Guide RNA, and composition thereof, and use thereof in editing PCSK9 genes

Abstract

The present invention relates to the field of gene editing, and specifically relates to a guide RNA, and a composition thereof, and the use thereof in editing PCSK9 genes. In the present invention, the guide RNA is further optimized to achieve a low off-target efficiency while maintaining an on-target editing efficiency, thereby facilitating a balance between efficacy and safety. By using the composition containing the guide RNA, the expression of the PCSK9 genes can be safely, effectively, and conveniently regulated for a long time, so that a sustained LDL-C lowering effect can be achieved, which is beneficial to avoiding side effects and compliance issues of traditional PCSK9-targeting drugs.

Description

A guide RNA, its composition and its application in editing the PCSK9 gene

[0001] Cross-references to related applications

[0002] This invention claims priority to Chinese Patent Application No. 2025101080675, filed January 23, 2025, and Chinese Patent Application No. 2025111345155, filed August 13, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of gene editing, and more specifically, to a guide RNA, a composition thereof, and its application in editing the PCSK9 gene. Background Technology

[0004] PCSK9 (proprotein convertase subtilisin 9) is a protein that plays a crucial role in cholesterol metabolism, inhibiting the degradation of low-density lipoprotein cholesterol (LDL-C) and increasing its levels. It has become a popular lipid-lowering target in recent years. The potential applications of PCSK9-targeted drugs in the treatment of hypercholesterolemia have been explored and are gradually being expanded to the treatment of other PCSK9-related diseases. For example, antibody-based therapies Alirocumab and Evolocumab, as well as RNA interference (RNAi)-based drugs Inclisiran, have all been approved for marketing. While these methods are effective, they have certain side effects, such as muscle pain and immune responses; and patient adherence issues—existing PCSK9 inhibitors require long-term injections, which are inconvenient and make it difficult for patients to adhere to long-term medication.

[0005] LNP delivery vectors can precisely deliver drug-active molecules to target organs, currently primarily the liver. Gene-editing drugs have the potential to achieve lifelong control / reversal of disease states with a single dose. However, there are currently no approved gene-editing drugs targeting PCSK9, and publicly available information on other PCSK9 gene-editing drug compositions in the development stage (such as VERVE-101 / 102) shows issues such as narrow therapeutic windows and high safety risks.

[0006] Off-target efficiency is a crucial safety indicator for in vivo gene-editing drugs based on CRISPR technology. Currently, among clinical trial drugs using Cas9 double-strand cutting (DSB) for in vivo gene editing, NTLA-2001 (10.1056 / NEJMoa2107454) and NTLA-2002 (10.1056 / NEJMoa2309149) both exhibited off-target editing rates exceeding 0.1% near the effective dose. VERVE-101 / 102, developed by Verve Therapeutics, is the first in vivo base-editing therapy to enter clinical trials, designed to treat heterozygous familial hypercholesterolemia (HeFH). This therapy delivers base-editing tools via lipid nanoparticles (LNPs) to permanently shut down PCSK9 gene expression, thereby reducing LDL-C (low-density lipoprotein cholesterol) levels and lowering disease risk. However, its drawbacks include poor safety profile and a narrow therapeutic window. While this drug can reduce PCSK9 protein levels to some extent, its efficacy has not yet reached saturation in the high-dose group, and significant safety issues have been observed. In contrast, the low-dose group has experienced death and numerous other serious adverse events. Meanwhile, the patent application PCT / CN2023 / 120234 also mentions a guide RNA targeting the PCSK9 gene; however, there is still room for improvement in balancing efficacy and off-target efficiency. Summary of the Invention

[0007] The first object of the present invention is to provide a guide RNA comprising a spacer sequence comprising nucleotides 4 to 20, 3 to 20, 2 to 20, or 1 to 20 of any of the sequences shown in SEQ ID No. 1 to 25.

[0008] The present invention also provides a guide RNA comprising a backbone sequence wherein the backbone sequence does not contain 2'-O-methyl modification at nucleotide positions 9, 49 and 62 of the nucleotide sequence shown in SEQ ID No. 51, and contains 2'-O-methyl modified nucleotides at nucleotide positions 8, 21 and 22 of the nucleotide sequence shown in SEQ ID No. 51.

[0009] The present invention also provides a composition comprising: (i) the guide RNA, or a nucleic acid encoding the guide RNA; and at least one selected from (ii) to (iii) below: (ii) an effector protein, or a nucleic acid encoding the effector protein; and (iii) a delivery vector.

[0010] The present invention also provides a pharmaceutical formulation comprising any one of (1) or (2) below and a pharmaceutically acceptable adjuvant thereof: (1) the guide RNA; (2) the composition.

[0011] A second objective of the present invention is to provide a method for editing the PCSK9 gene, comprising: delivering one or more of the guide RNA, the composition, and the pharmaceutical preparation to a cell.

[0012] A third object of the present invention is to provide the use of one or more of the described guide RNA, the described composition, and the described pharmaceutical preparation in the prevention or treatment of PCSK9-related diseases.

[0013] The present invention also provides the use of one or more of the described guide RNA, the described composition, and the described pharmaceutical formulation in the preparation of a medicament for the prevention or treatment of PCSK9-related diseases.

[0014] This invention further optimizes the guide RNA, enabling it to maintain on-target editing efficiency while exhibiting low off-target efficiency, thus balancing efficacy and safety. Compositions containing this guide RNA can safely, effectively, and conveniently regulate PCSK9 gene expression over the long term, achieving a sustained reduction in LDL-C levels, which helps avoid the side effects and compliance issues associated with traditional PCSK9 targeted drugs. Attached Figure Description

[0015] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0016] Figure 1 shows the on-target efficiency and off-target efficiency of sgRNAs containing different leader sequences in PHH cells of RAS donors in Example 2 of the present invention.

[0017] Figure 2 shows the on-target efficiency and off-target efficiency of sgRNAs containing different leader sequences in PHH cells of XWP donors in Example 2 of the present invention.

[0018] Figure 3 shows the gene editing efficiency of sgRNAs containing different backbone sequences in mice in vivo in Example 7 of the present invention.

[0019] Figure 4 shows the gene editing efficiency of sgRNAs containing different backbone sequences in mice in vivo experiments in Example 10 of the present invention.

[0020] Figure 5 shows the gene editing efficiency of sgRNAs containing different backbone sequences in mice in vivo experiments in Example 11 of the present invention. Detailed Implementation

[0021] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0022] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0023] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0024] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0025] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM can fluctuate within ranges of ±1%, ±2%, ±5%, etc.

[0026] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0027] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0028] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this invention.

[0029] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, then each "optional" or "optional" term is independent.

[0030] In this invention, the term "guide RNA" (gRNA) is a key molecule in the CRISPR-Cas system used to guide Cas enzymes (such as Cas9, Cas12a, Cas13, etc.) to recognize and cleave target DNA or RNA. In some embodiments, the guide RNA is a single-stranded guide RNA (sgRNA). In some specific embodiments, the guide RNA (sgRNA) contains the leader sequence and the backbone sequence sequentially from the 5' end to the 3' end. In some embodiments, the guide RNA is a double-stranded RNA (dsRNA), comprising crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA). In some specific embodiments, the guide RNA (dsRNA) comprises crRNA containing the leader sequence and tracrRNA containing the backbone sequence. In some embodiments, the guide RNA refers to crRNA containing the leader sequence.

[0031] In this invention, the term "PCSK9" refers to proprotein convertase subtilisin / kexin type 9, a protein that plays a key role in cholesterol metabolism and affects plasma LDL cholesterol levels by regulating LDLR levels.

[0032] In the sequence of this invention, the uppercase letters “A”, “U”, “C”, and “G” represent adenosine, uridine, cytidine, and guanosine, respectively; “dA”, “dT”, “dC”, and “dG” represent deoxyadenosine, deoxythymidine, deoxycytidine, and deoxyguanosine, respectively; * indicates that the adjacent nucleotides on both sides are connected by a phosphate thioester (PS) bond; and the lowercase letter “m” indicates that the adjacent nucleotide on the right is modified with 2'-O-methyl (2'-O-Me).

[0033] In this invention, when referring to "the ...th nucleotide position in the nucleotide sequence shown in SEQ ID No. 51," it means using the nucleotide position of that sequence as a reference to facilitate identification of modification and / or mutation positions by those skilled in the art. It does not only refer to modifications and / or mutations based on the sequence shown in SEQ ID No. 51, but also includes further modifications and / or mutations at corresponding positions based on other preferred sequences of this invention. Even after a deletion, insertion, or mutation occurs based on the sequence shown in SEQ ID No. 51, those skilled in the art can still identify the nucleotide positions in the new sequence corresponding to the sequence shown in SEQ ID No. 51.

[0034] While it is known in the art that DNA modification of the spacer region may reduce off-target editing strategies, it is also well known that this modification leads to a decrease in on-target editing efficiency, as demonstrated in publications 10.1038 / s41467-017-01732-9 (2017) and 10.1038 / nchembio.2559 (2018). This invention, through extensive experimentation, has discovered a modification scheme that achieves lower off-target efficiency while maintaining (without affecting or only slightly affecting) on-target editing.

[0035] Based on the above findings, the present invention first provides a guide RNA comprising a spacer sequence, wherein the spacer sequence comprises the 4th to 20th, or the 3rd to 20th, or the 2nd to 20th, or the 1st to 20th nucleotide sequences of any of the sequences shown in SEQ ID No. 1 to 25 (see Table 1).

[0036] In some preferred embodiments, the leader sequence comprises the 4th to 20th, 3rd to 20th, 2nd to 20th, or 1st to 20th nucleotide sequences of any of the sequences shown in SEQ ID No. 1-2, 4-6, 8-10, 12-13, 15-22, 24-25.

