CN118599845A

CN118599845A - Nucleic acid targeting human angiopoietin-like protein 3 and use thereof

Info

Publication number: CN118599845A
Application number: CN202410843642.1A
Authority: CN
Inventors: 蒋伟文; 郁东; 兰涛
Original assignee: Shi Nengkang Pharmaceutical Technology Suzhou Co ltd
Current assignee: Shi Neng Kang Biomedical Suzhou Co ltd
Priority date: 2023-06-27
Filing date: 2024-06-27
Publication date: 2024-09-06
Also published as: WO2025002247A1

Abstract

The present invention relates to the field of biotechnology, and in particular, to nucleic acids targeting human angiopoietin-like protein 3 and uses thereof. The nucleic acids of the present invention can effectively reduce the level of ANGPTL3 and effectively inhibit the accumulation of fat in the blood, and thus can be used to prevent or treat related diseases, such as lipid metabolism disorders.

Description

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

Cross Reference to Related Applications

The present invention claims priority from chinese patent application number 202310764185.2 filed on 27, 2023, 06, the entire contents of which are incorporated herein by reference.

Technical Field

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

Background

Human angiopoietin-like protein 3 (ANGPTL 3, also known as FHBL, ANL3, ANGPT5, ANG-5) is a 45kDa protein expressed in hepatocytes. ANGPTL3 and ANGPTL8 form a protein complex that inhibits lipoprotein lipase (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.)., an important rate-limiting enzyme for triglyceride metabolism in humans, and catalyzes the hydrolysis of triglycerides (Kersten s. Physiological regulation of lipoprotein lipase. Biochem biophysis Acta 2014; 1841:919-933). The decrease in lipoprotein lipase may cause fat to accumulate in the blood, leading to atherosclerosis. Reducing or increasing ANGPTL3 expression in animal models regulates triglyceride levels in mice, while reducing ANGPTL3 in APOE knockout mice models reduces atherosclerosis (Ando Y,Shimizugawa T,Takeshita S,et al.Adecreased expression of angiopoietin-like 3is protective against atherosclerosis in apoE-deficient mice.J Lipid Res 2003;44:1216–1223.).ANGPTL3 knockout mice, which also find increased lipoprotein lipase activity with concomitant reduction in blood lipid (Fujimoto K,Koishi R,Shimizugawa T,Ando Y.Angptl3-null mice show low plasma lipid concentrations by enhanced lipoprotein lipase activity.Exp Anim2006;55:27–34.).

ANGPTL3 loss of function due to ANGPTL3 gene mutation was found in human genetic studies to 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 studies of cardiovascular disease and ANGPTL3 association, ANGPTL3 loss of function was found to significantly reduce the incidence of atherosclerosis 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.), while inhibiting ANGPTL3 with antibodies significantly reduced low density lipoprotein and triglyceride levels. Currently, an antibody Evinacumab targeting ANGPTL3 has been approved for use in homozygous familial hypercholesterolemia patients.

RNA interference (RNA INTERFERENCE, RNAI) refers to the phenomenon of highly conserved and highly efficient specific degradation of homologous mRNA induced by double-stranded small interfering ribonucleic acid (SMALL INTERFERENCE RNA, SIRNA) during evolution, and RNAi drugs have the advantage of longer drug effect time compared with antibodies. Therefore, research and development of siRNA targeting ANGPTL3 is of great significance.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provides a novel nucleic acid targeting human angiopoietin-like protein 3 and application thereof.

The first aspect of the present invention provides a nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand comprises a sequence identical to SEQ ID NO: 1-33, said antisense strand comprising a sequence having more than 80% sequence identity to a sequence set forth in any one of SEQ ID NOs: 34 to 66, and the nucleotide sequence at positions 1 to 21 of the sequence shown in any one of the above items has a sequence identity of 80% or more.

In a second aspect the invention provides a targeted drug delivery system comprising a targeting group, a linking group and a nucleic acid as described above linked to the targeting group by the linking group.

In a third aspect the invention provides an ex vivo cell comprising a nucleic acid as described above.

In a fourth aspect the invention provides a pharmaceutical composition comprising a nucleic acid or targeted drug delivery system as described above and a pharmaceutically acceptable carrier.

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

In a sixth aspect the invention provides the use of a nucleic acid, targeted drug delivery system or pharmaceutical composition as described above in any of the following aspects: 1) Treating and/or preventing a disease associated with ANGPTL 3; 2) Preparing a medicament for treating and/or preventing diseases related to ANGPTL 3.

The nucleic acid of the invention can effectively reduce the level of ANGPTL3 and effectively inhibit the accumulation of fat in blood, thus being used for preventing or treating related diseases, such as lipid metabolism disorder and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the levels of ANGPTL3 protein in day 10 blood after 1mg/kg siRNA was injected into human ANGPTL3 transgenic mice in examples of the present invention.

FIG. 2 shows the levels of ANGPTL3 protein in day 10 blood after 3mg/kg siRNA was injected into human ANGPTL3 transgenic mice in the examples of the present invention.

FIG. 3 is a graph showing the efficacy of an siRNA drug targeting ANGPTL3 in a human ANGPTL3 transgenic mouse according to an embodiment of the present invention.

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

Detailed Description

The following describes specific embodiments of the present invention in detail. It will be understood that the embodiments described herein are for the purpose of illustration and explanation only and are not intended to limit the present invention, as many modifications and variations of the present invention may be made by those skilled in the art without departing from the scope or spirit thereof. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Interpretation of the terms

Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical schemes of all "logical or" connections), also include any and all combinations of A, B, C, D, i.e., the combinations of any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical schemes of all "logical and" connections).

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

The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.

