Composition and method for preventing or treating cardiovascular disease

Abstract

The present invention provides a composition and method for preventing or treating a cardiovascular disease. The composition can reduce the Lp(a) level by editing an LPA gene, thereby reducing the risk of developing a cardiovascular disease or treating the cardiovascular disease. The composition comprises: (i) a nucleic acid encoding one or more guide RNAs, or a vector comprising the nucleic acid encoding the one or more guide RNAs; and (ii) an RNA-guided DNA binder, a nucleic acid encoding the RNA-guided DNA binder, or a vector comprising the nucleic acid encoding the RNA-guided DNA binder.

Description

Compositions and methods for the prevention or treatment of cardiovascular diseases Technical Field

[0001] This disclosure pertains to the biomedical field. It relates to compositions and methods for the prevention or treatment of cardiovascular diseases. Specifically, the compositions of this disclosure can significantly reduce the risk of developing cardiovascular diseases or effectively treat cardiovascular diseases by editing the LPA gene to lower Lp(a) levels. Background Technology

[0002] Lipoprotein(a) (abbreviated as Lp(a)) is a lipoprotein structurally similar to low-density lipoprotein (LDL), containing a unique apolipoprotein, Apo(a). Apo(a) is primarily produced in the liver and is covalently linked to the Apo(b) moiety via disulfide bonds. Apo(a) is encoded by the LPA gene. Apo(a) contains multiple repeats of Kringle IV (K-IV), one Kringle V (KV), and an inactivated protease domain. More than 40 Apo(a) protein isoforms have been identified, which can directly affect Lp(a) levels. 90% of human Lp(a) levels are genetically determined and almost entirely unaffected by environmental or lifestyle factors; postmenopausal women have 17% higher Lp(a) levels than men. G Nordestgaard, Anne Langsted, Lancet. 2024 Sep 28; 404(10459):1255-1264. doi:10.1016 / S0140-6736(24)01308-4.). Plasma Lp(a) levels are mainly determined by the repeat polymorphism of K-IV on Apo(a), and are negatively correlated with the number of K-IV repeats. Some functional mutations can significantly affect Lp(a) levels.

[0003] Studies have shown that elevated Lp(a) is an independent risk factor for various cardiovascular diseases (CVDs, including myocardial infarction, ischemic stroke, and aortic stenosis). Its pathogenic mechanisms may involve processes such as atherosclerosis, inflammatory responses, and thrombosis. Lp(a) levels vary greatly in the population, ranging from 0.01 mg / dL to 300 mg / dL. Currently, many national guidelines set the risk threshold for Lp(a) at 50 mg / dL (Florian Kronenberg et al., European Heart Journal (2022) 43, 3925–3946. doi:10.1093 / eurheartj / ehac361.). It has been reported that approximately 1.4 billion people worldwide have Lp(a) levels exceeding 50 mg / dL, meaning that about 20% of the population faces lifetime risk for cardiovascular disease.

[0004] Currently, there are no approved drugs that can effectively and specifically lower Lp(a) levels. Several related specific treatments are under active development. Small interfering RNA (siRNA) and antisense oligonucleotide (ASO) therapies are among the main strategies. These therapies reduce Lp(a) synthesis by targeting the mRNA of the LPA gene, thereby decreasing its levels in the blood. Related phase 3 clinical trials are currently underway. While these therapies have yielded encouraging results, therapies that can produce durable inhibition of the LPA gene are still needed to lower Lp(a) levels for long-term prevention or treatment of cardiovascular disease. Summary of the Invention

[0005] The rapidly developing in vivo gene editing therapy in recent years has brought hope for the treatment and cure of many gene-related diseases. For subjects with high Lp(a) levels, it is possible to steadily reduce Lp(a) levels with a single dose, thereby significantly reducing cardiovascular risk or effectively treating cardiovascular diseases. Therefore, the inventors of this invention sought to find compositions and methods that can reduce Lp(a) levels by editing the LPA gene.

[0006] The purpose of this disclosure is to provide compositions and methods for the prevention or treatment of cardiovascular diseases.

[0007] The purpose of this disclosure is achieved by providing a composition for gene editing of the LPA gene. The composition of this disclosure can reduce Lp(a) levels by editing the LPA gene, thereby significantly reducing the risk of cardiovascular disease or effectively treating cardiovascular disease.

[0008] In a first aspect, this disclosure provides a guide RNA comprising:

[0009] a) A sequence selected from any of SEQ ID NO: 1-155 and 168-337, preferably selected from SEQ ID NO: 35, 34, 38, 39, 42, 37, 41, 40, 33, 28, 14, 3, 24, 18, 46, 15, 29, 16, 19, 55, 66, 68, 88, 96, 94, 97, 82, 83, 84, 86, 95, 93, 105, 106, 107, 109, 129, 130, 135, 138, 140, 141, 62, 183, 184. Any sequence of 187, 191, 193, 196, 215, 219, 220, 224, 235, 241, 245, 225, 243, 233, 246, 247, 252, 267, 251, 277, 278, 279, 282, 283, 285, 299, 294, 317, 303, 298, 306, 325, 320, 330, or 333-337;

[0010] b) At least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides selected from any sequence of SEQ ID NO: 1-155 and 168-337; or

[0011] c) A sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any sequence selected from SEQ ID NO:1-155 and 168-337.

[0012] In some implementations, the guide RNA comprises:

[0013] a) Selected from SEQ ID NO: 35, 34, 38, 39, 42, 37, 41, 40, 33, 28, 14, 3, 24, 18, 46, 15, 29, 16, 19, 55, 66, 68, 88, 96, 94, 97, 82, 83, 84, 86, 95, 93, 105, 106, 107, 109, 129, 130, 135, 138, 140, 141, 62, 183, 184. Any sequence of 187, 191, 193, 196, 215, 219, 220, 224, 235, 241, 245, 225, 243, 233, 246, 247, 252, 267, 251, 277, 278, 279, 282, 283, 285, 299, 294, 317, 303, 298, 306, 325, 320, 330, or 333-337;

[0014] b) Selected from SEQ ID NO: 35, 34, 38, 39, 42, 37, 41, 40, 33, 28, 14, 3, 24, 18, 46, 15, 29, 16, 19, 55, 66, 68, 88, 96, 94, 97, 82, 83, 84, 86, 95, 93, 105, 106, 107, 109, 129, 130, 135, 138, 140, 141, 62, 183, 184, 187, 191, 193, 196 At least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides of any sequence of the following sequences: 215, 219, 220, 224, 235, 241, 245, 225, 243, 233, 246, 247, 252, 267, 251, 277, 278, 279, 282, 283, 285, 299, 294, 317, 303, 298, 306, 325, 320, 330, or 333-337; or

[0015] c) and selected from SEQ ID NO: 35, 34, 38, 39, 42, 37, 41, 40, 33, 28, 14, 3, 24, 18, 46, 15, 29, 16, 19, 55, 66, 68, 88, 96, 94, 97, 82, 83, 84, 86, 95, 93, 105, 106, 107, 109, 129, 130, 135, 138, 140, 141, 62, 183, 184, 187, 191, 193, 196, 215, 219 The sequence 220, 224, 235, 241, 245, 225, 243, 233, 246, 247, 252, 267, 251, 277, 278, 279, 282, 283, 285, 299, 294, 317, 303, 298, 306, 325, 320, 330 or 333-337 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

[0016] In some embodiments, the guide RNA is at least partially complementary to a target sequence present in the human LPA gene. In some embodiments, the target sequence is located in any one of exons 2, 8, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 31, 32, 33, 34, 35, 36, 37, or 38 of the human LPA gene. In some embodiments, the target sequence is located at the junction of an exon and an intron of the human LPA gene. In some embodiments, the guide RNA sequence is complementary to a target sequence in the positive strand of the LPA gene. In some embodiments, the guide RNA sequence is complementary to a target sequence in the negative strand of the LPA gene. In some embodiments, the guide RNA comprises a first guide sequence and a second guide sequence, wherein the first guide sequence is complementary to a first target sequence in the positive strand of the LPA gene, and the second guide sequence is complementary to a second target sequence in the negative strand of the LPA gene.

[0017] In some embodiments, the guide RNA comprises crRNA, and optionally also comprises tracrRNA (trRNA) or a portion thereof, wherein the trRNA is operatively linked to the crRNA.

[0018] In some implementations, the guide RNA is a dual guide RNA (dgRNA). In other implementations, the guide RNA is a single guide RNA (sgRNA).

[0019] In some embodiments, the guide RNA comprises at least one modification. In some embodiments, said at least one modification includes a 2'-O-methyl (2'-O-Me) modified nucleotide, an internucleotide phosphate-thioester (PS) bond, a 2'-fluorine (2'-F) modified nucleotide, or a DNA-RNA hybrid. In some embodiments, said at least one modification is performed at one or more of the first 22 nucleotides at the 5' end of the guide RNA and / or at one or more of the last 22 nucleotides at the 3' end of the guide RNA. In some embodiments, at least 50% of the nucleotides in the guide RNA are modified.

[0020] In some embodiments, the guide RNA comprises the sequence shown in SEQ ID NO:41, and one or more bases at positions 8-11 are modified with 2'-fluorination. In other embodiments, the guide RNA comprises the sequence shown in SEQ ID NO:34, and one or more bases at positions 4, 5, 11, and 12 are deoxyribonucleotides.

[0021] In some implementations, the guide RNA or the nucleic acid encoding it is associated with or encapsulated within lipid nanoparticles (LNPs).

[0022] In a second aspect, this disclosure provides a vector comprising one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise:

[0023] a) A sequence selected from any of SEQ ID NO: 1-155 and 168-337, preferably selected from SEQ ID NO: 35, 34, 38, 39, 42, 37, 41, 40, 33, 28, 14, 3, 24, 18, 46, 15, 29, 16, 19, 55, 66, 68, 88, 96, 94, 97, 82, 83, 84, 86, 95, 93, 105, 106, 107, 109, 129, 130, 135, 138, 140, 141, 62, 183, 184. Any sequence of 187, 191, 193, 196, 215, 219, 220, 224, 235, 241, 245, 225, 243, 233, 246, 247, 252, 267, 251, 277, 278, 279, 282, 283, 285, 299, 294, 317, 303, 298, 306, 325, 320, 330, or 333-337;

[0024] b) At least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides selected from any sequence of SEQ ID NO: 1-155 and 168-337; or

[0025] c) A sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any sequence selected from SEQ ID NO:1-155 and 168-337.

