WO2025002247A1 - Nucleic acid targeting human angiopoietin-like protein 3 and use thereof - Google Patents

Abstract

Provided are a nucleic acid targeting human angiopoietin-like protein 3 (ANGPTL3) and a use thereof. The nucleic acid can effectively reduce the ANGPTL3 level, and can effectively inhibit the accumulation of fat in blood, so that the nucleic acid can be used for preventing or treating related diseases, such as a lipid metabolism disorder.

Description

Nucleic acid targeting human angiopoietin-like protein 3 and use thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims the priority of Chinese patent application No. 202310764185.2 filed on June 27, 2023, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention relates to the field of biotechnology, in particular to a nucleic acid targeting human angiopoietin-like protein 3 and uses thereof.

Background Art

Human angiopoietin-like protein 3 (ANGPTL3, also known as FHBL2, ANL3, ANGPT5, ANG-5) is a 45kDa protein expressed in hepatocytes. ANGPTL3 and ANGPTL8 form a protein complex that can inhibit lipoprotein lipase (Shimizugawa T, Ono M, Shimamura M, et al. ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase. J Biol Chem 2002; 277: 33742–33748.). Lipoprotein lipase is an important rate-limiting enzyme in triglyceride metabolism in the human body, which can catalyze the hydrolysis of triglycerides (Kersten S. Physiological regulation of lipoprotein lipase. Biochim Biophys Acta 2014; 1841: 919–933). The decrease of lipoprotein lipase can cause fat to accumulate in the blood, leading to atherosclerosis. In animal models, decreasing or increasing the expression of ANGPTL3 can regulate the triglyceride levels in mice, while decreasing ANGPTL3 in the APOE knockout mouse model can alleviate atherosclerosis (Ando Y, Shimizugawa T, Takeshita S, et al. A decreased expression of angiopoietin-like 3 is protective against atherosclerosis in apoE-deficient mice. J Lipid Res 2003; 44: 1216–1223.). It was also found that the activity of lipoprotein lipase increased in ANGPTL3 knockout mice, accompanied by a decrease in blood lipids (Fujimoto K, Koishi R, Shimizugawa T, Ando Y. Angptl3-null mice show low plasma lipid concentrations by enhanced lipoprotein lipase activity. Exp Anim 2006; 55: 27–34.).

Human genetic surveys found that loss of ANGPTL3 function caused by ANGPTL3 gene mutations can lead to hypocholesterolemia (Romeo S, Yin W, Kozlitina J, et al. Rare loss-of-function mutations in ANGPTL family members contribute to plasma triglyceride levels in humans. J Clin Invest 2009; 119: 70-79. and Musunuru K, Pirruccello JP, Do R, et al. Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia. N Engl J Med 2010; 363: 2220-2227.). In the study of the association between cardiovascular disease and ANGPTL3, it was found that the loss of ANGPTL3 function can significantly reduce the incidence of atherosclerotic cardiovascular disease (Dewey FE, Gusarova V, et al. Genetic and pharmacologic inactivation of ANGPTL3 and cardiovascular disease. N Engl J Med 2017; 377: 211-221.), and the use of antibodies to inhibit ANGPTL3 can significantly reduce low-density lipoprotein and triglyceride levels. Currently, an antibody targeting ANGPTL3, Evinacumab, has been approved for use in patients with homozygous familial hypercholesterolemia.

RNA interference (RNAi) refers to the highly conserved phenomenon in the evolutionary process, which is induced by double-stranded small interfering RNA (siRNA) to efficiently and specifically degrade homologous mRNA. RNAi drugs also have the advantage of having a longer duration of efficacy than antibodies. Therefore, it is of great significance to study and develop siRNA targeting ANGPTL3.

Summary of the invention

The purpose of the present invention is to overcome the problems existing in the prior art and provide a novel nucleic acid targeting human angiopoietin-like protein 3 and its use.

The first aspect of the present invention provides a nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand contains a sequence having more than 80% of the sequence shown in any one of SEQ ID NOs: 1 to 33. The antisense strand contains a sequence having a sequence identity of more than 80% with the 1st to 21st nucleotide sequence of any one of SEQ ID NOs: 34 to 66.

A second aspect of the present invention provides a targeted drug delivery system, which comprises a targeting group, a linking group, and the nucleic acid as described above connected to the targeting group via the linking group.

The third aspect of the present invention provides an isolated cell, which contains the nucleic acid as described above.

The fourth aspect of the present invention provides a pharmaceutical composition, which contains the nucleic acid or targeted drug delivery system as described above and a pharmaceutically acceptable carrier.

A fifth aspect of the present invention provides a method for inhibiting the expression of ANGPTL3 in a cell, the method comprising: contacting the cell with the nucleic acid, targeted drug delivery system or pharmaceutical composition as described above, so as to inhibit the expression of ANGPTL3 in the cell.

The sixth aspect of the present invention provides the use of the nucleic acid, targeted drug delivery system or pharmaceutical composition as described above in any of the following aspects: 1) treating and/or preventing diseases associated with ANGPTL3; 2) preparing drugs for treating and/or preventing diseases associated with ANGPTL3.

The nucleic acid of the present invention can effectively reduce the level of ANGPTL3 and effectively inhibit the accumulation of fat in the blood, and can therefore be used to prevent or treat related diseases, such as lipid metabolism disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 shows the ANGPTL3 protein level in the blood of human ANGPTL3 transgenic mice on the 10th day after subcutaneous injection of 1 mg/kg siRNA in an example of the present invention.

Figure 2 shows the ANGPTL3 protein level in the blood of human ANGPTL3 transgenic mice on the 10th day after subcutaneous injection of 3 mg/kg siRNA in an embodiment of the present invention.

FIG. 3 shows the efficacy of siRNA drugs targeting ANGPTL3 in human ANGPTL3 transgenic mice according to an embodiment of the present invention.

FIG. 4 shows the efficacy of SN-682210 targeting ANGPTL3 in human ANGPTL3 transgenic mice according to an example of the present invention.

DETAILED DESCRIPTION

The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Those skilled in the art may make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or illustrated as part of one embodiment may be used in another embodiment to produce a further embodiment.

Explanation of terms

Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

The terms "and/or", "or/and", and "and/or" used in this article have a selection range that includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, wherein the any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and/or", "or/and", and "and/or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and/or B" It includes three parallel solutions: A, B and A+B. For another example, the technical solution of "A, and/or, B, and/or, C, and/or, D" includes any one of A, B, C, and D (i.e., all of them are connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, i.e., any combination of any two or any three of A, B, C, and D, and also includes a combination of four of A, B, C, and D (i.e., all of them are connected by "logical AND").