Concentration values are referred to in this invention, the meaning of which includes fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may allow fluctuations within + -0.1%. For values that are larger or do not require finer control, it is also permissible for the meaning to include larger fluctuations. For example, 100mM, fluctuations in the range of.+ -. 1%,.+ -. 2%,.+ -. 5%, etc. can be tolerated. Molecular weight is referred to, allowing its meaning to include fluctuations of + -10%.

In the present invention, the terms "plurality", and the like refer to, unless otherwise specified, 2 or more in number.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present invention.

In the present invention, "optionally", and "optionally", "optional", refers to the existence or nonexistence, namely to any one of two parallel schemes of 'with' or 'without'. If multiple "optional" or "optional" items are present in a single embodiment, each "optional" or "optional" item is independent of the other, unless specified otherwise, and without conflict or limitation.

In the present invention, the term "nucleic acid" refers to a composition comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting (e.g., degrading or inhibiting under appropriate conditions) the translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence-specific manner. The nucleic acid may act by an RNA interference mechanism (i.e., by interacting with the RNA interference pathway mechanism of mammalian cells (RNA-induced silencing complex or RISC) to induce RNA interference), or by any alternative mechanism or pathway. The following defined ranges of nucleic acids including sense and antisense strands disclosed herein include, but are not limited to: short (or small) interfering RNAs (sirnas), double-stranded RNAs (dsRNA), micrornas (mirnas), short hairpin RNAs (shrnas), and dicer (dicer) substrates.

In the present invention, when referring to expression of a given gene, the terms "silence," "decrease," "inhibit," "down-regulate," or "knock-down" mean that expression of the gene is reduced when the cell, cell population, tissue, organ, or subject is treated with a nucleic acid as described herein, as measured by the level of RNA transcribed from the gene or the level of a polypeptide, protein, or protein subunit translated from mRNA in the cell, cell population, tissue, organ, or subject in which the gene is transcribed, as 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 a first oligonucleotide hybridize to the same number of bases in the contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise 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 a first oligonucleotide hybridize to the same number of bases in the contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or part of the first nucleotide sequence or the second nucleotide sequence.

In the present invention, "substantially complementary" means that at least 85% but not all of the bases in the contiguous sequence of a first oligonucleotide hybridize to the same number of bases in the contiguous sequence of a second oligonucleotide in a hybridization pair of nucleobase or nucleotide sequence molecules. 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" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, 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 alleviation of the number, severity and/or frequency of one or more disease symptoms in a subject. The treatment may include the prevention, management, prophylactic treatment, and/or inhibition or reduction of the number, severity, and/or frequency of one or more disease symptoms in the subject.

In the present invention, the term "linked" means that two compounds or molecules are joined by a covalent bond. As used herein, the term "linked" may refer to a linkage between a first compound and a second compound, with or without any intervening atoms or groups of atoms, unless otherwise indicated.

Nucleic acid

The present invention provides a (modified or unmodified) nucleic acid comprising a sense strand and an antisense strand, said sense strand comprising a sequence identical to SEQ ID NO: 1-33 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and the antisense strand comprises a sequence having more than 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO: 34-66, and the nucleotide sequence at positions 1-21 of the sequence shown in any one of claims (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) or more.

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 particular embodiments, one skilled in the art can combine the sequences provided in the present invention in combination with the complementarity considerations of the sense strand and the antisense strand to obtain a combined nucleic acid (siRNA).

In a preferred embodiment of the invention, as shown in table 1, the nucleic acid is selected from the group consisting of the sense strand sequence of SEQ ID NO:1 and the antisense strand sequence is SEQ ID NO:34, the sequence of the siRNA-1 and the sense strand of SEQ ID NO:2 and the antisense strand sequence is SEQ ID NO:35, the sequence of the siRNA-2 and the sense strand of the sequence SEQ ID NO:3 and the antisense strand sequence is SEQ ID NO:36, the sequence of the siRNA-3 and the sense strand of the sequence SEQ ID NO:4 and the antisense strand sequence is SEQ ID NO:37, the sequence of the siRNA-4 and the sense strand of the sequence SEQ ID NO:5 and the antisense strand sequence is SEQ ID NO:38, at least one of siRNA-5 … …, siRNA-31, siRNA-32, and siRNA-33.

In some preferred embodiments, the antisense strand comprises a sequence identical to SEQ ID NO: 34-66, said sense strand comprising a nucleotide sequence that is at least partially complementary (e.g., partially complementary, substantially complementary, or fully complementary) to said antisense strand, by at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that differ by no more than 0, 1,2, or 3 nucleotides.

In some preferred embodiments, the sense strand comprises a nucleotide sequence that hybridizes to SEQ ID NO: at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides differing by no more than 0, 1,2 or 3 nucleotides from the sequences indicated in any one of 1 to 33.

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

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

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

In some preferred embodiments, the antisense strand comprises a sequence identical to SEQ ID NO: 34. 35, 38, 39, 40, 41, 43, 45, 50, 51, 54, 56, 57, 59, said sense strand comprising a nucleotide sequence that is at least partially complementary (e.g., partially complementary, substantially complementary, or fully complementary) to said antisense strand. When the antisense strand has the sequence, the double-stranded RNA has a significantly better inhibitory effect on ANGPTL 3.

In some preferred embodiments, the sense strand comprises a nucleotide sequence that hybridizes to SEQ ID NO: 1.2, 5,6, 7, 8, 10, 12, 17, 18, 21, 23, 24, 26 by a nucleotide sequence of 0,1 or 2 nucleotides.