[0026] In a third aspect, this disclosure provides a composition comprising:

[0027] (i) a nucleic acid encoding one or more guide RNAs, or a vector containing a nucleic acid encoding one or more guide RNAs, wherein the guide RNA comprises:

[0028] a) A sequence selected from any of SEQ ID NO: 1-155 and 168-337, preferably selected from SEQ ID NO: 35, 34, 38, 39, 42, 37, 41, 40, 33, 28, 14, 3, 24, 18, 46, 15, 29, 16, 19, 55, 66, 68, 88, 96, 94, 97, 82, 83, 84, 86, 95, 93, 105, 106, 107, 109, 129, 130, 135, 138, 140, 141, 62, 183, 184. Any sequence of 187, 191, 193, 196, 215, 219, 220, 224, 235, 241, 245, 225, 243, 233, 246, 247, 252, 267, 251, 277, 278, 279, 282, 283, 285, 299, 294, 317, 303, 298, 306, 325, 320, 330, or 333-337;

[0029] b) At least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides selected from any sequence of SEQ ID NO: 1-155 and 168-337; or

[0030] c) A sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any sequence selected from SEQ ID NO: 1-155 and 168-337; and

[0031] (ii) RNA-guided DNA binders, nucleic acids encoding RNA-guided DNA binders, or vectors containing nucleic acids encoding RNA-guided DNA binders.

[0032] In some embodiments, the composition comprises nucleic acids encoding two guide RNAs, preferably, the two guide RNAs being at least partially complementary to different target sequences present in the human LPA gene, and more preferably, the two guide RNAs being at least partially complementary to different target sequences present in the same exon of the human LPA gene.

[0033] In some implementations, the RNA-guided DNA binder comprises a Cas nuclease or a Cas nickase.

[0034] In some embodiments, the Cas nuclease is a type II Cas nuclease. In some embodiments, the Cas nuclease is derived from a type II CRISPR / Cas system. In some embodiments, the Cas nuclease is Cas9, Cpfl, C2cl, C2c2, C2c3, Cas12, or a modified protein thereof. In some embodiments, the Cas nuclease is *Streptococcus pyogenes* Cas9 nuclease, *Faecalibaculum rodentium* Cas9 nuclease, *Staphylococcus haemolyticus* Sha2Cas9 nuclease, or *Staphylococcus aureus* Cas9 nuclease, Cas12i, or a modified protein thereof; for example, the RNA-guided DNA binder comprises any amino acid sequence of SEQ ID NO: 156, 158, 160, 162, 338, 340, or 342.

[0035] In some embodiments, the composition is a pharmaceutical preparation and also contains pharmaceutically acceptable excipients or carriers.

[0036] In some implementations, the vector is an expression vector, such as a viral vector, including but not limited to: adeno-associated virus (AAV) vectors, retroviral vectors, adenovirus vectors, lentiviral vectors, Sendai virus vectors, and herpesvirus vectors.

[0037] In some implementations, the polynucleotide may be codon-optimized for better expression in host cells.

[0038] In some embodiments, the composition further comprises:

[0039] (iii) Cytidine deaminase or adenosine deaminase, or a nucleic acid encoding cytidine deaminase or a carrier containing a nucleic acid encoding cytidine deaminase or a nucleic acid encoding cytidine deaminase.

[0040] In some embodiments, the compositions of this disclosure are encapsulated within an LNP. For example, one or more guide RNAs combined with a DNA binding agent are encapsulated within an LNP.

[0041] In a fourth aspect, this disclosure provides a nucleic acid molecule that encodes a nucleotide sequence of one or more guide RNAs as described in the first aspect, or comprises a nucleotide sequence encoding one or more guide RNAs as described in the first aspect and an RNA-guided DNA binder.

[0042] In some implementations, the RNA-guided DNA binder is as described in the third aspect.

[0043] In a fifth aspect, this disclosure provides an expression vector comprising the nucleic acid molecule described in the fourth aspect.

[0044] In a sixth aspect, this disclosure provides a cell comprising the nucleic acid molecule described in the fourth aspect or the expression vector described in the fifth aspect.

[0045] In some embodiments, the cell may be a prokaryotic cell or a eukaryotic cell, such as a bacterial cell (e.g., Escherichia coli cell), a fungal cell (e.g., yeast cell), or a mammalian cell (e.g., human cell).

[0046] In a seventh aspect, this disclosure provides a lipid nanoparticle (LNP) that is associated with one or more guide RNAs or nucleic acids encoding the same as described in the first aspect, or the LNP encapsulates one or more guide RNAs or nucleic acids encoding the same as described in the first aspect.

[0047] Optionally, the LNP is associated with two or more guide RNAs or nucleic acids encoding the first aspect, or encapsulates two or more guide RNAs or nucleic acids encoding the first aspect.

[0048] In some implementations, when LNP encapsulates two or more guide RNAs, the two or more guide RNAs may each be present in equal amounts.

[0049] In some embodiments, the LNP further encapsulates an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder. In some embodiments, the nucleic acid encoding the RNA-guided DNA binder may be present in a suitable mass ratio to the total guide RNA, which can be determined by those skilled in the art as needed, for example, in a mass ratio of 1:1 to 2:1 (e.g., 1:1 or 2:1) in the LNP. In some embodiments, the DNA binder, as described in the third aspect, optionally comprises any amino acid sequence from SEQ ID NO: 156, 158, 160, 162, 338, 340, or 342.

[0050] In some embodiments, the LNP encapsulates two guide RNAs described in the first aspect. Preferably, the two guide RNAs are at least partially complementary to different target sequences present in the human LPA gene. More preferably, the two guide RNAs are at least partially complementary to different target sequences present in the same exon of the human LPA gene.

[0051] Optionally, the two guide RNAs each contain:

[0052] (i) the sequences shown in SEQ ID NO:34 and SEQ ID NO:42, or

[0053] (ii) the sequences shown in SEQ ID NO:34 and SEQ ID NO:41, or

[0054] (iii) The sequences shown in SEQ ID NO:40 and SEQ ID NO:42, or

[0055] (iv) The sequences shown in SEQ ID NO:42 and SEQ ID NO:44;

[0056] Optionally, the two guide RNAs are present in a 1:1 mass ratio.

[0057] In some embodiments, the LNP comprises ALC0315, DSPC, cholesterol, and DMG-PEG2000. In some embodiments, the N / P ratio of the LNP is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the N / P ratio is approximately 3-7. In some embodiments, ALC0315 comprises 40 mol% to 60 mol% of the total lipids present in the particles, DSPC comprises 5 mol% to 15 mol% of the total lipids present in the particles, cholesterol comprises 30 mol% to 50 mol% of the total lipids present in the particles, and DMG-PEG2000 comprises 1 mol% to 5 mol% of the total lipids present in the particles. In some embodiments, the LNP comprises ALC0315, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 50:9.5:37.5:3.

[0058] In an eighth aspect, this disclosure provides a method for editing a human LPA gene, the method comprising applying a composition described in the third aspect of this disclosure or an LNP described in the seventh aspect to a cell, the composition or LNP recognizing and editing a target sequence in the LPA gene.

[0059] In some implementations, the method is capable of modifying the human LPA gene and / or inducing double-strand breaks (DSBs) within the human LPA gene.

[0060] In some embodiments, the method is performed in vitro or in vivo. In some embodiments, the method is used for therapeutic purposes or for non-therapeutic purposes.

[0061] In some embodiments, editing is calculated as the percentage of the cell population that was edited (editing percentage). In some embodiments, approximately 30% to 99% of the cell population was edited. In some embodiments, the editing percentage is 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% of the cell population.

[0062] In a ninth aspect, this disclosure provides a method for reducing lipoprotein (a) levels in a subject, the method comprising administering to the subject the composition described in the third aspect of this disclosure or the LNP described in the seventh aspect of this disclosure.

[0063] In some embodiments, after administration of the composition or LNP of this disclosure, the lipoprotein(a) level in the subject may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to the corresponding negative control or the lipoprotein(a) level measured in the subject before administration of the composition or LNP.

[0064] In a tenth aspect, this disclosure provides a method for preventing or treating cardiovascular disease in a subject, the method comprising administering to the subject an effective amount of the composition described in the third aspect of this disclosure or the LNP described in the seventh aspect.

[0065] In some embodiments, the method further includes administering an additional therapy to the subject. In some embodiments, the additional therapy is administered before or after administration of the composition of this disclosure or LNP. In some embodiments, the additional therapy is a therapy for cardiovascular disease. In some embodiments, the therapy for cardiovascular disease is administration of a cardiovascular disease treatment agent, such as a statin, for example, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, or simvastatin; a cholesterol absorption inhibitor, for example, ezetimibe; or a bile acid conjugate, for example, cholestyramine, colesvelam, or colestipol.

[0066] In some implementations, the cardiovascular disease includes, but is not limited to: myocardial infarction, ischemic stroke, aortic stenosis, coronary artery stenosis, carotid artery stenosis, heart failure, peripheral artery disease, atherosclerosis, or thrombosis.

[0067] In some embodiments, the composition or LNP can be administered by injection, such as intravenous injection, infusion, subcutaneous injection, intramuscular injection, intradermal injection, arterial injection, intraperitoneal injection, etc.

[0068] In some embodiments, the composition or LNP may be applied or delivered at least once. In some embodiments, the application or delivery is performed at the following intervals: (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days; (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks; (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months; or (d) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0069] In some embodiments, administration of the composition or LNP results in the deletion, insertion, or substitution of one or more nucleotides in the LPA gene. In some embodiments, the deletion, insertion, or substitution of one or more nucleotides induces a frameshift, nonsense, or missense mutation in the LPA gene. In some embodiments, a frameshift, nonsense, or missense mutation is induced in the LPA gene of about 20% to about 30% of cells. In some embodiments, the cells are hepatocytes. In some embodiments, the cells are kidney cells or adrenal cells. In some embodiments, the deletion, insertion, or substitution of one or more nucleotides in the LPA gene occurs at least 50 times more than at off-target sites.

[0070] In some embodiments, the composition or LNP reduces the level of lipoprotein(a) in the subject's cells. In some embodiments, the level of lipoprotein(a) in plasma is measured. In some embodiments, the subject's plasma lipoprotein(a) level is reduced by approximately 30% after 8 weeks of administration of the composition or LNP, compared to the subject's plasma lipoprotein(a) level measured before administration of the composition or LNP.