As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuations within the range of ±0.1%. For values that are large or do not require too fine control, its meaning is also allowed to include greater fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, its meaning is allowed to include fluctuations of ±10%.

In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

In the present invention, "optionally", "optional", "optionally", "optionally", "optional", "optional", "optional" means optional, that is, it means to be selected from any one of the two parallel schemes of "yes" or "no". If multiple "optional" or "optional" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" or "optional" is independent.

In the present invention, the term "nucleic acid" refers to a composition containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that can be degraded or modified in a sequence-specific manner. Inhibit (e.g., degrade or inhibit under appropriate conditions) the translation of messenger RNA (mRNA) transcripts of target mRNA. The nucleic acid can act through an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway mechanism of mammalian cells (RNA-induced silencing complex or RISC)), or through any alternative mechanism or approach. The following limited range of nucleic acids including sense strands and antisense strands disclosed herein includes, but is not limited to: short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and dicer substrates.

As used herein, the terms "silencing", "reducing", "inhibiting", "downregulating" or "knockdown" when referring to the expression of a given gene, mean that the expression of the gene is reduced when the cell, cell population, tissue, organ or subject is treated with a nucleic acid described herein, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein or protein subunit translated from mRNA in a cell, cell population, tissue, organ or subject in which the gene is transcribed, compared to a second cell, cell population, tissue, organ or subject not so treated.

In the present invention, "fully complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, all (100%) bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may include all or part of the first nucleotide sequence or the second nucleotide sequence.

In the present invention, "partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 70% but not all of the bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may include all or part of the first nucleotide sequence or the second nucleotide sequence.

In the present invention, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 85% but not all of the bases in the contiguous sequence of the first oligonucleotide hybridize with the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first nucleotide sequence or the second nucleotide sequence.

In the present invention, when referring to "at least partially complementary" it means that the nucleic acid base or nucleotide sequence of the molecule In the hybridization pair, the first oligonucleotide is partially complementary, substantially complementary, or fully complementary to the second oligonucleotide.

In the present invention, the term "treatment" means a method or step taken to provide relief or reduction in the number, severity and/or frequency of one or more disease symptoms in a subject. The treatment may include prevention, management, prophylactic treatment, and/or inhibition or reduction of the number, severity and/or frequency of one or more disease symptoms in a subject.

In the present invention, the term "connection" means that two compounds or molecules are joined by a covalent bond. Unless otherwise specified, as used herein, the term "connection" may refer to a connection between a first compound and a second compound with or without any intermediate atoms or atomic groups.

Nucleic Acids

The present invention provides a (modified or unmodified) nucleic acid, the nucleic acid comprising a sense strand and an antisense strand, the sense strand containing 80% (such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and more than 1% of the sequence shown in any one of SEQ ID NOs: 1 to 33. The antisense strand contains a sequence having a sequence identity of 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) or more to the nucleotide sequence at positions 1 to 21 of the sequence shown in any one of SEQ ID NOs: 34 to 66.

In some embodiments, the antisense strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides (bases).

In some embodiments, the sense strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides (bases).

In specific implementation, those skilled in the art can combine the sequences provided in the present invention in consideration of the complementarity of the sense strand and the antisense strand, thereby obtaining a combined nucleic acid (siRNA).

In a preferred embodiment of the present invention, as shown in Table 1, the nucleic acid is selected from siRNA-1, whose sense strand sequence is SEQ ID NO: 1 and whose antisense strand sequence is SEQ ID NO: 34, At least one of siRNA-2 having a sequence of SEQ ID NO: 2 and an antisense chain sequence of SEQ ID NO: 35, siRNA-3 having a sense chain sequence of SEQ ID NO: 3 and an antisense chain sequence of SEQ ID NO: 36, siRNA-4 having a sense chain sequence of SEQ ID NO: 4 and an antisense chain sequence of SEQ ID NO: 37, siRNA-5 having a sense chain sequence of SEQ ID NO: 5 and an antisense chain sequence of SEQ ID NO: 38..., siRNA-31, siRNA-32, and siRNA-33.

In some preferred embodiments, the antisense strand contains at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ from the sequence shown in any one of SEQ ID NOs: 34 to 66 by no more than 0, 1, 2 or 3 nucleotides, and the positive strand contains a nucleotide sequence that is at least partially complementary (such as partially complementary, substantially complementary or completely complementary) to the antisense strand.

In some preferred embodiments, the positive strand contains at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from the sequence shown in any one of SEQ ID NOs: 1 to 33 by no more than 0, 1, 2 or 3 nucleotides.

In the present invention, the sense strand and the antisense strand may have the same length or different lengths.

All nucleotide groups in the above nucleic acid may be chemically unmodified, or may contain at least one modified nucleotide group, and the modification may be on the nucleotide at any position.

In some embodiments, the sense and antisense strands may be partially complementary, substantially complementary, or fully complementary to each other.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from any one of SEQ ID NOs: 34, 35, 38, 39, 40, 41, 43, 45, 50, 51, 54, 56, 57, 59 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that is at least partially complementary (such as partially complementary, substantially complementary or completely complementary) to the antisense strand. When the antisense strand has the above sequence, the double-stranded RNA has a significantly better inhibitory effect on ANGPTL3.

In some preferred embodiments, the sense strand contains the same sequence as SEQ ID NO: 1, 2, 5, A nucleotide sequence in which the sequence shown in any one of 6, 7, 8, 10, 12, 17, 18, 21, 23, 24, and 26 differs by 0, 1 or 2 nucleotides.

In some further preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NOs: 34, 35, 38, 39, 40, 41, 43, 45, 50, and 51 by 0, 1, or 2 nucleotides, and the sense strand contains a nucleotide sequence that is at least partially complementary (such as partially complementary, substantially complementary, or completely complementary) to the antisense strand. When the antisense strand has the above sequence, the double-stranded RNA has a further better inhibitory effect on ANGPTL3. In some further preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NOs: 1, 2, 5, 6, 7, 8, 10, 12, 17, and 18 by 0, 1, or 2 nucleotides.

In some further preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NOs: 35, 38, 39, 45, and 50 by 0, 1, or 2 nucleotides, and the sense strand contains a nucleotide sequence that is at least partially complementary (such as partially complementary, substantially complementary, or completely complementary) to the antisense strand. When the antisense strand has the above sequence, the double-stranded RNA has a further better inhibitory effect on ANGPTL3. In some further preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NOs: 2, 5, 6, 12, and 17 by 0, 1, or 2 nucleotides.