In some further preferred embodiments, the antisense strand comprises a sequence identical to SEQ ID NO: 34. 35, 38, 39, 40, 41, 43, 45, 50, 51, said sense strand comprising a nucleotide sequence that is at least partially complementary (e.g., partially complementary, substantially complementary, or fully complementary) to said antisense strand. When the antisense strand has the above sequence, the double-stranded RNA has a further more excellent inhibitory effect on ANGPTL 3. In some further preferred embodiments, the sense strand comprises a nucleotide sequence that hybridizes to SEQ ID NO: 1.2, 5, 6, 7, 8, 10, 12, 17, 18, and a nucleotide sequence differing by 0, 1, or 2 nucleotides.

In some further preferred embodiments, the antisense strand comprises a sequence identical to SEQ ID NO: 35. 38, 39, 45, 50, said sense strand comprising a nucleotide sequence that is at least partially complementary (e.g., partially complementary, substantially complementary, or fully complementary) to said antisense strand. When the antisense strand has the above sequence, the double-stranded RNA has a further more excellent inhibitory effect on ANGPTL 3. In some further preferred embodiments, the sense strand comprises a nucleotide sequence that hybridizes to SEQ ID NO: 2.5, 6, 12, 17 differ by a nucleotide sequence of 0, 1 or 2 nucleotides.

In some further preferred embodiments, the antisense strand comprises a sequence identical to SEQ ID NO: 45. 50, the sense strand comprising a nucleotide sequence that is at least partially complementary (e.g., partially complementary, substantially complementary, or fully complementary) to the antisense strand. When the antisense strand has the above sequence, the double-stranded RNA has a further more excellent inhibitory effect on ANGPTL 3. In some further preferred embodiments, the sense strand comprises a nucleotide sequence that hybridizes to SEQ ID NO: 12. 17 differ by a nucleotide sequence of 0, 1 or 2 nucleotides.

In some preferred embodiments, the antisense strand comprises a sequence identical to SEQ ID NO:45, and the sense strand comprises a nucleotide sequence differing by 0,1 or 2 nucleotides from the sequence set forth in SEQ ID NO:12 differ by a nucleotide sequence of 0,1 or 2 nucleotides.

In some preferred embodiments, the antisense strand comprises a sequence identical to SEQ ID NO:50, and the sense strand comprises a nucleotide sequence differing by 0,1 or 2 nucleotides from the sequence set forth in SEQ ID NO:17 differ by a nucleotide sequence of 0,1 or 2 nucleotides.

In some embodiments, the sense strand or antisense strand in the nucleic acid has less than 100% sequence identity or more than 1 nucleotide difference to the corresponding sequence referred to herein, yet has an inhibitory effect on ANGPTL3 that is similar to (e.g., still has an efficacy equivalent to 80-120%, 85-115%, or 90-110% of the corresponding sequence) or equivalent to (e.g., still has 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 (e.g., the sequence set forth 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 nucleic acid sequences are also within the scope of the present invention.

The above-mentioned technical solutions for naked sequences (i.e. unmodified sequences) referred to in the present invention have the effect advantage of not depending on the modification method or the choice of targeting vector. Modifications to which it may be applied and further preferred modifications are described below:

The nucleic acid according to the present invention, wherein the nucleic acid contains a nucleotide group as a basic structural unit, the nucleotide group containing a phosphate group, a ribose group and a base, preferably the nucleic acid contains at least one modified nucleotide group. The inhibition efficiency of the modified nucleic acid to the ANGPTL3 is not lower than 50 percent (such as 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 percent).

The nucleic acid according to the present invention, wherein the modified nucleotide group is a nucleotide group modified with a phosphate group and/or a ribose group. The site with modification may 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 nucleotides of the sense strand and/or the antisense strand at positions 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.

For example, modification of the phosphate group refers to modification of oxygen in the phosphate group, including phosphorothioate modification (Phosphorthioate), boronated phosphate modification (Boranophosphate), and the like. Replacement of oxygen in the phosphate group with sulfur, borane, amine groups, alkyl groups, or alkoxy groups, respectively, is shown in the following formula. These modifications all stabilize the structure of the nucleic acid, maintaining high specificity and high affinity for base pairing.

In the above formulae, BASE represents BASE A, U, C, G or T. X may be oxygen (O) or sulfur (S). R may be the same or different in the above structure, for example: hydrogen (H), fluorine (F), methoxy (OME) or Methoxyethyl (MOE), hydroxy, allyl, ethylamino, propargyl, amino, cyanoethyl, acetyl, etc., R' and R "may each independently be hydrogen (H), methyl (CH 3), ethyl (CH 2CH 3), propyl (CH 2CH 3), isopropyl (CH 3) 2), allyl, propargyl, acyloxybenzyl, acyloxyethyl.

The modification of the ribose group refers to the modification of the 2 '-hydroxy group (2' -OH) in the ribose group. After introducing certain substituent groups such as methoxy or fluorine at the 2' -hydroxyl position of the ribosyl group, the nucleic acid is not easily cut by ribonuclease, so that the stability of the nucleic acid is improved, and the nucleic acid has stronger resistance to nuclease hydrolysis. Modifications to the 2 '-hydroxyl group in the nucleotide pentose include 2' -fluoro modifications (2 '-fluoromodification, such as 2' -arabino-fluoro modification), 2 '-methoxy modifications (2' -OME), 2 '-methoxyethyl modifications (2' -MOE), 2'-2, 4-dinitrophenol modifications (2' -DNP modification), ring-locked ethyl modifications (2 ',4' -constrained ethylmodification), 2'-amino modifications (2' -Amino modification), 2'-deoxy modifications (2' -Deoxy modification), BNA, acyclic nucleic acid modifications, misplaced nucleic acid modifications, L-type nucleic acid modifications, and the like. BNA (internal loop bridging nucleotide) refers to a constrained or inaccessible nucleotide. BNA may contain a five-, six-, or seven-membered ring bridging structure with "fixed" C3' -endo-saccharides tucked. The bridge is typically incorporated at the 2'-, 4' -position of the ribose ring to provide 2',4' -BNA nucleotides, such as locked ethyl modification (LNA), ring locked ethyl modification (ENA), and ethyl locked nucleic acid modification (cET BNA). Acyclic nucleic acids are nucleotides in which the sugar ring of a nucleotide is opened, such as Unlocking Nucleic Acid (UNA) nucleotides and Glycerol Nucleic Acid (GNA) nucleotides. Misplaced nucleic acid modification refers to the replacement of the 3',5' -phosphate linkage by a 2',5' -phosphate linkage. L-nucleic acid modification refers to the replacement of a naturally occurring D-nucleic acid with its mirror-image stereo-counterpart L-nucleic acid.