[0037] In some preferred embodiments, the lead sequence comprises nucleotides 4-20, 3-20, 2-20, or 1-20 of the sequence shown in any of SEQ ID Nos. 1-2, 4-6, 9-10, and 15. Such guide RNAs can better balance on-target and off-target efficiency.

[0038] In some further preferred embodiments, the lead sequence comprises nucleotides 4–20, 3–20, 2–20, or 1–20 of the sequence shown in SEQ ID No. 1. Guide RNAs containing this lead sequence exhibit significantly lower off-target efficiency while maintaining target editing efficiency comparable to or similar to that before modification.

[0039] Table 1

[0040] In some embodiments, at least the first three nucleotides at the 5' end of the leader sequence are modified nucleotides.

[0041] In some embodiments, the modified nucleotide includes one or more of 2′-O-methyl (2′-OMe), 2′-O-(2-methoxyethyl) (2′-O-moe), and 2′-fluoro (2′-F).

[0042] In some embodiments, the modified nucleotide is a 2'-O-methyl modified nucleotide.

[0043] In some embodiments, the first four nucleotides at the 5' end of the leader sequence are linked by phosphate thioester bonds.

[0044] In some embodiments, the leader sequence comprises the 4th to 20th, or the 3rd to 20th, or the 2nd to 20th, or the 1st to 20th nucleotide sequences of any of the sequences shown in SEQ ID No. 26 to 50 (see Table 2).

[0045] In some preferred embodiments, the leader sequence comprises the 4th to 20th, 3rd to 20th, 2nd to 20th, or 1st to 20th nucleotide sequences of any of the sequences shown in SEQ ID No. 26–27, 29–31, 33–35, 37–38, 40–47, 49–50.

[0046] In some preferred embodiments, the leader sequence comprises the 4th to 20th, 3rd to 20th, 2nd to 20th, or 1st to 20th nucleotide sequences of any of the sequences shown in SEQ ID No. 26-27, 29-31, 34-35, 41.

[0047] In some further preferred embodiments, the leader sequence comprises nucleotides 4 to 20, 3 to 20, 2 to 20, or 1 to 20 of the sequence shown in SEQ ID No. 26.

[0048] Table 2

[0049] In some specific embodiments, those skilled in the art can further replace one or more ribonucleotides in the lead sequence of the guide RNA (especially the nucleotide positions where deoxyribonucleotide substitution occurs as described in the above sequence) with deoxyribonucleotides based on the above sequence, and obtain guide RNAs with technical effects (such as target efficiency and off-target efficiency) equivalent to or similar to those of the sequences in this invention. Such guide RNAs are also within the scope of protection of this invention.

[0050] In some specific embodiments, those skilled in the art can further modify or demodify one or more ribonucleotides in the lead sequence of the guide RNA based on the above sequence (such as adding or removing a small amount of 2′-O-methyl modification or adding a small amount of one or two of 2′-O-(2-methoxyethyl) and 2′-fluorine modification) to obtain guide RNA with technical effects (such as target efficiency and off-target efficiency) equivalent to or similar to those of the sequence in this invention. Such guide RNA is also within the protection scope of this invention.

[0051] In some specific embodiments, those skilled in the art can combine the above-described lead sequence with different structural sequences (such as backbone sequences, repetitive sequences, etc.) to obtain guide RNAs targeting the PCSK9 gene that match different CRISPR systems (such as CRISPR-Cas9 system, CRISPR-Cas12a system, CRISPR-Cas12b system, CRISPR-Cas13 system, etc.).

[0052] In some implementations, the guide RNA also includes a scaffold sequence.

[0053] In some embodiments, the backbone sequence comprises a nucleotide sequence as shown in SEQ ID No. 51.

[0054] When the aforementioned leader sequence is combined with the backbone sequence, the resulting guide RNA exhibits better stability and efficacy in the CRISPR-Cas9 system.

[0055] The present invention also provides a guide RNA comprising a backbone sequence as described in any of the following embodiments. In some embodiments, the guide RNA further comprises a conventional leader sequence or a leader sequence as described in any of the preceding embodiments.

[0056] In some embodiments, the backbone sequence does not contain 2'-O-methyl modification at nucleotide positions 9, 49, and 62 of the nucleotide sequence shown in SEQ ID No. 51, and contains 2'-O-methyl modified nucleotides at nucleotide positions 8, 21, and 22 of the nucleotide sequence shown in SEQ ID No. 51. This is beneficial for further improving the editing efficiency of the guide RNA.

[0057] In some embodiments, one or more nucleotides (e.g., position 1, 2, 3, 4, or 5) at nucleotide positions 31, 42, 44, 45, and 48 of the nucleotide sequence shown in SEQ ID No. 51 are each independently a 2'-O-methyl modified nucleotide or a 2'-deoxynucleotide. After the above modifications to the backbone sequence, the in vitro and in vivo efficacy of the guide RNA remains unchanged or is slightly enhanced.

[0058] In some preferred embodiments, the backbone sequence has one or more (e.g., position 1, 2, 3 or 4) nucleotides at positions 31, 42, 45 and 48 of the nucleotide sequence shown in SEQ ID No. 51 that are 2'-O-methyl modified nucleotides.

[0059] In some preferred embodiments, the backbone sequence has one or more (e.g., position 1, position 2, position 3 or position 4) nucleotides at positions 42, 44, 45 and 48 of the nucleotide sequence shown in SEQ ID No. 51, each of which is an independent 2'-deoxynucleotide.

[0060] In some embodiments, the backbone sequence does not contain or is not entirely composed of 2'-O-methyl modified nucleotides at nucleotide positions 49-60 (hairpin1 region) and 62-76 (hairpin2 region) of the nucleotide sequence shown in SEQ ID No. 51.

[0061] In some embodiments, the backbone sequence does not contain or is not entirely composed of 2'-O-methyl modified nucleotides at positions 50-52, 57-59, 63-66, and 72-75 of the nucleotide sequence shown in SEQ ID No. 51.

[0062] Compared to guide RNAs whose hairpin1 and hairpin2 regions in the backbone sequence are entirely composed of nucleotides modified with 2'-O-methyl, the in vivo and in vitro efficacy of guide RNAs containing the backbone sequence of the present invention remains unchanged or is slightly improved.

[0063] In some embodiments, the backbone sequence is a nucleotide modified with 2′-deoxyribose at position 42 of the nucleotide sequence shown in SEQ ID No. 51. This invention has found that the above optimization further enhances the in vitro and in vivo efficacy of the guide RNA.

[0064] In some embodiments, the backbone sequence further includes at least one of the following mutations: (I) an insertion of 1 to 4 nucleotides (e.g., 1, 2, 3, or 4) between nucleotides 12 and 13, and between nucleotides 16 and 17, as shown in SEQ ID No. 51; (II) an insertion of 1 to 4 nucleotides (e.g., 1, 2, 3, or 4) between nucleotides 52 and 53, and between nucleotides 56 and 57, as shown in SEQ ID No. 51; (III) a deletion and / or substitution of 1 to 4 nucleotides (e.g., 1, 2, 3, or 4) at nucleotides 49 to 52, and at nucleotides 57 to 60, as shown in SEQ ID No. 51. This invention has found that the above mutations further enhance the in vitro and in vivo efficacy of the guide RNA.

[0065] In some embodiments, the backbone sequence includes at least one mutation selected from (I) above.

[0066] In some embodiments, the backbone sequence includes at least one mutation selected from (II) above.

[0067] In some embodiments, the backbone sequence includes at least one mutation selected from (III) above.

[0068] In some embodiments, the backbone sequence includes at least one mutation selected from (I) above and selected from (II) or (III) above.

[0069] In some embodiments, as described above (I), specifically, there are insertions of one, two, three, or four nucleotides between the 12th and 13th nucleotide positions and between the 16th and 17th nucleotide positions of the nucleotide sequence shown in SEQ ID No. 51.

[0070] In some embodiments, as described above (II), there are insertions of one, two, three, or four nucleotides between the 52nd and 53rd nucleotide positions and between the 56th and 57th nucleotide positions of the nucleotide sequence shown in SEQ ID No. 51.

[0071] In some embodiments, as described above (III), specifically, one, two, three, or four nucleotides are simultaneously deleted at positions 49-52 and 57-60 of the nucleotide sequence shown in SEQ ID No. 51.

[0072] In some embodiments, as described above (III), specifically, at nucleotide positions 49-52 and 57-60 of the nucleotide sequence shown in SEQ ID No. 51, there is a deletion of 3 nucleotides and a substitution of 1 nucleotide, respectively.

[0073] This invention has found that introducing the mutation in (I) above further improves the on-target efficiency of the guide RNA; introducing the mutation in (III) above further improves the on-target efficiency of the guide RNA, and also improves the off-target efficiency to some extent. By combining (I) above with at least one mutation selected from (II) or (III) above, it is possible to improve the on-target efficiency of the guide RNA while taking into account the off-target efficiency.

[0074] In some embodiments, the backbone sequence comprises any of the nucleotide sequences shown in SEQ ID No. 108 to 131 (see Table 3).

[0075] In some preferred embodiments, the backbone sequence comprises a nucleotide sequence as shown in any of SEQ ID No. 108, 114, 115, 121, 122, 124, 127, 130.

[0076] In some further preferred embodiments, the backbone sequence comprises a nucleotide sequence as shown in SEQ ID No. 108.

[0077] Table 3

[0078] In some embodiments, the backbone sequence comprises a nucleotide sequence as shown in any of SEQ ID No. 52-80, 84-107.

