Improved siRNAs for silencing pcsk9 expression and uses thereof

Abstract

Disclosed herein are improved siRNA compositions having sequences and modifications that mediate enhanced targeting of PCSK9 mRNA and methods of use thereof.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Applications Nos. 63 / 546,217, 63 / 546,215 and 63 / 546,216, filed on October 29, 2023, the disclosures of which are incorporated herein by reference in their entirety.

[0002] sequence list The contents of the electronic sequence list (129293021501_SL.xml; size: 739,907 bytes; creation date: October 28, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] This article discloses a double-stranded RNA molecule targeting the proprotein convertase subtilisin / kexin type 9 (PCSK9) and a method for using it to treat conditions regulated by PCSK9. Background Technology

[0004] The concept of gene silencing achieved through RNA interference (RNAi) was discovered in 1998, for example, as reported by Hu et al. (“Therapeutic siRNA: state of the art,”). Signal Transduction and Targeted Therapy (2020) 5:101). Article published by Tuschl et al. (Elbashir, SM) et al. , “Duplexes of 21±nucleotide RNAs mediate RNA interference in cultured mammalian cells,” Nature(2001) 411, 494-498) reported the use of chemically synthesized siRNA to silence specific genes in cultured mammalian cells. PCSK9 protein is primarily synthesized and secreted by the liver. PCSK9 plays a role in regulating the activity of low-density lipoprotein (LDL) receptor (LDLR) in hepatocytes, and LDLR binds PCSK9 on the cell membrane surface and / or in the cytoplasm. LDLR is also primarily responsible for competitively clearing circulating low-density lipoprotein cholesterol (LDL-C); therefore, a reduction in PCSK9 protein (e.g., by inhibiting its synthesis) can lead to increased LDL-C uptake and degradation. When PCSK9 binds to LDLR on the cell membrane, the PCSK9 / LDLR complex is internalized into the endosome, resulting in the degradation of both. Conversely, when LDL-C binds to LDLR and the complex is internalized, LDLR dissociates from LDL-C and can be recycled to the cell surface, while LDL-C is degraded (Chen...). et al ., “AReview of PCSK9 Inhibitors and their Effects on Cardiovascular Diseases,” Current Topics in Medicinal Chemistry, 2019, 19(20): 1790-1817). Therefore, the main objective of this invention is to silence the production of PCSK9 protein in hepatocytes by interfering with the translation of PCSK9 mRNA, thereby increasing the uptake of serum LDL-C by these cells more effectively than prior art.

[0005] Inclisiran, currently known as Leqvio in the United States and Europe. ® The first-generation synthetic siRNA to be marketed uses a conjugated ligand—a tri-antenna type. N -Acetylglucosamine (GalNAc)3—is conjugated to the 3' end of the positive strand (SS) of siRNA, thereby targeting hepatocytes. N Ac binds to the asialic acid glycoprotein receptor (ASGPR), which is primarily located on the surface of hepatocytes in the liver, and the conjugate is subsequently internalized. Inxil is usually administered subcutaneously, then enters the bloodstream, circulates systemically, and is taken up by hepatocytes. Once internalized into the cytoplasm of hepatocytes or other cells, inxil acts via the natural cellular pathway by binding to and cleaving PCSK9 messenger RNA (mRNA) to reduce PCSK9 expression. Because the antisense strand (AS) of the siRNA binds sequence-specifically to the target mRNA, theoretically only PCSK9 mRNA is affected, thereby reducing (i.e., silencing) the expression of that specific coding region and protein product.

[0006] The invention of inxile is a major therapeutic breakthrough for patients with hypercholesterolemia, particularly those who cannot tolerate statins (the previous treatment option) or whose response to statins is insufficient. While inxile currently offers a relatively new and important advance in the treatment of such patients, there remains a need for a significantly more effective PCSK9-targeting siRNA form. Summary of the Invention

[0007] This document discloses a double-stranded ribonucleic acid molecule comprising a sense strand and an antisense strand. The sense strand comprises the nucleic acid sequence of SEQ ID NO:1 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:2. The sense strand comprises the nucleic acid sequence of SEQ ID NO:3 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:4. The sense strand comprises the nucleic acid sequence of SEQ ID NO:5 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:6, or the sense strand comprises the nucleic acid sequence of SEQ ID NO:7 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:8. Attached Figure Description

[0008] The invention and the following detailed description can be better understood when read in conjunction with the accompanying drawings. To illustrate the disclosed double-stranded ribonucleic acid molecule and its methods of use, the drawings show exemplary embodiments of the compositions and methods; however, the compositions and methods are not limited to the specific embodiments disclosed. In the drawings: Figure 1 The modified sequence of the original inxlane is shown.

[0009] Figure 2 The dose-response curve for XD-44731 is shown.

[0010] Figure 3 The dose-response curve for XD-44725 is shown.

[0011] Figure 4 The dose-response curve for XD-44701 is shown.

[0012] Figure 5 The dose-response curve for XD-44695 is shown.

[0013] Figure 6 The dose-response curve for XD-44719 is shown.

[0014] Figure 7 The dose-response curve for XD-42182 is shown.

[0015] Figure 8 The dose-response curve for XD-44689 is shown.

[0016] Figure 9 The dose-response curve for XD-44682 is shown.

[0017] Figure 10 The dose-response curve of XD-44722 (2) is shown.

[0018] Figure 11 The dose-response curve for XD-44728 is shown.

[0019] Figure 12 The dose-response curve for XD-44698 is shown.

[0020] Figure 13 The dose-response curve for XD-44716 is shown.

[0021] Figure 14 The dose-response curve for XD-44692 is shown.

[0022] Figure 15 The dose-response curve for XD-44704 is shown. Detailed Implementation

[0023] The disclosed double-stranded RNA molecule and its methods of use can be more readily understood by referring to the following detailed description, which forms part of this disclosure, in conjunction with the accompanying drawings. It should be understood that the disclosed double-stranded RNA molecule and its methods of use are not limited to the specific compositions and methods described and / or shown herein, and the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the scope of the claimed double-stranded RNA molecule and its methods of use.

[0024] Unless otherwise expressly stated, any description of possible mechanisms, modes of action, or reasons for improvement is illustrative only, and the disclosed double-stranded RNA molecules and their methods of use are not subject to the validity of any such proposed mechanisms, modes of action, or reasons for improvement.

[0025] Throughout this document, a description relates to double-stranded RNA molecules and methods of using said double-stranded RNA molecules. Where the disclosure describes or claims features or embodiments related to double-stranded RNA molecules, those features or embodiments also apply to the methods of using said double-stranded RNA molecules. Similarly, where the disclosure describes or claims features or embodiments related to methods of using double-stranded RNA molecules, those features or embodiments also apply to the double-stranded RNA molecules themselves.

[0026] As used herein, the singular forms “a,” “an,” and “the / that” include plural references.

[0027] When a list is presented, unless otherwise stated, it should be understood that each individual element in the list and each combination of the list is a separate implementation. For example, a list of implementations presented as “A, B or C” should be interpreted as including implementations: “A”; “B”; “C”; “A or B”; “A or C”; “B or C”; or “A, B or C”.

[0028] When a numerical range is described or established herein, the range includes its endpoints and all individual integers and fractions within that range, and also includes each narrower range formed by all possible combinations of these endpoints and internal integers and fractions to form a subgroup of the stated larger range of values, as if each narrower range were explicitly described. When a numerical range is stated herein as being greater than a certain value, the range remains finite, and its upper limit is defined by a value operable within the context of the disclosure herein. When a numerical range is stated herein as being less than a certain value, the range remains bounded by its lower limit being a non-zero value. The scope of these compositions and methods is not intended to be limited to the specific numerical values ​​described when defining the range. All ranges are inclusive and composable.

[0029] When a numerical value is expressed as an approximation using the preposition "approximately," it should be understood that the specific value constitutes another implementation. Unless the context explicitly states otherwise, references to a specific numerical value must include at least that specific numerical value.

[0030] Various terms used in connection with the description of various aspects are used throughout the specification and claims. Unless otherwise stated, these terms shall have their ordinary meaning in the art. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein.