Wherein BASE represents BASE A, U, C, G or T. R may be the same or different in the above structure, for example: hydrogen (H), fluorine (F), methoxy (OME) or Methoxyethyl (MOE), hydroxy, allyl, ethylamino, propargyl, cyanoethyl, acetyl, etc.

The nucleic acid according to the present invention, wherein the nucleotide group whose ribose group is modified is preferably a nucleotide group whose 2' -OH group of ribose group is replaced with methoxy or fluoro.

According to a particularly preferred embodiment of the invention, the nucleotide group containing a uracil base or a cytosine base in the sense strand of the nucleic acid is a nucleotide group in which the ribose group is modified, i.e. the 2' -OH of the ribose group in the nucleotide group containing a uracil base or a cytosine base in the sense strand of the nucleic acid is substituted with methoxy or fluoro. More preferably, dTdT may be attached to the 3' end of both the sense and antisense strands of the nucleic acid; alternatively, the 3' end of the antisense strand of the nucleic acid may be linked with AA or UU or a combination of any two nucleic acids (which may be, but are not limited to CC, GG or UG) to confer sequence specificity to cause mRNA degradation. The nucleic acid having the above modification exhibits a more excellent in vivo inhibitory effect, and the above modification can further reduce the immunogenicity of the nucleic acid of the present invention in vivo.

The nucleic acids of the invention may also include modifications to ligate a nucleoside monophosphate to the 5' end of the antisense strand. Since the 5' -monophosphate of the siRNA guide terminal is important for RISC recognition. Wherein phosphorylation of the 5' -hydroxyl group plays a role in whether the siRNA can be efficiently loaded on Ago2 inside the cell. The guide chain 5' monophosphate in siRNA has H bond interaction with Argonaute-2 (Ago 2), thereby ensuring accurate positioning and accurate cutting of mRNA target. The derivatives of nucleoside 5' -monophosphates commonly used are those which have been shown to have stability in biological metabolic media and have an effect on Ago2 which facilitates loading of siRNA guide strands into cells (Nucleic ACIDS RESEARCH,2015,43,2993-3011). The nucleic acid according to the invention, wherein preferably the trans-Vinylphosphate (VP) is the most preferred, may also comprise derivatives of nucleoside monophosphates other than those described above.

In the above structures, BASE represents BASE A, U, C, G or T. R may be the same or different in the above structure, for example: hydrogen (H), fluorine (F), methoxy (OME) or Methoxyethyl (MOE), hydroxy, allyl, ethylamino, propargyl, cyanoethyl, amino, acetyl, etc.

In the present invention,Meaning that a chemical element X is attached 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 inter-bond.

In some preferred embodiments, the modified nucleotide is selected from one or more of a2 '-O-methyl nucleotide, a 2' -fluoro nucleotide, a2 '-deoxy nucleotide, a 2',3 '-ring opened nucleotide mimetic, a locked nucleotide, a 2' -F-arabinose nucleotide, a2 '-methoxyethyl nucleotide, an abasic nucleotide, a ribitol, an inverted nucleotide, an inverted 2' -O-methyl nucleotide, an inverted 2 '-deoxy nucleotide, a 2' -amino modified nucleotide, a2 '-alkyl modified nucleotide, a morpholino nucleotide, a vinyl phosphonate containing nucleotide, a cyclopropyl phosphonate containing nucleotide, and a 3' -O-methyl nucleotide; the modified nucleotide is further preferably selected from one or more of 2 '-O-methyl nucleotide and 2' -fluoro nucleotide.

In some preferred embodiments, the modified internucleotide linkages are preferably selected from one or more of phosphorothioate internucleotide linkages and methylphosphonate internucleotide linkages; the modified internucleotide linkages are further preferably selected from one or more of phosphorothioate monoester internucleotide linkages, phosphorothioate diester internucleotide linkages.

In some embodiments, the antisense strand contains 2 phosphorothioate internucleotide linkages, and 4 to 62 '-fluoro nucleotides, respectively, at the 5' and 3 'ends, with the remaining nucleotides being 2' -O-methyl nucleotides.

In some embodiments, the 5' end of the sense strand contains 2 phosphorothioate internucleotide linkages, and contains 3 to 52 ' -fluoro nucleotides, with the remaining nucleotides being 2' -O-methyl nucleotides.

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

In some preferred embodiments, the sense strand is 2 '-fluoro at nucleotides 7, 9, 10 and 11 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 contiguous nucleotides that differ by no more than 0,1, 2, or 3 nucleotides from the antisense strand sequences shown in any of table 2, and the sense strand has at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than 0,1, 2, or 3 nucleotides from the sense strand sequences shown in any of table 2.

In some preferred embodiments, the sense strand and the antisense strand form an siRNA having any one of the structures shown in table 3.

In some embodiments, the antisense strand comprises from the 5 'end to the 3' end a nucleotide sequence 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 comprises 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 as 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 nucleotide sequences that differ by 0, 1 or 2 nucleotides in sequence as shown by SN-52197, SN-52201, SN-52203, SN-52210 or SN-52218.