[0071] In some embodiments, the subject is at risk of developing cardiovascular disease, for example, by having high lipoprotein(a) levels or a family history of cardiovascular disease. In some embodiments, administration of the compositions of this disclosure or LNP can reduce the subject's risk of developing vascular disease.

[0072] In some embodiments, the subject is a patient with cardiovascular disease. In some embodiments, the cardiovascular disease may be one or more selected from myocardial infarction, ischemic stroke, aortic stenosis, coronary artery stenosis, carotid artery stenosis, heart failure, peripheral artery disease, atherosclerosis, and thrombosis.

[0073] In some embodiments, after administration of the compositions of this disclosure or LNP, the subjects exhibited improvement, stabilization, or slowed change in cardiovascular disease symptoms.

[0074] In an eleventh aspect, this disclosure provides a pharmaceutical formulation or pharmaceutical composition comprising the composition described in the third aspect of this disclosure, the nucleic acid molecule described in the fourth aspect, the expression vector described in the fifth aspect, or the LNP described in the seventh aspect, and a pharmaceutically acceptable excipient or carrier.

[0075] In a twelfth aspect, this disclosure provides a kit comprising the composition described in the third aspect, the nucleic acid molecule described in the fourth aspect, the expression vector described in the fifth aspect, or the LNP described in the seventh aspect, and instructions for use.

[0076] In a thirteenth aspect, this disclosure provides the use of the guide RNA described in the first aspect, or the vector described in the second aspect, or the composition described in the third aspect, or the nucleic acid molecule described in the fourth aspect, or the expression vector described in the fifth aspect, or the LNP described in the seventh aspect in the preparation of a medicament or kit, wherein the medicament or kit is used to edit the human LPA gene, reduce lipoprotein(a) levels in a subject, or prevent or treat cardiovascular disease in a subject.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Methods and materials used in this disclosure are described herein; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification (including definitions) shall prevail.

[0078] Other features and advantages of this disclosure will be apparent from the following detailed description, the accompanying drawings, and the claims. Attached Figure Description

[0079] Figures 1A, 1B, and 1C show the activity assays of spCas9 mRNA and sgRNA encapsulated in LNP as described in Example 13 in primary human hepatocytes (PHH cells).

[0080] Figure 2 shows the decrease in plasma lipoprotein (a) levels in mice after LNP injection.

[0081] Figures 3A and 3B show the activity assay of modified sgRNA encapsulated in LNP in PHH cells.

[0082] Figure 4 shows the activity assay of the combined sgRNA encapsulated in LNP in PHH cells.

[0083] Figure 5 shows the activity assay of Cas12i mRNA and sgRNA encapsulated in LNP in PHH cells. Detailed Implementation

[0084] This disclosure describes compositions and methods for editing the human LPA gene. The compositions and methods described herein are intended for the prevention or treatment of cardiovascular disease in subjects associated with the LPA gene.

[0085] definition

[0086] Unless otherwise stated, the following terms and phrases used herein shall have the following meanings:

[0087] As used herein, the term "guide RNA" refers to a combination of CRISPR RNA (crRNA) and tracr RNA (trRNA). "Guide RNA" is used interchangeably with "gRNA" or "guide". crRNA and trRNA can associate as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" can refer to each type, i.e., sgRNA or dgRNA. trRNA can be a naturally occurring sequence, or the trRNA sequence can be modified or varied compared to a naturally occurring sequence. Guide RNA can include the modified RNAs described herein. Additionally, guide RNA for Cas12i refers only to crRNA, as Cas12i does not require a corresponding trRNA.

[0088] As used herein, a “guide sequence” refers to a sequence within a guide RNA that is complementary to the target sequence and serves to guide the guide RNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binder. A “guide sequence” may also be referred to as a “target sequence” or a “spacer sequence.” The guide sequence can be approximately 20 base pairs long, for example, in the case of *Streptococcus pyogenes* (i.e., Spy Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences may also be used as guide sequences, for example, 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the guide sequence and the target sequence may be 100% complementary or identical to each other. In other embodiments, the guide sequence and the target sequence may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, wherein the total length of the target sequence is at least 17, 18, 19, 20, 21, or more base pairs. In some embodiments, the guide sequence and target sequence may contain 1-4 mismatches, wherein the guide sequence contains at least 17, 18, 19, 20, 21 or more nucleotides. In some embodiments, the guide sequence and target sequence may contain 1, 2, 3 or 4 mismatches, wherein the guide sequence contains at least 20 nucleotides.

[0089] In some embodiments, the guide RNA comprises crRNA having a guide sequence. In some embodiments, the guide RNA comprises crRNA and also comprises a tracrRNA (trRNA) sequence.

[0090] As used in this article, "target sequence" refers to a nucleic acid sequence in the target gene that is complementary to the guide sequence of the gRNA. The interaction between the target sequence and the guide sequence guides the RNA-guided DNA binder to bind within the target sequence and may create a nick or cleavage within the target sequence (depending on the activity of the binder).

[0091] The target sequences of Cas proteins include both the positive and negative strands of genomic DNA (i.e., a given sequence and its inverse complement), because the nucleic acid substrate of Cas proteins is double-stranded. Therefore, when a guide sequence is described as "complementary to the target sequence," it should be understood that the guide sequence can guide the guide RNA to bind to the inverse complement of the target sequence. Thus, in some embodiments where the guide sequence binds to the inverse complement of the target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence excluding the protospacer adjacent motif (PAM)) except that T is replaced with U in the guide sequence.

[0092] As used herein, a PAM, also known as a protospacer adjacent motif, is a short sequence located immediately adjacent to the target DNA sequence and is essential for the recognition and binding of Cas nucleases. For example, for Cas9 (including SpCas9, Sha2Cas9, and FrCas9), the PAM is located approximately 2–8 nucleotides downstream of the DNA sequence targeted by the guide RNA, and Cas cleaves it 3–4 nucleotides upstream. For Cas12, the PAM is located approximately 1–6 nucleotides upstream of the DNA sequence targeted by the guide RNA. PAM sequences are illustrated in Tables 1, 8, 10, 12, 14, 16, 18, 20, and 22. The PAM in this disclosure may be any of the sequences in Tables 1, 8, 10, 12, 14, 16, 18, 20, and 22 or any other sequence known in the art.

[0093] As used herein, "RNA-guided DNA binder" refers to a polypeptide or polypeptide complex having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binders (such as those described in International Patent Application No. WO2020198697, the entire contents of which are incorporated herein) include Cas nucleases or Cas nickases and their inactivated forms, such as dCas DNA binders.

[0094] As used herein, the term “Cas” refers to any Cas protein that can be operatively used to perform gene editing using a guide molecule. “Cas nuclease” also encompasses Cas nickases and Cas (dCas) DNA binders that are endonuclease-deficient or inactivated. Cas nickases and dCas DNA binders can include: Csm or Cmr complexes of type III CRISPR systems, with their Cas10, Csml, or Cmr2 subunits; Cascade complexes of type I CRISPR systems, with their Cas3 subunits; and class 2 Cas nucleases. As used herein, “class 2 Cas nucleases” are single-stranded polypeptides with RNA-guided DNA-binding activity, such as Cas9 or Cpfl nucleases. Class 2 Cas nucleases include class 2 Cas nickases (e.g., variants of H840A, D10A, or N863A) that also have RNA-guided DNA nickase activity, and class 2 dCas DNA binders in which the nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, C2c3, Cas12i, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and their modifications. The Cpfl protein (Zetsche et al., Cell, 163:1-13 (2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain. The Cpfl sequence from Zetsche et al.'s work is incorporated herein by reference in its entirety. See, for example, Zetsche et al., Tables S1 and S3. “Cas9” encompasses Spy Cas9, the Cas9 variants listed herein, and their equivalents. See, for example, Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).Cas12i (including Cas12i1 and Cas12i2) refers to the VI subtype CRISPR system, which is functionally similar to Cas12a. It can self-process precursor crRNA and does not require tracrRNA (trRNA) when cleaving target DNA (dsDNA). Compared to SpCas9 and Cas12a, Cas12i effectors are smaller (10³³-10⁹³ Aa), and with the short mature crRNA (40-43 nt) used for it, Cas12i is particularly suitable for multiplex genome editing and viral vector-based delivery (Colin McGaw et al., NATURE COMMUNICATIONS (2022) 13:2833, https: / / doi.org / 10.1038 / s41467-022-30465-7; Winston X. Yan et al., Science 6 Dec 2018, Vol 363, Issue). 6422, pp.88-91, DOI:10.1126 / science.aav7271).

[0095] As used herein, if an alignment of a first sequence with a second sequence shows that the second sequence as a whole matches the first sequence at X% or more positions, then the first sequence is considered to "contain at least X% of the same sequence as the second sequence". For example, the sequence AAGA contains a sequence with 100% identity to the sequence AAG because the alignment would produce 100% identity due to matching at all three positions of the second sequence. Differences between RNA and DNA (often the exchange of uridine with thymidine or vice versa) and the presence of nucleoside analogs (such as modified uridine) do not result in differences in identity or complementarity between polynucleotides, provided that the relevant nucleotide (such as thymidine, uridine, or modified uridine) binds to the same complementary nucleotide (e.g., adenosine is the complement of all thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which are complements of guanosine or modified guanosine). Therefore, for example, the sequence 5'-AXG (where X is any modified uridine, such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine) is considered 100% identical to AUG because both are perfectly complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. Those skilled in the art will understand which algorithm and parameter settings are appropriate for a given sequence pair to be aligned; for sequences that are generally similar in length and have an expected amino acid identity >50% or an expected nucleotide identity >75%, the Needleman-Wunsch algorithm using the default settings of the Needleman-Wunsch algorithm interface provided by EBI on the www.ebi.ac.uk web server is generally appropriate.

[0096] As used herein, the term "mRNA" refers to a polynucleotide that is RNA or modified RNA, including an open reading frame (i.e., a substrate that can be translated into a polypeptide) via ribosomes and aminoacylated tRNA. mRNA may include a phosphate-sugar backbone having ribose residues or analogues thereof (e.g., 2'-methoxyribose residues). In some embodiments, the sugars of the nucleic acid phosphate-sugar backbone are substantially composed of ribose residues, 2'-methoxyribose residues, or combinations thereof.

