif (PAM) and the second protospacer-adjacent motif (PAM) on the lower and upper strands of the gene (e.g., LPA). Thus, in Figure 1A, the two PAMs illustrated in Figure 1B are distal to each other and adjacency to the two protospacer sequences in the "PAM-out" configuration. Figure 1C shows a system and configuration using a nickase Cas9 protein with a RuvC domain mutation, exemplified by Streptococcus pyogenes Cas9 with the D10A mutation, together with two guide oligonucleotides. The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the upper chain, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the lower chain, with the 3' ends of the first spacer sequence and the 3' ends of the second spacer sequence being closer to each other compared to the 5' ends of the spacers. Cas9 nickase with the D10A mutation, in the "PAM-in" configuration, works with the first and second guide oligonucleotides to introduce two nicks on the lower and upper target strands of the LPA gene, respectively, outside the first and second protospacer-adjacent motifs (PAMs), where the PAMs of the two protospacer sequences are in close proximity to each other and adjacent to the protospacer sequences.Figure 1D shows a system and configuration using a nickase Cas9 protein with a mutation in the HNH domain, exemplified by Streptococcus pyogenes Cas9 with the H840A mutation, together with two guide oligonucleotides. The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the upper chain, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the lower chain, with the 3' ends of the first spacer sequence and the 3' ends of the second spacer sequence being closer to each other compared to the 5' ends of the spacers. Cas9 nickase with the H840A mutation, in a "PAM-in" configuration, works with the first and second guide oligonucleotides to puncture a first nick on the first protospacer sequence on the upper and lower strands of the LPA gene, respectively, outside the first and second protospacer-adjacent motifs (PAMs), where the PAMs of the two protospacer sequences are close to each other and adjacent to the protospacer sequences. The arrows in Figures 1A to 1D indicate the location of the nicks. It should be understood that D10A nickase represents a nickase that punctures the target strand, while H840A nickase represents a nickase that punctures the non-target strand (e.g., the strand containing the protospacer). Therefore, it is intended that other nickases can be used with the nickase-based editing systems described herein. [Figure 1B]This is a schematic diagram illustrating a nickase-based editing system, specifically a double nickase editing system and its configuration. The components of the gene editing substance (editor) and guide oligonucleotide (indicated as gRNA) are identified and described operationally. In each configuration, the editing system introduces a single nick on opposing DNA strands, which mobilizes DNA repair enzymes to facilitate non-homologous end joining (NHEJ) repair and produce editing (e.g., producing an indel variant or non-synonymous variant). Figure 1A shows one system and configuration using a nickase Cas9 protein with a RuvC domain mutation, exemplified by Streptococcus pyogenes (S. pyogenes) Cas9 with the D10A mutation, together with two guide oligonucleotides. The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the lower strand, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the upper strand, with the 5' ends of the first spacer sequence and the 5' ends of the second spacer sequence being close to each other compared to the 3' ends of the spacers. Cas9 nickase with the D10A mutation, in conjunction with the first and second guide oligonucleotides, in a "PAM-out" configuration, inserts two nicks on the target sequences on the upper and lower strands of the gene (e.g., LPA) between the first protospacer-adjacent motif (PAM) and the second protospacer-adjacent motif (PAM), respectively, where the two protospacer-adjacent motifs (PAMs) of the two protospacer sequences are separated by the length of the region covered by the protospacer sequences. That is, in a "PAM-out" configuration, the two PAMs are distal to each other and adjacent to the two protospacer sequences. Figure 1B shows a system and configuration using a nickase Cas9 protein with a mutation in the HNH domain, exemplified by Streptococcus pyogenes Cas9 with the H840A mutation, along with two guide oligonucleotides.The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the lower strand, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the upper strand, with the 5' ends of the first spacer sequence and the 5' ends of the second spacer sequence being closer to each other than the 3' ends of the spacers. In the "PAM-out" configuration, Cas9 nickase with the H840A mutation works together with the first and second guide oligonucleotides to introduce a first nick on the first protospacer sequence and a second nick on the second protospacer sequence between the first protospacer-adjacent motif (PAM) and the second protospacer-adjacent motif (PAM) on the lower and upper strands of the gene (e.g., LPA). Thus, in Figure 1A, the two PAMs illustrated in Figure 1B are distal to each other and adjacency to the two protospacer sequences in the "PAM-out" configuration. Figure 1C shows a system and configuration using a nickase Cas9 protein with a RuvC domain mutation, exemplified by Streptococcus pyogenes Cas9 with the D10A mutation, together with two guide oligonucleotides. The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the upper chain, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the lower chain, with the 3' ends of the first spacer sequence and the 3' ends of the second spacer sequence being closer to each other compared to the 5' ends of the spacers. Cas9 nickase with the D10A mutation, in the "PAM-in" configuration, works with the first and second guide oligonucleotides to introduce two nicks on the lower and upper target strands of the LPA gene, respectively, outside the first and second protospacer-adjacent motifs (PAMs), where the PAMs of the two protospacer sequences are in close proximity to each other and adjacent to the protospacer sequences.Figure 1D shows a system and configuration using a nickase Cas9 protein with a mutation in the HNH domain, exemplified by Streptococcus pyogenes Cas9 with the H840A mutation, together with two guide oligonucleotides. The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the upper chain, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the lower chain, with the 3' ends of the first spacer sequence and the 3' ends of the second spacer sequence being closer to each other compared to the 5' ends of the spacers. Cas9 nickase with the H840A mutation, in a "PAM-in" configuration, works with the first and second guide oligonucleotides to puncture a first nick on the first protospacer sequence on the upper and lower strands of the LPA gene, respectively, outside the first and second protospacer-adjacent motifs (PAMs), where the PAMs of the two protospacer sequences are close to each other and adjacent to the protospacer sequences. The arrows in Figures 1A to 1D indicate the location of the nicks. It should be understood that D10A nickase represents a nickase that punctures the target strand, while H840A nickase represents a nickase that punctures the non-target strand (e.g., the strand containing the protospacer). Therefore, it is intended that other nickases can be used with the nickase-based editing systems described herein. [Figure 1C]This is a schematic diagram illustrating a nickase-based editing system, specifically a double nickase editing system and its configuration. The components of the gene editing substance (editor) and guide oligonucleotide (indicated as gRNA) are identified and described operationally. In each configuration, the editing system introduces a single nick on opposing DNA strands, which mobilizes DNA repair enzymes to facilitate non-homologous end joining (NHEJ) repair and produce editing (e.g., producing an indel variant or non-synonymous variant). Figure 1A shows one system and configuration using a nickase Cas9 protein with a RuvC domain mutation, exemplified by Streptococcus pyogenes (S. pyogenes) Cas9 with the D10A mutation, together with two guide oligonucleotides. The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the lower strand, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the upper strand, with the 5' ends of the first spacer sequence and the 5' ends of the second spacer sequence being close to each other compared to the 3' ends of the spacers. Cas9 nickase with the D10A mutation, in conjunction with the first and second guide oligonucleotides, in a "PAM-out" configuration, inserts two nicks on the target sequences on the upper and lower strands of the gene (e.g., LPA) between the first protospacer-adjacent motif (PAM) and the second protospacer-adjacent motif (PAM), respectively, where the two protospacer-adjacent motifs (PAMs) of the two protospacer sequences are separated by the length of the region covered by the protospacer sequences. That is, in a "PAM-out" configuration, the two PAMs are distal to each other and adjacent to the two protospacer sequences. Figure 1B shows a system and configuration using a nickase Cas9 protein with a mutation in the HNH domain, exemplified by Streptococcus pyogenes Cas9 with the H840A mutation, along with two guide oligonucleotides.The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the lower strand, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the upper strand, with the 5' ends of the first spacer sequence and the 5' ends of the second spacer sequence being closer to each other than the 3' ends of the spacers. In the "PAM-out" configuration, Cas9 nickase with the H840A mutation works together with the first and second guide oligonucleotides to introduce a first nick on the first protospacer sequence and a second nick on the second protospacer sequence between the first protospacer-adjacent motif (PAM) and the second protospacer-adjacent motif (PAM) on the lower and upper strands of the gene (e.g., LPA). Thus, in Figure 1A, the two PAMs illustrated in Figure 1B are distal to each other and adjacency to the two protospacer sequences in the "PAM-out" configuration. Figure 1C shows a system and configuration using a nickase Cas9 protein with a RuvC domain mutation, exemplified by Streptococcus pyogenes Cas9 with the D10A mutation, together with two guide oligonucleotides. The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the upper chain, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the lower chain, with the 3' ends of the first spacer sequence and the 3' ends of the second spacer sequence being closer to each other compared to the 5' ends of the spacers. Cas9 nickase with the D10A mutation, in the "PAM-in" configuration, works with the first and second guide oligonucleotides to introduce two nicks on the lower and upper target strands of the LPA gene, respectively, outside the first and second protospacer-adjacent motifs (PAMs), where the PAMs of the two protospacer sequences are in close proximity to each other and adjacent to the protospacer sequences.Figure 1D shows a system and configuration using a nickase Cas9 protein with a mutation in the HNH domain, exemplified by Streptococcus pyogenes Cas9 with the H840A mutation, together with two guide oligonucleotides. The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the upper chain, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the lower chain, with the 3' ends of the first spacer sequence and the 3' ends of the second spacer sequence being closer to each other compared to the 5' ends of the spacers. Cas9 nickase with the H840A mutation, in a "PAM-in" configuration, works with the first and second guide oligonucleotides to puncture a first nick on the first protospacer sequence on the upper and lower strands of the LPA gene, respectively, outside the first and second protospacer-adjacent motifs (PAMs), where the PAMs of the two protospacer sequences are close to each other and adjacent to the protospacer sequences. The arrows in Figures 1A to 1D indicate the location of the nicks. It should be understood that D10A nickase represents a nickase that punctures the target strand, while H840A nickase represents a nickase that punctures the non-target strand (e.g., the strand containing the protospacer). Therefore, it is intended that other nickases can be used with the nickase-based editing systems described herein. [Figure 1D]This is a schematic diagram illustrating a nickase-based editing system, specifically a double nickase editing system and its configuration. The components of the gene editing substance (editor) and guide oligonucleotide (indicated as gRNA) are identified and described operationally. In each configuration, the editing system introduces a single nick on opposing DNA strands, which mobilizes DNA repair enzymes to facilitate non-homologous end joining (NHEJ) repair and produce editing (e.g., producing an indel variant or non-synonymous variant). Figure 1A shows one system and configuration using a nickase Cas9 protein with a RuvC domain mutation, exemplified by Streptococcus pyogenes (S. pyogenes) Cas9 with the D10A mutation, together with two guide oligonucleotides. The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the lower strand, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the upper strand, with the 5' ends of the first spacer sequence and the 5' ends of the second spacer sequence being close to each other compared to the 3' ends of the spacers. Cas9 nickase with the D10A mutation, in conjunction with the first and second guide oligonucleotides, in a "PAM-out" configuration, inserts two nicks on the target sequences on the upper and lower strands of the gene (e.g., LPA) between the first protospacer-adjacent motif (PAM) and the second protospacer-adjacent motif (PAM), respectively, where the two protospacer-adjacent motifs (PAMs) of the two protospacer sequences are separated by the length of the region covered by the protospacer sequences. That is, in a "PAM-out" configuration, the two PAMs are distal to each other and adjacent to the two protospacer sequences. Figure 1B shows a system and configuration using a nickase Cas9 protein with a mutation in the HNH domain, exemplified by Streptococcus pyogenes Cas9 with the H840A mutation, along with two guide oligonucleotides.The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the lower strand, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the upper strand, with the 5' ends of the first spacer sequence and the 5' ends of the second spacer sequence being closer to each other than the 3' ends of the spacers. In the "PAM-out" configuration, Cas9 nickase with the H840A mutation works together with the first and second guide oligonucleotides to introduce a first nick on the first protospacer sequence and a second nick on the second protospacer sequence between the first protospacer-adjacent motif (PAM) and the second protospacer-adjacent motif (PAM) on the lower and upper strands of the gene (e.g., LPA). Thus, in Figure 1A, the two PAMs illustrated in Figure 1B are distal to each other and adjacency to the two protospacer sequences in the "PAM-out" configuration. Figure 1C shows a system and configuration using a nickase Cas9 protein with a RuvC domain mutation, exemplified by Streptococcus pyogenes Cas9 with the D10A mutation, together with two guide oligonucleotides. The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the upper chain, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the lower chain, with the 3' ends of the first spacer sequence and the 3' ends of the second spacer sequence being closer to each other compared to the 5' ends of the spacers. Cas9 nickase with the D10A mutation, in the "PAM-in" configuration, works with the first and second guide oligonucleotides to introduce two nicks on the lower and upper target strands of the LPA gene, respectively, outside the first and second protospacer-adjacent motifs (PAMs), where the PAMs of the two protospacer sequences are in close proximity to each other and adjacent to the protospacer sequences.Figure 1D shows a system and configuration using a nickase Cas9 protein with a mutation in the HNH domain, exemplified by Streptococcus pyogenes Cas9 with the H840A mutation, together with two guide oligonucleotides. The first guide oligonucleotide has a spacer sequence identical to the first protospacer sequence on the upper chain, and the second guide oligonucleotide has a spacer sequence identical to the second protospacer sequence on the lower chain, with the 3' ends of the first spacer sequence and the 3' ends of the second spacer sequence being closer to each other compared to the 5' ends of the spacers. Cas9 nickase with the H840A mutation, in a "PAM-in" configuration, works with the first and second guide oligonucleotides to puncture a first nick on the first protospacer sequence on the upper and lower strands of the LPA gene, respectively, outside the first and second protospacer-adjacent motifs (PAMs), where the PAMs of the two protospacer sequences are close to each other and adjacent to the protospacer sequences. The arrows in Figures 1A to 1D indicate the location of the nicks. It should be understood that D10A nickase represents a nickase that punctures the target strand, while H840A nickase represents a nickase that punctures the non-target strand (e.g., the strand containing the protospacer). Therefore, it is intended that other nickases can be used with the nickase-based editing systems described herein.

[0050] [Figure 2]This report summarizes the percentage of LPA alleles edited in primary human hepatocytes (also known as editing efficiency or edit %) for selected guide RNA pairs (first gRNA and second gRNA) at high and low doses, and summarizes the LPA editing efficiency in primary human hepatocytes using a gene editing system that includes Cas9 nikkase along with the identified first and second guide RNA pairs. Specific guide RNA pairs are ranked by their editing efficiency at the higher total RNA dose of 2500 ng / mL. The lower total RNA dose is 312.5 ng / mL. Cas9 nikkase is encoded within mRNA (MS029) transfected into primary human hepatocytes at a total mRNA:total gRNA weight ratio of 1:1.

[0051] [Figure 3] Figure 2 shows dose-response curves in immortalized human hepatocellular carcinoma cells (HuH-7) for five pairs of first and second guide RNAs using a gene editing system including a dual nickase Cas9 system. The Cas9 nickase is encoded in mRNA (MS029) transfected into HuH-7 cells in a total mRNA:total gRNA weight ratio of 1:1. As shown in Figure 3, each of the five guide oligonucleotide pairs showed a dose-response that gradually increased with increasing concentration.

[0052] [Figure 4A]This is a schematic diagram showing the LPA gene sequences corresponding to the spacers of guides GA1183, GA1184, GA1264, and GA1266. The corresponding amino acid sequences are provided below the gene sequences and are numbered. Guide pairs for double nicking of the LPA gene (GA1183 / GA1184, GA1264 / GA1184, GA1266 / GA1184) are shown. The guide pairs were formulated into lipid nanoparticles (LNPs) with SpCas9-D10A nickase mRNA (MS029) and tested for editing efficiency. LNP1 corresponds to guide pair GA1183 / GA1184, LNP2 corresponds to guide pair GA1264 / GA1184, and LNP3 corresponds to guide pair GA1266 / GA1184. The results are shown in Figure 4B.

[0053] [Figure 4B] This plot shows the editing efficiency (edited %) of primary human hepatocytes (PHH) cells incubated with LNPs (LNP1, LNP2, and LNP3, as briefly described in Figure 4A) in a dose-response pattern for total RNA (guide and mRNA) in the range of 0 to 40,000 ng / mL. As shown in Figure 4B, the gene editing system (guide pair and mRNA encoding SpCas9-D10A nickase) produced a gradually increasing response with increasing concentration.

[0054] [Figure 5]This plot shows the percentage of apo(a) protein secreted from HuH-7 reporter cell lines exposed to LNPs at various concentrations (total RNA) ranging from 0 to 5000 ng / mL. The LNP corresponds to LNP1, described in the brief explanation in Figures 4A and 4B, and contains the GA1183 / GA1184 guide pair and mRNA (MS029) encoding SpCas9-D10A nickase. Apo(a) protein concentrations were evaluated using a validated Lp(a) ELISA kit from Mercodia. HuH-7 reporter cell lines were prepared by (i) lentiviral infection containing an expression cassette containing an LPA open reading frame (ORF) followed by an internal ribosome entry site (IRES) and puromycin N-acetyltransferase (puro), driven by a cytomegalovirus (CMV) promoter, and (ii) selection with puromycin. As shown in Figure 5, the relative percentage reduction in secreted apo(a) protein from the reporter cell line was reduced in a dose-dependent manner by LNP.

[0055] [Figure 6A] This graph shows the LPA gene editing efficiency (%) of indels in livers collected from transgenic mice expressing the human LPA gene 14 days after administration of the gene editing system (guide GA1296, guide GA1295, and MS029 mRNA). The gene editing system was formulated into lipid nanoparticles (LNPs) and delivered to transgenic mice via post-orbital injection at various concentrations. The concentrations shown in Figures 6A and 6B correspond to mg of total RNA (guide pair and mRNA) per kilogram (based on mouse body weight). As shown in Figures 6A and 6B, the gene editing efficiency increased in a dose-dependent manner with increasing dose of the gene editing system. [Figure 6B]This graph shows the LPA gene editing efficiency (%) of indels in livers collected from transgenic mice expressing the human LPA gene 14 days after administration of the gene editing system (guide GA1296, guide GA1295, and MS029 mRNA). The gene editing system was formulated into lipid nanoparticles (LNPs) and delivered to transgenic mice via post-orbital injection at various concentrations. The concentrations shown in Figures 6A and 6B correspond to mg of total RNA (guide pair and mRNA) per kilogram (based on mouse body weight). As shown in Figures 6A and 6B, the gene editing efficiency increased in a dose-dependent manner with increasing dose of the gene editing system.

[0056] [Figure 7] This graph shows the percentage change in plasma apo(a) protein levels (measured as a percentage change from baseline) in transgenic mice expressing the human LPA gene 14 days after administration of a gene editing system (guide GA1296, guide GA1295, and MS029 mRNA). The gene editing system was formulated into lipid nanoparticles (LNPs) and delivered to transgenic mice via post-orbital injection at various concentrations. The concentrations shown in Figure 7 correspond to mg of total RNA (guide pair and mRNA) per kilogram (based on mouse body weight). Plasma apo(a) concentrations were determined at baseline (-7 days) and 14 days after administration of the gene editing system. As shown in Figure 7, the percentage reduction in plasma apo(a) protein levels increased in a dose-dependent manner with increasing doses of the gene editing system.

[0057] [Figure 8A]This graph shows the changes in plasma apo(a) protein levels (measured as a percentage change from baseline) in transgenic mice expressing the human LPA gene 7 and 14 days after administration of a gene editing system (guide GA1296, guide GA1295, and MS029 mRNA). The gene editing system was formulated into lipid nanoparticles (LNPs) and delivered to transgenic mice via post-orbital injection at various concentrations. The concentrations shown in Figure 8A correspond to mg of total RNA (guide pair and mRNA) per kilogram (based on mouse body weight). Plasma apo(a) concentrations were determined at baseline (-7 days) and 7 and 14 days after administration of the gene editing system. As shown in Figure 8A, the percentage reduction in plasma apo(a) protein levels increased in a dose-dependent manner with increasing doses of the gene editing system.

[0058] [Figure 8B] This graph shows the changes in plasma apo(a) protein levels (measured as a percentage change from baseline) in transgenic mice expressing the human LPA gene 14 days after administration of various gene editing systems (guide oligonucleotide pairs and MS029 mRNA). The gene editing systems were formulated into lipid nanoparticles (LNPs) and delivered to transgenic mice via post-orbital injection at a dose of 0.5 mg / kg. The dose corresponds to mg of total RNA (guide pairs and mRNA) per kilogram (based on mouse body weight). Plasma apo(a) concentrations were determined at baseline (-7 days) and 14 days after administration of the gene editing system.