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

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

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, the nucleotide represented by the lower case letter represents that the nucleotide is a 2' -O-methyl nucleotide; f represents that one nucleotide adjacent to the left thereof is a 2' -fluoro nucleotide; s represents a phosphorothioate linkage between two adjacent nucleotides.

In particular implementations, one skilled in the art can combine the sequences of the sense and antisense strands mentioned above in combination with consideration of complementarity of the sense and antisense strands. In a preferred embodiment, the sense strand and antisense strand sequences, which are identical in number by the reciprocal, are combined to obtain the corresponding nucleic acid (siRNA). For example, SN-22194 and SN-52194 will be combined, and SN-22197 and SN-52197 will be combined to obtain the corresponding nucleic acids (siRNA).

In some preferred embodiments, the antisense strand contains a nucleotide sequence that differs by 0, 1, or 2 nucleotides from the sequence shown by SN-52210, and the sense strand contains a nucleotide sequence that differs by 0, 1, or 2 nucleotides from the sequence shown by 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 by SN-52218, and the sense strand contains a nucleotide sequence that differs by 0, 1, or 2 nucleotides from the sequence shown by SN-22218.

The nucleic acids according to the invention can be obtained by methods conventional in the art, for example by solid phase synthesis, which is already commercially available for subscription services, and liquid phase synthesis, and thus commercially available. The modified nucleotide groups may be introduced by nucleotide monomers having corresponding modifications.

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 siRNA described above, and the method for constructing the expression plasmid is well known to those skilled in the art and will not be described herein.

The invention also provides a target gene locus of the nucleic acid. In some embodiments, the targeted genetic locus is as noted in any one of column 1 of table 1.

TABLE 1

Note that: column 1 refers to the position of the first base of the targeted gene in the ANGPTL3 gene sequence, and so on; the numbers in columns 3, 5 represent sequence numbers, e.g. "1" represents SEQ ID NO:1.

Wherein the reference sequence of the target gene is the coding sequence NM_014495.4 of human ANGPTL 3.

Targeted drug delivery systems

The invention also provides a targeted drug delivery system comprising a targeting group, a linking group and a nucleic acid as described above linked to the targeting group via the linking group.

The nucleic acid (siRNA) of the present invention has superior inhibition effect when applied to different targeted drug delivery systems in combination with common knowledge in the art. In other words, the effect advantage of the naked sequence and the modified sequence in the present invention is not dependent on the choice of targeting vector. In order to further improve the bioavailability and the therapeutic effect of the siRNA, the invention also optimizes a targeted drug delivery system and obtains the following technical scheme.

In some embodiments, the linking group is attached to the 3 'or 5' end of the sense or antisense strand of the nucleic acid.

In some embodiments, the linking group 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 said nucleic acid linked to said ligand.

In some embodiments, the ligand is a GalNAc derivative.

In some embodiments, the ligand is one or more GalNAc derivatives linked by a single-, double-, or triple-chain branched linker.

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

In formula I, nu represents the nucleic acid (siRNA). In some embodiments, the compound moiety in the system can be linked to the 5 'or 3' end of the sense strand of the siRNA via a phosphodiester linkage, or can be linked to the 5 'or 3' end of the antisense strand of the siRNA via a phosphodiester linkage. The targeted drug delivery system can improve the cell penetration capability of nucleic acid drugs (Nu) by utilizing the structural characteristics of the left side of the targeted drug delivery system, enhance the stability of the targeted drug delivery system in cells, and has simple preparation process and strong practicability.

Cells

The invention also provides an isolated cell comprising a nucleic acid as described above.

The cell can be used for gene function research, disease model research or drug screening and other purposes.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising a nucleic acid or targeted drug delivery system as described above and a pharmaceutically acceptable carrier.

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

The pharmaceutical composition according to the present invention, wherein the amount of the nucleic acid or the targeted drug delivery system and the pharmaceutically acceptable carrier is not particularly limited, and generally, the pharmaceutically acceptable carrier may be contained in an amount of 1 to 100000 parts by weight (e.g., 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 or more) relative to 1 part by weight of the nucleic acid (or 1 part by weight of the targeted drug delivery system calculated on the nucleic acid).

The pharmaceutical composition according to the present invention, wherein the pharmaceutically acceptable carrier may be various carriers conventionally employed in the art, for example, may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator. The pH buffer solution can be a tris hydrochloride buffer solution with the pH value of 7.5-8.5 and/or a phosphate buffer solution with the pH value of 5.5-8.5, preferably a phosphate buffer solution with the pH value of 5.5-8.5. The protective agent may be at least one of inositol, sorbitol, and sucrose. The protective agent may be present in an amount of 0.01 to 30% by weight (e.g., 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30% by weight, or any value between any two of the above), based on the total weight of the pharmaceutical composition. The osmolality adjusting agent may be sodium chloride and/or potassium chloride. The osmolality adjusting agent is present in an amount such that the osmolality of the pharmaceutical composition is 200-700 milliosmol/kg. The amount of osmolality adjusting agent can be determined by one skilled in the art based on the desired osmolality.

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

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

Method and use

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

In some embodiments, the cells are in a subject, e.g., a human subject, e.g., a subject suffering from an ANGPTL 3-related disease, or a subject in need of prophylaxis of risk of an ANGPTL 3-related disease.

In some embodiments, the cell is located in vitro. The method is based on research purposes or used to construct animal models.

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., as compared to the expression level of ANGPTL3 prior to the cell first being contacted with the nucleic acid; e.g., prior to administration of a first dose of the nucleic acid to the subject). In certain embodiments, inhibiting expression of ANGPTL3 reduces the level of ANGPTL3 protein in a serum sample of a subject by at least 50%, 60%, 70%, 80%, 90% or 95%, e.g., as compared to the expression level of ANGPTL3 prior to the first contact of the cell with the nucleic acid.