[0031] When used to refer to a range of values, cutoff values, or specific values, the term "about" is used to indicate that the listed values ​​may vary by a maximum of 10%. Since many of the values ​​used herein were determined experimentally, those skilled in the art will understand that such determination may vary between different experiments. The values ​​used herein should not be considered unduly restrictive due to this inherent variation. Therefore, the term "about" is used to include variations of ±10%, ±5%, ±1%, ±0.5%, or ±0.1% or less compared to the specified value.

[0032] The terms “treatment,” “management,” and similar terms refer to both therapeutic treatment and preventative or preventative measures, and include reducing the severity and / or frequency of symptoms, eliminating symptoms and / or the underlying cause of symptoms, reducing the frequency or likelihood of symptoms and / or their underlying cause, and improving or repairing damage caused directly or indirectly by double-stranded RNA molecules. Treatment also includes prolonging survival compared to the expected survival of untreated subjects. Subjects to be treated include those who have a condition or disorder, those who are susceptible to a condition or disorder, or those whose condition or disorder needs to be prevented.

[0033] As used herein, "administered to the patient" and similar terms refer to a procedure of injecting a double-stranded RNA molecule into a patient, such that the subject's target cells, tissues, or body segments come into contact with the double-stranded RNA molecule.

[0034] As used herein, “pharmaceuticalally acceptable carrier” or “pharmaceuticalally acceptable excipient” includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and to be unresponsive to the subject’s immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline or physiological saline (0.9%). Compositions containing such carriers are formulated using well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

[0035] As used herein, the term "subject" is intended to refer to any animal, particularly a mammal. Although this document exemplifies the treatment of mice and non-human primates with double-stranded RNA molecules, the disclosed methods can be used to treat any type of mammal. Therefore, the methods are applicable to both human and non-human animals, but are preferably used for mice, non-human primates, and humans, with humans being the most preferred application. The terms "subject" and "patient" are used interchangeably herein.

[0036] The term “comprising” is intended to include instances covered by the terms “substantially consisting of” and “consisting of”; similarly, the term “substantially consisting of” is intended to include instances covered by the term “consisting of”.

[0037] RNA interference (RNAi) is a naturally occurring biological phenomenon in which siRNA functions by interacting with endogenous cellular proteins to form a structure called the RNA-induced silencing complex (RISC). siRNA contains a sense strand (SS) and an antisense strand (AS) with at least partially paired bases, which may or may not be covalently linked. In some embodiments, the siRNA enters the RISC, which separates the two strands and uses the antisense strand (complementary to the target mRNA) to guide the complex to the target mRNA transcribed from the source gene, whereupon the mRNA is cleaved. As a result, the fragmented target mRNA cannot be translated into its encoded protein, thus “silencing” its expression.

[0038] Every protein produced by a cell has a unique sequence translated from the unique gene that encodes it. Therefore, at least in theory, the translation of any gene into the protein it encodes can be silenced.

[0039] The intracellular mechanisms by which siRNA achieves gene silencing have recently been reviewed (e.g., Hu...). et al ., “Therapeutic siRNA: state of the art,” Signal Transduction and Targeted Therapy (2020) 5:101). Synthetic siRNA comprises double-stranded RNA (dsRNA) containing SS and AS, wherein each or nearly each ribonucleic acid is modified at the 2' position, for example, by an F or O-methyl group or other modification. siRNA is introduced into cells via one or more suitable delivery methods that bring the dsRNA into contact with the cell, allowing the dsRNA to enter the cytoplasm. Transmembrane transport of dsRNA can be active or passive. As those skilled in the art will understand, targeting siRNA-containing dsRNA to specific cell types and / or body tissues may be desirable for treating specific pathologies, reducing or eliminating adverse side effects (such as immune responses), and, among other practical considerations, minimizing the effective dose of the siRNA drug required to effectively treat a subject.

[0040] Just like Hu et al As noted by others, the synthesis and synthetic modification of siRNAs have been introduced due to the poor stability of unmodified siRNAs caused by degradation by RNases and phosphatases. U.S. Patent No. 10,851,377 (and in particular Table B therein, which is incorporated herein by reference) includes many examples of synthetically produced modified nucleotides now known in the art.

[0041] This article discloses siRNAs with modified ribonucleic acid (RNA), each ribonucleic acid containing modifications such as 2'-F or 2'-O-methyl groups, having one or more phosphate thioester (PS) bonds in its backbone, and also containing biostable phosphate mimics such as vinyl-phosphonate caps, Gal... N Ac and other modifications. The latter are included in the design of these synthetic siRNAs because they have been shown to confer some resistance to endonuclease cleavage of PS bonds. In some implementations, Gal N Ac and / or vinyl-phosphonate caps are optional.

[0042] The modification sequence of the original inclisiran is shown in Figure 1 See Table 1.

[0043] Table 1

[0044] For information on letter code modifications, please refer to Table 3.

[0045] This disclosure provides one or more chemically engineered macromolecules that are similar to but different from inxlane in sequence and modifications, mimicking and further enhancing the activity of inxlane small interfering RNA (siRNA). Various sequences of the unmodified improved siRNAs disclosed herein are shown in Table 2.

[0046] Table 2: Sequence-level improved siRNAs

[0047] in: A' is a nucleoside containing adenosine. C' is a cytosine-containing nucleoside. G' is a nucleoside containing guanine. T' is a thymine-containing nucleoside, and U' is a nucleoside containing uracil.

[0048] Table 3: Keys for Synthesized and Modified siRNA Structures Unless otherwise stated, all nucleosides are linked by phosphodiester bonds.

[0049] L96 is .

[0050] As used herein, bases such as adenine, cytosine, guanine, uracil, and thymine, including their modified and deoxygenated forms, can refer to heterocyclic bases themselves or nucleotides or nucleosides containing that base.

[0051] In some embodiments of the present invention, the double-stranded siRNA conjugate comprises a positive strand of 21 nucleotides in length, wherein the conjugate is a triantral type N -acetylglucosamine (Gal) N The 2'-O-methyladenine conjugated to the Ac)3 ligand is located at position 21, starting from the 5' end. The antisense strand of the dsRNA conjugate contains 23 modified nucleotides. In some embodiments of the invention, the nucleotides comprising the sense and antisense strands are modified compared to naturally occurring RNA. In several embodiments of the invention, phosphate thioester bonds link nucleotides 1=2 and 2=3 of the sense strand, and nucleotides 1=2, 2=3, 21=22, and 22=23 of the antisense strand (Formula III), wherein nucleotides 22 and 23 of the antisense strand are generally unpaired. (The nucleotide numbering here is indicated starting from the 5' end of each strand).

[0052] Three antennae N -acetylglucosamine (Gal) N The Ac)3 ligand, also known as L96, is fused to the 3' end of the positive-sense chain. The structure of the L96 ligand is shown, for example, in U.S. Patent Nos. 8,273,866 and 8,828,956 and Formula I. (Gal) N Ac), with single, double, and triple tentacles, is a well-defined liver-targeting motif, benefiting from its high affinity for the desialyl glycoprotein receptor (ASGPR). By directly conjugating it to oligonucleotides or modifying it into certain delivery systems as a targeting motif, Gal... N Ac has achieved remarkable success in the development of nucleic acid therapeutics in recent years. Several oligonucleotide drugs (modalities) are undergoing pivotal clinical trials.

[0053] In some embodiments, the antisense strand nucleotide 1 is 2'-O-methyluracil, which has a 5'-( E )-Vinylphosphonate (vinyl-phosphonate cap). For double-stranded siRNAs, it has been shown that the vinyl-phosphonate cap at this position protects the 5' end of the AS from degradation by phosphatases and 5'-3' exonucleases (see, e.g., Haraszti). et al .,“5’-Vinylphosphonate improves tissue accumulation and efficacy of conjugatedsiRNAs in vivo “, Nucleic Acids Research, 7 June 2017, 45(13): 7581-7592.” This protective effect can lead to increased stability of siRNA conjugates in vivo and has the potential to improve the expression of the silencing target gene (PCSK9 in this article) in clinical efficacy.