[0097] As used herein, “knockdown” refers to a reduction in the expression of a specific gene product (e.g., protein, mRNA, or both). Protein knockdown can be measured by detecting proteins secreted by a tissue or cell population (e.g., in serum or cell culture medium) or by detecting the total cellular amount of protein from the tissue or cell population of interest before and after knockdown. Methods for measuring mRNA knockdown are known in the art and include sequencing mRNA isolated from the tissue or cell population of interest. In some embodiments, “knockdown” can refer to some loss of expression of a specific gene product, such as a reduction in the amount of transcribed mRNA or a reduction in the amount of protein expressed or secreted by a cell population (including in vivo cell populations, such as those found in tissues).

[0098] As used herein, "knockout" refers to an experimental method that specifically renders a target gene in an organism inactive through genetic engineering techniques. This method typically utilizes gene editing tools (such as CRISPR-Cas9, TALENs, or homologous recombination) to directionally delete, insert, or modify the sequence of the target gene, causing it to be unable to express normally or produce functional proteins. In this disclosure, "knockout" refers to using the CRISPR-Cas system to introduce double-strand breaks into the DNA sequence of a specific gene (e.g., the LPA gene), leading to insertions or deletions through cellular repair mechanisms (such as non-homologous end joining), thereby disrupting the normal function of the gene; or using a base editor to introduce single-base substitutions, disrupting mRNA splicing or prematurely introducing stop codons to achieve gene inactivation.

[0099] As used herein, “prevention” means that, for subjects at risk of developing a certain disease (e.g., having a family history of cardiovascular disease or having elevated lipoprotein(a) levels), administration of the compositions of this disclosure significantly reduces the risk of developing the disease (e.g., significantly reduces lipoprotein(a) levels).

[0100] As used herein, "treatment" means that at least one symptom of the disclosed condition is improved, relieved, or alleviated after administration or application of a therapeutic agent for the disclosed condition. This term includes suppressing the condition or disease, preventing its progression, relieving one or more symptoms of the condition or disease, curing the condition or disease, or preventing the recurrence of one or more symptoms of the condition or disease. In the context of this disclosure, treatment of cardiovascular disease may include relieving the symptoms of cardiovascular disease. If treatment results in a reduction in the pathology of the condition, the use of the compositions of this disclosure is said to effectively "treat" the condition.

[0101] As used herein, the term "lipid nanoparticle (LNP)" refers to a particle containing multiple (i.e., more than one) lipid molecules physically associated with each other by intermolecular forces. An LNP can be, for example, a microsphere (including monolayer and multilayer vesicles, such as "liposomes"—a lamellar lipid bilayer, substantially spherical in some embodiments—and in more specific embodiments, may contain an aqueous core, e.g., containing predominantly RNA molecules), a dispersed phase in an emulsion, a micelle, or an inner phase in a suspension. See also, for example, WO2015006747, WO2016118724, WO2021026358, WO2017173054, and WO2019067992, the contents of which are incorporated herein by reference in their entirety. Any LNP known to those skilled in the art capable of delivering nucleotides to a subject may be used in conjunction with a guide RNA and a nucleic acid encoding an RNA-guided DNA binder as described herein.

[0102] As used herein, the term “pharmaceutical acceptable” means a biologically acceptable gaseous, liquid, or solid formulation, or a mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A “pharmaceutical acceptable” composition is a material that will not cause biological or other adverse effects; for example, the material can be administered to a subject without causing significant adverse biological effects.

[0103] As used herein, “injection” or “infusion” means the active administration of one or more agents over a period of time, such as approximately 30 minutes to 12 hours. In some embodiments, one or more agents include LNPs, such as mRNA having encoding an RNA-guided DNA binder (such as Cas9) as described herein and gRNA as described herein.

[0104] The terms “about” or “approximately” refer to an acceptable error in a particular value as determined by a person skilled in the art, depending in part on how the value is measured or determined. In some embodiments, “about” means, for example, a difference of less than 5% (e.g., less than 1%, less than 0.5%, or less than 0.1%). A range of numbers includes the number that defines the range. Measured and measurable values ​​should be understood as approximate values, taking into account significant figures and errors associated with the measurement.

[0105] RNA-guided DNA binders

[0106] Any nucleic acid having an open reading frame encoding an RNA-guided DNA binder (e.g., Cas9 nuclease, such as Streptococcus pyogenes Cas9) may be combined with any gRNA disclosed herein in a composition or method. In some embodiments, the nucleic acid having an open reading frame encoding an RNA-guided DNA binder is mRNA. In some embodiments, the RNA-guided DNA binder is administered in its amino acid form (i.e., as a protein). In some embodiments, the nucleic acid encoding an RNA-guided DNA binder is part of the vector described herein. The nucleic acid encoding an RNA-guided DNA binder may have any of the features described in WO2020198697, which is incorporated herein by reference in its entirety.

[0107] In some embodiments, the RNA-guided DNA binder used in the compositions and methods described herein is a class 2 Cas nuclease. In some embodiments, the RNA-guided DNA binder has double-stranded endonuclease activity. In some embodiments, the RNA-guided DNA binder comprises a Cas nuclease, such as a class 2 Cas nuclease (which may be, for example, type II, V, or VI Cas nucleases). Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, C2c3, or Cas12 proteins and modified versions thereof. In some embodiments, the Cas nuclease is *Streptococcus pyogenes* Cas9 nuclease, *Faecalibaculum rodentium* Cas9 nuclease, *Staphylococcus haemolyticus* Sha2Cas9 nuclease, or *Staphylococcus aureus* Cas9 nuclease, Cas12i, or a modified protein thereof. In some embodiments, the Cas nuclease used is a modified Cas12i protein, for example, with the amino acid sequence shown in SEQ ID NO:162.

[0108] Examples of Cas9 nucleases include those from type II CRISPR systems of Streptococcus pyogenes, Staphylococcus aureus, and other prokaryotes, and their modified (e.g., engineered or mutant) versions. See, for example, US2016 / 0312198A1; US2016 / 0312199A1. Other examples of Cas nucleases include the Csm or Cmr complex of type III CRISPR systems or its Cas10, Csml, or Cmr2 subunits; and the Cascade complex of type I CRISPR systems or its Cas3 subunit. In some embodiments, the Cas nuclease may be derived from type IIA, type IIB, or type IIC systems. For discussions of various CRISPR systems and Cas nucleases, see, for example, Makarova et al., Nat. Rev. Microbiol. 9:467-477 (2011); Makarova et al., Nat. Rev. Microbiol, 13:722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015). In some embodiments, the RNA-guided DNA binder is a Cas nickase, such as Cas9 nickase. In some embodiments, the RNA-guided DNA binder is Streptococcus pyogenes Cas9 nuclease. In some embodiments, the RNA-guided DNA binder is Cas12i. Cas12i (including Cas12i1 and Cas12i2) refers to the VI subtype CRISPR system (belonging to class 2 Cas nucleases). Functionally similar to Cas12a, it can self-process precursor crRNA and does not require tracrRNA (trRNA) when cleaving target DNA (dsDNA). Compared with SpCas9 and Cas12a, Cas12i effectors are smaller (1033-1093 Aa), and with the short mature crRNA (40-43 nt) used for it, Cas12i is particularly suitable for multiplex genome editing and viral vector-based delivery (Colin McGaw et al., NATURE COMMUNICATIONS (2022) 13:2833, https: / / doi.org / 10.1038 / s41467-022-30465-7; Winston X. Yan et al., Science 6 Dec 2018, Vol 363, Issue). 6422, pp.88-91, DOI:10.1126 / science.aav7271).

[0109] Non-limiting exemplary species from which RNA-guided DNA binders (e.g., Cas nucleases) originate include, but are not limited to, *Streptococcus pyogenes*, *Streptococcus thermophilus*, and *Streptococcus sp.*.The following bacteria were listed: Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter Jejuni, Pasteurella multocida, Fibrobacter succinogenes, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, and Streptomyces viridans. viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, naphthalene-degrading polar monoclonal bacteria, Polaromonas sp., Crocosphaera *Cyanothece* sp.Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter *Nitrosococcus halophilus*, *Nitrosococcus watsoni*, *Pseudoalteromonas haloplanktis*, *Ktedonobacter racemifer*, *Methanohalobium evestigatum*, *Anabaena variabilis*, *Nodularia spumigena*, *Nostoc sp.*, *Arthrospira maxima*, *Arthrospira platensis*, *Arthrospira sp.*, *Lyngbya sp.*, *Microcoleus chthonoplastes*, *Oscillatoria sp.*The bacteria included include: *Petrotoga mobilis*, *Thermosipho africanus*, *Streptococcus pasteurianus*, *Neisseria cinerea*, *Campylobacter Zari*, *Parvibaculum lavamentivorans*, *Corynebacterium diphtheria*, *Acidaminococcus sp.*, and bacteria from the family Lachnospiraceae, including ND2006 and *Acaryochloris marina*.

[0110] In some embodiments, the Cas nuclease is a Cas9 nuclease from *Streptococcus pyogenes*. In some embodiments, the Cas nuclease is a Cas9 nuclease from *Streptococcus thermophilus*. In some embodiments, the Cas nuclease is a Cas9 nuclease from *Neisseria meningitidis*. In some embodiments, the Cas nuclease is a Cas9 nuclease from *Staphylococcus aureus*. In some embodiments, the Cas nuclease is a Cpfl nuclease from *Francisella catarrhalis*. In some embodiments, the Cas nuclease is a Cpfl nuclease from *Aminococcus*. In some embodiments, the Cas nuclease is a Cpfl nuclease from *N. spp.* (a bacterium in the family Trichophyceae). In some implementations, the Cas nuclease is derived from *Francisella tularensis*, bacteria of the family *Trichophyceae*, *Butyrivibrio proteoclasticus*, *Peregrinibacteria bacterium*, *Parcubacteria bacterium*, *Smithella*, *Acidaminococcus*, *Candidatus Methanoplasma termitum*, *Eubacterium eligens*, *Moraxella bovoculi*, *Leptospira inadai*, *Porphyromonas crevioricanis*, *Prevotella disiens*, or *Porphyromonas rhesus*. Cpfl nucleases (from *Macacae*). In some embodiments, the Cpfl nuclease is a Cpfl nuclease derived from *Aminococcus* or *Trichophyton*.

[0111] Wild-type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, while the HNH domain cleaves the target DNA strand. In some embodiments, the Cas9 nuclease contains more than one RuvC domain and / or more than one HNH domain. In some embodiments, the Cas9 nuclease is wild-type Cas9. In some embodiments, Cas9 is capable of inducing double-strand breaks in the target DNA. In some embodiments, the Cas nuclease can cleave one or both strands of dsDNA. In some embodiments, the Cas nuclease can cleave single-stranded DNA. In some embodiments, the Cas nuclease may not have DNA cleavage enzyme activity.