[0059] [Figure 9-1] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-2]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-3] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-4] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-5] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-6] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-7]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-8] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-9] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-10] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-11] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-12]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-13] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-14] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-15] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-16] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-17]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-18] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-19] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-20] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-21] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-22]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-23] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-24] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-25] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-26] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-27]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-28] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-29] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-30] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-31] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-32]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-33] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-34] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-35] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-36] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-37]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-38] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-39] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-40] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-41] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-42]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-43] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-44] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-45] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-46] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-47]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-48] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-49] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-50] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-51] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-52]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-53] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-54] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-55] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-56] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-57]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-58] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-59] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-60] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-61] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-62]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-63] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-64] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-65] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-66] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-67]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-68] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-69] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-70] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-71] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-72]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-73] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-74] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-75] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-76] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-77]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-78] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-79] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-80] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-81] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-82]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-83] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-84] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-85] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-86] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-87]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-88] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-89] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-90] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-91] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-92]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-93] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-94] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-95] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-96] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-97]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-98] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-99] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-100] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-101] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-102]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-103] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-104] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-105] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-106] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-107]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-108] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-109] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-110] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-111] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-112]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-113] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-114] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-115] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-116] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-117]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-118] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-119] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-120] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-121] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-122]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-123] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-124] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-125] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-126] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-127]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-128] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-129] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-130] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-131] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-132]A figure showing the positions where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thereby hybridize to the gene. Due to the property that some sequences of the LPA gene are repeated (e.g., kringle IV type repeats), some spacers of the selected gRNAs are complementary to two or more positions in the LPA gene. [Figure 9-133] A figure showing the positions where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thereby hybridize to the gene. Due to the property that some sequences of the LPA gene are repeated (e.g., kringle IV type repeats), some spacers of the selected gRNAs are complementary to two or more positions in the LPA gene. [Figure 9-134] A figure showing the positions where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thereby hybridize to the gene. Due to the property that some sequences of the LPA gene are repeated (e.g., kringle IV type repeats), some spacers of the selected gRNAs are complementary to two or more positions in the LPA gene. [Figure 9-135] A figure showing the positions where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thereby hybridize to the gene. Due to the property that some sequences of the LPA gene are repeated (e.g., kringle IV type repeats), some spacers of the selected gRNAs are complementary to two or more positions in the LPA gene. [Figure 9-136] A figure showing the positions where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thereby hybridize to the gene. Due to the property that some sequences of the LPA gene are repeated (e.g., kringle IV type repeats), some spacers of the selected gRNAs are complementary to two or more positions in the LPA gene. [Figure 9-137]A figure showing the positions where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thereby hybridize to the gene. Due to the property that some sequences of the LPA gene are repetitive (e.g., kringle IV type repeats), some spacers of the selected gRNAs are complementary to two or more positions in the LPA gene. [Figure 9-138] A figure showing the positions where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thereby hybridize to the gene. Due to the property that some sequences of the LPA gene are repetitive (e.g., kringle IV type repeats), some spacers of the selected gRNAs are complementary to two or more positions in the LPA gene. [Figure 9-139] A figure showing the positions where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thereby hybridize to the gene. Due to the property that some sequences of the LPA gene are repetitive (e.g., kringle IV type repeats), some spacers of the selected gRNAs are complementary to two or more positions in the LPA gene. [Figure 9-140] A figure showing the positions where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thereby hybridize to the gene.Due to the property that some sequences of the LPA gene are repetitive (e.g., kringle IV type repeats), some spacers of the selected gRNAs are complementary to two or more positions in the LPA gene. [Figure 9-141] A figure showing the positions where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thereby hybridize to the gene. Due to the property that some sequences of the LPA gene are repetitive (e.g., kringle IV type repeats), some spacers of the selected gRNAs are complementary to two or more positions in the LPA gene. [Figure 9-142]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-143] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-144] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-145] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-146] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-147]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-148] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-149] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-150] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-151] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-152]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-153] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-154] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-155] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-156] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-157]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-158] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-159] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-160] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-161] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-162]This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-163] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-164] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-165] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-166] This diagram illustrates the locations where the spacers of the selected gRNAs described herein are complementary to the LPA gene and can thus hybridize to that gene. Due to the repeating nature of some sequences in the LPA gene (e.g., Kringle type IV repeats), some spacers of the selected gRNAs are complementary to two or more locations in the LPA gene. [Figure 9-167]A figure showing the positions where the spacer of the selected gRNA described herein is complementary to the LPA gene and can thereby hybridize to the gene. Due to the property that some sequences of the LPA gene are repeated (e.g., kringle IV type repeats), some spacers of the selected gRNA are complementary to two or more positions in the LPA gene. [Figure 9-168] A figure showing the positions where the spacer of the selected gRNA described herein is complementary to the LPA gene and can thereby hybridize to the gene. Due to the property that some sequences of the LPA gene are repeated (e.g., kringle IV type repeats), some spacers of the selected gRNA are complementary to two or more positions in the LPA gene.

Modes for Carrying Out the Invention

[0060] This specification provides, inter alia, compounds and compositions for the modification or editing of the LPA gene, and methods of using them. In embodiments, the method results in a reduction in blood Lp(a) concentration by inactivating the LPA gene. In embodiments, editing of the LPA gene results in indel variants and non-synonymous variants in the LPA sequence. Compositions and methods are disclosed that are directed to editing the LPA gene using an editing system capable of introducing the editing, such as a Cas nickase (e.g., Cas9 nickase) and two guide oligonucleotides (e.g., gRNA).

[0061] For convenience, this detailed description is organized into the following sections. I. Definitions II. Apolipoprotein(a) Protein and LPA Gene III. Gene Editing / Gene Modification IV. Guide Nucleic Acid and Target DNA Sequence V. Gene Editing Substance (editor) System VI. Therapeutic Uses VII. Pharmaceutical Compositions A. Lipid nanoparticle (LNP) composition 1. Aminolipids a) Equation (I) b) Equation (Ia) c) Modified forms of equations (I) and (Ia) 2. LNP composition containing different aminolipids 3. Additional aminolipid embodiments 4. PEG-Lipids 5. Phospholipids 6. Cholesterol 7. GalNAc-lipids 8. Phosphate charge neutralizing agent 9. Antioxidants 10. Other lipids 11. LNP preparations 12. Payload VIII. Kit IX. Medication X. Means XI. Examples XII. Other Embodiments

[0062] I. Definition The following provides definitions of some terms presented through this disclosure. In some cases, terms are defined in parts of this specification other than this “Definitions” section.

[0063] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple objects unless otherwise clearly indicated by the context. It should also be noted that the term “or” is generally used in its sense to include “and / or” unless otherwise clearly indicated by the context. The terms “and / or” and “any combination thereof,” as used herein, and their grammatical equivalents, may be used interchangeably. These terms may convey that any combination is specifically intended. For illustrative purposes only, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” may mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” may be used conjunctively or disjunctively unless the context specifically refers to disjunctive use. The use of the term "or" implies that a disjunctive meaning is intended. That is, "A, B, or C" can mean "A, B, and / or C" or "A, B, C, or any combination thereof," while "A, B, or C" also includes "A or B or C, but does not include (A and B), (A and C), (B and C), and (A, B, and C)."

[0064] The terms “about” or “approximately” may mean within an acceptable margin of error for a particular value as determined by those skilled in the art, which depends in part on how that value is measured or determined, i.e., on the limits of the measuring system. For example, “about” may mean a standard deviation of 1 or more than 1, depending on the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean within one order of magnitude, up to five times, more preferably up to two times a given value. Where a particular value is described in this application and claims, unless otherwise stated, the term “about” means that it should be assumed to be within an acceptable margin of error for that particular value.

[0065] As used herein and in the claims, the terms “comprising” (and any other form of “comprise” and “comprises”), “having” (and any other form of “have” and “has”), “including” (and any other form of “includes” and “include”), or “containing” (and any other form of “contains” and “contain”) are comprehensive or open-ended and do not exclude additional unlisted elements or steps of methods. Any embodiment discussed herein may be implemented with respect to any method or composition of the Disclosure, and vice versa. Furthermore, the compositions of the Disclosure may be used to achieve the methods of the Disclosure.

[0066] Articles, compositions, methods, etc., comprising one or more elements may consist of one or more elements, or may be essentially composed of one or more elements. As used herein and in the claims, "consisting of" (and any other form of "consisting of," such as "consists of" and "consist of") means including and being limited to. As used herein and in the claims, "consisting essentially of" (and any other form of "consisting essentially of," such as "consisting essentially of" and "consist essentially of") means that the article, composition, method, etc., may include additional elements that include specified enumerated elements, such as components, compounds, materials, processes, etc., and that do not substantially affect the basic and novel features of the article, composition, method, etc.

[0067] References in this specification to “several embodiments,” “a certain embodiment,” “one embodiment,” “one or more embodiments,” “embodiments,” or “other embodiments” mean that certain features, structures, or characteristics described in relation to that embodiment are included in at least one of the embodiments of this disclosure, but not necessarily in all embodiments. To the extent that this disclosure describes in more detail or broadly aspects of aspects, components, or elements related to a particular embodiment, it is intended that such aspects, components, or elements related to that embodiment should be understood to encompass the additional details and broadness described in this disclosure.

[0068] The terms “preferred” and “preferred” refer to embodiments of the present invention that may provide specific benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the enumeration of one or more preferred embodiments does not imply that other embodiments are unhelpful, nor is it intended to exclude other embodiments from the scope of the present invention.

[0069] As used herein, the term “nucleic acid” refers to a polymer comprising at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, including DNA and RNA. A “nucleotide” comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked to each other via phosphate groups. A “base” includes purines and pyrimidines, which further include the natural compounds adenine ("A"), thymine ("T"), guanine ("G"), cytosine ("C"), uracil ("U"), inosine ("I"), as well as natural analogs and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that introduce novel reactive groups, such as, but are not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. Nucleic acids include synthetic, naturally occurring, and non-naturally occurring nucleic acids, which have similar binding properties to a reference nucleic acid and include known nucleotide analogs, or modified skeleton residues or bonds, or modified sugar residues, or non-canonical / chemically modified nucleic acid bases, and combinations thereof. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphoramides, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0070] The term "nucleic acid" includes any oligonucleotide (e.g., gRNA) or polynucleotide (e.g., mRNA, genomic DNA). Fragments containing up to 150 nucleotides are generally called oligonucleotides, and longer fragments are called polynucleotides. Deoxyribooligonucleotides consist of a pentose sugar called deoxyribose, which is covalently bonded to phosphate at its 5' and 3' carbons to form alternating, unbranched polymers. DNA can be, for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these. Ribooligonucleotides consist of similar repeating structures where the pentose sugar is ribose. Therefore, the terms "polynucleotide" and "oligonucleotide" may refer to polymers or oligomers of nucleotides or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (skeletal) bonds. The terms "polynucleotide" and "oligonucleotide" may also include polymers or oligomers, or portions thereof, that contain naturally occurring monomers that function similarly. Such modified or substituted oligonucleotides are often preferred over their native forms due to properties such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases. It should be understood that the terms “polynucleotide” and “oligonucleotide” may also include polymers or oligomers containing combinations of both deoxynucleotides and ribonucleotides, or variants thereof, in combination with skeletal modifications such as those described herein.

[0071] As used herein, “nucleic acids” may include one or more nucleotide variants, including nonstandard nucleotides, non-natural nucleotides, nucleotide analogs, and / or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, Examples include 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), weybutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. In some cases, nucleotides may include modifications to their phosphate moieties, including modifications to the triphosphate moieties. Non-limiting examples of such modifications include longer phosphate chains (e.g., phosphate chains with 4, 5, 6, 7, 8, 9, 10 or more phosphate groups) and modifications with thiol groups (e.g., alpha-thiotriphosphate and beta-thiotriphosphate).

[0072] The nucleic acids described herein may be modified in the base moiety (e.g., one or more atoms typically available to form hydrogen bonds with complementary nucleotides, and / or one or more atoms not typically available to form hydrogen bonds with complementary nucleotides), the sugar moiety, or the phosphate skeleton. Examples of skeleton modifications include, but are not limited to, phosphorothioate bonds, phosphorodithioate bonds, phosphoroselenoate bonds, phosphorodiselenoate bonds, phosphoranilothioate bonds, phosphoraniladet bonds, phosphoramidate bonds, and phosphorodiamidate bonds. Phosphothioate bonds replace unbridged oxygen atoms in the phosphate skeleton with sulfur atoms, delaying nuclease degradation of oligonucleotides. Phosphorodiamidate bonds (N3'→P5') inhibit nuclease recognition and degradation. Skeletal modifications may also include peptide bonds in place of phosphorus in the skeletal structure (e.g., N-(2-aminoethyl)-glycine units linked by peptide bonds in peptide nucleic acids), or linking groups containing carbamates, amides, and linear and cyclic hydrocarbon groups. Oligonucleotides with modified skeletons are outlined in Micklefield, Curr. Med. Chem., 8(10):1157-79, 2001 and Lyer et al., Curr. Opin. Mol. Ther., 1(3):344-358, 1999. Nucleic acid molecules described herein may include sugar moieties containing ribose or deoxyribose present in naturally occurring nucleotides, or modified sugar moieties or sugar analogs. Examples of modified sugars, though not limited to them, include 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-aminoethyl, 2'-fluoro, N3'→P5' phosphoramide, 2'-dimethylaminooxyethoxy, 2'2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic-modified sugars.2'-O-methyl or 2'-O-methoxyethyl modifications may be included to promote "type A" or "RNA-like" conformation in oligonucleotides, increasing binding affinity to RNA and enhancing nuclease resistance. The modified sugar moiety may also contain extra crosslinks (e.g., methylene crosslinks linking the 2'-O and 4'-C atoms of ribose in locked nucleic acids) or sugar analogs such as morpholine rings (e.g., as in phosphorodiamidate morpholino).

[0073] This disclosure encompasses artificial, isolated, or substantially purified nucleic acid molecules and compositions containing such molecules. As used herein, “isolated” or “purified” DNA or RNA molecules are DNA or RNA molecules that exist away from their natural environment. Isolated DNA or RNA molecules may exist in a purified form or in a non-natural environment, such as a transgenic host cell. For example, “isolated” or “purified” nucleic acid molecules or their biologically active portions, when produced by recombinant technology, substantially contain no other cellular material or culture medium, or when chemically synthesized, substantially contain no chemical precursors or other chemicals. In one embodiment, the “isolated” nucleic acid does not contain sequences that are naturally adjacent to the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0074] As used herein, the terms “protein,” “polypeptide,” and “peptide” are interchangeable and refer to polymers of amino acid residues linked via peptide bonds, which may consist of two or more polypeptide chains. The terms “polypeptide,” “protein,” and “peptide” refer to polymers of at least two amino acid monomers linked to one another via amide bonds. Amino acids may be L-optical isomers or D-optical isomers. More specifically, the terms “polypeptide,” “protein,” and “peptide” refer to molecules consisting of two or more amino acids in a specific order, for example, the order determined by the base sequence of nucleotides in a protein-coding gene or RNA. Proteins are essential for the structure, function, and regulation of cells, tissues, and organs of the body, and each protein has a unique function. Examples of proteins include hormones, enzymes, antibodies, and any fragments thereof. In some cases, a protein may be a part of a protein, for example, a protein domain, subdomain, or motif. In some cases, a protein may be a variant (or mutant) of a protein, where one or more amino acid residues are inserted into, deleted from, and / or substituted within the naturally occurring (or at least known) amino acid sequence of the protein. The protein or its variant may be naturally occurring or recombinant. Methods for detecting and / or measuring polypeptides in biological materials are well known in the art and are not limited to, but include Western blotting, flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and various proteomics techniques, such as mass spectrometry. Exemplary methods for measuring or detecting polypeptides include immunoassays such as ELISA. This type of protein quantification may be based on an antibody capable of capturing a specific antigen and a secondary antibody capable of detecting the captured antigen.

[0075] The terms “subject” or “patient” encompass mammals. Examples of mammals include, but are not limited to, mammals, i.e., non-human primates such as humans and chimpanzees, as well as other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs; and any member of the same class.

[0076] "Subjects requiring it" refers to individuals who have a disease, symptoms of a disease, or a predisposition to a disease, and whose purpose is to cure, heal, alleviate, mitigate, change, correct, improve, or influence that disease, symptoms of a disease, or predisposition to a disease. In one or more embodiments, the subject has a high blood Lp(a) concentration.

[0077] As used herein, “administer” and its grammatical equivalents may mean providing one or more active pharmaceutical ingredients (e.g., mRNA encoding an editor protein, guide oligonucleotide), pharmaceuticals (e.g., LNPs encapsulating the active pharmaceutical ingredient for delivery to target cells / tissues), or pharmaceutical compositions thereof to a subject or patient. For example, but not limited to, “administer” may be carried out by intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, intramuscular (im) injection, intravascular injection, intraventricular (icv) injection, intrathecal (it) injection, infusion (inf.), oral (po) route, topical (top.) administration, or rectal (pr) administration. One or more such routes may be used.

[0078] As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intraventricular, intrathecal, intralesional, and intracranial injection or infusion techniques. Parenteral administration may be, for example, by bolus injection or by time-delayed, stepwise perfusion. Furthermore, it may be administered to subjects via injectable depot administration routes, such as those allowing for depot injections at 1 month, 3 months, or 6 months, or using biodegradable materials and methods.

[0079] As used herein, the terms “to treat,” “to treat,” or “treatment,” and their grammatical equivalents may include ameliorating, reducing, or improving at least one symptom of a disease or condition, preventing further symptoms, inhibiting a disease or condition, for example, stopping the onset of a disease or condition, mitigating a disease or condition, causing the disease or condition to subside, causing the disease or condition to resolve, mitigating a condition caused by a disease or condition, or preventing and / or therapeutically blocking the symptoms of a disease or condition. “To treat” may mean administering to a subject a composition containing nanoparticles, such as lipid nanoparticles (LNPs), before or after the onset or suspected onset of a disease or condition. “To treat” includes the concept of “to mitigate,” which means reducing the frequency or severity of the occurrence or recurrence of any symptom or other adverse effect associated with a disease or condition and / or adverse effects related to a disease or condition. The term “to treat” also encompasses the concept of “to manage,” which refers to reducing the severity of a particular disease or disorder in a patient, or delaying its relapse, for example, extending the period of remission in a patient suffering from the disease. The term “to treat” further encompasses the concepts of “prevention,” “prevention,” and “prevention.” It is recognized, though not excluded, that treating a disorder or condition does not require the complete elimination of the associated disorder, condition, or associated symptoms.

[0080] As used herein, terms such as “prevent,” “prevention,” and “prevention” refer to reducing the probability of developing a disease or condition in subjects who do not have the disease or condition but are at risk of developing it or are prone to developing it.

[0081] As used herein, the term “ameliorate” may mean to reduce, suppress, weaken, decrease, halt, or stabilize the onset or progression of a disease.

[0082] As used herein, “delaying” the onset of a disease means delaying, preventing, slowing, stabilizing, and / or postponing the progression of the disease. This delay can be of varying lengths depending on the disease history and / or the individual being treated. A method for “delaying” or mitigating the onset of a disease, or delaying the onset of a disease, is a method that reduces the probability of developing one or more symptoms of the disease within a given time frame and / or reduces the severity of symptoms within a given time frame compared to not using the method. Such comparisons are typically based on clinical trials using a sufficient number of subjects to yield statistically significant results.

[0083] The “onset” or “progression” of a disease means the initial appearance and / or subsequent progression of the disease. Disease onset may be detectable and assessed using standard clinical techniques well known in the art. However, onset also refers to progression that may be undetectable. For the purposes of this disclosure, onset or progression refers to the biological course of symptoms. “Onset” includes occurrence, recurrence, and onset.

[0084] As used herein, “onset” or “occurrence” of a disease includes the first onset and / or recurrence.

[0085] The terms “therapeutic agent” or “active pharmaceutical ingredient” may refer to any agent that, when administered to a subject, has therapeutic, diagnostic, and / or prophylactic effects and / or induces a desired biological and / or pharmacological effect. A therapeutic agent may also be called an “active substance” or “activator.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids such as guide oligonucleotides and mRNA.

[0086] As used herein, the term “pharmaceutical composition” and its grammatical equivalents may refer to a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients, carriers, and / or a target requiring them, such as a therapeutic agent administered to a human being.

[0087] As used herein, the term “pharmaceutically acceptable” and its grammatical equivalents may refer to the attributes of a material useful for preparing a pharmaceutical composition that is generally safe, non-toxic, not biologically or otherwise undesirable, and acceptable for veterinary and human medicinal use. “pharmaceutically acceptable” may refer to a material such as a carrier or diluent that does not neutralize the biological activity or properties of a compound and is relatively non-toxic; that is, the material can be administered to a subject without causing undesirable biological effects or interacting in a harmful manner with any of the components of the pharmaceutical composition in which it is contained.

[0088] "Pharmacologically acceptable excipients, carriers, or diluents" refer to excipients, carriers, or diluents that can be administered to a subject together with a drug, and which, when administered in a dose sufficient to deliver a therapeutic dose of the drug, do not impair its pharmacological activity and are non-toxic.

[0089] A “pharmaceutically acceptable salt” can be a salt of an acid or base that is generally considered in the art to be suitable for use in contact with human or animal tissue without causing excessive toxicity, irritation, allergic reactions, or other problems or complications. Those skilled in the art will recognize from this disclosure and the knowledge of the art that further pharmaceutically acceptable salts are those listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985).

[0090] As used herein, the term “therapeutic dose” means the amount of a delivered agent (e.g., nucleic acids, drugs, payloads, compositions, therapeutic agents, diagnostic agents, prophylactic agents, etc.) that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, improve, diagnose, prevent, and / or delay the onset of that infection, disease, disorder, and / or condition.

[0091] The ranges provided herein are understood to be abbreviated representations of all values ​​within the range. For example, the range 1–50 is understood to include any number, combination of numbers or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values ​​between the aforementioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, a concrete concept is conceivable of "nested subranges" extending from any endpoint of the range. For example, a nested subrange of the exemplary range 1-50 may include 1-10, 1-20, 1-30, and 1-40 in one direction, or 50-40, 50-30, 50-20, and 50-10 in the other direction.

[0092] All numbers used herein and in the claims to represent quantities, molecular weights, etc., of components should be understood in all cases to be modified with the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described herein and in the claims are approximations that may vary depending on the desired properties sought by the invention. At the very least, and not as an attempt to limit the doctrine of equivalents of the claims, each numerical parameter should be interpreted at least in light of the number of significant figures reported and by general rounding techniques.

[0093] Although the numerical ranges and parameters describing the broad scope of the present invention are approximations, the numerical values ​​described in specific examples are reported as accurately as possible. However, all numerical values ​​inherently include a range that necessarily derives from the standard deviation observed in their respective test measurements.

[0094] The term “complementary” is used throughout this application to describe two related nucleic acid sequences that can form a double-stranded complex of a first 5' to 3' “upper” strand and a second 3' to 5' “lower” strand. A “spacer” sequence of a guide nucleic acid is considered “complementary” to the sequence of the target nucleic acid. In relation to a guide oligonucleotide, a sequence can be considered sufficiently “complementary” to the target sequence if it can hybridize sufficiently to the intended DNA strand so that it can be used to guide an editor protein to the target sequence to cause the intended edit. Thus, a guide oligonucleotide is complementary if, for example, it can hybridize sufficiently to the intended DNA strand so that it can operatively position an editor at a desired location to facilitate the intended edit.