[0054] In some embodiments of the invention, modifications to the nucleotides may include baseless ribose rings (1'-H), locked nucleic acids (LNA; see Formula IV), or deoxyribonucleic acid (DNA), with or without further modifications. In some embodiments of the invention, the identity of each nucleotide (whether modified or not) can be any natural or synthetic nucleotide, deoxyribonucleotide, etc., with the only desired limitation being that the opposing regions of the sense and antisense strands are largely complementary, such that opposing nucleotides can form base pairs between the sense and antisense strands. Any single phosphate thioester (PS) bond within either strand of the dsRNA conjugate can be R p or S p This is because the chirality of the bond does not appear to affect the activity of the complex, and it is difficult to separate during synthesis.

[0055] As shown in the figure, the SS of inclisiran contains 21 modified ribonucleic acid nucleotides. On the other hand, the AS contains 23 modified ribonucleic acid nucleotides, where the nucleotide at the 3' end of the sense strand (or position 21 from the 5' end) is complementary to the nucleotide at position 1 of the 5' end of the sense strand, position 20 of the SS from the 5' end is complementary to position 2 of the AS from the 5' end, and so on. Therefore, when the two strands pair at other positions, the two nucleotides at positions 22 and 23 of the antisense strand are unpaired.

[0056] As those skilled in the art recognize, each complementary siRNA strand (SS and AS) must pair with a specific sequence of at least a certain length of the other strand of the dsRNA, and any modifications therein can affect the activity of the siRNA in its intended and unintended functions in unpredictable ways. When siRNA strands are modified, it may be useful to experimentally determine, under specific conditions, whether the designed siRNA interacts with the endogenous cellular mechanisms required to silence or reduce the expression of the target gene mRNA, and further, whether and how the siRNA (antisense strand) effectively recognizes the target mRNA based on its specific composition. Another consideration is the rate of degradation of the therapeutic siRNA en route to and within the target cell. Recognizing mRNAs other than the target gene has the potential to cause a variety of off-target side effects. Modified siRNAs also have the potential to trigger undesirable systemic effects, such as immune responses, which must be experimentally detected in vivo. After identifying a specific gene as a target, the challenges of therapeutic gene silencing include delivery, stability, and specificity.

[0057] This article discloses a double-stranded ribonucleic acid molecule comprising a sense strand and an antisense strand. The sense strand contains the nucleic acid sequence of SEQ ID NO:1 and the antisense strand contains the nucleic acid sequence of SEQ ID NO:2, the sense strand contains the nucleic acid sequence of SEQ ID NO:3 and the antisense strand contains the nucleic acid sequence of SEQ ID NO:4, the sense strand contains the nucleic acid sequence of SEQ ID NO:5 and the antisense strand contains the nucleic acid sequence of SEQ ID NO:6, or the sense strand contains the nucleic acid sequence of SEQ ID NO:7 and the antisense strand contains the nucleic acid sequence of SEQ ID NO:8.

[0058] In some embodiments, the positive strand contains the nucleic acid sequence of SEQ ID NO: 1, and the negative strand contains the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the positive strand contains the nucleic acid sequence of SEQ ID NO: 3, and the negative strand contains the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the positive strand contains the nucleic acid sequence of SEQ ID NO: 5, and the negative strand contains the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the positive strand contains the nucleic acid sequence of SEQ ID NO: 7, and the negative strand contains the nucleic acid sequence of SEQ ID NO: 8.

[0059] The sense strand of any double-stranded nucleic acid molecule disclosed herein may contain an L96. The L96 may be conjugated to the 5' end of the sense strand. The L96 may be conjugated to the 3' end of the sense strand.

[0060] The antisense strand of any double-stranded nucleic acid molecule disclosed herein may contain a biologically stable phosphate mimic, such as a vinyl-phosphonate cap. In some embodiments, the vinyl-phosphonate cap is (vinu). (vinu) may be conjugated to the 5' end of the antisense strand.

[0061] The double-stranded ribonucleic acid molecules disclosed herein may comprise a sense strand and / or an antisense strand, which contain one or more modified nucleotides. In some embodiments, the sense strand comprises one or more modified nucleotides. In some embodiments, the antisense strand comprises one or more modified nucleotides. In some embodiments, both the sense strand and the antisense strand comprise one or more modified nucleotides.

[0062] The one or more modified nucleotides include, but are not limited to, the modifications described in Table 3. For example, a double-stranded RNA molecule may contain one or more modified nucleotides selected from 2'-O-methyladenosine, 2'-fluoroadenosine, 2'-O-methylcytidine, 2'-fluorocytidine, 2'-O-methylguanosine, 2'-fluoroguanosine, 2'-O-methyluridine, and 2'-fluorouridine, or any combination thereof. A double-stranded RNA molecule may contain 2'-O-methyladenosine. A double-stranded RNA molecule may contain 2'-fluoroadenosine. A double-stranded RNA molecule may contain 2'-O-methylcytidine. A double-stranded RNA molecule may contain 2'-fluorocytidine. A double-stranded RNA molecule may contain 2'-O-methylguanosine. A double-stranded RNA molecule may contain 2'-fluoroguanosine. A double-stranded RNA molecule may contain 2'-O-methyluridine. A double-stranded RNA molecule may contain 2'-fluorouridine.

[0063] The double-stranded ribonucleic acid (BRNA) molecules disclosed herein may contain one or more 2'-deoxynucleotides selected from 2'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxythymidine, and 2'-deoxyguanosine, or any combination thereof. Double-stranded BRNA molecules may contain 2'-deoxyadenosine. Double-stranded BRNA molecules may contain 2'-deoxycytidine. Double-stranded BRNA molecules may contain 2'-deoxythymidine. Double-stranded BRNA molecules may contain 2'-deoxyguanosine.

[0064] The double-stranded ribonucleic acid (BRNA) molecules disclosed herein may contain nucleotides linked by one or more phosphodiester bonds, one or more thiophosphate bonds, or any combination of phosphodiester bonds and thiophosphate bonds. Double-stranded BRNA molecules may contain one or more phosphodiester bonds. Double-stranded BRNA molecules may contain one or more phosphodiester bonds and one or more thiophosphate bonds.

[0065] The double-stranded ribonucleic acid molecules disclosed in this article may contain one or more glycol nucleic acid (GNA) nucleotides.

[0066] The double-stranded ribonucleic acid (RNA) molecules disclosed herein may contain one or more 2'-4'-locked nucleic acid (LNA) nucleotides. The double-stranded RNA molecules may contain adenine 2'-4'-LNA nucleotides, cytosine 2'-4'-LNA nucleotides, guanine 2'-4'-LNA nucleotides, thymine 2'-4'-LNA nucleotides, or any combination thereof. In some embodiments, the double-stranded RNA molecule contains one or more adenine 2'-4'-LNA nucleotides. In some embodiments, the double-stranded RNA molecule contains one or more cytosine 2'-4'-LNA nucleotides. In some embodiments, the double-stranded RNA molecule contains one or more guanine 2'-4'-LNA nucleotides. In some embodiments, the double-stranded RNA molecule contains one or more thymine 2'-4'-LNA nucleotides.

[0067] This article discloses a double-stranded ribonucleic acid molecule comprising a sense strand and an antisense strand, wherein the sense strand and antisense strand are respectively SEQ ID NO: 9 and 10; SEQ ID NO: 11 and 12; SEQ ID NO: 13 and 14; SEQ ID NO: 15 and 16; SEQ ID NO: 17 and 18; SEQ ID NO: 19 and 20; SEQ ID NO: 21 and 22; SEQ ID NO: 23 and 24; SEQ ID NO: 25 and 26; SEQ ID NO: 27 and 28; SEQ ID NO: 29 and 30; SEQ ID NO: 31 and 32; SEQ ID NO: 33 and 34; SEQ ID NO: 35 and 36; SEQ ID NO: 37 and 38; SEQ ID NO: 39 and 40; and SEQ ID NO: 39 and 40. 41 and 42; containing SEQ ID NO: 43 and 44 respectively; containing SEQ ID NO: 45 and 46 respectively; containing SEQ ID NO: 47 and 48 respectively; containing SEQ ID NO: 49 and 50 respectively; containing SEQ ID NO: 51 and 52 respectively; containing SEQ ID NO: 53 and 54 respectively; containing SEQ ID NO: 55 and 56 respectively; containing SEQ ID NO: 57 and 58 respectively; containing SEQ ID NO: 59 and 60 respectively; containing SEQ ID NO: 61 and 62 respectively; containing SEQ ID NO: 63 and 64 respectively; containing SEQ ID NO: 65 and 66 respectively; containing SEQ ID NO: 67 and 68 respectively; containing SEQ ID NO: 69 and 70 respectively; or containing SEQ ID NO: 71 and 72 respectively.