[0112] The amino acid sequences of exemplary Cas nucleases used in some embodiments of this disclosure are shown below:

[0113] SpCas9 (SEQ ID NO:156)

[0114] FrCas9 (SEQ ID NO:158)

[0115] Sha2Cas9 (SEQ ID NO:160)

[0116] Cas12i_AE(SEQ ID NO:162)

[0117] The amino acid sequences of the exemplary cytidine deaminase and adenosine deaminase used in some embodiments of this disclosure are shown below:

[0118] ART-CBE1 (SEQ ID NO:164)

[0119] ART-ABE1 (SEQ ID NO:166)

[0120] The coding nucleotide sequences for these Cas nucleases or deaminases are shown below:

[0121] The encoding nucleotide sequence of SpCas9 (SEQ ID NO:157)

[0122] The encoding nucleotide sequence of FrCas9 (SEQ ID NO:159)

[0123] The encoding nucleotide sequence of Sha2Cas9 (SEQ ID NO:161)

[0124] The encoding nucleotide sequence of Cas12i_AE (SEQ ID NO:163)

[0125] The encoding nucleotide sequence of ART-CBE1 (SEQ ID NO:165)

[0126] The encoding nucleotide sequence of ART-ABE1 (SEQ ID NO:167)

[0127] Prevention or treatment methods

[0128] This disclosure provides a method for preventing or treating cardiovascular disease in a subject, the method comprising administering the composition or LNP described in this disclosure to the subject.

[0129] The method disclosed herein can reduce lipoprotein(a) levels in subjects, thereby reducing the risk of developing cardiovascular disease and achieving a preventive effect.

[0130] The method disclosed herein can modify the human LPA gene and / or induce double-strand breaks (DSBs) within the human LPA gene, thereby editing the human LPA gene and effectively preventing or treating hereditary cardiovascular diseases related to the LPA gene.

[0131] In some embodiments, the preventive or therapeutic effect can be determined by measuring the plasma lipoprotein(a) level of the subject some time after administration of the composition or LNP and comparing it with the plasma lipoprotein(a) level of the subject in a corresponding negative control or before administration of the composition or LNP.

[0132] In some embodiments, the composition or LNP comprising guide RNA and nucleic acid encoding a nuclease can be administered intravenously. In some embodiments, the composition or LNP comprising guide RNA and nucleic acid encoding a nuclease can be administered into the hepatic circulation.

[0133] In some embodiments, the composition or LNP can be administered by injection, such as intravenous injection, infusion, subcutaneous injection, intramuscular injection, intradermal injection, arterial injection, intraperitoneal injection, etc.

[0134] In some embodiments, a single application of a composition or LNP containing the guide RNA and nucleic acid encoding a nuclease provided herein is sufficient to knock down lipoprotein(a) expression. In some embodiments, a single application of a composition or LNP containing the guide RNA and nucleic acid encoding a nuclease provided herein is sufficient to knock out lipoprotein(a) expression. In other embodiments, multiple applications of a composition or LNP containing the guide RNA and nucleic acid encoding a nuclease provided herein may facilitate maximizing editing through a cumulative effect. For example, the composition or LNP provided herein may be applied 2, 3, 4, 5 or more times, such as twice. Application may be spaced at intervals, for example, (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days; (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 weeks; (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 months; or (d) 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.

[0135] In some embodiments, the effective amount of the applied composition or LNP is in the range of 0.01 to 20 mg / kg body weight (mpk), for example, 0.01 to 0.1 mpk, 0.1 to 0.3 mpk, 0.3 to 0.5 mpk, 0.5 to 1 mpk, 1 to 2 mpk, 2 to 3 mpk, 3 to 5 mpk, 5 to 10 mpk, or 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5, 6, 8, 10, 15, or 20 mpk. In some embodiments, the amount of the applied composition or LNP is 0.1-1 mg / kg body weight. In some embodiments, the amount of the applied composition or LNP is 2-4 mg / kg body weight, such as 2.5-3.5 mg / kg body weight. In some embodiments, the amount of the applied composition or LNP is about 3 mg / kg body weight.

[0136] In some embodiments, the efficacy of treatment with the composition or LNP described herein is evaluated at 1, 2, 3, 4, 5, or 10 years after delivery. In some embodiments, the efficacy of treatment with the composition or LNP described herein is evaluated by measuring lipoprotein(a) levels in the subject's plasma before and after treatment. In some embodiments, the efficacy of treatment with the composition or LNP, evaluated by a reduction in lipoprotein(a) levels in the subject's plasma, is observed at 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or 11 months. In some embodiments, the lipoprotein(a) levels in the subject's plasma are reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0137] Additional Therapy

[0138] In some embodiments, additional therapy may be administered to the subject before or after administration of the composition or LNP disclosed herein.

[0139] In some embodiments, the additional therapy is a treatment for cardiovascular disease. In some embodiments, the treatment for cardiovascular disease is the administration of cardiovascular disease treatment agents, such as statins, for example, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, or simvastatin; cholesterol absorption inhibitors, such as ezetimibe; and bile acid conjugates, such as cholestyramine, colesevelam, or colestipol.

[0140] In some embodiments, the adjunctive therapy is the administration of an antibody targeting lipoprotein (a). In some embodiments, the adjunctive therapy is the administration of any siRNA capable of reducing LPA gene expression. In some embodiments, the adjunctive therapy is the administration of any antisense nucleotide capable of targeting the LPA gene. In some embodiments, the adjunctive therapy is administered periodically following treatment with the compositions of this disclosure or LNPs.

[0141] Delivery of nucleic acid compositions

[0142] In some embodiments, the nucleic acid compositions described herein comprise gRNA and nucleic acids encoding RNA-guided DNA binders (either as RNA or encoded on one or more vectors), said nucleic acid compositions may be formulated in or administered via lipid nanoparticles (LNPs); see, for example, WO2017173054A1 and WO2019067992A1, the contents of which are incorporated herein by reference in their entirety. For further information on the selection of LNP components and formulation methods, see also WO2023 / 185697A2, particularly the section entitled “Delivery of Nucleic Acid Compositions,” which is incorporated herein by reference in its entirety. Any LNP known to those skilled in the art capable of delivering nucleotides to a subject may be used in conjunction with the guide RNA and nucleic acids encoding RNA-guided DNA nucleases described herein.

[0143] In some embodiments, the guide RNA and the nucleic acid encoding the RNA-guided DNA nuclease are administered in the LNP described herein, such as an LNP containing CCD lipids (e.g., amine lipids), accessory lipids (e.g., cholesterol), stealth lipids (e.g., PEG lipids, such as PEG2k-DMG), and optionally neutral lipids (e.g., DSPC).

[0144] This document discloses various embodiments of LNP formulations for RNA (including CRISPR / Cas payloads). Such LNP formulations may include (i) CCD lipids, such as amine lipids, (ii) neutral lipids, (iii) accessory lipids, and (iv) occult lipids, such as PEG lipids, and may also include a targeting moiety, such as GalNAc lipids. Some embodiments of LNP formulations include amine lipids, as well as accessory lipids, neutral lipids, and occult lipids (such as PEG lipids). In some embodiments, the LNP formulation includes less than 1% neutral phospholipids. In some embodiments, the LNP formulation includes less than 0.5% neutral phospholipids. “Lipid nanoparticles” can be particles containing multiple (i.e., more than one) lipid molecules physically linked together by intermolecular forces. The CCD lipids, amine lipids, neutral lipids and other lipids that can be used in LNP formulations disclosed herein are described in WO2020198697, WO2015006747, WO2016118724 and WO2021026358, all of which are incorporated herein by reference in their entirety.

[0145] In some embodiments, the nucleic acid compositions described herein (e.g., nucleic acids comprising one or more guide RNAs, encoding DNA binding agents (e.g., nucleases)) may be formulated in or administered via lipid nanoparticles (LNPs) comprising ALC0315, DSPC, cholesterol, and DMG-PEG2000. In some embodiments, the N / P ratio of the LNP is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the N / P ratio is about 3 to about 7. The N / P ratio is the molar ratio between an amine (N, which becomes a cation at low pH) on an ionizable lipid and a phosphate (P, anion) on the RNA backbone. The N / P ratio can affect the encapsulation efficiency and bioactivity of LNP formulations containing RNA.

[0146] In some embodiments, ALC0315 comprises 40 mol% to 60 mol% of the total lipids present in the particles, DSPC comprises 5 mol% to 15 mol% of the total lipids present in the particles, cholesterol comprises 30 mol% to 50 mol% of the total lipids present in the particles, and DMG-PEG2000 comprises 1 mol% to 5 mol% of the total lipids present in the particles. In one embodiment, LNP comprises ALC0315, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 50:9.5:37.5:3.

[0147] Further techniques that can be used to deliver the compositions of this disclosure include encapsulation techniques utilizing biodegradable polymers, liposomes, virus-like particles, or nanoparticles. In some embodiments, the compositions of this disclosure are administered with any suitable delivery medium, including but not limited to polymers, engineered viral particles (e.g., adeno-associated virus), exosomes, liposomes, supercharged proteins, implantable devices, or red blood cells. Suitable delivery methods are described in US10851357, US10709797, and US20170349914, each of which is incorporated herein by reference in its entirety.

[0148] Example

[0149] The compositions and methods disclosed herein are further described in the following examples, which are merely exemplary and do not imply that the scope of this disclosure is limited thereto. The scope of this disclosure is defined by the claims.

[0150] Example 1. Screening for SpCas9 sgRNAs targeting LPA in Huh7 cells

[0151] To evaluate the CRISPR-Cas9 method for knocking out LPA, sgRNAs targeting LPA were designed for use with SpCas9 (amino acid sequence SEQ ID NO:156, nucleotide sequence SEQ ID NO:157). The sequences, positions, and bioinformatics-predicted mismatch (MM) sites of different sgRNAs were statistically analyzed, as shown in Table 1 below.