[0095] As used herein, a nucleic acid sequence that is "substantially identical" to another nucleic acid sequence is a nucleotide sequence having 70% or more sequence identity with the other nucleic acid sequence. In some embodiments, a nucleic acid sequence that is "substantially identical" to another nucleic acid sequence has 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the other nucleic acid sequence.

[0096] For the purpose of achieving percent sequence identity between RNA and DNA sequences, uracil bases in RNA are considered to be identical to thymine bases in DNA.

[0097] As used herein, “sequence identity” refers to the degree to which two optimally aligned nucleic acid sequences remain invariant throughout the entire alignment window of their constituent elements, e.g., nucleotides. “Identity” can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0098] As used herein, the terms “percent sequence identity” or “percent identity” refer to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference (“inquiry”) nucleic acid (or its complementary strand) compared to a test (“subject”) nucleic acid (or its complementary strand) when the two sequences are optimally aligned. The sequence identity percentage may be determined when the sequences to be compared are aligned to the greatest match, using the sequence comparison algorithms described below and known in the art, or measured by visual inspection.

[0099] For sequence comparison, typically one sequence serves as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence(s) to the reference sequence based on the specified program parameters. Optimal sequence alignment for aligning the comparison window is well known to those skilled in the art and can be achieved by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA, available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, California). Sequence alignment can be analyzed using the Burrows-Wheeler transformation, such as the BOWTIE open-source software available at https: / / github.com / BenLangmead / bowtie. The "identity fraction" for aligned segments of the test and reference sequences is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. Percent sequence identity is expressed as the percentage of identity multiplied by 100.

[0100] "Percent identity" can also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves initially identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in the data system sequence, either match or satisfy a certain positive threshold score T. T is called the neighbor word score threshold (Altschul et al., 1990). These initial neighbor word hits serve as seeds to initiate a search for longer HSPs that contain them. Word hits are then extended bidirectionally along each sequence as long as the cumulative alignment score can increase. The cumulative score is calculated for nucleotide sequences using the parameter M (reward score for matching residue pairs; always >0) and the parameter N (penalty score for mismatched residues; always <0). Word hit extension in each direction stops when the cumulative alignment score decreases by X from its maximum achieved value, when the cumulative score becomes zero or less due to the accumulation of one or more negative score residue alignments, or when the end of any sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses the following default settings: word length (W) 11, expected value (E) 10, cutoff 100, M=5, N=-4, and comparison of both strands.

[0101] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide or amino acid sequences may occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if its smallest sum probability in comparison to the reference nucleotide sequence is less than approximately 0.1 to less than approximately 0.001.

[0102] Sequence "identity" (or, for example, "identicality" or "identical to"), as used herein, refers to the amount of nucleotides or amino acids that exactly match between two different sequences. When comparing RNA and DNA sequences, uracil bases and thymine bases are considered to be the same base. Gaps are not counted, and the measurement is typically relating to the shorter of the two sequences.

[0103] For example, for nucleotide sequences A:AAGGCTT;B:AAGGC; and C:AAGGCAT, the identity of sequence A compared to reference sequence B is 100% (5 identical nucleotides / minimum (length(A), length(B))), the identity of sequence B to reference sequence C is 100%, but the identity of sequence A to reference sequence C is 85% (6 identical nucleotides / 7).

[0104] Sequence “similarity” (or, for example, “similar” sequences), as used herein, may be described as the optimal matching problem (edit distance) that finds the minimum number of editing operations (insertions, deletions, and substitutions) required to transform a first sequence into an exact copy of a second sequence to be aligned. If the lengths of the first and second sequences are different, the similarity is determined against the length of the shortest sequence. That is, sequence similarity is [1 - (number of editing operations / length of the shortest sequence)]. Using this, the percentage sequence similarity for nucleotide sequences A:AAGGCTT; B:AAGGC; and C:AAGGCAT is 60% similarity between sequence A and sequence B, 60% similarity between sequence B and sequence C, and 86% similarity between sequence A and sequence C.

[0105] It should be understood that sequence comparisons can be determined with or without chemical modifications. Therefore, when comparing a modified oligonucleotide with a reference unmodified oligonucleotide, the percentage sequence identity is determined based on the sequence of nucleic acid bases in the modified oligonucleotide, where the modified nucleic acid base is considered equivalent to the unmodified nucleic acid base from which it is derived. For example, 2,4-dichlorotoluene, 2,4-dibromotholuene, and 2,4-diiodotolume are considered equivalent to thymine for the purpose of comparing the sequence identity of a modified oligonucleotide with a reference unmodified oligonucleotide. As another example, 4-methylbenzimidazole and 9-methylimidazo are considered equivalent to adenine for the purpose of comparing the sequence identity of a modified oligonucleotide with a reference unmodified oligonucleotide. If a modified oligonucleotide includes modifications to the binding site (e.g., other than the phosphate linker) and / or sugar (e.g., 2'-modified ribose or dideoxyribose), sequence identity is determined by comparing the sequence of nucleic acid bases of the modified oligonucleotide with the sequence of nucleic acid bases in the reference unmodified sequence. For example, nucleotide sequence:A:mA * AGGCmT *For mT;B:AAGGCTT; and C:AAGGCAT, the identity of sequence A to reference sequence B is 100% (7 identical nucleotides out of 7), and the identity of sequence A to reference sequence C is 85% (6 identical nucleotides out of 7). The percentage of sequence similarity in the above example is as follows: the similarity between sequence A and sequence B is 100%, and the similarity between sequence A and sequence C is 86%.

[0106] When chemical modifications are involved in determining identity, sequence identity is determined based on the exact modifications of each nucleotide in the reference modified oligonucleotide (e.g., based on any modifications to the linker, sugar moiety, and nucleic acid base). Therefore, an unmodified (or differently modified) nucleotide of an oligonucleotide cannot be considered identical to the corresponding modified oligonucleotide of the reference modified oligonucleotide. Thus, for example, for the purpose of identity, consider the following two sequences: A: AAGGCTTC; B: mA * AGGCmT * When comparing mT sequences, if chemical modifications are not excluded, the identity of sequence A to reference sequence B is 57% (4 / 7 identical nucleotides), while if chemical modifications are excluded, the identity of sequence A to reference sequence B is 100% (7 / 7 identical nucleotides).

[0107] The comparison of any modified sequences may be determined with or without taking chemical modifications into account, for example, as described in the claims.

[0108] As used herein, a spacer sequence of a guide nucleic acid is considered "homologous" to a protospacer sequence of a target nucleic acid if a gene editing system (editor) containing a guide oligonucleotide having the spacer sequence can modify (e.g., nick) the target nucleic acid (e.g., a nick on the sense or antisense strand of the target nucleic acid). A spacer sequence homologous to a protospacer sequence may be identical or substantially identical to the protospacer sequence.

[0109] In some embodiments, a first nucleotide sequence homologous to a second nucleotide sequence can hybridize to a complementary sequence of the second nucleotide sequence under stringent or highly stringent conditions. “Stringent hybridization conditions” and “stringent hybridization washing conditions” in relation to nucleic acid hybridization are sequence-dependent and differ under different environmental parameters. Extensive guidance on nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part I chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier, New York (1993). Generally, highly stringent hybridization and washing conditions are selected to be approximately 5°C lower than the melting point Tm of a particular sequence at a given ionic strength and pH. Tm is the temperature (under specified ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Highly stringent conditions are selected to be equal to the Tm of a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences with more than 100 complementary residues on the filter in Southern or Northern blotting is 50% formamide with 1 mg of heparin at 42°C, with hybridization performed overnight. An example of highly stringent washing conditions is 0.15 M NaCl at 72°C for approximately 15 minutes. An example of stringent washing conditions is 0.2 × SSC washing at 65°C for 15 minutes (for a description of SSC buffer, see Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3 rdSee ed., Cold Spring Harbor Laboratory Press, 2001. In many cases, a low-stringency wash is performed before a high-stringency wash to remove background probe signals. For example, an example of a medium-stringency wash for double helixes with more than 100 nucleotides is 1×SSC for 15 minutes at 45°C. For example, an example of a low-stringency wash for double helixes with more than 100 nucleotides is 4-6×SSC for 15 minutes at 40°C. For short probes (e.g., about 10-50 nucleotides), stringent conditions typically include a pH of 7.0-8.3, a sodium ion salt concentration of less than about 1.0 M, typically about 0.01-1.0 M (or other salt), and a temperature of at least about 30°C. Stringent conditions can also be achieved by adding an destabilizer such as formamide.

[0110] The terms “effect” or “effectuate” and their grammatical equivalents are used throughout this disclosure to refer to actions performed that are expected to induce the creation of an intended result. The entire process by which the result is produced does not necessarily have to be carried out by the system used to bring about the editing. Cellular processing may, for example, play a role in completing the intended result. However, the intended result will not occur without the system; that is, the system is necessary for the intended result to be brought about. For example, a double nickase in vivo editing system, such as those shown in Figures 1A–1D, which include Cas9 nickase and two guide oligonucleotides, can bring about editing in a gene without performing a final processing step that results in the editing being fully incorporated into both strands of the gene. Cellular DNA repair enzymes may perform the final processing step. Not all steps necessary to complete the editing are carried out by the double nickase editing system, but the system is still said to have brought about the editing.

[0111] As used herein, “inactivating the LPA gene” and its grammatical equivalent refer to editing of the LPA gene by a gene editing system that results in reduced expression of apo(a), expression of a nonfunctional variant of apo(a), and / or expression of an apo(a) variant with reduced function compared to wild-type apo(a). Editing of the LPA gene by the gene editing systems described herein may result in indel variants and / or nonsynonymous variants. Indel variants and / or nonsynonymous variants of the LPA gene may result in the production of premRNA that is degraded, for example, via nonsense mutation-dependent degradation. Indel variants and / or nonsynonymous variants of the LPA gene may result in the production of apo(a) variants with reduced function, and in some cases non-functional apo(a) variants, compared to wild-type apo(a).

[0112] Throughout this application, in several places, guidance is provided by examples, which can be used in various combinations, including their specific embodiments, and may be the subject of the claims. In each embodiment, the enumerated elements function only as a representative group and should not be construed as an exclusive list. It should be understood that specific embodiments, materials, quantities, and procedures should be interpreted broadly in accordance with the scope and concepts of the invention as described herein, and it is intended that the embodiments and various embodiments described herein may be combined and described in separate patentable claims.

[0113] With respect to any method disclosed herein that includes individual steps, the steps may be performed in any executable order, and any combination of two or more steps may be performed simultaneously, if necessary.

[0114] II. Apolipoprotein (A) protein and LPA gene The LPA gene is remarkably similar to the PLG (plasminogen) gene, which evolved in primates, and the protein products of these two genes share structural similarities. They each possess a Kringle type IV (KIV) domain, a Kringle type V (KV) domain, and a protease-like domain, respectively. Apo(a) has 10 types of KIV domains (KIV1 to KIV) with a variable number of repeating KIV2 domains ranging from 2 to over 40. 10 It contains ), and nine other single domains, a single KV domain, and an inactive protease-like domain. Serum Lp(a) concentration is largely genetically determined, and it varies inversely to the number of KIV2 repeats, as LPA alleles with fewer repeats result in smaller apo(a) isoforms that are produced and processed more rapidly in hepatocytes. There is a wide inter-individual variability in serum Lp(a) concentration, with one in five individuals having high concentrations that indicate a substantial increase in the risk of cardiovascular disease. Conversely, individuals with very low serum Lp(a) concentrations caused by naturally occurring null variants in LPA are protected from ASCVD and do not suffer serious adverse consequences.

[0115] Apolipoprotein (a) [apo(a)] is a definitive component of Lp(a). Apo(a) is understood to be primarily synthesized in hepatocytes of the liver. Apo(a) is synthesized from the KIV1 domain and KIV3 into KIV 10 It contains a single copy of the domain, a variable number of repeating KIV2 domains ranging from 2 to over 40, a single KV domain, and an inactive protease-like domain. The wild-type apo(a) protein annotated in the reference human genome (GRCh38 / hg38, NCBI) has six repeating KIV2 domains, is 2040 amino acids long, and has the following amino acid sequence. It should be understood that the following sequence represents one of the many isoforms of apo(a) present in the human population.

[0116] The aforementioned sequence uses a standard, conventional nomenclature well known in the art, and it should be understood that each letter represents an amino acid. [Table 1]

[0117] The LPA gene, annotated in the reference human genome (GRCh38 / hg38, NCBI), consists of 39 exons and is located on chromosome 6, 6q25.3, at nucleotides 160,664,275 to 160,531,482.

[0118] The complete sequence of the human LPA gene is available in UniProtKB-P08519(APOA_HUMAN).

[0119] High blood Lp(a) concentrations are a well-established incidental risk factor for ASCVD and calcified aortic valve disease. Individuals with very low blood Lp(a) concentrations, caused by naturally occurring null variants in LPA, are protected from ASCVD and do not suffer serious adverse consequences.

[0120] The inventors demonstrate below that it is possible to edit the coding sequence of the LPA gene using a gene editing approach to introduce indel variants or non-synonymous variants, providing a one-time therapeutic approach for patients with high blood Lp(a) concentrations.

[0121] Any subject with elevated blood Lp(a) levels can be treated using the gene editing approaches described herein. Subjects with elevated blood Lp(a) levels may be identified by clinical tests, genetic screening, or a combination thereof.

[0122] In embodiments, the method includes identifying a subject having high blood Lp(a) concentrations and administering a composition to the subject that contains components capable of introducing an indelian variant or a non-synonymous variant into the coding sequence of the LPA gene. Administering the composition to the subject may include administering a therapeutically effective dose of the composition. The resulting LPA gene editing may result in reduced functionality of the apo(a) protein. The resulting LPA gene editing may result in a decrease in blood Lp(a) concentrations.

[0123] The compositions described herein, including gene editing systems, comprise one or more guide nucleic acids designed to target one or more protospacer regions on the LPA gene.

[0124] III. Gene editing / genetic modification As used herein, the terms “gene editing” or “genetic modification” and their grammatical equivalents refer to genetic manipulations that modify, insert, substitute, or remove one or more nucleotides from a genome. Gene editing may be carried out using artificial, non-naturally occurring gene editing systems comprising one or more nucleases, which may be derived from naturally occurring nucleases or be artificially engineered. Genetic modification may include introducing double-strand breaks, nonsense variants, frameshift variants, splice site alterations, or inversions in a polynucleotide sequence, such as a target polynucleotide sequence. Genetic modification may also be achieved using other editing systems, such as double nickase editing systems, as shown and described in Figures 1A–1D.

[0125] Editing of the LPA gene can be induced using any suitable gene editing system. The gene editing system may include a gene editing substance (editor) and a suitable guide nucleic acid for use with the gene editing substance (editor). For the purposes of this disclosure, a “gene editing substance (editor) system” including a nucleic acid that is translated into a gene editing substance (editor) protein or a protein component of a gene editing substance (editor), or that can be transcribed and translated, is considered a gene editing substance (editor) system including a gene editing substance (editor). The nucleic acid may be, for example, plasmid DNA or mRNA. The mRNA may be mature mRNA or premRNA, which may be processed into mature mRNA, for example, in a cell, and then translated, for example, in a cell, to produce a gene editing substance (editor). For the purposes of this disclosure, a “gene editing substance (editor) system” including a nucleic acid that can be transcribed to produce gRNA is considered a gene editing substance (editor) system including gRNA. The nucleic acid may be, for example, plasmid DNA. In the embodiments, the gene editing substance (editor) is a Cas9 variant or an associated gene editing substance (editor). In the embodiments, the gene editing substance (editor) is a nickasase, such as those shown in Figures 1A to 1D.

[0126] A. Cas9 and related nucleic acid-directed editing proteins A ribonucleoprotein complex derived from Streptococcus pyogenes, capable of introducing double-strand breaks into double-stranded DNA, was published in 2012 by Jennifer Doudna's group (Jinek et al. Science 2012 Aug 17;337(6096):816-21). In this context, the term "ribonucleoprotein" is used to refer to a non-covalent assembly of RNA and protein. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have been described. It has been shown that the CRISPR region can be transcribed into a short RNA sequence (crRNA), which can then form a hairpin with another short RNA sequence called trans-activating CRISPR RNA (tracrRNA) to form a guide RNA (gRNA). Operationally, crRNA contains a nucleotide sequence (known as a "spacer" or "spacer sequence") that is complementary to the target strand of DNA, while tracrRNA functions as a binding scaffold for the Cas protein. The Cas protein can then complex with gRNA, and the ribonucleoprotein is targeted to a sequence substantially identical to the crRNA sequence. The sequence substantially identical to the crRNA is known as the "protospacer" sequence and is directly adjacent to the "protospacer flanking motif" (PAM). In the case of Streptococcus pyogenes (S. pyogenes) Cas9, the PAM is the "NGG" sequence, where N represents any standard nucleotide and G represents guanine.

[0127] The Cas protein contains two nuclease domains, called the HNH domain and the RuvC domain, which can cleave the phosphate backbone of nucleic acids. The HNH domain cleaves the backbone of the strand complementary to the gRNA, called the "target strand," while the RuvC domain cleaves the backbone of the opposite strand, also called the "non-target strand" or "substitution strand" (i.e., the strand containing the protospacer). When the gRNA-Cas complex binds to a double-stranded DNA sequence, each nuclease domain cleaves the backbone of one strand of DNA, causing a double-strand break (DSB). DSBs can be repaired by cells in several ways, including the creation of insertions or deletions (indels), homology-directed repair (HDR), microhomology-mediated end joining (MMEJ), or mismatch repair (MMR). Multiple Cas-based systems have been engineered to utilize each of these repair pathways. Guide RNA designs have been further characterized and engineered to improve editing efficiency and obtain different editing results.

[0128] Other variants of the Cas9 protein and Cas-type proteins have also been characterized. Furthermore, by mutating the amino acid sequence of each nuclease domain of Cas9, it is possible to obtain “nickase” Cas9 proteins (nCas9) that impair the nuclease activity of the Cas9 protein and introduce single-strand breaks instead of double-strand breaks in the target DNA sequence. The “D10A” mutation in the RuvC domain results in a Cas9 protein that cleaves only the strand complementary to the gRNA (i.e., the target strand). Such nickases based on the “D10A” mutation in the RuvC domain of the Cas9 protein are used in base editing systems to introduce nicks in the target strand, which is the strand opposite the strand containing the protospacer. The “H840A” mutation in the HNH domain results in a Cas9 protein that cleaves only the substituted strand (i.e., the strand containing the protospacer sequence). Such nickases based on the “H840A” mutation in the HNH domain of the Cas9 protein are used in template-based editing systems to introduce nicks in the non-target strand or the protospacer sequence. Mutations that impair both nuclease domains result in a Cas9 that has lost its catalytic function (also known as "dCas9"), which can still bind to gRNA and target gene regions, but does not essentially alter the target region. Various modified and unmodified Cas proteins have been described (see, for example, Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013); Hwang et al., Nature Biotechnology 31, 227-229 (2013); Jinek et al., eLife 2, e00471 (2013); Dicarlo et al., Nucleic Acids Research (2013); and Jiang et al., Nature Biotechnology 31, 233-239 (2013)).Examples of Cas9 proteins and Cas-type proteins include, but are not limited to, SpCas9 (e.g., dCas9 and nCas9), SaCas9 (e.g., SaCas9d, SaCas9d, SaKKH Cas9), NmeCas9, CasX, CasY, Cas12a / Cpf1, C2c1, C2c2, C2c3, and Argonaut. The gene editing materials (editors) described herein may include Cas9 or Cas-related domains or proteins, or preferred variants of the domains or proteins.

[0129] B. Double Nickase Editing System A dual nickase gene editing system comprises one or more nickases, a first guide oligonucleotide, and a second guide oligonucleotide. At least one of the one or more nickases is configured to interact with the first guide oligonucleotide. In its interaction with the first guide oligonucleotide, the nickase is manipulated to introduce a nick into either the first or second strand of the LPA gene. At least one of the one or more nickases is configured to interact with the second guide oligonucleotide. In its interaction with the second guide oligonucleotide, the nickase is manipulated to introduce a nick into either the first or second strand of the LPA gene. The nickases interacting with the first and second guide oligonucleotides may be the same or different. The nicks created on opposing strands in the LPA gene can activate cellular DNA repair mechanisms, which can result in indelvariants or nonsynonymous variants in the LPA gene. Therefore, the dual nickase system can result in indelvariants or nonsynonymous variants.

[0130] Figures 1A–1D show a double nickase editing system containing a single nickase editor. A nickase editor is a protein or protein complex that, using two guide nucleic acids, can introduce an indel variant or a non-synonymous variant into a target DNA site. The nickase editor may include the nickase Cas9.

[0131] A double nickase editing system (a nickase protein and two guide nucleic acids) may be capable of locating a specific target site in a gene or genome and nicking both strands at separate locations on the target DNA site. The nickase protein may be catalytically inactivated or impaired to nick or cleave at most one strand of a double-stranded nucleic acid target. The “D10A” mutation in the RuvC domain of the Cas9 protein and the “H840A” mutation in the HNH domain of the Cas9 protein are examples of impaired nucleases that can be used in a double nickase editing system.

[0132] Editing of the LPA gene in cells or subjects can be induced using any suitable double nickase editing material (editor) system. Cells or subjects may be treated with assembled lipid nanoparticles (LNPs) that encapsulate or otherwise contain two guide nucleic acids, such as a nickase protein or mRNA encoding a nickase protein, in order to achieve editing.