[0068] This article discloses a double-stranded ribonucleic acid molecule comprising a sense strand and an antisense strand. The sense strand contains the nucleic acid sequence of SEQ ID NO:15, and the antisense strand contains the nucleic acid sequence of SEQ ID NO:16.

[0069] This article discloses a double-stranded ribonucleic acid molecule comprising a sense strand and an antisense strand. The sense strand contains the nucleic acid sequence of SEQ ID NO:49, and the antisense strand contains the nucleic acid sequence of SEQ ID NO:50.

[0070] This article discloses a double-stranded ribonucleic acid molecule comprising a sense strand and an antisense strand. The sense strand contains the nucleic acid sequence of SEQ ID NO:21, and the antisense strand contains the nucleic acid sequence of SEQ ID NO:22.

[0071] This article discloses a double-stranded ribonucleic acid molecule comprising a sense strand and an antisense strand. The sense strand contains the nucleic acid sequence of SEQ ID NO:47, and the antisense strand contains the nucleic acid sequence of SEQ ID NO:48.

[0072] This article discloses a double-stranded ribonucleic acid molecule comprising a sense strand and an antisense strand. The sense strand contains the nucleic acid sequence of SEQ ID NO:57, and the antisense strand contains the nucleic acid sequence of SEQ ID NO:58.

[0073] This article discloses a double-stranded ribonucleic acid molecule comprising a sense strand and an antisense strand. The sense strand contains the nucleic acid sequence of SEQ ID NO:53, and the antisense strand contains the nucleic acid sequence of SEQ ID NO:54.

[0074] This document discloses a method for treating a PCSK9-regulated condition in a subject, the method comprising administering to the subject an effective amount of the double-stranded RNA molecule as described in any of the preceding claims. The PCSK9-regulated condition may be hypercholesterolemia or mixed dyslipidemia. In some embodiments, the PCSK9-regulated condition is hypercholesterolemia. In some embodiments, the PCSK9-regulated condition is mixed dyslipidemia. The treatment methods disclosed herein can result in a decrease in PCSK9 protein levels and / or a decrease in low-density lipoprotein (LDL) levels.

[0075] The treatment methods disclosed herein may include administering to a subject any disclosed double-stranded RNA molecule at a dose of about 0.25 mg / kg to about 5 mg / kg. In some embodiments, the dose is about 0.25 mg / kg. In some embodiments, the dose is about 0.5 mg / kg. In some embodiments, the dose is about 1.0 mg / kg. In some embodiments, the dose is about 2.0 mg / kg. In some embodiments, the dose is about 5 mg / kg. This dose may be administered as a pharmaceutical composition. This dose may be administered by injection. In some embodiments, the injection is a subcutaneous injection.

[0076] This document discloses pharmaceutical compositions comprising any double-stranded RNA disclosed herein and a pharmaceutically acceptable carrier.

[0077] The siRNA disclosed in this paper serves as an incoxlane substitute in terms of both sequence and novel, non-obvious chemical modifications, producing a lower IC50 compared to the original incoxlane. 50Therefore, the PCSK9-targeting siRNA of this invention is completely different from those of inxlane.

[0078] Example The following examples are provided to further describe some embodiments disclosed herein. These examples are intended to illustrate, and not limit, the disclosed embodiments.

[0079] The siRNA of this invention is related to incoherent but has novel and non-obvious chemical modifications, producing, in particular, a lower IC50 than that of the original incoherent under the same experimental conditions ("for comparison with similar organisms"). 50 .

[0080] Example 1. Screening scheme for modified siRNA conjugates 1. siRNA production Synthesis of 2'-modified oligonucleotides Single-chain oligonucleotides were synthesized using standard phosphoramidyl oligomerization chemistry according to standard solid-phase oligonucleotide synthesis techniques, wherein the oligonucleotides were assembled on a solid support using a 96-well Mermade 96E synthesizer (LGC Bioautomation) controlled by a Poseidon software package.

[0081] Synthesis was carried out on a solid support made of controlled-pore glass (CPG). Specifically, the guide chain was assembled on a 500 Å general-purpose solid support (#DS0500, 500 Å porosity) purchased from Biocomma. For the guest chain modified with a tri-antennae GalNAc cluster ligand (L96-tris-N-acetylgalactosamine (GalNAc)-cluster (J. Am. Chem. Soc. 2014, 136, 16958)) at the 3' end, synthesis was carried out on an L96-GalNAc-cluster (#ON-469, 500 Å porosity, 55 µmol / g loading) immobilized on a CPG solid support purchased from Hongene biotech, China.

[0082] All 2'-modified RNA phosphoramidites and most auxiliary reagents were purchased from SAFC, Proligo, and SigmaAldrich (now Merck, Hamburg, Germany). Specifically, the following 2'-O-methylphosphoramidites were used: (5'-O-dimethoxytriphenylmethyl-N6-(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramid, (5'-O-dimethoxytriphenylmethyl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramid, (5'-O-dimethoxytriphenylmethyl-N2-(isobutyryl)-2 '-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide and 5'-O-dimethoxytriphenylmethyl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphamide. 2'-Deoxy-2'-fluorophosphamide has the same protecting group as 2'-O-methylRNAphosphamide. Other phosphoramids were purchased from the following sources: 5'-(E)-vinylphosphonate-2'-OMe-U (POM (Protective) (#PR5-032) phosphorous amide and (S)-ethylene glycol nucleic acid phosphorous amide (GNA-A(bz) (#OP-033), GNA-C(ac) (#OP-041), GNA-G(ibu) (#OP-194), GNA-U (#OP-193) and GNA-T (#OP-032)) were purchased from Hongene biotech, Hamburg; 5'-O-dimethoxytriphenylmethyl-2'-O-deoxy-thymidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphorous amide was purchased from SAFC, Proligo, SigmaAldrich (now Merck, Hamburg, Germany). The phosphorous amide solution (50 mM) used was prepared from the corresponding commercially available phosphorous amide in anhydrous acetonitrile with the addition of molecular sieves (3 Å, 8-12 mesh, Car Roth, #N893.1).

[0083] 5-Ethiotetrazole (ETT, 500 mM acetonitrile solution, 99.8%, Biosolve #0022112402BS) was used as the activating agent solution. The coupling time was 6 minutes. To introduce the thiophosphate bond, a solution of 100 mM 3-amino-1,2,4-dithiazol-5-thione (or xanthate hydrazine, purchased from TCI Chemicals, Germany, (purity: >96.0% (HPLC) cas:6846-35-1)) dissolved in ACN-pyridine (2:3 v / v) was used as the sulfiding agent. Other auxiliary reagents used were as follows: iodine oxidant (50 mM I2 (dissolved in pyridine-H2O (9:1 v / v))), TCA (3%, dissolved in DCM) for deblocking, and Cap A (acetic anhydride in THF (9.1:90.9 v / v)) and Cap B (THF, N-methylimidazole, and pyridine (8:1:1 v / v / v)) as capping agents. All oligonucleotides were synthesized with the final DMT protecting group removed (“DMT-Off”).

[0084] Cleavage and deprotection of support-bound oligomers After solid-phase synthesis, oligonucleotides were cleaved from the solid support by adding AMA (a 1:1 (v / v) mixture of concentrated ammonia and 40% methylamine aqueous solution, both purchased from Sigma Aldrich) and collected in 96-well plates. To achieve quantitative removal of all protecting groups, the solution was incubated with shaking at 33 °C. The sample was then dried under reduced pressure (using a SpeedVac concentrator (ThermoFisher)), and the solid residue was redissolved in 250 mM tris(hydroxymethyl)aminomethane (TRIS) (pH 7.0) to obtain a crude sample solution for subsequent purification.