[0152] The editing activity of different sgRNAs was evaluated in the human hepatocellular carcinoma cell line Huh7, as described below. Huh7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum. 20–24 hours before transfection, cells were seeded at a density of 8,000 cells / well in 96-well plates (Corning). Following the manufacturer's protocol, 100 ng of SpCas9 mRNA and 100 ng of sgRNA were transfected into each well using Lipofectamine MessengerMAX (ThermoFisher, catalog number LMRNA003). After 72 hours, Huh7 cells were lysed using QuickExtract DNA extraction solution (Lucigen, catalog number QE09050) to release genomic DNA. Primers were designed around the target sites, and the genomic regions of interest were amplified, preferentially using NGS sequencing. Editing efficiency was calculated using CRISPResso2 software. For target sites where it is difficult to design suitable NGS primers, Sanger primers were designed and amplified. The raw sequencing files (.ab1) were analyzed using online analysis tools (e.g., TIDE: tipe.nki.nl / , ICE: ice.synthego.com / ) to determine editing efficiency. Editing data are listed in Table 2, arranged from highest to lowest efficiency.

[0153] As shown in Table 2, the editing efficiency ranking reveals that, except for LPA-h-07, LPA-h-08, and LPA-h-09 (SEQ ID NO:7-9), the remaining sgRNAs all achieved high editing efficiency in the human hepatocellular carcinoma cell line Huh7. Among them, 15 sgRNAs had an editing efficiency greater than 75%, with LPA-h-24 (SEQ ID NO:24) exhibiting the highest editing efficiency (88.47%).

[0154] Table 2. Evaluation of SpCas9 sgRNA editing efficiency in Huh7 cells

[0155] Example 2. Evaluation of SpCas9 sgRNA activity in PHH cells

[0156] sgRNAs with editing efficiency exceeding 75% in Huh7 cells were selected, and the editing efficiency of LPA-hc-01 to LPA-h-04 targeting the second exon was further evaluated in primary human hepatocytes (PHH cells). PHH cells were cultured according to the manufacturer's protocol. Cells were thawed and resuspended in hepatocyte thawing medium supplemented with saturates, and then centrifuged at 100g for 10 minutes for concentration. The supernatant was discarded, and the precipitated cells were resuspended in hepatocyte seeding medium supplemented with saturates. Cells were counted and seeded into 48-well plates (ThermoFisher, catalog number 877272) coated with Bio-coat collagen I at a density of 132,000 cells / well. The seeded cells were allowed to settle and adhere in a tissue culture incubator at 37°C and a 5% CO2 atmosphere for 4–6 hours. Following the manufacturer's protocol, 125 ng of SpCas9 mRNA and 125 ng of sgRNA were transfected into each well using Lipofectamine MessengerMAX (ThermoFisher, catalog number LMRNA003). After 24 hours, the culture medium was replaced with fresh medium. 72 hours after transfection, genomic DNA was obtained from the cells, and editing efficiency was analyzed using NGS sequencing. Table 3 shows the editing efficiencies achieved by different sgRNAs in PHH.

[0157] Table 3. Evaluation of SpCas9 sgRNA editing efficiency in PHH cells

[0158] To further compare the activities of these sgRNAs, the top 10 sgRNAs with the highest editing efficiency and LPA-h-03 targeting the second exon were selected for dose-dependent transfection experiments. Four doses were set up, with the first dose consisting of 125 ng mRNA + 125 ng sgRNA, followed by 2-fold serial dilutions. The results are shown in Table 4.

[0159] The results in Table 4 indicate that the sgRNA disclosed herein works in a largely dose-dependent manner.

[0160] Table 4. Evaluation of editing efficiency at different doses in PHH cells

[0161] Example 3. Off-target risk assessment in PHH cells

[0162] Potential off-target sites (OTs) for SpCas9 and its corresponding gRNA cleavage were screened using a double-stranded DNA (dsDNA) insertion detection method (GUIDE-seq). Approximately 500,000 PHH cells were seeded in 24-well plates, and 0.0125 μg of dsDNA, 0.3 μg of SpCas9 mRNA, and 0.3 μg of sgRNA were transfected into the cells using Lipofectamine MessengerMAX. Three days later, genomic DNA was extracted and subjected to library construction and next-generation sequencing (NGS) analysis. Sites with dsDNA insertion detected by NGS were identified as potential off-target sites. The sum of the readings of the top 30 off-target sites was divided by the readings at the target sites (top 30 OT / ON) as a semi-quantitative indicator for assessing the potential off-target effects of sgRNA. The top five sites with the highest dsDNA insertion readings are listed in Table 5.

[0163] To address the potential off-target sites identified above, genomic DNA (125ng + 125ng highest dose group) obtained from the dose transfection experiment in Example 2 was validated using NGS-based amplicon sequencing. The sequencing values ​​obtained from the control sample without transfected sgRNA were used as background values. The sample values ​​were then subtracted from the background values, and the results are shown in Table 6.

[0164] The results in Table 6 show that most sgRNAs (e.g., LPA-h-03, LPA-h-28, LPA-h-34, LPA-h-40, LPA-h-41, LPA-h-42) showed no significant off-target effects under hypersaturation editing.

[0165] Table 6. Validation of Potential Off-Target Sites

[0166] Example 4. Detection of in vitro protein knockdown effect

[0167] To assess the in vitro protein knockdown effects of different sgRNAs, PHH cells were transfected with a combination of 125 ng SpCas9 mRNA and 125 ng sgRNA. Fresh culture medium was introduced on days 1 and 4 post-transfection, and cell culture supernatant was collected on day 7. Lp(a) levels in different samples were detected using the Lp(a) ELISA kit (Abcam, catalog number ab108878) according to the manufacturer's instructions, with blank transfection wells containing Lipofectamine as a control. The protein knockdown levels are shown in Table 7.

[0168] The results in Table 7 demonstrate that sgRNA editing can be effectively translated into protein knockdown.

[0169] Table 7. Protein knockdown effects of different sgRNAs transfected in PHH cells

[0170] Example 5. Screening for FrCas9 sgRNAs targeting LPA

[0171] To evaluate whether other different Cas9 sequences could be used for LPA knockout, sgRNAs targeting FrCas9 (amino acid sequence SEQ ID NO:158, nucleotide sequence SEQ ID NO:159) were designed, as shown in Table 8 below. The editing activity of different sgRNAs was evaluated in PHH cells, and the experimental procedures were similar to those in Example 2. The results are shown in Table 9.

[0172] The results in Table 9 show that when using FrCas9 as a nuclease, Fr-h-16 (SEQ ID NO:68) had the highest editing efficiency (79.23%), while Fr-hc-14 (SEQ ID NO:66) had an editing efficiency of 57.33%.

[0173] Table 9. Detection of FrCas9 sgRNA editing efficiency in PHH cells

[0174] Example 6. Screening for Sha2Cas9 sgRNAs targeting LPA

[0175] sgRNAs targeting Sha2Cas9 (amino acid sequence SEQ ID NO:160, nucleotide sequence SEQ ID NO:161) were designed, as shown in Table 10 below. First, the editing activity of different sgRNAs was evaluated in the Huh7 cell line, following experimental procedures similar to Example 1, and the results are shown in Table 11. Then, the top 10 sgRNAs with the highest editing activity in the Huh7 cell line were further evaluated in PHH cells, following experimental procedures similar to Example 2, and the results are shown in Table 11.

[0176] The results in Table 11 show that when Sha2Cas9 is used as a nuclease, a variety of sgRNAs can achieve gene editing in Huh7 and PHH cell lines.

[0177] Table 11. Editing efficiency of Sha2Cas9 sgRNA in Huh7 and PHH cells

[0178] Example 7. Screening for Cas12i_AE gRNAs targeting LPA

[0179] To evaluate whether the Cas12 protein could be used for LPA knockout, gRNA was designed targeting Cas12i_AE (amino acid sequence SEQ ID NO:162, nucleotide sequence SEQ ID NO:163) (see Table 12 below). Editing activity was evaluated in Huh7 cells, and the experimental procedures were as described in Example 2. The results are shown in Table 13.

[0180] As shown in Table 13, when using Cas12i_AE as a nuclease, most gRNAs were able to achieve effective gene editing in Huh7 cells. The top 5 gRNAs in terms of editing efficiency were: 12i-h-9 (SEQ ID NO:106, 86.82%), 12i-h-33 (SEQ ID NO:130, 85.32%), 12i-h-32 (SEQ ID NO:129, 83.03%), 12i-h-10 (SEQ ID NO:107, 81.48%), and 12i-h-8 (SEQ ID NO:105, 79.44%).

[0181] Table 13. Editing efficiency of Cas12i_AE gRNA in Huh7 cells

[0182] Example 8. Screening for base editor sgRNAs targeting LPA

[0183] To evaluate base editing strategies for LPA knockout, we designed sgRNAs for the Sha2Cas9-based base editor (Sha2-BE) and used them in conjunction with either the cytidine base editor (CBE) or the adenosine base editor (ABE) to disrupt splice sites or introduce stop codons prematurely to achieve gene knockout. A list of sgRNAs is shown in Table 14, indicating the editor pairings used to achieve gene knockout and the target nucleotide positions (guide sequences target 21 bases, and PAM positions are defined as 22-25).

[0184] Table 14. sgRNAs designed based on the Sha2-BE system

[0185] To assess the editing activity of sgRNA in PHH cells, ART-CBE1 (amino acid sequence SEQ ID NO:164, nucleotide sequence SEQ ID NO:165) and ART-ABE1 (amino acid sequence SEQ ID NO:166, nucleotide sequence SEQ ID NO:167) mRNAs were transfected with their corresponding sgRNAs, and the experimental procedures were similar to those in Example 2. NGS sequencing was used to analyze the editing efficiency of sgRNAs at the target nucleotide sites, and the results are shown in Table 15.

[0186] The results in Table 15 show that most of the sgRNAs constructed in this disclosure achieve good editing efficiency when used in conjunction with the cytidine base editor (CBE). For example, the editing efficiencies of NNGG-C-47 (SEQ ID NO:138), NNGG-C-53 (SEQ ID NO:140), and NNGG-C-55 (SEQ ID NO:141) in the presence of CBE are 59.92%, 55.77%, and 54.28%, respectively. However, when used in conjunction with the adenosine base editor (ABE), a small number of sgRNAs show lower editing efficiency, with NNGG-AC-67 (SEQ ID NO:151) and NNGG-AC-71 (SEQ ID NO:154) showing the highest editing efficiencies at 26.70% and 26.43%, respectively.

[0187] Table 15. Editing efficiency of Sha2-BE sgRNA at target nucleotides

[0188] Example 9. Expanding the screening of spCas9 sgRNAs targeting LPA

[0189] More spCas9 sgRNAs targeting LPA were designed, as shown in Table 16 below. Similar to Example 1, the editing activity of these sgRNAs was evaluated in the Huh7 cell line. Two transfection doses were used: mRNA:sgRNA of 100 ng:100 ng and 6.25 ng:6.25 ng. Editing efficiencies are shown in Table 17.