In some further preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NOs: 45 and 50 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that is at least partially complementary (such as partially complementary, substantially complementary or completely complementary) to the antisense strand. When the antisense strand has the above sequence, the double-stranded RNA has a further better inhibitory effect on ANGPTL3. In some further preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NOs: 12 and 17 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO:45 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO:12 by 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 50 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 17 by 0, 1 or 2 nucleotides.

In some embodiments, when the sequence identity of the sense strand or antisense strand in the nucleic acid with the corresponding sequence mentioned in the present invention is less than 100% or differs by more than 1 nucleotide, it still has an inhibitory effect on ANGPTL3 that is similar to the corresponding sequence (such as still having an efficacy equivalent to 80-120%, 85-115% or 90-110% of the corresponding sequence) or equivalent (such as still having an efficacy equivalent to 95-105% of the corresponding sequence). For example, the two bases at the 3' end of the antisense strand of the nucleic acid (such as the sequence shown in any one of SEQ ID NOs: 34-66) are replaced with UU, AA, CC, GG or UG, etc., or a combination of any two nucleic acids. Such a nucleic acid sequence also falls within the scope of protection of the present invention.

The above-mentioned technical solution for naked sequence (i.e., unmodified sequence) mentioned in the present invention has an effect advantage that does not depend on the selection of modification method or targeting vector. The following is an introduction to the applicable modification scheme and further preferred modification scheme:

According to the nucleic acid of the present invention, the nucleic acid contains a nucleotide group as a basic structural unit, and the nucleotide group contains a phosphate group, a ribose group and a base. Preferably, the nucleic acid contains at least one modified nucleotide group. The modified nucleic acid has an inhibition efficiency of ANGPTL3 of no less than 50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).

According to the nucleic acid of the present invention, wherein the modified nucleotide group is a nucleotide group in which a phosphate group and/or a ribose group is modified. The modified site can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 of the 1st, 2nd, 3rd, 4th, 5th, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30th nucleotide of the sense strand and/or the antisense strand.

For example, modification of the phosphate group refers to modification of the oxygen in the phosphate group, including phosphorothioate modification and boranophosphate modification. As shown in the following formula, the oxygen in the phosphate group is replaced by sulfur, borane, amine, alkyl or alkoxy. These modifications can stabilize the structure of nucleic acids and maintain high specificity and high affinity of base pairing.

In the above structural formula, BASE represents the base A, U, C, G or T. X can be oxygen (O) or sulfur (S). R in the above structure can be the same or different, such as: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, amino, cyanoethyl, acetyl, etc., R' and R" can each independently be hydrogen (H), methyl (CH3), ethyl (CH2CH3), propyl (CH2CH2CH3), isopropyl (CH(CH3)2), allyl, propargyl, acyloxybenzyl, acyloxyethyl.

The modification of the ribose group refers to the modification of the 2′-hydroxyl (2′-OH) in the ribose group. After introducing certain substituents such as methoxy or fluorine at the 2′-hydroxyl position of the ribose group, the nucleic acid is not easily cut by ribonucleases, thereby increasing the stability of the nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis. The modification of the 2′-hydroxyl in the pentose of nucleotides includes 2′-fluoro modification (2′-fluoromodification, such as 2′-arabino-fluoro modification), 2′-methoxy modification (2′-OME), 2′-methoxyethyl modification (2′-MOE), 2′-2,4-dinitrophenol modification (2′-DNP modification), cyclolocked ethyl modification (2′,4′-constrained ethyl modification), 2'-Amino modification, 2'-deoxy modification, BNA, acyclic nucleic acid modification, misplaced nucleic acid modification, L-type nucleic acid modification, etc. BNA (endocyclic bridged nucleotide) refers to a constrained or inaccessible nucleotide. BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C 3'-endo sugar condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose ring to provide a 2', 4'-BNA nucleotide, such as locked ethyl modification (LNA), ring locked ethyl modification (ENA) and ethyl locked nucleic acid modification (cET BNA). Acyclic nucleic acids are nucleotides formed by opening the sugar ring of the nucleotide, such as unlocked nucleic acid (UNA) nucleotides and glycerol nucleic acid (GNA) nucleotides. Misplaced nucleic acid modification refers to the replacement of the 3', 5'-phosphate bond link by a 2', 5'-phosphate bond chain. L-type nucleic acid modification refers to the replacement of the naturally occurring D-type nucleic acid with its mirror stereo equivalent L-type nucleic acid.

Wherein, BASE represents the base A, U, C, G or T. R in the above structure can be the same or different, such as: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, acetyl, etc.

According to the nucleic acid of the present invention, preferably, the nucleotide group in which the ribose group is modified is a nucleotide group in which the 2'-OH of the ribose group is substituted by a methoxy group or a fluorine group.

According to a particularly preferred embodiment of the present invention, the nucleotide group containing uracil base or cytosine base in the sense strand of the nucleic acid is a nucleotide group in which the ribose group is modified, that is, the nucleotide group containing uracil base or cytosine base in the sense strand of the nucleic acid The 2'-OH of the ribose group in the nucleic acid is replaced by a methoxy group or a fluorine group. More preferably, the 3' end of the sense strand and the antisense strand of the nucleic acid can be connected to dTdT; or, the 3' end of the antisense strand of the nucleic acid can be connected to AA or UU or a combination of any two nucleic acids (which can be but is not limited to CC, GG or UG), so that the sequence has a specific inducement to mRNA degradation. The nucleic acid with the above modifications shows a more excellent in vivo inhibitory effect, and the above modifications can further reduce the immunogenicity of the nucleic acid of the present invention in vivo.

The nucleic acid of the present invention may also include a modification of a monophosphate nucleoside connected to the 5' end of the antisense strand. Since the 5'-monophosphate at the terminal of the siRNA guide strand is important for RISC recognition. Among them, the phosphorylation of the 5'-hydroxyl group plays a certain role in whether the siRNA can be effectively loaded into the Ago2 inside the cell. The 5'-terminal monophosphate of the guide strand in the siRNA has an H bond interaction with Argonaute-2 (Ago2), thereby ensuring accurate positioning and precise cutting of the mRNA target. Commonly used 5'-monophosphate nucleoside derivatives are as follows. Such phosphate nucleoside derivatives have been proven to have certain stability in biological metabolic media and have a certain effect on promoting the loading of the siRNA guide strand into the Ago2 inside the cell (Nucleic Acids Research, 2015, 43, 2993-3011). According to the nucleic acid of the present invention, wherein, preferably, trans-vinyl phosphate (VP) is the first choice, and monophosphate nucleoside derivatives other than those described above may also be included.

In the above structure, BASE represents the base A, U, C, G or T. R in the above structure can be the same or different, such as hydrogen (H), fluorine (F), methoxy (OME) Or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, amino, acetyl, etc.