[0085] Purification of oligonucleotides The crude extract was then purified by anion exchange chromatography using a Dionex DNA Pac100 (9 x 250 mm) column (ThermoFisher, Dreieich, Germany) on an ÄKTA Purifier system (GE Healthcare, Freiburg, Germany) equipped with an autosampler (A905) and a fraction collector (Frac-950). Buffer A was a 20 mM TRIS pH 7.4 solution containing 20% ​​acetonitrile, and buffer B was a 500 mM sodium perchlorate solution of buffer A. A flow rate of 5 mL / min and a gradient from 10% to 100% buffer B were used within 15 column volumes (CV). UV traces at 260 nm and 280 nm were recorded to monitor product elution. Appropriate fractions were combined and precipitated overnight in a refrigerator using 3 M NaOAc (pH=5.2) in ethanol (1:32 v / v). The precipitate was collected by centrifugation and redissolved in purified water. The purified sense and antisense strands were quantitatively analyzed by measuring UV absorption at 260 nm. Subsequently, the materials were evaluated to ensure they met pre-agreed MS identification (calculated mass + / - 0.05% (by ESI-MS)) and purity (single-strand purity >85%, based on UV signal integration from the analytical AEX trace) specifications.

[0086] Oligonucleotide annealing to generate siRNA The purified complementary strands were then mixed in an equimolar ratio and dried in a SpeedVac concentrator. They were subsequently prepared in annealing buffer (40 mM NaH₂PO₄·H₂O + Na₂HPO₄ buffer, pH 6.8, 0.2 M NaCl) and annealed (the double-stranded solution was placed in a 70 °C water bath and then cooled to room temperature over 3 hours) to produce the required amount (in nmol or mg) of siRNA for subsequent experiments. The obtained siRNA duplexes were characterized by size exclusion chromatography (SEC) to determine if they met the pre-agreed purity specifications (double-stranded purity >90%, based on the UV signal integral of the analytical SEC trace).

[0087] 2. Cell culture work Cell culture and transfection HuH-7 cells were purchased from CLS (through the German distributor Hoelzel Diagnostics, catalog number #300156) and cultured in DMEM medium (Biochrom #F0435). Hep3B cells were purchased from ATCC (ATCC in collaboration with LGC Standards, Wesel, Germany, catalog number #HB-8064) and cultured in EMEM medium (#ATCC #30-2003). Both media were supplemented with 10% fetal bovine serum (#1248D, Biochrom GmbH, Berlin, Germany) and 100 U / ml penicillin / 100 µg / ml streptomycin (#A2213, Biochrom GmbH, Berlin, Germany). Cells were cultured in a humidified incubator at 37 °C with a 5% CO2 atmosphere. For siRNA transfection, cells were seeded at 15,000 cells / well in 96-well tissue culture plates.

[0088] Transfection of siRNA was performed using Lipofectamine 2000 (0.5 µl / well for HuH7 cells) and Lipofectamine RNAiMax (0.3 µl / well for Hep3B cells), both purchased from Invitrogen / Thermo (Karlsruhe, Germany), following the manufacturer's reverse transfection instructions. Dual-dose selection was performed in HuH7 cells, with siRNA at concentrations of 20 nM and 0.3 nM in quadruple replicates. As controls, siRNAs targeting Ahsa1, firefly luciferase, and FVII were used as nonspecific controls and as a mock transfection. Ahsa1 siRNA was also used as a positive control for transfection efficiency (detecting Ahsa1 mRNA levels instead of PCSK9). Dosage-response experiments were performed with 10 concentrations in quadruple replicates, starting at 30 nM and decreasing to approximately 15 fM in 5-fold dilution steps. After incubation for 24 hours, the culture medium was removed, and the cells were lysed in 150 µl of a culture medium-lysis mixture (1 volume of lysis buffer, 2 volumes of cell culture medium), and then incubated at 53 °C for 30 minutes. The lysates were frozen for analysis.

[0089] Direct uptake in primary hepatocytes Cryopreserved primary hepatocytes were purchased from Primacyt (Schwerin, Germany, human hepatocyte batch CHM2221-HE-C, cynomolgus monkey hepatocyte batch # CH141204). Dosage-response experiments were performed in quadruple replicates at 10 concentrations, starting at 5 µM and decreasing to approximately 20 pM with 4-fold dilutions. After 5 hours, the siRNA-containing plating medium was replaced with maintenance medium (Primacyt catalog number #HHMM). The medium was changed every 24 hours. After 48 hours of incubation, the medium was removed, and cells were lysed in 200 μl of medium-lysis mixture (1 volume of lysis mixture, 2 volumes of cell culture medium containing 1 μL / mL proteinase K) and incubated at 53°C for 30 minutes. The lysates were cryopreserved until further analysis.

[0090] mRNA quantification mRNA levels were quantified post-transfection using a branched DNA assay (Quantigene Singleplex version (Thermo, #QS0014)). The assay was performed according to the manufacturer's instructions, using probe sets for PCSK9 and GAPDH specific to their respective species, both designed by ThermoFisher Scientific and synthesized by Metabion (Planegg, Germany).

[0091] Table 4: Probe sets for PCS97 and human GAPDH

[0092] After incubation at room temperature in the dark for 30 minutes, the luminescence values ​​were read using a 1420 luminescence detector (WALLAC VICTOR Light, Perkin Elmer, Rodgau-Jügesheim, Germany). For each well, the PCSK9 mRNA level was normalized relative to the corresponding GAPDH mRNA level. The activity of a given PCSK9 siRNA is expressed as the percentage of PCSK9 mRNA concentration (normalized to GAPDH mRNA) in treated cells relative to the average PCSK9 mRNA concentration (normalized to GAPDH mRNA) in control wells.

[0093] 3. RNA-Seq specificity analysis In vitro working and library preparation Cell culture in HepB3 cells:For RNA-Seq analysis of cell lines, Hep3B cells purchased from ATCC (ATCC in collaboration with LGC Standards, Wesel, Germany, catalog number #HB-8064) were used. For RNA-Seq experiments, Hep3B cell lines were transfected with Lipofectamine RNAiMAX (4.5 µL / well, Thermo Fisher Scientific, Karlsruhe, Germany) in 6-well plates at a final siRNA concentration of 10 nM for 24 hours, with triple transfection performed.

[0094] Library preparation: Following siRNA treatment, Hep3B cells were lysed in RNA tissue lysis buffer (Mannheim, Germany, catalog number #03604721001), and total RNA was isolated using the MagNa Pure 96 Cell RNA Mass Enlargement Kit (Mannheim, Germany, catalog number #05467535001) on a Roche MagNa Pure 96 system (Roche, Mannheim, Germany). RNA quality was monitored using the RNA ScreenTape assay kit (Santa Clara, USA, catalog numbers #50675576; 50675578; 50675577) on an Agilent TapeStation system (Santa Clara, USA). RIN only. eRNA with a concentration >8 was used for subsequent RNA-Seq library preparation. RNA concentration was quantified on a Qubit® analyzer (Karlsruhe, Germany) using the Qubit® RNA Wide Range Kit (Thermo Fisher Scientific, Karlsruhe, Germany, Catalog No. #Q10211). Sequencing libraries were prepared using the TruSeq stranded mRNA Kit (Illumina, Berlin, Germany, Catalog No. #20020595) on a fully automated NGS Star (Hamilton, Switzerland), following the manufacturer's instructions, by inputting 500 ng of RNA. After preparation, the library was quantified on a Qubit® analyzer using the Qubit® dsDNA HS Quantification Kit (Thermo Fisher Scientific, Karslruhe, Germany, Catalog No. #Q32854), and the library size distribution was analyzed on an Agilent TapeStation system (Santa Clara, USA) using the DNA ScreenTape Analysis Kit (Santa Clara, USA, Catalog Nos. #50675586; 50675583; 50675582). After calculating the final library concentration, the libraries were normalized to 10 nM and pooled for subsequent single-end sequencing. The pooled libraries were denatured, diluted to 1.7 pM, and loaded onto reagent cartridges for sequencing according to the manufacturer's instructions. Sequencing was performed on a NextSeq 550 sequencer (Illumina, Darmstadt, Germany) using the High Output Kit v2.5 75 cycles (Darmstadt, Germany, catalog number #20024906). Single-end sequencing of 75 bp was performed, estimating 20 to 30 million reads per library. The adapter-trimmed raw reads were submitted for bioinformatics analysis.