[0190] As can be seen from the editing efficiencies shown in Table 17, multiple sgRNAs exhibited high editing activity at both doses. For example, LPA-h-65 (SEQ ID NO:183), LPA-h-66 (SEQ ID NO:184), LPA-h-69 (SEQ ID NO:187), LPA-h-76 (SEQ ID NO:191), LPA-h-78 (SEQ ID NO:193), and LPA-h-81 (SEQ ID NO:196) showed editing efficiencies greater than 60% at the low dose of 6.25 ng: 6.25 ng.

[0191] Table 16. sgRNAs designed for SpCas9

[0192] Table 17. Evaluation of editing efficiency in the Huh7 cell line

[0193] Example 10. Expanding the screening of Sha2Cas9 sgRNAs targeting LPA

[0194] More Sha2Cas9 sgRNAs targeting LPA were designed, as shown in Table 18. Similar to Example 1, the editing activity of these sgRNAs was evaluated in the Huh7 cell line using two transfection doses: mRNA:sgRNA of 100 ng:100 ng and 6.25 ng:6.25 ng. The editing efficiency is shown in Table 19.

[0195] As can be seen from the editing efficiencies shown in Table 19, multiple sgRNAs exhibited high editing activity at both doses. For example, Sha2-hc-28 (SEQ ID NO:215), Sha2-h-32 (SEQ ID NO:219), and Sha2-h-33 (SEQ ID NO:220) showed editing efficiencies greater than 40% at the low dose of 6.25 ng:6.25 ng.

[0196] Table 18. sgRNAs designed for Sha2Cas9

[0197] Table 19. Evaluation of editing efficiency in the Huh7 cell line

[0198] Example 11. Expanding the screening of Cas12i gRNAs targeting LPA

[0199] More Cas12i gRNAs targeting LPA were designed, as shown in Table 20 below. Similar to Example 1, the editing activity of these gRNAs was evaluated in the Huh7 cell line using two transfection doses: mRNA:sgRNA of 100 ng:100 ng and 6.25 ng:6.25 ng. The editing efficiency is shown in Table 21. As can be seen from the editing efficiencies shown in Table 21, multiple sgRNAs exhibited high editing activity at both doses. For example, 12i-h-37 (SEQ ID NO:224), 12i-h-48 (SEQ ID NO:235), 12i-h-54 (SEQ ID NO:241), 12i-h-58 (SEQ ID NO:245), 12i-h-38 (SEQ ID NO:225), 12i-h-56 (SEQ ID NO:243), and 12i-h-46 (SEQ ID NO:233) showed editing efficiencies greater than 40% at the low dose of 6.25 ng: 6.25 ng.

[0200] Table 20. gRNAs designed for Cas12i

[0201] Table 21. Evaluation of editing efficiency in the Huh7 cell line

[0202] Example 12. Screening for sgRNAs targeting LPA and NAG PAM

[0203] sgRNAs were designed targeting the enzymes spAGA (amino acid sequence SEQ ID NO:338, nucleotide sequence SEQ ID NO:339) with NAGA PAM cleavage activity and spAGY (amino acid sequence SEQ ID NO:340, nucleotide sequence SEQ ID NO:341) with NAGY PAM cleavage activity. sgRNA information is shown in Table 22 below. Similar to Example 1, editing activity was evaluated in Huh7 cells using two mRNA:sgRNA transfection doses: 100 ng:100 ng and 6.25 ng:6.25 ng. Editing efficiency is shown in Table 23.

[0204] As shown in Table 23, the editing efficiencies indicate that multiple sgRNAs exhibited high editing activity at both dosages. Examples include LPA-h-128 (SEQ ID NO:246), LPA-h-129 (SEQ ID NO:247), LPA-h-134 (SEQ ID NO:252), LPA-h-149 (SEQ ID NO:267), LPA-hc-133 (SEQ ID NO:251), LPA-hc-159 (SEQ ID NO:277), LPA-h-160 (SEQ ID NO:278), LPA-h-161 (SEQ ID NO:279), LPA-h-164 (SEQ ID NO:282), LPA-hc-165 (SEQ ID NO:283), LPA-h-167 (SEQ ID NO:285), LPA-h-181 (SEQ ID NO:299), and LPA-h-176 (SEQ ID NO:299). LPA-h-199 (SEQ ID NO:317), LPA-h-185 (SEQ ID NO:303), LPA-h-180 (SEQ ID NO:298), and LPA-h-188 (SEQ ID NO:306) showed editing efficiency greater than 20% at low doses of 6.25 ng:6.25 ng.

[0205] Table 22. sgRNAs designed for NAG PAM

[0206] Table 23. Evaluation of editing efficiency in the Huh7 cell line

[0207] Example 13. Testing the activity of SpCas9 sgRNA in PHH cells in the form of LNPs

[0208] To further evaluate the activity of sgRNA, spCas9 mRNA and modified sgRNA (Finn et al., 2018) were combined (with a mass ratio of spCas9 mRNA to modified sgRNA of 1:1 or 2:1) and encapsulated in lipid nanoparticles (LNPs) according to the method outlined in patent application WO2023 / 185697A2. Primary human hepatocytes (PHH cells) were resuscitated according to the method in Example 2. After 20 to 24 hours, different doses of LNPs were transfected into PHH cells. After 72 hours of transfection, genomic DNA was obtained from the cells, and the editing efficiency was detected using NGS. The results are shown in Figures 1A, 1B, and 1C.

[0209] Three LNPs, spCas9 mRNA and LPA-h-34, spCas9 mRNA and LPA-h-41, and spCas9 mRNA and LPA-h-76 sgRNA respectively, were selected for editing potential off-target sites. As shown in Table 24, LPA-h-76 exhibited significant off-target editing at potential off-target sites OT1, OT2, and OT3, while the editing efficiencies of LPA-h-34 and LPA-h-41 at the detected potential off-target sites OT1 to OT4 were all less than 0.1%.

[0210] Table 24. Evaluation of off-target editing of different LNPs in PHH cells

[0211] Example 14. Activity assessment of LNPs encapsulating SpCas9 sgRNA in humanized mice

[0212] LNPs encapsulated with spCas9 mRNA and LPA-h-41, and LNPs encapsulated with spCas9 mRNA and LPA-h-34 were selected for activity assessment in LPA-humanized mice. The prepared LNPs were administered to LPA-humanized mice (source: GemPharmatech, mice were 7-9 weeks old and weighed 18-23g at the time of the experiment) via tail vein injection at different doses (0.5 or 0.25 mg / kg body weight). Peripheral blood was collected from the mice after 1 or 2 weeks, and lipoprotein (a) expression was detected using an ELISA kit (brand: Abcam, catalog number Ab108878). As shown in Figure 2, at a dose of 0.5 mg / kg, both LNPs achieved approximately 90% protein knockdown.

[0213] Example 15. Activity-enhancing modification of LPA-h-41

[0214] To further enhance the activity of LPA-h-41 (SEQ ID NO:41), its guide sequence region was modified with 2'-fluorine (2'-F), as shown in Table 25 below (PAM is 1 at the distal end, with position numbers increasing from the 5' end to the 3' end). Activity was assessed in the Huh7 cell line. mRNA:sgRNA was transfected at a 1:1 mass ratio in four gradients, with sgRNA doses of 33.3 ng, 16.67 ng, 8.33 ng, and 4.17 ng for each gradient. Editing efficiency results are shown in Table 26. One or more 2'-fluorine modifications at positions 8, 9, 10, and 11 of the guide sequence increased the activity of the fluorinated sgRNA at different doses compared to the unfluorinated sgRNA (i.e., LPA-h-41-FS01).

[0215] Table 25. Correspondence between sgRNA names and modifications Note: fN represents a nucleotide modified with 2'-fluorine (2'-F); mN represents a nucleotide modified with 2'-O-methyl (2'-O-Me); * represents a phosphate thioester (PS) bond between nucleotides.

[0216] Table 26. Evaluation of the editing of sgRNAs with different modifications in Huh7 cells.

[0217] Based on the results in Table 26, 2'-fluorinated sgRNAs were synthesized and encapsulated with spCas9 mRNA for LNP. The 2'-fluorinated sgRNA LPA-h-41-FS01 served as a control. LNP activity was evaluated in PHH cells from different donors, and the results are shown in Figures 3A and 3B. As shown in Figures 3A and 3B, among the eight sgRNAs tested, the fluorinated sgRNAs LPA-h-41-FS09 (SEQ ID NO:325), FS03 (SEQ ID NO:320), and FS14 (SEQ ID NO:330) exhibited significantly better editing activity than the fluorinated sgRNA LPA-h-41-FS01.

[0218] Example 16. Modification of LPA-h-34

[0219] The guide sequence of LPA-h-34 (SEQ ID NO:34) was modified with DNA, as shown in Table 27 below. spCas9 mRNA and modified sgRNA were transfected into PHH cells, and the editing efficiency at target and potential off-target sites was detected. The transfection dose of spCas9 mRNA:sgRNA was 320 ng:80 ng, and the results are shown in Table 28.

[0220] The results shown in Table 28 indicate that DNA modification successfully reduced the off-target efficiency of sgRNA.

[0221] Table 27. Correspondence between sgRNA names and modifications

[0222] Note: dN represents deoxyribonucleotide; mN represents nucleotide modified with 2'-O-methyl (2'-O-Me); * represents phosphate thioester (PS) bond between nucleotides.

[0223] Table 28. Assessment of the on-target and off-target activities of DNA-modified sgRNAs in PHH cells.

[0224] Example 17. LNP activity assay of dual sgRNAs

[0225] To investigate whether the simultaneous addition of two sgRNAs could improve overall editing activity, different sgRNAs were mixed in a 1:1 mass ratio. During LNP encapsulation, the mass ratio of spCas9 mRNA to total sgRNA (1:1) was maintained. Editing activity was detected in PHH cells, and the results are shown in Figure 4. Compared with the single sgRNA LPA-h-34, the dual sgRNA combination further improved editing efficiency. The combination of LPA-h-34 and LPA-h-42 showed the highest editing efficiency.

[0226] Example 18. Testing the activity of Cas12i sgRNA in PHH cells in the form of LNP.