In the present invention, The meaning is consistent with that of , which means that a chemical element X is connected to any one or more groups.

In some embodiments, at least one nucleotide in the nucleic acid is a modified nucleotide or includes a modified linkage.

In some preferred embodiments, the modified nucleotide is selected from one or more of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-open ring nucleotide mimics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitols, reverse nucleotides, reverse 2'-O-methyl nucleotides, reverse 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, nucleotides containing vinyl phosphonate, nucleotides containing cyclopropyl phosphonate and 3'-O-methyl nucleotides; the modified nucleotide is further preferably selected from one or more of 2'-O-methyl nucleotides and 2'-fluoro nucleotides.

In some preferred embodiments, the modified inter-linkage is preferably selected from one or more of phosphorothioate inter-nucleotide linkages and methylphosphonate inter-nucleotide linkages; the modified inter-linkage is further preferably selected from one or more of phosphorothioate monoester inter-nucleotide linkages and phosphorothioate diester inter-nucleotide linkages.

In some embodiments, the antisense strand contains two phosphorothioate internucleotide bonds at the 5' end and the 3' end, respectively, and contains 4 to 6 2'-fluoro nucleotides, and the remaining nucleotides are 2'-O-methyl nucleotides.

In some embodiments, the 5' end of the sense strand contains two phosphorothioate internucleotide bonds and 3 to 5 2'-fluoro nucleotides, and the remaining nucleotides are 2'-O-methyl nucleotides.

In some preferred embodiments, the antisense strand has 2'-fluoro nucleotides at the 2nd, 4th, 6th, 12th and 14th nucleotides counted from the 5' end.

In some preferred embodiments, the sense strand is 2'-fluoro nucleotides at the 7th, 9th, 10th and 11th nucleotides counted from the 5' end.

In some preferred embodiments, the antisense strand has at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ from the antisense strand sequence of any one of Table 2 by no more than 0, 1, 2 or 3 nucleotides, and the sense strand has at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from the sense strand sequence of any one of Table 2 by no more than 0, 1, 2 or 3 nucleotides.

In some preferred embodiments, the sense strand and the antisense strand form a siRNA having any one of the sequences shown in Table 3.

In some embodiments, the antisense strand contains a nucleotide sequence from the 5' end to the 3' end that differs from the following nucleotide sequence by 0, 1 or 2 nucleotides:

SN-52194: asGfsuAfgAfauuuuUfuCfuucuaggsasg;

SN-52197:usAfsaGfuUfaguuaGfuUfgcucuucsusa;

SN-52201:usUfsuUfaAfgugaaGfuUfacuucugsusu;

SN-52203:usAfsuUfuCfuuuuaUfuUfgacuaugscsu;

SN-52204:usUfsuCfuAfuuucuUfuUfauuugacsusa;

SN-52205: asAfsgAfuAfgagaaAfuUfucuguggsusu;

SN-52208: asGfsuUfuUfgugauCfcAfucuauucsgsa;

SN-52210:usUfsuCfaUfugaagUfuUfugugaucscsa;

SN-52218: asAfsaAfgAfauauuCfaAfuauaaugsusu;

SN-52219: asUfsaGfuUfgguuuCfgUfgauuuccsusu;

SN-52222: asGfsaUfgUfagcguAfuAfguugguususc;

SN-52224:usAfsuAfaCfcuuccAfuUfuugagacsusu;

SN-52226:usUfsgAfuUfuuauaGfaGfuauaaccsusu;

SN-52228:usCfsaUfuCfaaagcUfuUfcugaaucsusg;

The sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs by 0, 1, or 2 nucleotides from the sequence shown in SN-52194, SN-52197, SN-52201, SN-52203, SN-52204, SN-52205, SN-52208, SN-52210, SN-52218, or SN-52219.

In some further preferred embodiments, the antisense strand contains a nucleotide sequence that differs by 0, 1 or 2 nucleotides from the sequence shown in SN-52197, SN-52201, SN-52203, SN-52210 or SN-52218.

In some most preferred embodiments, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SN-52210 or SN-52218 by 0, 1 or 2 nucleotides.

In some embodiments, the sense strand contains a nucleotide sequence from the 5' end to the 3' end that differs by 0, 1 or 2 nucleotides from any of the following nucleotide sequences:

SN-22194:cscsuagaAfgAfAfAfaaauucuacu;

SN-22197:gsasagagCfaAfCfUfaacuaacuua;

SN-22201: csasgaagUfaAfCfUfucacuuaaaa;

SN-22203: csasuaguCfaAfAfUfaaaagaaaua;

SN-22204:gsuscaaaUfaAfAfAfgaaauagaaa;

SN-22205: cscsacagAfaAfUfUfucucuaucuu;

SN-22208:gsasauagAfuGfGfAfucacaaaacu;

SN-22210:gsasucacAfaAfAfCfuucaaugaaa;

SN-22218: csasuuauAfuUfGfAfauauucuuuu;

SN-22219:gsgsaaauCfaCfGfAfaaccaacuau;

SN-22222:asasccaaCfuAfUfAfcgcuacaucu;

SN-22224: gsuscucaAfaAfUfGfgaagguuaua;

SN-22226:gsgsuuauAfcUfCfUfauaaaaucaa;

SN-22228: gsasuucaGfaAfAfGfcuuugaauga.

In each sequence of the present invention, a nucleotide represented by a lowercase letter represents that the nucleotide is a 2'-O-methyl nucleotide; f represents that the adjacent nucleotide on its left is a 2'-fluoro nucleotide; s represents that the two adjacent nucleotides on the left and right are connected by a phosphorothioate diester bond.

In specific implementation, those skilled in the art can combine the sense strand and antisense strand sequences mentioned above in consideration of the complementarity of the sense strand and the antisense strand. In a preferred embodiment, the sense strand and antisense strand sequences with the same last three digits of the number are combined to obtain the corresponding nucleic acid (siRNA). For example, SN-22194 is combined with SN-52194, and SN-22197 is combined with SN-52197 to obtain the corresponding nucleic acid (siRNA).

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs by 0, 1 or 2 nucleotides from the sequence shown in SN-52210, and the sense strand contains a nucleotide sequence that differs by 0, 1 or 2 nucleotides from the sequence shown in SN-22210.

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs by 0, 1 or 2 nucleotides from the sequence shown in SN-52218, and the sense strand contains a nucleotide sequence that differs by 0, 1 or 2 nucleotides from the sequence shown in SN-22218.

The nucleic acid according to the present invention can be obtained by conventional methods in the art, such as solid phase synthesis and liquid phase synthesis, and the solid phase synthesis has commercial custom services and can therefore be obtained commercially. The modified nucleotide group can be introduced by a nucleotide monomer having a corresponding modification.