[0079] In some embodiments, the backbone sequence comprises a nucleotide sequence as shown in any of SEQ ID No. 54–60, 69, 71, 74–75, 77–80, 89–91, 97, 98, 100, 103, 106.

[0080] In some preferred embodiments, the backbone sequence comprises a nucleotide sequence as shown in SEQ ID No. 56, 60 or 77.

[0081] In some further preferred embodiments, the backbone sequence comprises a nucleotide sequence as shown in SEQ ID No. 60 or 77.

[0082] In some further preferred embodiments, the backbone sequence comprises a nucleotide sequence as shown in any of SEQ ID Nos. 89–91, 97, 98, 100, 103, and 106.

[0083] In some further preferred embodiments, the backbone sequence comprises a nucleotide sequence as shown in SEQ ID No. 89.

[0084] In some preferred embodiments, the backbone sequence comprises a nucleotide sequence such as 60, 77, or 89.

[0085] In specific implementations, those skilled in the art can further replace parts (one or more of the nucleotide positions 50-52, 57-59, 63-66, and 72-75 of the nucleotide sequence shown in SEQ ID No. 51) or all of the 2'-O-methyl modified nucleotides in the hairpin1 and hairpin2 regions with unmodified nucleotides, based on the aforementioned advantageous backbone sequences (such as SEQ ID No. 56, 60, 77, or 89), to obtain guide RNAs that are comparable to or slightly improved in terms of technical effects (such as in vivo and in vitro editing efficiency) with the aforementioned advantageous backbone sequences. Such RNAs are also within the scope of protection of this invention.

[0086] In specific implementations, those skilled in the art can further replace one or more nucleotides at positions 31, 42, 44, 45, and 48 of the nucleotide sequence shown in SEQ ID No. 51 with 2'-O-methyl modified nucleotides or 2'-deoxynucleotides, based on the aforementioned advantageous backbone sequences (such as SEQ ID No. 56, 60, 77, or 89), to obtain guide RNAs that are comparable to or slightly improved in terms of technical effects (such as in vivo and in vitro editing efficiency) with the aforementioned advantageous backbone sequences. Such RNAs are also within the scope of protection of this invention.

[0087] In some specific embodiments, those skilled in the art can further modify or demodify one or more ribonucleotides in the backbone sequence of the guide RNA based on the above sequence (such as adding or removing a small amount of 2′-O-methyl modification or adding a small amount of one or two of 2′-O-(2-methoxyethyl) and 2′-fluorine modification) to obtain guide RNA with technical effects (such as in vivo and in vitro editing efficiency) equivalent to or similar to those of the sequence in this invention. Such guide RNA is also within the protection scope of this invention.

[0088] In some embodiments, the guide RNA has a maximum off-target efficiency of less than 0.1% at the effective dose.

[0089] In some preferred embodiments, the guide RNA (e.g., a guide RNA containing P9-hc-023-seq22) has a maximum off-target efficiency of less than 0.1% at three times the EC90 dose.

[0090] The present invention also provides a composition comprising: (i) the guide RNA, or a nucleic acid encoding the guide RNA; and at least one selected from (ii) to (iii) below: (ii) an effector protein, or a nucleic acid encoding the effector protein; and (iii) a delivery vector.

[0091] In some embodiments, the effector protein is a nuclease.

[0092] In some embodiments, the nuclease is a nicking enzyme.

[0093] In some embodiments, the effector protein is a Cas protein; for example, Cas9, Cas12, Cas13, etc.

[0094] In some embodiments, the effector protein is the Cas9 protein; for example, the Streptococcus pyogenes Cas9 protein.

[0095] In some embodiments, the nucleic acid encoding the effector protein is mRNA encoding the Cas protein.

[0096] In some embodiments, the nucleic acid encoding the effector protein is mRNA encoding the Cas9 protein.

[0097] In some specific implementations, the specific nucleotide sequence of the nucleic acid encoding the effector protein can be determined by referring to the disclosures in relevant patent documents in the field, such as PCT / CN2023 / 120234 and US11697806B2.

[0098] In some embodiments, the nucleic acid encoding the effector protein comprises a nucleotide sequence as shown in SEQ ID No. 81 or SEQ ID No. 82.

[0099] Specifically, SEQ ID No. 81 contains a 5'-UTR, CDS, 3'-UTR, and polyA sequence sequentially from the 5' end to the 3' end. The 5'-UTR sequence (nucleotides 1-73 of SEQ ID No. 81) corresponds to SEQ ID NO: 961 in PCT / CN2023 / 120234. In practice, any one of the sequences SEQ ID NO: 32, 34, 36, 38, 41, or 75-77 in US11697806B2 can be selected to achieve a similar function. The CDS sequence (nucleotides 74-4129 of SEQ ID No. 81) corresponds to SEQ ID NO: 959 in PCT / CN2023 / 120234. Alternatively, any other sequence from SEQ ID NO: 954-959 in PCT / CN2023 / 120234 can be selected to achieve a similar function. The 3'-UTR sequence (nucleotides 4130-4414 in SEQ ID No. 81) corresponds to SEQ ID NO: 962 in PCT / CN2023 / 120234. Alternatively, any sequence from SEQ ID NO: 33, 35, 37, 39, or 40 in US11697806B2 can be selected to obtain a similar function. The polyA sequence (nucleotides 4415-4523 in SEQ ID No. 81) corresponds to SEQ ID NO: 970 in PCT / CN2023 / 120234. Alternatively, any sequence from SEQ ID NO: 965-970 in PCT / CN2023 / 120234 can be selected to obtain a similar function.

[0100] SEQ ID No. 82 contains a 5'-UTR, a CDS, and a 3'-UTR sequence from its 5' end to its 3' end. The 5'-UTR sequence (nucleotides 1-73 of SEQ ID No. 82) corresponds to SEQ ID NO: 961 in PCT / CN2023 / 120234. In practice, any one of the sequences SEQ ID NO: 32, 34, 36, 38, 41, or 75-77 in US11697806B2 can be selected to achieve a similar function. The CDS sequence (nucleotides 74-4210 of SEQ ID No. 82) corresponds to SEQ ID NO: 956 in PCT / CN2023 / 120234. Alternatively, any other sequence from SEQ ID NO: 954-959 in PCT / CN2023 / 120234 can be selected to achieve a similar function. The 3'-UTR sequence (nucleotides 4211-4405 in SEQ ID No. 82) corresponds to SEQ ID NO: 962 in PCT / CN2023 / 120234. Alternatively, any one of the sequences SEQ ID NO: 33, 35, 37, 39, or 40 in US11697806B2 can be selected to achieve a similar function. In some specific embodiments, a polyA sequence is further included at the 3' end of the 3'-UTR sequence shown in SEQ ID No. 82. This polyA sequence can be any one of the sequences SEQ ID NO: 965-970 in PCT / CN2023 / 120234 to achieve a similar function.

[0101] In some embodiments, the delivery carrier is LNP (Lipid Nanoparticles).

[0102] In some embodiments, the LNP includes the following components: ionizable lipids, phospholipids, cholesterol, and polyethylene glycol ester conjugates.

[0103] In some embodiments, the ionizable lipid is a cationic lipid, such as ALC-0315.

[0104] In some embodiments, the ionizable lipid can be determined with reference to the contents of Chinese patent application number 2024116579272. As an example, the ionizable lipid (cationic lipid) can be one or more of ILL-N-41, ILL-N-42, ILL-N-43, ILL-N-44, ILL-N-45, ILL-N-46, ILL-N-47, ILL-N-48, ILL-N-49, ILL-N-50, ILL-N-51, and ILL-N-52 from Chinese patent application number 2024116579272.

[0105] In some embodiments, the ionizable lipid is ILL-N-43, which has the following structure:

[0106] In some embodiments, the phospholipid is DSPC (disteaaroyl-sn-glycero-3-phosphocholine).

[0107] In some embodiments, the polyethylene glycol ester conjugate is PEG-DMG (polyethylene glycol dimyristylglycerol).

[0108] In some specific embodiments, the LNP includes the following components: one selected from ALC-0315 or ILL-N-43, DSPC, cholesterol, and PEG2k-DMG.

[0109] The composition of this invention can precisely edit the PCSK9 gene, thereby significantly reducing the level of low-density lipoprotein (LDL-C) in the blood. Compared with traditional LNP-based gene-editing drugs, the composition of this invention can effectively carry and precisely deliver the gene-editing system (CRISPR / Cas9 system) to liver cells, significantly improving delivery efficiency and liver targeting, and has lower toxicity and a wider therapeutic window. Compared with other PCSK9-targeting drugs (such as RNAi or antibodies), the composition of this invention precisely edits the PCSK9 gene with a single dose, eliminating the need for long-term repeated administration, thereby reducing side effects and providing a more convenient, safe, and efficient treatment option.

[0110] In some embodiments, the delivery carrier may also be selected from polymer nanoparticles, cell-penetrating peptides (CPPs), viral vectors (such as adenovirus, adeno-associated virus, lentivirus, etc.), polymer carriers (such as polyethyleneimine (PEI), polylactic acid-glycolic acid copolymer (PLGA), etc.), nanogels, liposomes, cationic nanoparticles, microvesicles, exosomes, microcapsules, and nanocapsules.

[0111] In some embodiments, the composition is a lipid nanoparticle (LNP) composition containing the guide RNA.

[0112] In some embodiments, the composition comprises the guide RNA in conjunction with lipid nanoparticles (LNPs).

[0113] In some embodiments, the composition comprises guide RNA and an effector protein or mRNA encoding an effector protein.

[0114] In some specific embodiments, the composition includes sgRNA and mRNA encoding the Cas protein.

[0115] In some specific embodiments, the composition includes sgRNA and LNP.