The invention also provides the use of a nucleic acid as described above, a targeted drug delivery system as described above or a pharmaceutical composition as described above in any of the following aspects: 1) Treating and/or preventing a disease associated with ANGPTL 3; 2) Preparing a medicament for treating and/or preventing diseases related to ANGPTL 3.

In some embodiments, the disease is: (i) diseases associated with ANGPTL3 enhancement or elevation; or (ii) a disease that would benefit from reduced ANGPTL3 expression.

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 (e.g., type 2 diabetes), obesity, fatty liver (e.g., nonalcoholic fatty liver), knee injury, osteoarthritis, chylomicronemia syndrome, familial Partial Lipodystrophy (FPLD).

In the present invention, the subject may be a mammal, including a primate (e.g., human, non-human primate, e.g., monkey and chimpanzee), a non-primate (e.g., cow, pig, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, or mouse), or a bird. In some embodiments, the subject is preferably a primate, more preferably a human.

Administration may be by a variety of routes, depending on whether local or systemic treatment is desired. The amount of the drug to be administered may be as described above, and will not be described in detail herein.

In some embodiments, the administration may 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, transdermal, oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subsurface, e.g., via a grafting 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, intravenous, and/or intramuscular administration.

Examples

Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present invention, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

Example 1 in vitro screening of siRNA

After the sense strand and antisense strand sequences shown in Table 1 were obtained by screening, the sense strand and antisense strand were 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 the antisense strand in Table 2 were formed into double-stranded siRNA modified in Table 3 by solid phase synthesis.

TABLE 3 Table 3

0.5 Ml of cell culture medium (DMEM, 10% calf serum, 1% penicillin+streptomycin solution) containing 10 ⁴ Hep3B (Procell, cat#CL-0102) cells was added to a 96-well cell culture dish and incubated overnight in a 5% CO2 cell incubator at 37 ℃. RNAiMAX (1.5. Mu.l/well) and small interfering nucleic acid (siRNA) of Table 3 were added to Opti-MEM medium and the cell culture wells were added to a final concentration of 1nM or 10nM per well and the culture was continued for 48 hours at 37℃for a cell culture period of 5% CO 2. To extract RNA, the cell culture supernatant was blotted, rinsed with PBS, after blotting, 50. Mu.L of the prepared lysate (suggested by cell-to-CT kit (ThermoFisher Scientific, cat#4391851 c)) was added, mixed well, allowed to stand for 10min, and 2.5. Mu.L of Stop solution was added to terminate for 2min. RT-PCR was performed according to the proposal of HIGH CAPACITY CDNA REVERSE Transcription Kits (Thermo Fisher, cat. No.: 4368814) and contained 10. Mu.L of post-lysis liquid per reaction. The quantitative gene expression was determined by real-time fluorescent PCR, the TaqMan probe of human ANGPTL3 was Hs00205581_m1, and the probe of the internal reference gene (human HPRT 1) was Hs02800695_m1 (Thermo FISHER SCIENTIFIC, waltham, mass., USA). The PCR conditions were 95℃for 20 seconds for 1 cycle, 95℃for 1 second and 60℃for 20 seconds for 40 cycles, and the real-time fluorescent PCR instrument was QuantStudio ^TM Pro real-time fluorescent quantitative PCR system (Thermo Fisher). ANGPTL3 gene expression is calculated by 2-delta Ct, and human HPRT1 gene expression is used as an internal reference. ANGPTL3 gene expression levels were expressed relative to the RNAiMAX-only cell group as a control. The results are shown in Table 4.

TABLE 4 inhibition of ANGPTL3 Gene expression by siRNA in Hep3B cells

Numbering device	1nM	Standard deviation of	10nM	Standard deviation of
					SN-252194	87.5％	1.0％	84.8％	2.7％
SN-252197	89.5％	9.2％	96.8％	0.0％
					SN-252198	77.2％	2.4％	81.1％	2.0％
SN-252199	71.4％	3.7％	80.6％	1.1％
					SN-252201	91.4％	2.7％	92.6％	1.1％
SN-252203	85.0％	0.4％	89.5％	3.3％
					SN-252204	88.8％	3.8％	88.8％	0.4％
SN-252205	91.4％	0.3％	95.5％	1.0％
					SN-252207	76.4％	2.1％	79.7％	0.8％
SN-252208	81.7％	4.3％	83.9％	1.8％
					SN-252209	79.2％	1.7％	71.8％	1.0％
SN-252210	80.4％	0.4％	84.8％	2.7％
					SN-252212	53.9％	4.8％	56.6％	0.4％
SN-252214	69.8％	1.7％	70.5％	2.5％
					SN-252216	65.0％	4.1％	89.4％	0.3％
SN-252217	76.3％	0.3％	72.6％	1.9％
					SN-252218	89.4％	2.4％	89.7％	0.8％
SN-252219	83.3％	4.5％	76.4％	0.7％
					SN-252220	76.0％	4.7％	72.4％	0.7％
SN-252221	67.0％	1.7％	62.0％	5.9％
					SN-252222	79.3％	3.1％	82.7％	1.1％
SN-252223	70.0％	1.4％	67.0％	4.4％
					SN-252224	78.9％	1.5％	82.7％	0.4％
SN-252226	85.7％	1.3％	81.6％	1.3％
					SN-252227	61.2％	2.4％	45.7％	11.6％
SN-252228	78.8％	1.0％	86.8％	3.0％
					SN-252229	58.1％	6.4％	38.2％	6.5％
SN-252230	43.2％	3.9％	29.0％	23.8％
					SN-252232	60.8％	2.5％	54.9％	5.8％
SN-252233	48.5％	2.4％	53.2％	5.7％
					SN-252234	49.7％	5.6％	48.5％	17.0％
SN-252235	58.4％	1.2％	55.9％	0.2％
					SN-252236	62.3％	7.3％	65.0％	3.9％

From the results, the siRNA of the invention can precipitate the ANGPTL3 gene to different degrees, wherein ,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、SN-252228 has better inhibition effect, and the average inhibition effect is more than 78% at the concentration of 1 nM. And after comparing the silencing effect of siRNA with different sequences under the same modification on the expression of the ANGPTL3 gene in cells, the siRNA can be confirmed to have obvious effect advantage at the naked sequence level.