Based on the nucleic acid (siRNA) synthesized as above, the present invention can further construct an shRNA expression plasmid having the same or similar function as the above siRNA. The method for constructing the expression plasmid is well known to those skilled in the art and will not be described in detail here.

The present invention also provides a targeted gene site of the nucleic acid as described above. In some embodiments, the targeted gene site is marked as any one of the items in column 1 of Table 1.

Table 1

Note: The first column refers to the position of the first base of the targeted gene in the ANGPTL3 gene sequence, and so on; the numbers in the third and fifth columns represent the sequence numbers, for example, "1" represents SEQ ID NO: 1.

Among them, the reference sequence of the targeted gene is the coding sequence NM_014495.4 of human ANGPTL3.

Targeted drug delivery system

The present invention also provides a targeted drug delivery system, which comprises a targeting group, a linking group and the above-mentioned nucleic acid connected to the targeting group via the linking group.

In combination with common sense in the art, the nucleic acid (siRNA) of the present invention has a better inhibitory effect when applied to different targeted drug delivery systems. In other words, the effect advantage of the naked sequence and the modified sequence in the present invention does not depend on the selection of the targeting carrier. In order to further improve the bioavailability and therapeutic effect of siRNA, the present invention also optimizes the targeted drug delivery system and obtains the following technical scheme.

In some embodiments, the linker is linked to the 3' end or the 5' end of the sense strand or the antisense strand of the nucleic acid.

In some embodiments, the linker is attached to the 3' end of the sense strand of the nucleic acid.

In some embodiments, the targeted drug delivery system comprises a ligand and the nucleic acid linked to the ligand.

In some embodiments, the ligand is a GalNAc derivative.

In some embodiments, the ligand is one or more GalNAc derivatives connected by single-stranded, double-stranded, or triple-stranded branched linkers.

In some preferred embodiments, the structure of the targeted drug delivery system is shown in Formula I below:

In formula I, Nu represents the nucleic acid (siRNA). In some embodiments, the compound part in the system can be connected to the 5' end or 3' end of the sense strand of the siRNA through a phosphodiester bond, and can also be connected to the 5' end or 3' end of the antisense strand of the siRNA through a phosphodiester bond. The targeted drug delivery system utilizes the structural characteristics on its left side to improve the cell penetration ability of the nucleic acid drug (Nu), enhance its stability in the cell, and has a simple preparation process and strong practicality.

cell

The present invention also provides an in vitro cell comprising the nucleic acid as described above.

The cells can be used for gene function research, disease model research, drug screening and the like.

Pharmaceutical composition

The present invention also provides a pharmaceutical composition, which contains the nucleic acid or targeted drug delivery system as described above and a pharmaceutically acceptable carrier.

The pharmaceutical composition can be prepared from the nucleic acid and the pharmaceutically acceptable carrier by conventional methods. For example, the pharmaceutical composition can be an injection. The injection can be used for subcutaneous, intramuscular or intravenous injection.

According to the pharmaceutical composition of the present invention, there is no special requirement for the amount of the nucleic acid or the targeted drug delivery system and the pharmaceutically acceptable carrier. Generally, relative to 1 part by weight of the nucleic acid (or 1 part by weight of the targeted drug delivery system calculated as nucleic acid), the content of the pharmaceutically acceptable carrier can be 1-100000 parts by weight (such as 1 part by weight, 5 parts by weight, 10 parts by weight, 50 parts by weight, 100 parts by weight, 500 parts by weight, 1000 parts by weight, 5000 parts by weight, 10000 parts by weight, 50000 parts by weight, 100000 parts by weight or any value between any two of the above values).

According to the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier may be any of the conventional carriers in the art, for example, it may include at least one of a pH buffer, a protective agent and an osmotic pressure regulator. The pH buffer may be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, preferably a phosphate buffer with a pH of 5.5-8.5. The protective agent may be a mixture of inositol, sorbitol and sucrose. At least one of. Based on the total weight of the pharmaceutical composition, the content of the protective agent can be 0.01-30 weight % (such as 0.01 weight %, 0.05 weight %, 0.1 weight %, 0.5 weight %, 1 weight %, 5 weight %, 10 weight %, 15 weight %, 20 weight %, 25 weight %, 30 weight % or any value between any two of the above values). The osmotic pressure regulator can be sodium chloride and/or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmole/kg. According to the desired osmotic pressure, those skilled in the art can determine the content of the osmotic pressure regulator.

According to a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is a liposome. The liposome can be any liposome capable of encapsulating nucleic acid, and its diameter can be 25-1000 nm, and can include but not limited to cholesterol and its analogs or derivatives.

The dosage of the pharmaceutical composition of the present invention can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially according to the age, weight and gender of the subject. For example, for female, 3-4 month old mice weighing 25-30 g, based on the amount of the nucleic acid in the pharmaceutical composition, the dosage of the pharmaceutical composition can be 0.01-100 mg/kg body weight, preferably 1-10 mg/kg body weight.

Methods and uses

The present invention also provides a method for inhibiting the expression of ANGPTL3 in a cell, the method comprising: contacting the cell with the nucleic acid as described above, the targeted drug delivery system as described above, or the pharmaceutical composition as described above, so as to inhibit the expression of ANGPTL3 in the cell.

In some embodiments, the cell is in a subject, e.g., a human subject, e.g., a subject having an ANGPTL3-associated disease, or a subject in need of prevention of risk of an ANGPTL3-associated disease.

In some embodiments, the cell is located in vitro. The method is for research purposes or for constructing an animal model.

In some embodiments, contacting the cell with the nucleic acid inhibits expression of ANGPTL3 by at least 50%, 60%, 70%, 80%, 90%, 95% (e.g., when the cell is first contacted with the nucleic acid). In some embodiments, inhibiting the expression of ANGPTL3 reduces the level of ANGPTL3 protein in a serum sample of the subject by at least 50%, 60%, 70%, 80%, 90% or 95%, e.g., compared to the expression level of ANGPTL3 before the cell is first contacted with the nucleic acid; e.g., before administering a first dose of the nucleic acid to the subject. In some embodiments, inhibiting the expression of ANGPTL3 reduces the level of ANGPTL3 protein in a serum sample of the subject by at least 50%, 60%, 70%, 80%, 90% or 95%, e.g., compared to the expression level of ANGPTL3 before the cell is first contacted with the nucleic acid.

The present invention also provides uses of the nucleic acid as described above, the targeted drug delivery system as described above, or the pharmaceutical composition as described above in any of the following aspects: 1) treating and/or preventing diseases associated with ANGPTL3; 2) preparing drugs for treating and/or preventing diseases associated with ANGPTL3.