[0116] In some specific embodiments, the composition includes sgRNA, mRNA encoding the Cas protein, and LNP.

[0117] In some specific embodiments, the composition includes sgRNA and Cas protein.

[0118] In some specific embodiments, the composition includes sgRNA, Cas protein, and lipid nanoparticles.

[0119] In some specific embodiments, the composition includes a plasmid containing an sgRNA sequence and a plasmid containing a Cas protein-encoded nucleic acid.

[0120] In some specific embodiments, the composition includes an AAV vector containing an sgRNA sequence and an AAV vector containing a Cas protein-encoded nucleic acid.

[0121] The present invention also provides a pharmaceutical formulation comprising any one of (1) or (2) below and a pharmaceutically acceptable adjuvant thereof: (1) the guide RNA; (2) the composition.

[0122] In some specific embodiments, the pharmaceutically acceptable adjuvant is selected from one or more of stabilizers (protectants), transfection agents, immunosuppressants, buffers, solvents, preservatives, pH adjusters, osmotic pressure adjusters, and surfactants.

[0123] The present invention also provides a method for editing the PCSK9 gene, comprising: delivering one or more of the guide RNA, the composition, and the pharmaceutical preparation to a cell.

[0124] In some implementations, the method is performed within the body.

[0125] In some implementations, the method is performed outside the body.

[0126] In some embodiments, the method results in an insertion or deletion in the PCSK9 gene. In some embodiments, the insertion or deletion of nucleotides in the target gene leads to a frameshift mutation or premature stop codon, which produces a non-functional protein. In some embodiments, the insertion or deletion of nucleotides in the target gene leads to the knockout or elimination of target gene expression.

[0127] In some embodiments, the method further includes delivering a template to a cell, at least a portion of which is incorporated into a sequence at or near a double-strand break site induced by the Cas protein.

[0128] The present invention also provides the use of one or more of the described guide RNA, the described composition, and the described pharmaceutical preparation in the prevention or treatment of PCSK9-related diseases. Specifically, a method for preventing or treating PCSK9-related diseases includes: delivering one or more of the described guide RNA, the described composition, and the described pharmaceutical preparation to cells.

[0129] The present invention also provides the use of one or more of the described guide RNA, the described composition, and the described pharmaceutical formulation in the preparation of a medicament for the prevention or treatment of PCSK9-related diseases.

[0130] In some embodiments, the PCSK9-related diseases include one or more of the following: lipid metabolism disorders (such as hypercholesterolemia), cardiovascular diseases, atherosclerosis, liver diseases, diabetes, kidney diseases, neurodegenerative diseases, tumors, infectious diseases, and autoimmune diseases.

[0131] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0132] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0133] To facilitate comparison, the following examples all use sgRNA consisting of a leader sequence and a backbone sequence from the 5' end to the 3' end.

[0134] The skeleton sequences used in the following embodiments are shown in Table 4. To facilitate comparison of on-target efficiency and off-target efficiency, a uniform skeleton sequence is used in each embodiment for screening and optimizing the lead sequence. However, in the embodiments for screening and optimizing the lead sequence, the difference between on-target efficiency and off-target efficiency is mainly determined by the different lead sequences; the limitation of the skeleton sequence should not be construed as a limitation on the scope of protection of the invention.

[0135] Table 4

[0136] The following is a detailed description of the general experimental methods involved in the embodiments of the present invention:

[0137] Cell culture

[0138] The Huh7 cell line was used. The human hepatocellular carcinoma cell line Huh7 was cultured in DMEM medium supplemented with 10% fetal bovine serum. Twenty-four hours before transfection, cells were seeded in 96-well plates at a density of 8,000 cells / well. Cells were transfected with Lipofectamine RNAiMAX (ThermoFisher, catalog number 13778030) according to the supplier's protocol. Cells were transfected with a lipid complex containing 1-500 ng Cas9 mRNA, 1-500 ng sgRNA, and Lipofectamine RNAiMAX.

[0139] Primary human hepatocytes. Culture primary human hepatocytes (PHH) (Lonza or BioIVT) according to the supplier's protocol. In short, thaw the cells and resuspend them in hepatocyte thawing medium supplemented with the supplement. Then, centrifuge the human hepatocytes at 100g for 10 minutes, count the cells, and seed them at a density of 120,000 cells / well in 48-well Bio-coat collagen I-coated plates (ThermoFisher, catalog number 877272). Allow the seeded cells to settle and adhere in a tissue culture incubator at 37°C and 5% CO2 atmosphere for 4 hours. After culture, check for cell monolayer formation and replace the medium with hepatocyte culture medium containing a serum-free supplement packet. On the same day, cells were transfected using Lipofectamine MessengerMAX (ThermoFisher, catalog number LMRNA003) according to the supplier's protocol, using a lipid complex containing 1-500 ng Cas9 mRNA, 1-500 ng sgRNA, and Lipofectamine MessengerMAX. Alternatively, the cells were transfected the following day using lipid nanoparticles containing Cas9 mRNA and sgRNA, with a total RNA amount of 1-3000 ng.

[0140] Genomic DNA isolation

[0141] Transfected cells were harvested 72 hours post-transfection. Genomic DNA was extracted from each well of a 24-well plate using the FastPure Cell / Tissue DNA Extraction Kit (Novozymes, catalog number DC102-01) or from a 48 / 96-well plate using QuickExtract DNA Extraction Solution (LGC Lucigen, catalog number QE09050) according to the supplier's protocol. All DNA samples were then used for library construction and sequencing analysis.

[0142] Next-generation sequencing (“NGS”) analysis

[0143] To quantitatively determine the editing efficiency and patterns at target sites in the genome, sequencing is used to identify the presence of substitutions, insertions, and deletions introduced by gene editing.

[0144] NGS is used to assess editing efficiency and patterns at specific genomic target sites. This method allows for the quantitative detection of any insertions, deletions, or substitutions introduced during gene editing.

[0145] Primers are designed around target sites within genes of interest (e.g., PCSK9) to amplify the genomic regions of interest.

[0146] Following the Illumina vendor's protocol, additional PCR was performed to add adapter sequences for sequencing. The amplicon was then sequenced using an Illumina NovaSeq 6000 instrument. The resulting reads were aligned to a reference genome, which could be a human reference genome (hg38), a cynomolgus monkey reference genome (mf5), a rat reference genome (rn6), or a mouse reference genome (mm39). Low-quality reads were eliminated before alignment. The reads were mapped to the reference genome, and only those overlapping with the target region of interest were selected. Wild-type reads and reads containing insertions, substitutions, or deletions were identified and counted.

[0147] Edit percentage, also known as “edit efficiency,” “edit percentage,” or “insertion / deletion frequency,” is determined by dividing the total number of sequence readings with insertions, deletions, or substitutions by the total number of sequence readings, including wild-type readings.

[0148] LNP in vivo delivery

[0149] There is a need for compositions for delivering CRISPR / Cas protein and nucleic acid components to cells, such as cells in a patient's body. In particular, compositions with useful properties for in vitro and in vivo delivery, capable of stabilizing and delivering RNA components, are of interest.

[0150] Here, we provide compositions of lipid nanoparticles with useful properties, particularly compositions for delivering CRISPR / Cas gene editing components. The LNP composition comprises: an RNA component; and a lipid component, wherein the lipid component comprises: (1) about 45-55 mol% amine lipids (ALC-0315 unless otherwise specified); (2) about 9-11 mol% neutral lipids; and (3) about 1-5 mol% PEG lipids, wherein the remainder of the lipid component is accessory lipids, and wherein the N / P ratio of the LNP composition is about 3 to about 8. The proportions of the substances in the LNP used in the following examples are set according to the disclosure of PCT / CN2023 / 120234. In practice, those skilled in the art can also set the specific proportions of the substances in the LNP based on common sense, all of which can achieve the technical effects mentioned in this invention. The limitation on the proportions of the substances in the LNP should not be construed as a limitation on the scope of protection of the invention.

[0151] Unless otherwise stated, each study used 6-8 week old female CD-1 mice or PCSK9 humanized mice. Animals were weighed and grouped according to body weight to prepare the dosing solution based on the group average body weight. LNP was administered via tail vein at a dose of 0.2 ml per animal (approximately 10 ml / kg body weight). Animals were observed daily for adverse reactions. Body weight was measured every other day after administration. Blood was collected at different time points via orbital or cardiac puncture. For studies involving in vivo editing, animals were euthanized and liver tissue was collected for DNA extraction and analysis.

[0152] The ILL-N-43 used in the following embodiments was synthesized by the following method:

[0153] At 20 °C, 3-pyrrolidone-1-yl-1-propanol (503.58 mg, 3.90 mmol) was added to a DCM (40 mL) solution of intermediate B (1.7 g, 1.95 mmol), triethylamine (591.61 mg, 5.85 mmol, 813.77 μL), and DMAP (23.81 mg, 194.89 μmol). The mixture was stirred at 50 °C for 12 hours. The reaction mixture was then stirred at 80 °C for another 4 hours, and the reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by preparative HPLC (FA) under the following conditions: column: X-Select CSH phenylhexyl column, 100*30mm, 5μm; mobile phase: [H2O(0.2%FA)-ACN:THF=1:1]; gradient: 1-20min, 42%-62% (B), flow rate: 130-150mL / min; ILL-N-43 white solid was obtained (458.23mg, yield 27.26%).

[0154] LC-MS: RT = 1.877 min, [M+H]+ = 862.9, Method: LIPID_50-100AB_6 min.