[0133] The double nickase editing process can occur as shown in Figure 1A or Figure 1C. The target DNA site is engaged by the Cas9-D10A protein via a spacer region of a first guide nucleic acid that is substantially identical to the protospacer sequence on one strand of the target DNA site. The Cas9-D10A domain nicks the target strand of the first guide nucleic acid (i.e., the strand hybridized to the first spacer sequence). Separately, the target DNA site is engaged by the Cas9-D10A protein via a spacer region of a second guide nucleic acid that is substantially identical to the protospacer sequence on the other strand of the target DNA site. The Cas9-D10A domain nicks the target strand of the second guide nucleic acid (i.e., the strand hybridized to the second spacer sequence). The orientation of the protospacer sequences relative to each other determines whether the two nick sites are somewhat separated (Figure 1A) or close to each other (Figure 1C). Two nicks on opposing DNA strands can subsequently be repaired by the cell via non-homologous end joining, potentially resulting in an indel variant or a non-synonymous variant.

[0134] The double nickase editing process can occur as shown in Figure 1B or Figure 1D. The target DNA site is engaged by the Cas9-H840A protein via a spacer region of a first guide nucleic acid that is substantially identical to the protospacer sequence on one strand of the target DNA site. The Cas9-H840A domain nicks the non-target strand of the first guide nucleic acid (i.e., the strand with the first protospacer sequence). Separately, the target DNA site is engaged by the Cas9-H840A protein via a spacer region of a second guide nucleic acid that is substantially identical to the protospacer sequence on the other strand of the target DNA site. The Cas9-H840A domain nicks the non-target strand of the second guide nucleic acid (i.e., the strand with the second protospacer sequence). The orientation of the protospacer sequences relative to each other determines whether the two nick sites are close together (Figure 1B) or somewhat separated (Figure 1D). Two nicks on opposing DNA strands can subsequently be repaired by the cell via non-homologous end joining, potentially resulting in an indel variant or a non-synonymous variant.

[0135] In some embodiments, Cas9 nickase (or any other suitable nickase) working with a first guide nucleotide and a second guide nucleotide nicks the opposing strands of DNA to produce a 5' overhang (e.g., as shown in Figures 1A and 1D). In some embodiments, Cas9 nickase (or any other suitable nickase) working with a first guide nucleotide and a second guide nucleotide nicks the opposing strands of DNA to produce a 3' overhang (e.g., as shown in Figures 1B and 1C). The overhang may have any length suitable for enabling non-homologous end joining. It will be understood that the length of the overhang suitable for enabling non-homologous end joining may vary, among other things, based on the composition of the overhang. In some embodiments, the overhang may have a length of 1 to 200 nucleotides, e.g., 10 to 150 nucleotides, 15 to 100 nucleotides, or 20 to 50 nucleotides. In some embodiments, the overhang has a length of 10 nucleotides or more, for example, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, or 50 nucleotides or more.In some embodiments, the overhang has a length of 200 nucleotides or less, for example, 190 nucleotides, 180 nucleotides, 170 nucleotides, 160 nucleotides, 150 nucleotides, 140 nucleotides, 130 nucleotides, 120 nucleotides, 110 nucleotides, 100 nucleotides, 90 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, or 50 nucleotides or less. In some embodiments, the overhang has a length of 20 to 50 nucleotides, for example, 23 to 45 nucleotides, 30 to 40 nucleotides, 31 to 40 nucleotides, 32 to 40 nucleotides, 33 to 40 nucleotides, 31 to 39 nucleotides, 32 to 39 nucleotides, 33 to 39 nucleotides, or 34 to 38 nucleotides.

[0136] One implementation of the double nickase editing system is the D10A mutation (SpCas9-D10A) in the RuvC domain of the Streptococcus pyogenes Cas9 protein. The protein and cDNA sequences of SpCas9-D10A are shown in Table 1. Also shown in Table 1 are chemically modified mRNA sequences (MS029) manipulated to express SpCas9-D10A in cells, including those related to editing in the LPA gene, as further described herein.

[0137] One implementation of the double nickase editing system is the H840A mutation (SpCas9-H840A) in the RuvC domain of the Streptococcus pyogenes Cas9 protein. The protein and cDNA sequences of SpCas9-H840A are shown in Table 1. Also shown in Table 1 are chemically modified mRNA sequences engineered to express SpCas9-H840A in cells, including those related to editing in the LPA gene, as further described herein. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

[0138] In some embodiments, the mRNA encoding nickase has the mRNA sequences listed in Table 1. In some embodiments, the mRNA encoding nickase has sequences that are at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the mRNA sequences listed in Table 1.

[0139] In some embodiments, the mRNA encoding nickase has a coding sequence of the mRNAs listed in Table 1. In some embodiments, the mRNA encoding nickase has a coding sequence that is at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the coding sequences of the mRNA sequences listed in Table 1.

[0140] The mRNA coding sequences listed in Table 1 may be modified or optimized, and it will be understood that the untranslated region (UTR), and / or the 5' and 3' ends (e.g., within the last 3 or 5 nucleotides) may be modified or optimized to enhance or alter mRNA stability and / or expression. In some embodiments, mRNA may contain one or more stabilizing motifs, such as 5' and / or 3' end stabilizing motifs.

[0141] A dual nickase editing system for achieving editing in the LPA gene is disclosed herein, as described herein.

[0142] IV. Guide nucleic acids and target DNA sequences In some embodiments, the dual nickase editing system includes two or more guide nucleic acids. In some embodiments, the dual nickase editing system includes two guide nucleic acids.

[0143] Guide nucleic acid sequences direct gene editing substances (editors) to target genomic locations. Guide nucleic acids can vary depending on the gene editing substance (editor). This disclosure refers to guide nucleic acids as guide nucleic acid sequences, guide RNA, gRNA, and / or guide oligonucleotides. As described herein, guide RNA may include an RNA sequence that is or may not be chemically modified. While gRNA generally includes an RNA sequence, it should be understood that a portion of the guide RNA may not be ribonucleic acid and may include other chemical substitutions, including deoxyribonucleic acid or nucleotide analogs.

[0144] Each guide nucleic acid may contain a nucleotide sequence complementary to a target site in chromosomal DNA. The portion of the guide nucleic acid that identifies the target site is referred to herein as the “spacer.” The spacer is typically located at the 5’ end of the guide nucleic acid. The strand of chromosomal DNA containing the sequence complementary to the spacer is referred to herein as the “target strand.” The non-target strand of chromosomal DNA, also called the “substitution strand,” has a sequence identical or substantially identical to the spacer, referred to herein as the “protospacer.”

[0145] In one or more embodiments, each guide nucleic acid includes a spacer sequence that is identical or substantially identical to the protospacer sequence of the LPA gene. In embodiments, the gene editing system includes two guide nucleic acids, each having a different spacer sequence, where each spacer sequence is identical or substantially identical to the protospacer sequence specified in Table 2 or Table 5. In some embodiments, the guide oligonucleotide includes a spacer that includes sequences identical or substantially identical to nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the protospacer sequence specified in Table 2 or Table 5. In some embodiments, the guide oligonucleotide includes a spacer that includes sequences identical or substantially identical to nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the protospacer sequence specified in Table 2 or Table 5, having 0, 1, 2, 3, 4, or 5 mismatches. In some embodiments, the guide oligonucleotide includes a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the protospacer sequence identified in Table 2 or Table 5.

[0146] In embodiments, the gene editing systems described herein include a first guide oligonucleotide and a second guide oligonucleotide, wherein the first guide oligonucleotide includes a spacer sequence identical or substantially identical to the guide 1 protospacer sequence specified in Table 2 or Table 5, and the second guide oligonucleotide includes a spacer sequence identical or substantially identical to the corresponding guide 2 protospacer sequence (in the same row) specified in Table 2 or Table 5. In some embodiments, the first guide oligonucleotide includes a spacer sequence identical or substantially identical to nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the guide 1 protospacer sequence specified in Table 2 or Table 5, and the second guide oligonucleotide includes a spacer sequence identical or substantially identical to nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the corresponding guide 2 protospacer sequence (in the same row) specified in Table 2 or Table 5. In some embodiments, the first guide oligonucleotide comprises spacer sequences identical or substantially identical to nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the guide 1 protospacer sequence specified in Table 2 or Table 5, having 0, 1, 2, 3, 4, or 5 mismatches, and the second guide oligonucleotide comprises spacer sequences identical or substantially identical to nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the corresponding guide 2 protospacer sequence (in the same row) specified in Table 2 or Table 5, having 0, 1, 2, 3, 4, or 5 mismatches. In some embodiments, the first guide oligonucleotide includes a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the guide 1 protospacer sequence identified in Table 2 or Table 5, and the second guide oligonucleotide includes a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the corresponding guide 2 protospacer sequence (in the same row) identified in Table 2 or Table 5.In some embodiments, the first guide oligonucleotide includes the same spacer sequence as the guide 1 protospacer sequence specified in Table 2 or Table 5, and the second guide oligonucleotide includes the same spacer sequence as the corresponding guide 2 protospacer sequence (in the same row) specified in Table 2 or Table 5. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] PAM sequences are shown in bold and underlined text, and the corresponding spacer may have the same sequence as the protospacer sequence (if the spacer sequence is RNA, "T" is replaced with "U").

[0147] Each guide nucleic acid may include a portion that is recognized and bound to a gene editing substance (editor) (e.g., nickase). The portion of the guide nucleic acid that recognizes and binds to the gene editing substance (editor) is sometimes referred to herein as a “scaffold” region. The scaffold region may form one or more stem-loop structures recognized by the gene editing substance (editor). The lengths of the loop and stem may vary. In one or more embodiments, the loop may be in the range of about 3 to about 10 nucleotides in length. In one or more embodiments, the stem may be in the range of about 6 to about 20 nucleotides in length. The stem may include one or more bulges of 1 to 10 nucleotides or about 10 nucleotides. In one or more embodiments, the total length of the second region may be in the range of about 16 to 60 nucleotides in length. In one or more embodiments, the loop may be about 4 nucleotides in length. In one or more embodiments, the stem may be about 12 nucleotides in length. The scaffold region may include regions that do not form a stem-loop structure. This portion, which lacks substantial secondary structure, can have any preferred length, such as in the range of about 3 to about 100 nucleotides. The portion of the scaffold region lacking substantial secondary structure may be located at the 3' end of the guide nucleic acid. In some embodiments, the scaffold region includes a tracr sequence. In some embodiments, the scaffold region consists of or is essentially derived from a tracr sequence.

[0148] In one or more embodiments, the arrangement of the scaffolding regions is as follows: 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3', or 5'-GUUUGAGAGCUAUGCUGGAAACAGCAUAGCAAGUUCAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' It includes one of the following.

[0149] In one or more embodiments, the scaffold region may have a sequence that is at least 75%, at least 85%, at least 90%, or at least 95% identical to one of the sequences described herein. It will be obvious to those skilled in the art that the sequence and / or length of a suitable scaffold region may vary depending on several factors, such as the edited protein used. In one or more embodiments, the scaffold region may comprise DNA and / or RNA nucleotides. The scaffold region may be modified as described herein.

[0150] In one or more embodiments, the arrangement of scaffold regions includes, essentially consists of, or is composed of, 5'-mGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmAmGmUmGGmCmAmCmCmGmAmGmUmCmGmGmUmGmC 3', where mN is 2'-O-methylribose.

[0151] In one or more embodiments, the guide oligonucleotide may include an unstructured RNA motif or a structured RNA motif designed to prevent the degradation of the nucleic acid. The RNA motif may be located at the 3' end of the nucleic acid. The nucleic acid may include any suitable RNA motif, such as an RNA motif containing the sequences listed below.

[0152] In one or more embodiments, the guide oligonucleotide contains an RNA motif at the 5' end of the nucleic acid. In one or more embodiments, the guide oligonucleotide contains RNA motifs at both the 5' and 3' ends. When two or more RNA stabilizing motifs are incorporated into the guide oligonucleotide, they may have the same sequence or they may have different sequences.

[0153] The guide oligonucleotide may include an unstructured RNA motif containing, essentially having, or consisting of the sequence 5'-UUU-3'. In embodiments, the unstructured motif may have the following modifications, i.e., 5'-* mU * mU * including mU-3’, where “mU * ” represents a phosphorothioated 2’-O-methyluracil base, and “mU” represents a 2’-O-methyluracil base.

[0154] The guide oligonucleotide may contain a tevopreQ1 motif. The tevopreQ1 motif can be modified from the prequeosine 1 (prequeosine1)-1 riboswitch aptamer. The guide oligonucleotide may contain, consist essentially of, or consist of a tevopreQ1 motif having the sequence 5’-CGCGGUUCUAUCUAGUUACGCGUUAAACCAACUAGAA-3’.

[0155] The guide oligonucleotide may contain an mpknot motif. The mpknot motif can be modified from the frameshift pseudoknot from Moloney murine leukemia virus. The guide oligonucleotide may contain, consist essentially of, or consist of an mpknot motif having the sequence 5’-GGGUCAGGAGCCCCCCCCCUGAACCCAGGAUAACCCUCAAAGUCGGGGGGCAACCC-3’.

[0156] In some embodiments, the gRNA consists of or consists essentially of the following sequence: 5’-mN * mN * mN * NNNNNNNNNNNNNNNNNmGUUUUAGmAmGmCmUmAGmAmAmAmUmAmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUGmAmAmAmAmAmGmUmGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU * mU * mU * mU-3’, where N refers to any nucleotide, and m in mN is 2’-O-methyl ribose, *This refers to a phosphorothioate bond. The first 20 nucleotides may correspond to a spacer sequence. The spacer sequence may be identical or substantially identical to the protospacers listed in Table 2 or Table 5.

[0157] The guide nucleic acid in a dual nickase editing system may have any preferred length. The length may depend on the CRISPR Cas components of the gene editing system (editor) and the components used. For example, different Cas proteins from different bacterial species have a variety of optimal spacer sequence lengths. Therefore, the spacer sequence may include nucleotide lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50. In one or more embodiments, the spacer sequence includes a length of 15 to 24 nucleotides. In embodiments, the spacer sequence includes a length of 18 to 24 nucleotides. In one or more embodiments, the spacer sequence comprises 19 to 21 nucleotides in length. In one or more embodiments, the spacer sequence comprises 20 nucleotides in length + / -1 nucleotide, + / -2 nucleotides, or + / -3 nucleotides.

[0158] In one or more embodiments, each guide nucleic acid includes a spacer sequence, otherwise it corresponds to a conventional 100-nucleotide Streptococcus pyogenes CRISPR gRNA sequence.

[0159] Each guide nucleic acid (e.g., guide RNA) may be approximately 15 to 300 nucleotides long and may contain a sequence of at least 10 consecutive nucleotides that are substantially identical to the protospacer sequence at the target DNA site. In one or more embodiments, the 3' end of the protospacer sequence is directly adjacent to a canonical PAM sequence (e.g., NGG). In one or more embodiments, the 3' end of the protospacer sequence is not directly adjacent to a canonical PAM sequence. In one or more embodiments, the 3' end of the protospacer sequence is directly adjacent to a NAG, NGA, NGC, NGT, or other non-NGG sequence.

[0160] In one or more embodiments, the guide nucleic acid comprises DNA nucleotides. In one or more embodiments, the guide nucleic acid comprises DNA and RNA. In one or more embodiments, the guide nucleic acid is RNA, also referred herein as guide RNA or gRNA. In one or more embodiments, the guide nucleic acid is a modified nucleic acid. As used herein, a modified nucleic acid is a nucleic acid comprising at least one modified nucleic acid base, sugar, or backbone (e.g., binding) moiety.

[0161] In one or more embodiments, the guide nucleic acid may be synthesized. The guide nucleic acid may include, for example, a spacer sequence configured to hybridize to a complementary sequence on the target chain under intracellular conditions. In embodiments, the guide nucleic acid includes a spacer sequence homologous to a protospacer sequence in the LPA gene. The guide nucleic acid may include a spacer sequence identical or substantially identical to a protospacer sequence. In one or more embodiments, the guide nucleic acid includes a spacer having a sequence identical or substantially identical to a protospacer described in Table 2 or Table 5.

[0162] In one or more embodiments, the guide oligonucleotide may have a sequence as shown in Table 3, which may be modified by zero, one, or two or more nucleotides. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0163] Some of the guide nucleic acids described herein have been experimentally tested and are known to produce the desired edits, but several other guide nucleic acid sequences may be suitable for the desired edits. The experimentally tested guide oligonucleotides contained modified nucleotides. Some examples of modified guide oligonucleotide sequences are provided in Table 4, some of which have been experimentally tested as described in the Examples section below. It will be understood that guide oligonucleotides modified in other ways may also be suitable for producing the desired edits. For example, the guide oligonucleotides listed in Table 3 may be modified in a similar manner to those listed in Table 4, or in any other preferred manner.

[0164] In some embodiments, the guide oligonucleotide comprises one of the more modified nucleotides. The nucleotide may be modified in any preferred manner. In some embodiments, the nucleotide comprises a modified sugar moiety. The sugar moiety may be modified in any preferred manner. In embodiments, the modified sugar moiety comprises 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-aminoethyl, 2'-fluoro, N3'→P5' phosphoramide, 2'-dimethylaminooxyethoxy, 2'2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O-guanidinium ethyl, carbamate-modified sugar, or bicyclic-modified sugar. In some embodiments, the modified nucleotide comprises 2'-O-methyl modification. In some embodiments, the nucleotide comprises skeletal modification. In some embodiments, the skeletal modification includes phosphorothioate bonds, phosphorodithioate bonds, phosphoroselenoate bonds, phosphorodiselenoate bonds, phosphoroanilothioate bonds, phosphoraniladet bonds, phosphoramidate bonds, or phosphorodiamidate bonds. In some embodiments, the skeletal modification includes phosphorothioate bonds.

[0165] In some embodiments, the spacer sequence of the guide oligonucleotide contains 1 to 20 modified nucleotides. In some embodiments, the spacer sequence contains 1 to 10 modified nucleotides. In some embodiments, the spacer sequence contains 1 to 5 modified nucleotides. In some embodiments, the spacer sequence contains 1 to 3 modified nucleotides. In some embodiments, one or more of the five nucleotides closest to the 5' end of the spacer sequence are modified. In some embodiments, one or more of the five nucleotides closest to the 5' end of the spacer sequence are modified to include a 2'-O-methyl group, a phosphorothioate bond, or a combination thereof. In some embodiments, one or more of the three nucleotides closest to the 5' end of the spacer sequence are modified. In some embodiments, one or more of the three nucleotides closest to the 5' end of the spacer sequence are modified to include a 2'-O-methyl group, a phosphorothioate bond, or a combination thereof. In some embodiments, the three nucleotides closest to the 5' end of the spacer sequence are modified. In some embodiments, the three nucleotides on the 5' end of the spacer sequence are modified to include a 2'-O-methyl group, a phosphorothioate bond, or a combination thereof.

[0166] In some embodiments, the scaffold sequence of the guide oligonucleotide includes 1 to 76 modified nucleotides, for example, 1 to 70 modified nucleotides, 1 to 60 modified nucleotides, or 1 to 55 modified nucleotides. In some embodiments, the scaffold sequence of the guide oligonucleotide includes 10 to 76 modified nucleotides, for example, 20 to 76 modified nucleotides, for example, 30 to 76 modified nucleotides, 40 to 76 modified nucleotides, or 50 to 76 modified nucleotides. In some embodiments, the scaffold sequence of the guide oligonucleotide includes 20 to 70 modified nucleotides, for example, 30 to 65 modified nucleotides, 40 to 60 modified nucleotides, or 50 to 55 modified nucleotides.

[0167] In some embodiments, 20% to 95% of the nucleotides in the scaffold sequence are modified. In some embodiments, 30% to 90% of the nucleotides in the scaffold sequence are modified, for example, 40% to 85%, 45% to 80%, 50% to 75%, 60% to 75%, or 65% to 75%.

[0168] In some embodiments, the modified scaffold nucleotides include 2'-sugar modifications. In some embodiments, the modified scaffold nucleotides include a 2'-O-methyl group. In some embodiments, 20% to 95% of the nucleotides in the scaffold sequence include 2'-sugar modifications. In some embodiments, 30% to 90% of the nucleotides in the scaffold sequence, for example 40% to 85%, 45% to 80%, 50% to 75%, 60% to 75%, or 65% to 75%, include 2'-sugar modifications. In some embodiments, 20% to 95% of the nucleotides in the scaffold sequence include a 2'-O-methyl group. In some embodiments, 30% to 90% of the nucleotides in the scaffold sequence, for example 40% to 85%, 45% to 80%, 50% to 75%, 60% to 75%, or 65% to 75%, include a 2'-O-methyl group.

[0169] Table 5 summarizes the percentage of LPA alleles edited in primary human hepatocytes (also known as editing efficiency or editing %) for selected guide RNA pairs (first gRNA and second gRNA) at high and low doses, and summarizes the LPA editing efficiency in primary human hepatocytes using a gene editing system that includes Cas9 nikkase along with the first and second guide RNA pairs identified.

[0170] Table 6 shows the number of nucleotides between nicks (overhang length) and the target exons for some of the gRNA pairs shown in Table 5. All nicking in Table 5 was in the PAM-out configuration.

[0171] In some embodiments, the distance between the first nick and the second nick is approximately 1 to 200 nucleotides, for example, 10 to 150 nucleotides, 15 to 100 nucleotides, or 20 to 50 nucleotides. In some embodiments, the distance between the first nick and the second nick is 10 nucleotides or more, for example, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, or 50 nucleotides or more. In some embodiments, the distance between nicks is 200 nucleotides or less, for example 190 nucleotides, 180 nucleotides, 170 nucleotides, 160 nucleotides, 150 nucleotides, 140 nucleotides, 130 nucleotides, 120 nucleotides, 110 nucleotides, 100 nucleotides, 90 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, or 50 nucleotides or less. In some embodiments, the distance between nicks is 20 to 50 nucleotides, for example 23 to 45 nucleotides, 30 to 40 nucleotides, 31 to 40 nucleotides, 32 to 40 nucleotides, 33 to 40 nucleotides, 31 to 39 nucleotides, 32 to 39 nucleotides, 33 to 39 nucleotides, or 34 to 38 nucleotides.