The preferred siRNAs in Table 4 were further dose-dependent tested in Hep3B cells. Specifically, RNAiMAX (1.5. Mu.l/well) and small interfering nucleic acid (siRNA) were mixed in Opti-MEM medium and added to the cell culture well to a final concentration of 0.005,0.01,0.04,0.12,0.37,1.1,3.3 or 10nM per well, followed by culturing at 37℃for 48 hours in a 5% CO2 cell culture period. RNA extraction and relative quantification were as described previously. IC ₅₀ of siRNA compounds inhibiting ANGPTL3 expression is shown in Table 5.

TABLE 5 IC ₅₀ values for inhibition of ANGPTL3 Gene expression by siRNA Compounds in Hep3B cells

Numbering device	IC50(nM)
		SN-252194	0.066
SN-252197	0.017
		SN-252201	0.004
SN-252203	0.014
		SN-252204	0.024
SN-252205	0.037
		SN-252208	0.029
SN-252210	<0.01
		SN-252218	0.015
SN-252219	0.048
		SN-252222	0.236
SN-252224	0.216
		SN-252226	0.136
SN-252228	0.158

To determine the activity of siRNA, siRNA preferred in table 5 (IC 50 less than 0.1 nM) was conjugated to hepatocyte-targeting compound Tri-GalNAc (see formula I above for specific structure, see table 6 for sequence involved) and free uptake experiments were performed in monkey primary hepatocytes, siRNA-GalNAc samples were dissolved in 100 μl of sterile water to 10000 μl of a solution, respectively, and 10 μl of 10000 μl of a test solution was diluted to 1000 μΜ of a solution as a 1000nM final concentration group working solution by adding 90 μ L PMonH plating medium; the final working solution was diluted 3-fold at 8 concentration points with PMonH plating medium to a final working solution concentration of 0.5,1.4,4, 12, 37, 111, 333, 1000nM. Removing monkey primary liver cells from liquid nitrogen, thawing at 37deg.C, recovering, washing with serum-containing PMonH plating medium, counting, centrifuging, removing supernatant, diluting cells to 250k/mL with new serum-containing PMonH plating medium, spreading 90 μl diluted cell solution onto 96-well cell culture plate to obtain cell number of 25k per well, adding prepared sample working solution to cell solution to obtain final concentration of 0.5,1.4,4, 12, 37, 111, 333, 1000nM, placing in 5% carbon dioxide incubator, The culture was carried out at 37℃for 48 hours. after 48 hours all medium in the 96 well plates was aspirated, washed with 1 XPBS buffer, mixed well by adding 50. Mu.L of the prepared Cellsto CT lysate (according to manufacturer's recommendations), left to stand for 10min, and terminated by adding 2.5. Mu.L of termination solution for 2min. RT-PCR was performed according to the proposal of HIGH CAPACITY CDNA REVERSE Transcription Kits (Thermo Fisher, cat. No.: 4368814) and contained 10. Mu.L of post-lysis liquid per reaction. The quantitative gene expression was measured by real-time fluorescence PCR, the TaqMan probe of monkey ANGPTL3 was Mf04384789_m1, and the probe of the internal reference gene (monkey PPIB) was Mf02802985_m1 (ThermoFisher Scientific, waltham, mass., USA). the PCR conditions were 95℃for 20 seconds for 1 cycle, 95℃for 1 second and 60℃for 20 seconds for 40 cycles, and the real-time fluorescent PCR instrument was QuantStudio ^TM 6. 6 Pro real-time fluorescent quantitative PCR system (Thermo Fisher). ANGPTL3 gene expression was calculated as 2- ΔΔct, PPIB gene expression as an internal reference. The siRNA concentration (IC 50) values for 50% reduction of ANGPTL3 expression were calculated as the percentage of ANGPTL3 gene silencing expression compared to the cell line with culture medium alone, as shown in table 7.

TABLE 6

Numbering device	Sense strand (5 '-3')	Antisense strand (5 '-3')
			SN-682194	cscsuagaAfgAfAfAfaaauucuacu-Tri-galNac	asGfsuAfgAfauuuuUfuCfuucuaggsasg
SN-682197	gsasagagCfaAfCfUfaacuaacuua-Tri-galNac	usAfsaGfuUfaguuaGfuUfgcucuucsusa
			SN-682201	csasgaagUfaAfCfUfucacuuaaaa-Tri-galNac	usUfsuUfaAfgugaaGfuUfacuucugsusu
SN-682203	csasuaguCfaAfAfUfaaaagaaaua-Tri-galNac	usAfsuUfuCfuuuuaUfuUfgacuaugscsu
			SN-682204	gsuscaaaUfaAfAfAfgaaauagaaa-Tri-galNac	usUfsuCfuAfuuucuUfuUfauuugacsusa
SN-682205	cscsacagAfaAfUfUfucucuaucuu-Tri-galNac	asAfsgAfuAfgagaaAfuUfucuguggsusu
			SN-682208	gsasauagAfuGfGfAfucacaaaacu-Tri-galNac	asGfsuUfuUfgugauCfcAfucuauucsgsa
SN-682210	gsasucacAfaAfAfCfuucaaugaaa-Tri-galNac	usUfsuCfaUfugaagUfuUfugugaucscsa
			SN-682218	csasuuauAfuUfGfAfauauucuuuu-Tri-galNac	asAfsaAfgAfauauuCfaAfuauaaugsusu
SN-682219	gsgsaaauCfaCfGfAfaaccaacuau-Tri-galNac	asUfsaGfuUfgguuuCfgUfgauuuccsusu