[0155] 1H NMR (400MHz, CDCl3) δ = 5.10 (br d,J=9.2Hz,2H),4.74-4.62(m,1H),4.19(t,J=6.4Hz,2H),4.00-3.84(m,2H),2.62-2.46(m,6H), 2.15(t,J=7.6Hz,4H),1.92(quin,J=7.2Hz,2H),1.83-1.75(m,4H),1.65-1.45(m,12H),1.28(br d, J=5.6Hz, 56H), 0.88 (t, J=6.4Hz, 12H).

[0156] Example 1

[0157] In the huh7 cell line, sgRNAs containing different spacer sequences were screened. Each sgRNA consists of a spacer sequence and a backbone sequence from the 5' end to the 3' end. The spacer sequences corresponding to each name are shown in Table 5. The backbone sequences are all the sequences shown in SEQ ID No. 52.

[0158] Specifically, 2.2 ng sgRNA and 17.8 ng Cas9 mRNA (the nucleotide sequence of which is shown in SEQ ID No. 81) were co-delivered into Huh7 cells in 96-well plates using Lipofectamine RNAiMAX. Cells were harvested 48 hours after transfection, and genomic DNA was extracted for analysis and evaluation of its target efficiency and off-target efficiency.

[0159] The results are shown in Table 5 below.

[0160] Table 5

[0161] Example 2

[0162] Further screening of sgRNAs containing different leader sequences was conducted in human primary hepatocytes (PHH). These sgRNAs consisted of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequences available for screening included P9-hc-023-seq1, P9-hc-023-seq8, P9-hc-023-seq15, P9-hc-023-seq16, P9-hc-023-seq17, P9-hc-023-seq20, P9-hc-023-seq21, and P9-hc-023-seq22. The specific sequences corresponding to each name are shown in Table 4. The backbone sequences were all as shown in SEQ ID No. 60.

[0163] Specifically, sgRNA and Cas9 mRNA (the nucleotide sequence of which is shown in SEQ ID No. 81) were delivered via LNP to PHH cells from donors RAS and XWP, respectively. The mass ratio of sgRNA to Cas9 mRNA was 1:2. Different experimental groups were set up with sgRNA concentrations of 0.25 nM, 1 nM, 4 nM, 16.2 nM, and 64.6 nM. 72 hours after transfection, PHH cells from both RAS and XWP donors were harvested, and genomic DNA was extracted. The on-target efficiency and off-target efficiency were then analyzed and evaluated.

[0164] The detection results in PHH cells from RAS donors and PHH cells from XWP donors are shown in Figures 1 and 2, respectively. These results show that, compared to P9-hc-023-seq1, the sgRNA containing P9-hc-023-seq22 has significantly lower off-target efficiency while maintaining on-target editing efficiency.

[0165] Example 3

[0166] Based on GUIDE-seq technology, the off-target editing efficiency of sgRNAs containing P9-hc-023-seq1 and P9-hc-023-seq22 was analyzed. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequences corresponding to each name are shown in Table 4, and the backbone sequences are all the sequences shown in SEQ ID No. 61.

[0167] Specifically, 100 ng sgRNA, 500 ng Cas9 mRNA (its nucleotide sequence is shown in SEQ ID No. 81), and 50 ng double-stranded oligonucleotides were co-delivered into 24-well PHH cells of donor number HUM183001 using Lipofectamine RNAiMAX. Library construction and sequencing were performed according to the method described in the literature (doi:10.1038 / nbt.3117), and then the off-target efficiency was analyzed and evaluated.

[0168] The results are shown in Table 6.

[0169] Table 6

[0170] Specifically, sgRNA and Cas9 mRNA (the nucleotide sequence of which is shown in SEQ ID No. 81) were delivered via LNP to PHH cells from donors MFS and XWP, respectively. The mass ratio of sgRNA to Cas9 mRNA was 1:2. An experimental group with 64.6 nM of sgRNA was used. 72 hours after transfection, PHH cells from donors MFS and XWP were harvested, and genomic DNA was extracted. The on-target efficiency and off-target efficiency were then analyzed and evaluated. Off-target sites 1–3 were derived from the first three off-target sites in GUIDE-Seq. Off-target sites 4–13 were supplemented from the bioinformatics prediction website (http: / / www.rgenome.net / cas-offinder / ). The spacer sequence of P9-hc-023-seq1 was input, the PAM sequence was selected as spCas9 NGG, the base mismatch number was selected as less than or equal to 3, and the DNA or RNA protrusion was selected as 0. A total of 11 sites were predicted, one of which was the first off-target site ranked in GUIDE-Seq, so a total of 13 off-target sites were identified. As shown in Table 7, the values ​​in Table 7 are the results after subtracting the values ​​of the negative control samples; negative numbers are indicated by "-".

[0171] Table 7

[0172] The results showed greater off-target editing efficiency, indicating that, except for the first off-target site, off-target editing was very low (or lower than the negative control).

[0173] Example 4

[0174] Based on GUIDE-seq technology, the off-target editing efficiency of sgRNAs containing P9-hc-023-seq1 and P9-hc-023-seq22 was analyzed. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequences corresponding to each name are shown in Table 4, and the backbone sequences are all the sequences shown in SEQ ID No. 60.

[0175] Specifically, 100 ng sgRNA, 500 ng Cas9 mRNA (its nucleotide sequence is shown in SEQ ID No. 81), and 50 ng double-stranded oligonucleotides were co-delivered into 24-well PHH cells of donor number HUM183001 using Lipofectamine RNAiMAX. Library construction and sequencing were performed according to the methods described in the literature, and then the off-target efficiency was analyzed and evaluated.

[0176] The results are shown in Table 8.

[0177] Table 8

[0178] Specifically, sgRNA and Cas9 mRNA (the nucleotide sequence of which is shown in SEQ ID No. 81) were delivered via LNP to PHH cells from donors RAS and XWP, respectively. The mass ratio of sgRNA to Cas9 mRNA was 1:2. An experimental group with 64.6 nM of sgRNA was used. 72 hours after transfection, PHH cells from both donors (RAS and XWP) were harvested, and genomic DNA was extracted. The on-target efficiency and off-target efficiency were then analyzed and evaluated. Off-target sites 1–3 were derived from the first three off-target sites in GUIDE-Seq. Off-target sites 4–13 were supplemented from the bioinformatics prediction website (http: / / www.rgenome.net / cas-offinder / ). The spacer sequence of P9-hc-023-seq22 was input, the PAM sequence was selected as spCas9 NGG, the base mismatch number was selected as less than or equal to 3, and the DNA or RNA protrusion was selected as 0. A total of 11 sites were predicted, one of which was the first off-target site ranked in GUIDE-Seq, so a total of 13 off-target sites were identified. The results are shown in Table 9. The values ​​in Table 9 are the results after subtracting the values ​​of the negative control samples; negative numbers are indicated by "-".

[0179] Table 9

[0180] The results showed greater off-target editing efficiency, with low (or lower than) off-target editing at all sites.

[0181] Example 5

[0182] Based on in vivo experiments in PCSK9 humanized mice, the on-target and off-target efficiencies of sgRNAs containing P9-hc-023-seq1, P9-hc-023-seq21, or P9-hc-023-seq22 were analyzed. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequences corresponding to each name are shown in Table 4, and the backbone sequences are all the sequences shown in SEQ ID No. 53.

[0183] In this embodiment, 6- to 15-week-old humanized PCSK9 mice were used. These PCSK9 humanized mice were genetically engineered to delete a region of the endogenous mouse Pcsk9 locus and replace it with a homologous human PCSK9 sequence, thereby encoding the human PCSK9 protein at that locus. These humanized mice received LNP drugs (as shown in Table 10) encapsulating different sgRNAs and Cas9 mRNAs (the nucleotide sequences of which are shown in SEQ ID No. 81), with a weight ratio of mRNA to sgRNA of 2:1. The LNPs contained ALC-0315, DSPC, cholesterol, and PEG2k-DMG. The dosage was 0.6 / 0.4 / 0.2 mg / kg (based on total RNA content). Mice were randomly divided into groups, with the number of mice in each group shown as N in Table 10, and administered via tail vein, with an injection volume of approximately 0.2 ml per mouse (approximately 10 ml per kilogram of body weight). Blood samples were collected from the heart of the mice within 7 days after administration. Immediately after blood collection, mouse liver tissue was collected and placed at -80℃ for DNA extraction and NGS sequencing. The mouse-specific off-target sites were chr17:71475477-71475499, which contained a 3-base mismatch.

[0184] Table 10

[0185] The results showed that, in in vivo experiments, the on-target editing efficiencies of sgRNAs containing P9-hc-023-seq1 and P9-hc-023-seq22 were similar. Although off-target sites on the mouse genome and on the human genome differ due to genomic sequence differences, it was still evident that LNPs containing P9-hc-023-seq22 exhibited lower off-target editing in mice.

[0186] Example 6

[0187] Based on in vivo experiments in WT mice, the gene editing efficiency of sgRNAs containing different backbone sequences was analyzed. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is C188 (its sequence is 5'-mA*mC*mA*CAAAUACCAGUCCAGCG-3'), and the backbone sequences are shown in Table 11.

[0188] In this embodiment, 6- to 9-week-old C57BL / 6J WT mice were used. WT mice received LNP drugs encapsulated with different sgRNAs and Cas9 mRNAs (the nucleotide sequences of which are shown in SEQ ID No. 81), with a weight ratio of mRNA to sgRNA of 2:1. The LNPs contained ALC-0315, DSPC, cholesterol, and PEG2k-DMG. The dosage was 0.3 / 0.1 mg / kg (based on total RNA content). Mice were randomly divided into groups of one, and the drug was administered via tail vein, with an injection volume of approximately 0.2 mL per mouse (approximately 10 mL per kilogram of body weight). Blood samples were collected from the heart of the mice within 7 days after administration. Immediately after blood collection, liver tissue was collected from the mice and subjected to DNA extraction and NGS sequencing at -80°C.