[0172] Table 4 provides the complete guide gRNA sequences tested to obtain the results shown in Table 5. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11] [Table 5-12] [Table 5-13] [Table 5-14] [Table 5-15] m=2'O-methyl analog; * =3' phosphorothioate; the first 20 nucleotides of each sequence correspond to the spacer sequence; no guide ID indicates that a guide ID has not yet been assigned. [Table 6-1] [Table 6-2] [Table 6-3] The tested guide oligonucleotides are identical to the protospacer sequences shown in the table above, but include an RNA spacer sequence in which "T" is replaced with "U". The edited results are shown for gRNA pairs evaluated by MessengerMax transfection. * The edited results of in vitro LNP delivery tests for guide pairs with spacer arrangements corresponding to GA1264 / GA1184 (GA1297 / GA1296) and GA1266 / GA1184 (GA1298 / GA1296) are shown in Figure 4B and were similar to those for guide pairs with spacer arrangements corresponding to GA1183 / GA1184 (GA1295 / GA1296) (see also Tables 4 and 7B). ** The spacers of guide GA1265 overlap with the spacers of GA1264 (and GA1297), GA1266 (and GA1298), and GA1184 (and GA1295) (see Figure 4A for the alignment of the spacer / protospacer arrays of GA1297, GA1298, and GA1295), and the protospacer of GA1265 is located between the protospacers of GA1264 (and GA1297) and GA1266 (and GA1298) (and is offset by one space from them). [Table 7-1] [Table 7-2]

[0173] In embodiments, the gene editing system described herein includes a first guide oligonucleotide and a second guide oligonucleotide, wherein the first guide oligonucleotide includes a spacer sequence identical or substantially identical to the guide 1 protospacer sequence identified in Table 5, and the second guide oligonucleotide includes a spacer sequence identical or substantially identical to the corresponding guide 2 protospacer sequence (in the same row) identified in Table 5. In the embodiment, the first guide oligonucleotide comprises a spacer sequence identical or substantially identical to the guide 1 protospacer sequence identified in Table 5, and the second guide oligonucleotide comprises a spacer sequence identical or substantially identical to the corresponding guide 2 protospacer sequence identified in Table 5, wherein the tested guide pairs in Table 5 exhibit editing of 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more at a concentration of 2500 ng / mL, or the gRNA pairs correspond to GA1264 / GA1184, GA1265 / GA1184, or GA1266 / GA1184. In the embodiment, the gene editing system includes a first guide oligonucleotide and a second guide oligonucleotide, wherein the first guide oligonucleotide includes a spacer sequence identical or substantially identical to the guide 1 protospacer sequence identified in Table 5, and the second guide oligonucleotide includes a spacer sequence identical or substantially identical to the corresponding guide 2 protospacer sequence identified in Table 5, and the tested guide pairs in Table 5 show editing of 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more at a concentration of 2500 ng / mL, or the gRNA pairs correspond to GA1264 / GA1184, GA1265 / GA1184, or GA1266 / GA1184.In the embodiment, the first guide oligonucleotide comprises a spacer sequence identical or substantially identical to the guide 1 protospacer sequence identified in Table 5, and the second guide oligonucleotide comprises a spacer sequence identical or substantially identical to the corresponding guide 2 protospacer sequence identified in Table 5, wherein the tested guide pair exhibits 70% or more, 80% or more, 85% or more, or 90% or more editing at a concentration of 2500 ng / mL, or the guide pair corresponds to GA1264 / GA1184, GA1265 / GA1184, or GA1266 / GA1184.

[0174] In some embodiments, the first guide oligonucleotide includes a spacer sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CUGUCACCAGGCAUUGUGUC-3', and the second guide oligonucleotide includes a spacer sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-UGUCCUUGCAACUCUCACGG-3'. For clarity, an oligonucleotide containing nucleotides 6-20 of 5'-CUGUCACCAGGCAUUGUGUC-3' would contain 5'-ACCAGGCAUUGUGUC-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CUGUCACCAGGCAUUGUGUC-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-UGUCCUUGCAACUCUCACGG-3'.

[0175] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-GUAGUAGCAGUCCUGUACCC-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CAUUAUGGACAGAGUUACCG-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to 5'-GUAGUAGCAGUCCUGUACCC-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to 5'-CAUUAUGGACAGAGUUACCG-3'.

[0176] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-AGGACACUCGAUUCUGUCAC-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CACAACUCCCACAGUGGCCC-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-AGGACACUCGAUUCUGUCA-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CACAACUCCCACAGUGGCCC-3'.

[0177] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CUGUCACUGGACAUUGUGUC-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-AAGUGUCCUUGCGACGUCCA-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CUGUCACUGGACAUUGUGUC-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-AAGUGUCCUUGCGACGUCCA-3'.

[0178] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-GGAGCAAAGCCCCACAGUCC-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-GUUGGUGCUGAAAUUCAAAG-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-GGAGCAAAGCCCCACAGUCC-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-GUUGGUGCUGAAAUUCAAAG-3'.

[0179] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-GGAGCAAAGCCCCGGGGUCC-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-GUUGGUGCUGAAAUUCAAAG-3'. In some embodiments, the first guide oligonucleotide includes a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-GGAGCAAAGCCCCGGGGUCC-3', and the second guide oligonucleotide includes a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-GUUGGUGCUGAAAUUCAAAG-3'.

[0180] In some embodiments, the first guide oligonucleotide includes a spacer sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CUGGAACUGGGACCACCGU-3', and the second guide oligonucleotide includes a spacer sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-ACAGAGCUUCCUUCUGAAGA-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CUGGAACUGGGACCACCGU-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-ACAGAGCUUCCUUCUGAAGA-3'.

[0181] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-AUGCCAGUGUGGUGUCAUAG-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-ACCACAGAAUACUACCCAAA-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-AUGCCAGUGUGGUGUCAUAG-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-ACCACAGAAUACUACCCAAA-3'.

[0182] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-GGAGCCAGAAUAACAUUCGG-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CUAGAGGCUUUUUUUGAACA-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-GGAGCCAGAAUAACAUUCGG-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CUAGAGGCUUUUUUUGAACA-3'.

[0183] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CAGAUGCUGAGAUUAGUCCU-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-UGGAUUCCUGCAGUAGUUCC-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CAGAUGCUGAGAUUAGUCCU-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-UGGAUUCCUGCAGUAGUUCC-3'.

[0184] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-UGACACCACAUUGGCAUCGG-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-ACAUGUUCUUCCUGUGAUAG-3'. In some embodiments, the first guide oligonucleotide includes a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-UGACACCACAUUGGCAUCGG-3', and the second guide oligonucleotide includes a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-ACAUGUUCUUCCUGUGAUAG-3'.

[0185] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CAUAGAUGACCAAGAUUGAC-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-UGAUACCACACUGGCAUCAG-3'. In some embodiments, the first guide oligonucleotide includes a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CAUAGAUGACCAAGAUUGAC-3', and the second guide oligonucleotide includes a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-UGAUACCACACUGGCAUCAG-3'.

[0186] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CCAUCACUGGACAUUGCGUC-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-AACUCUCCUCACAACUCCCA-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CCAUCACUGGACAUUGCGUC-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-AACUCUCCUCACAACUCCCA-3'.

[0187] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CUGCAUCUGAGCAUCGUGUC-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CGUCCCUCCGAAUGUUAUUC-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CUGCAUCUGAGCAUCGUGUC-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CGUCCCUCCGAAUGUUAUUC-3'.

[0188] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-AAACAGCCGUGGACGUCGCA-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-UGAACAAGGUAAGAAGUCUC-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-AAACAGCCGUGGACGUCGCA-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-UGAACAAGGUAAGAAGUCUC-3'.

[0189] In some embodiments, the first guide oligonucleotide includes a spacer sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-ACAGAGGCUCCUUCUGAACA-3', and the second guide oligonucleotide includes a spacer sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-GCUUGGAACCGGGGCCACUG-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-ACAGAGGCUCCUUCUGAACA-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-GCUUGGAACCGGGGCCACUG-3'.

[0190] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-AUGCCAGUGUGGUGUCAUAG-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-ACAACAGAAUAUUAUCCAAA-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-AUGCCAGUGUGGUGUCAUAG-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-ACAACAGAAUAUUAUCCAAA-3'.

[0191] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CUAUGACACCACAUUGGCAU-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-ACAUGUUCUUCCUGUGAUAG-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CUAUGACACCACAUUGGCAU-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-ACAUGUUCUUCCUGUGAUAG-3'.

[0192] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-AAUAACAUUCGGAGGGACGA-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-UAUUCUGGCUCCAAGCCUAG-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-AAUAACAUUCGGAGGGACGA-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-UAUUCUGGCUCCAAGCCUAG-3'.

[0193] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-GUAGCAGUCCUGUACCCCGG-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CAUUAUGGACAGAGUUACCG-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-GUAGCAGUCCUGUACCCCGG-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CAUUAUGGACAGAGUUACCG-3'.

[0194] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-AGUAGCAGUCCUGUACCCCG-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CAUUAUGGACAGAGUUACCG-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-AGUAGCAGUCCUGUACCCCG-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CAUUAUGGACAGAGUUACCG-3'.

[0195] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-UAGUAGCAGUCCUGUACCCC-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-CAUUAUGGACAGAGUUACCG-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-UAGUAGCAGUCCUGUACCCC-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CAUUAUGGACAGAGUUACCG-3'.

[0196] In some embodiments, the first guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-UGGACCACAUGGCUUUGCUC-3', and the second guide oligonucleotide includes a spacer sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence, 5'-ACGUACUCCACCACUGUCAC-3'. In some embodiments, the first guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-UGGACCACAUGGCUUUGCUC-3', and the second guide oligonucleotide comprises a spacer having a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-ACGUACUCCACCACUGUCAC-3'.

[0197] Table 7A summarizes the dose-response curves in immortalized human hepatocellular carcinoma cells (HuH-7) for five pairs of primary and secondary guide RNAs using a gene editing system including a dual nickase Cas9 system. The Cas9 nickase is encoded in mRNA (MS029) transfected into HuH-7 cells at a total mRNA:total gRNA weight ratio of 1:1. Table 7B summarizes the dose-response curves in HuH-7 cells tested in separate experiments under similar conditions to those used to obtain the results shown in Table 7A. [Table 8] [Table 9]

[0198] In one or more embodiments, the guide oligonucleotide or a portion thereof is chemically modified. Chemical modification of the guide oligonucleotide may provide improved stability when transfected into mammalian cells. For example, gRNA may be chemically modified to include a combination of 2'-O-methylribosugar modification and phosphorothioate backbone modification on at least one 5' nucleotide and at least one 3' nucleotide of each gRNA. In one or more embodiments, three terminal 5' nucleotides and three terminal 3' nucleotides are chemically modified to include a combination of 2'-O-methylribosugar modification and phosphorothioate modification.

[0199] This disclosure also intends to include guide nucleic acids comprising the component portions of the guide nucleic acids identified above (e.g., crRNA, tracrRNA, or scaffold regions that may include tracrRNA, and connector regions between them).

[0200] The guide oligonucleotides described herein can be synthesized chemically, enzymatically, or in combination thereof. For example, guide oligonucleotides can be synthesized using conventional phosphoramidite-based solid-phase synthesis methods. Alternatively, gRNA can be synthesized in vitro by operably ligating the DNA encoding the gRNA to a promoter control sequence recognized, for example, by phage RNA polymerase. Examples of suitable phage promoter sequences include, but are not limited to, T7 promoter sequences, T3 promoter sequences, SP6 promoter sequences, or variations thereof. In one or more embodiments, the guide oligonucleotide comprises two distinct molecules (e.g., crRNA (including a spacer) and tracrRNA or a scaffold region that may contain tracrRNA). One molecular guide oligonucleotide portion (e.g., tracrRNA or a scaffold region that may contain tracrRNA) can be synthesized chemically, and the other molecule (e.g., crRNA) can be synthesized enzymatically. Portions of the guide oligonucleotide can be ligated together by sprint ligation or other preferred methods to form a larger, integrated guide oligonucleotide. The full-length purity of synthesized and / or ligated guide oligonucleotides can be increased for use as suitable active pharmaceutical ingredients in pharmaceutical compounds by using an ion-pairing anion exchange chromatography and / or reverse-phase chromatography process, performed alone or sequentially with or without filtration, and a subsequent purification process including lyophilization and / or dispensing.

[0201] In some embodiments, the guide oligonucleotide may include DNA nucleotides and / or nucleotide analogs in addition to RNA nucleotides. The gRNA may include molecules other than RNA.

[0202] In the embodiment, three or more guide nucleic acids may be used simultaneously to introduce edits to one or more target genomic locations. Using three or more guide nucleic acids with different sequences may improve the editing efficacy.

[0203] The gene editing and delivery systems described herein may include a guide oligonucleotide having a spacer region operated to bind to a target sequence on a strand of the human LPA gene opposite a protospacer in the LPA gene. In embodiments, the gene editing system is configured such that a nickase cooperating with the guide oligonucleotide nicks one strand of the LPA gene within or adjacent to the target sequence or protospacer. In some embodiments, the gene editing system is operated so that the nickase nicks within 5 nucleotides, 4 nucleotides, 3 nucleotides, 2 nucleotides, 1 nucleotide, or between the 3' end of the protospacer and the PAM, or at a corresponding position in the target sequence. In embodiments, the gene editing system is operated so that the gene editing system nicks within 2 or 3 nucleotides at the 3' end of the protospacer or at a corresponding position in the target sequence. In embodiments, the gene editing system is operated so that the gene editing system nicks (i) within 2 or 3 nucleotides within the protospacer and the 3' end of the protospacer, or (ii) at a corresponding position in the target sequence.

[0204] In some embodiments, the target sequence to which the spacer region of the guide oligonucleotide binds corresponds to (i.e., is complementary to) the protospacer sequences listed in Table 2 or Table 5. In some embodiments, the spacer sequence is identical to, or has at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity with, the protospacers listed in Table 2 or Table 5. In some embodiments, the guide oligonucleotide includes a spacer sequence that is identical or substantially identical to, the protospacers listed in Table 2 or Table 5 or their 3' portion, having 0, 1, 2, 3, 4, or 5 mismatches. In some embodiments, the spacer region of the guide oligonucleotide includes a sequence identical to the 15, 16, 17, 18, 19, or 20 nucleotides on the outermost 3' side of the protospacers listed in Table 2 or Table 5. For example, the spacer may contain the same sequence as the 15, 16, 17, 18, 19, or 20 nucleotides (5'-GGAGCCAGAATAACATTCGG-3') on the 3' end of the protospacers in the first row and first column of Table 2, meaning that the spacer may contain the sequences 5'-GGAGCCAGAAUAACAUUCGG-3', 5'-GAGCCAGAAUAACAUUCGG-3', 5'-AGCCAGAAUAACAUUCGG-3', 5'-GCCAGAAUAACAUUCGG-3, 5'-CCAGAAUAACAUUCGG-3', or 5'-CAGAAUAACAUUCGG-3'. In some embodiments, the guide oligonucleotide includes a spacer having a sequence identical to the protospacers listed in Table 2 or Table 5.

[0205] The gene editing and delivery systems described herein may include a first guide oligonucleotide and a second guide oligonucleotide. In embodiments, the first guide oligonucleotide binds to a first target sequence on the first strand of the human LPA gene, and the second guide oligonucleotide binds to a second target sequence on the second strand of the LPA gene. The first target sequence is opposite and complementary to a first protospacer in the LPA gene. The second target sequence is opposite and complementary to a second protospacer in the LPA gene. In embodiments, the gene editing system is operated such that one or more nickases cooperate with the first guide oligonucleotide to nick one strand of the LPA gene in or near the first target sequence or first protospacer, and cooperate with the second guide oligonucleotide to nick the other strand of the LPA gene in or near the second target sequence or second protospacer. In one embodiment, the gene editing system is operated such that one or more nicks cooperate with a first guide oligonucleotide to insert a nick into the LPA gene strand at a position of up to 5 nucleotides, up to 4 nucleotides, up to 3 nucleotides, up to 2 nucleotides, up to 1 nucleotide, or between the 3' end of the first protospacer and PAM, or at a corresponding position in the first target sequence, and cooperate with a second guide oligonucleotide to insert a nick into the other strand of the LPA gene at a position of up to 5 nucleotides, up to 4 nucleotides, up to 3 nucleotides, up to 2 nucleotides, up to 1 nucleotide, or between the 3' end of the second protospacer and PAM, or at a corresponding position in the second target sequence. In another embodiment, the gene editing system is operated such that the gene editing system inserts a nick into the LPA gene strand at a position of up to 2 or 3 nucleotides at the 3' end of the first protospacer or at a corresponding position in the first target sequence, and inserts a nick into the other strand of the LPA gene at a position of up to 2 or 3 nucleotides at the 3' end of the second protospacer or at a corresponding position in the second target sequence.In this embodiment, the gene editing system is operated to insert a nick into the LPA gene strand (i) within a protospacer and within 2 or 3 nucleotides of the 3' end of the first protospacer, or (ii) at a corresponding position in the first target sequence, and into the other strand of the LPA gene (i) within a second protospacer and within 2 or 3 nucleotides of the 3' end of the second protospacer, or (ii) at a corresponding position in the second target sequence.

[0206] In some embodiments, the target sequence to which the spacer region of the first guide oligonucleotide binds corresponds to the guide 1 protospacer sequence listed in Table 2 or Table 5, and the target sequence to which the spacer region of the second guide oligonucleotide binds corresponds to the guide 2 protospacer sequence listed in Table 2 or Table 5, where the guide 1 protospacer and the guide 2 protospacer are in the same row in Table 2 or Table 5. In some embodiments, the spacer sequence of the first guide oligonucleotide is identical to, or has at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity with, the spacer sequence of the second guide oligonucleotide is identical to, or has at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity with, the guide 1 protospacer and the guide 2 protospacer are in the same row in Table 2 or Table 5. In some embodiments, the first guide oligonucleotide includes a spacer sequence that is identical or substantially identical to the guide 1 protospacer sequence listed in Table 2 or Table 5, having 0, 1, 2, 3, 4, or 5 mismatches, or a 3' portion thereof, and the second guide oligonucleotide includes a spacer sequence that is identical or substantially identical to the guide 2 protospacer sequence listed in Table 2 or Table 5, having 0, 1, 2, 3, 4, or 5 mismatches, or a 3' portion thereof, where the guide 1 protospacer and the guide 2 protospacer are in the same row in Table 2 or Table 5.In some embodiments, the spacer region of the first guide oligonucleotide contains the same sequence as the 15, 16, 17, 18, 19, or 20 nucleotides on the outermost 3' of the guide 1 protospacer listed in Table 2 or Table 5, and the spacer region of the second guide oligonucleotide contains the same sequence as the 15, 16, 17, 18, 19, or 20 nucleotides on the outermost 3' of the guide 2 protospacer listed in Table 2 or Table 5, where the guide 1 protospacer and the guide 2 protospacer are in the same row in Table 2 or Table 5. In some embodiments, the first guide oligonucleotide contains a spacer having the same sequence as the guide 1 protospacer listed in Table 2 or Table 5, and the second guide oligonucleotide contains a spacer having the same sequence as the guide 2 protospacer listed in Table 2 or Table 5, where the guide 1 protospacer and the guide 2 protospacer are in the same row in Table 2 or Table 5.

[0207] V. Gene editing (EDITOR) systems The term “gene-editing substance (editor)” is used throughout this disclosure to refer to a protein or protein complex that, in the presence of or in conjunction with guide nucleic acids, can insert, substitute, delete, or nick a DNA sequence in a genome. Guide nucleic acids and gene-editing substances (editors) are collectively referred herein to as a “gene-editing substance (editor) system” or “gene-editing system.” In some gene-editing systems, intracellular enzymes may facilitate the incorporation of edits into the genome, such as DNA repair enzymes, or otherwise, intracellular enzymes may be required to complete them. In some embodiments, a gene-editing substance (editor) that nicks a DNA strand may, during operation, interact with two different guide oligonucleotides to nick opposing strands of genomic DNA. Nicks on opposing strands may facilitate one or more DNA repair mechanisms to trigger edits (e.g., trigger indel variants or non-synonymous variants).

[0208] Gene editing systems are nucleotide-directed. These systems generally include at least an editing protein and a guide nucleic acid. The proteins and systems described above as Cas9, nucleotide-directed editing proteins, and nickases are encompassed within the term “gene editing system.” The guide nucleic acids mentioned above may also be components of a “gene editing system.”

[0209] The editing proteins described herein are nickases, which work in conjunction with guide oligonucleotides to nick a single strand of genomic DNA. Given a nickase for nicking opposing strands of genomic DNA, the nickase works with a first guide oligonucleotide to nick one strand of genomic DNA and with a second guide oligonucleotide to nick the other strand. The first and second guide oligonucleotides contain spacer sequences complementary to the target sequences on the opposing strands of genomic DNA. Such nickases are in contrast to nucleases (e.g., Cas9 nuclease) that interact with a single guide RNA to catalyze double-strand breaks in DNA.

[0210] The edited proteins described herein may include, but are not limited to, Cas nickases such as Streptococcus pyogenes Cas9 variants, Staphylococcus aureus Cas9 variants, or Cas12a / Cpf1 variants. The edited proteins may be provided as recombinant proteins. Alternatively, the edited proteins may be transcribed and / or translated from provided nucleotides such as mRNA or plasmid DNA.

[0211] The guide nucleic acid may include, but is not limited to, spacer sequences and scaffold regions. The guide nucleic acid components may be covalently bonded to each other, assembled into a complex, or provided as individual strands. The nucleotide components may, alternatively, be transcribed from a provided nucleic acid such as plasmid DNA.

[0212] In one or more embodiments, nucleotide components and gene-editing substances (editors) may be transcribed from a single nucleic acid, such as plasmid DNA or linear DNA. Protein components may then be translated from the transcript. In one or more embodiments, the nucleic acid encoding the gene-editing substance (editor) is mRNA. In one or more embodiments, the mRNA, when translated in target cells or subjects after administration, generates the gene-editing substance (editor). In one or more embodiments, the gene-editing substance (editor) forms a ribonucleoprotein (RNP) complex in the target cells or subjects.