[0095] Data Analysis Original reading segment quality check: The raw reads generated on the Illumina NextSeq550 (fastq.gz file format) were used as input for quality checks (PHRED score, reads per library, read length) using the software FastQC (version 0.11.0) (bioinformatics.babraham.ac_uk / projects / fastqc / ).

[0096] Reading quality trimming: Use the software TrimGalore (version 0.6.4) (bioinformatics.babraham.ac_uk / projects / trim_galore / ) to remove raw reads with a PHRED score < 28. Then, repeat FastQC (version 0.11.0) (bioinformatics.babraham.ac_uk / projects / fastqc / ) on the pruned reads.

[0097] Read segments were mapped to the human reference genome: The pruned reads were aligned to the human genome using the software STAR (version 2.7.3) (STAR: Dobin, Alexander, et al. "STAR: ultrafast universal RNA-seq aligner." Bioinformatics 29.1 (2013): 15-21). (ftp.ncbi.nlm.nih_gov / genomes / all / annotation_releases / 9606 / GCF_000001405.40-RS_2023_03 / GRCh38_major_release_seqs_for_alignment_pipelines / GCA_000001405.1) The BAM file is generated by combining the data from 5_GRCh38_full_analysis_set.fna.gz and the corresponding reference annotation from NCBI (ftp.ncbi.nlm.nih_gov / genomes / all / annotation_releases / 9606 / GCF_000001405.40-RS_2023_03 / GRCh38_major_release_seqs_for_alignment_pipelines / GCA_000001405.1 5_GRCh38_full_analysis_set.refseq_annotation.gtf.gz).

[0098] Segment counting and normalization:Using the BAM file generated from read alignment, the read counts for each gene were calculated using the software featureCounts (version 2.0.3) (featureCounts (subread): Liao, Yang, Gordon K. Smyth, and Wei Shi. "featureCounts: an efficient general purpose program for assigning sequencereads to genomic features." Bioinformatics 30.7 (2014): 923-930), generating a read count matrix. The read counts were then normalized using the DESeq2 method (DESeq2: Love, Michael I., WolfgangHuber, and Simon Anders. "Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2." Genome biology 15 (2014): 1-21).

[0099] Principal component analysis and differential expression analysis: Normalized read counts were used as input to the DESeq2 software for principal component analysis (PCA) and differential expression analysis (DEA). For PCA, variance stabilization transformation (VST) was performed as recommended in the manual. DEA was performed according to the DESeq2 manual. Here, log fold change shrinkage was applied to account for low-expression and potentially noisy genes (method: asher).

[0100] Further analysis and data visualization: All further analyses and visualizations were generated using R statistical software.

[0101] Dual-dose screening was performed using GalNAc-conjugated PCSK9 siRNA, with quadruple incubations at concentrations of 5 µM and 50 nM, respectively. A positive control siRNA targeting PCSK9 (XD-15878 (original inxilelan) or its derivative XD-36954 (shown in Table 5)) was used as the gold standard, exhibiting equivalent activity at its respective concentration on each cell culture plate. Untreated cells served as a negative control.

[0102] Table 5

[0103] Preliminary dose-response experiments (data not shown) were conducted using designed PCSK9 siRNA conjugates obtained from the first initial applications, with quadruple incubations at five concentrations (10.0, 1.429, 0.204, 0.029, and 0.004 µM). Notably, the original inxile was also tested in the same experiments at these concentrations.

[0104] Subsequently, experiments were conducted using PCSK9 siRNA at 10 concentrations, with quadruple incubation starting at 10 µM and then sequentially diluted to approximately 500 pM or 40 pM using 3-fold or 4-fold dilution steps.

[0105] In both DRC experiments, inxril was used as a positive control on each plate, and untreated cells were used as a negative control.

[0106] Sequential optimization of modifications to improve the efficacy of inxlane Example 2. Vinu-modified Inxlane [XD-42180] The nucleic acid sequences of the sense strand (SS 5'-3') and antisense strand (AS 5'-3') of the designed XD-42180 siRNA duplex are shown in Table 6 according to the symbols provided in Table 3.

[0107] Table 6

[0108] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data are shown in Tables 7 and 8. IC50 was determined based on dose-response curves (DRC). 50 The concentration was 0.015 µM, while the IC50 of the original incoherent was 0.015 µM under the same experimental conditions (hepatocyte batches). 50 =0.059 µM, showing an approximately 4-fold increase in activity.

[0109] Table 7

[0110] Table 8

[0111] Example 3. DV18-Vinu modified Inxlane [XD-44683] The nucleic acid sequences of the sense strand (SS 5'-3') and antisense strand (AS 5'-3') of the designed siRNA conjugate duplex are shown in Table 9 according to the symbols provided in Table 3. The IC50 of DV18-vinu modified inxlane was determined based on the dose-response curve (DRC).50 The value was 0.005 µM, while the IC50 of Inxlane measured under the same conditions was... 50 = 0.033 µM, showing an activity increase of more than 6 times.

[0112] Table 9

[0113] Table 10: DV18-Vinu modified Inkstone

[0114] Table 11

[0115] Example 4. Exemplary modified inxile DV18_vinu_4F_SS11 DNA [XD-44687] The nucleic acid sequences of the sense strand (SS 5'-3') and antisense strand (AS 5'-3') of the double-stranded siRNA conjugate designed for the second-phase test are shown in Table 12 according to the symbols provided in Table 3.

[0116] Table 12

[0117] Table 13: Incoceles modified with DV18 + 4F_SS11 DNA

[0118] Table 14

[0119] An empty cell represents an outlier removed from a quadruple.

[0120] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data are shown in Tables 13 and 14. IC50 was determined based on dose-response curves (DRC). 50 The value was 0.03 µM, while the IC50 of the original Inxlane measured under the same conditions was 0.03 µM. 50 = 0.37 µM, showing an activity increase of more than 12-fold.

[0121] Example 5. Unmodified Inxlane The nucleic acid sequences of the sense strand (SS 5'-3') and antisense strand (AS 5'-3') of the double-stranded design siRNA conjugates tested are shown in Table 1. As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes. Data are shown in Table 14. IC50 was determined based on the dose-response curve (DRC). 50 For IC 50 = 0.37 µM.

[0122] The nucleic acid sequences of the sense strand (SS 5'-3') and antisense strand (AS 5'-3') of the designed siRNA conjugate double strands for the second-phase testing are shown in Table 15 according to the symbols provided in Table 3. If the sequence contains one or more locked nucleic acids (LNAs), the strand containing this modification (SS or AS) is identified by the modified nucleotide number starting from the 5' end and "LNA" after the name (short format sequence modification). For example, "3512_4F_AS6OMe_16LNA" contains the LNA at position 16 of AS. Other modifications containing corresponding sequence information are indicated by the sequence symbol itself.

[0123] Table 15: Sequences based on 3512

[0124] The nucleic acid sequences of the sense strand (SS 5'-3') and antisense strand (AS 5'-3') of the designed siRNA conjugate duplexes for the second-phase testing are shown in Table 16 according to the symbols provided in Table 3. If the sequence contains one or more locked nucleic acids (LNAs), the strand containing this modification (SS or AS) is identified by the modified nucleotide number starting from the 5' end and "LNA" after the name (short format sequence modification). For example, "3545_4F_AS6OMe_16LNA" contains the LNA at position 16 of AS. Other modifications containing corresponding sequence information are indicated by the sequence symbol itself.

[0125] Table 16. Sequences based on 3545

[0126] Example 6. 3512_4F_AS6OMe_16LNA [XD-44731] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 2 And Table 17. IC50 determined based on dose-response curve (DRC). 50The value was 0.11 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing more than 3-fold increase in activity.

[0127] Table 17

[0128] data MV quadruple [% residual target mRNA] data SD quadruple Example 7. 3512_4F_AS6DNA [XD-44725] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 3 And Table 18. IC50 determined based on dose-response curve (DRC). 50 The value was 0.05 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing more than 7-fold improvement in the in vitro activity of the designed siRNA.