[0189] Table 11

[0190] In summary, the results show that the sgRNA containing the backbone sequence shown in SEQ ID No. 56 has better editing efficiency than the backbone sequence shown in SEQ ID No. 53 (an existing backbone sequence).

[0191] Example 7

[0192] Based on in vitro experiments in PHH (using the same method as in Example 2), the gene editing efficiency of sgRNAs containing different backbone sequences was analyzed. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is P9-hc-023-seq1, and the backbone sequences are shown as shown in SEQ ID No. 53 and SEQ ID No. 60, respectively.

[0193] The results are shown in Figure 3. The results show that, compared with the backbone sequence shown in SEQ ID No. 53 (an existing backbone sequence), the sgRNA containing the backbone sequence shown in SEQ ID No. 60 has better editing efficiency.

[0194] Example 8

[0195] Based on in vivo experiments in PCSK9 humanized mice, the gene editing efficiency of sgRNAs containing different backbone sequences was analyzed. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is P9-hc-023seq1, and the backbone sequences are shown in Table 12.

[0196] In this embodiment, 6- to 15-week-old PCSK9 humanized mice, as described above, were used. These mice received LNP drugs encapsulating sgRNA and Cas9 mRNA (nucleotide sequences shown in SEQ ID No. 81) with different backbones, wherein the weight ratio of mRNA to sgRNA was 2:1. The LNP contained ALC-0315, DSPC, cholesterol, and PEG2k-DMG. The dosage was 0.6 / 0.2 mg / kg (based on total RNA content). Mice were randomly divided into groups, with the number of mice in each group as shown by the N values ​​in Table 12. The drug was administered via tail vein, with an injection volume of approximately 0.2 ml per mouse (approximately 10 ml per kilogram of body weight). Blood samples were collected from the heart of the mice within 7 days after administration. Immediately after blood collection, liver tissue was collected from the mice and subjected to DNA extraction and NGS sequencing at -80°C.

[0197] Table 12

[0198] The results show that, compared with the backbone sequence shown in SEQ ID No. 53 or the backbone sequence shown in SEQ ID No. 61 (existing backbone sequences), the sgRNA containing the backbone sequence shown in SEQ ID No. 60 has better editing efficiency.

[0199] Example 9

[0200] Based on in vivo experiments in WT mice, the gene editing efficiency of sgRNAs containing different backbone sequences was analyzed. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is C188 in all cases, and the backbone sequences are shown in Table 13.

[0201] In this embodiment, 6- to 9-week-old C57BL / 6J WT mice were used. These WT mice received LNP drugs encapsulating different backbone sgRNAs and Cas9 mRNAs (the nucleotide sequences of which are shown in SEQ ID No. 81), with a mRNA to sgRNA weight ratio of 2:1. The LNPs contained ALC-0315, DSPC, cholesterol, and PEG2k-DMG. The dosage was 0.25 / 0.15 mg / kg (based on total RNA content). Mice were randomly divided into groups of two and administered the drug via tail vein, with each mouse receiving approximately 0.2 mL (approximately 10 mL per kg body weight). Blood samples were collected from the heart via biopsy within 7 days post-administration. Immediately after blood collection, liver tissue was collected and subjected to DNA extraction and NGS sequencing at -80°C.

[0202] Table 13

[0203] The results show that after removing the 2OMe modification in hairpin1 and / or hairpin2 in the skeleton sequence, its editing efficiency is close to or better than that of the skeleton sequence before removing the modification, as shown in SEQ ID No. 56.

[0204] Example 10

[0205] This embodiment analyzes the gene editing efficiency of sgRNAs containing different backbone sequences. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is P9-hc-023seq22, and the backbone sequences are shown in Table 14.

[0206] Specifically, 17.8 ng sgRNA and 142.2 ng Cas9 mRNA (the nucleotide sequence of which is shown in SEQ ID No. 81) were co-delivered into 48-well PHH cells (donor number HUM18300) using Lipofectamine RNAiMAX. Cells were harvested 72 hours after transfection, and genomic DNA was extracted for analysis and evaluation of its target efficiency and off-target efficiency.

[0207] Table 14

[0208] The results indicate that sgRNAs containing backbone sequences such as SEQ ID No. 69, 71, 74, and 75 exhibit superior editing advantages on PHH compared to backbone sequences such as those shown in SEQ ID No. 60. The backbone sequence shown in SEQ ID No. 73 can be used as a negative control.

[0209] Furthermore, 6- to 15-week-old PCSK9 humanized mice were used. These mice were genetically engineered to delete a region of the endogenous mouse Pcsk9 locus and replace it with a homologous human PCSK9 sequence, thereby encoding the human PCSK9 protein at that locus. These humanized mice received LNP drugs encapsulating different backbones (as shown in SEQ ID No. 60, 69, 71, 73, 74, and 75) of sgRNA and Cas9 mRNA (the nucleotide sequence of which is shown in SEQ ID No. 81), with a weight ratio of mRNA to sgRNA of 2:1. The LNPs contained ALC-0315, DSPC, cholesterol, and PEG2k-DMG. The dosage was 0.3 / 0.4 / 0.6 mg / kg (based on total RNA content). The administration was via tail vein, with an injection volume of approximately 0.2 mL per mouse (approximately 10 mL per kg body weight). Blood samples were collected from the heart of the mice within 7 days after administration. Immediately after blood collection, mouse liver tissue was collected and placed at -80℃ for DNA extraction and NGS sequencing.

[0210] The results are shown in Figure 4. These results indicate that, compared to the backbone sequence shown in SEQ ID No. 60, sgRNAs containing backbone sequences shown in SEQ ID Nos. 69, 71, 74, and 75 did not exhibit an editing advantage in mice. The backbone sequence shown in SEQ ID No. 73 remained a negative control.

[0211] Example 11

[0212] This embodiment analyzes the gene editing efficiency of sgRNAs containing different backbone sequences. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is P9-hc-023seq22, and the backbone sequences are shown in Table 15.

[0213] Specifically, 20 ng sgRNA and 160 ng Cas9 mRNA (the nucleotide sequence of which is shown in SEQ ID No. 81) were co-delivered into 48-well PHH cells (donor number HUM18300) using Lipofectamine RNAiMAX. Cells were harvested 72 hours after transfection, and genomic DNA was extracted for analysis and evaluation of its target efficiency and off-target efficiency.

[0214] Table 15

[0215] These results indicate that sgRNAs containing the backbone sequence shown in SEQ ID No. 77 have a superior editing advantage on PHH compared to backbone sequences shown in SEQ ID No. 60. The backbone sequence shown in SEQ ID No. 76 serves as a negative control.

[0216] Furthermore, 6- to 15-week-old PCSK9 humanized mice were used. These mice were genetically engineered to delete a region of the endogenous mouse Pcsk9 locus and replace it with a homologous human PCSK9 sequence, thereby encoding the human PCSK9 protein at that locus. These humanized mice received LNP drugs encapsulating sgRNA and Cas9 mRNA (nucleotide sequence shown in SEQ ID No. 81) with different backbones (as shown in SEQ ID No. 60, 77, 78, 79, and 80, respectively), with a weight ratio of mRNA to sgRNA of 2:1. The LNPs contained ALC-0315, DSPC, cholesterol, and PEG2k-DMG. The dosage was 0.4 / 0.2 mg / kg (based on total RNA content). Administration was via tail vein, with an injection volume of approximately 0.2 mL per mouse (approximately 10 mL per kg body weight). Blood samples were collected from the heart via biopsy within 7 days post-administration. Immediately after blood collection, mouse liver tissue was collected and placed at -80℃ for DNA extraction and NGS sequencing.

[0217] The results are shown in Figure 5. The results show that, compared with the backbone sequence shown in SEQ ID No. 60, sgRNA containing the backbone sequence shown in SEQ ID No. 77 has a better editing advantage in mice.

[0218] Example 12

[0219] Based on in vivo experiments in WT mice, the gene editing efficiency of LNP drugs containing different cations (as shown in Table 16) was analyzed. The sgRNA used consisted of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence was P9-hc-023seq22 and the backbone sequence was the sequence shown in SEQ ID No. 60.

[0220] In this embodiment, 6- to 15-week-old humanized PCSK9 mice were used. These PCSK9 humanized mice were genetically engineered to delete a region of the endogenous mouse Pcsk9 locus and replace it with a homologous human PCSK9 sequence, thereby encoding the human PCSK9 protein at that locus. These humanized mice received an LNP drug encapsulating the same sgRNA (P9-hc-023seq22 leader sequence, backbone sequence as shown in SEQ ID No. 60) and Cas9 mRNA (containing the nucleotide sequence as shown in SEQ ID No. 82), wherein the weight ratio of mRNA to sgRNA was 2:1. The LNP contained ALC-0315 or ILL-N-43, DSPC, cholesterol, and PEG2k-DMG. The dosage was 0.2–0.8 mg / kg (based on total RNA content). Mice were divided into groups, with the number of mice in each group as shown by the N values ​​in Table 16. The drug was administered via tail vein, with each mouse receiving approximately 0.2 ml (about 10 ml per kilogram of body weight). Blood samples were collected from the heart of the mice within 7 days of drug administration. Immediately after blood collection, liver tissue was collected and placed at -80°C for DNA extraction and NGS sequencing.

[0221] Table 16

[0222] In this table, "NA" indicates that the experiment was not completed and there is no data available.

[0223] The results show that when the sgRNA with the lead sequence P9-hc-023seq22 and the backbone sequence shown in SEQ ID No. 60 is combined with ALC-0315 or ILL-N-43 cationic lipids, the combination can induce dose-dependent editing of the PCSK9 target gene in the liver of mice. Saturation editing of the PCSK9 gene in the liver of mice can be achieved at a dose of 0.6 mg / kg.