In some embodiments, the disease is: (i) a disease associated with enhanced or elevated ANGPTL3; or (ii) a disease that would benefit from decreased expression of ANGPTL3.

In some embodiments, the disease is a lipid metabolism disorder.

In some embodiments, the disease is selected from one or more of the following: hyperlipidemia, hypertriglyceridemia, cardiovascular disease, atherosclerosis, hypercholesterolemia, familial hypercholesterolemia, diabetes (such as type 2 diabetes), obesity, fatty liver (such as non-alcoholic fatty liver disease), knee injury and osteoarthritis, chylomicronemia syndrome, familial partial lipodystrophy (FPLD).

In the present invention, the subject can be a mammal, including primates (such as humans, non-human primates, such as monkeys and chimpanzees), non-primates (such as cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats or mice) or birds. In some embodiments, the subject is preferably a primate, more preferably a human.

The drug can be administered via a variety of routes, depending on whether local or systemic treatment is required. The dosage can be referred to above and will not be repeated here.

In some embodiments, administration can be topical (e.g., transdermal patch), pulmonary, e.g., via inhalation or insufflation of a powder or spray, including via a nebulizer; intratracheal, nasal, epidermal, and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, e.g., via an implant device; or intracranial, e.g., via intraparenchymal, Intrathecal or intraventricular administration.

In some embodiments, the nucleic acid, the targeted drug delivery system or the pharmaceutical composition is administered to the subject by subcutaneous administration, intravenous administration and/or intramuscular administration.

Example

Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

Example 1 In vitro screening of siRNA

After screening to obtain the sense strand and antisense strand sequences shown in Table 1, each sense strand and antisense strand was modified, and the modified sequences are shown in Table 2.

Table 2

In the table, a/c/g/u = 2'-OMe nucleotides; Af/Cf/Gf/Uf = 2'-F nucleotides; s = phosphorothioate diester bond.

The sense strand and antisense strand in Table 2 were synthesized into the modified double-stranded siRNA in Table 3 by solid phase synthesis.

Table 3

In the table, a/c/g/u = 2'-OMe nucleotides; Af/Cf/Gf/Uf = 2'-F nucleotides; s = phosphorothioate diester bond.

0.5 ml of cell culture medium (DMEM, 10% calf serum, 1% penicillin + streptomycin solution) containing 10 ⁴ Hep3B (Procell, Cat#CL-0102) cells were added to a 96-well cell culture dish and cultured overnight in a cell culture incubator at 37°C, 5% CO2. RNAiMAX (1.5 μL/well) and small interfering nucleic acids (siRNA) listed in Table 3 were added to the Opti-MEM culture medium and added to the cell culture wells to make the final concentration of each well 1 nM or 10 nM. The cells were cultured for 48 hours at 37°C, 5% CO2. To extract RNA, the cell culture supernatant was aspirated and washed with PBS. After aspiration, 50 μL of the prepared lysis solution (as recommended by the Cells-to-CT kit (ThermoFisher Scientific, Cat#4391851c)) was added and mixed. After standing for 10 minutes, 2.5 μL of Stop solution was added to terminate the process for 2 minutes. RT-PCR was performed according to the recommendations of High Capacity cDNA Reverse Transcription Kits (Thermo Fisher, Cat. No. 4368814), with 10 μL of lysed liquid in each reaction. Gene expression was measured quantitatively by real-time fluorescence PCR, with the TaqMan probe for human ANGPTL3 being Hs00205581_m1 and the probe for the internal reference gene (human HPRT1) being Hs02800695_m1 (Thermo Fisher Scientific, Waltham, MA, USA). PCR conditions were 1 cycle at 95°C for 20 seconds, 40 cycles at 95°C for 1 second and 60°C for 20 seconds, and the real-time fluorescence PCR instrument was QuantStudio ^TM 6 Pro Real-time fluorescence quantitative PCR system (Thermo Fisher). ANGPTL3 gene expression was calculated by 2^-ΔΔCt, and human HPRT1 gene expression was used as an internal reference. ANGPTL3 gene expression was expressed as a relative value to the control group with only RNAiMAX. The results are shown in Table 4.

Table 4. Inhibitory effect of siRNA on ANGPTL3 gene expression in Hep3B cells

From the results, it can be seen that the siRNAs of the present invention can silence the ANGPTL3 gene to varying degrees, among which SN-252194, SN-252197, SN-252201, SN-252203, SN-252204, SN-252205, SN-252208, SN-252210, SN-252218, SN-252219, SN-252222, SN- 252224, SN-252226, and SN-252228 have better inhibitory effects, with an average inhibitory effect of more than 78% at a concentration of 1 nM. By comparing the silencing effects of siRNAs with different sequences under the same modification on ANGPTL3 gene expression in cells, it can be confirmed that the above siRNAs have obvious effect advantages at the naked sequence level.

The preferred siRNA in Table 4 was further tested for dose dependence in Hep3B cells. Specifically, RNAiMAX (1.5 μl/well) and small interfering nucleic acid (siRNA) were mixed in Opti-MEM culture medium and added to cell culture wells to make the final concentration per well 0.005, 0.01, 0.04, 0.12, 0.37, 1.1, 3.3 or 10 nM, and the cells were cultured for 48 hours at 37°C and 5% CO2. RNA extraction and relative quantification were as described above. The IC ₅₀ of siRNA compounds for inhibiting ANGPTL3 expression is shown in Table 5.

Table 5. IC ₅₀ values of siRNA compounds for inhibition of ANGPTL3 gene expression in Hep3B cells