TABLE 7 silencing IC50 values of siRNA on ANGPTL3 Gene expression in monkey primary hepatocytes

Numbering device	IC50,nM	Numbering device	IC50,nM
				SN-682194	35.6	SN-682205	14.8
SN-682197	12.8	SN-682208	25.9
				SN-682201	3.0	SN-682210	3.3
SN-682203	9.5	SN-682218	10.6
				SN-682204	17.6	SN-682219	25.6

Therefore, the siRNA can better silence the ANGPTL3 gene, wherein the silencing effects of SN-682197, SN-682201, SN-682203, SN-682210 and SN-682218 are better, and the silencing IC50 value of the siRNA on the expression of the ANGPTL3 gene in the monkey primary liver cells is lower than 13nM.

Example 2 in vivo efficacy validation of siRNA

To further determine the activity of siRNA, experiments were performed in human ANGPTL3 transgenic mice with the preferred sirnas in table 7 as experimental groups and PBS as control group. 1 or 3mg/kg of siRNA-Tri-galNac compound or PBS was subcutaneously injected into mice on day 0, and the decrease effect was expressed as a percentage compared to the pre-injection ANGPTL3 protein in blood plasma measured on day 10, as shown in FIG. 1 (1 mg/kg) and FIG. 2 (3 mg/kg).

From FIGS. 1-2, it can be seen that the siRNA of the present invention can effectively reduce the protein level of ANGPTL3 in mice, wherein the inhibition effect of SN-682210 and SN-682218 is significantly better.

To further verify the activity of siRNA, human ANGPTL3 transgenic mice were subcutaneously injected with 1 or 3mg/kg of PBS, SN-682210, or SN-682218 drug on day 0, respectively, and the human ANGPTL3 protein levels in the blood were observed in a follow-up, as shown in fig. 3.

As can be seen from fig. 3, the ANGPTL3 protein level in blood of the human ANGPTL3 transgenic mice can still be effectively reduced after SN-682210 or SN-682218 targeting drugs are injected for more than 20 days. Wherein, compared with SN-682218, the drug effect of SN-682210 is further and better. Therefore, the efficacy of the experimental group injected with SN-682210 was continuously observed, and the results are shown in FIG. 4.

As can be seen from fig. 4, the SN-682210 targeting drug can still effectively reduce the ANGPTL3 protein level in blood of human ANGPTL3 transgenic mice for more than 40 days after injection.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.

Claims

1. A nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand contains a sequence having a sequence identity of more than 80% with a sequence shown in any one of SEQ ID NOs: 1 to 33, and the antisense strand contains a sequence having a sequence identity of more than 80% with a sequence of nucleotides 1 to 21 of a sequence shown in any one of SEQ ID NOs: 34 to 66.

2. 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.

3. The nucleic acid according to claim 1, wherein the antisense strand comprises 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, 51, 54, 56, 57, and 59 by 0, 1 or 2 nucleotides, and the sense strand comprises a nucleotide sequence that is at least partially complementary to the antisense strand; optionally, the sense strand comprises 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, 18, 21, 23, 24, and 26 by 0, 1 or 2 nucleotides.

4. The nucleic acid of claim 1, wherein the antisense strand comprises a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 45 by 0, 1 or 2 nucleotides, and the sense strand comprises 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.

5. The nucleic acid of claim 1, wherein at least one nucleotide in the nucleic acid is a modified nucleotide or comprises a modified internucleotide;

The modified nucleotide is preferably selected from one or more of 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2',3'-open ring nucleotide mimic, locked nucleotide, 2'-F-arabino nucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, reverse nucleotide, reverse 2'-O-methyl nucleotide, reverse 2'-deoxy nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide, vinyl phosphonate containing nucleotide, cyclopropyl phosphonate containing nucleotide and 3'-O-methyl nucleotide; the modified nucleotide is further preferably selected from one or more of 2'-O-methyl nucleotide and 2'-fluoro nucleotide;

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.

6. The nucleic acid according to claim 5, wherein 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; the 5' end of the sense strand contains two 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.

7. 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.

8. The nucleic acid according to claim 1, wherein the antisense strand comprises 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.

9. 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.

10 . 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.

12. according to the targeted drug delivery system described in claim 10 or 11, wherein, this targeted drug delivery system comprises the described nucleic acid connected with the part and the part; Preferably the described part is a GalNAc derivative; More preferably the described part is one or more GalNAc derivatives connected by single-stranded, double-stranded or triple-stranded branched linkers.

13. The targeted drug delivery system according to claim 10, wherein the structure of the targeted drug delivery system is as shown below:

In the formula, Nu represents the nucleic acid.

14. An isolated cell, characterized in that the cell contains the nucleic acid according to any one of claims 1 to 9.

15. A pharmaceutical composition, characterized in that it 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.

16. A method for inhibiting ANGPTL3 expression in a cell, the method comprising: contacting the cell with the nucleic acid of any one of claims 1 to 9, the targeted drug delivery system of any one of claims 10 to 13, or the pharmaceutical composition of claim 15, to inhibit ANGPTL3 expression in the cell.

17. 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.

18. 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.

19. The use according to claim 17, wherein the disease is 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.

20. The use according to claim 17, wherein the nucleic acid, the targeted drug delivery system or the pharmaceutical composition is administered to a subject by subcutaneous administration, intravenous administration and/or intramuscular administration.