[0224] Example 13

[0225] In this embodiment, cynomolgus monkeys aged 3 to 4 years and weighing between 2.5 and 4 kg were used. These animals received an LNP drug encapsulating the same sgRNA (P9-hc-023seq22 leader sequence, as shown in the backbone sequence of SEQ ID No. 60) and Cas9 mRNA, wherein the weight ratio of mRNA to sgRNA was 2:1. The LNP contained cationic lipids (ILL-N-43 or other cationic lipids with similar structures as described in Chinese patent application No. 2024116579272), DSPC, cholesterol, and PEG2k-DMG. Administration was via intravenous infusion over 3 to 4 hours, with an injection volume of 10 mL / kg body weight per animal. Blood samples and liver biopsies were collected some time after administration and stored at -80°C for subsequent blood testing, protein analysis, or DNA extraction and NGS sequencing.

[0226] Example 14

[0227] This embodiment analyzes the gene editing efficiency of sgRNAs containing different backbone sequences. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is mU*mG*mG*UGCAGUAAUGACUACCU (SEQ ID No. 83), and the backbone sequence is shown in Table 17.

[0228] Specifically, 62.5 ng sgRNA and 62.5 ng Cas9 mRNA (the nucleotide sequence of which is shown in SEQ ID No. 81) were co-delivered into 48-well PHH cells (donor number 24TL015215, LONZA) using Lipofectamine RNAiMAX. Cells were harvested 72 hours after transfection, and genomic DNA was extracted. Its on-target efficiency and off-target efficiency were then analyzed and evaluated. The results are shown in Table 17.

[0229] Table 17

[0230] The results show that, compared with the backbone sequence shown in SEQ ID No. 52, sgRNAs containing backbone sequences shown in SEQ ID No. 84, 86, and 88 have higher on-target and off-target editing in PHH, while sgRNAs containing backbone sequences shown in SEQ ID No. 87 have lower on-target and off-target editing in PHH.

[0231] Example 15

[0232] This embodiment analyzes the gene editing efficiency of sgRNAs containing different backbone sequences. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is P9-hc-023seq22, and the backbone sequences are shown in Table 18.

[0233] Specifically, 20 ng sgRNA and 80 ng Cas9 mRNA (the nucleotide sequence of which is shown in SEQ ID No. 81) were co-delivered into 48-well PHH cells (donor number HUM180812C) using Lipofectamine RNAiMAX. Cells were harvested 72 hours after transfection, and genomic DNA was extracted for analysis and evaluation of its on-target and off-target efficiencies.

[0234] Table 18

[0235] The results show that, compared with the backbone sequence shown in SEQ ID No. 77, sgRNAs containing backbone sequences shown in SEQ ID Nos. 89–98 and 100–102 have higher on-target editing at PHH, and off-target editing is not increased.

[0236] Example 16

[0237] This embodiment is based on in vivo experiments in PCSK9 humanized mice to analyze the gene editing efficiency of sgRNAs containing different backbone sequences. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is P9-hc-023seq22, and the backbone sequences are shown in Table 19.

[0238] In this embodiment, 6- to 15-week-old PCSK9 humanized mice were used as described above. These mice received LNP drugs encapsulating sgRNA and Cas9 mRNA (containing the nucleotide sequence shown in SEQ ID No. 82) with different backbones, wherein the weight ratio of mRNA to sgRNA was 2:1. The LNP contained ALC-0315, DSPC, cholesterol, and PEG2k-DMG. The dosage was 0.4 or 0.2 mg / kg (based on total RNA content). Mice were randomly divided into groups, with the number of mice in each group as shown by the N values ​​in Table 19, and administered via tail vein, with an injection volume of approximately 0.2 mL per mouse (approximately 10 mL per kilogram of body weight). Blood samples were collected from the heart of the mice within 7 days after administration. Immediately after blood collection, liver tissue was collected from the mice and subjected to DNA extraction and NGS sequencing at -80°C to evaluate its on-target efficiency and off-target efficiency.

[0239] Table 19

[0240] The results show that, compared with the backbone sequence shown in SEQ ID No. 77, the sgRNA containing the backbone sequence shown in SEQ ID No. 98 has higher off-target editing, while the sgRNA containing the backbone sequence shown in SEQ ID No. 94 has lower on-target editing.

[0241] Example 17

[0242] This embodiment is based on in vivo experiments in PCSK9 humanized mice to analyze the gene editing efficiency of sgRNAs containing different backbone sequences. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is P9-hc-023seq22, and the backbone sequences are shown in Table 20.

[0243] In this embodiment, 6- to 15-week-old PCSK9 humanized mice were used as described above. These mice received LNP drugs encapsulating sgRNA and Cas9 mRNA (containing the nucleotide sequence shown in SEQ ID No. 82) with different backbones, wherein the weight ratio of mRNA to sgRNA was 2:1. The LNP contained ALC-0315, DSPC, cholesterol, and PEG2k-DMG. The dosage was 0.2 mg / kg (based on total RNA content). Mice were randomly divided into groups, with the number of mice in each group as shown by the N values ​​in Table 20. The drug was administered via tail vein, with an injection volume of approximately 0.2 mL per mouse (approximately 10 mL per kilogram of body weight). Blood samples were collected from the heart of the mice within 7 days after administration. Immediately after blood collection, liver tissue was collected from the mice and subjected to DNA extraction and NGS sequencing at -80°C to evaluate its on-target efficiency and off-target efficiency.

[0244] Table 20

[0245] The results show that, compared with the backbone sequence shown in SEQ ID No. 77, sgRNAs containing the backbone sequences shown in SEQ ID No. 89, 91, and 100 have higher on-target editing, sgRNAs containing the backbone sequence shown in SEQ ID No. 100 have higher off-target editing, and sgRNAs containing the backbone sequence shown in SEQ ID No. 99 have lower on-target editing.

[0246] Example 18

[0247] In this embodiment, sgRNAs containing different backbone sequences were further screened in PHH. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is P9-hc-023-seq22, and the backbone sequences are shown in Table 21.

[0248] Specifically, sgRNA and Cas9 mRNA were delivered to PHH cells (donor ID HUM180812C(LONZA)) via LNP. The mass ratio of sgRNA to Cas9 mRNA was 1:2. Different experimental groups were set up with sgRNA concentrations of 16.2 nM and 64.6 nM. PHH cells were harvested 72 hours after transfection, and genomic DNA was extracted. The on-target efficiency and off-target efficiency were then analyzed and evaluated.

[0249] Table 21

[0250] The results show that, compared with the backbone sequence shown in SEQ ID No. 77, the sgRNA containing the backbone sequence shown in SEQ ID No. 98 has higher on-target editing, while the sgRNA containing the backbone sequence shown in SEQ ID No. 94 has lower on-target editing.

[0251] Example 19

[0252] In this embodiment, sgRNAs containing different backbone sequences were further screened in PHH. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is P9-hc-023-seq22. The backbone sequences are shown in Table 22.

[0253] Specifically, sgRNA and Cas9 mRNA (containing the nucleotide sequence shown in SEQ ID No. 82) were delivered separately to PHH cells of donor number HUM181301B via LNP. The mass ratio of sgRNA to Cas9 mRNA was 1:2. Different experimental groups were set up with sgRNA concentrations of 0.25 nM, 1 nM, 4 nM, 16.2 nM, and 64.6 nM. PHH cells from donor HUM181301B were harvested 72 hours after transfection, and genomic DNA was extracted. Its on-target efficiency and off-target efficiency were then analyzed and evaluated.

[0254] Table 22

[0255] The detection results of PHH cells from the HUM181301B donor are shown in Table 21. Compared with the backbone sequence shown in SEQ ID No. 77, sgRNAs containing the backbone sequences shown in SEQ ID No. 89, 90, 91, and 100 exhibit higher on-target editing performance.

[0256] Example 20

[0257] In this embodiment, sgRNAs containing different backbone sequences were further screened in PHH. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is P9-hc-023-seq22, and the backbone sequences are shown in Table 23.

[0258] Specifically, sgRNA and Cas9 mRNA (containing the nucleotide sequence shown in SEQ ID No. 82) were delivered separately to PHH cells of donor number 23TL240143 (LONZA) via LNP. The mass ratio of sgRNA to Cas9 mRNA was 1:2. Different experimental groups were set up with sgRNA concentrations of 16.2 nM and 64.6 nM. 72 hours after transfection, PHH cells from donor 23TL240143 were harvested, and genomic DNA was extracted. Its on-target efficiency and off-target efficiency were then analyzed and evaluated.

[0259] Table 23

[0260] The detection results of PHH cells from donor 23TL240143 are shown in Table 22. These results show that, compared to the backbone sequence shown in SEQ ID No. 77 (an existing backbone sequence), sgRNA containing the backbone sequence shown in SEQ ID No. 103 exhibits higher on-target editing, while sgRNA containing the backbone sequences shown in SEQ ID Nos. 101, 104, and 105 exhibit lower on-target editing.

[0261] Example 21

[0262] This embodiment is based on in vivo experiments in PCSK9 humanized mice to analyze the gene editing efficiency of sgRNAs containing different backbone sequences. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is P9-hc-023seq22, and the backbone sequences are shown in Table 24.