[0129] Table 18

[0130] data MV (mean) quadruple replicates [% residual target mRNA] data SD (standard deviation) four replicates Example 8. 3512_4F_SS7+10LNA [XD-44701] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 4 And Table 19. IC50 determined based on dose-response curve (DRC). 50 The value was 0.08 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing more than 4-fold increase in activity.

[0131] Table 19

[0132] data MV quadruple [% residual target mRNA] data SD quadruple Example 9. 3512_4F_SS11ab [XD-44695] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 5 And Table 20. IC50 determined based on dose-response curve (DRC). 50 The value was 0.10 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing more than 3-fold increase in activity.

[0133] Table 20

[0134] data MV quadruple [% residual target mRNA] data SD quadruple Example 10. 3512_4F_AS6OMe [XD-44719] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 6 And Table 21. IC50 determined based on dose-response curve (DRC). 50 The value was 0.02 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing an activity increase of more than 18 times.

[0135] Table 21

[0136] data MV quadruple [% residual target mRNA] data SD quadruple Example 11. 3512_DV18_vinu [XD-42182] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 7 And Table 22. IC50 determined based on dose-response curve (DRC). 50 The value was 0.07 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing more than 5-fold increase in activity.

[0137] Table 22

[0138] data MV quadruple [% residual target mRNA] data SD quadruple Example 12. 3512_4F_SS11 DNA [XD-44689] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 8 And Table 23. IC50 determined based on dose-response curve (DRC). 50 The value was 0.14 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing more than 2-fold increase in activity.

[0139] Table 23

[0140] data MV quadruple [% residual target mRNA] data SD quadruple Example 13. 3545_DV18_vinu [XD-44682] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 9 And Table 24. IC50 determined based on dose-response curve (DRC). 50 The value was 0.03 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing an activity increase of more than 12-fold.

[0141] Table 24

[0142] data MV quadruple [% residual target mRNA] data SD quadruple Example 14. 3545_4F_AS6DNA [XD-44722] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 10 And Table 25. IC50 determined based on dose-response curve (DRC). 50 This is the most significant improvement for both datasets, where the IC50 is 0.01 µM. This is comparable to the IC50 of the original inxlane measured in the same experiment. 50 Compared to 0.37 µM, this variant showed an approximately 37-fold increase in activity.

[0143] Table 25

[0144] data MV quadruple [% residual target mRNA] data SD quadruple Example 15. 3545_4F_AS6OMe_16LNA [XD-44728] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 11 And Table 26. IC50 determined based on dose-response curve (DRC). 50 The value was 0.02 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing an over 18-fold increase in the activity of the modified siRNA.

[0145] Table 26

[0146] data MV quadruple [% residual target mRNA] data SD quadruple Example 16. 3545_4F_SS7+10LNA [XD-44698] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 12 And Table 27. IC50 determined based on dose-response curve (DRC). 50 The value was 0.07 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing more than 5-fold increase in activity.

[0147] Table 27

[0148] data MV quadruple [% residual target mRNA] data SD quadruple Example 17. 3545_4F_AS6OMe [XD-44716] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 13 And Table 28. IC50 determined based on dose-response curve (DRC). 50 The value was 0.02 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing an activity increase of more than 18 times.

[0149] Table 28

[0150] data MV quadruple [% residual target mRNA] data SD quadruple Example 18. 3545_4F_SS11ab [XD-44692] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 14 And Table 29. IC50 determined based on dose-response curve (DRC). 50 The value was 0.08 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing more than 4-fold increase in activity.

[0151] Table 29

[0152] data MV quadruple [% residual target mRNA] data SD quadruple Example 19. 3545_4F_SS7+10LNA_9+11DNA [XD-44704] As described in Example 1, the activity of the designed siRNA complex in reducing PCSK9 mRNA expression was determined in cultured primary human hepatocytes at a series of 10 concentrations. Data were presented in... Figure 15 And Table 30. IC50 determined based on dose-response curve (DRC). 50 The value was 0.13 µM, while the IC50 of the original inxlane was measured in the same experiment. 50 = 0.37 µM, showing a nearly 3-fold increase in activity.

[0153] Table 30

[0154] data MV quadruple [% residual target mRNA] data SD quadruple Example 20. Sequence Optimization Seven siRNAs and incoherent were analyzed in one experiment to ensure proper head-to-head comparisons were performed.

[0155] Table 31: Dose-response analysis of seven selected siRNAs and inxilelan in primary human hepatocytes and Huh7 cells:

[0156] Three siRNAs were selected for further development and experimentation, as shown in Tables 32 to 35.

[0157] Table 32: Activity analysis of selected siRNAs in primary cynomolgus monkey hepatocytes

[0158] Table 33: Synthesis of two GNA-modified variants for each parental sequence, and dose-response analysis of nine siRNAs and incoherent in Huh7 cells.

[0159] Table 34: RNA-Seq analysis of six siRNAs and incoherent.

[0160] Example 21. Mouse Experiment Mice. Human PCSK9-exon 12 (UTR) knock-in mice were generated in a C57BL6 / N background using CRISPR-Cas9. The knock-in strategy aimed to replace the entire mouse exon 12 (1230 bp, encoding 70 C-terminal amino acids and a 1020 bp 3' untranslated region) with human exon 12 (1484 bp in total length, encoding 71 C-terminal amino acids and a 1271 bp 3' untranslated region).

[0161] Male homozygous human PCSK9-exon 12 (UTR) knock-in mice were randomly fed a 2018 Teklad global 18% protein rodent diet (Inotiv, catalog number #2018). Each group of mice (n=4-5) received a single subcutaneous injection of a specified dose of inxexlan or COR-1003. Tail tip blood was collected (using EDTA-treated blood collection tubes) before injection (day 0) or on days 14 and 28 post-injection. After centrifugation at 3500g for 10 minutes at 4°C, plasma was transferred to fresh tubes and stored at -80°C. Plasma PCSK9 levels were determined by ELISA as previously described (PNAS 105:11915-20, 2008. pubmed.ncbi.nlm.nih_gov / 18695239 / ). Plasma PCSK9 levels in mice treated with specified doses of control, inxexlan, or XD-78827 at specified time points are shown in Table 35.

[0162] Table 35: PCSK9 levels in mice (ng / mL)

[0163] Example 22. Non-human primate experiments The pharmacodynamic effects of XD-44719, XD-44722, XD-44687, and XD-78827 on reducing PCSK9 and LDL cholesterol levels were evaluated at Charles River Laboratories (Study No. NC-PCS-24-001). Circulating plasma PCSK9 protein and serum LDL cholesterol levels were measured after a single subcutaneous injection in four groups of animals (n=3–4 / group). Briefly, plasma PCSK9 protein levels (ng / mL) were measured using the R&D System's Human PCSK9 Quantikine ELISA Kit. PCSK9 protein levels were quantified using a Molecular Devices SPECTRAmax® M@ or SPECTRAmax® M5 microplate reader. Serum LDL cholesterol levels (mg / dL) were measured using a Roche Diagnostics Cobas® 6000 analyzer.

[0164] The PCSK9 protein levels of monkeys treated with specified doses of XD-44719, XD-44722, XD-44687, or XD-78827 at specified time points are shown in Table 36. The LDL levels of monkeys treated with specified doses of XD-44719, XD-44722, XD-44687, or XD-78827 at specified time points are shown in Table 37.

[0165] Those skilled in the art will understand that many changes and modifications can be made to the preferred embodiments disclosed herein, and that such changes and modifications can be made without departing from the spirit of the invention. Therefore, the appended claims are intended to cover all equivalent variations that fall within the true spirit and scope of the invention.

[0166] The disclosure of every patent, patent application, and publication cited or described in this document is incorporated herein by reference in its entirety.

[0167] Table 38: Sequences

[0168] Implementation Plan The following list of implementation schemes is intended to supplement, rather than replace or exceed, the preceding description.

[0169] Implementation scheme 1A. Any new PCSK9 siRNA whose IC50 is at least 25%, 50%, 75%, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times lower than that of the original incoherent under the same conditions.

[0170] Implementation Scheme 2A. A method of using PCSK9 siRNA of Implementation Scheme 1, comprising administering the siRNA to a subject, such as a human, thereby treating a disease or condition.

[0171] Implementation Scheme 3A. A method for preparing PCSK9 siRNA, comprising synthesizing the siRNA.