In order to determine the activity of siRNA, the preferred siRNA in Table 5 (IC50 less than 0.1nM) was coupled to the hepatocyte targeting compound Tri-GalNAc (its specific structure is shown in the aforementioned formula I, and the sequences involved are shown in Table 6) and a free uptake experiment was carried out in monkey primary hepatocytes. The siRNA-GalNAc samples were dissolved into 10000μM solution with 100μL enzyme-free sterile water, and 10μL of 10000μM test solution was added to 90μL PMonH plating medium to dilute it to 1000μM solution as a 1000nM final concentration group working solution for standby use; the 1000μM test solution was then diluted 3 times with PMonH plating medium at 8 concentration points, so that the final working solution concentrations were 0.5, 1.4, 4, 12, 37, 111, 333, 1000nM. The monkey primary hepatocytes were taken out of liquid nitrogen, thawed and revived at 37°C, washed and counted with serum-containing PMonH plating medium and centrifuged, the supernatant was removed and the cells were diluted to 250k/mL with new serum-containing PMonH plating medium, and then 90μL of the diluted cell solution was plated on a 96-well cell culture plate so that the number of cells in each well was 25k. The prepared sample working solution was added to the cell solution to make the final concentration of 0.5, 1.4, 4, 12, 37, 111, 333, 1000nM, and then placed in a 5% carbon dioxide incubator and cultured at 37°C for 48 hours. After 48 hours, all the culture medium in the 96-well culture plate was aspirated, washed with 1×PBS buffer, 50μL of the prepared Cells to CT lysis solution (as recommended by the manufacturer) was added and mixed, and after standing for 10 minutes, 2.5μL of stop solution was added to stop for 2 minutes. RT-PCR was performed according to the recommendations of High Capacity cDNA Reverse Transcription Kits (Thermo Fisher, Cat. No. 4368814), with 10 μL of lysate in each reaction. Gene expression was measured by real-time fluorescence PCR, with the TaqMan probe for monkey ANGPTL3 being Mf04384789_m1 and the probe for the internal reference gene (monkey PPIB) being Mf02802985_m1 (Thermo Fisher Scientific, Waltham, MA, USA). PCR conditions were 1 cycle at 95°C for 20 seconds, 40 cycles at 95°C for 1 second and 60°C for 20 seconds, and the real-time fluorescence PCR instrument was the QuantStudio ^TM 6Pro Real-Time Fluorescence Quantitative PCR System (Thermo Fisher). ANGPTL3 gene expression was calculated as 2^-ΔΔCt, with PPIB gene expression as the internal reference. The silencing expression of the ANGPTL3 gene was calculated as a percentage of the control group with only culture medium. The siRNA concentration (IC50) that reduces the expression of ANGPTL3 by 50% is shown in Table 7.

Table 6

In the table, a/c/g/u = 2'-OMe nucleotides; Af/Cf/Gf/Uf = 2'-F nucleotides; s = dithiophosphate Ester bond.

Table 7. IC50 values of siRNA silencing ANGPTL3 gene expression in monkey primary hepatocytes

It can be seen that the above siRNAs can silence the ANGPTL3 gene well, among which SN-682197, SN-682201, SN-682203, SN-682210 and SN-682218 have better silencing effects, and the IC50 values for silencing ANGPTL3 gene expression in primary monkey hepatocytes are lower than 13nM.

Example 2 Verification of the in vivo effect of siRNA

To further determine the activity of siRNA, experiments were conducted in human ANGPTL3 transgenic mice using the preferred siRNA in Table 7 as the experimental group and PBS as the control group. 1 or 3 mg/kg siRNA-Tri-galNac compound or PBS was subcutaneously injected into mice on day 0, and blood was collected on day 10 to measure the ANGPTL3 protein level in plasma. The reduction effect was expressed as a percentage compared with the ANGPTL3 protein before injection. The results are shown in Figures 1 (1 mg/kg) and 2 (3 mg/kg).

As shown in Figures 1 and 2, the siRNA of the present invention can effectively reduce the level of ANGPTL3 protein in mice, among which SN-682210 and SN-682218 have significantly better inhibitory effects.

To further verify the activity of siRNA, human ANGPTL3 transgenic mice were subcutaneously injected with 1 or 3 mg/kg of PBS, SN-682210 or SN-682218 on day 0, and the level of human ANGPTL3 protein in the blood was tracked and observed. The results are shown in Figure 3.

As shown in Figure 3, more than 20 days after the injection of SN-682210 or SN-682218 targeted drugs, the ANGPTL3 protein level in the blood of human ANGPTL3 transgenic mice can still be effectively reduced. Among them, compared with SN-682218, the efficacy of SN-682210 is further superior. Therefore, the efficacy of the experimental group injected with SN-682210 was further observed, and the results are shown in Figure 4.

As shown in FIG4 , more than 40 days after the injection of the targeted drug SN-682210, the ANGPTL3 protein level in the blood of human ANGPTL3 transgenic mice can still be effectively reduced.

The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims (20)

A nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand contains a sequence having more than 80% sequence identity with a sequence shown in any one of SEQ ID NOs: 1 to 33, and the antisense strand contains a sequence having more than 80% sequence identity with a nucleotide sequence at positions 1 to 21 of a sequence shown in any one of SEQ ID NOs: 34 to 66. The nucleic acid according to claim 1, wherein the antisense strand contains at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the sequence shown in any one of SEQ ID NOs: 34 to 66, and the sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand; optionally, the sense strand contains at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the sequence shown in any one of SEQ ID NOs: 1 to 33. The nucleic acid according to claim 1, wherein the antisense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NO: 34, 35, 38, 39, 40, 41, 43, 45, 50, 51, 54, 56, 57, 59 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand; optionally, the sense strand contains a nucleotide sequence that differs from the sequence shown in any one of SEQ ID NO: 1, 2, 5, 6, 7, 8, 10, 12, 17, 18, 21, 23, 24, 26 by 0, 1 or 2 nucleotides. The nucleic acid according to claim 1, wherein the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 45 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 12 by 0, 1 or 2 nucleotides; Or, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 50 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 17 by 0, 1 or 2 nucleotides. The nucleic acid according to claim 1, wherein at least one nucleotide in the nucleic acid is a modified nucleotide or comprises a modified internucleotide; The modified nucleotides are preferably selected from 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-open ring nucleotide mimics, locked nucleotides, 2'-F-arabinose nucleotides, 2'- One or more of methoxyethyl nucleotides, abasic nucleotides, ribitols, inverted nucleotides, inverted 2'-O-methyl nucleotides, inverted 2'-deoxynucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, vinyl phosphonate containing nucleotides, cyclopropyl phosphonate containing nucleotides and 3'-O-methyl nucleotides; the modified nucleotides are further preferably selected from one or more of 2'-O-methyl nucleotides and 2'-fluoro nucleotides; The modified internucleotide bond is preferably selected from one or more of phosphorothioate internucleotide bonds and methylphosphonate internucleotide bonds; the modified internucleotide bond is further preferably selected from one or more of phosphorothioate monoester internucleotide bonds and phosphorothioate diester internucleotide bonds. The nucleic acid according to claim 5, wherein the antisense strand contains 2 phosphorothioate internucleotide bonds at the 5' end and the 3' end, respectively, and contains 4 to 6 2'-fluoro nucleotides, and the remaining nucleotides are 2'-O-methyl nucleotides; the 5' end of the sense strand contains 2 phosphorothioate internucleotide bonds, and contains 3 to 5 2'-fluoro nucleotides, and the remaining nucleotides are 2'-O-methyl nucleotides; preferably, the 2nd, 4th, 6th, 12th and 14th nucleotides of the antisense strand counting from the 5' end are 2'-fluoro nucleotides, and the 7th, 9th, 10th and 11th nucleotides of the sense strand counting from the 5' end are 2'-fluoro nucleotides. The nucleic acid according to claim 1, wherein the antisense strand has at least 15 consecutive nucleotides with a difference of no more than 3 nucleotides from the antisense strand sequence shown in any one of Table 2, and the sense strand has at least 15 consecutive nucleotides with a difference of no more than 3 nucleotides from the sense strand sequence shown in any one of Table 2; preferably, the sense strand and the antisense strand form an siRNA shown in any one of Table 3. The nucleic acid according to claim 1, wherein the antisense strand contains a nucleotide sequence from the 5' end to the 3' end that differs from the following nucleotide sequence by 0, 1 or 2 nucleotides:
SN-52194: asGfsuAfgAfauuuuUfuCfuucuaggsasg;
SN-52197:usAfsaGfuUfaguuaGfuUfgcucuucsusa;
SN-52201:usUfsuUfaAfgugaaGfuUfacuucugsusu;
SN-52203:usAfsuUfuCfuuuuaUfuUfgacuaugscsu;
SN-52204:usUfsuCfuAfuuucuUfuUfauuugacsusa;
SN-52205: asAfsgAfuAfgagaaAfuUfucuguggsusu;
SN-52208: asGfsuUfuUfgugauCfcAfucuauucsgsa;
SN-52210:usUfsuCfaUfugaagUfuUfugugaucscsa;
SN-52218: asAfsaAfgAfauauuCfaAfuauaaugsusu;
SN-52219: asUfsaGfuUfgguuuCfgUfgauuuccsusu;
SN-52222: asGfsaUfgUfagcguAfuAfguugguususc;
SN-52224:usAfsuAfaCfcuuccAfuUfuugagacsusu;
SN-52226:usUfsgAfuUfuuauaGfaGfuauaaccsusu;
SN-52228:usCfsaUfuCfaaagcUfuUfcugaaucsusg; The sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand; Preferably, the sense strand contains a nucleotide sequence from the 5' end to the 3' end that differs by 0, 1 or 2 nucleotides from any of the following nucleotide sequences:
SN-22194:cscsuagaAfgAfAfAfaaauucuacu;
SN-22197:gsasagagCfaAfCfUfaacuaacuua;
SN-22201:csasgaagUfaAfCfUfucacuuaaaa;
SN-22203: csasuaguCfaAfAfUfaaaagaaaua;
SN-22204:gsuscaaaUfaAfAfAfgaaauagaaa;
SN-22205:cscsacagAfaAfUfUfucucuaucuu;
SN-22208:gsasauagAfuGfGfAfucacaaaacu;
SN-22210:gsasucacAfaAfAfCfuucaaugaaa;
SN-22218:csasuuauAfuUfGfAfauauucuuuu;
SN-22219:gsgsaaauCfaCfGfAfaaccaacuau;
SN-22222:asasccaaCfuAfUfAfcgcuacaucu;
SN-22224: gsuscucaAfaAfUfGfgaagguuaua;
SN-22226:gsgsuuauAfcUfCfUfauaaaaucaa;
SN-22228: gsasuucaGfaAfAfGfcuuugaauga; In each sequence, a nucleotide represented by a lowercase letter indicates that the nucleotide is a 2'-O-methyl nucleotide; f indicates that the nucleotide adjacent to its left is a 2'-fluoro nucleotide; and s indicates that the two adjacent nucleotides on the left and right are connected by a phosphorothioate diester bond. The nucleic acid of claim 8, wherein the antisense strand comprises a nucleotide sequence that differs by 0, 1, or 2 nucleotides from the sequence set forth in SN-52210, and the sense strand comprises a nucleotide sequence that differs by 0, 1, or 2 nucleotides from the sequence set forth in SN-22210; Alternatively, the antisense strand contains a nucleotide sequence that differs from the sequence shown in SN-52218 by 0, 1 or 2 nucleotides, and the sense strand contains a nucleotide sequence that differs from the sequence shown in SN-22218 by 0, 1 or 2 nucleotides. A targeted drug delivery system, characterized in that the targeted drug delivery system comprises a targeting group, a linking group and the nucleic acid according to any one of claims 1 to 9 connected to the targeting group via the linking group. The targeted drug delivery system according to claim 10, wherein the linking group is connected to the 3' end or the 5' end of the sense strand or the antisense strand of the nucleic acid. The targeted drug delivery system according to claim 10 or 11, wherein the targeted drug delivery system comprises a ligand and the nucleic acid connected to the ligand; preferably the ligand is a GalNAc derivative; more preferably the ligand is one or more GalNAc derivatives connected by single-stranded, double-stranded or triple-stranded branched linkers. The targeted drug delivery system according to claim 10, wherein the structure of the targeted drug delivery system is shown as follows:
In the formula, Nu represents the nucleic acid. An isolated cell, characterized in that the cell contains the nucleic acid according to any one of claims 1 to 9. A pharmaceutical composition, characterized in that the pharmaceutical composition contains the nucleic acid according to any one of claims 1 to 9 or the targeted drug delivery system according to any one of claims 10 to 13 and a pharmaceutically acceptable carrier. A method for inhibiting ANGPTL3 expression in a cell, the method comprising: contacting the cell with the nucleic acid according to any one of claims 1 to 9, the targeted drug delivery system according to any one of claims 10 to 13, or the pharmaceutical composition according to claim 15, so as to inhibit the expression of ANGPTL3 in the cell. Use of the nucleic acid according to any one of claims 1 to 9, the targeted drug delivery system according to any one of claims 10 to 13, or the pharmaceutical composition according to claim 15 in any of the following aspects: 1) Treating and/or preventing diseases associated with ANGPTL3; 2) Preparation of drugs for treating and/or preventing diseases associated with ANGPTL3. The use according to claim 17, wherein the disease is: (i) a disease associated with enhancement or elevation of ANGPTL3; or (ii) Diseases that would benefit from reduced ANGPTL3 expression. The use according to claim 17, wherein the disease is a lipid metabolism disorder; Optionally, the disease is selected from one or more of the following: hyperlipidemia, hypertriglyceridemia, cardiovascular disease, atherosclerosis, hypercholesterolemia, familial hypercholesterolemia, diabetes, obesity, fatty liver, knee injury and osteoarthritis, chylomicronemia syndrome, familial partial lipodystrophy. The use according to claim 17, wherein the nucleic acid, the targeted drug delivery system or the pharmaceutical composition is administered to the subject by subcutaneous administration, intravenous administration and/or intramuscular administration.