[0227] Three sgRNAs (12i-h-33, 12i-h-37, and 12i-h-48) were selected and encapsulated in LNPs with the highly active Cas12i nuclease variant Cas12i-AE_v1 (from patent application PCT / CN2026 / 075196, amino acid sequence SEQ ID NO:342, nucleotide sequence SEQ ID NO:343) (the mass ratio of sgRNA to Cas12i nuclease variant mRNA in the LNP was 1:1). Editing activity was detected in PHH cells, and the results are shown in Figure 5. All three sgRNAs exhibited high editing activity.

[0228] Table 29. Amino acid sequences of the nucleases used in this disclosure

[0229] Table 30. Encoding nucleotide sequences of nucleases used in this disclosure

[0230] Other implementation plans

[0231] It should be understood that although this application has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative and not to limit the scope of this application, which is defined by the scope of the appended claims. Other aspects, advantages, and improvements are within the scope of the following claims.

Claims

1. A guide RNA comprising: a) A sequence selected from any of SEQ ID NO: 1-155 and 168-337, preferably selected from SEQ ID NO: 35, 34, 38, 39, 42, 37, 41, 40, 33, 28, 14, 3, 24, 18, 46, 15, 29, 16, 19, 55, 66, 68, 88, 96, 94, 97, 82, 83, 84, 86, 95, 93, 105, 106, 107, 109, 129, 130, 135, 138, 140, 141, 62, 183, 184. Any sequence of 187, 191, 193, 196, 215, 219, 220, 224, 235, 241, 245, 225, 243, 233, 246, 247, 252, 267, 251, 277, 278, 279, 282, 283, 285, 299, 294, 317, 303, 298, 306, 325, 320, 330, or 333-337; b) At least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides selected from any sequence of SEQ ID NO: 1-155 and 168-337; or c) A sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any sequence selected from SEQ ID NO:1-155 and 168-337.

2. A vector comprising one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise: a) A sequence selected from any of SEQ ID NO: 1-155 and 168-337, preferably selected from SEQ ID NO: 35, 34, 38, 39, 42, 37, 41, 40, 33, 28, 14, 3, 24, 18, 46, 15, 29, 16, 19, 55, 66, 68, 88, 96, 94, 97, 82, 83, 84, 86, 95, 93, 105, 106, 107, 109, 129, 130, 135, 138, 140, 141, 62, 183, 184. Any sequence of 187, 191, 193, 196, 215, 219, 220, 224, 235, 241, 245, 225, 243, 233, 246, 247, 252, 267, 251, 277, 278, 279, 282, 283, 285, 299, 294, 317, 303, 298, 306, 325, 320, 330, or 333-337; b) At least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides selected from any sequence of SEQ ID NO: 1-155 and 168-337; or c) A sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any sequence selected from SEQ ID NO:1-155 and 168-337.

3. A composition comprising: (i) a nucleic acid encoding one or more guide RNAs, or a vector containing a nucleic acid encoding one or more guide RNAs, wherein the guide RNA comprises: a) A sequence selected from any of SEQ ID NO: 1-155 and 168-337, preferably selected from SEQ ID NO: 35, 34, 38, 39, 42, 37, 41, 40, 33, 28, 14, 3, 24, 18, 46, 15, 29, 16, 19, 55, 66, 68, 88, 96, 94, 97, 82, 83, 84, 86, 95, 93, 105, 106, 107, 109, 129, 130, 135, 138, 140, 141, 62, 183, 184. Any sequence of 187, 191, 193, 196, 215, 219, 220, 224, 235, 241, 245, 225, 243, 233, 246, 247, 252, 267, 251, 277, 278, 279, 282, 283, 285, 299, 294, 317, 303, 298, 306, 325, 320, 330, or 333-337; b) At least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides selected from any sequence of SEQ ID NO: 1-155 and 168-337; or c) A sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any sequence selected from SEQ ID NO: 1-155 and 168-337; and (ii) RNA-guided DNA binders, nucleic acids encoding RNA-guided DNA binders, or vectors containing nucleic acids encoding RNA-guided DNA binders.

4. The composition according to claim 3, wherein the RNA-guided DNA binder comprises a Cas nuclease or a Cas nickase; preferably, the Cas nuclease is a type 2 Cas nuclease; more preferably, the Cas nuclease is Cas9, Cpfl, C2cl, C2c2, C2c3, or Cas12 or a modified protein thereof; most preferably, the Cas nuclease is *Streptococcus pyogenes* Cas9 nuclease, *Faecalibaculum rodentium* Cas9 nuclease, *Staphylococcus haemolyticus* Sha2Cas9 nuclease, or *Staphylococcus aureus* Cas9 nuclease, Cas12i, or a modified protein thereof; for example, the RNA-guided DNA binder comprises any amino acid sequence of SEQ ID NO: 156, 158, 160, 162, 338, 340, or 342.

5. The composition according to claim 3, wherein the guide RNA is at least partially complementary to a target sequence present in the human LPA gene, preferably, the target sequence is located in any one of exons 2, 8, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 31, 32, 33, 34, 35, 36, 37 or 38 of the human LPA gene, or at the junction of exons and introns of the human LPA gene.

6. The composition according to claim 3, wherein the guide RNA is complementary to the target sequence in the positive strand of the LPA gene, or the guide RNA sequence is complementary to the target sequence in the negative strand of the LPA gene.

7. The composition of claim 3, wherein the guide RNA comprises a first guide sequence and a second guide sequence, wherein the first guide sequence is complementary to a first target sequence in the positive strand of the LPA gene, and the second guide sequence is complementary to a second target sequence in the negative strand of the LPA gene.

8. The composition of claim 3, wherein the guide RNA comprises crRNA, and optionally further comprises tracrRNA (trRNA) or a portion thereof, wherein the trRNA is operatively linked to the crRNA.

9. The composition according to claim 3, wherein the guide RNA is a dual guide RNA (dgRNA) or a single guide RNA (sgRNA).

10. The composition of claim 3, wherein the guide RNA comprises at least one modification, optionally, wherein the at least one modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide, an internucleotide phosphate thioester (PS) bond, a 2'-fluorine (2'-F) modified nucleotide, or a DNA-RNA hybrid; More preferably, wherein (i) The guide RNA comprises the sequence shown in SEQ ID NO:41, and one or more bases at positions 8-11 are modified with 2'-fluorination; or (ii) The guide RNA comprises the sequence shown in SEQ ID NO:34, and one or more bases at positions 4, 5, 11 and 12 are deoxyribonucleotides.

11. The composition of claim 3, wherein the composition further comprises: (iii) Cytidine deaminase or adenosine deaminase, or a nucleic acid encoding cytidine deaminase or a carrier containing a nucleic acid encoding cytidine deaminase or a nucleic acid encoding cytidine deaminase.

12. The composition according to any one of claims 3-11, wherein the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable excipient or carrier.

13. The composition according to any one of claims 3-11, wherein the guide RNA is associated with lipid nanoparticles (LNPs), or the composition is encapsulated within an LNP, optionally, one or more guide RNAs are encapsulated within an LNP in combination with the DNA binding agent.

14. A nucleic acid molecule comprising a nucleotide sequence encoding one or more guide RNAs as claimed in claim 1, or comprising a nucleotide sequence encoding one or more guide RNAs as claimed in claim 1 and an RNA-guided DNA binder.

15. An expression vector comprising the nucleic acid molecule of claim 14.

16. A cell comprising the nucleic acid molecule of claim 14 or the expression vector of claim 15.

17. A lipid nanoparticle (LNP) associated with one or more guide RNAs of claim 1 or nucleic acids encoding therethe thereof, or encapsulating one or more guide RNAs of claim 1 or nucleic acids encoding therethe thereof; Optionally, it is associated with two or more guide RNAs of claim 1 or nucleic acids encoding therethe, or it encapsulates two or more guide RNAs of claim 1 or nucleic acids encoding therethe, optionally wherein the two or more guide RNAs are present in equal amounts.

18. The lipid nanoparticles (LNPs) according to claim 17, further encapsulating an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder; Optionally, the DNA binder comprises any amino acid sequence from SEQ ID NO: 156, 158, 160, 162, 338, 340 or 342.

19. The lipid nanoparticles (LNPs) according to claim 17 or 18, wherein the two guide RNAs of claim 1 are encapsulated, preferably, the two guide RNAs are at least partially complementary to different target sequences present in the human LPA gene, more preferably, the two guide RNAs are at least partially complementary to different target sequences present in the same exon of the human LPA gene. Optionally, the two guide RNAs each contain: (i) the sequences shown in SEQ ID NO:34 and SEQ ID NO:42, or (ii) the sequences shown in SEQ ID NO:34 and SEQ ID NO:41, or (iii) The sequences shown in SEQ ID NO:40 and SEQ ID NO:42, or (iv) The sequences shown in SEQ ID NO:42 and SEQ ID NO:

44.

20. A method for editing the human LPA gene, the method comprising administering to cells the composition of any one of claims 3-13 or the lipid nanoparticles (LNPs) of any one of claims 17-19.

21. A method for preventing or treating cardiovascular disease in a subject, the method comprising administering to the subject an effective amount of the composition of any one of claims 3-13 or lipid nanoparticles (LNPs) of any one of claims 17-19.

22. The method according to claim 20 or 21, wherein the composition or LNP is applied or delivered at least once, preferably, the composition is applied by injection.

23. The method of claim 22, wherein the application or delivery is performed at the following intervals: (a) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days; (b) Weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; (c) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 months; or (d) 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.

24. The method of claim 21, wherein the subject has a high lipoprotein(a) level or a family history of cardiovascular disease, or the subject suffers from one or more cardiovascular diseases selected from myocardial infarction, ischemic stroke, aortic stenosis, coronary artery stenosis, carotid artery stenosis, heart failure, peripheral artery disease, atherosclerosis, and thrombosis.

25. The method according to claim 21, further comprising: The cardiovascular disease treatment agent is administered to the subject before or after the administration of the composition.

26. A kit comprising the composition of any one of claims 3-12, or the nucleic acid molecule of claim 14, or the expression vector of claim 15, or the lipid nanoparticles (LNPs) of any one of claims 17-19, and instructions for use.

27. A pharmaceutical composition comprising the composition of any one of claims 3-11, or the nucleic acid molecule of claim 14, or the expression vector of claim 15, or the lipid nanoparticles (LNPs) of any one of claims 17-19, and a pharmaceutically acceptable excipient or carrier.

28. Use of the composition of any one of claims 3-11, or the nucleic acid molecule of claim 14, or the expression vector of claim 15, or the lipid nanoparticles (LNP) of any one of claims 17-19 in the preparation of a medicament for the prevention or treatment of cardiovascular disease in a subject.

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