[0172] Implementation Scheme 1B. A double-stranded ribonucleic acid molecule comprising: The sense strand contains the nucleic acid sequence of SEQ ID NO: 1 and the antisense strand contains the nucleic acid sequence of SEQ ID NO: 2; The sense strand contains the nucleic acid sequence of SEQ ID NO: 3 and the antisense strand contains the nucleic acid sequence of SEQ ID NO: 4; The sense strand contains the nucleic acid sequence of SEQ ID NO: 5 and the antisense strand contains the nucleic acid sequence of SEQ ID NO: 6; or The sense strand contains the nucleic acid sequence of SEQ ID NO: 7 and the antisense strand contains the nucleic acid sequence of SEQ ID NO: 8.

[0173] Implementation Scheme 2B. A double-stranded ribonucleic acid molecule as described in Implementation Scheme 1B, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 1 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 2.

[0174] Implementation Scheme 3B. A double-stranded ribonucleic acid molecule as described in Implementation Scheme 1B, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 3 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 4.

[0175] Implementation Scheme 4B. A double-stranded ribonucleic acid molecule as described in Implementation Scheme 1B, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 5 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 6.

[0176] Implementation Scheme 5B. A double-stranded ribonucleic acid molecule as described in Implementation Scheme 1B, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 7 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 8.

[0177] Implementation Scheme 6B. A double-stranded ribonucleic acid molecule as described in any of the preceding implementation schemes, wherein the positive strand comprises L96.

[0178] Implementation Scheme 7B. A double-stranded ribonucleic acid molecule as described in any of the preceding embodiments, wherein the antisense strand comprises (vinu).

[0179] Implementation Scheme 8B. A double-stranded ribonucleic acid molecule as described in any of the preceding embodiments, wherein the sense strand and / or the antisense strand comprises one or more modified nucleotides.

[0180] Implementation Scheme 9B. A double-stranded ribonucleic acid molecule as described in Implementation Scheme 8B, wherein one or more modified nucleotides are selected from 2'-O-methyladenosine, 2'-fluoroadenosine, 2'-O-methylcytidine, 2'-fluorocytidine, 2'-O-methylguanosine, 2'-fluoroguanosine, 2'-O-methyluridine, and 2'-fluorouridine.

[0181] Implementation Scheme 10B. A double-stranded ribonucleic acid molecule as described in any of the preceding implementation schemes, comprising one or more deoxynucleotides selected from deoxyadenosine, deoxycytidine, and deoxythymidine.

[0182] Implementation Scheme 11B. A double-stranded ribonucleic acid molecule as described in any of the preceding embodiments, comprising nucleotides linked by one or more phosphodiester bonds, one or more thiophosphate bonds, or any combination of phosphodiester bonds and thiophosphate bonds.

[0183] Implementation scheme 12B. A double-stranded ribonucleic acid molecule as described in any of the preceding implementation schemes, comprising one or more glycol nucleic acid (GNA) nucleotides.

[0184] Implementation scheme 13B. A double-stranded ribonucleic acid molecule as described in any of the preceding implementation schemes, comprising one or more 2'-4'-locked nucleic acid (LNA) nucleotides.

[0185] Implementation Scheme 14B. A double-stranded ribonucleic acid molecule as described in Implementation Scheme 1B, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 15 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 16.

[0186] Implementation Scheme 15B. A double-stranded ribonucleic acid molecule as described in Implementation Scheme 1B, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 49 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 50.

[0187] Implementation Scheme 16B. A double-stranded ribonucleic acid molecule as described in Implementation Scheme 1B, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 21 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 22.

[0188] Implementation Scheme 17B. A double-stranded ribonucleic acid molecule as described in Implementation Scheme 1B, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 47 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 48.

[0189] Implementation Scheme 18B. A double-stranded ribonucleic acid molecule as described in Implementation Scheme 1B, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 57 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 58.

[0190] Implementation Scheme 19B. A double-stranded ribonucleic acid molecule as described in Implementation Scheme 1B, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 53 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO: 54.

[0191] Implementation Scheme 20B. A method for treating a PCSK9-regulated condition in a subject, the method comprising administering to the subject an effective amount of a double-stranded ribonucleic acid molecule as described in any of the preceding embodiments.

[0192] Implementation Scheme 21B. The method as described in Implementation Scheme 20B, wherein the condition regulated by PCSK9 is hypercholesterolemia or mixed dyslipidemia.

[0193] Implementation Scheme 22B. A pharmaceutical composition comprising a double-stranded ribonucleic acid molecule as described in any of the preceding embodiments and a pharmaceutically acceptable carrier.

Claims

1. A double-stranded ribonucleic acid molecule comprising: The sense strand containing the nucleic acid sequence of SEQ ID NO: 1 and the antisense strand containing the nucleic acid sequence of SEQ ID NO: 2; The sense strand containing the nucleic acid sequence of SEQ ID NO: 3 and the antisense strand containing the nucleic acid sequence of SEQ ID NO: 4; The sense strand containing the nucleic acid sequence of SEQ ID NO: 5 and the antisense strand containing the nucleic acid sequence of SEQ ID NO: 6; or The sense strand containing the nucleic acid sequence of SEQ ID NO: 7 and the antisense strand containing the nucleic acid sequence of SEQ ID NO:

8.

2. The double-stranded ribonucleic acid molecule of claim 1, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 1 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:

2.

3. The double-stranded ribonucleic acid molecule of claim 1, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 3 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:

4.

4. The double-stranded ribonucleic acid molecule of claim 1, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 5 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:

6.

5. The double-stranded ribonucleic acid molecule of claim 1, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 7 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:

8.

6. The double-stranded RNA molecule as claimed in any of the preceding claims, wherein the positive strand comprises L96.

7. The double-stranded ribonucleic acid molecule as claimed in any of the preceding claims, wherein the antisense strand comprises (vinu).

8. The double-stranded ribonucleic acid molecule as claimed in any of the preceding claims, wherein the sense strand and / or the antisense strand comprises one or more modified nucleotides.

9. The double-stranded ribonucleic acid molecule of claim 8, wherein one or more modified nucleotides are selected from 2'-O-methyladenosine, 2'-fluoroadenosine, 2'-O-methylcytidine, 2'-fluorocytidine, 2'-O-methylguanosine, 2'-fluoroguanosine, 2'-O-methyluridine, and 2'-fluorouridine.

10. The double-stranded ribonucleic acid molecule as claimed in any of the preceding claims, comprising one or more deoxynucleotides selected from deoxyadenosine, deoxycytidine, and deoxythymidine.

11. The double-stranded ribonucleic acid molecule as claimed in any of the preceding claims, comprising nucleotides linked by one or more phosphodiester bonds, one or more thiophosphate bonds, or any combination of phosphodiester bonds and thiophosphate bonds.

12. The double-stranded ribonucleic acid molecule as claimed in any of the preceding claims, comprising one or more glycol nucleic acid (GNA) nucleotides.

13. The double-stranded ribonucleic acid molecule as claimed in any of the preceding claims, comprising one or more 2'-4'-locked nucleotides.

14. The double-stranded ribonucleic acid molecule of claim 1, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 15 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:

16.

15. The double-stranded ribonucleic acid molecule of claim 1, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 49 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:

50.

16. The double-stranded ribonucleic acid molecule of claim 1, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 21 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:

22.

17. The double-stranded ribonucleic acid molecule of claim 1, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 47 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:

48.

18. The double-stranded ribonucleic acid molecule of claim 1, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 57 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:

58.

19. The double-stranded ribonucleic acid molecule of claim 1, wherein the sense strand comprises the nucleic acid sequence of SEQ ID NO: 53 and the antisense strand comprises the nucleic acid sequence of SEQ ID NO:

54.

20. A method of treating a PCSK9-regulated condition in a subject, the method comprising administering to the subject an effective amount of a double-stranded RNA molecule as described in any of the preceding claims.

21. The method of claim 20, wherein the condition regulated by PCSK9 is hypercholesterolemia or mixed dyslipidemia.

22. A pharmaceutical composition comprising a double-stranded ribonucleic acid molecule as described in any of the preceding claims and a pharmaceutically acceptable carrier.

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