[0263] In this embodiment, 6- to 15-week-old PCSK9 humanized mice were used as described above. These mice received LNP drugs encapsulating sgRNA and Cas9 mRNA (containing the nucleotide sequence shown in SEQ ID No. 82) with different backbones, wherein the weight ratio of mRNA to sgRNA was 2:1. The LNP contained ALC-0315, DSPC, cholesterol, and PEG2k-DMG. The dosage was 0.4 / 0.2 mg / kg (based on total RNA content). Mice were randomly divided into groups, with the number of mice in each group as shown by the N values ​​in Table 24, and administered via tail vein, with an injection volume of approximately 0.2 ml per mouse (approximately 10 ml per kilogram of body weight). Blood samples were collected from the heart of the mice within 7 days after administration. Immediately after blood collection, liver tissue was collected from the mice and subjected to DNA extraction and NGS sequencing at -80°C.

[0264] Table 24

[0265] The results show that, compared with the backbone sequence shown in SEQ ID No. 77, sgRNAs containing the backbone sequences shown in SEQ ID No. 89 and 91 have lower off-target editing, sgRNAs containing the backbone sequences shown in SEQ ID No. 97 and 106 have higher off-target editing, sgRNAs containing the backbone sequence shown in SEQ ID No. 106 also have higher on-target editing, and sgRNAs containing the backbone sequence shown in SEQ ID No. 107 have lower on-target editing.

[0266] Example 22

[0267] In this embodiment, sgRNAs containing different backbone sequences were further screened in PHH. The sgRNA consists of a leader sequence and a backbone sequence from the 5' end to the 3' end. The leader sequence is P9-hc-023-seq22. The backbone sequences are shown in Table 25.

[0268] Specifically, sgRNA and Cas9 mRNA (containing the nucleotide sequence shown in SEQ ID No. 82) were delivered separately to PHH cells from donor HUM17299A via LNP. The mass ratio of sgRNA to Cas9 mRNA was 1:2. Different experimental groups were set up with sgRNA concentrations of 16.2 nM and 64.6 nM. PHH cells from donor HUM17299A were harvested 72 hours after transfection, and genomic DNA was extracted. The on-target efficiency and off-target efficiency were then analyzed and evaluated.

[0269] Table 25

[0270] The detection results of PHH cells from the HUM17299A donor are shown in Table 24. These results show that, compared to the backbone sequence shown in SEQ ID No. 77, sgRNAs containing the backbone sequences shown in SEQ ID No. 89 and 97 exhibit higher on-target editing, while sgRNAs containing the backbone sequence shown in SEQ ID No. 91 exhibit lower on-target editing.

[0271] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of protection of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A guide RNA comprising a spacer sequence, said spacer sequence comprising nucleotides 4 to 20, 3 to 20, 2 to 20, or 1 to 20 of any of the sequences shown in SEQ ID No. 1 to 25.

2. The guide RNA according to claim 1, wherein, The leader sequence comprises nucleotides 4 to 20, 3 to 20, 2 to 20, or 1 to 20 of any of the sequences shown in SEQ ID No. 1 to 2, 4 to 6, 9 to 10, and 15; more preferably, the leader sequence comprises nucleotides 4 to 20, 3 to 20, 2 to 20, or 1 to 20 of the sequence shown in SEQ ID No.

1.

3. The guide RNA according to claim 1 or 2, wherein, At least the first three nucleotides at the 5' end of the leader sequence are modified nucleotides; optionally, the modified nucleotides are nucleotides modified with 2'-O-methyl.

4. The guide RNA according to any one of claims 1 to 3, wherein, The first four nucleotides at the 5' end of the leader sequence are linked by phosphate thioester bonds.

5. The guide RNA according to claim 1, wherein, The leader sequence comprises nucleotides 4 to 20, 3 to 20, 2 to 20, or 1 to 20 of any of the sequences shown in SEQ ID No. 26 to 50; preferably, the leader sequence comprises nucleotides 4 to 20, 3 to 20, 2 to 20, or 1 to 20 of any of the sequences shown in SEQ ID No. 26 to 27, 29 to 31, 34 to 35, and 41; more preferably, the leader sequence comprises nucleotides 4 to 20, 3 to 20, 2 to 20, or 1 to 20 of the sequence shown in SEQ ID No.

26.

6. The guide RNA according to any one of claims 1 to 5, wherein, The guide RNA also includes a scaffold sequence comprising a nucleotide sequence as shown in SEQ ID No.

51.

7. The guide RNA according to claim 6, wherein, The backbone sequence does not contain 2'-O-methyl modification at positions 9, 49, and 62 of the nucleotide sequence shown in SEQ ID No. 51, and contains 2'-O-methyl modified nucleotides at positions 8, 21, and 22 of the nucleotide sequence shown in SEQ ID No.

51.

8. The guide RNA according to claim 6 or 7, wherein, The backbone sequence has one or more nucleotides at positions 31, 42, 44, 45 and 48 of the nucleotide sequence shown in SEQ ID No. 51, each of which is independently a 2'-O-methyl modified nucleotide or a 2'-deoxy nucleotide. Preferably, the backbone sequence has one or more nucleotides at positions 31, 42, 45 and 48 of the nucleotide sequence shown in SEQ ID No. 51 that are nucleotides modified with 2'-O-methyl; Preferably, one or more nucleotides at positions 42, 44, 45 and 48 of the nucleotide sequence shown in SEQ ID No. 51 are each independently a 2'-deoxynucleotide.

9. The guide RNA according to any one of claims 6 to 8, wherein, The backbone sequence does not contain or is not entirely composed of 2'-O-methyl modified nucleotides at positions 50-52, 57-59, 63-66, and 72-75 of the nucleotide sequence shown in SEQ ID No.

51.

10. The guide RNA according to any one of claims 6 to 9, wherein, The backbone sequence is a nucleotide modified with 2'-deoxyribose at the 42nd nucleotide position of the nucleotide sequence shown in SEQ ID No.

51.

11. The guide RNA according to any one of claims 6 to 10, wherein, The backbone sequence also includes at least one of the following mutations: (I) An insertion of 1 to 4 nucleotides is present between the 12th and 13th nucleotide positions and between the 16th and 17th nucleotide positions in the nucleotide sequence shown in SEQ ID No. 51; (II) An insertion of 1 to 4 nucleotides is present between the 52nd and 53rd nucleotide positions and between the 56th and 57th nucleotide positions of the nucleotide sequence shown in SEQ ID No. 51; (III) The nucleotide sequence shown in SEQ ID No. 51 has a deletion and / or substitution of 1 to 4 nucleotides at positions 49 to 52 and 57 to 60.

12. The guide RNA according to any one of claims 6 to 11, wherein, The backbone sequence comprises any of the nucleotide sequences shown in SEQ ID No. 108 to 131; preferably, the backbone sequence comprises any of the nucleotide sequences shown in SEQ ID No. 108, 114, 115, 121, 122, 124, 127, and 130.

13. The guide RNA according to claim 6, wherein, The backbone sequence comprises any of the nucleotide sequences shown in SEQ ID No. 52-80, 84-107; preferably, the backbone sequence comprises any of the nucleotide sequences shown in SEQ ID No. 54-60, 69, 71, 74-75, 77-80, 89-91, 97, 98, 100, 103, 106; more preferably, the backbone sequence comprises the nucleotide sequence shown in SEQ ID No. 60, 77 or 89.

14. The guide RNA according to any one of claims 1 to 13, wherein, The guide RNA has a maximum off-target efficiency of less than 0.1% at the effective drug dose.

15. A guide RNA comprising a backbone sequence, said backbone sequence being as described in any one of claims 7 to 14.

16. A composition comprising: (i) the guide RNA of any one of claims 1 to 15, or the nucleic acid encoding said guide RNA; and Select from at least one of (ii) to (iii) below: (ii) an effector protein, or a nucleic acid encoding the effector protein; (iii) Delivery carrier.

17. The composition according to claim 16, wherein, The effector protein is a Cas protein; optionally, the effector protein is a Cas9 protein.

18. The composition according to claim 16, wherein, The nucleic acid encoding the effector protein is mRNA encoding a Cas protein; optionally, the nucleic acid encoding the effector protein is mRNA encoding a Cas9 protein; optionally, the nucleic acid encoding the effector protein includes a nucleotide sequence as shown in SEQ ID No. 81 or SEQ ID No.

82.

19. The composition according to any one of claims 16 to 18, wherein, The delivery carrier is an LNP; preferably, the ionizable lipid in the LNP has the following structure:

20. A pharmaceutical preparation comprising either (1) or (2) below and a pharmaceutically acceptable adjuvant thereof: (1) The guide RNA according to any one of claims 1 to 15; (2) The composition according to any one of claims 16 to 19.

21. Methods for editing the PCSK9 gene, including: Deliver one or more of the guide RNA of any one of claims 1 to 15, the composition of any one of claims 16 to 19, and the pharmaceutical formulation of claim 20 to cells.

22. The method for editing the PCSK9 gene according to claim 21, wherein, The method results in insertions or deletions in the PCSK9 gene.

23. The method for editing the PCSK9 gene according to claim 21, wherein, The method further includes delivering a template to a cell, at least a portion of which is incorporated into a sequence at or near a double-strand break site induced by the Cas protein.

24. The use of one or more of the guide RNA of any one of claims 1 to 15, the composition of any one of claims 16 to 19, and the pharmaceutical preparation of claim 20 in the prevention or treatment of PCSK9-related diseases.

25. The use of one or more of the guide RNA of any one of claims 1 to 15, the composition of any one of claims 16 to 19, and the pharmaceutical preparation of claim 20 in the preparation of a medicament for the prevention or treatment of PCSK9-related diseases.

26. The application according to claim 24 or 25, wherein, The PCSK9-related diseases include one or more of the following: lipid metabolism disorders, cardiovascular diseases, atherosclerosis, liver diseases, diabetes, kidney diseases, neurodegenerative diseases, tumors, infectious diseases, and autoimmune diseases.

Images