[0213] It should be recognized that the editing of this disclosure may include one or more additional features. For example, in one or more embodiments, the gene editing material (editor) may include cytoplasmic localization sequences, exotoperation sequences such as nuclear export sequences, or other localization sequences, and sequence tags useful for solubilization, purification, or detection of fusion proteins. Suitable protein tags provided herein, but not limited to, include biotin carboxylase carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags, hemagglutinin (HA) tags, polyhistidine tags also called histidine tags or His tags, maltose-binding protein (MBP) tags, nus tags, glutathione-S-transferase (GST) tags, green fluorescent protein (GFP) tags, thioredoxin tags, S tags, Softag (e.g., Softag1, Softag3), strep tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP tags. Further suitable sequences will be apparent to those skilled in the art. In one or more embodiments, the gene editing substance (editor) includes one or more His tags.

[0214] VI. Therapeutic applications The guide nucleic acids and compositions described herein may be administered in therapeutically effective doses to target cells or subjects in need of them for the treatment or prevention of cardiovascular disease. In one or more embodiments, the subject has a cardiovascular disease at least partially attributable to a high blood Lp(a) concentration that may directly correlate with apo(a) concentration or inversely correlated with the size of the apo(a) protein. In one or more embodiments, the subject has atherosclerotic cardiovascular disease at least partially attributable to a high blood Lp(a) concentration that may directly correlate with apo(a) concentration or inversely correlated with the size of the apo(a) protein. In one or more embodiments, the subject has calcified aortic valve disease at least partially attributable to a high blood Lp(a) concentration that may directly correlate with apo(a) concentration or inversely correlated with the size of the apo(a) protein.

[0215] Through such administration, the guide nucleic acid instructs a gene-editing substance (editor) to modify the LPA gene, thereby reducing blood Lp(a) concentration in the subject that may directly correlate with apo(a) concentration or inversely correlate with the size of the apo(a) protein. In one or more embodiments, the gene modification occurs in the liver cells (hepatocytes) of the subject.

[0216] For example, a gene editing system comprises an editing protein and a guide nucleic acid, which may be introduced and / or expressed in cells where editing of a target gene is desired, such as liver cells (or hepatocytes), thereby enabling contact between the target gene and the guide nucleic acid, such as gRNA, and the gene editing protein. In one or more embodiments, the binding of the editing protein to a target polynucleotide sequence in the target gene is directed by the guide nucleic acid, where the spacer sequence of each guide nucleic acid hybridizes with a complementary sequence in the target chain of the target gene. Thus, the guide nucleic acid guides the editing protein to edit the polynucleotide sequence in the target gene. In one or more embodiments, the guide nucleic acid is co-introduced into the cell where editing is desired, together with the editing protein or together with the nucleic acid encoding the editing protein.

[0217] In one or more embodiments, the methods and compositions disclosed herein impair the function of the apo(a) protein encoded by the LPA gene for constituting Lp(a) particles. The impairment of function can be measured by the blood Lp(a) concentration in a subject to whom the methods or compositions disclosed herein are administered. For example, the methods and compositions disclosed herein can reduce blood Lp(a) concentration by at least 10 to 95 percent compared to a control. For example, the methods and compositions disclosed herein can reduce blood Lp(a) concentration by at least 10 percent, at least 15 percent, at least 20 percent, at least 25 percent, at least 30 percent, at least 35 percent, at least 40 percent, at least 45 percent, at least 50 percent, at least 55 percent, at least 60 percent, at least 65 percent, at least 70 percent, at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent compared to a control.

[0218] In one or more embodiments, the methods described herein for treating or preventing cardiovascular disease in a target subject include (i) administering a guide nucleic acid and (ii) a nucleic acid encoding an edited protein to the target subject.

[0219] In one or more embodiments, a method for treating or preventing cardiovascular disease in a target subject as described herein comprises administering lipid nanoparticles (LNPs) that encapsulate or otherwise deliver (i) a guide nucleic acid or nucleic acid encoding a guide nucleic acid, and / or (ii) an edited protein containing a programmable DNA-binding domain or nucleic acid encoding such an edited protein, such guide nucleic acids and edited proteins or nucleic acids encoding such an edited protein are described herein. In one or more embodiments, (i) the guide nucleic acid or nucleic acid encoding such an edited protein, and (ii) the edited protein containing a programmable DNA-binding domain or nucleic acid encoding such an edited protein are encapsulated in the same LNP. In one or more embodiments, they are encapsulated in separate LNPs.

[0220] VII. Pharmaceutical Compositions In one or more embodiments, pharmaceutical compositions comprising a guide oligonucleotide or gene editing material (editor) system provided herein and a pharmaceutically acceptable carrier or excipient are provided herein. In one or more embodiments, pharmaceutical compositions for genetic modification comprising one or more guide nucleic acids, such as gRNAs, described herein, an edited protein or nucleic acid sequence encoding an edited protein, and a pharmaceutically acceptable carrier are provided herein. The pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inert components that facilitate the processing of the active compound into a pharmaceutically usable preparation. Suitable pharmaceutically acceptable salts are generally known in the art. The appropriate formulation depends on the selected route of administration. An overview of the pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0221] The pharmaceutical composition may contain guide nucleic acids, such as gRNAs, or nucleic acid sequences encoding guide RNAs, as described herein, in any preferred molar ratio to each other. In embodiments, the weight ratio of one guide nucleic acid (or nucleic acid sequence encoding one guide RNA) to the other guide nucleic acid (or nucleic acid sequence encoding the other guide RNA) is 10:1 to 1:10, for example, 5:1 to 1:5, 3:1 to 1:3, or 2:1 to 1:2. In some embodiments, the weight ratio is 1:1 to 1:3. In some embodiments, the weight ratio is approximately 1:2. In some embodiments, the weight ratio is 1:1.5 to 1:2.5. Therefore, for example, the weight ratio of one guide RNA to the other guide RNA may be 1:1 to 1:3 or 3:1.

[0222] The pharmaceutical composition may contain a guide nucleic acid, such as gRNA as described herein, or a nucleic acid sequence encoding guide RNA, in a molar ratio suitable for the edited protein or the nucleic acid sequence encoding the edited protein. In embodiments, the weight ratio of the guide nucleic acid (or nucleic acid sequence encoding the guide nucleic acid) to the edited protein (or nucleic acid encoding the edited protein) is 10:1 to 1:10, for example, 5:1 to 1:5, 3:1 to 1:3, or 2:1 to 1:2. In some embodiments, the weight ratio is 1:1 to 1:3. In some embodiments, the weight ratio is approximately 1:2. In some embodiments, the ratio is 1:1.5 to 1:2.5. Therefore, for example, the weight ratio of the guide RNA to the mRNA encoding the edited protein may be 1:1 to 1:3 or 3:1.

[0223] A pharmaceutical composition may be a mixture of a guide nucleic acid such as gRNA described herein or a nucleic acid sequence encoding a guide RNA, an edited protein or a nucleic acid sequence encoding an edited protein, and one or more other chemical components (i.e., pharmaceutically acceptable components), such as carriers, excipients, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, moistening agents, plasticizers, stabilizers, penetration enhancers, wetting agents, defoaming agents, antioxidants, preservatives, or one or more of these. The pharmaceutical composition facilitates administration to an organism or subject requiring it.

[0224] The pharmaceutical compositions of this disclosure can be administered to a subject by any preferred method known in the art. The pharmaceutical compositions described herein can be administered to a subject by a variety of methods, including parenteral, intravenous, intradermal, intramuscular, intracolonic, intrarectal, or intraperitoneal. In one or more embodiments, the pharmaceutical composition can be administered to the subject by intraperitoneal, intramuscular, subcutaneous, or intravenous injection. In one or more embodiments, the pharmaceutical composition can be administered parenterally, intravenously, intramuscularly, or orally. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable solution containing one or more gRNAs and an LNP encapsulating an mRNA encoding an editor protein(s) manipulated to result in editing of the LPA gene, as described herein, and is administered intravenously to a subject requiring it, wherein the LNP may or may not contain GalNAc (e.g., GalNAc-lipid) as described herein.

[0225] In one or more embodiments, the pharmaceutical composition for genetic modification includes a further therapeutic agent. The additional therapeutic agent modulates different aspects of the disease, disorder, or condition being treated and may provide a greater overall benefit than the administration of the therapeutic agent alone. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, hormonal therapy agents, and / or immunotherapeutic agents. In one or more embodiments, the therapeutic agent may be a radiotherapeutic agent. In one or more embodiments, the therapeutic agent may be a hormonal therapy agent. In one or more embodiments, the therapeutic agent may be an immunotherapeutic agent. In one or more embodiments, the therapeutic agent may be a chemotherapeutic agent. The preparation and administration schedule of the additional therapeutic agent may be used according to the manufacturer's instructions or as determined empirically by a skilled practitioner.

[0226] A. Lipid nanoparticle (LNP) composition The pharmaceutical compositions for genetic modification described herein may be encapsulated in or contain lipid nanoparticles (LNPs). As used herein, “lipid nanoparticle (LNP) composition” or “nanoparticle composition” is a composition comprising one or more described lipids. The LNP compositions or LNP formulations intended herein are typically on the order of micrometers or less in size and may contain a lipid bilayer. The nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, the nanoparticle compositions or nanoparticle formulations intended herein may be liposomes having a lipid bilayer and a diameter of 500 nm or less. The LNPs described herein may have an average diameter of approximately 1 nm to approximately 2500 nm, approximately 10 nm to approximately 1500 nm, approximately 20 nm to approximately 1000 nm, approximately 30 nm to approximately 150 nm, approximately 40 nm to approximately 150 nm, approximately 50 nm to approximately 150 nm, approximately 60 nm to approximately 130 nm, approximately 50 nm to approximately 90 nm, approximately 55 nm to approximately 85 nm, approximately 55 nm to approximately 75 nm, approximately 50 nm to approximately 80 nm, approximately 60 nm to approximately 80 nm, approximately 70 nm to approximately 110 nm, approximately 70 nm to approximately 100 nm, approximately 80 nm to approximately 100 nm, approximately 90 nm to approximately 100 nm, approximately 70 nm to approximately 90 nm, approximately 80 nm to approximately 90 nm, or approximately 70 nm to approximately 80 nm. The LNPs described herein may have an average diameter of approximately 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, or greater.

[0227] In one embodiment, the average diameter of the LNPs is approximately 70 nm ± 20 nm, 70 nm ± 10 nm, and 70 nm ± 5 nm. In another embodiment, the average diameter of the LNPs is approximately 60 nm ± 20 nm, 60 nm ± 10 nm, and 60 nm ± 5 nm. In yet another embodiment, the average diameter of the LNPs is approximately 50 nm ± 20 nm, 50 nm ± 10 nm, and 50 nm ± 5 nm. The LNPs described herein may be substantially nontoxic.

[0228] Lipid nanoparticles (LNPs) utilize a nonviral drug delivery mechanism that allows them to pass through blood vessels and reach hepatocytes (Am.J.Patel.2010,176,14-21). Apolipoprotein E (apoE) protein can bind to LNPs after PEG-lipid diffusion from the LNP surface, which has a nearly neutral charge in the bloodstream, thereby functioning as an endogenous ligand for hepatocytes expressing low-density lipoprotein receptor (LDLR) (Mol.Ther.,2010,18,1357-1364). Necessary conditions for efficient LNP delivery to the liver include 1) effective PEG-lipid detachment from the LNP surface in serum, and 2) apoE binding to LNPs. The endogenous apoE-mediated LDLR-dependent LNP delivery pathway is either unusable or less effective for achieving LNP-based gene delivery to the liver in patient populations with LDLR deficiency.

[0229] Efficient delivery to cells requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins. One way to achieve specific targeting is to conjugate the targeting moiety to an activator or pharmaceutical effector, such as a nucleic acid agent, thereby directing the activator or pharmaceutical effector to specific cells or tissues depending on the specificity of the targeting moiety. One way in which the targeting moiety can improve delivery is through receptor-mediated endocytosis activity. This uptake mechanism involves the movement of a nucleic acid agent bound to a membrane receptor into the membrane-enclosed region via invagination of the membrane structure or fusion of the delivery system with the cell membrane. This process is initiated via activation of the cell surface or membrane receptor after a specific moiety, such as a ligand, binds to the receptor. Examples of receptor-mediated endocytosis systems recognize sugars such as galactose, mannose, and mannose-6-phosphate, as well as peptides and proteins such as transferrin, asialoglycoprotein, vitamin B12, insulin, and epidermal growth factor (EGF). Lipophilic moieties, such as cholesterol or fatty acids, can substantially enhance plasma protein binding and consequently substantially increase circulating half-life when bound to highly hydrophilic molecules such as nucleic acids. Lipophilic conjugates can also be used in combination with targeting moieties to improve intracellular transport in targeted delivery approaches.

[0230] The asialoglycoprotein receptor (ASGPR) is a high-capacity receptor that is abundant on hepatocytes. ASGPR has 50 times the affinity for N-acetyl-D-galactosamine (GalNAc) compared to D-galactose. LNPs containing receptor-targeting conjugates can be used to facilitate targeted delivery of the active pharmaceutical ingredients described herein. LNPs may contain one or more receptor-targeting moieties on the surface or periphery of a particle at a specified or manipulated surface density ranging from relatively low to relatively high surface densities. The receptor-targeting conjugate may include a targeting moiety (such as a ligand), a linker, and a lipophilic moiety linked to the targeting moiety. In one or more embodiments, the receptor-targeting moiety (such as a ligand) targets a lectin receptor. In one or more embodiments, the lectin receptor is the asialoglycoprotein receptor (ASGPR). In one or more embodiments, the receptor-targeting moiety is GalNAc or a GalNAc derivative that targets ASGPR. In one embodiment, the receptor-targeting conjugate comprises one GalNAc moiety or a derivative thereof. In another embodiment, the receptor-targeting conjugate comprises two different GalNAc moieties or derivatives thereof. In yet another embodiment, the receptor-targeting conjugate comprises three different GalNAc moieties or derivatives thereof. In yet another embodiment, the receptor-targeting conjugate is lipophilic. In one or more embodiments, the receptor-targeting conjugate comprises one or more GalNAc moieties and one or more lipid moieties (i.e., GalNAc-lipids). In one or more embodiments, the receptor-targeting conjugate is a GalNAc-lipid.

[0231] This specification describes (i) LNP compositions comprising aminolipids, phospholipids, PEG-lipids, cholesterol or cholesterol derivatives, payloads, or any combination thereof, and (ii) LNP compositions comprising aminolipids, phospholipids, PEG-lipids, cholesterol, GalNAc-lipids or their derivatives, payloads, or any combination thereof. Each component is described in more detail below.

[0232] In the preparation of an LNP composition containing aminolipids, phospholipids, PEG-lipids, and cholesterol as excipients, the four excipients are dissolved in a water-miscible organic solvent, such as ethanol, in a desired molar ratio. The homogeneous lipid solution is then rapidly mixed in-line with an aqueous buffer having an acidic pH in the range of 4 to 6.5 containing a nucleic acid payload to form lipid nanoparticles (LNPs) that encapsulate the nucleic acid payload(s). After rapid in-line mixing, the thus formed LNPs are subjected to further downstream processing, including concentration and buffer exchange, to obtain a final LNP pharmaceutical composition with a nearly neutral pH for administration to cell lines or animal disease models for evaluation, or for administration to human subjects.

[0233] The preparation of the GalNAc-LNP pharmaceutical composition involves mixing GalNAc-lipids with four lipid excipients in a water-miscible organic solvent before preparing the GalNAc-LNP. Subsequently, the preparation of the GalNAc-LNP pharmaceutical composition follows the same procedure as described for the LNP pharmaceutical composition. The molar percentage of GalNAc-lipids in the GalNAc-LNP preparation is in the range of 0.001 to 2.0 of the total excipients.

[0234] For the preparation of both LNP and GalNAc-LNP, the payload includes a guide nucleic acid, such as a guide RNA that targets the LPA gene, and mRNA encoding a gene-editing (editor) protein. In one or more embodiments, the weight ratio of guide nucleic acid to mRNA in the acidic aqueous buffer and the final formulation is 6:1, 5:1, 4:1, 3:1, 2.5:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:5, or 1:6 by weight.

[0235] In one or more embodiments, the LNP composition may be prepared as described in U.S. Patent Application No. 17 / 192,709, filed on March 4, 2021, entitled “COMPOSITIONS AND METHODS FOR TARGETED RNA DELIVERY,” invented by Kallanthottathil G. Rajeev and applied for by Verve Therapeutics, Inc., claiming the benefits of U.S. Provisional Patent Applications No. 62 / 984,866 (filed March 4, 2020) and No. 63 / 078,982 (filed September 16, 2020), which is incorporated herein by reference in its entirety.

[0236] 1. Aminolipids a) Equation (I) In one or more embodiments, the LNP composition comprises an aminolipid. In one embodiment, an aminolipid having the structure of formula (I), or a pharmaceutically acceptable salt or solvate thereof, is disclosed herein. [ka] During the ceremony, R 1 and R 2 Each of these is independent of C3~C 22 Alkyl, C3~C 22 Alkenyl, C3-C8 cycloalkyl, -C2-C 10 Alkylene-LR 6 , or [ka] Alkyl, alkylene, alkenyl, and cycloalkyl are each independently substituted or unsubstituted. X, Y, and Z are each independently -C(=O)NR 4 -, -NR 4 C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -NR 4 C(=O)O-, -OC(=O)NR 4 -, -NR 4 C(=O)NR 4-, -NR 4 C(=NR 4 )NR 4 -, -C(=S)NR 4 -, -NR 4 C(=S)-, -C(=O)O-, -OC(=S)-, OC(=S)O-, -NR 4 C(=S)O-, -OC(=S)NR 4 -, -NR 4 C(=S)NR 4 -, -C(=O)S-, -SC(=O)-, -OC(=O)S-, -NR 4 C(=O)S-. -SC(=O)NR 4 -, -C(=S)S-, -SC(=S)-, -SC(=S)O-, -NR 4 C(=S)S-, -SC(=S)NR 4 -, -C(=S)S-, -SC(=S)-, -SC(=O)S-, -SC(=S)S-, -NR 4 C(=S)S-, -SC(=S)NR 4 -O, S, or bond, Each L is independently -C(=O)NR 4 -, -NR 4 C(=O)-, -C(=O)O-. -OC(=O)O-, -NR 4 C(=O)O-, -OC(=O)NR 4 -, -NR 4 C(=O)NR 4 -, -NR 4 C(=NR 4 )NR 4 -, -C(=S)NR 4 -, -NR 4 C(=S)-, -C(=O)O-, -OC(=S)-, OC(=S)O-, -NR 4 C(=S)O-, -OC(=S)NR 4 -, -NR 4 C(=S)NR 4 -, -C(=O)S-, SC(=O)-, -OC(=O)S-, -NR 4 C(=O)S-, -SC(=O)NR 4 -, -C(=S)S-, -SC(=S)-, -SC(=S)O-, -NR 4 C(=S)S-, -SC(=S)NR 4-, -C(=S)S-, -SC(=S)-, -SC(=O)S-, -SC(=S)S-, -NR 4 C(=S)S-, -SC(=S)NR 4 -, O, S, -C1~C 10 Alkylene-O-,-C1~C 10 Alkylene -C(=O)O-, -C1~C 10 The alkylene is -OC(=O)- or a bond, and the alkylene can be substituted or unsubstituted. R 3 is -C0~C 10 Alkilen-NR 7 R 8 , -C0~C 10 Alkylene-heterocycloalkyl, or -C0~C 10 Alkylene-heterocycloaryl, where alkylene, heterocycloalkyl, and heterocycloaiyl are independently substituted or unsubstituted, R 4 Each of these is independently hydrogen, or a substituted or unsubstituted C1-C6 alkyl group. R 5 is hydrogen, or a substituted or unsubstituted C1-C6 alkyl group. R 6 Each of these can be independently substituted or non-substituted C3~C 22 Alkyl, or substituted or unsubstituted C3-C 22 It is alkenyl, R 7 and R 8 Each of these is independently hydrogen, or a substituted or unsubstituted C1-C6 alkyl, or R together with the bonded nitrogen. 7 and R 8 However, it forms substituted or unsubstituted C2-C6 heterocyclines. p is an integer selected from 1 to 10. n, m, and q are each independently 0, 1, 2, 3, 4, or 5.

[0237] In one or more embodiments of formula (I), when the structure has two or more asymmetric C atoms, each asymmetric C atom independently represents a racemate, a chirally pure R and / or a chirally pure S isomer, or a combination thereof.

[0238] In one or more embodiments, n, m, and q in formula (I) are each independently 0, 1, 2, or 3. In one or more embodiments, n, m, and q in formula (I) are each 1.

[0239] b) Formula (Ia) 0In one or more embodiments, the compound of formula (1) is of formula (Ia),

Chemical formula

Chemical formula

[0240] In one or more embodiments of formula (Ia), if the structure has two or more chiral carbon atoms, each chiral carbon atom independently represents a racemate, a chiral pure R and / or a chiral pure S isomer, or a combination thereof.

[0241] c) Modified forms of equations (I) and (Ia) In one or more embodiments, R in formulas (I) and (Ia) 1 and R 2 These are, independently, C3~C 22 Alkyl, C3~C 22 Alkenyl, -C2~C 10 Alkylene-LR 6 , or [ka] Alkyl, alkylene, alkenyl, and cycloalkyl are each independently substituted or unsubstituted. In one or more embodiments, R in formulas (I) and (la) 1 and R 2 Independently, C 10 ~C 20 Alkyl, C 10 ~C 20 Alkenyl. -C8~C7 Alkilen-LR 6 , or [ka] Alkyl, alkylene, alkenyl, and cycloalkyl are each independently substituted or unsubstituted. In one or more embodiments, R in formulas (I) and (la) 1 teeth, [ka] That is the case.

[0242] In one or more embodiments, L in formula (I) and formula (Ia) is independently O, S, -C1~C 10 Alkylene-O-,-C1~C 10 Alkylene -C(=O)O-, -C1~C 10The alkylene is alkylene-OC(=O)- or a bond, and the alkylene is substituted or unsubstituted. In one or more embodiments, L in formulas (I) and (Ia) is independently O, S, -C1~C3 alkylene-O-, -C1~C3 alkylene-C(=O)O-, -C1~C3 alkylene-OC(=O)-, or a bond, and the alkylene is substituted or unsubstituted. In one or more embodiments, L in formulas (I) and (Ia) is independently O, S, -C1~C3 alkylene-O-, -C1~C3 alkylene-C(=O)O-, -C1~C3 alkylene-OC(=O)-, or a bond, and the alkylene is linear or branched unsubstituted alkylene.

[0243] In one or more embodiments, R in formulas (I) and (Ia) 6 Each of these independently comprises substituted or unsubstituted linear C3-C3. 22 Alkyl, or substituted or unsubstituted linear C3-C 22 It is an alkenyl. In one or more embodiments, R in formulas (I) and (Ia) 6 Each of these can be independently substituted or non-substituted C3~C 20 Alkyl, or substituted or unsubstituted C3-C 20 It is an alkenyl. In one or more embodiments, R in formulas (I) and (Ia) 6 Each of these can be independently substituted or non-substituted C3~C 10 Alkyl, or substituted or unsubstituted C3-C 10 It is an alkenyl. In one or more embodiments, R in formulas (I) and (Ia) 6 Each of these can be independently substituted or non-substituted C3~C 10 It is alkyl. In one or more embodiments, R in formula (I) and formula (la) 6 Each of these can be independently substituted or non-substituted C3~C 10 It is alkyl. In one or more embodiments, R in formula (I) and formula (la) 6Each of these is independently a substituted or unsubstituted n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, or n-dodecyl. In one or more embodiments, R in formulas (I) and (Ia) 6 Each of these is independently a substituted or unsubstituted n-octyl. In one or more embodiments, R in formulas (I) and (Ia) 6 Each of these is an n-octyl.

[0244] In one or more embodiments, L in formula (I) and formula (Ia) are independently -C(=O)O-, -OC(=O)-, and -C1~C 10 It is alkylene-O- or O. In one or more embodiments, L in formula (I) and formula (Ia) is O. In one or more embodiments, L in formula (I) and formula (Ia) is -C1~C3 alkylene-O-. In one or more embodiments, p in formula (I) and formula (Ia) is 1, 2, 3, 4, or 5. In one or more embodiments, p in formula (I) and formula (Ia) is 2.

[0245] In one or more embodiments, R in formulas (I) and (Ia) 1 teeth, [ka] That is the case.

[0246] In one or more embodiments, R in formulas (I) and (Ia) 1 R 2 That is the case.

[0247] In one or more embodiments, R in formulas (I) and (Ia) 4 Each of these is independently H, or a substituted or unsubstituted C1-C4 alkyl group. In one or more embodiments, R in formulas (I) and (Ia) 4 Each of these is independently a substituted or unsubstituted linear C1-C4 alkyl group. In one or more embodiments, R in formula (1) and formula (la) 4Each of these is H. In one or more embodiments, R in formulas (I) and (Ia) 4 Each of these is independently H, -CH3, -CH2CH3, -CH2CH2CH3, or -CH(CH3)2. In one or more embodiments, R in formulas (I) and (Ia) 4 Each is independently H or -CH3. In one or more embodiments, R in formulas (I) and (Ia) 4 These are each -CH3.

[0248] In one or more embodiments, X in formulas (I) and (Ia) is -C(=O)O- or -OC(=O))-. In one or more embodiments, X in formulas (I) and (Ia) is -C(=O)NR 4 or -NR 4 C(=O)-. In one or more embodiments, X in formulas (I) and (Ia) is -C(=O)N(CH3)-, -N(CH3)C(=O)-, -C(=O)NH-, or -NHC(=O)-. In one or more embodiments, X in formulas (I) and (Ia) is -C(=O))NH-, -C(=O)N(CH3)-, -OC(=O))-, -NHC(=O)-, -N(CH3)C(=O))-, -C(=O)O-, -OC(=O)O-, -NHC(=O)O-, -N(CH3)C(=O)O-, -OC(=O))NH-, -OC(=O)N(CH3)-, -NHC(=O)NH-, -N(CH3)C(=O) )NH-, -NHC(=O)N(CH3)-, -N(CH3)C(=O)N(CH3)-, NHC(=NH)NH-, -N(CH3)C(=NH)NH-, -NHC(=NH)N(CH3)-, -N(CH 3) C(=NH)N(CH3)-, NHC(=NMe)NH-, -N(CH3)C(=NMe)NH-, -NHC(=NMe)N(CH3)-, or -N(CH3)C(=NMe)N(CH3)-.

[0249] In one or more embodiments, R in formulas (I) and (Ia) 2 C3~C 22 Alkyl, C3~C 22 Alkenyl, -C2~C 10 Alkylene-LR 6, or [ka] Alkyl, alkylene, alkenyl, and cycloalkyl are each independently substituted or unsubstituted. In one or more embodiments, R in formulas (I) and (Ia) 2 This is either substituted or non-substituted C7~C 22 Alkyl, or substituted or unsubstituted C3-C 22 It is an alkenyl. In one or more embodiments, R in formulas (I) and (la) 2 This is either substituted or non-substituted C7~C 22 Alkyl, or substituted or unsubstituted linear C3-C3 chains 22 It is an alkenyl. In one or more embodiments, R in formulas (I) and (Ia) 2 is a substitution or non-substitution C 10 ~C 20 Alkyl, or substituted or unsubstituted C 10 ~C 20 It is an alkenyl. In one or more embodiments, R in formulas (I) and (Ia) 2 is unsubstituted C 10 ~C 20 It is alkyl. In one or more embodiments, R in formula (I) and formula (Ia) 2 is unsubstituted C 10 ~C 20 It is an alkenyl. In one or more embodiments, R in formulas (I) and (Ia) 2 is -C2~C 10 Alkylene-LR 6 In one or more embodiments, R in formulas (I) and (Ia) 2 is -C2~C 10 Alkylene C(=O)OR 6 or -C2~C 10 Alkylene-OC(=O)-R 6 That is the case.

[0250] In one or more embodiments, R in formulas (I) and (Ia) 2 teeth, [ka] That is the case.

[0251] In one or more embodiments, R in formulas (I) and (Ia) 1 R 1 That is the case.

[0252] In one or more embodiments, Y in formulas (I) and (Ia) is -C(=O)O- or -OC(=O)-. In one or more embodiments, Y in formulas (I) and (Ia) is -C(=O)NR 4 - or -NR 4 C(=O)-. In one or more embodiments, Y in formulas (I) and (Ia) is -C(=O)N(CH3)-, -N(CH3)C(=O)-, -C(=O)NH-, or -NHC(=O)-. In one or more embodiments, Y in formulas (I) and (Ia) is -OC(=O)O-, -NR 4 C(=O)O-, -OC(=O)NR 4 -, or -NR 4 C(=O)NR 4 -. In one or more embodiments, Y in formulas (I) and (la) is -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -N(CH3)C(=O)O-, -OC(=O)N(CH3)-, -N(CH3)C(=O)N(CH3)-, or -N(CH3)C(=O)NH-. In one or more embodiments, Y in formulas (I) and (Ia) is -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, or -NHC(=O)NH-.

[0253] In one or more embodiments, R in formulas (I) and (Ia) 3 is -C0~C 10 Alkilen-NR 7 R 8 or -C0~C 10 The alkylene-heterocycloalkyl group is alkylene and heterocycloalkyl are independently substituted or unsubstituted. In one or more embodiments, R in formulas (I) and (Ia) 3 is -C0~C 10 Alkilen-NR 7 R8 In one or more embodiments, R in formulas (I) and (Ia) 3 -C1~C6 alkylene-NR 7 R 8 In one or more embodiments, R in formulas (I) and (Ia) 3 -C1~C4 alkylene-NR 7 R 8 In one or more embodiments, R in formulas (I) and (Ia) 3 is -C1-alkylene-NR 7 R 8 In one or more embodiments, R in formulas (I) and (Ia) 3 is -C2--alkylene-NR 7 R 8 In one or more embodiments, R in formulas (I) and (Ia) 3 is -C3-alkylene-NR 7 R 8 In one or more embodiments, R in formulas (I) and (Ia) 3 is -C4-alkylene-NR 7 R 8 In one or more embodiments, R in formulas (I) and (Ia) 3 is -C5-alkylene-NR 7 R 8 In one or more embodiments, R in formulas (I) and (Ia) 3 is -C0~C 10 It is an alkylene-heterocycloalkyl. In one or more embodiments, R in formulas (I) and (Ia) 3 R is a -C1~C6 alkylene-heterocycloalkyl, where the heterocycloalkyl contains 1~3 nitrogen atoms and 0~2 oxygen atoms. In one or more embodiments, R in formulas (I) and (la) 3 These are -C1~C6 alkylene heterocycloaryl compounds.

[0254] In one or more embodiments, R in formulas (I) and (Ia) 7 and R 8 Each of these is independently hydrogen, or a substituted or unsubstituted C1-C6 alkyl group. In one or more embodiments, R7 and R 8 Each of these is independently hydrogen, or a substituted or unsubstituted C1-C3 alkyl group. In one or more embodiments, R 7 and R 8 Each of these is independently a substituted or unsubstituted C1-C3 alkyl group. In one or more embodiments, R 7 and R 8 Each of these is independently -CH3, -CH2CH3, -CH2CH2CH3, or -CH(CH3)2. In one or more embodiments, R and R 8 Each of these is CH3. In one or more embodiments, R 7 and R 8 These are -CH2CH3, respectively.

[0255] In one or more embodiments, the R in formulas (I) and (Ia) is combined with the bonded nitrogen. 7 and R 8 This forms a substituted or unsubstituted C2-C6 heterocycline. In one or more embodiments, R is bound together with the nitrogen. 7 and R 8 This forms a substituted or unsubstituted C2-C6 heterocycloalkyl group. In one or more embodiments, R is bound together with the nitrogen atom. 7 and R 8 These form substituted or unsubstituted 3- to 7-membered heterocycloalkyl groups.

[0256] In one or more embodiments, R in formulas (I) and (la) 3 teeth, [ka] That is the case.

[0257] In one or more embodiments, R in formulas (I) and (Ia) 3 teeth, [ka] That is the case.

[0258] In one or more embodiments, R in formulas (1) and (la) 3 teeth, [ka] That is the case.

[0259] In one or more embodiments, Z in formulas (I) and (Ia) is -C(=O)O- or -OC(=O)-.

[0260] In one or more embodiments, Z in formulas (I) and (Ia) is -C(=O)NR 4 - or -NR 4 C(=O)-

[0261] In one or more embodiments, Z in formulas (I) and (Ia) is -C(=O)N(CH3)-, -N(CH3)C(=O)-, -C(=O)NH-, or -NHC(=O)-.

[0262] In one or more embodiments, Z in formulas (I) and (Ia) is -OC(=O)O-, -NR 4 C(=O)O-, -OC(O)NR 4 -, or -NR 4 C(=O)NR 4 - is

[0263] In one or more embodiments, Z in formulas (I) and (Ia) is -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -N(CH3)C(=O)O-, -OC(=O)N(CH3)-, -N(CH3)C(=O)N(CH3)-, -NHC(=O)N(CH3)-, or -N(CH3)C(=O)NH-.

[0264] In one or more embodiments, Y in formulas (I) and (Ia) is -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, or -NHC(=O)NH-.

[0265] In one or more embodiments, R in formulas (I) and (Ia) 5This is hydrogen, or a substituted or unsubstituted C1-C3 alkyl group.

[0266] In one or more embodiments, R in formulas (I) and (Ia) 5 These are H, -CH3, -CH-)CH3, -CH2CH2CH3, or -CH(CH3)2.

[0267] In one or more embodiments, R in formulas (I) and (Ia) 5 H is H.

[0268] 2. LNP composition containing different aminolipids In one or more embodiments, the LNP comprises a plurality of aminolipids having different formulas. For example, an LNP composition may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more aminolipids. In another example, an LNP composition may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 20 aminolipids. In yet another example, an LNP composition may contain up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 20, or up to 30 aminolipids.

[0269] In one or more embodiments, the LNP composition contains a first aminolipid. In one or more embodiments, the LNP composition contains a first aminolipid and a second aminolipid. In one or more embodiments, the LNP composition contains a first aminolipid, a second aminolipid, and a third aminolipid. In one or more embodiments, the LNP composition contains a first aminolipid, a second aminolipid, a third aminolipid, and a fourth aminolipid. In one or more embodiments, the LNP composition does not contain a fourth aminolipid. In one or more embodiments, the LNP composition does not contain a third aminolipid. In one or more embodiments, the molar ratio of the first aminolipid to the second aminolipid is about 0.1 to about 10. In one or more embodiments, the molar ratio of the first aminolipid to the second aminolipid is about 0.20 to about 5. In one or more embodiments, the molar ratio of the first aminolipid to the second aminolipid is about 0.25 to about 4. In one or more embodiments, the molar ratio of the first aminolipid to the second aminolipid is about 0.25, about 0.33, about 0.5, about 1, about 2, about 3, or about 4.

[0270] In one or more embodiments, the molar ratio of the first aminolipid:second aminolipid:third aminolipid is approximately 4:1:1. In one or more embodiments, the molar ratio of the first aminolipid:second aminolipid:third aminolipid is approximately 1:1:1. In one or more embodiments, the molar ratio of the first aminolipid:second aminolipid:third aminolipid is approximately 2:1:1. In one or more embodiments, the molar ratio of the first aminolipid:second aminolipid:third aminolipid is approximately 2:2:1. In one or more embodiments, the molar ratio of the first aminolipid:second aminolipid:third aminolipid is approximately 3:2:1. In one or more embodiments, the molar ratio of the first aminolipid:second aminolipid:third aminolipid is approximately 3:1:1. In one or more embodiments, the molar ratio of the first aminolipid:second aminolipid:third aminolipid is approximately 5:1:1. In one or more embodiments, the molar ratio of the first aminolipid:second aminolipid:third aminolipid is approximately 3:3:1. In one or more embodiments, the molar ratio of the first aminolipid:second aminolipid:third aminolipid is approximately 4:4:1.

[0271] 3. Additional aminolipid embodiments In one or more embodiments, the LNP composition comprises one or more aminolipids. In one or more embodiments, one or more aminolipids account for about 40 mol% to about 65 mol% of the total lipids present in the particles. In one or more embodiments, one or more aminolipids account for about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, or about 65 mol% of the total lipids present in the particles. In one or more embodiments, the first aminolipid accounts for approximately 1 mol% to approximately 99 mol% of the total aminolipids present in the particles. In one or more embodiments, the first aminolipid accounts for approximately 16.7 mol% to approximately 66.7 mol% of the total aminolipids present in the particles. In one or more embodiments, the first aminolipid accounts for approximately 20 mol% to approximately 60 mol% of the total aminolipids present in the particles.

[0272] In one or more embodiments, the aminolipid is an ionizable lipid. The ionizable lipid may contain one or more ionizable nitrogen atoms. In one or more embodiments, at least one of the one or more ionizable nitrogen atoms is positively charged. In one or more embodiments, at least 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, 90 mol%, 95 mol%, or 99 mol% of the ionizable nitrogen atoms in the LNP composition are positively charged. In one or more embodiments, the aminolipid contains a primary amine, a secondary amine, a tertiary amine, an imine, an amide, a guanidine moiety, a histidine residue, a lysine residue, an arginine residue, or any combination thereof. In one or more embodiments, the aminolipid contains a primary amine, a secondary amine, a tertiary amine, a guanidine moiety, or any combination thereof. In one or more embodiments, the aminolipid contains a tertiary amine.

[0273] In one or more embodiments, the aminolipid is a cationic lipid. In one or more embodiments, the aminolipid is an ionizable lipid. In one or more embodiments, the aminolipid contains one or more nitrogen atoms. In one or more embodiments, the aminolipid contains one or more ionizable nitrogen atoms. Examples of cationic lipids and / or ionizable lipids include, but are not limited to, 3-(didodecylamino)-N1,N1,4-tridodecyl-l-piperazinediethanamine (KL10), N142-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), and heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC 3-DMA), 2,2-dioleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propane-l-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-yloxy]octyl}oxy) Examples include (octyl-CLinDMA(2R))-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA(2R)) and (2S)-2-({8-[(3β)-cholest-5-ene-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-l-yloxy]propan-l-amine(octyl-CLinDMA(2S)).

[0274] In one or more embodiments, the aminolipids described herein may take the form of salts, such as pharmaceutically acceptable salts. All pharmaceutically acceptable salts of aminolipids are incorporated herein. As used herein, the term “aminolipid” also includes its pharmaceutically acceptable salts, as well as its diastereomers, enantiomers, and epimers.

[0275] In one or more embodiments, the aminolipids described herein have one or more stereocenters, each stereocenter independently existing in either an R or S configuration. The lipids presented herein include all diastereomers, enantiomers, and epimers, as well as suitable mixtures thereof. The lipids provided herein include all cis isomers, trans isomers, syn isomers, anti isomers, entgegen (E) isomers, and zusammen (Z) isomers, as well as suitable mixtures thereof. In certain embodiments, the lipids described herein are prepared as their individual stereoisomers by reacting a racemic mixture of the compounds with an optically active resolving agent to form diastereoisomer compound / salt pairs, separating the diastereomers, and recovering the optically pure enantiomers. In one or more embodiments, the resolving of enantiomers is carried out using covalent diastereomer derivatives of the compounds described herein. In another embodiment, the diastereomers are separated by separation / resolving techniques based on solubility differences. In other embodiments, the separation of stereoisomers is carried out by chromatography, or by separation by the formation and recrystallization of diastereomer salts, or by chromatography, or by any combination thereof. Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions," John Wiley and Sons, Inc., 1981. In one embodiment, the stereoisomers are obtained by stereoselective synthesis.

[0276] In one or more embodiments, lipids such as aminolipids are substituted based on the structures disclosed herein. In one or more embodiments, lipids such as aminolipids are unsubstituted. In another embodiment, the lipids described herein are isotoped (e.g., with radioactive isotopes) by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0277] The lipids described herein include isotope-labeled compounds, which are identical to those listed in the various formulas and structures presented herein, except that one or more atoms are replaced by atoms having atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that may be incorporated into these lipids include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, and chlorine, for example, 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, and 36 Examples include Cl. In one embodiment, isotope-labeled lipids as described herein, for example 3 H and 14 Substances incorporating radioactive isotopes such as 13C are useful in drug and / or substrate tissue distribution assays. In one embodiment, isotope substitution, such as deuterium, provides specific therapeutic benefits resulting from greater metabolic stability, such as an increased in vivo half-life or a reduced required dose.

[0278] In one or more embodiments, the chiral carbon atoms of the aminolipids exist in enantiomerically concentrated forms. In some embodiments, the chiral carbon atoms of the aminolipids have an enantiomer excess of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% in the (S)- or (R)-configuration.

[0279] In one or more embodiments, the disclosed aminolipids can be converted to N-oxides. In one or more embodiments, the N-oxides are formed by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid and / or hydrogen peroxide). Accordingly, NO or N, when permissible by valence and structure, are used herein. + -O - N-oxide compounds of the aminolipids described herein, which may be named as such, are disclosed. In one or more embodiments, the nitrogen in the compounds of this disclosure may be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine with an oxidizing agent such as ra-CPBA. All nitrogen-containing compounds shown are also considered. Accordingly, N-hydroxy and N-alkoxy (e.g., N-OR) derivatives of the aminolipids described herein are also disclosed.

[0280] In one or more embodiments, one or more aminolipids account for approximately 40 mol% to approximately 65 mol% of the total lipids present in the particles.

[0281] 4. PEG-Lipids In one or more embodiments, the described LNP composition comprises one or more PEG-lipids. As used herein, “PEG-lipid” or “PEG-lipid” refers to a lipid containing a polyethylene glycol component. Suitable examples of PEG-lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, one or more PEG-lipids may include PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, or combinations thereof.

[0282] In one or more embodiments, PEG-lipids account for approximately 0.1 mol% to approximately 10 mol% of the total lipids present in the particles.

[0283] 5. Phospholipids In one or more embodiments, the described LNP composition comprises one or more phospholipids.

[0284] In one or more embodiments, phospholipids account for approximately 5 mol% to approximately 15 mol% of the total lipids present in the particles.

[0285] 6. Cholesterol In one or more embodiments, the LNP composition comprises cholesterol or a derivative thereof.

[0286] 7. GalNAc-lipids In one or more embodiments, the LNP composition is of formula (V), [ka] The formula includes a receptor-targeting conjugate containing the compound, Multiple A groups together constitute the receptor targeting moiety. L 1 , L 2 , L 3 , L 4 , L 5 , L6 , L 7 , L 8 , L 9 , L 10 , and L 12 Each of these can be independently substituted or not substituted C1-C 12 Alkylene, substituted or unsubstituted C1-C 12 Heteroalkylene, substituted or unsubstituted C2-C 12 Alkenylene, substituted or unsubstituted C2-C 12 Alkynylene, -(CH2CH2O) m -,-(OCH2CH2) m -, -O-, -S-, -S(=O)-, -S(=O)2-, -S(=O)(=NR 1 )-, -C(=O)-, -C(=N-OR 1 )-, -C(=O)O-, -OC(=O)-, -C(=O)C(=O)-, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR 1 C(=O)NR 1 -, -C(=O)NR 1 C(=O)-, -S(=O)2NR 1 -, -NR 1 S(=O)²⁻, -NR 1 -, or -N (OR 1 )- and, L 11 is either substituted or unsubstituted -(CH2CH2O) n -, substitution or non-substitution - (OCH2CH2) n -, or substituted or unsubstituted -(CH2) n -and, R 1 Each of these is independently H, or a substituted or unsubstituted C1-C6 alkyl group. R is a lipid, nucleic acid, amino acid, protein, or lipid nanoparticle. m is an integer selected from 1 to 10. n is an integer selected from 1 to 200.

[0287] In one or more embodiments, L 1 , L 4 , and L 7 Each of these can be independently substituted or non-substituted C1-C 12 It is an alkylene. In one or more embodiments, L 1 , L 4 , and L 7 Each of these is independently a substituted or unsubstituted C2-C6 alkylene. In one or more embodiments, L 1 , L 4 , and L 7 Each of these is a C4 alkylene. In one or more embodiments, L 2 , L 5 , and L 8 Each of these is independently -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -NR1C(=O)NR 1 -, or -C(=O)NR 1 C(=O)-. In one or more embodiments, L 2 , L 5 , and L 8 Each of these is independently -C(=O)NR 1 - or -NR 1 C(=O)-. In one or more embodiments, L 2 , L 5 , and L 8 Each of these is -C(=O)NH-. In one or more embodiments, L 3 , L 6 , and L 9 Each of these can be independently substituted or non-substituted C1-C 12 It is an alkylene. In one or more embodiments, L 3 Each of these is a substituted or unsubstituted C2-C6 alkylene. In one or more embodiments, L 3 is a C4 alkylene. In one or more embodiments, L 6 and L 9 Each of these can be independently substituted or non-substituted C2~C 10 It is an alkylene. In one or more embodiments, L 6 and L9 Each of these is independently a substituted or unsubstituted C2-C6 alkylene. In one or more embodiments, L 6 and L 9 Each of these is a C3 alkylene. In one or more embodiments, A binds to a lectin. In one or more embodiments, the lectin is an asialoglycoprotein receptor (ASGPR). In one or more embodiments, A is N-acetylgalactosamine (GalNAc) or [ka] or its derivatives. A is N-acetylgalactosamine (GalNAc). [ka] or a derivative thereof.

[0288] Examples of such GalNAc lipids include the following: [ka] In the formula, p and q are independent integers between 1 and 5, and n is an integer between 1 and 50. [ka] In the formula, n is an integer between 1 and 50; [ka] In the formula, p and q are independent integers between 1 and 5, and n is an integer between 1 and 50. [ka] In the formula, n is an integer between 1 and 50; [ka] In the formula, p and q are independent integers between 1 and 5, and n is an integer between 1 and 50. [ka] In the formula, p and q are independent integers between 1 and 5, and n is an integer between 1 and 50. [ka] In the formula, p and q are independent integers between 1 and 5, and n is an integer between 1 and 50. [ka] In the formula, p and q are independent integers between 1 and 5, and n is an integer between 33 and 39; [ka] In the formula, n is an integer between 1 and 50; [ka]

[0289] In one or more embodiments, the receptor-targeting conjugate accounts for approximately 0.001 mol% to approximately 20 mol% of the total lipid content present in the nanoparticle composition.

[0290] 8. Phosphate charge neutralizing agent In one or more embodiments, the LNP described herein includes a phosphate ...

Claims

1. A pharmaceutical composition for in vivo editing of the LPA gene in mammals, (i) A modified, non-naturally occurring gene editing system, (a) one or more polynucleotides (mRNAs) that encode nicasse, (b) A first guide oligonucleotide (gRNA) comprising a first spacer sequence including a region complementary to the first strand of the LPA gene in the first target sequence, and a first scaffold region that functions as a binding scaffold for the nickase, and (c) A gene editing system comprising a second guide oligonucleotide (gRNA) including a second spacer sequence having a region complementary to the second strand of the LPA gene in a second target sequence, and a second scaffold region having a binding scaffold for the nickase, (ii) comprising lipid nanoparticles (LNPs) encapsulating the gene editing system, The first chain and the second chain are opposing chains, A pharmaceutical composition wherein the first spacer has an arrangement that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% similar to, or identical to, or identical to, the guide 1 protospacers listed in Table 2 or Table 5, and the second spacer has an arrangement that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% similar to, or identical to, or identical to, the guide 2 protospacers listed in Table 2 or Table 5.

2. The pharmaceutical composition according to claim 1, wherein the first gRNA and the nickase are manipulated such that the nickase inserts a nick into one of the first or second strands of the LPA gene at a first position between positions 160,664,275 and 160,531,482 of chromosome 6, and the second gRNA and the nickase are manipulated such that the nickase inserts a nick into the other of the first or second strands of the LPA gene at a second position between positions 160,664,275 and 160,531,482 of chromosome 6.

3. The pharmaceutical composition according to claim 1 or 2, wherein the first spacer includes the same or substantially the same arrangement as the guide 1 protospacers listed in Table 2 or Table 5, and the second spacer includes the same or substantially the same arrangement as the corresponding (in the same row) guide 2 protospacers listed in Table 2 or Table 5.

4. The first spacer has an arrangement that is identical to or is identical to 5'-CUGUCAACCAGGCAAUUGUGUC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-UGUCCUUGCAACUCUCACGG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-GUAGUAGCAGUUCCUGUACCC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-CAUUAUGGACACAGUUACCG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-AGGACACAUCUCGAAUUCUGUCA-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-CACAACUCCCCACAGUGGCCCC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-CUGUCACUGGGACAUUGUGUC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-AAGUGUCCCUUGCGGACGUCCA-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-GGAGCAAAAGCCCCCCAGUCC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-GUUGGUGCUGAGAAAAUUCAAAG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-GGAGCAAAAGCCCGGGGGUCC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-GUUGGUGCUGAGAAAAUUCAAAG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-CUGGAACUGGGGACCACGU-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-ACAGAGCUUCCUUCUGAAGA-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-AUGCCAGUGUGUGUGUCAAUAG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-ACCACAGAAUACUCCCAAA-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-GGAGCCCAGAAUACAUUCGG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-CUAGAGCUUUUUUUGAACA-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-CAGAUGCUGAGAUUAGUCCU-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-UGGAAUUCCUUGCAGUAGUUCC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-UGACACCACAUUGGCAUUCGG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-ACAUGUUCUUCCUGUGAUAG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-CAAUAGAUGAACCAAGAUUGAC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-UGAUACCACACUGGCAUCAG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-CCAUCACUGGGACAAUUGCGUC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-ACUCUCUCUCACACUCUCCA-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-CUGCAUCUGAGCCAUCGUGUUC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-CGUCCCCUCCGAAUGUUAUUC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-AAACAGCCCGUGGGACGCA-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-UGAACAAGGUAAAGAAGUCUC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-ACAGAGGCUCUCCUUCUGACA-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-GCUUGGAACCGGGGCCACUG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-AUGCCAGUGUGUGUGUCAAUAG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-ACACAGAAUAUAUAUCCAAA-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-CUAUGAACACCACAUUGGCAU-3', and the second spacer has an arrangement that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% identical to or identical to 5'-ACAUGUUCUUCCUGUGAAUAG-3', The first spacer has an arrangement that is identical to or is identical to 5'-AAUAACAUUCGGGAGGGAACGA-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-UAUUCUGGCUCCAAGCUAG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-GUAGCAGUCCCUGUACCCCGG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-CAUUAUGGACACAGUUACCG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-AGUAGCAGUCCCUGUACCCCCG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-CAUUAUGGACACAGUUACCCG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, The first spacer has an arrangement that is identical to or is identical to 5'-UAGUAGCAGUCCUGUACCCC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, and the second spacer has an arrangement that is identical to or is identical to 5'-CAUUAUGGACACAGUUACCG-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, or The pharmaceutical composition according to claim 1, wherein the first spacer has an arrangement that is identical or identical to 5'-UGGACCACAUGGCUUUGUCUC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%, and the second spacer has an arrangement that is identical or identical to 5'-ACGUACUCCAACCAUGUGUCAAC-3' by at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95%.

5. The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CUGUCAACCAGGCAAUUGUGUC-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-UGUCCUUGCAACUCUCACGG-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-GUAGUAGCAGUUCCUGUACCC-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CAUUAUGGACACAGUUACCG-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-AGGACACAUCUCGAAUUCUGUCCA-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CACAACUCCCCACAGUGGCCCC-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CUGUCACUGGGACAUUGUGUC-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-AAGUGUCCCUUGCGGACGUCCA-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-GGAGCAAAAGCCCCCCAGUCC-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-GUUGGUGCUGAGAAAAUUCAAAG-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-GGAGCAAAAGCCCGGGGGUCC-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-GUUGGUGCUGAGAAAAUUCAAAG-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CUGGAACUGGGGACCACCGU-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-ACAGAGCUUCCUUCUGAAGA-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-AUGCCAGUGUGUGUGUCAAUAG-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-ACCACAGAAUACUCCCAAA-3', The first has a spacer sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-GGAGCCCAGAAUACAUUCGG-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CUAGAGCUUUUUUUGACA-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CAGAUGCUGAGAUUAGUCCU-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-UGGAAUUCCUUGCAGUAGUUCC-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-UGACACCACAUUGGCAUCGGG-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-ACAUGUUCUUCCUGUGAUAG-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CAUAGAUGAACCCAAGAUUGAC-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-UGAUACCACACUGGCACAG-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CCAUCACUGGGACAAUUGCGUC-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-ACUCUCCUCACAACUCCCCA-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CUGCAUCUGAGCCAUCGUGUUC-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CGUCCCUCUCGAAUGUUAUUC-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-AAACAGCCGUGUGACGUCGCA-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-UGAAACAGGUAAAGAGUCUC-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-ACAGAGGCUCCUUCUGAACA-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-GCUUGGAACCGGGGCCACUG-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-AUGCCAGUGUGUGUGUCAAUAG-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-ACACAGAAUAUAUAUCCAAA-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CUAUGAACACCACAUUGGCAU-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-ACAUGUUCUUCCUGUGAAUAG-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-AAAUACAUUCGGGAGGGAACGA-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-UAUUCUGGCUCCAAGCUAG-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-GUAGCAGUCCCUGUACCCCGG-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CAUUAUGGACACAGUUACCG-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-AGUAGCAGUCCUGUACCCCG-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CAUUAUGGACACAGUUACCG-3', The first spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-UAGUAGCAGUCCUACCCC-3', and the second spacer has a sequence containing nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-CAUUAUGGACACAGUUACCG-3', or The pharmaceutical composition according to claim 1, wherein the first spacer has a sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-UGGACCACAUGGCUUUGUCUC-3', and the second spacer has a sequence comprising nucleotides 6-20, 5-20, 4-20, 3-20, 2-20, or 1-20 of the following sequence 5'-ACGUACUCCAACCAUGUGUCAAC-3'.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the first spacer and / or the second spacer comprises a modified nucleotide.

7. The pharmaceutical composition according to claim 6, wherein one or more nucleotides within 5 nucleotides from the 5' end of the first spacer and / or the second spacer are modified nucleotides.

8. The pharmaceutical composition according to claim 6 or 7, wherein the nucleotides within 3 nucleotides at the 5' end of the first spacer and / or the second spacer are each modified nucleotides.

9. The pharmaceutical composition according to any one of claims 6 to 8, wherein the modified nucleotide comprises a 2'-OMe modification and / or a phosphorothioate group.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein one or more of the aforementioned nickases include CRSPR Cas nickases.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein at least one of the one or more Cas nickases is manipulated to introduce a nick into the opposite strand of the LPA gene with which the operable guide oligonucleotide (gRNA) hybridizes when it operably interacts with the first guide oligonucleotide or the second guide oligonucleotide.

12. The pharmaceutical composition according to claim 11, wherein at least one of the one or more polynucleotides (mRNAs) encoding the one or more Cas nickases encodes the Cas9 nickase of Streptococcus pyogenes having the H840A mutation.

13. The pharmaceutical composition according to any one of claims 1 to 10, wherein at least one of the one or more Cas nickases is manipulated to introduce a nick into the same strand of the LPA gene that the operable guide hybridizes when it operably interacts with the first guide oligonucleotide (gRNA) or the second guide oligonucleotide (gRNA).

14. The pharmaceutical composition according to claim 13, wherein at least one of the one or more polynucleotides (mRNAs) encoding the one or more Cas nickases encodes the Cas9 nickase of Streptococcus pyogenes having the D10A mutation.

15. At least one of the one or more polynucleotides (mRNAs) is (a) 5' Untranslated region (UTR) and (b) 3'UTR region and (c) A poly(A) tail that is closer to the 3'UTR than the 5'UTR, A poly(A)tail containing a chain of 80 to 150 nucleotides in length, including adenine nucleotides, (d) Damaged CRISPR Cas9 A gene editing substance (editor) coding region encoding an endonuclease domain and a polymerase domain, the gene editing substance (editor) coding region located between the 5'UTR and the 3'UTR, A pharmaceutical composition according to any one of claims 1 to 14, comprising:

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein at least one of the one or more polynucleotides is selected from mRNAs having at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity with any of the mRNA sequences listed in Table 1.

17. The pharmaceutical composition according to any one of claims 1 to 15, wherein at least one of the one or more polynucleotides includes a coding sequence of any of the mRNA sequences listed in Table 1, or includes a coding sequence having at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity with any of the coding sequences of the nickase mRNA sequences listed in Table 1.

18. The aforementioned LNP is (a) One or more ionizable lipids, (b) Cholesterol and, (c) One or more PEG-lipids, (d) Phospholipids and (e) The pharmaceutical composition according to any one of claims 1 to 17, optionally comprising a targeted portion such as a GalNAc lipid.

19. The aforementioned LNP is (a) 40 to 60 mole percent of one or more ionizable lipids, 28 to 48 mole percent of cholesterol, 5 to 13 mole percent of phospholipids, and 2 to 5 mole percent of PEG-lipids, (b) 50+ / -10% mole percent of the one or more ionizable lipids, 38+ / -10% mole percent of the cholesterol, 9+ / -10% mole percent of the phospholipids, and 3+ / -10% mole percent of the PEG-lipids, (c) 40 to 60 mole percent of one or more ionizable lipids, 27.95 to 47.95 mole percent of cholesterol, 5 to 13 mole percent of phospholipids, 2 to 5 mole percent of PEG-lipids, and 0.02 to 0.09 mole percent of GalNAc-lipids, (d) The pharmaceutical composition according to claim 18, formulated to contain 50+ / -10% mole percent of one or more ionizable lipids, 37.95+ / -10% mole percent of cholesterol, 9+ / -10% mole percent of phospholipids, 3+ / -10% mole percent of PEG-lipids, and 0.05+ / -10% mole percent of GalNAc-lipids.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the arrangement of the first scaffold region and the second scaffold region is the same.

21. The scaffold region sequences of the first guide oligonucleotide (gRNA) and the second guide oligonucleotide (gRNA) are independently the following sequences: 5'-GUUUUAGAGCUAGAAAUAGCAAGUAAAAAUAGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3', and A pharmaceutical composition according to any one of claims 1 to 20, selected from one of the following sequences: 5'-GUUUGAGAGCUAUGCUGGAAAACAGCCAAUUGCAAAAAUAGCUAGUCCGUUAUCAACUUGAGAGCAACCGAGUGCGUGC-3', or a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% similarity or identity with one of the above sequences.

22. The pharmaceutical composition according to any one of claims 1 to 21, wherein the first scaffold and / or the second scaffold comprises a modified nucleotide.

23. The aforementioned scaffold has the following nucleotide sequence: A pharmaceutical composition according to any one of claims 1 to 22, comprising one or more of the modified nucleotides in 5'-mGUUUUAGmAmGmCmUmAGmAmAmUmAmGmGmCmAGUUMAAAmAmUAmGmGmCmUmAGUUMCmCGUUAmUmCAAamCmUmUGmAmAmAmAmGmUmGGmCmAmCmCmGmAmGmUmCmGmC3' (wherein mN is 2'-O-methylribose).

24. The aforementioned scaffolding is arranged as follows: A pharmaceutical composition according to any one of claims 1 to 23, comprising 5'-mGUUUUAGmAmGmCmUmAGmAmAmUmAmGmCmAmAGUUmAAAmUAmAmGmGmCmUmAGUUmCAMUmCAAmCmUmUGmAmAmAmAmAmGmUmGGmCmAmCmCmGmAmGmUmCmGmC3 (wherein mN is 2'-O-methylribose), or a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity with the above sequence.

25. The pharmaceutical composition according to any one of claims 1 to 24, wherein the first guide oligonucleotide and / or the second guide oligonucleotide contains an RNA motif at the 3' end of the guide oligonucleotide.

26. The pharmaceutical composition according to claim 25, wherein the RNA motif includes, is essentially composed of, or consists of 5'-UUU-3'.

27. The aforementioned RNA motif is 5'- ＊ mU ＊ mU ＊ containing, essentially having, or being having, mU-3' in the formula, where "mU ＊ The pharmaceutical composition according to claim 25, wherein " represents a phosphorothioate-modified 2'-O-methyluracil base and "mU" represents a 2'-O-methyluracil base.

28. The pharmaceutical composition according to any one of claims 1 to 27, wherein the weight ratio of the total weight of the first guide oligonucleotide and the second guide oligonucleotide to the weight of the mRNA is 1:1 ± 25%.

29. The pharmaceutical composition according to any one of claims 1 to 27, wherein the weight ratio of the first guide oligonucleotide to the second guide oligonucleotide is 1:1 ± 25%.

30. A gene editing system according to claim 2, or any one of claims 3 to 29 dependent on claim 2, wherein the first position and the second position are separated by less than 200 nucleotides.

31. The gene editing system according to claim 30, wherein the first position and the second position are separated by 20 to 50 nucleotides.

32. The pharmaceutical composition according to any one of claims 1 to 31, wherein administration of the composition to hepatocytes results in an editing efficiency of 40% or more.

33. The pharmaceutical composition according to claim 32, wherein the liver cells are primary liver cells.

34. The pharmaceutical composition according to claim 32 or 33, wherein the liver cells are human liver cells.

35. A method for treating and preventing cardiovascular disease in mammals by inactivating the LPA gene in vivo, the method comprising the step of administering to the subject a pharmaceutical composition according to any one of claims 1 to 34.

36. A method for treating and preventing cardiovascular disease in a mammal by reducing blood Lp(a) concentration, comprising the step of administering to the subject a pharmaceutical composition according to any one of claims 1 to 34.

37. A method for treating and / or preventing cardiovascular disease associated with the LPA gene in a mammal, comprising the step of administering to the subject a pharmaceutical composition according to any one of claims 1 to 34.

38. A method for in vivo editing of the LPA gene in a mammal, comprising the step of administering to the subject a pharmaceutical composition according to any one of claims 1 to 34.

39. A method for in vivo editing of the LPA gene in mammals, The aforementioned target, (i) One or more polynucleotides (mRNAs) encoding one or more CRISPR Cas nickase, (ii) A first guide oligonucleotide (gRNA) comprising a first spacer sequence and a scaffold region, (iii) A second guide oligonucleotide (gRNA) comprising a second spacer sequence and a scaffold region, (iv) Administering a pharmaceutical composition comprising a delivery system configured to deliver one or more mRNAs, the first gRNA, and / or the second gRNA individually or collectively to the liver, The first gRNA and at least one of the one or more Cas nickases are manipulated such that at least one of the one or more Cas nickases causes a nick to be inserted into either the first or second strand of the LPA gene at a first position between positions 160,664,275 and 160,531,482 of chromosome 6. A method wherein the second gRNA and at least one of the one or more Cas nickases are manipulated to cause at least one of the one or more Cas nickases to insert a nick into the other of the first or second strand of the LPA gene at a second position between positions 160,664,275 and 160,531,482 of chromosome 6.

40. A gene editing system for editing the LPA gene, which is produced by expressing one or more exogenous polynucleotides (mRNAs) encoding one or more CRISPR Cas nickase within a cell, and introducing a first gRNA and a second gRNA into the cell, The first guide oligonucleotide (gRNA) comprises (i) a first spacer sequence complementary to the first strand of the LPA gene in a first target sequence, and (ii) a first scaffold region that functions as a binding scaffold for at least one of the one or more Cas nickases. The second guide oligonucleotide (gRNA) comprises (i) a second spacer sequence complementary to the second strand of the LPA gene in the second target sequence, and (ii) a second scaffold region that functions as a binding scaffold for at least one of the one or more Cas nickases. The first gRNA and at least one of the one or more Cas nickases are manipulated to cause at least one of the one or more Cas nickases to insert a nick into either the first or second strand of the LPA gene at a first position between positions 160,664,275 and 160,531,482 of chromosome 6. A gene editing system in which the second gRNA and at least one of the one or more Cas nickases are manipulated to cause at least one of the one or more Cas nickases to insert a nick into the other of the first or second strand of the LPA gene at a second position between positions 160,664,275 and 160,531,482 of chromosome 6.

41. It is a gene editing system, A method for expressing one or more CRISPR Cas nickases within a cell, A gene editing system comprising means for directing one or more Cas nickases to a first position and a second position within the LPA gene, for causing one or more Cas nickases to introduce a nick into the first strand of the LPA gene, and for causing a nick into the second strand of the LPA gene.

42. A pharmaceutical composition comprising a gene editing system according to claim 41 and a delivery system.

43. The pharmaceutical composition according to claim 42, wherein the delivery system includes means for delivering to the cell the means for expressing one or more CRISPR Cas nickases, and the means for directing the one or more Cas nickases to a first position and a second position within the LPA gene.

44. A gene editing system for editing the aforementioned LPA gene, Cas nickas or nucleic acid encoding the said Cas nickas, (i) a first spacer sequence complementary to the first strand of the LPA gene in a first target sequence, and (ii) a first scaffold region that functions as a binding scaffold for the Cas nickase, (i) a second spacer sequence complementary to the second strand of the LPA gene in a second target sequence, and (ii) a second guide oligonucleotide comprising a second scaffold region that functions as a binding scaffold for the Cas nickase, The first guide oligonucleotide and the Cas nickase are manipulated such that the Cas nickase causes a nick to be inserted into either the first or second strand of the LPA gene at a first position. The second guide oligonucleotide and the Cas nickase are manipulated such that the Cas nickase causes a nick to be inserted at a second position into the other of the first or second strands of the LPA gene. A gene editing system in which the first position and the second position are separated by 1 to 200 nucleotides.

45. The gene editing system according to claim 44, wherein the first position and the second position are separated by 20 to 50 nucleotides.

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